Systemic Lupus Erythematosus: A Companion to Rheumatology

Other companion titles in the Rheumatology series:

Ankylosing Spondylitis and the Spondyloarthropathies Psoriatic and Reactive Arthritis Osteoporosis and the Osteoporosis of Rheumatic Diseases Osteoarthritis

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SYSTEMIC LUPUS ERYTHEMATOSUS

ISBN 13: 978-0-323-04434-9 ISBN 10: 0-323-04434-4

Copyright © 2007 by Mosby, Inc., an affiliate of Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Health Sciences Rights Department in Philadelphia, PA, USA: phone: (+1) 215 239-3804, fax: (+1) 215 239-3805, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://www.elsevier.com), by selecting "Customer Support" and then "Obtaining Permissions."

Notice Emergency Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the Publisher nor the editors assume any liability for any injury and/or damage to person or property arising from this publication. The Publisher

Library of Congress Cataloging-in-Publication Data Systemic lupus erythematosus: a companion to Rheumatology / editors, George C. Tsokos, Caroline Gordon, Josef Smolen. – 1st ed. p.; cm. ISBN 0-323-04434-4 1. Systemic lupus erythematosus. I. Tsokos, George C. II. Gordon, Caroline, 1956- III. Smolen, Josef S., 1950IV. Rheumatology. [DNLM: 1. Lupus Erythematosus, Syetmic. WR 152 S99492 2007] RC924.5.L85S967 2007 616.7’7—dc22 2006046716

Acquisitions Editor: Kim Murphy Developmental Editor: Matthew Ray Project Manager: Bryan Hayward

Printed in the United States of America Last digit is the print number: 9

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To all the patients who teach, inspire, and motivate us

Contributing Authors

Nabih I. Abdou, MD, PhD Clinical Professor of Medicine Division of Allergy, Immunology, and Rheumatology St. Luke’s Hospital University of Missouri Kansas City, Missouri Matthew Adler, MRCP Professor of Rheumatology University College London London, United Kingdom Joseph M. Ahearn, MD Associate Professor of Medicine Division of Clinical Immunology and Rheumatology University of Pittsburgh School of Medicine Co-Director, Lupus Center of Excellence University of Pittsburgh School of Health Sciences Pittsburgh, Pennsylvania Graciela S. Alarcón, MD, MPH Jane Knight Lowe Chair of Medicine Division of Rheumatology The University of Alabama at Birmingham Birmingham, Alabama Mustafa Al-Maini, MD Anca D. Askanase, MD, MPH Associate Professor of Clinical Medicine Division of Rheumatology New York University School of Medicine Attending Physician Department of Rheumatology New York University Hospital for Joint Diseases New York, New York

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James E. Balow, MD Professor of Medicine Uniformed Services University of the Health Sciences Clinical Director, National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland Jacques Banchereau, MD Director, Baylor Institute for Immunology Research Dallas, Texas Adjunct Professor of Biomedical Studies Baylor University Medical Center Waco, Texas Ana M. Bertoli, MD Mary Kirkland Post-doctoral Research Fellow of Medicine Division of Rheumatology The University of Alabama at Birmingham Birmingham, Alabama Dan J. Birmingham, PhD Associate Professor of Medicine The Ohio State University Columbus, Ohio Markus Böhm, MD Professor of Dermatology University of Münster Münster, Germany Stefano Bombardieri, MD Professor of Rheumatology Department of Internal Medicine University of Pisa Pisa, Italy Gisela Bonsmann, MD Department of Dermatology University of Münster Münster, Germany

Dimitrios Boumpas, MD, FACP Professor of Medicine and Chief Department of Internal Medicine Division of Rheumatology, Clinical Immunology and Allergy University Hospital of Crete Heraklion, Greece Jill P. Buyon, MD Professor of Medicine Division of Rheumatology New York University School of Medicine Vice Chairman Department of Rheumatology New York University Hospital for Joint Diseases New York, New York Edward K.L. Chan, PhD Professor of Oral Biology Professor of Anatomy and Cell Biology Member, Center for Orphan Autoimmune Disorders Member, Shands Cancer Center University of Florida Gainesville, Florida Bhabadeb Chowdhury, PhD Scientist Laboratory of Molecular Immunology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland Robert M. Clancy, PhD Associate Professor of Medicine Division of Rheumatology New York University School of Medicine New York, New York Ann E. Clark, MD, MSc Associate Professor of Medicine McGill University Health Centre Quebec, Canada

Bruce N. Cronstein, MD Professor of Medicine, Pathology, and Pharmacology Department of Medicine Division of Clinical Pharmacology New York University School of Medicine Attending Physician Department of Medicine Bellevue Hospital New York, New York Erika Darrah, BS Graduate Student Department of Immunology Johns Hopkins University School of Medicine Baltimore, Maryland Alastair Denniston, MRCP, MRCOphth Clinical Lecturer Academic Unit of Ophthalmology University of Birmingham Birmingham and Midland Eye Center Birmingham, United Kingdom Steven G. Dimitriou, DO Department of Nephrology and Transplantation Temple University Hospital Philadelphia, Pennsylvania Ron du Bois, MD Professor of Medicine Division of Occupational and Environmental Medicine Imperial College London, United Kingdom Jennifer R. Elliot, MD Third Year Rheumatology Research Fellow Department of Rheumatology and Clinical Immunology University of Pittsburgh Pittsburgh, Pennsylvania Marvin J. Fritzler, MD, PhD Professor of Medicine University of Calgary Alberta, Canada Bill Giannakopoulos, MB, BS, FRACP NH&MRC Funded Research Scholar (Autoimmunity) Department of Medicine University of New South Wales New South Wales, Australia

Gary S. Gilkeson, MD Professor of Medicine Division of Rheumatology Medical University of South Carolina Chief, Rheumatology Service Medical Service Ralph H. Johnson VA Medical Center Charleston, South Carolina William R. Gilliland, MD Associate Professor of Medicine Division of Immunology and Rheumatology Uniformed Services University of Health Sciences Bethesda, Maryland Dafna D. Gladman, MD, FRCPC Professor of Medicine Division of Rheumatology University of Toronto Senior Scientist Toronto Western Research Institute Toronto Western Hospital Ontario, Canada Caroline Gordon, MA, MD, FRCP Reader in Rheumatology, University of Birmingham Consultant Rheumatologist, City Hospital University Hospital Birmingham NHS Trust Foundation Birmingham, United Kingdom John G. Hanly, MD Professor of Rheumatology Department of Medicine and Pathology Dalhousie University Rheumatologist Queen Elizabeth II Health Sciences Centre Nova Scotia, Canada John B. Harley, MD, PhD Professor of Medicine & James R. McEldowney Chair in Immunology University of Oklahoma Member & Program Head Department of Arthritis and Immunology Oklahoma Medical Research Foundation Oklahoma City, Oklahoma E. Nigel Harris, MD Vice Chancellor and Professor of Medicine Office of the Vice Chancellor The University of the West Indies Kingston, Jamaica

Georges Hauptmann, MD Professor of Medicine Institute of Immunology Louis Pasteur University Strasbourg, France Lee A. Hebert, MD Professor of Nephrology Department of Internal Medicine Division of Nephrology The Ohio State University Columbus, Ohio

CONTRIBUTING AUTHORS

Megan E.B. Clowse, MD, MPH Assistant Professor of Medicine Division of Rheumatology and Immunology Duke University Medical Center Durham, North Carolina

Robert Hoffman, DO Professor of Medicine Division of Microbiology and Immunology University of Miami Chief, Division of Rheumatology and Immunology Jackson Memorial Hospital Miami, Florida Gabor G. Illei, MD Head, Sjogren's Syndrome Clinic Gene Therapy and Therapeutics Branch National Institute of Dental and Craniofacial Research National Institutes of Health Bethesda, Maryland David Isenberg, MD, FRCP ARC Diamond Jubilee Professor of Rheumatology Centre for Rheumatology University College London London, United Kingdom Shozo Izui, MD Professor of Medicine Department of Pathology and Immunology Centre Médicale Universitaire University of Geneva Geneva, Switzerland Judith A. James, MD, PhD Lou Kerr Chair in Biomedical Research Department of Rheumatology and Immunology Oklahoma Medical Research Foundation Associate Professor of Medicine Adjunct Associate Professor of Pathology Department of Medicine Oklahoma University Medical Center Oklahoma City, Oklahoma

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CONTRIBUTING AUTHORS

Yuang-Taung Juang Amy H. Kao, MD, MPH Assistant Professor of Medicine Division of Clinical Immunology and Rheumatology University of Pittsburgh School of Medicine Assistant Professor Lupus Center for Excellence University of Pittsburgh School of Health Sciences Pittsburgh, Pennsylvania Jennifer A. Kelly, MPH Senior Research Assistant Department of Arthritis and Immunology Oklahoma Medical Research Foundation Oklahoma City, Oklahoma Munther A. Khamashta, MD, FRCP, PhD Senior Lecturer Lupus Research Unit The Rayne Institute King’s College School of Medicine London, United Kingdom Steve Krillis, MB, BS, PhD Professor of Medicine University of New South Wales Professor and Director Department of Immunology, Allergy, and Infectious Disease St. George Hospital New South Wales, Australia Sandeep Krishnan, MD, PhD Research Associate Cellular Injury Walter Reed Army Institute of Research Silver Spring, Maryland Annegret Kuhn, MD Department of Dermatology University of Münster Münster, Germany Vasileios C. Kyttaris, MD Instructor of Medicine Harvard Medical School Attending Physician Department of Medicine Division of Rheumatology Beth Israel Deaconess Medical Center Boston, Massachusetts

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Robert G. Lahita, MD, PhD Professor of Medicine Mount Sinai School of Medicine New York, New York Chairman Department of Medicine Jersey Medical Center Jersey City, New Jersey

Larissa Lapteva, MD Sjorgren’s Syndrome Clinic National Institute of Dental and Craniofacial Research National Institute of Health Bethesda, Maryland

Michael P. Madaio MD Renal Electrolyte and Hypertension Division University of Pennsylvania Medical Center Philadelphia, Pennsylvania

Xiaolan Li, MD Associate Professor of Medicine Department of Dermatology The Second Medical School of Kunming Medical College Vice-Doctor-in-Chief Department of Dermatology and Rheumatology The Second Affiliated Hospital of Kunming Medical College Kunmig-Yunnan, China

Marcos E. Maldonado, MD Assistant Professor of Medicine Division of Rheumatology and Immunology Department of Medicine Miller School of Medicine University of Miami Miami, Florida

Stamatis-Nick Liossis, MD Lecturer Department of Medicine University of Patras Medical School Attending Phsyician Division of Rheumatology Patras University Hospital Patras, Greece Chau-Ching Liu, MD, PhD Professor of Medicine Division of Clinical Immunology and Rheumatology University of Pittsburgh School of Medicine Lupus Center for Excellence University of Pittsburgh School of Health Sciences Pittsburgh, Pennsylvania Kui Liu, PhD Division of Rheumatology and Center for Immunology University of Texas Southwestern Medical Center Dallas, Texas Michael D. Lockshin, MD Professor of Medicine Division of Obstetrics and Gynecology Weill Medical College of Cornell University Attending Physician Department of Medicine Division of Rheumatology New York Presbyterian Hospital New York, New York Thomas A. Luger, MD Professor of Dermatology University of Münster Münster, Germany

Susan Manzi, MD, MPH Associate Professor of Medicine Division of Clinical Immunology and Rheumatology University of Pittsburgh School of Medicine Co-Director Lupus Center for Excellence University of Pittsburgh School of Health Sciences Pittsburgh, Pennsylvania Rapti Mediwake, MD Research Registrar National Heart & Lung Institute Imperial College Honorary Registrar Interstitial Lung Disease Unit Royal Brompton Hospital London, United Kingdom Joan Merrill, MD Head, Clinical Pharmacology Research Program Oklahoma Medical Research Foundation OMRF Professor of Medicine University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma Chandra Mohan, MD, PhD Professor of Medicine University of Texas Southwestern Medical Center Dallas, Texas Chi Chiu Mok, MD, FRCP Consultant Department of Medicine Tuen Mun Hospital Hong Kong SAR, China

Marta Mosca, MD Assistant Professor of Medicine Rheumatology Unit University of Pisa Pisa, Italy Phillip Murray, PhD, FRCOphth Professor of Ophthalmology Academic Unit of Ophthalmology University of Birmingham Birmingham and Midland Eye Center Birmingham, United Kingdom Bahram Namjou, MD Senior Research Scientist Department of Arthritis and Immunology Oklahoma Medical Research Foundation Oklahoma City, Oklahoma Jeannine S. Navratil, MS Professor of Medicine Division of Clinical Immunology and Rheumatology University of Pittsburgh School of Medicine Lupus Center of Excellence University of Pittsburgh School of Health Sciences Pittsburgh, Pennsylvania Johannes C. Nossent, MD Professor of Medicine Department of Rheumatology Institute of Clinical Medicine University of Tromso Rheumatology Consultant University Hospital North Norway Tromso, Norway James C. Oates, MD Assistant Professor of Medicine Division of Rheumatology Medical University of South Carolina Consultant and Attending Physician Department of Medical Service Ralph H. Johnson VA Medical Center Charleston, South Carolina Karolina Palucka, MD Adjunct Professsor of Biomedical Studies Baylor University Medical Center Waco, Texas

Panetelis Panopalis, MD Research Fellow Department of Medicine McGill University Health Centre Quebec, Canada Eva D. Papadimitraki, MD Senior Fellow Department of Internal Medicine Division of Rheumatology, Clinical Immunology, and Allergy University Hospital of Crete Heraklion, Greece Virginia Pascual, MD

Baylor Institute for Immunology Research Dallas, Texas Carol Peebles, MS, MT INOVA Diagnostics, Inc. San Diego, California Andras Perl, MD, PhD Professor and Chief of Rheumatology Department of Medicine State University of New York Syracuse, New York Michelle Petri, MD, MPH Director, Lupus Center Professor of Medicine Johns Hopkins University School of Medicine Baltimore, Maryland Silvia S. Pierangeli, PhD Professor of Internal Medicine Division of Rheumatology University of Texas Galveston, Texas David S. Pisetsky, MD, PhD Professor of Medicine and Immunology Chief, Division of Rheumatology and Immunology Duke University Medical Center Durham VA Medical Center Durham, North Carolina Brian D. Poole, PhD Associate Research Scientist Arthritis and Immunology Oklahoma Medical Research Foundation Oklahoma City, Oklahoma Rosalind Ramsey-Goldman, MD, DrPH Professor of Medicine Division of Rheumatology Northwestern University Feinberg School of Medicine Attending Physician Northwest Memorial Hospital Chicago, Illinois

Bruce Richardson, MD, PhD Professor of Medicine Chief, Section of Rheumatology Ann Arbor VA Medical Center University of Michigan Ann Arbor, Michigan Virginia Rider, PhD Professor of Biology Pittsburg State University Pittsburg, Kansas

CONTRIBUTING AUTHORS

Yair Molad, MD Director, Rheumatology Unit Rabins Medical Center Beilinson Campus Petah Tiqwa Senior Lecturer Sackler Faculty of Medicine Tel Aviv University TEl Aviv, Israel

Antony Rosen, MD Professor of Medicine Director, Division of Rheumatology Johns Hopkins University School of Medicine Baltimore, Maryland Brad H. Rovin, MD Professor of Medicine Chief, Division of Nephrology The Ohio State University Violeta Rus, MD Division of Rheumatology University of Maryland School of Medicine Baltimore, Maryland Minoru Satoh, MD, PhD Associate Professor of Medicine University of Florida Gainesville, Florida Amr H. Sawalha, MD Assistant Professor of Medicine Division of Rheumatology University of Oklahoma Attending Physician Division of Rheumatology Veterans Affairs Medical Center Oklahoma City, Oklahoma Georg Schett, MD Professor and Chair Department of Internal Medicine III and Institute of Clinical Immunology University of Erlangen-Nuremberg Nuremberg, Germany Josef S. Smolen, MD Professor of Medicine Chair, Division of Rheumatology Vienna General Hospital University of Vienna Vienna, Austria Günter Steiner, MD Department of Internal Medicine III Division of Rheumatology Medical University of Vienna Vienna, Austria

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CONTRIBUTING AUTHORS

Tom J.G. Swaak, MD Department of Rheumatology Ikazia Hospital Rotterdam, the Netherlands Tsutomu Takeuchi, MD Professor of Medicine Rheumatology/Clinical Immunology Faculty of Medicine Saitama Medical University Saitama, Japan George C. Tsokos, MD Visiting Professor of Medicine Division of Rheumatology Beth Israel Deaconess Medical Center Harvard Medical School Boston, Massachusetts Murray Urowitz, MD Professor of Medicine University of Toronto Director, Centre for prognosis Studies in the Rheumatic Diseases Toronto Western Hospital Ontario, Canada Charles S. Via, MD Professor of Medicine Department of Pathology Uniformed Services University of Health Sciences Bethesda, Maryland

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Mark H. Wener, MD Professor of Laboratory Medicine Division of Immunology Division of Rheumatology University of Washington Attenting Physician, Rheumatology University of Washington Medical Center Seattle, Washington Alan Wilk, MD, PhD Wendell A. Wilson, MD Department of Medicine Division of Rheumatology Louisiana State University School of Medicine New Orleans, Louisanna Yee Ling Wu, BS, BA Graduate Research Associate Integrated Biochemical Graduate Program The Ohio State University Investigator Center for Molecular and Human Genetics Columbus Children’s Research Institute Columbus Children’s Hospital Columbus, Ohio

Xiaokai Yan, PhD Academic Visiting Fellow Department of Medicine University of New South Wales New South Wales, Australia Yan Yang, MD, PhD Research Scientist Center for Molecular and Human Genetics Columbus Children’s Research Institute Columbus, Ohio C. Yung Yu, DPhil Associate Professor of Medicine Department of Pediatrics The Ohio State University Columbus, Ohio

Preface

Few diseases have as mystic an origin and present with as fascinating a complexity as Systemic Lupus Erythematosus (SLE). Physicians studying the clinical presentation of the disease have either acquired skills in a variety of subspecialties such as rheumatology, nephrology, dermatology, and neurology or they have developed close partnerships with the respective specialists. Investigators of its pathogenesis are scattered throughout diverse disciplines including molecular biology, immunology, and genetics. The editors of this volume have assumed the task of producing a manual that will bring together the current knowledge and experience of experts working on various aspects of the pathogenesis and clinical manifestations and treatment of SLE. In many ways, the volume has been compiled using a systems biology approach by converging basic and clinical concepts to a unified goal: understanding the disease so we may treat it optimally. The book has been organized into five sections. The first presents a group of articles that define the disease, provide measures of its activity, and discuss its impact on the life of the patient. In the second section, distinct chapters discuss the pathogenesis of the disease with emphasis on the cellular, molecular, genetic and environmental factors that instigate and drive the disease. A unique feature of this volume is the editors’ decision to present a section where expert authors discuss the processes that are responsible for tissue injury. The emphasis on the mechanisms that lead to organ damage is deliberate, as the editors believe that they may serve as

the source for new ideas for the treatment of the disease. In the fourth section, physicians and researchers discuss current issues that relate to the presentation of the disease. In the last section, expert authors discuss current and evolving approaches to the treatment of the disease. This volume on SLE does not replace existing comprehensive compendiums on the disease. Instead, it has been designed to present, within the limits of a manageable volume, current concepts of the disease in a comprehensive rather than encyclopedic manner. The editors believe that this volume will contribute to the overall battle to conquer the disease by bringing together diverse knowledge. This knowledge will prove useful to everyone interested in the study of SLE and systemic autoimmunity. Rheumatologists, nephrologists, dermatologists, neurologists, hematologists, and other specialists will find this volume useful in delivering what all of them aspire to: a better and more enjoyable life for our patients. Basic researchers will find in this volume a brief definitive presentation of concepts pursued by other colleagues. Clinical researchers will benefit similarly by the views of fellow clinicians and basic researchers. And likewise, young students and fellows starting a career in the field will find this to be a unique resource covering all aspects of SLE in a readily absorbable form. Thus, the current state of the art of SLE is condensed in this small book, but it should promote improvement in the understanding and treatment of this complex disease.

xi

Acknowledgements

We are grateful to our esteemed colleagues who embraced our concept for this book and took time out of overburdened schedules to accept our invitation to contribute state-of-the-art chapters. The editors wish to acknowledge the expert professional help they received

xii

from Matthew Ray, Kim Murphy, and Bryan Hayward. They are responsible for the high quality of this book and without their hard work this project would not have been completed.

EPIDEMIOLOGY AND DIAGNOSIS

1

Epidemiology of Systemic Lupus Erythematosus Ana M. Bertoli, MD and Graciela S. Alarcón, MD, MPH

INTRODUCTION Systemic lupus erythematosus (SLE) is one of the most common autoimmune diseases.1 It often associates with severe morbidity; mortality rates higher than those of the general population are well recognized.2 Understanding the distribution of SLE across different populations may help estimate the burden that it imposes at individual and societal levels. Given that SLE affects mainly individuals during their adult years,3-9 it has the potential to account for years of loss productivity10-12 as well as for functional losses affecting the same, and therefore, the quality of these patients’ lives.13-17 During the past few decades different SLE studies5,8,9,18-24 have provided valuable information about the distribution of SLE, and its course and outcome. While some studies have addressed the impact certain nonmodifiable factors such as age at disease presentation, gender and ethnicity may exert, others have addressed the role that socioeconomic factors have in outcomes such as damage accrual and mortality. In the following, we address lupus worldwide, the impact of age at disease onset, the impact of gender, the impact of ethnicity, and mortality trends.

Lupus Worldwide SLE has been recognized in all five continents, although it appears to be more common in Europe, the Americas, and Asia than in Australia25 and Africa.26 Of interest, in individuals of African ancestry, the disease appears to be quite rare in Africa but common in individuals of African ancestry living the United States, the Caribbean Islands, the United Kingdom, and Continental Europe. Analyses of the population burden imposed by SLE are hampered by the different sampling and recruitment methodologies used in the studies reporting prevalence and incidence rates. Several issues need to be addressed to better understand the differences in the rates reported. First, a case definition should be included in the report. Studies conducted prior to the establishment of classification criteria by the

American Rheumatism Association (ARA), now the American College of Rheumatology27 (revised in 198228 and modified in 199729), used various different case definitions; it is more than likely these studies could not have captured cases of mild SLE. The ACR criteria are now widely used in the clinical setting despite the fact that they were intended for the research setting; moreover, they were validated using prevalent rather than incident cases of SLE, because some time may elapse between the first disease manifestation and the accrual of four ACR criteria, which is a requirement for a patient to be classified as having SLE.30 Second, the method used to gather data may yield different rates, as they have variable case-capture sensitivity and specificity.31,32 Patient questionnaires,31,33-35 self-reported physician diagnosis,36-38 medical records review,39 or multiple assessment methods with capturerecapture techniques2 have been used. Third, it is important to distinguish between community-based and hospital-based studies. Community-based studies can provide more accurate incidence and prevalence rates; hospital-based studies may be flawed because more severe cases are likely to be included while milder cases are not. This may result in relatively lower incidence rates in hospital-based studies; however, higher morbidity and mortality indicators will emanate from such studies. Table 1.1 depicts SLE incidence rates reported over the last three decades. Although difficult to compare as already noted, it seems that there is a trend toward an increase in the incidence of SLE2,40; if this increment is real, or if it simply reflects a more accurate case ascertainment or the inclusion of milder cases is difficult to determine. As shown in Table 1.2, SLE prevalence rates vary widely around the world; higher rates have been reported in the United States2,36,37,41 than in countries in Europe,34,42-45 Asia,32,38 and Oceania.25 There is also a tendency toward higher prevalence rates now than in the past.2,46 Possible

1

America

Continent

1965–1973 1970–1977

Fessel106

Hochberg40

Vilar and Sato177 2000

1950–1992

2

Uramoto et al.

1985–1990

1980–1989

McCarty et al.39

Nossent

176

Michet et al.

1950–1979

1956–1965

Siegel and Lee174

175

1956–1975

Study Year(s)

Siegel et al.173

Author(s)

Brazil

United States

United States

Curacao

United States

United States

United States

United States

United States

Country

Community based

Community based

Community based

Mixed

Community based

Hospital based

Community based

Community based

Community based

Study Type

Physician self-reported diagnosisc

Medical records

c

Medical recordsd

Medical records and death certificatesd

Medical records

c

Hospital recordsb

Practice clinic filesb

Hospital and clinic files and death certificatesa

Hospital and clinic files and death certificatesa

Case Definition

TABLE 1.1 WORLDWIDE INCIDENCE OF SYSTEMIC LUPUS ERYTHEMATOSUS (1950–2000)

H

C

AA, C

AC

C

AA, C

AA, C

AA, C

AA, C

Ethnic Group(s)

8.7

5.6

2.4

4.6

1.8

4.6

7.6

2.0

1.0

Incidence per 100,000 Inhabitants per Year

EPIDEMIOLOGY OF SYSTEMIC LUPUS ERYTHEMATOSUS

2

1990–1994

Voss et al.43

Nossent42

Pelkonen et al.

180

1978–1996

1983–1986

1991

1981–1991

Stahl-Hallengren et al.179

Johnson et al.

1987–1990

Hopkinson et al.45

73

1981–1982

Nived et al.178

Norway

Finland

UK

Denmark

Sweden

UK

Sweden

Community based

Hospital based

Community based

Community based

Community based

Community based

Community based

b

Multisystemic disease and immunological abnormalities applied as criteria. 1971 American Rheumatism Association (now the American College of Rheumatology) (ARA/ACR) criteria applied. c 1982 ARA/ACR criteria applied. d 1971 or 1982 ARA/ACR criteria applied. e Incidence in women; in men the incidence drops to 1.5/100,000 inhabitants. f Study limited to pediatric cases. A, Asian; AA, African American; AC, Afro Caribbean; C, Caucasian; H, Hispanic.

a

Europe

Hospital records and mortality databasec

National patient registry and hospital recordsc

Multiple sources

Multiple sourcesc

Physician self-reported diagnosis, medical and laboratory records

Multiple sourcesb

Inpatient and outpatient medical recordsd

C

AC, C, A

C

C

AC, C

C

2.6

0.37f

3.8

2.5

4.8

6.5e

4.8

INTRODUCTION

3

Europe

America

Continent

36

1981–1982 1974–1983

Hochberg182

2000

Ward37

Nived et al.178

1997

1995

1950–1992

Balluz et al.41

Hochberg et al.

Uramoto et al.2

1980–1989

1951–1967

Michet et al.175

Nossent

1965–1973

Fessel106

176

1956–1965

1956–1975

1951–1967

Study Year(s)

Siegel and Lee174

Siegel et al.

173

Kurland et al.181

Author(s)

UK

Sweden

United States

United States

United States

United States

Curacao

United States

United States

United States

United States

United States

Country

Community based

Community based

Community based

Community based

Community based

Community based

Mixed

Community based

Community based

Community based

Community based

Community based

Study Type

Physician self-reported diagnosis

Inpatient and outpatient medical records

Physician self-reported diagnosis

Physician self-reported diagnosis

Physician self-reported diagnosis

Medical recordsc

Medical records and death certificates

Medical recordsc

Practice plan medical recordsb

Hospital and clinic files and death certificatesa

Hospital and clinic files and death certificatesa

Medical records

Case definition

TABLE 1.2 WORLDWIDE PREVALENCE OF SYSTEMIC LUPUS ERYTHEMATOSUS (1950—2004)

AA, C

C

AA, C

H

AA, C

C

AC

C

AA, C

AA, C

C

Ethnic Group(s)*

13

39

241

103

124

122

47

40

51

6

15

48

Prevalence per 100,000 Inhabitants

EPIDEMIOLOGY OF SYSTEMIC LUPUS ERYTHEMATOSUS

4

1995–1999

Huang et al.38

1996–1998

1992

Al-Arfaj et al.184

Bossingham25

~1990

1978–1996

Malaviya et al.32

Nossent42

Voss et al.

43

Australia

Taiwan

Saudi Arabia

India

Norway

Denmark

Ireland

UK

UK

Finland

UK

Sweden

Community based

Community based

Community based

Community based

Community based

Community based

Community based

Community based

Community based

Hospital based

Community based

Community based

c

b

Multisystemic disease and immunological abnormalities applied as criteria. 1971 American Rheumatism Association (now the American College of Rheumatology) (ARA/ACR) criteria applied. 1982 ARA/ACR criteria applied. d Study limited to women, aged 18-65. e Study limited to pediatric cases. *A, Asian; AA, African American; AAu, Aboriginal Australian; AC, Afro-Caribbean; C, Caucasian; H, Hispanic.

a

Oceania

Asia

1993

Gourley et al.44 1990–1994

1994

Johnson et al.

34

1987–1990

1972–1978

Helve183

Hopkinson et al.

1992

Johnson et al.73

45

1981–1991

Stahl-Hallengren et al.179

Multiple sourcesc

Patient registry

Surveyc

Mailed survey and laboratory testing

Hospital records and mortality database

Multiple sources

Multiple sources

Mailed survey and protocol assessment

Multiple sources

c

Discharge diagnosis

Multiple sources

Physician self-reported diagnosis and medical and laboratory records

AAu, C

A (Chinese)

A (Arabic)

A (Indian)

C

C

C

AC, C, A

AC, C

C

AC, C, A

C

45

6e

19

3

45

22

25

54d

25

28

28

68

INTRODUCTION

5

EPIDEMIOLOGY OF SYSTEMIC LUPUS ERYTHEMATOSUS

6

reasons underlying this phenomenon are the inclusion of milder cases, improved case ascertainment and capture methods, and finally, the accrual of cases over time as a result of improved survival rates and consequently of patients experiencing a longer disease duration.46,47

these relatively less-serious disease manifestations, late-onset lupus patients may have a poor outcome, both in terms of morbidity (damage accrual)22,71,71a and mortality.23,50,64,70 The disease itself, plus other agerelated factors (comorbidities) probably exert a synergistic negative effect explaining these findings.

Impact of Age at Disease Onset Although SLE is generally regarded a disease of women of childbearing age, it can affect individuals of all ages, from the newborn to the very old. It has been suggested that age at disease presentation has a modifying effect in both the course and outcome of the disease. Recognizing SLE subsets according to age at disease onset has important clinical and therapeutic implications. Pediatric lupus, that is SLE with onset or diagnosis before age 16 years, accounts for approximately 8% to 15% of all SLE cases48-51; yet SLE rarely occurs before age 5 years, being more common after age 10.52-55 The female-to-male ratio seems to be lower than among the adult-onset lupus (6:1), yet a female-to-male ratio closer to that of the adult-onset lupus population ranging from 7 to 18:1 has been reported in some studies from Western and Middle Eastern countries.51,54,55 However, gender does not seem to impose a different prognosis in pediatric SLE.53 Lupus in the pediatric age group usually presents with severe disease manifestations including a high proportion of patients with major organ system involvement; this is particularly the case for renal51,54,56,57 and neuropsychiatric involvement.57-60 Not surprisingly, therefore, the domains of the damage index most frequently affected are the renal and neuropsychiatric domains.52 Few studies have addressed survival in pediatric-onset lupus; in the few studies addressing this, survival rates as high as 80% at 10 years have been reported.60,61 Once thought to be a rare condition, late-onset lupus (generally considered if onset occurs at age 50 and later),62-65 is being increasingly recognized beyond the sixth decade of life. According to a meta-analysis conducted in the late 1980s that included nine studies, lateonset lupus comprised up to 18% of all patients.66 Given that life expectancy has increased significantly over the last century, and it is expected to continue to increase,67 the increased occurrence of late-onset lupus is not surprising as there are more individuals at risk of developing it. The female-to-male ratio also tends to be lower than among adult early-onset patients with a female-to-male ratio of 2.6 to 5.5:1.63-65,68,69 Late-onset lupus patients tend to have a more insidious onset,62,70 to be less likely to have major organ system involvement,65 and to have lower degrees of disease activity.63 In the meta-analysis mentioned, the authors concluded that these patients have more serositis, interstitial lung disease, Sjögren’s syndrome, and anti-La antibody positivity than their younger counterparts.66 Despite

Impact of Gender The most striking gender-related difference in SLE is in its incidence (and prevalence). SLE is much more frequent among women than among men with ratios of 6 to 14:1 reported in the literature.6,39,72 These ratios vary significantly, however, probably as a result of other variables such as ethnicity and age at disease presentation and the relative underascertainment of the disease in men.73 The female preponderance observed probably reflects the role that sex hormones have in the pathogenesis of the disease.74 This relationship has been well demonstrated in murine models of SLE75-78; furthermore, the peak incidence in women occurs during their reproductive years. The disease also tends to flare up during periods of hormonal changes, especially during pregnancy,79-81 or with the use of oral contraceptives and hormone replacement therapy.82-86 The hypoestrogenemic state that occurs during menopause appears, however, not to be protective of disease activity and damage accrual; in fact, it has been suggested that age rather than menopausal status is a strong independent predictor of damage accrual and of vascular events in women with lupus.87 Similarly, the use of hormone replacement therapy appears not to be an independent predictor of disease activity, severe flareups, and damage accrual88; however, mild to moderate flare-ups have been reported in the SELENA trial.89 Data on the relationship among gender, clinical manifestations, and disease outcome are somewhat more controversial. Women tend to develop lupus at a younger age than men, while men tend to have serosal and renal involvement more frequently than women. Finally, men tend to accrue more damage and to experience lower survival rates than women, although there is some degree of variability across studies, as noted in Table 1.3.5,6,53,68,72,90-101 It should be emphasized that some of these studies include a relatively small number of patients (20 or less) making their conclusions less reliable.

Impact of Ethnicity Genetic (inherited) and nongenetic (acquired) factors are known to predispose to, and to modulate, the course and outcome of diseases. However, many of these predisposing factors are unknown; thus epidemiologists aim at categorizing individuals by using surrogate variables such as race and ethnicity. Terms such as race, ethnicity, or ancestry are not interchangeable. Race implies genetic homogeneity, which

Author(s)

Study Year(s)

Country

n

Female/Male Ratio

Hochberg et al.90

1980–1984

United States

150

11.5:1

Younger age at diagnosis in women; clinical manifestations similar in both genders

Ward and Studenski91

1969–1983

United States

62

4.8:1

Clinical manifestations similar in both genders

Font et al.68

1980–1990

Spain

30

8.7:1

Discoid lupus more frequent in men; no differences in major organ system involvement

Pande et al.92

1986–1983

India

39

NA

Renal involvement and infectious complications more frequent in women

Specker et al.96

1986–1991

Germany

21

3.9:1

Renal and cardiovascular involvement, especially thromboembolic events, more frequent in men

Lo et al.53

1989–1998

Taiwan

24

4.6:1

No differences in major organ system involvement and in survival rates

Mok et al.93

NA

Hong Kong

51

11.4:1

No differences in major organ involvement; less relapses in men; renal and cardiovascular damage more frequent in men

Petri5

1989–1999

United States

41

11.8:1

Seizures, hemolytic anemia, pulmonary fibrosis, renal insufficiency, and myocardial infarction more frequent in men

Aranow et al.99

?–1994

United States

18

NA

Cerebritis and thromboembolic events more common in men; no differences in damage accrual

Molina et al.101

1972– 1993

Latin America

107

11.3:1

Renal disease, vascular thrombosis, and anti-dsDNA antibodies more frequent in men

Voulgari et al.6

1981–2000

Greece

68

6.2:1

Serosal and renal involvement more frequent in men; no differences in disease activity and damage accrual

Miettunen et al.100,a

1980–1997

Canada

13

2.9:1

No differences in damage accrual and survival rates

Manger et al.94

1985–1999

Germany

47

6.2:1

Male gender a risk factor for mortality

95

1997–1999

United States

25

9.6:1

Renal and hematologic involvement more frequent in men

Mayor and Vila98

?–2001

Puerto Rico (United States)

12

19.6:1

Male gender a risk factor for damage accrual and mortality

Lopez et al.97

1992–2002

Spain

43

50:1

Younger age at diagnosis in women

Soto et al.

1998–2002

Mexico

33

4.8:1

Discoid lupus, psychosis, pericarditis, and renal involvement more frequent in men; no differences in survival rates

Andrade et al72a

1994-2005

United States

63

8.8:1

Accelerated damage development, particularly in the disease course

Cooper et al.

72

Findings

INTRODUCTION

TABLE 1.3 GENDER DIFFERENCES IN CLINICAL MANIFESTATIONS AND OUTCOME OF SYSTEMIC LUPUS ERYTHEMATOSUS (1969–2002)

a

Study limited to pediatric cases. NA, not available.

7

EPIDEMIOLOGY OF SYSTEMIC LUPUS ERYTHEMATOSUS

8

TABLE 1.4 ETHNIC DISPARITIES IN INCIDENCE AND PREVALENCE OF SYSTEMIC LUPUS ERYTHEMATOSUS Frequency Variable

Author(s)

Study Year(s)

Location

Ethnic Group(s)

Findings

Anstey et al.105

1984–1991

Australia

Aboriginal and nonAboriginal Australian

Incidence in Aboriginal Australians is two times higher

McCarty et al.39

1985–1990

United States

African American and Caucasian

Incidence in African Americans is two times higher

Johnson et al.73

1991

UK

Afro-Caribbean, Asian, and Caucasian

Incidence in AfroCaribbeans and Asians is five to six times higher than that of Caucasians

Fessel106

1965–1973

United States

African American and Caucasian

Prevalence in African American is four times higher

Serdula and Rhoads107

1970–1975

Hawaii (United States)

Chinese, Filipino, Japanese, and white

Prevalence in non-whites is four times higher

Hart et al.108

1975–1980

New Zealand

Polynesian and Caucasian

Prevalence in Polynesians is three times higher

Samanta et al.109

1979–1988

UK

Asian (Indian) and Caucasian

Prevalence in Asians is two times higher

Boyer et al.110

1970–1984

Alaska (United States)

Native Alaskan and U.S. Prevalence in Native population Alaskans is two times higher

Johnson et al.73

1991–1992

UK

Afro-Caribbean, Asian, and Caucasian

Incidence

Prevalence

does not exist in humans; for example, Hispanics in the United States and Asians in England and in the United States include different subgroups or categories of individuals within each group.18,19,102,103 Individuals categorized as Caucasians or white are equally heterogeneous. The Institute of Medicine (United States) has recommended that the term “race” be banned from the scientific literature, and that ethnicity be used instead. This term is a much broader self-defined construct. Ethnic groups are defined on the basis of geographic, social, cultural, and religious characteristics; patients of the same ethnic group have the potential to exhibit a similar genetic background, particularly within ethnic subgroups. Thus, not surprisingly, the variable phenotypic expression of several disorders, SLE among them, among individuals of different ethnic groups has been recognized. This variability cannot be solely explained by genetics-related factors, given the tight association between some socioeconomic indicators of disadvantageous status and defined ethnic groups. In SLE, differences among ethnic groups can be found in the incidence and prevalence of the disease,39,73,104-110 in its course (disease activity and clinical

Prevalence in Asians and Afro-Caribbeans is two to five times higher than in Caucasians

manifestations)4,5,8,18,90,109,111-114 and in its mediate (damage accrual)22 and long-term (mortality)9,47,60,105,115-141 outcomes.142,143 As noted in Table 1.4, it has been shown that patients from minority populations of African (living in the United States, Caribbean Islands, United Kingdom, or Continental Europe) or Asian ancestry tend to show a higher SLE incidence and prevalence, along with a more severe disease course and outcome.4,5,90,111,112,114,140,144 Similarly, the disease is more frequent among Aboriginal than non-aboriginal Australians.105 These patients as a group tend to have more abrupt disease onset, more severe clinical manifestations, and an overall higher degree of disease activity.145 Hispanics, African Americans, and Asians also tend to have more hematologic, serosal, neurologic, and renal involvement, regardless of age and gender.4,8,18,109,112,113 Patients of non-Caucasian ethnicity also accrue more damage over time22 and faster146 than Caucasians; they also develop specific damage more often (renal and integument)22,147 and exhibit higher mortality rates when compared with Caucasians.23,107,140 These data are summarized in Table 1.5.

Variable

Author(s)

Study Year(s)

n

Location

Ethnic Group(s)

Findings

Disease manifestations, activity and criteria accrual

Hochberg et al.90

1980–1984

1875

United States

African American and Caucasian

Renal and lung involvement more frequent in African Americans

Ward and Studenski112

1983–1989

258

United States

African American and Caucasian

Renal, neurological, and serosal involvement more frequent in African Americans

Gioud-Paquet et al.111

1976–1986

80

France

African (North), Afro-Caribbean and Caucasian

Overall more severe disease in African descendants, particularly renal involvement

Samanta et al.109

1979–1988

87

UK

Asian and Caucasian

Renal and neuropsychiatric involvement more frequent in Asians

Petri5

1989–1999

525

United States

African American and Caucasian

Renal, serosal, and muscular involvement more frequent in African Americans

Alarcón et al.4

1994–1996

229

United States

Hispanic, African American, and Caucasian

Higher disease activity in nonCaucasians

Bastian et al.113

1994–2000

353

United States

Hispanic, African American, and Caucasian

Renal involvement more frequent in non-Caucasians

Alarcón et al.8

1989–2000

568

United States

Hispanic, African American, and Caucasian

Renal involvement more frequent in non-Caucasians

Arbuckle et al.114

1988–1996

130

United States

African American and European American

African Americans accrue diagnostic criteria factor faster than other groups

Alarcón et al.30

1994–2002

471

United States

Hispanic, African American, and Caucasian

Hispanics accrue criteria faster than other groups

Pons-Estel et al.18

1997–2000

1214

Latin America

Mestizo, African-Latin American, and white

Renal involvement more frequent in among non-whites

Alarcón et al.22

1994–1998

258

United States

Hispanic, African American, and Caucasian

Hispanics accrue damage more rapidly

Rivest et al.147

1990’s

200

United States

African American and Caucasian

African Americans exhibit more renal damage

INTRODUCTION

TABLE 1.5 ETHNIC DISPARITIES IN DISEASE COURSE AND OUTCOME OF SYSTEMIC LUPUS ERYTHEMATOSUS

Damage

Continued

9

EPIDEMIOLOGY OF SYSTEMIC LUPUS ERYTHEMATOSUS

TABLE 1.5 ETHNIC DISPARITIES IN DISEASE COURSE AND OUTCOME OF SYSTEMIC LUPUS ERYTHEMATOSUS—cont’d Variable

Author(s)

Study Year(s)

Toloza et al.146

1994–2003

Serdula and Rhoads107

n

Location

Ethnic Group(s)

Findings

152

United States

Hispanic, African American, and Caucasian

Hispanics (Texas orgin) accrue new damage more rapidly

1970–1975

107

Hawaii Chinese, (United States) Filipino, Japanese, and white

Higher mortality rates in non-whites

Ward et al.140

1969–1983

408

United States

African American and Caucasian

Lower socioeconomic status (not ethnicity) predicts mortality

Alarcón et al.23

1994–1999

288

United States

Hispanic, African American, and Caucasian

Higher mortality rate in African Americans, but poverty (not ethnicity) explains findings

Fernández et al.141

1994–2004

578

United States

Hispanic, African American, and Caucasian

Higher mortality rate in African Americans, but poverty (not ethnicity) explains findings

Mortality

It is very likely that the differences observed among ethnic groups early in the course of the disease reflect the genetic component of ethnicity, whereas the ones observed later in its course reflect nongenetic factors such as those of a socioeconomic-demographic, behavioral, and/or psychological nature.

Mortality Trends

10

Considered in the first half of the twentieth century a uniformly fatal disorder, patients with lupus are now expected to live years if not decades after diagnosis. Survival analyses were first used in medicine in the early 1950s when Merrell and Shulman115 published their landmark paper demonstrating a survival rate of less than 50% at 5 years (pre-corticosteroid patients). This figure contrasts with those reported over the last two decades showing survival rates at 5 years of 90% and above.94,131,139,148 This improved survival can be explained by earlier diagnosis of the disease, the diagnosis of milder cases (who may not have been diagnosed in years past), the introduction of glucocorticoids and possibly of immunosuppressants and the availability of effective therapeutic interventions for comorbid conditions such as dialysis, antibiotics, and antihypertensive agents. Furthermore, during the same period of time, the improvement in survival in lupus has been greater than the one observed in the general population,149 as

evidenced by standardized mortality ratios (SMR) that have declined from 10.1 in the 1970s, to 4.8 in the early 1980s and to 3.3 in the 1990s.150 However, life expectancy in SLE patients is still below that of the general population,2,151,152 which means that efforts need to be directed toward unraveling the pathogenesis of the disease—the factors affecting its course and outcome—particularly those affecting survival. Table 1.6 summarizes various SLE survival studies. As noted before, the reasons underlying the differences in survival rates between older and more recent publications are probably multiple; however, differences in survival rates still persist even in later reports. The basis for such discrepancies relates mainly to characteristics of the cohort being studied including the time at which patients are recruited into the cohort, the patients’ sociodemographic background, the length of follow-up, and the method used for the analyses. For example, mortality in SLE has been reported to be higher during the first few years of the disease132,152,153; thus, inception cohorts, which include patients who otherwise will be censored in other cohorts, will provide lower survival rates than noninception cohorts. Although in many cases disease onset and disease diagnosis are not the same, it is very hard to clearly establish disease onset in patients unless disease manifestations evolve over a relatively short time period; thus, disease

United States

United States

116

117

47

Australia

Curaçao

Nossent133

India

Anstey et al.105

Kumar et al.

United States

Zeleznick and Fries129

130

United States

Finland

United States

Taiwan

UK

Holland

India

Pistiner et al.128

Gripenberg and Helve

Reveille et al.

126

Wang et al.60,a

Worrall et al.

Swaak et al.

125

Malaviya et al.124

Jonsson et al.

Sweden

United States

122

123

United States

Wallace et al.121

Ginzler et al.

Singapore

United States

Canada

Boey120

127

117

Urman and Rothfield

Urowitz et al.

126

Estes and Christian118

Urman and Rothfield

United States

United States

Merrel and Shulman115

Kellum and Haserick

Country

Author(s)

68

21

288

310

570

66

389

153

100

110

101

NA

1103

609

183

156

81

150

209

299

99

n

1980–1990

1984–1991

1981–1990

1970–1982

1980–1989

1980–1987

1975–1985

1980–1990

1970–1989

1970–1986

1986

1986

1965–1978

1950–1980

1970–1980

1968–1976

1970–1974

1961–1969

1957–1968

1949–1960

1949–1953

Study Year(s)

AC

AAu

A (Indian)

C, AA, H

C, AA

C

C, AA

A

C, AC, Af, A (Indian)

C

A (Indian)

C

C, AA

C, AA, H

A

C, AA

C

C, AA

C, AA

C, AA

C, AA

Ethnic Group(s)*

56

60

78

88

97

—

89

60

88

92

68

97

86

88

70

93

75

77

70

69

50

5

—

—

—

64

93

91

83

44

—

87

50

—

76

79

60

84

63

60

63

—

—

10

—

—

—

—

83

81

79

—

—

—

—

—

—

74

—

—

53

50

—

—

—

15

Survival Probability (%), Year

TABLE 1.6 WORLDWIDE SURVIVAL RATES IN SYSTEMIC LUPUS ERYTHEMATOSUS (1949–2001)

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

20

Continued

INTRODUCTION

11

132

1000

178

153

349

86

513

306

98

539

665

408

218

n

1990–2000

1982–2001

1991–2001

1986–2000

1992–1999

1975–1995

1975–1993

1981–1993

1974–1990

1970–1993

1969–1983

1970–1991

Study Year(s)

Study limited to pediatric cases. *A, Asian; AA, African American; Af, African; AAu, Aboriginal Australian; C, Caucasian; H, Hispanic; NA, not available.

a

Cervera et al.

Europe (multinational)

Greece

Alamanos et al.139

9

Taiwan

Thailand

Hong Kong

Wang60,a

Kasitanon et al.

Mok et al.

131

Jacobsen et al.151

Denmark

Spain

138

Blanco et al.

India

Murali et al.137

Canada

United States

Malaysia

135

Wang et al.136

Abu-Shakra et al.

Ward et al.

Chile

Massardo et al.134

140

Country

Author(s)

C

C

A

A

A

C

C

A (Indian)

A

C, AA, A

C, AA

H

Ethnic Group(s)*

—

97

85

84

93

91

90

89

82

93

82

92

5

92

90

76

75

—

76

85

77

70

85

71

77

10

—

—

—

—

—

64

80

60

—

79

63

66

15

Survival Probability (%), Year

TABLE 1.6 WORLDWIDE SURVIVAL RATES IN SYSTEMIC LUPUS ERYTHEMATOSUS (1949–2001)—cont’d

—

—

—

—

—

53

—

—

—

68

—

—

20

EPIDEMIOLOGY OF SYSTEMIC LUPUS ERYTHEMATOSUS

12

A large body of literature deals with factors predictive of mortality. Variables from the socioeconomicdemographic, clinical, and psychological domains have been implicated as predictors of mortality. Data from various countries have shown that patients of non-Caucasian ethnicity have higher mortality rates, even at younger ages, when compared with Caucasian patients.23,105,107,108,130,140,156,157 Moreover, data from the United States have shown that mortality is not only higher in African American women, but that it is also increasing over time when compared to Caucasian women87,158,159; these data are depicted in Fig. 1.1. Information on other minority groups is scarce, particularly for Hispanics. However, interpretation of the role that ethnicity has in predicting mortality must be done with great caution given that, as already discussed, “ethnicity” could be a proxy for socioeconomic-demographic variables and psychological factors that could be the actual reasons for the differences observed. For example, in the LUMINA (LUpus in MInorities, NAture versus Nurture) cohort, African-American patients exhibited a lower probability of survival in univariable analyses. However, in multivariable analyses, poverty rather than ethnicity has been consistently found to be an independent predictor of mortality; this was the case in analyses performed in a prevalent and relatively young cohort,23 but has been corroborated recently.141 The impact of other socioeconomic variables has also been underscored in other studies; for example, fewer years of formal education have been found to be associated with higher mortality rates in Caucasian patients160 and the lack of health insurance has been found to independently predict mortality in the GLADEL (for Grupo Latino Americano de Estudio de Lupus or

INTRODUCTION

diagnosis is the starting point in most studies to date even though this may artifactually shorten disease duration and affect survival rates. Another issue to consider is the length of follow-up of patients in the cohort as well as the rates of loss to follow-up; the longer the follow-up and the higher the retention rates in the cohort, the more accurate the data will be. The patients’ sociodemographic background is also important when comparing results from different cohorts, given that ethnicity and age, for example, are well-recognized factors influencing survival. Finally, and as noted in Table 1.6, contemporary survival rates in developing countries are comparable to survival rates of years past in developed countries, further emphasizing the importance that socioeconomic factors have in the ultimate outcome of SLE. A very valuable tool to estimate improvement in survival rates is the estimation of such rates within the same cohort over time, avoiding some of the above mentioned problems; for example Urowitz et al.150 compared SMRs in patients from the Toronto cohort at three different time periods. In such analyses, the SMR decreased from 10.1 in the oldest cohort (1970-1977), to 4.8 in the intermediate (1978-1985), and to 3.3 in the most recent (1986-1994). In the 1970s, Urowitz et al. described a bimodal pattern of mortality in lupus119; in that study the authors reported an early mortality peak due mainly to active disease and a later peak due to cardiovascular complications. This bimodal pattern has been later corroborated in other studies; currently, the most important causes of death among SLE patients are still considered to be active disease and infections during the first few years of the disease and complications derived from accelerated atherosclerosis later in the disease course.7,60,135,151,152,154,155

Black aged ≥65 yrs Black aged 45–64 yrs Black aged 15–44 yrs White aged ≥65 yrs White aged 45–64 yrs White aged 15–44 yrs

Fig. 1.1 Death rates (x100,000 inhabitants) among women with systemic lupus erythematosus by ethnic group. (From Sacks JJ, Helmick CG, Langmaid G, Sniezek JE. MMWR Morb Mortal Wkly Rep 2002;51:371-4.)

13

EPIDEMIOLOGY OF SYSTEMIC LUPUS ERYTHEMATOSUS

Latino American Group for the Study of Lupus) study, a multinational Latin American cohort.18 Ward et al.140 have also found several indicators of socioeconomic status, such as income and type of medical insurance, to be associated with mortality. Among the clinical variables, renal involvement,9,94,161,162 disease activity over time,23,24,163,164 and damage accrued23,94,141,165,166 have been found to be predictors of mortality.141 Not only disease characteristics, but also treatment modalities have been related to a worse outcome, especially glucocorticoids. These compounds have been found to predict not only damage accrual22,131,167,168 but also mortality.131 In contrast, antimalarials, mainly hydroxycholoquine, have recently been found to be protective of both damage accrual169 and mortality.170 Finally, variables somewhat unrelated to the patient, such as greater hospital experience in treating SLE patients171 and a higher volume of patients per physician have been proven to be associated with lower risk of in-hospital mortality.172

Conclusions SLE is one of the most common autoimmune diseases. Incidence and prevalence rates are difficult to compare because of methodologic differences in the studies reported (e.g., case ascertainment, sampling frame). Nevertheless, the incidence of the disease appears

to be increasing, and since SLE patients are living longer, the prevalence of the disease is, likewise, increasing. Age at disease onset has a modulating effect in SLE. Children are more likely to have more severe disease and major organ system involvement at onset. In contrast, patients with late-onset lupus tend to have less severe clinical manifestations; however, these patients accrue more damage and show higher mortality rates than younger patients. Male gender appears to be a risk factor for renal involvement, damage accrual, and mortality. SLE tends to be more frequent and more severe in minority population groups. Although mortality rates in SLE patients have remarkably improved during the last five decades, they are still above those of the general population; this is especially true for African American women. Patients with more severe disease, in terms of organ system involvement, disease activity, and damage accrued, as well as those with a less favorable socioeconomic-demographic background, are at higher risk of succumbing earlier from the disease or its treatments. Assessments of the underlying reasons for the discrepancies in the course and outcome of SLE and the existing inequities in access to health care are of utmost importance if the prognosis of SLE is going to be substantially modified worldwide.

REFERENCES

14

1. Jacobson DL, Gange SJ, Rose NR, Graham NM. Epidemiology and estimated population burden of selected autoimmune diseases in the United States. Clin Immunol Immunopathol 1997;84: 223-43. 2. Uramoto KM, Michet CJ Jr, Thumboo J, Sunku J, O’Fallon WM, Gabriel SE. Trends in the incidence and mortality of systemic lupus erythematosus, 1950-1992. Arthritis Rheum 1999;42: 46-50. 3. Rabbani MA, Siddigui BK, Tahir MH, Ahmad B, Shamim A, Shah SM, et al. Systemic lupus erythematosus in Pakistan. Lupus 2004; 13:820-25. 4. Alarcón GS, Friedman AW, Straaton KV, Moulds JM, Lisse J, Bastian HM, et al. Systemic lupus erythematosus in three ethnic groups: III. A comparison of characteristics early in the natural history of the LUMINA cohort. LUpus in MInority populations: NAture vs. Nurture. Lupus 1999;8:197-209. 5. Petri M. Hopkins lupus cohort: 1999 update. Rheum Dis Clin North Am 2000;26:199-213. 6. Voulgari PV, Katsimbri P, Alamanos Y, Drosos AA. Gender and age differences in systemic lupus erythematosus. A study of 489 Greek patients with a review of the literature. Lupus 2002;11: 722-29. 7. Klippel JH. Systemic lupus erythematosus: demographics, prognosis, and outcome. J Rheumatol 1997;48:67-71. 8. Alarcón GS, McGwin G Jr, Petri M, Reveille JD, Ramsey-Goldman R, Kimberly RP, et al. Baseline characteristics of a multiethnic lupus cohort: PROFILE. Lupus 2002;11:95-101. 9. Cervera R, Khamashta MA, Font J, Sebastiani GD, Gil A, Lavilla P, et al. Morbidity and mortality in systemic lupus erythematosus

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148. Trager J, Ward MM. Mortality and causes of death in systemic lupus erythematosus. Curr Opin Rheumatol 2001;13:345-51. 149. Abu-Shakra M, Gladman DD, Urowitz MB. Mortality studies in SLE: how far can we improve survival of patients with SLE. Autoimmun Rev 2004;3:418-20. 150. Urowitz MB, Gladman DD, Abu-Shakra M, Farewell VT. Mortality studies in systemic lupus erythematosus. Results from a single center. III. Improved survival over 24 years. J Rheumatol 1997;24:1061-5. 151. Jacobsen S, Petersen J, Ullman S, Junker P, Voss A, Rasmussen JM, et al. Mortality and causes of death of 513 Danish patients with systemic lupus erythematosus. Scand J Rheumatol 1999;28:75-80. 152. Moss KE, Ioannou Y, Sultan SM, Haq I, Isenberg DA. Outcome of a cohort of 300 patients with systemic lupus erythematosus attending a dedicated clinic for over two decades. Ann Rheum Dis 2002;61:409-13. 153. Hochberg MC. Mortality from systemic lupus erythematosus in England and Wales, 1974-1983. Br J Rheumatol 1987;26:437-41. 154. Tsokos GC, Wong HK, Enyedy EJ, Nambiar MP. Immune cell signaling in lupus. Curr Opin Rheumatol 2000;12:355-63. 155. Ward MM, Pyun E, Studenski S. Causes of death in systemic lupus erythematosus: Long-term followup of an inception cohort. Arthritis Rheum 1995;38:1492-9. 156. Walsh SJ, Dechello LM. Geographical variation in mortality from systemic lupus erythematosus in the United States. Lupus 2001;10:637-46. 157. Peschken CA, Esdaile JM. Systemic lupus erythematosus in North American Indians: a population based study. J Rheumatol 2000;27:1884-91. 158. Walsh SJ, Algert C, Gregorio DI, Reisine ST, Rothfield NF. Divergent racial trends in mortality from systemic lupus erythematosus. J Rheumatol 1995;22:1663-8. 159. Sacks JJ, Helmick CG, Langmaid G, Sniezek JE. Trends in deaths from systemic lupus erythematosus-United States, 1979-1998. MMWR Morb Mortal Wkly Rep 2002;51:371-4. 160. Ward MM. Education level and mortality in systemic lupus erythematosus (SLE): Evidence of under ascertainment of deaths due to SLE in ethnic minorities with low education levels. Arthritis Rheum (Arthritis Care Res) 2004;51:616-24. 161. Grodstein F, Stampfer MJ, Colditz GA, Willett WC, Manson JW, Joffe M, et al. Postmenopausal hormone therapy and mortality. N Engl J Med 1997;336:1769-75. 162. Lee PT, Fang HC, Chen CL, Chiou YH, Chou KJ, Chung HM. Poor prognosis of end-stage renal disease in systemic lupus erythematosus: a cohort of Chinese patients. Lupus 2003;12:827-32. 163. Cook RJ, Gladman DD, Pericak D, Urowitz MB. Prediction of short term mortality in systemic lupus erythematosus with time dependent measures of disease activity. J Rheumatol 2000;27:1892-5. 164. Stoll T, Sutcliffe N, Mach J, Klaghofer R, Isenberg DA. Analysis of the relationship between disease activity and damage in patients with systemic lupus erythematosus—a 5-year prospective study. Rheumatology (Oxford) 2004;43:1039-44. 165. Nived O, Jonsen A, Bengtsson AA, Bengtsson C, Sturfelt G. High predictive value of the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index for survival in systemic lupus erythematosus. J Rheumatol 2002;29:1398-400. 166. Mok CC, Ho CT, Wong RW, Lau CS. Damage accrual in Southern Chinese patients with systemic lupus erythematosus. J Rheumatol 2003;30:1513-9.

167. Brunner HI, Silverman ED, To T, Bombardier C, Feldman BM. Risk factors for damage in childhood-onset systemic lupus erythematosus: Cumulative diseae activity and medication use predict disease damage. Arthritis Rheum 2002;46:436-44. 168. Zonana-Nacach A, Barr SG, Magder LS, Petri M. Damage in systemic lupus erythematosus and its association with corticosteroids. Arthritis Rheum 2000;43:1801-8. 169. Fessler BJ, Alarcón GS, McGwin G Jr, Roseman JM, Bastian HM, Friedman AW, et al. Systemic lupus erythematosus in a multiethnic group. XVI. Hydroxychloroquine usage is associated with a lower risk of damage accrual. Arthritis Rheum 2005;52:1473-80. 170. Ruiz-Irastorza G, Egurbide MV, Ibarra S, Garmendia M, Erdozain JG, Villar I, et al. Effect of antimalarials on long-term survival of patients with systemic lupus erythematosus. Lupus 2005;14:220. 171. Ward MM. Hospital experience and mortality in patients with systemic lupus erythematosus. Arthritis Rheum 1999;42:891-8. 172. Ward MM. Association between physician volume and inhospital mortality in patients with systemic lupus erythematosus. Arthritis Rheum 2005;52:1646-54. 173. Siegel M, Holley HL, Lee SL. Epidemiologic studies on systemic lupus erythematosus. Comparative data for New York City and Jefferson County, Alabama, 1956-1965. Arthritis Rheum 1970;13:802-11. 174. Siegel M, Lee SL. The epidemiology of systemic lupus erythematosus. Semin Arthritis Rheum 1973;3:1-54. 175. Michet CJ Jr, McKenna CH, Elveback LR, Kaslow RA, Kurland LT. Epidemiology of systemic lupus erythematosus and other connective tissue diseases in Rochester, Minnesota, 1950 through 1979. Mayo Clin Proc 1985;60:105-13. 176. Nossent JC. Systemic lupus erythematosus on the Caribbean island of Curacao: an epidemiological investigation. Ann Rheum Dis 1992;51:1197-201. 177. Vilar MJ, Sato EI. Estimating the incidence of systemic lupus erythematosus in a tropical region (Natal, Brazil). Lupus 2002;11:528-32. 178. Nived O, Sturfelt G, Wollheim F. Systemic lupus erythematosus in an adult population in Souther Sweden: Incidence, prevalence and validity of ARA revised classification criteria. Br J Rheumatol 1985;24:147-54. 179. Stahl-Hallengren C, Jonsen A, Nived O, Sturfelt G. Incidence studies of systemic lupus erythematosus in Southern Sweden: increasing age, decreasing frequency of renal manifestations and good prognosis. J Rheumatol 2000;27:685-91. 180. Pelkonen PM, Jalanko HJ, Lantto RK, Makela AL, Pietikainen MA, Savolainen HA, et al. Incidence of systemic connective tissue diseases in children: a nationwide prospective study in Finland. J Rheumatol 1994;21:2143-6. 181. Kurland LT, Hauser WA, Ferguson RH, Holley KE. Epidemiologic features of diffuse connective tissue disorders in Rochester, Minnesota, 1951 through 1967, with special reference to systemic lupus erythematosus. Mayo Clin Proc 1969;44:649-63. 182. Hochberg MC. Prevalence of systemic lupus erythematosus in England and Wales, 1981-2. Ann Rheum Dis 1987;46:664-6. 183. Helve T. Prevalence and mortality rates of systemic lupus erythematosus and causes of death in SLE patients in Finland. Scand J Rheumatol 1985;14:43-6. 184. Al-Arfaj AS, Al-Balla SR, Al-Dalaan AN, Al-Saleh Ss, Bahabri SA, Mousa MM, et al. Prevalence of systemic lupus erythematosus in central Saudi Arabia. Saudi Med J 2002;23:87-9.

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2

Assessment of Disease Activity in Systemic Lupus Erythematosus Marta Mosca, MD, Joan T. Merrill, MD, and Stefano Bombardieri, MD

Systemic lupus erythematosus (SLE) is a complex disease characterized by the occurrence of various clinical manifestations that can be related to acute disease activity or chronic damage. The distinction between active, potentially treatable manifestations and permanent damage is critical in routine clinical practice. Assessment of disease activity in SLE patients is a problem faced every day by treating physicians. The simplest disease activity scale in use is an analogue global assessment scale1,2 that consists of a line (usually 10 cm) along which the rater draws a perpendicular mark, reflecting his or her overall judgment of disease activity (far left usually indicates no disease activity, and far right the most severe). This allows for a fairly reliable indicator of the rater’s overall impression after factoring in the entire complexity of subjective and objective signs suggesting individual organ activity, worsening, or improvement. Although generally considered as the “gold standard” for disease activity, this scale has great inter- as well as intra-rater variability.2-5 This feature becomes problematic when the assessment of activity in many patients is required in clinical research or in drug efficacy trials. However, given the heterogeneity of signs and symptoms related to systemic lupus and the unpredictable fluctuations of the disease, the development of reliable and reproducible disease activity indices that can outperform the simple analogue scale has not been easy. The evolution of valid and sensitive methods to assess disease activity has progressed slowly in the past 25 years. Some indices such as the BILAG, ECLAM, SLAM, SLEDAI and LAI (Table 2.1) have been validated in prospective studies,6-8 and their reproducibility, validity, and sensitivity to change have been compared.8 These instruments are now widely used in clinical research, including randomized controlledtrials of investigational treatments, with varying degrees of success. Critical factors in the use of formulated disease activity indices in multicenter studies of lupus are investigator training and consensus among investigators

about how these instruments should be applied. Equally important is to understand from the beginning the strengths and limitations of each instrument so that each is applied along with an optimal study design. Most activity measures are useful mainly as scoring systems for overall (global) disease activity (ECLAM, SLAM, SLEDAI, LAI). Global scores are useful to compare variables or interventions that apply widely across entire cohorts of SLE patients, since they allow comparisons of patients with a variety of disease manifestations. Global indices may also be used to provide benchmarks to define criteria for entry in some clinical trials. However, a global score does not differentiate between modest activity in many organs and high activity in one single organ, and may tend to blur the line between prognostic categories. The BILAG instrument was designed to provide separate scores for individual organ systems but can be used as a global disease measure. Assessment of individual organs allows quantification of the number or organs involved, the variable severity with which each organ might be affected, and, the ability to sort large cohorts into prognostic or therapeutic subgroups.3-5 The most widely used activity indices in SLE are reviewed below.

BRITISH ISLES LUPUS ASSESSMENT GROUP INDEX First reported in 1988,6 scoring of the BILAG is anchored in the physician’s intention to treat and assesses overall disease activity during the month previous to examination in each of eight organ systems: general, mucocutaneous, neurological, musculoskeletal, cardiovascular/respiratory, renal, and hematological.6,7 Laboratory and diagnostic testing required at each visit are relatively simple and usually ordered in routine clinical practice. However, when clinically indicated, more sophisticated testing can provide diagnostic data that reflect on the BILAG score. These aspects allow the calculation of the index with minimal

19

ASSESSMENT OF DISEASE ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

TABLE 2.1 MAIN CHARACTERISTICS OF WIDELY USED ACTIVITY INDICES BILAG

ECLAM

SLAM

SLEDAI

LAI

Type of index

Individual organ or global

Global

Global

Global

Global

Weighted variables

Yes

Yes

No

Yes

No

Severity assessment

Yes

No

Yes

No

Yes

Immunological variables

No

Yes

No

Yes

Yes

Therapy

No

No

No

No

Yes

Retrospective calculation

No

Yes

Yes

Yes

Yes

Modified for pregnancy

No

Yes

Yes

Yes

Yes

Used in childhood SLE

Yes

Yes

Yes

Yes

No

expense and potentially in any clinical setting, but ensures that for very sick patients scoring can be made as accurate as possible. Activity in each organ system is scored as follows: A, most active disease; B, intermediate activity; C, mild, stable disease; D, inactive disease in a previously affected organ; and E, no history of any activity. These scores are derived from assessment of a number of potential clinical manifestations in each organ system as to whether during the past month each has been absent, new, getting worse, getting better, or remaining overall the same as in the previous month. Manifestations are further weighted by assigning more impact to severe forms of each disorder (e.g., polyarticular arthritis with loss of function receives an A score, and so is weighted to score more heavily than simple arthritis, which engenders a B score) (see Appendix E). By assigning numerical values to the activity scores for each organ system, (A=9, B=3, C=1, D or E=0) it is possible to calculate a global activity score ranging from 0 to 72 (albeit in practice scores as high as 27 are rare). The reliability, validity, and sensitivity to change of the BILAG have been evaluated both by the original study group and independent investigators.7-11 The index was reviewed in 2000 and in 2004.12,13 In the last version, two additional systems—gastrointestinal and ophthalmic—were added (see Appendices G, H, and I). In 2004 the validity of the BILAG in assessing disease activity in childhood onset SLE was also demontrated.14 The BILAG index can be calculated with a computerized program, the British Lupus Integrated Prospective System (BLIPS). This software can calculate, along with the BILAG index, SLAM, SLEDAI, SLICC/ACR Damage Index, and SF-36 patient Health Questionnaire.12

EUROPEAN CONSENSUS LUPUS ACTIVITY MEASUREMENT 20

The ECLAM index is the result of work done between 1990 and 1992 by a European Consensus Study Group

aimed at defining disease activity in SLE, distinguishing clinical and serological variables that most identify disease activity, and comparing disease activity scales.15-18 A standardized clinical chart was prepared to record data from consecutively observed SLE patients, and the physician global assessment (PGA) was considered as the reference “gold standard” for lupus activity. Data from 704 SLE patients from various European centers were obtained and examined. Univariate analysis was performed to select the symptoms and laboratory parameters that best predicted disease activity. Multivariate regression analyses were carried out to define the relative weight of each variable. Therefore, the ECLAM derives from the study of real patients.15-18 The index, a global activity score, assesses disease activity within the past month. It comprises 15 weighted clinical and serological items, and scores disease activity from 0 to 10 (see Appendix J). Autoantibody testing is not included in the index, and the serological variables required to calculate the ECLAM (ESR, blood count, serum creatinine, urine analysis, and complement levels) are those normally used in routine clinical practice, facilitating its use in any clinical setting and at any patient’s visit.15 The reliability, validity, and sensitivity to change of the ECLAM have been evaluated both by the original study group who designed the index as well as by independent investigators.10,17-19 In 2000 the ECLAM index was validated for the retrospective calculation of disease activity from the data provided in patients’ clinical charts. The instrument’s reliability is good depending on the quality of data recorded on clinical charts.20 The ECLAM has been validated for use in pediatric lupus, and recently the Pediatric Rheumatology International Trials Organization (PRINTO) included the ECLAM in a disease activity core set21,22 to be used in clinical trials in juvenile SLE. A modified version for use in pregnancy exists.23 After training, 5 minutes are required to complete the ECLAM, which makes the index very easy to be used

SYSTEMIC LUPUS ACTIVITY MEASURE Developed in 1988, the SLAM also measures disease activity in the last month. It is a global activity score and includes nonweighted clinical and laboratory manifestations, which are only graded for severity.8 In the revised version (SLAM R), disease activity ranges from a minimum of 0 to a maximum of 84 and is based on the evaluation of 32 variables regarding 11 organs/systems and 8 laboratory manifestations.25 Each variable is scored from 0 to 3 on the basis of its severity, but the same 0 to 3 points applies to all manifestations whether fatigue or cerebritis is being rated. The index includes some subjective variables, such as arthralgias, myalgias, abdominal pain, and fatigue that may not be directly related to disease activity (see Appendix L). The SLAM has been used in the assessment of skin manifestations in cutaneous LE.26 We suggested a revision of the cutaneous parameters, which group together manifestations such as diffuse discoid LE, localized discoid LE and subacute LE lesions, or scarring and nonscarring alopecia, even though these parameters are not equivalent and probably represent different conditions.26 The index has proved to be reliable, valid, and sensitive to change.8,10,25 A score above 6 is considered clinically important since it is associated with a greater than 50% probability of initiating treatment.27 The SLAM R correlates with several aspects of the patient’s perception of health, as evaluated with the SF36.28,29 This could be due to the inclusion of the abovementioned subjective manifestations. A version of the index modified for use in pregnancy is available. This version does not include weight loss, ESR, and the scale for miscellaneous disease manifestations. It does, however, include fatigue, myalgias, arthralgias, and abdominal pain, which can be symptoms associated with pregnancy.30 However, items should not be scored on the SLAM if they are not, in the rater’s opinion, due to active lupus disease.

SYSTEMIC LUPUS ERYTHEMATOSUS DISEASE ACTIVITY INDEX The SLEDAI measures disease activity within the last 10 days. A global index, it includes 24 clinical and

laboratory variables that are weighted by the type of manifestation, but not by severity. Thus, vasculitis engenders far more points than thrombocytopenia, but a platelet count of 80 renders the same score as a platelet count of 5. Disease activity may in theory range from 0 to 105, but when this index is properly scored it is rare to find a patient with a score over 20. The SLEDAI includes scoring for the presence of autoantibodies (anti-dsDNA antibodies titers) and low complement, as well as for some renal and hematologic parameters.31 The index has been validated, and demonstrated to be reliable and sensitive to change.10,32-35 New versions of the index have been developed (SLEDAI 2000 and SELENA SLEDAI) to score persistent active disease in manifestations that were scored in the previous version only if new or recurrent (proteinuria, rash, alopecia, mucocutaneous manifestations).36 See Appendix A. The SELENA SLEDAI also contains the Physician’s Global Assessment scale and a flare index (see Appendices B and C). This latter addition is a major improvement since it allows worsening disease to be counted, even when a parameter may have received a score at the beginning of a study. A Spanish version (Mex-SLEDAI) is also available.37 A modified version for use in pregnancy has been developed (SLE-P-DAI).38 SLEDAI scores above 5 are associated with a greater than 50% probability of initiating therapy.27 Activity categories have been defined on the basis of SLEDAI scores: no activity (SLEDAI=0), mild activity (SLEDAI=1 to 5), moderate activity (SLEDAI=6 to 10), high activity (SLEDAI=11 to 19), and very high activity (SLEDAI≥20).39 The following outcomes have been suggested on the basis of SLEDAI scores: SLE flare-up, increase in SLEDAI of more than 3; improvement, reduction in SLEDAI of more than 3; persistently active disease, change in SLEDAI of more than or less than 3; and remission, a SLEDAI of 0.40 The possibility of retrospective use of the SLEDAI has been assessed. Although direct and chart index scores were correlated, the chart scores tended to underestimate disease activity. Therefore, disease activity scores calculated retrospectively from the data in clinical charts can only provide a qualitative assessment and cannot be considered as a substitute for scoring based on direct clinical assessment.41

OTHER INDICES

in routine clinical settings. A computerized program (ACTICARD) is also available to record patient demographic and clinical data, collect all variables that contribute to the assessment of disease activity, and automatically calculate at each visit the most used activity indices in SLE (ECLAM, SLAM, SLEDAI). The computerized program requires 10 minutes to complete, and validity of ACTICARD in the retrospective calculation of the ECLAM index has been evaluated.24

OTHER INDICES The lupus activity index (LAI) is a global activity score assessing activity over the previous 2 weeks. The index consists of five sections and includes eight organ systems and three laboratory measures, including antidsDNA antibodies. The index includes the physician global assessment as well as a score for treatment with corticosteroids and immunosuppressive drugs. The index

21

ASSESSMENT OF DISEASE ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

allows grading for severity based on physician judgment. The overall score ranges from 0 to 3, and is the mean of the physician global assessment, physician judgment of the severity of clinical manifestations, degree of laboratory abnormalities, and treatment.42 There is a validated version used in pregnancy (LAI P) that excludes asthenia and physician global assessment, and takes proteinuria and renal involvement into consideration separately.43 The SLE Activity Index Score (SIS) was developed in Austria by Josef Smolen and colleagues. It consists of 21 clinical items and 10 laboratory items (see Appendix K) and has been validated as well.

CONCLUSIONS Several instruments to assess disease activity in SLE have been developed, validated, and found to be reliable and sensitive. All seem adequate, in an appropriate clinical setting, to measure changes in disease activity over time. Furthermore, all of these indices require a relatively short time to be completed after appropriate training is completed. All instruments are increasingly used in clinical research, as well as in randomized controlled trials.44,45 It has been suggested that some indices will perform better as static indices, others as transitional indices, and some may be more feasible than others in clinical practice.10 However, no data are available that support the use of one index over the others. A recent study has shown that the BILAG, ECLAM, SELENA SLEDAI, SLAM R, and RIFLE (Responder Index for Lupus

Erythematosus) have a similar discriminatory ability as to whether disease activity was modified during follow-up, and therefore we suggest that any of these indices could be be used in clinical trials.46 However, effective use of these instruments in the hands of experts does not guarantee acceptable consistency in large, multicenter international trials, particularly when interventions are being tested for as long as a year or more in disparate populations of lupus patients. Investigator training and consensus are extremely important to maintain the validity of these measures in large studies over time. Many studies employ more than one index as primary and secondary outcomes, taking into account their diverse characteristics that may have different strengths or weaknesses in the context of a particular trial design. Multiple instrument use may be made easier by the use of computerized databases that automatically calculate some activity indices, such as ACTICARD and BLIPS. These databases not only automatically calculate the indices, avoiding a major source of potential inaccuracy in a study, but also can be used to collect real-time data as the study progresses, thereby facilitating data management and interim analyses. However, employment of these very useful tools should not be considered a substitute for complete understanding of instrument scoring by clinical assessors. Without understanding the numerical changes that occur with each assessment change, investigators cannot reliably gauge the degree of worsening or improvement that is being translated from a clinic note to a score sheet.

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EPIDEMIOLOGY AND DIAGNOSIS

3

Disease Development and Outcome Rosalind Ramsey-Goldman, MD, DrPH and Dafna Gladman, MD

NATURAL HISTORY Systemic lupus erythematosus (SLE) has been recognized as a disease of exacerbations and remission with variable course and prognosis. Prior to the early 1970s there were no criteria to classify SLE, and until the 1980s, there was no common method to evaluate disease activity and define flares and remission in a standard way. Physicians would describe a patient as flaring when there was evidence of new onset or increase in certain manifestations such as more arthritis, more extensive rash, increased serum creatinine, or increased proteinuria. Likewise, it was difficult to define remission. A patient who had no complaints was considered to be doing well, and some might describe that as a remission. (See Chapter 2 for a detailed discussion of methods for measuring disease activity.) Prior to the discovery of corticosteroid therapy, patients with SLE had a very poor prognosis, with the 5-year survival rate being no more than 55%.1 Corticosteroid therapy was discovered in the same year as the lupus erythematosus cell test for SLE. These early milestones facilitated the diagnosis of SLE, allowing for earlier diagnosis and therefore treatment of these patients.

Modern Era

24

Although the diagnosis of SLE was facilitated by the discovery of the lupus erythematosus (LE) phenomenon,2 it was not until the introduction of the fluorescent antinuclear antibody (ANA) test approximately 10 years later that laboratory diagnostic emphasis focused on the determination of ANAs.3 Initially, a positive ANA test was considered to be virtually synonymous with the diagnosis of active disease.4 However, the later detection of anti-DNA antibodies and depressed serum complement levels in patients allowed for more precise and earlier detection of both onset and subsequent exacerbations of disease.5 The subsequent description of the classification criteria for SLE further enhanced the ability to diagnose patients with SLE, and recent criteria have included more autoantibody systems.6

However, as far back as the early 1970s it was noted that there are patients with clinical features of SLE who do not demonstrate antinuclear antibodies.7-9 Despite the lack of ANA detection, these patients ran a similar course to ANA-positive patients. Most series of ANA-negative lupus consisted of patients with a high frequency of a photosensitive skin rash and perhaps a milder form of renal disease and central nervous system (CNS) involvement. Nevertheless, individual patients may be otherwise typical of classical SLE. These patients may have other autoantibodies in their sera, such as antibodies to cytoplasmic components.10 Thus, a negative ANA should not deter the clinician from considering a diagnosis of lupus. A large proportion of patients with “ANA-negative” lupus did have other autoantibodies detected in their sera, including anti-DNA antibodies, and antibodies to extractable nuclear antigens, especially anti-Ro and anti-La8,9 (Box 3.1). Provost and Reichlin11 pointed out that among ANA-negative patients, anti-Ro and anti-La were much more common than among ANA-positive patients, and that these patients often present with the clinical picture of a photosensitive facial dermatitis, positive latex fixation, and Sjögren’s syndrome. Interstitial pneumonitis has been reported in two ANA-negative patients with SLE.11 Serologically negative disease has also been recognized among patients with complement deficiencies.12 Another group of patients labeled as having “latent” or “incomplete” lupus has been described. These patients present with a constellation of features suggestive of SLE, but do not qualify by “classification criteria” or by a rheumatologist’s intuition as having classic SLE.13-15

BOX 3-1 ANA-Negative SLE ●

●

●

Anti-SSA/Ro and Anit-SSB/La are the most frequent antibodies present when the ANA antibody test is negative. ANA may be negative in patients with complement deficiencies. Other antibodies are rarely present when ANA is negative; includes double stranded DNA, Sm, or RNP.

also many other diseases with multisystem organ involvement that may mimic SLE. Prolonged observation is often necessary prior to an unequivocal diagnosis. Clinical presentation along with medical and family history, medication review, and laboratory testing are essential elements used to confirm a diagnosis of SLE.

CLINICAL MANIFESTATIONS

CLINICAL MANIFESTATIONS

These patients usually present with either one or two of the American College of Rheumatology (ACR) classification criteria for SLE, plus a number of additional and much less specific clinical features suggestive of lupus such as lymphadenopathy, fever, headache, nodules, Sjögren’s syndrome, fatigue, neuropathy, and oligoarthritis. These patients may, in addition, display some laboratory abnormalities including an increase in partial prothrombin time, hypergammaglobulinemia, an increased erythrocyte sedimentation rate, depressed complement components, positive rheumatoid factor, or aspirin-induced hepatotoxicity. Many of these patients will persist with their constellation of signs and symptoms over many years, without ever developing classic lupus. Although a small number do eventually develop classic lupus, none of the presenting clinical or laboratory features are sufficiently predictive to identify such patients in advance. Patients with latent lupus tend to have a milder form of disease and do not present with CNS involvement or renal disease. See Chapter 43 for further discussion of latent or incomplete lupus. Two subsets of patients who do satisfy classification criteria for SLE have been described that underscore the importance of recognizing whether patients are concordant or discordant in their clinical serologic profile. Patients with serologically active clinically quiescent disease (SACQ) were first described by Gladman and colleagues in 1979 and constituted 12% of the cohort.16 These patients remained well despite elevated antiDNA antibodies and reduced complement levels for a mean of 2.5 years. Over a longer follow-up period, half of these patients eventually did flare.17 Thus, there is a group of patients who remain serologically active but clinically quiescent, and who do not develop a disease flare even after a prolonged period of observation. These patients may not need any corticosteroids or immunosuppressive therapy. Similarly, there is a group of patients with clinical SLE who do not demonstrate any serologic abnormalities. These patients, who also constitute about 12% of patients with SLE, have been labeled as clinically active serologically quiescent (CASQ).18 Despite the lack of serologic markers, these patients have severe lupus manifestations, and may have severe disease requiring therapy with high doses of corticosteroids and immunosuppressive medications. Patients with discordant and serologic activity challenge the role of these antibody systems in the direct pathogenesis of SLE, and also complicate the attempt to identify predictors of flares in patients with SLE. SLE disease activity is the prototypic inflammatory autoimmune disease with multiorgan involvement, a wide variety of manifestations, and an unpredictable clinical course. The dynamic nature of the disease, with variable and intermittent signs and symptoms, makes the diagnosis particularly challenging. There are

Constitutional complaints such as malaise, overwhelming fatigue, fever, and weight loss are common presenting features of SLE. The presence of these features does not help the physician in the diagnosis of the disease, or in the identification of a flare, because they are just as likely to represent other medical problems including the development of infection or of fibromyalgia. (See Chapter 29 for further discussion of constitutional factors.) Although some organ system manifestations such as skin disease or arthritis are common in SLE, any system may be involved and may present in variable combinations with other organ systems. Thus, SLE may have such diverse clinical presentations as rash, arthritis, pleurisy, proteinuria, Raynaud’s phenomenon, seizures, or pyrexia of unknown origin. It is only with a high index of suspicion, a careful history and physical examination, and by obtaining appropriate laboratory confirmation, that the diagnosis will be recognized. CNS dysfunction and renal disease are two of the most critical manifestations. Potential CNS abnormalities include seizure, psychosis, cognitive impairment, mood disorders, headache, strokes, movement disorders, and aseptic meningitis. There is great variability in the expression, histopathology, and clinical course of renal disease, with virtually all patients with SLE displaying some degree of glomerular abnormality by renal biopsy. However, only 50% have clinically apparent disease. Early detection of renal involvement is critical as early intervention may prevent or delay progression to end-stage renal disease.

Assessment of Disease Activity The assessment of disease activity in SLE has become easier with the development and validation of a number of instruments over the past several years (Box 3.2).

BOX 3-2 Approach to Patient with SLE At every clinic visit: ● Assess lupus disease activity using a validated instrument. ● Assess damage using the SLICC/ACR-DI. ● Base treatment decisions not only on current disease activity, but also directed towards preventing complications, that is, hypertension, hypercholesterolemia, ischemic heart disease, diabetes mellitus, osteoporosis, and infection.

25

DISEASE DEVELOPMENT AND OUTCOME

The most commonly used instruments include: the SLEDAI,19 the SLAM,20 the British Isles Lupus Assessment Group (BILAG),21 the Lupus Activity Index (LAI),22 and the European Consensus Lupus Activity Measurement (ECLAM).23 These indices have been shown to be comparable.24,25 Thus, overall disease activity now can be evaluated as a prognostic factor in SLE. (See Chapter 2 on disease activity.)

Disease Damage The health status of patients with SLE is related not only to disease activity, but to the damage that results from recurrent episodes of disease flare and treatment complications (Box 3.3). The Systemic Lupus International Collaborating Clinics (SLICC) group, in conjunction with ACR, developed a damage index for SLE. The SLICC/ACR Damage Index describes the accumulation of damage in patients with SLE since disease onset without attribution, and includes items that may have resulted from the inflammatory process, disease treatment, or intercurrent events.26 The SLICC/ACR Damage Index has been validated and used in a number of studies and has been found to predict mortality.27-29 The Damage Index thus provides an important outcome measure in SLE, both for studies of prognosis, and in the assessment of long-term effects of treatment. Factors contributing to the development of damage include ethnicity,30,31 disease activity at presentation and over time,31-34 older age at onset, and disease duration.30,35 Socioeconomic features have also been suggested to contribute to disease damage. Investigators in a multiethnic U.S. cohort of SLE (LUMINA–lupus in minorities, nature versus nurture) patients found poverty to be an important variable associated with disease damage and mortality.36 The course of SLE and common complications of the illness are best understood by reviewing the individual major areas of potential disease involvement. The spectrum of disease damage includes cardiovascular disease, renal complications, musculoskeletal complications, CNS dysfunction, infections, and malignancy.

BOX 3-3 Spectrum of Disease Damage in SLE ●

●

●

●

●

26

Cardiovascular complications occur late in the disease, but earlier than in the general population. Lupus nephritis, the most serious complication with potential for disease damage, occurs in 50% of patients. Neuropsychiatric problems occur in 50% of patients, and may be difficult to diagnose or attribute to lupus or other causes. Up to 50% of patients will be ill with at least one serious infection. There is a small association between SLE and malignancy; the increased risk is primarily with non-Hodgkin’s lymphoma.

Cardiovascular disease: Fatal and nonfatal cardiovascular outcomes, myocardial infarction, and stroke are increasingly reported in longitudinal lupus cohorts. From a clinical and epidemiologic perspective, traditional risk factors for cardiovascular disease such as smoking, obesity, hypertension, hypercholesterolemia, sedentary lifestyle, and diabetes mellitus are similar in SLE to those documented in the general population.37,38-40 In addition, even after adjusting for the effect of these known risk factors, the rate of risks of myocardial infarction and stroke remained increased compared with population-based data.41 Renal complications: Lupus nephritis is the most serious complication and the most important determinant of morbidity and mortality in SLE patients. Although pathologically the majority of patients with SLE may have glomerulopathy, clinically relevant kidney disease occurs in about 50% of patients. In a proportion of these patients, renal failure resulting in dialysis or transplantation may develop. While it has traditionally been considered that SLE patients develop renal disease within the first 5 years of disease, recent data suggest that new manifestations may appear well into the course of SLE.64 Musculoskeletal complications: Almost every patient with SLE will have at least one musculoskeletal complaint through his or her disease course. Over 25% of these patients develop damage from all components of the musculoskeletal system.43 These complications range from synovitis, myositis, and tendon involvement, which may lead to joint deformities, osteonecrosis, and osteoporosis. Such complications become major concerns as these patients age. CNS dysfunction: Neuropsychiatric complications occur in 50% of SLE patients and include acute and chronic, as well as focal and diffuse manifestations. Seizures complicate the course in 25% of patients with lupus. Diffuse cerebral dysfunction is manifest as an organic effective disorder, personality disorder, psychosis, or coma. Vascular or migraine headaches occur in 10% of lupus patients. Recurrent involvement of the CNS may result in organic brain syndrome and dementia. The presence of CNS disease has been found commonly in patients who die with active lupus,44 and has been found to be associated with decreased survival. Infections: At least 50% of patients have one or more serious infections during the course of their disease. The spectrum of infections is related to the severity of disease, treatment, and endemic organisms. However, emerging genetic studies indicate that other host factors, such as race/ethnicity, might modulate the susceptibility to infection and thus may be particularly relevant in these situations. Malignancy: SLE patients, by virtue of their disease, have basic defects in immune cell function, resulting

increased risk of developing a malignancy, but this risk has not been confirmed in rigorous epidemiologic studies.

MORTALITY

in immune dysregulation independent of immunosuppressive treatments. These immune defects could be potentiated by immunosuppressive therapy, leading to concern that this population may be at increased risk for developing cancer. Results of a study involving a multisite international cohort of SLE patients support the hypothesis of an association between SLE and cancer, and more precisely define the risk of non-Hodgkin’s lymphoma (NHL) in SLE. Data from the study also suggested an increased risk of lung cancer.45 Clinicians frequently inform patients with SLE, particularly those who require immunosuppressive therapy, of a potential

MORTALITY Once a disease with high mortality, SLE is now considered a chronic disease because of new treatment approaches. More than 90% of patients with SLE survive for at least 2 years after diagnosis, compared with 50% of such patients 50 years ago. More recent surveys reveal an 80% to 90% 10-year survival rate and 80% at 20 years (Table 3.1).36,42,44,46-68 The mechanism for

TABLE 3.1 SURVIVAL RATES IN SLE Author

n

Year

Center

Kellum and Haserick47

299

1964

Cleveland

69

54

—

—

Urman and Rothfield48

156

1968

New York

70

63

—

—

Estes and Christian49

150

1971

New York

77

60

50

—

209

1976

Farmington

93

84

—

—

609

1979

Los Angeles

88

79

74

—

183

1980

Singapore

70

60

—

—

1103

1982

U.S. multicenter

86

76

—

—

48

Urman and Rothfield Wallace et al.

50

Boey51 52

Ginzler et al.

53

5 Years

10 Years

15 Years

20 Years

Malaviya et al.

101

1986

India

68

50

—

—

Swaak et al.54

110

1989

Holland

92

87

—

—

55

389

1990

Alabama

89

83

79

—

56

570

1990

Los Angeles

97

93

83

—

310

1990

Stanford

88

64

—

—

286

1990

India

78

—

—

—

Wang et al.

539

1990

Malaysia

82

70

—

—

Ward et al.60

408

1991

Durham

82

71

63

—

218

1993

Chile

92

77

66

—

665

1993

Toronto

93

85

79

68

165

1993

London

93

86

78

—

Reveille et al. Pistiner et al.

Seleznick and Fries57 58

Kumar et al.

59

Massardo et al.61 Abu-Shakra et al.

44

Tucker et al.62 Blanco et al.63

306

1993

Spain

90

85

80

—

42

162

1994

Sweden

93

83

—

—

64

Peshcken and Esdaile

177

1996

Manitoba

98

96

90

—

Jacobsen et al.65

513

1999

Denmark

91

76

64

53

288

2001

U.S. multicenter

86

70

—

—

Bellomio et al.

366

2001

Argentina

91

85

—

—

Manger et al.67

338

2002

Germany

97

90

—

—

178

2003

Greece

97

90

—

—

Ståhl-Hallengen et al.

Alarcon et al.

36 66

Alamanos et al.68 46

Pons-Estel et al. a

1214

2003

Latin America

a

95

Four-year survival.

27

DISEASE DEVELOPMENT AND OUTCOME

improved survival over the past five decades is unclear. It may be concluded that the major contributing factors toward improved survival since 1950 are the availability of dialysis, corticosteroids, and improved antibiotic and antihypertensive agents. While further improvement in subsequent decades may have resulted from earlier diagnosis and the inclusion of milder cases in the more recent studies, authors of a Dutch study analyzed the pattern of clinical features in patients with SLE over a recent 24-year period and concluded that the use of ACR criteria for the classification of SLE, or the availability of laboratory tests for the diagnosis of the disease, have not led to earlier diagnosis of SLE nor to a change in its clinical pattern during that time.69 Likewise, a study from Toronto70 further investigated the reasons for the improved survival noted among patients with SLE over a 24-year period. These authors had documented the increased survival rates for their patients over that period of time and concluded that the improved rates in their patients were not the result of earlier diagnosis and/or a milder form of the disease. Because no new medications for SLE were instituted during the period of study, new treatments could not be considered as the reason for improvement. More appropriate use of conventional therapy was a more likely explanation. Our understanding of the prospects for recovery from SLE has evolved in the past several decades, both because of better understanding of the disease process itself and through the development of methods with which to assess outcome. However, despite improved survival, patients with SLE still die at a rate three times that of the general population.55,70,71 Several studies have implicated factors associated with mortality in SLE. A recent study in Japan evaluated the long-term prognosis of SLE patients divided according to organ involvement at the time of diagnosis. For example, the study found that patients with neuropsychiatric SLE (NPSLE), accompanied with acute confusional state/seizure disorder, cerebral vascular disease, or pneumonitis had poor survival rates, and the cause of death was related to their major organ involvement.73 The LUMINA study discovered demographic variation in survival associated with SLE, with mortality rates higher among Hispanics and African Americans than among whites.36

Causes of Death

28

The causes of death in patients with SLE may be divided into those related to the SLE disease process itself, those related to treatment, and deaths from unrelated causes. The causes related to the SLE include active disease, vasculitis leading to CNS disease or intestinal perforation, intractable bleeding, and endorgan failure, such as renal, cardiac or pulmonary.

A recent study examining trends in mortality rates from a large multicenter cohort of patients with SLE found increased mortality rates in SLE versus the general population, and importantly, cause-specific death rates due to infections and renal disease greatly elevated in earlier decades and decreased over the observation interval. Furthermore, important heightened risk was seen in SLE regarding death due to circulatory disease, respiratory disease, NHL, and lung cancer over the same time period when infections and renal disease as causes of death declined.73 Treatment for SLE may in itself result in fatal complications such as fulminant infection (which may just as likely be associated with active disease), perforation or peptic ulcer disease and, possibly, vascular disease.

Disease-Related Factors SLE-related factors that may affect prognosis include time between the onset of symptoms and the diagnosis of SLE, change in disease expression over time, presence of specific disease manifestations, overall disease activity, and use of therapeutic modalities. A bimodal mortality curve in SLE is prevalent. Patients who die within 5 years of disease onset usually have active SLE, high steroid requirements, and infections. Patients who die later usually have evidence of atherosclerotic cardiovascular disease; in contrast, active SLE, infection, and high steroid requirements are uncommon. Most patients with SLE die from active SLE, nephritis, sepsis, and cardiovascular disease. Mortality from CNS disease or malignancies rarely occurs.

Other Factors Several specific factors have been implicated as predisposing factors for mortality in patients with SLE (Table 3.2). These include general features that are unrelated to the disease process itself, such as race/ ethnicity, gender, age at onset, and socioeconomic status. Race/ethnicity: Black patients with SLE have been considered to have a poorer prognosis than white patients. Although race/ethnicity did not appear to be an important prognostic factor in a logistic regression analysis in the multicenter study published by Ginzler and colleagues,52 it was found to be a factor adversely affecting survival in SLE when a Cox multivariate analysis was applied to a group of 389 patients studied by Reveille and colleagues.55 It has been difficult to separate out the effects of race/ethnicity and socioeconomic status, particularly with reference to differences between black and white patients in the United States. In Reveille’s study, white patients with private insurance fared better than black patients with private insurance.55 However, there was no difference in

Study

Year

Time

Age

Estes and Christian49

1955

A

−

Wallace et al.50

1981

D

55

1990

Pistiner et al.56

1991 1993

Reveille et al.

Ward et al.

60

Race/Ethnicity

Sex

SES

Renal

CNS

BP

Plat

DA

SDI

−

−

?

+

+

?

?

?

?

+

−

−

?

+

−

−

−

?

?

D

+

+

−

−

+

?

+

+

?

?

D

−

−

+

−

+

?

?

+

?

?

A

+

−

−

+

?

?

?

?

?

?

Massadro et al.

1993

A

−

−

−

−

+

−

−

+

+*

?

Abu-Shakra et al.44

1993

A

+

−

−

−

+

−

−

+

+*

?

Blanco et al.63

1993

A

−

−

+

?

+

+

?

?

?

?

Jacobsen et al.65

1999

A

+

NA

+

?

−

+

?

?

?

?

36

2001

E

−

−

−

+

−

−

−

−

+

+

67

2002

D

+

−

−

?

+

+

?

?

?

+

61

Alarcon et al.

Manger et al.

CONCLUSIONS

TABLE 3.2 FACTORS AFFECTING MORTALITY IN SLE

A, any time before death; BP, increased blood pressure; CNS, central nervous system; D, at diagnosis; DA, disease activity; E, at study entry; NA, not available; Plat, decreased platelets; SES, socioeconomic status, −, no asscociation; +, association; ?, not studied. *At Study entry.

outcome when black patients with and without private insurance were compared. This supports the notion that there may be racial/ethnic differences in the expression of this disease and its outcome. However, Ward and colleagues60 demonstrated that, although survival was better for whites than for blacks, it was related to socioeconomic status, which was lower among blacks. Similarly, in the LUMINA study, poverty appeared to be one of the most important determinants of mortality, not race/ethnicity.36 Gender: The relationship between gender and prognosis has been controversial. Higher mortality in females than males with SLE has been suggested by some, whereas others demonstrated a better prognosis for women than for men. Gender did not appear as a significant predictor in the statistical analysis performed by Ginzler and colleagues,52 or in the University of Toronto cohort.44 Thus, the issue of the effect of gender on prognosis in SLE remains unanswered. Age at onset: Age at onset of SLE was found to be a significant predictor of survival at both 1 and 5 years in a multicenter study, with better survival in older patients.52 In contrast, Reveille and colleagues55 found that increasing age of onset adversely affected survival, and Abu-Shakra and colleagues44 found age greater than 50 years at diagnosis to be a risk for death. Onset of SLE in the pediatric age group has been associated with worse prognosis,

but a study of childhood SLE found the 5-year survival at 85.3% to be the same as for adult populations.75 Indeed, a comparison with the estimated survival of the age-matched segment of the U.S. population showed that SLE patients fared worse in all age groups.60 Socioeconomic factors: Patients with better education and higher socioeconomic status seem to fare better than the lower-status population. Patients with a lower education level, which may reflect lower socioeconomic status, do less well than those with more education.

CONCLUSIONS The outcomes in patients with SLE have changed over the years. Initially there was significantly decreased survival due to the disease process itself. Over the past five decades there has been improvement in survival such that in the current era the 20-year survival rate far exceeds the 5-year survival rate in the mid-1950s. However, patients with SLE still have three times the risk of death compared to the general population. The trend toward improved survival is counterbalanced with residual morbidity associated with organ damage due to the disease itself or as a consequence of treatment. Therefore, physicians caring for patients with SLE must address issues of inflammatory activity and be cognizant of the fact that damage must be kept to a minimum.

29

DISEASE DEVELOPMENT AND OUTCOME

30

REFERENCES 1. Merrell M, Shulman, LE. Determination of prognosis in chronic disease, illustrated by systemic lupus erythematosus. J Chronic Dis 1955;1:12-32. 2. Hargraves MM, Richmond H, Morton R. Presentation of two bone marrow elements: the “tart” cell and “LE” cell. Mayo Clin Proc 1948;23:25. 3. Friou CJ. Clinical application of lupus serum nucleoprotein reaction using fluorescent antibody technique. J Clin Invest 1957;36:890. 4. Notman DD, Kurata N, Tan EM. Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann Intern Med 1975;83:464. 5. Schur PH, Sandson J. Immunologic factors and clinical activity in systemic lupus erythematosus. N Engl J Med 1968;278:533. 6. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997:40:1725. 7. Fessel WJ. ANA-negative systemic lupus erythematosus. Am J Med 1978;64:80. 8. Gladman DD, Chalmers A, Urowitz MB. Systemic lupus erythematosus with negative LE cells and antinuclear factor. J Rheumatol 1978;5:142. 9. Maddison PJ, Provost TT, Reichlin M. Serologic findings in patients with “ANA-negative” systemic lupus erythematosus. Medicine 1981;60:87. 10. Provost TT, Ahmed AR, Maddison PJ, et al. Antibodies to cytoplasmic antigens in lupus erythematosus. Serologic marker for systemic disease. Arthritis Rheum 1977;20:1457. 11. Provost TT, Reichlin M. Antinuclear antibody-negative systemic lupus erythematosus. I. Anti-Ro (SSA) and anti-La (SSB) antibodies. J Am Acad Dermatol 1981;4:84. 12. Vandesteen PR, Provost TT, Jordon RE, et al. C2 deficient systemic lupus erythematosus. Its association with anti-Ro (SSA) antibodies. Arch Dermatol 1982;118:584. 13. Ganczarczyk L, Urowitz MB, Gladman DD. Latent lupus. J Rheumatol 1989;16: 475-478. 14. Swaak AHG, van de Brink H, Smeenk RJT, et al. Incomplete lupus erythematosus: results of a multicenter study under the supervision of EULAR Standing Committee on International Clinical Studies Including Therapeutic Trials (ESCISIT). Rheumatology 2001;40:89-94. 15. Stahl HC, Nived O, Sturfelt G. Outcome of incomplete systemic lupus erythematosus after 10 years. Lupus 2004;13(2):85-88. 16. Gladman DD, Urowitz MB, Keystone EC. Serologically active clinically quiescent systemic lupus erythematosus: a discordance between clinical and serologic features. Am J Med 1979;66:210-215. 17. Walz-Leblanc B, Gladman DD, Urowitz MB. Serologically active clinically quiescent SLE, long term follow-up. J Rheumatol 1994;21:174-175. 18. Gladman DD, Hirani N, Ibañez D, et al. Clinically active serologically quiescent (CASQ) SLE. J Rheumatol 2003;30:1960-1962. 19. Gladman DD, Ibañez D, Urowitz MB. SLE Disease Activity Index 2000. J Rheumatol 2002;29:288-291. 20. Liang MH, Socher SA, Larsen MG, et al. Reliability and validity of 6 systems for the clinical assessment of disease activity in SLE. Arthritis Rheum 1989;32:1107-1118. 21. Isenberg DA, Rahman A, Allen E, et al. BILAG 2004. Development and initial validation of an updated version of the British Isles Lupus Assessment Group’s disease activity index for patients with systemic lupus erythematosus. Rheumatology (Oxford) 2005;44:902-906. 22. Petri M, Hellmann D, Hochberg M. Validity and reliability of lupus activity measures in the routine clinic setting. J Rheumatol 1992;19:53-59. 23. Vitali C, Bencivelli W, Isenberg DA, et al. Disease activity in systemic lupus erythematosus: report of the Consensus Study Group of the European Workshop for Rheumatology Research. II. Identification of the variables indicative of disease activity and their use in the development of an activity score. The European Consensus Study Group for Disease Activity in SLE. Clin Exp Rheumatol 1992;10:541-547. 24. Gladman DD, Goldsmith CH, Urowitz MB, et al. Cross-cultural validation of three disease activity indices in systemic lupus erythematosus (SLE). J Rheumatol 1992;19:608-611.

25. Gladman D, Goldsmith C, Urowitz M, et al. Sensitivity to change of 3 SLE disease activity indices: international validation. J Rheumatol 1994;21:1468-1471. 26. Gladman D, Ginzler E, Goldsmith CH, et al. The development and initial validation of the SLICC/ACR damage index for SLE. Arthritis Rheum 1996;39:363-369. 27. Urowitz MB, Gladman DD. Assessment of disease activity and damage in SLE. Baillière’s Clin Rheumatol 1998;12:405-413. 28. Rahman P, Gladman DD, Urowitz MB, et al. Early damage as measured by the SLICC/ACR Damage Index is a predictor of mortality in SLE. Lupus 2001;10:93-96. 29. Nived O, Jonsen A, Bengtsson AA, et al. High predictive value of the Systemic Lupus International Collaborating Clinics/ American College of Rheumatology damage index for survival in systemic lupus erythematosus. J Rheumatol 2002;29: 1398-1400. 30. Sutcliffe N, Clarke AE, Gordon C, et al. The association of socioeconomic status, race, psychosocial factors and outcome in patients with systemic lupus erythematosus. Rheumatology 1999;38:1130-1137. 31. Mok CC, Ho CT, Wong RW, et al. Damage accrual in southern Chinese patients with systemic lupus erythematosus. J Rheumatol 2003;30:1513-1519. 32. Toloza SM, Roseman JM, Alarcon GS, et al. Systemic lupus erythematosus in a multiethnic US cohort (LUMINA): XXII. Predictors of time to the occurrence of initial damage. Arthritis Rheum 2004;50:3177-3186. 33. Stoll T, Sutcliffe N, Mach J, et al. Analysis of the relationship between disease activity and damage in patients with systemic lupus erythematosus–a 5-yr prospective study. Rheumatology (Oxford) 2004;43:1039-1044. 34. Ibanez D, Gladman DD, Urowitz MB. Adjusted mean Systemic Lupus Erythematosus Disease Activity Index-2K is a predictor of outcome in SLE. J Rheumatol 2005;32:824-827. 35. Maddison P, Farewell V, Isenberg D, et al. The rate and pattern of organ damage in late onset systemic lupus erythematosus. J Rheumatol 2002;29:913-917. 36. Alarcon GS, McGwin G Jr, Bastian HM, et al. Systemic lupus erythematosus in three ethnic groups. VII [correction of VIII]. Predictors of early mortality in the LUMINA cohort. LUMINA Study Group. Arthritis Rheum 2001;45(2):191-202. 37. Manzi S, Meilahn EN, Rairie JE, et al. Age-specific incidence rates of myocardial infarction and angina in women with systemic lupus erythematosus: comparison with the Framingham Study. Am J Epidemiol 1997;145(5):408-415. 38. Nikpour M, Urowitz MB, Gladman DD. Premature atherosclerosis in systemic lupus erythematosus. Rheum Dis Clin North Am 2005;31:329-354. 39. Petri M, Perez-Gutthann S, Spence D. Risk factors for coronary artery disease in patients with systemic lupus erythematosus. Am J Med 1992;93:513-519. 40. Manzi S. Prevalence and risk factors of carotid plaque in women with systemic lupus erythematosus. Arthritis Rheum 1999; 42:51-60. 41. Esdaile JM, Abrahamowicz M, Grodzicky T, et al. Traditional Framingham risk factors fail to fully account for accelerated atherosclerosis in systemic lupus erythematosus. Arthritis Rheum 2001;44:2331-2337. 42. Ståhl-Hallengren C, Nived O, Sturfelt G. Outcome of incomplete systemic lupus erythematosus after 10 years. Lupus 2004; 13(2):85-88. 43. Zonana-Nacach A, Barr SG, Magder LS, et al. Damage in systemic lupus erythematosus and its association with corticosteroids. Arthritis Rheum 2000;43:1801-1808. 44. Abu-Shakra M, Urowitz MB, Gladman DD, et al. Mortality studies in systemic lupus erythematosus. Results from a single centre. I. Causes of death. J Rheumatol 1995;22:1259-1264. 45. Bernatsky S, Boivin JF, Joseph L, et al. An international cohort study of cancer in systemic lupus erythematosus. Arthritis Rheum 2005;52:1481-1490. 46. Pons-Estel BA, Catoggio LJ, Cardiel MH, et al. The GLADEL multinational Latin American prospective inception cohort of 1,214 patients with systemic lupus erythematosus: ethnic and disease

48.

49. 50.

51. 52.

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59.

60. Ward MM, Pyun E, Studenski S. Long-term survival in systemic lupus erythematosus. Patient characteristics associated with poorer outcomes. Arthritis Rheum 1995;38:274-283. 61. Massardo L, Martinez ME, Jacobelli S, et al. Survival of Chilean patients with systemic lupus erythematosus. Semin Arthritis Rheum 1994;24:1-11. 62. Tucker LB, Menon S, Schaller JG, et al. Adult and childhood onset systemic lupus erythematosus: a comparison of onset, clinical features, serology and outcome. Br J Rheumatol 1995;34:866-872. 63. Blanco FJ, Gomez-Reino JJ, de la Mata J, et al. Survival analysis of 306 European Spanish patients with systemic lupus erythematosus. Lupus 1998;7:159-163. 64. Peschken CA, Esdaile JM. Systemic lupus erythematosus in North American Indians: a population based study. J Rheumatol 2000;27:1884-1891. 65. Jacobsen s, Petersen J, Ulman S, et al. Mortality and causes of death of 513 Danish patients with systemic lupus erythematosus. Scand J Rheumatol 1999;28:75-80. 66. Bellomio V, Spindler A, Lucero E, et al. Systemic lupus erythematosus: mortality and survival in Argentina. A multicenter study. Lupus 2000;9:377-381. 67. Manger K, Manger B, Repp R, et al. Definition of risk factors for death, end stage renal disease, and thromboembolic events in a monocentric cohort of 338 patients with systemic lupus erythematosus. Ann Rheum Dis 2002;61:1065-1070. 68. Alamanos Y, Voulgari PV, Siozos C, et al: Epidemiology of systemic lupus erythematosus in northwest Greece 1982-2001. J Rheumatol 2003;30:731-735. 69. Swaak AJG, Nieuwenhuis EJ, Smeenk RJT. Changes in clinical features of patients with systemic lupus erythematosus followed prospectively over 2 decades. Rheumatol Int 1992;12:71-75. 70. Urowitz MB, Abu-Shakra M, Gladman DD, et al. Mortality studies in systemic lupus erythematosus. Results from a single centre. III. Improved survival over 24 years. J Rheumatol 1997;24:1061-1065. 71. Moss KE, Ioannou Y, Sultan SM, et al. Outcome of a cohort of 300 patients with systemic lupus erythematosus attending a dedicated clinic for over two decades. Ann Rheum Dis 2002;61:409-413. 72. Tokano Y, Morimoto S, Amano H, et al. The relationship between initial clinical manifestation and long-term prognosis of patients with systemic lupus erythematosus. Mod Rheumatol 2005;15: 255-262. 73. Bernatksy S, Boivan J-F, Joseph L, et al. Mortality in systemic lupus erythematosus. Arthritis Rheum 2006;54:2550-2557.

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heterogeneity among “Hispanics.” Medicine (Baltimore) 2004; 83(1):1-17. Kellum RE, Haserick JR. Systemic lupus erythematosus. A statistical evaluation of mortality based on a consecutive series of 299 patients. Arch Intern Med 1964;113:200-207. Urman JD, Rothfield NF. Corticosteroid treatment in systemic lupus erythematosus: survival studies. JAMA 1977;238: 2272-2276. Estes D, Christian CL. The natural history of systemic lupus erythematosus by prospective analysis. Medicine 1971;50:85-95. Wallace DJ, Podell T, Weiner J, et al. Systemic lupus erythematosus survival patterns. Experience with 609 patients. JAMA 1981;245:934-938. Boey ML. Systemic lupus erythematosus in Singapore. Ann Acad Med Singapore 1998;27:35-41. Ginzler EM, Diamond HS, Weiner M, et al. A multicenter study of outcome of systemic lupus erythematosus. I. Entry variables as predictors of progress. Arthritis Rheum 1982;25:601-611. Malaviya AN, Misral R, Banerjee S, et al. Systemic lupus erythematosus in North Indian Asians: a prospective analysis of clinical and immunological features. Rheumatol Int 1986;6: 97-101. Swaak AJG, Nossent JC, Bronsveld W, et al. Systemic lupus erythematosus. I. Outcome and survival: Dutch experience with 110 patients studied prospectively. Ann Rheum Dis 1989; 48:447-454. Reveille JD, Bartolucci A, Alarcón-Segovia D. Prognosis in systemic lupus erythematosus. Negative impact of increasing age at onset, black race, and thrombocytopenia, as well as causes of death. Arthritis Rheum 1990;33:37-48. Pistiner M, Wallace DJ, Nessim S, et al. Lupus erythematosus in the 1980s: a survey of 570 patients. Semin Arthritis Rheum 1991;21:55-64. Seleznick MJ, Fries JF. Variables associated with decreased survival in systemic lupus erythematosus. Semin Arthritis Rheum 1991;21:73-80. Kumar A, Malaviya AN, Singh RR, et al. Survival in patients with systemic lupus erythematosus in India. Rheumatol Int 1992;122: 107-109. Wang F, Wang CL, Tan CT, et al. Systemic lupus erythematosus in Malaysia: a study of 539 patients and comparison of prevalence and disease expression in different racial and gender groups. Lupus 1997;6:248-253.

31

EPIDEMIOLOGY AND DIAGNOSIS

4

Quality of Life and Economic Aspects Ann E. Clarke, MD, MSc and Pantelis Panopalis, MD

INTRODUCTION Systemic lupus erythematosus (SLE) primarily affects young women in the prime of life and is characterized by variable and unpredictable manifestations. This pervasive disease can affect every aspect of a person’s life, including physical, psychological, and social functioning, and its impact can range anywhere from mild, benign symptoms to life-threatening complications. Even though considerable progress has been made in understanding SLE both in terms of disease activity and chronic damage, it has become increasingly clear that these physiologic consequences alone are not sufficient to characterize the wide spectrum of its effects on the patient. Given this realization, in recent years there has been increased emphasis in clinical trials on measuring both the health outcomes of direct importance to patients and the economic consequences. Quality of life (QoL) has, as a result, gained widespread acceptance as an important measure of efficacy in clinical trials. Economic evaluations are also being incorporated as a means of determining whether the benefits of an intervention are commensurate with its costs. The first portion of this chapter will focus on the psychological and social aspects of SLE and discuss measurement tools to characterize and quantify QoL in this disorder. The second part will provide an overview of the types of economic analyses and describe how they may be implemented in research on SLE.

QUALITY OF LIFE

Definition

32

Quality of life is an ill-defined term that means different things to different people. The concept is vague and multidimensional, and research in this area spans a wide range of disciplines. Of particular concern in the health sciences are those areas that are affected by disease and its treatment, and so to distinguish between QoL in its more general sense and to emphasize its relevance within the context of health, the term healthrelated quality of life (HRQOL) is frequently used.

The concept of HRQOL parallels the World Health Organization’s 1948 definition of health: “Health is a state of complete physical, mental and social wellbeing and not merely the absence of disease and infirmity.” Measures of the physiologic processes of disease alone cannot adequately capture the many dimensions of health embodied within this broad definition. Traditionally, health status assessments relied on a “disease” model, in which abnormalities are indicated by objective signs and symptoms. In contrast, the “illness” model relies on subjective feelings of pain and discomfort that may not necessarily result from a pathologic abnormality. Both of these concepts of health—disease and illness—must be taken into account in order to make comprehensive assessments of health status. Health-related quality of life comprises those parts of QoL that directly relate to a person’s health, and may be defined as a measure of a person’s sense of physical, emotional, and social well-being associated with a disease or its treatment. This definition may be broadened to include indirect consequences of disease such as unemployment or financial difficulties.1

Why Measure Quality of Life? Increasingly, HRQOL is being recognized as an important aspect of chronic diseases such as SLE and is being recognized as a relevant measure of efficacy in clinical trials. OMERACT (Outcomes Measures in Rheumatology Clinical Trials), an international network of experts and opinion leaders, has recommended the inclusion of three outcome measures in SLE clinical trials: (1) a disease activity score, (2) a damage index, and (3) a patient-assessed measure of health status, disability, and HRQOL.2 The importance of QoL assessment in health has been underscored by the need to assess the relative effectiveness and appropriateness of rival medical treatments in a context of increasing pressure on healthcare resources. Increased questioning of the use of various medical treatments and methods of organizing health services has led to a paradigm shift in the approach to measurement of health outcomes.3 Furthermore, measurement of QoL in

Measuring Quality of Life Although it has been debated whether QoL assessments should be made by the patient or by a health professional, there is now a general agreement among researchers that patients should complete questionnaires about their QoL themselves. Questionnaires may be administered to patients by trained interviewers in certain situations, such as when the patient is unable to read or write.3 The goal of QoL research is to determine health outcomes from the perspective of the patient, and numerous studies have shown that patients’ opinions may vary considerably from those of both healthcare professionals and patients’ relatives.1 Although one important criticism of patient selfassessment is the occurrence of subjectivity, this subjectivity should, in fact, be viewed as a strength, as it reflects the patient’s point of view. A plethora of instruments exist that aim to measure aspects of QoL regarded as pertinent to health status, such as life satisfaction, mental health, relationships, fatigue, energy, and vitality.4 Given its multidimensional nature, instruments developed for measuring HRQOL will likely be more accurate if they evaluate a number of dimensions. Most authors agree on the existence of four major domains of QoL: (1) physical status and functional abilities, (2) psychological status and well-being, (3) social interactions, and (4) economic and/or vocational status and factors.5 Tools that measure only one or two of these domains may fail to comprehensively assess an individual’s well-being. Two basic approaches have been used in the measurement of HRQOL: generic instruments and diseasespecific instruments.

Generic Instruments Assessment of HRQOL in patients with SLE has relied largely on the use of generic instruments. Generic instruments are intended for general use, apply to a wide variety of populations, and may be applicable to various illnesses and conditions. They allow for comparisons with other groups, including comparing the relative impact of various healthcare programs.5 It has been argued, however, that generic instruments may be less responsive in specific conditions and as a result will always require supplementation with disease-specific measures in order to detect important clinical changes.3

Earlier health assessment instruments tended to focus on physical symptoms, that is, measuring physical impairment, disability, and handicap. These instruments emphasized the measurement of general health, with the assumption that poorer health indicates poorer QoL.1 However, patients may not respond equally to similar levels of impairment or disability. Newer instruments, such as the Medical Outcomes Study 36-Item Short Form (SF-36), aim to better assess the subjective nonphysical aspects of QoL and place greater emphasis on emotional and social issues.

Disease-Specific Instruments Disease-specific questionnaires are designed to measure outcomes for a specific disease. In contrast to generic instruments, disease-specific instruments aim to identify issues pertinent to a specific condition and, as a consequence, may be more responsive in detecting differences in clinical outcomes. Two examples of disease-specific questionnaires include the Stanford Arthritis Centre Health Assessment Questionnaire (HAQ)6 and the Arthritis Impact Measurement Scales (AIMS)7; both were developed to assess health status in patients with arthritis and are discussed in detail in a subsequent section of this chapter. As of yet, there are no widely used disease-specific questionnaires for patients with SLE.

MEASUREMENT OF QUALITY OF LIFE IN PATIENTS WITH SLE

addition to more objective clinical indicators of disease allows for a more comprehensive assessment of the impact of disease and clinical therapies. Information about broader patient outcomes empowers physicians and patients when making decisions about the most appropriate health care. The challenge remains to identify instruments that will accurately and reliably assess these disease outcomes.

MEASUREMENT OF QUALITY OF LIFE IN PATIENTS WITH SLE The following instruments have been used to evaluate HRQOL in SLE studies (Table 4.1).

Generic Instruments The SF-36, developed by Ware and Sherbourne,8 is the most commonly used generic health status questionnaire. It has become the standard health status questionnaire in U.S. health policy research and it is increasingly being used worldwide.9 It is a concise 36-item questionnaire that was designed to be a short, psychometrically sound, generic measure of subjective health status applicable in a wide range of settings.10 It can either be self-assessed or administered by a trained interviewer. Eight domains are measured: physical functioning, social functioning, role limitations due to physical problems, role limitations due to emotional problems, mental health, energy/ vitality, pain, and general health perception. These subscales can be summarized into two component scores: the Physical Component Summary score (PCS) and the Mental Component Summary score (MCS), allowing for easier comparisons and reducing the probability of chance findings.11 The SF-36 has been translated into numerous languages and cultures, and

33

QUALITY OF LIFE AND ECONOMIC ASPECTS

TABLE 4.1 INSTRUMENTS USED IN ASSESSMENT OF HEALTH-RELATED QUALITY OF LIFE IN SLE Dimensions Assessed Physical/Functional Impact

Social/Emotional Impact

Self-Esteem/ Well-being

Medical Outcomes Study Short Form 36 (SF-36)8

Yes

Yes

Nottingham Health Profile (NHP)15

Yes

Sickness Impact Profile (SIP)16

Items

Validity studies in SLE

Yes

36

Yes, numerous studies

No

Yes

38

No

Yes

Yes

Yes

136

No

European QoL Scale (EuroQol)17

Yes

Yes

No

5 VAS

Yes18,19

World Health Organization QoL scale-Bref (WHOQOL-Bref)21

Yes

Yes

Yes

26

No

Stanford Arthritis Center Health Assessment Questionnaire (HAQ I)6

Yes

No

No

20

Yes23,24

Arthritis Impact Measurement Scale 2 (AIMS2)7

Yes

Yes

No

78

No

SLE Quality of Life Scale (SLEQOL)29

Yes

Yes

Yes

40

Yes29

SLE Symptom Checklist (SSC)30

Yes

Yes

No

38

Yes30

Lupus Quality of Life Scale (LupusQoL)a

Yes

Yes

Yes

34

Yes31

Generic

Disease-Specific

VAS, visual analogue scale. a. See Appendix M.

34

has been found to be valid and reliable in various conditions. The SF-36 has been shown to be a valid and reliable questionnaire in SLE.12 Patients with SLE have been shown to have a poorer QoL than people without chronic illness with respect to all aspects of health.12,13 In a British study of 150 patients with SLE, Stoll and colleagues12 showed that all of the QoL domains assessed by the SF-36, except for emotional role limitations, were significantly lower in patients with SLE than in a British control population of normal adults of working age. In another study, SF-36 results of 120 Canadian SLE patients were compared to normative data from Canadian women of the same age. The authors found that SLE has a negative influence on patients’ QoL, especially with regard to physical health status.14

Nottingham Health Profile The Nottingham Health Profile (NHP)15 is a 38-item questionnaire that assesses the domains of physical mobility, pain, sleep, social isolation, emotional reactions, and energy level. Its wording is simple and easily understood, and can be completed by patients in 5 minutes. The NHP cannot fully assess the impact of a condition such as SLE on QoL, and for this reason has been often used in combination with other measures, such as a functional disability scale and a measure of psychological disturbance.

Sickness Impact Profile The Sickness Impact Profile (SIP)16 aims to asses the impact of sickness on daily activities and behavior. It is much longer than the NHP and takes approximately 20 to 30 minutes to complete. It contains 312 items in

European QoL Scale The European QoL scale (EQ-5D)17 has been proposed as a potentially useful measure of QoL in SLE studies. It is a simple measure that assesses five dimensions of health status: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. In addition, a visual analog scale provides a self-rated assessment of health status. The EQ-5D was used in a study by Wang and colleagues18 of 54 patients with SLE, evaluating the relationship between self-reported QoL and disease activity, damage, impairment, disability, and handicap. In this study, the EQ-5D was shown to be a valid instrument for the measure of HRQOL. Luo and colleagues have used Singaporean English and Singaporean Chinese versions of the EQ-5D in patients with various rheumatic diseases, including SLE.19,20 Both versions were found to be valid measures of HRQOL in Singaporeans with rheumatic diseases; however, the reliability of these questionnaires requires further investigation.

World Health Organization Quality of Life Scale The World Health Organization Quality of Life-Bref (WHOQOL-Bref),21 a 26-item abbreviated version of the original 100-item WHOQOL, assesses four dimensions of QoL: physical, psychological, social, and environmental. Preferred because of its crosscultural applicability, Khanna and colleagues recently used the WHOQOL-Bref to assess QoL in SLE patients from India.22 In their study of 73 patients, the physical and psychological domains of QoL were impaired in patients with active disease, whereas the social and environmental domains of QoL were not found to correlate with disease activity.

Disease-Specific Instruments Stanford Health Assessment Questionnaire Disability Index The most commonly used measure of functioning in the rheumatic diseases is the Stanford HAQ Disability Index.6 This questionnaire was developed as an “arthritis-specific” instrument and places a significant emphasis on physical functioning. It has been used extensively in the evaluation of patients with rheumatoid

arthritis, and has also proven useful in the assessment of patients with other conditions. The HAQ is a 20item scale that assesses activities of daily living (ADL) in eight domains: dressing, arising, eating, walking, hygiene, reaching, gripping, and errands and chores.4 Each of these components consists of two or three relevant questions and assistance from others or the use of aids can also be incorporated in the final score. The HAQ is a reliable and valid instrument and has been used widely in clinical trials. In SLE, the validity of the HAQ has been demonstrated by Hochberg and Sutton23 and Milligan and colleagues.24 Hochberg and Sutton demonstrated significant correlations between increased disability and worse global assessment. Milligan and colleagues found that patients with inactive disease had less disability than active patients. A study by Lotstein and colleagues25 showed that women of lower socioeconomic status had more functional disability as measured by the HAQ. One limitation of the HAQ is that it only assesses physical functioning, and so it has been suggested that, for a more complete evaluation, additional questionnaires designed to assess psychosocial functioning should also be used. Two such surveys are the Hospital Anxiety and Depression (HAD) scale and the General Health Questionnaire (GHQ).9

MEASUREMENT OF QUALITY OF LIFE IN PATIENTS WITH SLE

various dimensions of physical and psychosocial functioning, including sleep and rest, eating, work, home management, recreation and pastimes, ambulation, mobility, body care and movement, social interaction, alertness behavior, emotional behavior, and communication. Both the NHP and SIP have been used in a variety of diseases and have been shown to be reliable and valid. Nevertheless, neither of these questionnaires has been validated in SLE; their use, therefore, cannot be recommended in clinical trials of SLE.

Arthritis Impact Measurement Scale The AIMS2,7 a revised version of the original AIMS,26 is a 78-item scale that asks respondents to report on physical functioning, ADL, social activities, social support, pain from arthritis, work, level of tension, mood, satisfaction with health status, general health perceptions, overall impact of arthritis, and medication usage.4 Although it assesses a wide range of physical and emotional problems, the AIMS was specifically designed for arthritis outcomes studies and has rarely been used in SLE. The original AIMS has been used in only one SLE study,27 in which 50 women with SLE were compared with age-matched women with rheumatoid arthritis.

SLE-Specific Instruments Generic measures have the advantage of permitting comparisons across diseases and interventions, an important consideration for policymakers in the allocation of resources. They also allow measurement of dysfunction for individuals experiencing more than one condition. Nonetheless, it has been suggested that such generic measures may not be able to capture elements specific to particular diseases and may not be sufficiently responsive in clinical trials. As noted by Patrick and Deyo,28 disease-specific measures may have greater salience for physicians and better focus on functional areas of particular concern, and may possess

35

QUALITY OF LIFE AND ECONOMIC ASPECTS

greater responsiveness to disease-specific interventions. As such, there has been considerable interest in the development of a disease-specific measure of QoL in SLE. Leong and colleagues29 recently developed and validated a new SLE-specific QoL instrument, the SLEQOL. The SLEQOL is a 40-item questionnaire consisting of six subsections: physical functioning, activities, symptoms, treatment, mood, and self-image. It was developed entirely in English and its performance was studied on 275 SLE patients in Singapore. It was shown to be valid, possessing construct validity, face and content validity, internal consistency, test–retest reliability, and responsiveness. The SLEQOL was found to be more responsive to change than the SF-36. Grootscholten and colleagues30 recently developed a disease-specific questionnaire for lupus patients, called the SLE Symptom Checklist (SSC), that assesses the presence and burden of 38 disease- and treatment-related symptoms. The questionnaire was developed in Dutch and has been translated into English. Reliability and reproducibility were tested in 87 and 28 stable SLE patients, respectively, and it was found to have satisfactory internal consistency and test–retest reliability. The Lupus Quality of Life Scale (LupusQoL),31 a new patientderived measure of health-related QoL, has also been recently developed. This is a 34-item instrument comprised of eight domains: physical functioning, pain, emotional functioning, fatigue, body image, sex, planning, and burden to others (see Appendix M). This questionnaire has been shown to possess internal consistency, test–retest reliability, and concurrent validity compared with the SF-36. Further evaluation of these instruments is necessary before they can be recommended for routine use. Furthermore, although the question of whether to use disease-specific versus generic measures has been widely debated, there now appears to be a general consensus that generic measures should be used preferentially, supplemented with disease-specific measures where applicable.

FACTORS ASSOCIATED WITH QUALITY OF LIFE IN SLE

Disease Activity

36

Health-related QoL questionnaires, such as the SF-36, have allowed the study of predictors and associations of impaired QoL in SLE.9 SLE disease activity, as evaluated by various measures, has been assessed in SLE and correlated with QoL. Stoll and colleagues,12 using the British Isles Lupus Assessment Group (BILAG) disease activity instrument, showed that disease activity was closely and significantly correlated with each domain of the SF-36. In fact, the authors of this study noted that even patients with minimal disease activity

had significantly impaired QoL. In another study, Sutcliffe and colleagues,13 using the Systemic Lupus Activity measure (SLAM)32 to measure disease activity, showed that a higher SLAM score (i.e., increased disease activity) was an important determinant of health status. Higher disease activity was associated with significantly poorer scores in the SF-36 domains of physical functioning, role limitations (physical), pain, general health, vitality, and social functioning. Fortin and colleagues 33 evaluated the association of two measures of disease activity, the Systemic Lupus Disease Activity Index (SLEDAI)34 and the SLAM-R, with health status as expressed on the SF-36. The SLAM-R was correlated with several aspects of the SF-36 while the SLEDAI was not. Gladman and colleagues35 have shown a similar lack of correlation between the SLEDAI and the SF-36. These findings suggest that important differences exist between these disease activity indices, possibly related to the SLAM-R capturing more patient-derived information on lupus activity.

Damage Damage, whether resulting from the disease process itself or as a result of treatment, is recognized as an important outcome in SLE. Studies that have assessed associations between disease damage and QoL have shown that the major effects in health status result from decreases in physical functioning. Fortin and colleagues,33 in a prospective study of 96 patients, measured disease damage using the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC/ACR DI), and correlated it with QoL assessed using the SF-36. Not surprisingly, as stated by the authors, the SLICC/ACR DI correlated with the SF-36 domain physical function at baseline and with its mean value over time. It appears that permanent damage will continue to interfere with physical performance and functioning, whereas other areas of health status such as emotional well-being and social functioning may, with time, adapt to a physical deficit. Other studies have shown no significant correlation between disease damage and health status. In one longitudinal observational study of 87 SLE patients, Gilboe and colleagues36 showed that the best predictors of SF-36 PCS and MCS scores were the respective baseline scores and no significant relationship was found between SLICC/ACR DI score and health status. Gladman and colleagues,35 in an earlier study, also showed no such correlation.

Psychosocial Aspects Psychological disorders occur frequently in SLE patients. Up to 40% of patients with SLE have diagnosable psychological disorders, most commonly anxiety disorders and depression.37 The importance of psychological

Fibromyalgia/Fatigue Fibromyalgia is a common rheumatologic disorder that is characterized by widespread pain and fatigue, and may be an important potential confounder in measurement of health status. The prevalence of fibromyalgia in the general population is approximately 2%45; in SLE it has been reported to be much more common, with estimates as high as 23%.46 A recent study of patients with SLE showed a strong association between the number of fibromyalgia tender points and health status as assessed by the HAQ.47 They noted that the number of tender points, and not just the absence or presence of fibromyalgia, was associated with health status in SLE. In a cross-sectional study of 119 outpatients with SLE, Gladman and colleagues showed that SF-36 scores reflected the presence of fibromyalgia, rather than disease activity or damage.48 Fibromyalgia, therefore, is likely a very important determinant of health status in SLE.

An important and common symptom in SLE is fatigue. One study of fatigue in SLE found that 80% to 90% of patients surveyed reported abnormal fatigue49; similar findings have been reported in other studies.50 Fatigue is a symptom that may result from many different processes: active lupus, mood disorders, fibromyalgia, and other comorbid illnesses. It is often difficult to determine the etiology of fatigue in any given case and just as difficult to quantify. Nevertheless, there is evidence that conditions commonly co-expressed in SLE, such as fibromyalgia and depression, may be more important in the development of fatigue than active lupus. In one study of 81 patients with SLE, fatigue was found to correlate moderately or strongly with all components of the SF-36; increased fatigue scores were associated with worse SF-36 scores. Fatigue did not correlate with disease activity or disease damage.51 In another study by Wang and colleagues,52 fatigue was found to be highly correlated with the presence of fibromyalgia and depression. There was no correlation between fatigue and disease activity. The authors of this study conclude that fatigue may reflect a decreased overall coping ability in these patients, rather than active disease itself. Studies of interventions aimed at improving disease activity may not have the desired effect on QoL indices if a significant number of patients have comorbid conditions such as fibromyalgia. Specific assessments for such conditions may need to be included in studies of QoL in order to better help understand the associations between lupus manifestations and health status.

FACTORS ASSOCIATED WITH QUALITY OF LIFE IN SLE

functioning in QoL was underscored in a study by Burckhardt and colleagues27 which showed that psychological distress alone was the best predictor of QoL among patients with SLE. Nevertheless, studies on the prevalence of psychological disorders in SLE and physically healthy controls have shown conflicting results, with one study showing higher rates of psychological disorders among patients than among controls,38 and another showing rates that were similar between patients and controls.39 Psychological functioning appears to worsen with increased disease activity and damage, particularly when the disease causes greater pain, helplessness, and physical disability.37,40,41 Given the pervasive nature of SLE and its course, its impact on mental health is not surprising. Moreover, poor baseline psychological functioning may, in turn, contribute to worse physiologic outcomes in SLE, as has been seen in a number of other diseases.42,43 The effect of psychological function on SLE outcomes requires further investigation. Social support has also been found to be an important determinant of health status. A cross-sectional study by Sutcliffe and colleagues13 showed that social support, as measured by the Interpersonal Support Evaluation List (ISEL), was one of the most consistent determinants of health status in their study of 195 patients with SLE. Their study showed that increasing total social support had a positive effect on all of the SF-36 subscales. The authors, therefore, suggest that increasing social support may have the potential to improve overall functioning. A study by Bae and colleagues44 also showed that higher social support influenced health status. However, the patients found to benefit most from social support were those who already possessed social, economic, and health advantages: white patients above poverty level who had health insurance and low premorbid disease activity.

Intervention Studies Studies examining the outcome of behavioral interventions in patients with SLE have shown variable results. Dobkin and colleagues evaluated the effect of brief supportive-expressive group psychotherapy in a randomized clinical trial of 133 SLE patients.53 Both treatment and control groups improved over time on measures of psychological distress, stress, and coping, but there was no differential improvement, suggesting that these changes could not be attributed to the intervention. Therefore, the authors concluded that their study did not support the referral of these patients for this type of intervention. Edworthy and colleagues54 evaluated illness intrusiveness as a secondary outcome in this trial. They found that the group psychotherapy subjects experienced significant reduction in illness intrusiveness, and concluded that this intervention may facilitate adaptation to SLE by assisting patients in reducing illness-induced disruptions into important domains of life experience. Haupt and colleagues55 evaluated a psychotherapeutic intervention specifically tailored to the needs of SLE patients, combining a psychoeducational approach (training programs,

37

QUALITY OF LIFE AND ECONOMIC ASPECTS

self-management courses) with group psychotherapy. The 34 patients enrolled improved significantly over a 6-month period in most psychological domains, including depression, anxiety, and overall mental burden. Karlson and colleagues56 evaluated a psychoeducational approach in a randomized clinical trial of 122 patients. The intervention group was designed to enhance self-efficacy, couple communication about lupus, social support, and problem solving. They found significantly better couple communication, selfefficacy, and mental health status, and less fatigue in the experimental group compared with the control group. Although the studies mentioned have shown promising results, the challenge remains to identify or develop appropriate psychological interventions for this patient group that consists largely of young women in the midst of trying to establish families and careers.53 More studies with longitudinal designs and larger and more diverse sample populations are needed to facilitate the identification of causal links and help guide the design of interventions.37

ECONOMIC ASSESSMENT This section has two main objectives. The first objective is to review the common types of economic evaluations and describe how they may be conducted in SLE; the second objective is to discuss the specific studies evaluating the costs of SLE. For a full discussion of economic evaluations, the reader is referred to the textbooks by Gold and colleagues,57 Drummond and colleagues,58 and Sloan.59 A standardized framework specific to the conduct of economic evaluations in the rheumatic diseases has also been developed by the Economics Working Group of OMERACT.60,61 Box 4.1 outlines the

BOX 4-1 CHECKLIST FOR ASSESSING ECONOMIC EVALUATIONS ●

●

●

●

● ● ● ● ●

38

●

What type of economic evaluation was conducted (cost-ofillness, cost-minimization, cost-effectiveness, cost-utility, or cost-benefit analysis)? Did it examine the costs and outcomes of competing interventions? How were health outcomes assessed (generic, rheumaticdisease specific, or SLE-specific QoL instruments or utility scores)? What costs were considered (direct, direct non–health care, and productivity costs)? What population was studied (clinic-based cohort, population data, administrative data, hypothetical cohort)? How were costs valued? Was the analytic perspective stated? Was the time horizon appropriate? Were future health outcomes and costs discounted? Was the uncertainty of the estimates considered and appropriate analyses conducted? How can the results influence decision making?

factors to consider when reviewing or conducting an economic evaluation (a more detailed list is provided in Drummond and colleagues62).

TYPES OF ECONOMIC EVALUATIONS

Cost of Illness This type of economic evaluation estimates the direct and indirect costs associated with a disease. Direct costs refer to all resources consumed in providing care to a patient; indirect costs represent the value of productivity losses due to the disease and both are discussed in subsequent sections of this chapter. In this type of evaluation, there is no consideration of the health effects of the health services consumed.

Cost Minimization Cost minimization studies compare the costs of competing interventions, but similar to cost of illness studies, they do not incorporate health outcomes. Health outcomes are not considered relevant because the interventions are assumed to produce similar effects.

Cost Effectiveness Cost-effectiveness studies also compare competing interventions, but they incorporate both costs and outcomes. Results are expressed as an incremental cost-effectiveness ratio where the differential costs and outcomes between the two treatment groups are compared: (ΔCost) / (ΔEffectiveness) = (Cost treatment—Cost control) / (Effectiveness treatment—Effectiveness control)

The effectiveness measure is often disease specific, and in SLE could be a disease activity or damage scale (i.e., SLEDAI or SLICC/ACR DI) or one of the QoL instruments discussed earlier in this chapter. In this type of analysis, there is no attempt to value the consequences or benefits of the health outcome by eliciting patient preferences for the outcome; the effectiveness measure merely characterizes the health state.

Cost Utility The cost-utility analysis also considers healthcare costs and health effects, but the effectiveness measure attempts to value the consequences of the health outcomes by adjusting the outcomes by health state preference scores or utility weights.63-66 Utilities attempt to aggregate the morbidity and mortality effects of an intervention into a single measure, usually a qualityadjusted life year. Utilities or health state preferences can be collected from either patients or the general public. They can be elicited either directly by asking respondents to reveal their preferences through techniques such as the standard gamble or time trade-off or

Cost Benefit Cost-benefit analyses also compare costs and effects, but express both in monetary terms. The conventional approach to cost-benefit analysis, the human capital approach, considers direct and productivity costs, but does not include intangible costs such as pain and anxiety when they are not associated with productivity loss. However, an alternative method, the willingnessto-pay approach, elicits respondents’ preferences for health interventions by asking them what they would be willing to pay,69 and theoretically provides a more comprehensive valuation. Yet, it is fraught with methodologic challenges related to the acceptability and feasibility of monetizing complex medical outcomes.

Other Considerations in Conduct of Economic Evaluations Since economic analyses are generally conducted to aid in societal decisions, it is recommended that they adopt a societal viewpoint or analytic perspective, that is, the costs and effects of all those affected by the intervention are considered whether or not they are the intended recipients.9,65,70 The time horizon of the analyses should extend sufficiently far in the future to capture all important costs and outcomes related to the intervention.65 This may not be possible with primary data collected from a real cohort and may require the creation of a hypothetical cohort through decision-analytic modeling. Decisionanalytic modeling will not be discussed further in this chapter; the reader is referred to reviews by Drummond and colleagues,71 Weinstein and Fineberg,72 and Buxton and colleagues.73 Although costs and effects may occur in the distant future, there is general agreement that both should be expressed in terms of their “present value,” that is, they should be discounted to the present, reflecting societal preference for present over future outcomes.58,65,74

In the conduct of an economic analysis, uncertainty will exist regarding the estimates of costs and effects as well as the method of modeling used to combine these parameters. Numerous approaches, including a sensitivity analyses, where parameters are varied across a range of possible alternatives, as well as statistical or probabilistic methods, have been recommended to incorporate uncertainty into the estimated cost-effectiveness ratios.58,65,75-77 Recently, cost-effectiveness acceptability curves have been recommended as an alternative approach to representing uncertainty; these plot the probability that an intervention is cost effective over a range of cost-effectiveness ratios.78,79 In attempt to translate the results of economic analyses into practice, “league tables” have been constructed which list incremental cost-utility ratios for varying interventions across numerous disease states, theoretically allowing the determination of a threshold value beyond which a particular intervention is considered unacceptable. However, the use of league tables to influence resource allocation has been criticized, both because of potential methodologic inconsistencies between studies and because of ethical and political considerations.80

CONDUCTING ECONOMIC EVALUATIONS IN SLE

indirectly by having respondents describe themselves using a health classification system where preferences have been pre-measured for defined health states. It has been recommended by the U.S. Panel on CostEffectiveness in Health and Medicine67,68 and the Canadian Coordinating Office of Health Technology Assessment65 that cost-utility analysis should supplant all other types of economic analyses. Cost-utility analyses, by providing a common metric in the denominator, allow comparisons of interventions across different disease states. Furthermore, it is recommended that only direct costs be considered in the numerator. Inclusion of indirect costs could lead to double counting because if the effectiveness measure is sufficiently comprehensive, it is believed to subsume the consequences of productivity loss.

CONDUCTING ECONOMIC EVALUATIONS IN SLE

Estimating Direct Costs Cost Domains When conducting an economic analysis, Luce and colleagues have recommended that cost domains be considered according to the following convention—direct healthcare costs, direct non-healthcare costs, and productivity costs.81 Merkesdal and colleagues,82 by thoroughly reviewing the economic evaluations published in the rheumatic diseases, have recommended a standardized set of items to be considered within each cost domain. The perspective adopted for the analysis will determine which are most appropriate to include. Direct healthcare costs refer to all the resources associated with the treatment of SLE and include both outpatient and inpatient services. Outpatient services include physicians, nonphysician healthcare professionals, outpatient surgery, emergency room visits, prescribed and nonprescribed medications, diagnostic and therapeutic procedures, assistive devices, and complementary and alternative therapies. Inpatient services refer to acute and nonacute hospital facilities. Given that the use of nontraditional therapies is likely to be extensive in chronic rheumatic illnesses such as SLE,83 which are often characterized by intractable fatigue and pain, this cost component merits consideration.

39

QUALITY OF LIFE AND ECONOMIC ASPECTS

Direct non-healthcare costs refer to transportation costs associated with obtaining medical care, the time the patient spends seeking and receiving medical care, and the time that family members or volunteers spend in assisting the patient to obtain medical care. Although time costs are conventionally considered as indirect costs, it is now recommended those directly associated with the delivery of health care be considered as direct costs and included in the numerator of the cost-effectiveness analysis.81 The term “productivity costs” is preferred by many experts to “indirect costs” because the latter can also refer to overhead or fixed costs associated with a medical service. Productivity costs are those associated with the inability or decreased ability to participate in either paid or unpaid labor or leisure activities due to illness or death. It is recommended that these costs be excluded from the denominator of a cost-effectiveness analysis because if the measure of effectiveness is sufficiently comprehensive, these costs, as well as the intangible costs associated with the physical and psychological suffering associated with the illness, are subsumed in the measure of effectiveness.81 The estimation of indirect costs will be discussed more fully in a following section. Given that SLE can cause such a spectrum of health problems, it is often difficult for either the patient or treating physician to determine if health resource use or productivity loss is directly ascribable to SLE. Hence, we recommend that in developing both direct and indirect cost estimates, all health services and all time loss should be incorporated without attempting to make attributions to SLE or comorbid conditions.9

Measuring Health Service Use

40

Health resource use and time loss can theoretically be assessed using clinic-based cohorts, population data, or a hypothetical cohort (creation of the latter type of cohort is reviewed elsewhere71-73). In SLE, resource use is most comprehensively assessed using participants from clinic-based cohorts where the diagnosis of SLE has been confirmed by a specialist.9 Although the use of national survey data or administrative claims data from a public or private payer may theoretically provide a more representative SLE population than a clinical cohort, the diagnosis of lupus is likely to be unreliable. Reliance on self-defined illness or diagnosis by a nonspecialist may result in the inclusion of patients with other inflammatory arthritic diseases and noninflammatory arthritic and nonrheumatic conditions. In addition, clinical cohorts are able to provide data on health resources not reimbursed by insurers and on disease characteristics and health status that are necessary in interpreting economic evaluations.

Numerous questionnaires have been developed to elicit resource use and time loss from patients with rheumatic diseases.84,85 Ruof and colleagues reviewed these instruments and found that they differed substantially, and underscored the need for the development of a core instrument with standardized cost domains supplemented by disease-specific components. Furthermore, only a few instruments have been demonstrated to be psychometrically sound.86-91 If possible, the validity and reliability of patient self-report over varying recall periods should be evaluated by comparison with medical chart audit, computerized provider utilization databases, and payer claims data. By comparing the self-reported utilization of rheumatoid arthritis (RA) patients with payer-reported utilization, Ruof and colleagues91 have developed a series of recommendations regarding the assessment of healthcare use. They favored the use of highly aggregated cost items and advised against differentiating between physician specialties and diagnostic and therapeutic procedures. They suggested eliciting only dichotomous (i.e., yes/no) responses for most items and requesting quantification only for physician visits and length of hospital stay.

Valuing Health Services A health resource should be valued at its opportunity cost, which refers to the “value of the resource in its next best alternative use.”81 If the market for health care were truly competitive, market prices should reflect opportunity costs. However, in many cases, market imperfections exist (e.g., regulated entry of physicians through compulsory licensure, government control of hospitals and physician fees, and discordance among provider chargers, third-party reimbursement, and economic costs), and therefore prices do not accurately reflect opportunity costs.9 The costing method must also consider the severity of the patient’s condition. For example, using average per diem costs to value a hospital day may underestimate costs incurred by severely ill SLE patients with multiorgan failure. Furthermore, cost estimates derived from a single institution or region may not be representative of costs incurred by patients serviced by other providers or institutions. National recommendations exist for valuing health resources with suggested adjustments for market distortions and other possible biases.68,81,92-94

Estimating Indirect Costs Types of Productivity Impairment Productivity costs result from lost or diminished ability to engage in paid and unpaid work and leisure activities. In calculating productivity costs, it is often helpful to consider three mutually exclusive groups of patients based on their employment status: (1) labor market

does not value non–labor market activity, and values productivity losses in the labor market only for a short period until the disabled worker can be replaced. An alternative method, the human capital approach, values time loss in either paid or unpaid work for the entire length of the impairment. Under this approach, it is recommended that time loss from paid work be valued using age and gender-matched employment income.68 Time loss from unpaid work can be valued using replacement or opportunity costs. The replacement cost method uses the market value of the services performed by the patient at home, whereas the opportunity cost method values time in the home as equivalent to time in the work force and uses employment income.

Measuring Productivity Impairment

CLINIC-BASED STUDIES ON THE COSTS OF SLE

As with health resource use, productivity loss is best assessed through patient self-report. However, instruments assessing productivity costs are less common, less standardized, and more difficult to validate than those evaluating direct costs.84,85 Validation of lost time in paid labor could theoretically be performed by comparing with insurer data, yet validation of lost time in unpaid labor and leisure activities is not possible because no gold standard exists. Only a single study in the rheumatic diseases has compared patient self-report of sick leave and work disability to that reported by insurers,96 and the authors concluded that when assessed every 3 months, patient report was a valid method of assessing productivity loss. To fully capture productivity costs, respondents should be queried on actual time loss in paid and unpaid labor and leisure activities, as well as on the additional time they anticipate they would devote to these activities if not ill. Even with consideration of both of these components of time loss, patient self-report yields anticipated limitations in productivity, whereas population surveys have the potential to reveal observed differences in productivity between the diseased and the nondiseased population.9

Valuing Productivity Impairment Estimating productivity costs in a disease such as SLE that affects women almost exclusively is particularly challenging. Productivity costs resulting from diminished labor market activity are usually represented by employment income. However, in SLE, much of the productivity loss will be in non–labor market activities such as childcare, household chores, and volunteering for which a wage is not received, and hence the valuation of time loss is less evident. Clarke and colleagues have calculated the productivity costs associated with time loss in both paid and unpaid labor under a variety of assumptions.95 The friction cost approach provides a lower limit for productivity costs because it

CLINIC-BASED STUDIES ON THE COSTS OF SLE

participants, (2) non–labor market participants who would be employed if not ill, and (3) non–labor market participants who would not be employed if not ill.95 For each patient group, certain types of productivity impairment are relevant. For participants in groups 1 and 2, time loss from paid work, activities of daily living such as household chores, and leisure activities should be considered, whereas for those in group 3, only time loss from activities of daily living and leisure activities should be included. As noted previously, it is recommended that time spent by the patient in receiving medical care or by the caregiver in assisting the patient in receiving care should be considered as a direct non-healthcare cost.81

Direct Costs Few economic studies have been conducted on patients with SLE.86,97-104 The most comprehensive of these costing studies involved two cohorts from each of three countries (Canada, United States, and the United Kingdom), and assessed direct and indirect costs as well as health status over a 4-year interval.100,105 Health resource use and diminished productivity in both paid and unpaid work were assessed through semiannual patient self-report and translated into costs using pricing and income data from a single country. Given that the price of health services and employment income differ across countries, using values from a single country allowed a comparison of resource use and productivity loss across countries. A total of 715 SLE patients (Canada 231, United States 269, United Kingdom 215) participated.100 Mean cumulative direct costs per patient over 4 years in Canada, the United States, and the United Kingdom were $15,845 (95% confidence interval [CI], $13,509–$18,182), $20,244 ($17,764–$22,724) and $17,647 ($15,557–$19,737) (expressed in 2002 Canadian dollars) (Table 4.2). After adjustment for important baseline patient covariates (potential covariates included demographics, disease characteristics, health status, and health expenditure), Canadian patients, on average, had 20% (95% CI, 8%–32%) lower costs than Americans at the study conclusion, and the British had 13% (1%–24%) lower costs than the Americans. Despite Canadian and British patients incurring lower health costs, on average, there were no differences in health outcomes expressed as accumulation of disease damage100 and change in QoL over 4 years105 (Table 4.2). After adjustment for baseline covariates, SLICC/ACR DI scores increased by 0.10 (95% CI, −0.03–0.23) units less in Canadians and by 0.12 (−0.01–0.26) units less in the British relative to the Americans at study conclusion.100 Quality of life

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QUALITY OF LIFE AND ECONOMIC ASPECTS

TABLE 4.2 CUMULATIVE DIRECT AND INDIRECT COSTS AND HEALTH OUTCOMES AT 4 YEARS IN PATIENTS WITH SLE Canada (n = 231) Mean (95% CI)

United States (n = 269) Mean (95% CI)

United Kingdom (n = 215) Mean (95% CI)

15,845 (13,509–18,182)

20,244 (17,764–22,724)

17,647 (15,557–19,737)

38,642 (32,785–44,500)

56,745 (49,919–63,571)

42,213 (35,859–48,567)

9976 (7363–12,589)

9833 (7230–12,435)

13,565 (10,232,16,898)

Total cumulative indirect cost

48,618 (42,170–55,066)

66,578 (59,879–73,276)

55,778 (48,501–63,054)

Change in SLICC/ACR DI100

0.49 (0.39–0.60)

0.63 (0.52–0.74)

0.48 (0.39–0.57)

PCS score annual change (units/yr)105

0.18 (−0.07–0.43)

−0.05 (−0.27–0.17)

0.03 (−0.20–0.27)a

MCS score annual change (units/yr)105

0.15 (−0.04–0.34)a

0.23 (0.09–0.37)a

0.08 (−0.10–0.27)a

Cumulative direct costs100 (2002 Canadian $) Cumulative indirect costs (2002 Canadian $) Labor market activity Non–labor market activity Replacement cost

a

a

a

Refers to 95% credible intervals. CI, confidence interval; SLICC/ACR DI, Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index; PCS, SF-36 Physical Component Summary; MCS, SF-36 Mental Component Summary.

remained stable across countries over the course of the study (Table 4.2).

Indirect Costs

42

This work by Clarke and colleagues is also the only research to evaluate indirect costs in patients with SLE.95 By incorporating patient self-report data on employment status, days lost from labor and non–labor market activities, and time lost by caregivers in delivering health care to the patient or aiding the patient in obtaining care, Clarke and colleagues estimated indirect costs according to the human capital approach. Four-year cumulative indirect costs due to diminished productivity in paid labor in Canada, the United States, and the United Kingdom were $38,642 (95% CI $32,785–$44,500), $56,745 ($49,919–$63,571), and $42,213 ($35,859–$48,567) (expressed in 2002 Canadian dollars) (Table 4.2). If lost time in unpaid labor was valued at replacement cost and included in the above estimates, cumulative indirect costs increased to $48,618 ($42,170–$55,066), $66,578 ($59,879–$73,276), and $55,778 ($48,501–$63,054) (Table 4.2). Indirect costs are thus substantial and exceed direct costs by threefold. After adjustment, cumulative indirect costs due to diminished paid work were $6750 ($580–$12,910) less in Canadian patients and $10,430 ($4050–$16,800) less in British patients relative to those in the United States. Indirect costs due to diminished unpaid work did not differ across countries. Therefore, despite the greater medical expenditure of the U.S. SLE patients, they did not

experience superior health outcomes expressed as either disease damage, QoL, or less productivity loss in paid or unpaid work. In earlier work on this cohort, using only the baseline data, Clarke and colleagues estimated annual indirect costs using a variety of assumptions for the value of labor and non–labor market activity,95 and showed that estimates could vary by as much as 15-fold. Indirect cost estimates that do not consider long-term productivity losses, omit lost time from unpaid work, and value unpaid work at replacement cost (i.e., the expected earnings of those performing the market counterpart of the nonmarket activity), tend to underestimate the economic burden of SLE. Although this study by Clarke and colleagues95 is the only one to provide estimates for indirect costs, there is a single cohort study that characterized the work disability experienced by patients with SLE without calculating costs.104 Partridge and colleagues reported that an average of 3.4 years after diagnosis, 40% of patients who had been employed at some time since diagnosis had become unemployed because of their illness. Predictors of unemployment were less education, no health insurance, having a physically demanding job, low income, and greater disease activity. Clarke and colleagues have shown that in Canada (mean patient age 43 years, mean SLE duration 10 years), the United States (mean age 39 years, mean duration 9 years), and the United Kingdom (mean age 41 years, mean duration 10 years), 48.7%, 45.3%, and 52.6%, respectively, of patients were employed.

Direct Costs There is a single population-based study evaluating the direct costs of SLE.101 Nichol and colleagues conducted a retrospective evaluation of California Medicaid claims data with the primary objective of examining the association between ethnicity and Medicaid eligibility, healthcare utilization, and direct medical costs. Medicaid is a government-sponsored health insurance providing healthcare coverage for the economically disadvantaged. They reported that although at study entry, whites, blacks, and Hispanics all incurred comparable monthly costs (approximately $900 expressed in 2002 U.S. dollars), over 3 years, the Hispanics generated increasingly lower costs (approximately $200 at 3 years), while the costs incurred by the other ethnic groups remained at the baseline level. The monthly costs incurred by the non-Hispanics exceed those in the study conducted by Clarke and colleagues described above,100 where patients were predominantly white, and average monthly costs were approximately $400 (2002 Canadian dollars). There are several possible explanations for this difference. Nichol and colleagues attempted to include only newly diagnosed SLE patients, whereas Clarke and colleagues studied patients with an average disease duration of about 10 years. Hence, it is likely that a greater frequency of patients in Nichol’s study presented with an acute exacerbation of SLE and required considerable health services for diagnosis and treatment. Furthermore, U.S. prices, which exceed Canadian prices for many healthcare services, were used in the Nichol study. Since Nichol examined administrative data only, he was unable to determine if the lower healthcare utilization by Hispanic patients was associated with poorer health outcomes.

Indirect Costs Two population-based studies characterized the work disability experienced by patients with SLE without calculating costs.102,103 Using the German rheumatologic

database, which includes a cohort of approximately 4000 SLE patients assembled from numerous outpatient practices throughout Germany, Zink and colleagues102 and Mau and colleagues103 described the employment rates in SLE and compared it with other rheumatic diseases102 and the general population.103 After matching for age, sex, disease duration, and kind of referral, an equal proportion of SLE and RA patients (46%) aged less than 65 years remained employed.102 When compared with the general population, matched for age, place of residence, education level, and calendar-year, the standardized employment ratios (i.e., SER, ratio of observed to expected number of patients employed) for SLE patients with a disease duration of less than 6 years did not differ from the general population.103 This contrasts with the study by Partridge and colleagues104 discussed above where 40% of SLE patients were disabled after 3.4 years of disease. However, Mau and colleagues reported that the SERs for women with disease duration of 6 to 10 years and more than 10 years were 0.80 (95% CI, 0.74–0.87) and 0.68 (0.63–0.73).

REFERENCES

POPULATION-BASED STUDIES ON COSTS OF SLE

CONCLUSIONS SLE is a pervasive and unpredictable disease that can impact every aspect of a person’s life. In addition to the well-known physiologic consequences, there are a multitude of other emotional, psychological, social, and financial effects that also require consideration. Comprehensive assessment requires proper evaluation and quantification of all dimensions of this disease so that informed decisions can be made regarding appropriate treatment and the equitable allocation of resources. With the anticipated emergence of novel biologicals for the treatment of SLE, which will most certainly be more expensive, yet potentially more effective and less toxic than current interventions, there will be an increasing need to determine if their benefits are commensurate with their costs. It is our hope that this chapter will serve as a guide for those interested in the review or conduct of QoL assessments and economic evaluations in SLE.

REFERENCES 1. Fayers PM, Machin D. Quality of Life: Assessment, Analysis and Interpretation. West Sussex, England: John Wiley & Sons, 2000. 2. Strand V, Gladman D, Isenberg D, et al. Endpoints: consensus recommendations from OMERACT IV. Lupus 9:322, 2000. 3. Bowling A. Measuring Disease. 2nd ed. Buckingham, England: Open University Press, 2001. 4. Wolfe F. Health-status questionnaires. Rheum Dis Clin North Am 21:445, 1995. 5. Cramer JA, Spilker B. Quality of Life and Pharmacoeconomics: An Introduction. Philadelphia: Lippincott-Raven, 1998.

6. Fries JF, Spitz PW, Kraines RG, et al. Measurement of patient outcome in arthritis. Arthritis Rheum 23:127, 1980. 7. Meenan RF, Mason JH, Anderson JJ, et al. Aims2 - the content and properties of a revised and expanded arthritis impact measurement scales health-status questionnaire. Arthritis Rheum 35:1, 1992. 8. Ware JE, Jr, Sherbourne CD. The MOS 36 item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care 30:473, 1992. 9. Gordon C, Clarke AE. Quality of life and economic evaluation in SLE clinical trials. Lupus 8:645, 1999.

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10. Bowling A. Research Methods in Health: Investigating Health and Health Services. Buckingham, England: Open University Press, 1997. 11. Ware JE, Jr, Kosinski M, Bayliss MS, et al. Comparison of methods for the scoring and statistical analysis of SF-36 health profile and summary measures: summary of results from the Medical Outcomes Study. Med Care 33:AS264, 1995. 12. Stoll T, Gordon C, Seifert B, et al. Consistency and validity of patient administered assessment of quality of life by the MOS SF-36; its association with disease activity and damage in patients with systemic lupus erythematosus. J Rheumatol 24:1608, 1997. 13. Sutcliffe N, Clarke AE, Levinton C, et al. Associates of health status in patients with SLE. J Rheumatol 26:2352, 1999. 14. Dobkin PL, DaCosta D, Fortin PR, et al. Living with lupus: a prospective Pan-Canadian study. J Rheumatol 28:2442, 2001. 15. Hunt SM, McKenna SP, McEwen J, et al. The Nottingham Health Profile: subjective health status and medical consultations. Soc Sci Med [A] 15:221, 1981. 16. Bergner M, Bobbitt RA, Carter WB, et al. The sickness impact profile - development and final revision of a health-status measure. Med Care 19:787, 1981. 17. EuroQol Group. EuroQol: a new facility for the measurement of health-related quality of life. Health Policy 16:199, 1990. 18. Wang C, Mayo NE, Fortin PR. The relationship between health related quality of life and disease activity and damage in systemic lupus erythematosus. J Rheumatol 28:525, 2001. 19. Luo N, Chew LH, Fong KY, et al. Validity and reliability of the EQ5D self-report questionnaire in English-speaking Asian patients with rheumatic diseases in Singapore. Qual Life Res 12:87, 2003. 20. Luo N, Chew LH, Fong KY, et al. Validity and reliability of the EQ5D self-report questionnaire in Chinese-speaking patients with rheumatic diseases in Singapore. Ann Acad Med Singapore 32:685, 2003. 21. WHOQOL Group. Development of the World Health Organization WHOQOL-BREF quality of life assessment. The WHOQOL Group. Psychol Med 28:551, 1998. 22. Khanna S, Pal H, Pandey RM, et al. The relationship between disease activity and quality of life in systemic lupus erythematosus. Rheumatology (Oxford) 43:1536, 2004. 23. Hochberg MC, Sutton JD. Physical disability and psychosocial dysfunction in systemic lupus erythematosus. J Rheumatol 15:959, 1988. 24. Milligan SE, Hom DL, Ballou SP, et al. An assessment of the Health Assessment Questionnaire functional ability index among women with systemic lupus erythematosus. J Rheumatol 20: 972, 1993. 25. Lotstein DS, Ward MM, Bush TM, et al. Socioeconomic status and health in women with systemic lupus erythematosus. J Rheumatol 25:1720, 1998. 26. Meenan RF, Gertman PM, Mason JH, et al. The arthritis impact measurement scales. Further investigations of a health status measure. Arthritis Rheum 25:1048, 1982. 27. Burckhardt CS, Archenholtz B, Bjelle A. Quality of life of women with systemic lupus erythematosus: a comparison with women with rheumatoid arthritis. J Rheumatol 20:977, 1993. 28. Patrick DL, Deyo RA. Generic and disease-specific measures in assessing health status and quality of life. Med Care 27:S217, 1989. 29. Leong KP, Kong KO, Thong BY, et al. Development and preliminary validation of a systemic lupus erythematosus-specific quality-of-life instrument SLEQOL. Rheumatology (Oxford) 44:1267, 2005. 30. Grootscholten C, Ligtenberg G, Derksen RHWM, et al. Healthrelated quality of life in patients with systemic lupus erythematosus: development and validation of a lupus specific symptom checklist. Qual Life Res 12:635, 2003. 31. Teh L, McElhone K, Bruce IN, et al. Development and validation of a disease specific quality of life measure for adults with systemic lupus erythematosus, the LupusQoL. Arthritis Rheum 52(Suppl 9):S188, 2005. 32. Liang MH, Socher SA, Larson MG, et al. Reliability and validity of six systems for the clinical assessment of disease activity in systemic lupus erythematosus. Arthritis Rheum 32:1107, 1989. 33. Fortin PR, Abrahamowicz M, Neville C, et al. Impact of disease activity and cumulative damage on the health of lupus patients. Lupus 7:101, 1998.

34. Bombardier C, Gladman DD, Urowitz MB, et al. The Committee on Prognosis Studies in SLE. Derivation of the SLEDAI. A disease activity index for lupus patients. Arthritis Rheum 35:630, 1992. 35. Gladman DD, Urowitz MB, Ong A, et al. Lack of correlation among the 3 outcomes describing SLE: disease activity, damage and quality of life. Clin Exp Rheumatol 14:305, 1996. 36. Gilboe IM, Kvien TK, Husby G. Disease course in systemic lupus erythematosus: Changes in health status, disease activity, and organ damage after 2 years. J Rheumatol 28:266, 2001. 37. Seawell AH, Danoff-Burg S. Psychosocial research on systemic lupus erythematosus: a literature review. Lupus 13:891, 2004. 38. Omdal R, Husby G, Mellgren SI. Mental health status in systemic lupus erythematosus. Scand J Rheumatol 24:142, 1995. 39. Lindal E, Thorlacius S, Steinsson K, et al. Psychiatric disorders among subjects with systemic lupus erythematosus in an unselected population. Scand J Rheumatol 24:346, 1995. 40. Dobkin PL, Fortin PR, Joseph L, et al. Psychosocial contributors to mental and physical health in patients with systemic lupus erythematosus. Arthritis Care Res 11:23, 1998. 41. Ward MM, Lotstein DS, Bush TM, et al. Psychosocial correlates of morbidity in women with systemic lupus erythematosus. J Rheumatol 26:2153, 1999. 42. Frasure-Smith N, Lesperance F. Depression—a cardiac risk factor in search of a treatment. JAMA 289:3171, 2003. 43. Black SA, Markides KS, Ray LA. Depression predicts increased incidence of adverse health outcomes in older Mexican Americans with type 2 diabetes. Diabetes Care 26:2822, 2003. 44. Bae SC, Hashimoto H, Karlson EW, et al. Variable effects of social support by race, economic status, and disease activity in systemic lupus erythematosus. J Rheumatol 28:1245, 2001. 45. Mease P. Fibromyalgia syndrome: Review of clinical presentation, pathogenesis, outcome measures, and treatment. J Rheumatol 32:6, 2005. 46. Middleton GD, McFarlin JE, Lipsky PE. The prevalence and clinical impact of fibromyalgia in systemic lupus erythematosus. Arthritis Rheum 37:1181, 1994. 47. Akkasilpa S, Goldman D, Magder LS, et al. Number of fibromyalgia tender points is associated with health status in patients with systemic lupus erythematosus. J Rheumatol 32:48, 2005. 48. Gladman DD, Urowitz MB, Gough J, et al. Fibromyalgia is a major contributor to quality of life in lupus. J Rheumatol 24:2145, 1997. 49. Tench CM, McCurdie I, White PD, et al. The prevalence and associations of fatigue in systemic lupus erythematosus. Rheumatology (Oxford) 39:1249, 2000. 50. Krupp LB, LaRocca NG, Muir J, et al. A study of fatigue in systemic lupus erythematosus. J Rheumatol 17:1450, 1990. 51. Bruce IN, Mak VC, Hallett DC, et al. Factors associated with fatigue in patients with systemic lupus erythematosus. Ann Rheum Dis 58:379, 1999. 52. Wang B, Gladman DD, Urowitz MB. Fatigue in lupus is not correlated with disease activity. J Rheumatol 25:892, 1998. 53. Dobkin PL, DaCosta D, Joseph L, et al. Counterbalancing patient demands with evidence: results from a Pan-Canadian randomized clinical trial of brief supportive expressive group psychotherapy for women with systemic lupus erythematosus. Ann Behav Med 24:88, 2002. 54. Edworthy S, Clarke AE, DaCosta D, et al. Group psychotherapy reduces illness intrusiveness in systemic lupus erythematosus. J Rheumatol 30:1011, 2003. 55. Haupt M, Millen S, Janner M, et al. Improvement of coping abilities in patients with systemic lupus erythematosus: a prospective study. Ann Rheum Dis 64:1618, 2005. 56. Karlson EW, Liang MH, Eaton H, et al. A randomized clinical trial of a psychoeducational intervention to improve outcomes in systemic lupus erythematosus. Arthritis Rheum 50:1832, 2004. 57. Gold MR, Siegel JE, Russell LB, et al. Cost-Effectiveness in Health and Medicine. New York: Oxford University Press, 1996. 58. Drummond MF, O’Brien BJ, Stoddart GL, et al. Methods for the Economic Evaluations of Health Care Programmes. 2nd ed. Oxford: Oxford University Press, 1997. 59. Sloan FA. Valuing Health Care: Costs, Benefits, and Effectiveness of Pharmaceuticals and Other Medical Technologies. Cambridge: Cambridge University Press, 1995. 60. Gabriel S, Drummond M, Maetzel A, et al. OMERACT 6 Economics Working Group Report: a proposal for a reference case for

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83. Moore AD, Petri MA, Manzi S, et al. The use of alternative medical therapies in patients with systemic lupus erythematosus. Arthritis Rheum 43:1410, 2000. 84. Ruof J, Hulsemann JL, Stucki, G. Evaluation of costs in rheumatic diseases: a literature review. Curr Opin Rheumatol 11:104, 1999. 85. Ruof J, Merkesdal S, Huelsemann JL, et al. Cost assessment instruments in rheumatology: evaluation of applied instrument characteristics. J Rheumatol 28:662, 2001. 86. Clarke AE, Esdaile JM, Bloch DA, et al. A Canadian study of the total medical costs for patients with systemic lupus erythematosus and the predictors of costs. Arthritis Rheum 36:1548, 1993. 87. Lubeck DP, Spitz PW, Fries JF, et al. A multicenter study of annual health service utilization and costs in rheumatoid arthritis. Arthritis Rheum 29:488, 1986. 88. Guzman J, Peloso P, Bombardier C. Capturing health care utilization after occupational low-back pain: development of an interviewer-administered questionnaire. J Clin Epidemiol 52:419, 1999. 89. Goossens MEJB, Rutten-van Molken MPH, Vlaeyen JWS, et al. The cost diary: a method to measure direct and indirect costs in cost-effectiveness research. J Clin Epidemiol 53:688, 2000. 90. Ritter PL, Stewart AL, Kaymaz H, et al. Self-reports of health care utilization compared to provider records. J Clin Epidemiol 54:136, 2001. 91. Ruof J, Huelsemann JL, Mittendorf T, et al. Patient-reported health care utilization in rheumatoid arthritis: what level of detail is required? Arthritis Rheum Arthritis Care Res 51:774, 2004. 92. Baladi JF. A Guidance Document for the Costing Process. Ottawa: Canadian Coordinating Office for Health Technology Assessment, 1996. 93. Drummond MF, O’Brien BJ, Stoddart GL, et al. Cost analysis. In: Methods for the Economic Evaluations of Health Care Programmes. 2nd ed. Oxford: Oxford University Press, 1997 94. Dranove D. Measuring costs. In: Costs, Benefits, and Effectiveness of Pharmaceuticals and Other Medical Technologies. Cambridge: Cambridge University Press, 1995. 95. Clarke AE, Petri MA, Manzi S, et al. Underestimating the value of women: assessing the indirect costs of women with systemic lupus erythematosus. J Rheumatol 27:2597, 2000. 96. Merkesdal S, Ruof J, Huelsemann JL, et al. Indirect cost assessment in patients with rheumatoid arthritis (RA): comparison of data from the health economic patient questionnaire HEQ-RA and insurance claims data. Arthritis Rheum Arthritis Care Res 53:234, 2005. 97. Gironimi G, Clarke AE, Hamilton VH, et al. Why health care costs more in the US: comparing health care expenditures between systemic lupus erythematosus patients in Stanford and Montreal. Arthritis Rheum 39:979, 1996. 98. Sutcliffe N, Clarke AE, Taylor R, et al. Total costs and predictors of costs in patients with systemic lupus erythematosus. Rheumatology 40:37, 2001. 99. Clarke AE, Petri MA, Manzi S, et al. An international perspective on the well-being and health care costs for patients with systemic lupus erythematosus. J Rheumatol 26:1500, 1999. 100. Clarke AE, Petri M, Manzi S, et al. The SLE Tri-Nation Study: absence of a link between health resource use and heath outcome. Rheumatology 43:1016, 2004. 101. Nichol MB, Shi S, Knight TK, et al. Eligibility, utilization, and costs in a California Medicaid lupus population. Arthritis Rheum 51:996, 2004. 102. Zink A, Fischer-Betz R, Thiele K, et al. Health care and burden of illness in systemic lupus erythematosus compared to rheumatoid arthritis: results from the national database of the German Collaborative Arthritis Centres. Lupus 13:529, 2004. 103. Mau W, Listing J, Huscher D, et al. Employment across chronic inflammatory rheumatic diseases and comparison with the general population. J Rheumatol 32:721, 2005. 104. Partridge AJ, Karlson EW, Daltroy LH, et al. Risk factors for early work disability in systemic lupus erythematosus—results from a multicenter study. Arthritis Rheum 40:2199, 1997. 105. Panopalis P, Petri M, Manzi S, et al. The systemic lupus erythematosus tri-nation study: longitudinal changes in physical and mental well-being. Rheumatology 44:751, 2005.

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economic evaluation in rheumatoid arthritis. J Rheumatol 30:886, 2003. Maetzel A, Tugwell P, Boers M, et al. Economic evaluation of programs or interventions in the management of rheumatoid arthritis: defining a consensus-based reference case. J Rheumatol 30:891, 2003. Drummond MF, O’Brien BJ, Stoddart GL, et al. Critical assessment of economic evaluation. In: Methods for the Economic evaluations of Health Care Programmes. 2nd ed. Oxford: Oxford University Press, 1997. Drummond MF, O’Brien BJ, Stoddart GL, et al. Cost-utility analysis. In: Methods for the Economic Evaluations of Health Care Programmes. 2nd ed. Oxford: Oxford University Press, 1997 Gold MR, Patrick DL, Torrance GW, et al. Identifying and valuing outcomes. In: Cost-Effectiveness in Health and Medicine. New York: Oxford University Press, 1996. Canadian Coordinating Office for Health Technology Assessment. Guidelines for Economic Evaluation of Pharmaceuticals. 2nd ed. Ottawa: Canadian Coordinating Office for Health Technology Assessment, 1997. Suarez-Almazor ME, Conner-Spady B. Rating of arthritis health states by patients, physicians, and the general public. Implications for cost-utility analyses. J Rheumatol 28:648, 2001. Russell LB, Gold MR, Siegel JE, et al. The role of cost-effectiveness analysis in health and medicine. JAMA 276:1172, 1996. Weinstein MC, Siegel JE, Gold MR, et al. Recommendations of the panel on cost-effectiveness in health and medicine. JAMA 276:1253, 1996. O’Brien BJ, Gafni A. When do the “dollars” make sense? Toward a conceptual framework for contingent valuation studies in health care. Med Decis Making 16:288, 1996. Torrance GW, Siegel JE, Luce BR. Framing and designing the cost-effectiveness analysis. In: Cost-Effectiveness in Health and Medicine. New York: Oxford University Press, 1996. Drummond MF, O’Brien BJ, Stoddart GL, et al. Collection and anaysis of data. In: Methods for the Economic Evaluations of Health Care Programmes. 2nd ed. Oxford: Oxford University Press, 1997 Weinstein MC, Fineberg HV. Clinical Decision Analysis. Philadelphia: WB Saunders, 1980. Buxton MJ, Drummond MF, Van Hout BA, et al. Modelling in economic evaluation: an unavoidable fact of life. Health Econ 6:217, 1997. Lipscomb J, Weinstein MC, Torrance GW. Time preference. In: Cost-Effectiveness in Health and Medicine. New York: Oxford University Press, 1996. Manning WG, Fryback DG, Weinstein MC. Reflecting uncertainty in cost-effectiveness analysis. In: Cost-Effectiveness in Health and Medicine. New York: Oxford University Press, 1996. Goeree R, O’Brien BJ. Cost-effectiveness modeling in rheumatology: toward principles of good practice. J Rheumatol 30(Suppl 68):21, 2003. Vanness DJ. Knowing what you don’t know: Bayesian approaches to uncertainty in economic evaluations. J Rheumatol 30(Suppl 68):23, 2003. Briggs AH, O’Brien BJ, Blackhouse G. Thinking outside the box: recent advances in the analysis and presentation of uncertainty in cost-effectiveness studies. Annu Rev Public Health 23:377, 2002. Fenwick E, Claxton K, Sculpher M. Representing uncertainty: the role of cost-effectiveness acceptability curves. Health Econ 10:779, 2001. Drummond MF, O’Brien BJ, Stoddart GL, et al. Presentation and use of economic evaluation results. In: Methods for the Economic Evaluations of Health Care Programmes. 2nd ed. Oxford: Oxford University Press, 1997 Luce BR, Manning WG, Siegel JE, et al. Estimating costs in costeffectiveness analysis. In: Cost-Effectiveness in Health and Medicine. New York: Oxford University Press, 1996. Merkesdal S, Ruof J, Huelsemann JL, et al. Development of a matrix of cost domains in economic evaluation of rheumatoid arthritis. J Rheumatol 28:657, 2001.

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EPIDEMIOLOGY AND DIAGNOSIS

5

Biomarkers of Disease Activity Larissa Lapteva, MD and Gabor G. Illei, MD

INTRODUCTION Systemic lupus erythematosus has a characteristically waxing and waning course that can range in severity from a relatively mild chronic condition to an acutely life-threatening disease. The clinical heterogeneity most likely reflects the combination of various pathogenetic events modified by the patient’s genetic background, environmental factors, and treatment. This complexity adds to the difficulty of diagnosing and optimally managing patients. Biologic markers that reliably describe various aspects of SLE could be used to improve diagnostic accuracy, predict prognosis, characterize disease activity and guide therapy. Markers that are closely associated with or predict important clinical outcomes could be used as surrogate endpoints in clinical trials to accelerate drug development. Numerous candidates have been proposed as biomarkers, primarily based on data reflecting various pathophysiologic abnormalities; however, there are no biomarkers that have been rigorously validated and are widely accepted in SLE. The need to use biomarkers as surrogate endpoints in clinical trials is becoming more pressing with the availability of several promising new therapeutic agents and the unwillingness of the pharmaceutical industry to embark on studies that may last 5 to 10 years to show a beneficial outcome using traditional clinical endpoints. In this chapter, we will provide a general overview of biomarkers and surrogate endpoints, and summarize our view on the most promising candidates of biomarkers in SLE with a focus on promising candidate biomarkers of disease activity.

BIOMARKERS AND SURROGATE ENDPOINTS

46

The terms biomarker and surrogate endpoint describe different entities. However, they are commonly used interchangeably, which has led to considerable confusion in the literature. In an attempt to prevent such confusion and standardize the nomenclature, the National Institutes of Health convened an expert panel in 1991. Their recommendations are summarized in

Table 5.1.1 Biomarker can be defined as a physical sign or cellular, biochemical, molecular, or genetic alteration by which a normal or abnormal biologic process can be recognized and/or monitored, and that may have diagnostic or prognostic utility. Biomarkers must measure an underlying biologic process reliably and reproducibly. Surrogate endpoint is a measurement that is intended to serve as a substitute for a clinically meaningful outcome and is expected to predict the effect of a therapeutic intervention. Both biomarkers and surrogate endpoints have to be validated to prove that they are measuring intended outcomes reliably. It is important to recognize that the requirements of surrogate markers are much more stringent and that only a small minority of biomarkers will fulfill the criteria of a surrogate endpoint. In order for a biomarker to be validated as a surrogate endpoint, it must be shown that the presence of or a change in the measurement predicts an important clinical endpoint.

VALIDATION OF BIOMARKERS Validation of biomarkers is a complex process.2,3 The criteria for validation should be defined by the nature of the question that the biomarker is intended to address, the degree of certainty required for the answer, and the assumptions between the biomarker and clinical endpoints. An ideal biomarker should measure a clinically relevant process, and be sensitive and specific for the measurement that it is intended for. Any biomarker should be validated for sensitivity, specificity, details of bioanalytical assessment, and the probability of false positives and false negatives. Sensitivity is the ability of a biomarker to reflect a meaningful change in important clinical and/or biological endpoints and describes the level of correlation between the magnitude of change in the biomarker and clinical/biological endpoint. However, even a strong correlation does not prove a cause–effect relationship. Specificity defines the extent to which a biomarker explains the changes in a clinical/biological endpoint.

Term

Definition1

Elements of Validation

Biomarker

A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.

Sensitivity: the level of correlation between the magnitude of change in the biomarker and clinical/biological endpoint Specificity: the extent to which a biomarker explains the changes in a clinical/biological endpoint Bioanalytical assessment: the laboratory test or measurement should include assessment of precision, reproducibility, range of use, variability, and practicality False positivity: a change in a biomarker is not reflected by a change in a clinical/biological endpoint False negativity: a biomarker does not change despite a change in the clinical/biological outcome

Surrogate endpoint

A surrogate endpoint is expected to predict clinical benefit or harm (or lack of benefit or harm) based on epidemiologic, therapeutic, pathophysiologic, or other scientific evidence. A biomarker may fulfill the criteria for a surrogate endpoint.

Requires evaluation and qualification1,2: a graded process by which evidence is acquired linking a biomarker with a clinical endpoint Biologic plausibility: a mechanistic basis for using a surrogate endpoint Statistical relationship between the biomarker and the clinical endpoint based on epidemiologic or observational studies of the natural history of the disease Estimate of the expected benefit based on adequate, well-controlled and appropriately powered clinical trials Ability to predict potential adverse reactions Consistency of effects following interventions with various drug classes and/or within different stages of disease

The bioanalytical assessment of the laboratory test or measurement should include assessment of precision, reproducibility, range of use, variability, and practicality. False positivity is the situation in which a desired change in a biomarker is not reflected by a positive change in a clinical/biological endpoint, or even worse, is associated with a negative change. False negativity is the opposite, when a biomarker does not change despite a change in the clinical/biological outcome (Table 5.1).

Qualification of Surrogate Endpoints An ideal surrogate endpoint can be thought of as a validated biomarker that can be definitively substituted for a clinically meaningful endpoint in an efficacy trial or clinical practice. To meet the most rigorous standards, the surrogate endpoint must correlate with the true clinical outcome and must fully capture the net clinical effect of treatment.4 This may be impossible to achieve for most biomarkers, but it is clear that extensive clinical evidence is needed, which is collected in a rigorous scientific process and analyzed by careful statistical assessment. There is no consensus on validation of surrogate endpoints, and some experts favor the term qualification to describe this process,1,5 which has to include the following elements. Biologic plausibility should provide a mechanistic basis for using a surrogate endpoint, and epidemiologic or observational studies of

VALIDATION OF BIOMARKERS

TABLE 5.1 VALIDATION OF BIOMARKERS AND SURROGATE ENDPOINTS

the natural history of the disease should establish the statistical relationship between the biomarker and the clinical endpoint. Adequate, well-controlled and appropriately powered clinical trials should provide an estimate of the expected benefit; ideally, an appropriate dose– or exposure–response relationship would provide additional support for surrogate status. The analysis should include a consideration of whether potential adverse reactions are predicted by the surrogate endpoint. It is essential that the development and validation of biomarkers and surrogate markers be built into the drug development process, starting from the preclinical phase. It may be helpful to conduct a metaanalysis of multiple clinical trials to determine the consistency of effects following interventions with various drug classes and within different stages of disease.2

Current State of Biomarkers in SLE A large number of studies described potential biomarkers in lupus, but none fulfills the criteria of a true validated biomarker. There are many reasons that account for the conflicting results of various studies. Most were not designed as biomarker validation studies; therefore, their study design may not be appropriate for this purpose. A cross-sectional study may show an association between a biomarker and disease activity in a group of patients, but longitudinal studies are

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BIOMARKERS OF DISEASE ACTIVITY

TABLE 5.2 POTENTIAL USES OF BIOMARKERS IN SLE Application

Potential Biomarkers

Predict SLE and/or organ Genetic markers involvement Autoantibodies Monitor disease activity

Soluble markers (blood, urine, cerebrospinal fluid) Autoantibodies Complement activation markers Cytokines Chemokines Mediators of inflammation Circulating subsets of inflammatory cells Effector T and B lymphocytes Regulatory T cells Complex gene expression or proteomic profiles

Predict response to therapya

Genetic markers Related to lupus Related to drug metabolism (pharmacogenomics) Changes in selected markers of diseases activity

Predict flarea

Changes in selected markers of diseases activity

Predict damagea

Genetic markers Autoantibodies Complex gene expression or proteomic profiles Lack of normalization of markers of disease activity

Describe damage

Physiologic measures Imaging

a

Therapeutic decision based on predictive biomarkers should be made only after a strong correlation between the biomarker and the clinical outcome has been established; that is, the biomarker is qualified as a surrogate endpoint.

necessary to evaluate whether the same marker can be used to monitor disease activity in individual patients. The patient population may differ among studies in several ways such as ethnicity, treatment, organ manifestation, and stage of disease (early vs. late). The choice of controls (healthy vs. other rheumatic diseases vs. subpopulations of lupus patients) is also critical, and varies substantially among studies. Most frequently, widely accepted disease activity indices, such as SLEDAI, SLAM, ECLAM, and BILAG, are used as outcome measures. Although all are valid tools, they do not capture exactly the same aspects of lupus.6 Therefore, in some studies selected biomarkers correlated with one but not another activity index. In some cases, organ-specific outcome measures may be more appropriate, but the lack of widely accepted clinical endpoints makes standardization difficult. A large number of studies lack the statistical rigor that is essential to draw valid conclusions. Last, but not least, the bioassays used to measure biomarkers are frequently not standardized, leading to conflicting results in different laboratories.

BIOMARKERS IN SLE Lupus is a chronic disease with various mechanisms dominating at various stages. Genetic predisposition and environmental factors are the most important for predicting or quantifying the risk of lupus. At the onset of clinical symptoms, classic biomarkers of autoreactivity such as autoantibodies are used to help to establish or confirm the diagnosis of SLE. During the acute phases of lupus, biomarkers of disease activity could be used to optimize risk–benefit assessment in individual patients. Eventually, the sustained autoimmune and inflammatory process leads to the next phase characterized by organ damage, when there is a much greater

TABLE 5.3 BIOMARKERS OF SLE DISEASE ACTIVITY Biomarkersa

Biomarkers of Overall SLE Disease Activity

Biomarkers of Proliferative Lupus Nephritis

Conventional biomarkers

C3,C4

anti-dsDNA, C3,C4

E-CR1, E-C4d, complement activation products sIL-2R, sTNF-R, IFN-α

anti- C1q

sVCAM-1, sTM,

sVCAM-1, uVCAM-1, sTM

Promising biomarkers

a

48

Complement components Cytokines and cytokine receptors Markers of endothelial activation Cellular markers

sIL-2R, uIL-6,

CD27+ B cells, activated T cells

None of these biomarkers has been validated in studies. E-C4d, erythrocyte C4d; E-CR-1, erythrocyte complement receptor 1; sIL-2R, soluble IL-2 receptor; sTM, soluble thrombomodulin; sTNF-R, soluble TNF receptor; sVCAM-1, soluble VCAM-1; uIL-6, urinary IL-6; uVCAM-1, urinary VCAM-1.

sensitivity (72%) and specificity (79%) in distinguishing SLE and other autoimmune diseases. The overall negative predictive value of the combination of the two tests was 92%.11-13 Platelet C4d was found in only 18% of lupus patients, but it had 100% specificity against normal subjects and 98% against patients with other autoimmune diseases.14 Both of these findings have to be confirmed in larger studies, and their applicability to early diagnosis needs to be formally tested.

BIOMARKERS IN SLE

reliance on physiologic measures of organ function, such as renal function measurements. Due to the complexity of the underlying pathogenesis, it is likely that any specific biomarker will perform best at certain stages of the disease and novel biomarkers have to be defined specifically as to what they are intended to reflect (prognosis, future organ involvement, severity, disease activity, risk of flare-up, etc.), and at what stage of the disease (Table 5.2). A detailed review of the literature on novel biomarkers in SLE was published recently.7,8 Other chapters in this book describe the genetics of lupus and the current use of various laboratory tests in diagnosing SLE and their relationship to specific clinical manifestations. Here we will provide a summary of both the conventional and the most promising biomarkers of disease activity. Recent studies have demonstrated that autoantibodies can be found in the majority of lupus patients years before the diagnosis or the onset of symptoms.9,10 Moreover, the appearance of antibodies seemed to follow a temporal sequence. In 88 of the 130 patients with SLE, at least one autoantibody tested was present before the diagnosis (up to 9.4 years earlier; mean, 3.3 years). High titer antinuclear antibodies were present in 78%; anti–double-stranded DNA and anti-Ro antibodies in about 50%; anti-La, anti-Sm, and antinuclear ribonucleoprotein in about 30%; and antiphospholipid antibodies in 18% of patients. Antinuclear, antiphospholipid, anti-Ro, and anti-La antibodies were present earlier than anti-Sm and antinuclear ribonucleoprotein antibodies (a mean of 3.4 years before diagnosis vs. 1.2 years). Anti–double-stranded DNA antibodies, with a mean onset 2.2 years before the diagnosis, were found later than antinuclear antibodies (p = 0.06) and earlier than antinuclear ribonucleoprotein antibodies. The finding that the appearance of autoantibodies in patients with SLE tends to follow a predictable course, with a progressive accumulation of specific autoantibodies before the onset of SLE raises the possibility of using autoantibody profiles as predictors of lupus. These observations are clearly exciting; however, their clinical applicability needs to be determined. Although they account only for a very small proportion of patients, deficiencies in early complement components, such as C4, C2, and C1q, are associated with a significantly increased susceptibility to lupus. Moreover, activation of the complement system plays a central role in the pathogenesis of lupus. Therefore, it is conceivable that subtle changes in the complement system could be used for early diagnosis of SLE. A recent study showed that combined detection of high levels of erythrocyte-bound C4d (E-C4d) and low levels of erythrocyte-complement receptor 1 (E-CR1) had a sensitivity of 81% and specificity of 91% in distinguishing SLE patients from normal controls and acceptable

Markers of Disease Activity Classical Markers of Lupus Activity Despite a large body of literature about the associations of various autoantibodies with clinical manifestations and/or disease activity, there is remarkably little consensus on the value of these examinations in specific situations in individual patients; even the most widely used tests, such as anti-dsDNA antibodies and complement levels, are controversial. The opinions about the utility of anti-dsDNA ranges from proponents in favor of preemptive treatment in response to increases in anti-dsDNA15,16 to believers that such changes have no value in predicting flare-ups.17,18 Several recent publications and reviews addressed this issue.16-22 It is clear that methodologic differences, such as the frequency of testing, the tools used to assess activity, the definition of flares and the statistical methods used all contributed to the conflicting results. Furthermore, a 1-year longitudinal study of 53 patients found a decrease, and not an increase, in anti-dsDNA levels at the time of flares,23 presumably due to deposition of anti-dsDNA in tissues. Interestingly, flares measured by some but not all disease activity measures were preceded by an increase in anti-dsDNA levels. The association between disease activity and dsDNA levels is strongest for proliferative lupus nephritis; other manifestations correlate only weakly or not at all. Even in lupus nephritis, changes in anti-dsDNA levels are more important than absolute levels. The available evidence is insufficient to warrant preventive therapy based on changes in anti-dsDNA levels alone, but patients with an increase in anti-dsDNAs antibodies should be monitored very closely for other signs and symptoms of increased lupus activity. Because of the strong association of anti-dsDNA and proliferative nephritis, careful urinalysis must be part of this monitoring. Complement has an important role in the pathogenesis of SLE. Traditional measures of complement activity, such as CH50, C3, and C4, have low sensitivity and specificity because plasma levels reflect the result of the dynamic state of complement synthesis and consumption, both of which are increased during inflammation. Activation of the complement system is characterized by the generation of activated breakdown products of

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precursor molecules. Complement activation products may be more specific for complement activation and there is a good rationale to use them as markers of disease activity. However, the available studies show conflicting results with markers of the classic, alternative, or common pathways showing correlation with activity in some but not in other studies. Some of this may result from methodologic differences, such as the use of plasma versus serum and differences in the definition of disease activity. The instability and high turnover of the complement products have also limited their use as potential biomarkers. One direction for investigations in this field was an attempt to measure erythrocyte bound isoforms of complement products and complement receptor on the surface of erythrocytes and reticulocytes. These complementsplit products are acquired on the surface of the red blood cells during activation of the classical pathway, and the accumulated change in their levels is thought to reflect the state of complement activation for as long as the life span of a normal erythrocyte.12 It has also been shown that patients with SLE have reduced clearance of immune complexes associated with decreased levels of complement receptor 1 (CR1) on erythrocytes.24 In a recent study, expression of erythrocyte bound C4d (E-C4d) and CR1 (E-CR1) was determined by flow cytometry in 100 patients with SLE, 133 patients with other diseases, and 84 normal controls.13 Patients with SLE had significantly higher levels of E-C4d and lower levels of E-CR1 than healthy controls or patients with other diseases. The two tests combined together could distinguish patients with SLE from healthy controls with 81% sensitivity and 91% specificity, and SLE from other diseases with 72% sensitivity and 79% specificity. Another two color flow–cytometric analysis of C4d and CR1 on the surface of reticulocytes performed by the same group of investigators in 156 SLE patients, 140 patients with other diseases, and 159 healthy controls demonstrated significantly higher levels of C4d in SLE when compared with the two other groups; lupus patients with reticulocyte C4d levels in the highest quartile had higher SELENASLEDAI and SLAM scores than patients whose reticulocyte C4d levels were in the lowest quartile.11 Further work and large-scale trials are needed in this area to help further define appropriate complement-split products for assessing lupus disease activity, and to determine whether any of these can be used as a reliable biomarker.

Promising Candidate Biomarkers for Disease Activity 50

Systemic autoimmunity leads to local inflammation during periods of active disease. During these periods most elements of the immune system are different

from normals and from lupus patients in remission. These differences led to many studies proposing various cytokines, chemokines, markers of endothelial activation, and selected cellular subsets as biomarkers of activity. These studies were critically reviewed recently7,8; for most of the targets, the data are either not conclusive or not supportive of their use as a biomarker of disease activity. Most analyzed the relationship between overall disease activity measured by various disease activity indices and the putative biomarkers. There are very few studies that look at organ specific biomarkers of disease activity and most involve patients with renal disease. We first review the state of potential biomarkers of overall disease activity and then summarize those that are promising specifically for renal disease.

Cytokine and Cytokine Receptors Cytokines play an important role in mediating the autoimmune response. From all cytokines and cytokine receptors tested, data are most promising for interferon-α, soluble IL-2 receptor (SIL-2R, sCD25), and the soluble TNF receptors. Type-1 interferons link innate to adaptive immunity and appear to play an important role in the development or maintenance of the immune responses in autoimmunity. Serum interferon-α (IFN-α) levels correlated with disease activity in both untreated patients25 and in sera of adult26,27 and pediatric lupus patients on various treatments.28 The central role of interferons is supported by recent findings that IFN-regulated genes are overexpressed in patients with SLE.29-32 This gene expression pattern seems to be associated with the presence of antibodies against nucleoproteins and patients with major organ involvement.30,31 Moreover, this “interferon signature” was also detected in patients who did not have detectable levels of serum interferons suggesting that microarray analysis can be used to detect the activation of distinct pathways that can lead to better understanding of pathogenesis and suggest the development of therapeutic targets. The utility of using microarray expression profiles to monitor disease activity remains to be determined. Soluble IL-2 receptor (sIL-2R, sCD25) is released by activated lymphocytes and may be a measure of lymphocyte activation. Several studies looked at serum sIL-2R levels as a marker of disease activity, and most found elevated levels in patients with active disease with an increase of sIL-2R levels during flares and a decrease with treatment and clinical improvement.33-37 Views on the role of TNF in the pathogenesis of SLE are controversial and reflect the spectrum of its effects in different tissues during various stages of the disease. It is still unclear whether changes in TNF levels reflect changes in clinical activity. Interestingly, circulating titers of p55 and p75 soluble TNF receptors were found

Markers of Endothelial Activation Inflammation leads to the activation of the endothelium with up-regulation of several adhesion molecules. Markers of endothelial activation are, therefore, attractive candidates as biomarkers of disease activity. Soluble VCAM-1 (sVCAM-1) levels are elevated in patients with active lupus and both serum and urine VCAM-1 levels correlate with global measures of disease activity.41-47 Thrombomodulin (TM) is expressed on the luminal surface of the vascular endothelium. Its soluble form (sTM) can be detected in the plasma and urine after endothelial cell injury. All studies found elevated levels of sTM in patients with active SLE.48-50 Moreover, sTM levels positively correlated with SLEDAI,41 SLAM, and ECLAM scores.42 The consistent results observed in all studies make VCAM-1 and sTM good candidates as biomarkers of overall SLE disease activity.

Cellular Markers of Disease Activity Lymphocytes play a major role in the immune dysregulation in SLE. Therefore, assessing the number of activated or abnormal lymphocyte subsets is an obvious choice to monitor disease activity. Several studies suggested a correlation of T-lymphocyte subsets expressing various markers of activation,51-53 but most studies were limited in size and need further confirmation. Recently, the role of various B-cell subsets were compared to laboratory and clinical measures of disease activity.54 The number and frequency of plasma cells strongly expressing CD27 significantly correlated with SLE disease activity indices (SLEDAI and ECLAM) and the titer of anti-dsDNA antibodies. Highly active patients (SLEDAI >8) had an increased frequency of CD19+ cells. Using a nonparametric data-sieving algorithm, these B-cell abnormalities exhibited predictive values for nonactive and active disease of 78% and 78.9%, respectively. The predictive value of the B-cell abnormalities was greater than that of the humoral/ clinical data pattern, making this B-cell subpopulation a promising candidate biomarker of disease activity.54

Promising Candidate Biomarkers for Lupus Nephritis In search for better indicators of lupus nephritis, some biomarkers have been reported in association with renal outcomes. Several studies have shown that increases in soluble IL-2 receptor (sIL-2R) correlated not only with overall disease activity but also with major flares of lupus nephritis,33-35 and increases in sIL-2R levels

preceded flares.36,37 These results suggest that sIL-2R can be considered as a promising candidate biomarker of activity in patients with kidney involvement. Markers of endothelial activation were shown to be elevated in patients with lupus nephritis. Higher levels of sVCAM-1 correlated with disease activity stronger in patients who had renal disease than in those who had only extrarenal manifestations.41,43,47 In one study, higher levels of sVCAM were found at the time of biopsy in patients with an activity index of more than 4 on kidney biopsy.43 Urinary VCAM-1 positively correlated with SLEDAI and SLICC scores, and negatively correlated with glomerular filtration rates.55 Soluble thrombomodulin (sTM) was elevated in patients with evidence of lupus nephritis.41,42,48,50,56 Von Willebrand factor (vWF) is a glycoprotein released by the activated vascular endothelium. Both plasma and urinary vWF levels are increased in lupus nephritis, and patients with rapidly progressive lupus nephritis had the highest levels of vWF in one study.57 The consistent increase of markers of endothelial activation may potentially reflect the presence of renal endothelial injury, with the evidence suggesting that they may be considered promising biomarkers of lupus nephritis. Abnormalities in the C1q and C1q receptors have long been found in association with lupus and lupuslike syndromes.58 Recently, two independent crosssectional studies demonstrated higher prevalence of anti-C1q antibodies in patients with lupus nephropathy,59,60 and found a positive association among overall disease activity measured by ECLAM,60 lupus nephritis activity measured by BILAG renal score,59 and presence anti-C1q antibodies. Longitudinal studies are needed to evaluate the role of anti-C1q antibodies as a possible biomarker of lupus nephritis. Of the multitude of cytokines, urinary IL-6 correlated with overall disease activity and presence of active urinary sediment in SLE.61 Increased IL-18 levels have been found to correlate with SLEDAI scores in several studies62,63; two small studies found positive association between IL-18 levels and renal disease,64,65 but a larger study62 did not reproduce these results. In one study, urine monocyte chemoattractant protein-1 (uMCP-1) and urine IL-8 were measured by ELISA as biomarkers of renal flare. The investigators observed a significant increase in uMCP-1 level in 25 patients with lupus nephritis before, during, and after renal flares when compared to 22 SLE patients with nonrenal flares, 28 healthy individuals, or 15 patients with other renal diseases. The level of uMCP-1 was higher in patients with proliferative glomerulonephritis or with impaired renal function and correlated with the increase in proteinuria.66 The role of cytokines and chemokines in lupus nephritis remains to be determined; it is possible that the pattern of disequilibrium

BIOMARKERS IN SLE

to be significantly increased in lupus patients and correlated with disease activity in most studies,33,38-40 making them promising candidates as biomarkers of lupus activity.

51

BIOMARKERS OF DISEASE ACTIVITY

in cytokine/chemokine production may be more important than measurements of isolated cytokines or chemokines. Microarray analyses of gene expression in glomeruli isolated by laser capture microscopy from clinical biopsies of patients with lupus nephritis were used in an attempt to identify different phenotypes of focal and diffuse proliferative lupus nephritis at the molecular level. One subgroup expressed fibrosis-related genes that correlated with presence of glomerulosclerosis; another subset showed high expression of type-I interferon and reduced expression of fibrosis-related genes in association with milder pathological features of nephritis.67 The small study sample and crosssectional design limited interpretation of the findings of this study, but the implications of molecular phenotyping in lupus nephritis clearly warrant further research in this direction.

CONCLUSION Biomarkers reliably reflecting disease activity would be useful in clinical practice to monitor therapy and in

clinical studies to evaluate the effect of drugs. If a direct relationship with a clinically relevant endpoint can be proven, they may qualify as surrogate endpoints and be used to make clinical decisions of starting or discontinuing treatment. Although the criteria for a biomarker and surrogate endpoint are different, both must pass a rigorous scientific evaluation process to fulfill their respective functions. Despite the vast literature on putative biomarkers in lupus, no biomarker of disease activity has been validated to date. Some of the widely used activity markers as well as certain novel targets look promising, but must be subjected to a validation process. Better understanding of the pathogenesis of lupus will no doubt generate a large number of new candidate markers. Improvements in various multiplex technologies allowing the simultaneous measurement of gene and protein expression, lipid levels, and metabolite concentrations will open a new era in which, instead of individual measurements, complex patterns can be recognized and related to clinical activity. The success of this approach depends on the establishment of a rigorous scientific framework and collaborative efforts that must include a uniform assessment of clinical activity.

REFERENCES

52

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13. Manzi S, Navratil JS, Ruffing MJ, Liu CC, Danchenko N, Nilson SE, et al. Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus. Arthritis Rheum 2004; 50(11):3596-3604. 14. Navratil JS, Manzi S, Kao AH, Krishnaswami S, Liu CC, Ruffing MJ, et al. Platelet C4d is highly specific for systemic lupus erythematosus. Arthritis Rheum 2006;54(2):670-674. 15. Bootsma H, Spronk P, Derksen R, de Boer G, Wolters-Dicke H, Hermans J, et al. Prevention of relapses in systemic lupus erythematosus. Lancet 1995;345(8965):1595-1599. 16. Spronk PE, Bootsma H, Kallenberg CG. Anti-DNA antibodies as early predictor for disease exacerbations in SLE. Guideline for treatment? Clin Rev Allergy Immunol 1998;16(3):211-218. 17. Esdaile JM, Abrahamowicz M, Joseph L, MacKenzie T, Li Y, Danoff D. Laboratory tests as predictors of disease exacerbations in systemic lupus erythematosus. Why some tests fail. Arthritis Rheum 1996;39(3):370-378. 18. Esdaile JM, Joseph L, Abrahamowicz M, Li Y, Danoff D, Clarke AE. Routine immunologic tests in systemic lupus erythematosus: is there a need for more studies? J Rheumatol 1996;23(11): 1891-1896. 19. Bootsma H, Spronk PE, ter Borg EJ, Hummel EJ, de Boer G, Limburg PC, et al. The predictive value of fluctuations in IgM and IgG class anti-dsDNA antibodies for relapses in systemic lupus erythematosus. A prospective long-term observation. Ann Rheum Dis 1997;56(11):661-666. 20. Kallenberg CG, Bootsma H, Spronk PE, ter Borg EJ, Derksen RH, Kater L. Laboratory tests as predictors of flares in systemic lupus erythematosus: comment on the article by Esdaile et al. Arthritis Rheum 1997;40(2):393-394. 21. Swaak AJ, Smeenk RJ. Following the disease course in systemic lupus erythematosus: are serologic variables of any use? J Rheumatol 1996;23(11):1842-1844. 22. Zonana-Nacach A, Sanchez L, Camargo-Coronel A, MartinezOsuna P, Jimenez-Balderas FJ. Laboratory abnormalities and systemic lupus erythematosus flare: comment on the article by Esdaile et al. Arthritis Rheum 1997;40(11):2092-2093.

42. Horak P, Scudla V, Hermanovo Z, Pospisil Z, Faltynek L, Budikova M, et al. Clinical utility of selected disease activity markers in patients with systemic lupus erythematosus. Clin Rheumatol 2001;20(5):337-344. 43. Ikeda Y, Fujimoto T, Ameno M, Shiiki H, Dohi K. Relationship between lupus nephritis activity and the serum level of soluble VCAM-1. Lupus 1998;7(5):347-354. 44. Janssen BA, Luqmani RA, Gordon C, Hemingway IH, Bacon PA, Gearing AJ, et al. Correlation of blood levels of soluble vascular cell adhesion molecule-1 with disease activity in systemic lupus erythematosus and vasculitis. Br J Rheumatol 1994;33(12): 1112-1116. 45. Spronk PE, Bootsma H, Huitema MG, Limburg PC, Kallenberg CG. Levels of soluble VCAM-1, soluble ICAM-1, and soluble E-selectin during disease exacerbations in patients with systemic lupus erythematosus (SLE); a long term prospective study. Clin Exp Immunol 1994;97(3):439-444. 46. Tesar V, Masek Z, Rychlik I, Merta M, Bartunkova J, Stejskalova A, et al. Cytokines and adhesion molecules in renal vasculitis and lupus nephritis. Nephrol Dial Transplant 1998;13(7):1662-1667. 47. Kaplanski G, Cacoub P, Farnarier C, Marin V, Gregoire R, Gatel A, et al. Increased soluble vascular cell adhesion molecule 1 concentrations in patients with primary or systemic lupus erythematosus-related antiphospholipid syndrome: correlations with the severity of thrombosis. Arthritis Rheum 2000;43(1):55-64. 48. Boehme MW, Nawroth PP, Kling E, Lin J, Amiral J, Riedesel J, et al. Serum thrombomodulin. A novel marker of disease activity in systemic lupus erythematosus. Arthritis Rheum 1994;37(4): 572-577. 49. Frijns R, Fijnheer R, Schiel A, Donders R, Sixma J, Derksen R. Persistent increase in plasma thrombomodulin in patients with a history of lupus nephritis: endothelial cell activation markers. J Rheumatol 2001;28(3):514-519. 50. Kotajima L, Aotsuka S, Sato T. Clinical significance of serum thrombomodulin levels in patients with systemic rheumatic diseases. Clin Exp Rheumatol 1997;15(1):59-65. 51. Anand A, Dean GS, Quereshi K, Isenberg DA, Lydyard PM. Characterization of CD3+ CD4- CD8- (double negative) T cells in patients with systemic lupus erythematosus: activation markers. Lupus 2002;11(8):493-500. 52. Su CC, Shau WY, Wang CR, Chuang CY, Chen CY. CD69 to CD3 ratio of peripheral blood mononuclear cells as a marker to monitor systemic lupus erythematosus disease activity. Lupus 1997;6(5):449-454. 53. Viallard JF, Bloch-Michel C, Neau-Cransac M, Taupin JL, Garrigue S, Miossec V, et al. HLA-DR expression on lymphocyte subsets as a marker of disease activity in patients with systemic lupus erythematosus. Clin Exp Immunol 2001;125(3):485-491. 54. Jacobi AM, Odendahl M, Reiter K, Bruns A, Burmester GR, Radbruch A, et al. Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus. Arthritis Rheum 2003;48(5):1332-1342. 55. Molad Y, Miroshnik E, Sulkes J, Pitlik S, Weinberger A, Monselise Y. Urinary soluble VCAM-1 in systemic lupus erythematosus: a clinical marker for monitoring disease activity and damage. Clin Exp Rheumatol 2002;20(3):403-406. 56. Frijns CJ, Derksen RH, De Groot PG, Algra A, Fijnheer R. Lupus anticoagulant and history of thrombosis are not associated with persistent endothelial cell activation in systemic lupus erythematosus. Clin Exp Immunol 2001;125(1):149-154. 57. Bobkova I, Lysenko L, Polyantseva L, Tareyeva I. Urinary von Willebrand factor as a marker of lupus nephritis progression. Nephron 2001;87(4):369-370. 58. Ghebrehiwet B, Peerschke EI. Role of C1q and C1q receptors in the pathogenesis of systemic lupus erythematosus. Curr Dir Autoimmun 2004;7:87-97. 59. Marto N, Bertolaccini ML, Calabuig E, Hughes GR, Khamashta MA. Anti-C1q antibodies in nephritis: correlation between titres and renal disease activity and positive predictive value in systemic lupus erythematosus. Ann Rheum Dis 2005;64(3):444-448. 60. Sinico RA, Radice A, Ikehata M, Giammarresi G, Corace C, Arrigo G, et al. Anti-C1q autoantibodies in lupus nephritis: prevalence and clinical significance. Ann N Y Acad Sci 2005;1050:193-200. 61. Peterson E, Robertson AD, Emlen W. Serum and urinary interleukin-6 in systemic lupus erythematosus. Lupus 1996;5(6): 571-575.

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23. Ho A, Magder LS, Barr SG, Petri M. Decreases in anti-doublestranded DNA levels are associated with concurrent flares in patients with systemic lupus erythematosus. Arthritis Rheum 2001;44(10):2342-2349. 24. de Carvalho Lins CE, Pereira Crott LS, Teixeira JE, Barbosa JE. Reduced erythrocyte complement receptor type 1 in systemic lupus erythematosus is related to a disease activity index and not to the presence or severity of renal disease. Lupus 2004; 13(7):517-521. 25. Kim T, Kanayama Y, Negoro N, Okamura M, Takeda T, Inoue T. Serum levels of interferons in patients with systemic lupus erythematosus. Clin Exp Immunol 1987;70(3):562-569. 26. Dall’era MC, Cardarelli PM, Preston BT, Witte A, Davis JC, Jr Type I interferon correlates with serological and clinical manifestations of SLE. Ann Rheum Dis 2005;64(12):1692-1697. 27. Bengtsson AA, Sturfelt G, Truedsson L, Blomberg J, Alm G, Vallin H, et al. Activation of type I interferon system in systemic lupus erythematosus correlates with disease activity but not with antiretroviral antibodies. Lupus 2000;9(9):664-671. 28. Blanco P, Palucka AK, Gill M, Pascual V, Banchereau J. Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science 2001;294(5546):1540-1543. 29. Kirou KA, Lee C, George S, Louca K, Papagiannis IG, Peterson MG, et al. Coordinate overexpression of interferon-alpha-induced genes in systemic lupus erythematosus. Arthritis Rheum 2004;50(12):3958-3967. 30. Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK. Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum 2005;52(5):1491-1503. 31. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A 2003;100(5):2610-2615. 32. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau J, et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 2003;197(6):711-723. 33. Davas EM, Tsirogianni A, Kappou I, Karamitsos D, Economidou I, Dantis PC. Serum IL-6, TNFalpha, p55 srTNFalpha, p75srTNFalpha, srIL-2alpha levels and disease activity in systemic lupus erythematosus. Clin Rheumatol 1999;18(1):17-22. 34. Dejica D. Serum soluble IL-2 receptor as a marker of lymphocyte activation in some autoimmune diseases. Effect of immunosuppressive therapy. Roum Arch Microbiol Immunol 2001;60(3): 183-201. 35. Laut J, Senitzer D, Petrucci R, Sablay LB, Barland P, Glicklich D. Soluble interleukin-2 receptor levels in lupus nephritis. Clin Nephrol 1992;38(4):179-184. 36. Swaak AJ, Hintzen RQ, Huysen V, van den Brink HG, Smeenk JT. Serum levels of soluble forms of T cell activation antigens CD27 and CD25 in systemic lupus erythematosus in relation with lymphocytes count and disease course. Clin Rheumatol 1995; 14(3):293-300. 37. ter Borg EJ, Horst G, Limburg PC, Kallenberg CG. Changes in plasma levels of interleukin-2 receptor in relation to disease exacerbations and levels of anti-dsDNA and complement in systemic lupus erythematosus. Clin Exp Immunol 1990;82(1):21-26. 38. Gabay C, Cakir N, Moral F, Roux-Lombard P, Meyer O, Dayer JM, et al. Circulating levels of tumor necrosis factor soluble receptors in systemic lupus erythematosus are significantly higher than in other rheumatic diseases and correlate with disease activity. J Rheumatol 1997;24(2):303-308. 39. Aderka D, Wysenbeek A, Engelmann H, Cope AP, Brennan F, Molad Y, et al. Correlation between serum levels of soluble tumor necrosis factor receptor and disease activity in systemic lupus erythematosus. Arthritis Rheum 1993;36(8):1111-1120. 40. Aringer M, Feierl E, Steiner G, Stummvoll GH, Hofler E, Steiner CW, et al. Increased bioactive TNF in human systemic lupus erythematosus: associations with cell death. Lupus 2002; 11(2):102-108. 41. Ho CY, Wong CK, Li EK, Tam LS, Lam CW. Elevated plasma concentrations of nitric oxide, soluble thrombomodulin and soluble vascular cell adhesion molecule-1 in patients with systemic lupus erythematosus. Rheumatology (Oxford) 2003;42(1): 117-122.

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62. Robak E, Robak T, Wozniacka A, Zak-Prelich M, SysaJedrzejowska A, Stepien H. Proinflammatory interferongamma–inducing monokines (interleukin-12, interleukin-18, interleukin-15)—serum profile in patients with systemic lupus erythematosus. Eur Cytokine Netw 2002;13(3):364-368. 63. Wong CK, Li EK, Ho CY, Lam CW. Elevation of plasma interleukin18 concentration is correlated with disease activity in systemic lupus erythematosus. Rheumatology (Oxford) 2000;39(10): 1078-1081. 64. Wong CK, Ho CY, Li EK, Tam LS, Lam CW. Elevated production of interleukin-18 is associated with renal disease in patients with systemic lupus erythematosus. Clin Exp Immunol 2002;130(2): 345-351.

65. Amerio P, Frezzolini A, Abeni D, Teofoli P, Girardelli CR, De Pita O, et al. Increased IL-18 in patients with systemic lupus erythematosus: relations with Th-1, Th-2, pro-inflammatory cytokines and disease activity. IL-18 is a marker of disease activity but does not correlate with pro-inflammatory cytokines. Clin Exp Rheumatol 2002;20(4):535-538. 66. Rovin BH, Song H, Birmingham DJ, Hebert LA, Yu CY, Nagaraja HN. Urine chemokines as biomarkers of human systemic lupus erythematosus activity. J Am Soc Nephrol 2005;16(2):467-473. 67. Peterson KS, Huang JF, Zhu J, D’Agati V, Liu X, Miller N, et al. Characterization of heterogeneity in the molecular pathogenesis of lupus nephritis from transcriptional profiles of lasercaptured glomeruli. J Clin Invest 2004;113(12):1722-1733.

PATHOGENESIS

6

Overview of the Pathogenesis of Systemic Lupus Erythematosus Sandeep Krishnan, MD, PhD, Bhabadeb Chowdhury, PhD, Yuang-Taung Juang, MD, PhD, and George C. Tsokos, MD

INTRODUCTION Systemic lupus erythematosus (SLE) is a complex autoimmune disease that affects multiple organ systems. It predominantly affects females (female:male ratio of 10:1) and certain racial/ethnic groups more than others, such as for example, African-American women more than white women. The etiopathogenesis of SLE is not clearly understood. SLE is considered as a complex genetic trait. However, despite advances in identification of several genes associated with SLE, it is not known what exactly confers “SLE susceptibility.” The current models of inheritance of disease susceptibility of multifactorial traits such as SLE favor the principle of “threshold liability.”1,2 According to this model, the genetic makeup of an individual who is predisposed to developing SLE comprises a certain number of SLE susceptibility genes that contribute to additive disease liability when their number exceeds a certain hypothetical threshold. This “additive inheritance” is possibly modified further by “multiplicative inheritance,” such as epistatic interactions among susceptibility alleles, and together they skew an individual’s disease liability toward a critical threshold at which point the disease manifests.1,2 Environmental and stochastic events experienced by an individual in his life and hormonal factors could also contribute to these factors. The pathogenesis of SLE is equally complex involving multiple immune abnormalities including abnormal B- and T-cell function that perpetuates autoantibody production by B cells and generates autoreactive T cells. In addition, abnormal clearance of immune complexes that results in their deposition in tissues, activation of complement and defective cellular apoptosis that generates a pool of potential autoantigens, are also integral components of the SLE pathology. The net result of these processes is induction of varying degrees of organ inflammation and failure, most importantly of the

kidneys, heart, skin and nervous system that ultimately result in various degrees of morbidity and mortality.3 In this section, we briefly consider the pathogenesis of SLE at the molecular level. The reader is referred to appropriate chapters where each topic is discussed in detail.

ETIOPATHOGENESIS OF SLE

Environmental Factors Environmental factors may be involved in triggering the onset of the autoimmune process in SLE in a genetically predisposed individual. These factors include drugs, UV rays in sunlight, heavy metals and chemicals, pathogenic organisms, and lifestyle, including diet. The role of these agents is considered in detail in Chapter 7. Several drugs have been implicated in drug-induced lupus, with procainamide and hydralazine being the best studied. These drugs modify epigenetic mechanisms that control gene expression in T cells such as inhibition of DNA methylation.4 DNA methylation is a mechanism employed by cells to regulate transcription of genes, and hypomethylation of DNA could result in abnormal expression of genes implicated in the pathogenesis of SLE. The molecules that become overexpressed in helper cells include LFA-1, CD70, and IFN-γ.4 As a result, there is loss of major histocompatibility complex (MHC) restriction to self-antigens by T cells, abnormal TCR signaling, and alteration in B-cell responses that include increased antibody production. Another environmental factor that has been known to trigger SLE flares or augment the pathologic process is UV ray exposure. Although the precise mechanism of action of UV rays is unclear, there is emerging evidence that similar to drugs that induce lupus, UV rays might also be involved in altering DNA methylation, and thus similarly alter the immune response. In addition,

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OVERVIEW OF THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

they might also play a role in increasing apoptosis in SLE patients and trigger the autoimmune process by unmasking potential autoantigens (reviewed in Mok and Lau5). Among numerous chemicals and heavy metals implicated in SLE pathogenesis, the most important are crystalline silica and mercury. Their precise role in initiating/augmenting the abnormal autoimmune response remains unclear (see Chapter 7). It is a common clinical observation that production of autoantbodies and subsequent development of SLE occurs following an infection. Several mechanisms are believed to be involved in virus-induced autoimmunity such as molecular mimicry (production of antigenic epitopes similar to self-antigens), alteration of the immune response, and other mechanisms. These mechanisms are discussed in detail in Chapters 2 and 11. Although several viruses have been implicated, the association of lupus with Epstein-Barr virus (EBV) is the most extensively studied. The characteristics of EBV that might possibly contribute to SLE pathogenesis include (1) establishment of a life-long infectious process in the host that is punctuated by periods of reactivation, (2) production of viral proteins similar to host molecules such as IL-10, CD40, and bcl-2 that have the potential to alter the immune response, (3) molecular mimicry that results in targeting of host proteins by autoantibodies, and (4) its effect on activation and immortalization of B cells.6 Cytomegalovirus (CMV), similar to EBV, is another herpes virus believed to be involved in the pathogenesis of SLE. CMV is believed to alter the immune process by multiple mechanisms such as (1) production of a protein similar to IL-10, (2) induction of cytokines such as TNF-α in massive amounts or (3) by triggering the generation of autoantibodies, the most significant among them being against the U1 small nuclear ribonucleoprotein.6 Other viruses implicated are retroviruses that share several features of herpes viruses, and parvovirus B19. However, further studies are necessary to fully ascertain their association with disease development in SLE. The role of diet and SLE remains unresolved. Flareup of lupus activity following ingestion of alfalfa has been reported in humans. Alfalfa contains L-canavanine that is implicated in mediating this effect, although these findings have not found sufficient support among studies in various population groups (reviewed in Mok and Lau5).

Genetic Factors

56

The observations that SLE aggregates in families and demonstrates a high sibling recurrence risk ratio and high disease concordance rates in identical twins, strongly support a genetic basis of susceptibility to SLE.

Over the past several years, association studies of candidate genes and linkage analysis have identified several genes with varying abilities to confer susceptibility to SLE (see Chapter 8 for full discussion). The genes coding for MHC have been associated with SLE for several decades. It has been reported that the HLA-DR2 and HLA-DR3 have been associated with a two- to three-fold relative risk conferred by each allele in the white population.7 While the mechanisms that are responsible for the contribution of these alleles to lupus pathogenesis remain unclear, the most plausible explanation is altered antigen presentation to CD4 T cells. Another set of alleles with strong association with SLE is the Fcγ receptor genes. Allelic variants of Fcγ receptor genes can contribute to SLE pathology by altering the functions of phagocytic cells via altered binding affinity to respective subclasses of IgG. Singlenucleotide-gene polymorphisms (SNP) of FcγRIIA, FcγRIIIA, FcγRIIIB, and FcγRIIB, a cluster of four genes at 1q23 encoding for low-affinity IgG receptors, have been found to be associated with SLE, with FcγRIIA and FcγRIIIA bearing the strongest association.8-10 Complement gene products participate in rapid clearance of apoptotic debris, thus masking potential autoantigens. Complete deficiency of complement components C1q, C4, and C2 tremendously increases the risk of developing SLE in an individual. In addition, deficiencies of C1r/s, C5, and C8 have also been reported to induce SLE-like syndromes.9 Cytotoxic T-lymphocyte antigen (CTLA-4) normally serves to dampen the immune response by acting as a negative regulator of T lymphocytes. A strong association of CTLA-4 gene polymorphisms with susceptibility to SLE has been reported. Specifically an allelic variation characterized by T/C substitution at the -1722 site has been shown to influence susceptibility to SLE (reviewed in Nath et al.9 and Croker and Kimberly10). Programmed cell death-1 (PDCD-1) is an immunoreceptor of the CD28 family normally expressed on the surface of activated T and B cells and regulates peripheral tolerance. In the European and Mexican populations, development of SLE has been attributed to a SNP in the intronic sequences of PDCD-1. This polymorphism has been shown to alter the binding site for the runt-related transcription factor 1 (RUNX1), and thus disrupt the regulation of expression of PDCD-1 protein. This process then triggers increased responsiveness of lymphocytes in SLE (reviewed in Nath et al.,9 Croker and Kimberly,10 and Shen and Tso11). SNP have been reported for several other genes in SLE that associate these genes with SLE. Among them are protein tyrosine phosphatase N22 (PTPN22),

Role of Hormones Given the strong gender bias observed in SLE, the role of hormonal influences in the pathogenesis of SLE has been long suspected. In general, it has been shown that androgens are immunoprotective whereas in a number of autoimmune diseases, estrogens are involved either as immunoprotective agent (especially the diseases demonstrating a Th2-type response) or as an agent involved in the disruption of tolerance. Estrogens act via ER-α and -β receptors that are expressed singly or in combination in cells of the immune system. Both T and B cells have been shown to express ER-α and -β.12 The precise mechanism by which estrogen exerts its role in SLE pathogenesis is unclear. Indeed, treatment of SLE T cells but not normal T cells results in increased activity of calcineurin phosphatase induction and expression of CD154. It has been shown that estrogen can bind to DNA directly to alter transcription of several genes and in addition may also indirectly modulate transcription via its association with other activators or repressors or through stimulation of the Erk (MAP kinase) pathway.12 Estrogen targets several genes including those that code for cytokines and molecules involved in the apoptotic factors.12 Extensive research on animal models and trials with receptor antagonists are expected to shed light on the precise role of estrogen in SLE pathogenesis. The role of other hormones is even less clear. Important among them is prolactin, which stimulates disease activity and enhances T-cell proliferation and B-cell maturation. Estrogen may also control prolactin, thereby aiding these events indirectly. These and other hormones are discussed in detail in Chapter 9.

IMMUNOPATHOLOGY

Lymphocyte Abnormalities T-Cell Abnormalities Several aspects of the abnormal regulation of T cells contribute to autoimmunity in SLE. These include disruption of immune tolerance, abnormal response to autoantigens, abnormal display of autoantigens, and pathologic alterations in signal transduction across the T-cell receptor (TCR). In contrast to T cells derived from a healthy individual, SLE T cells display an activated phenotype characterized by surface expression of activation markers, lowered threshold of activation, and altered co-stimulation requirements. The activated phenotype of T cells in lupus has been reported in both humans and mice.13,14 (See Chapter 10.) Studies that examined the structure and associations of the TCR/CD3 complex in SLE have shed light on some of the mechanisms behind the hyperexcitable phenotype of SLE T cells. These findings are summarized in Fig. 6.1. Specifically, in SLE T cells, the classical TCRs that contain TCR ζ chains are replaced by TCRs that associate with the TCR ζ homologue FcRγ chain, which becomes upregulated in SLE.15 Heightened amplification of signals emerging from this “rewired” TCR/CD3 complexes containing FcRγ is mediated via the association of FcRγ with Syk that is more potent enzymatically compared to ZAP-70 kinase, which traditionally associates with TCRs containing TCR ζ chains.13 Alterations in the lipid raft dynamics in SLE T cells also represent another checkpoint that determines the heightened TCR signaling. Lipid rafts are cholesterol/ ganglioside enriched signal-modulating compartments of T-cell membranes. SLE T cells have the inherent ability to produce more lipid rafts that are also increasingly mobile on the surface membrane of T cells. Thus, anti-CD3–induced capping of the receptor occurs more rapidly in SLE compared to normal T cells. The lipid rafts of SLE T cells also possess distinct protein composition compared to normal T cells and contain FcRγ and Syk, which could account for the heightened TCR-induced intracellular calcium flux observed in SLE T cells upon prior cross-linkage of lipid rafts.16 Table 6.1 outlines the main differences in the signaling molecules expressed in normal and SLE T cells. What leads to the down-regulation of TCR ζ and up-regulation of FcRγ chain remains unclear. However, down-regulation of TCR ζ in SLE appears to be regulated at multiple levels, including at the level of transcription, resulting in generation of abnormally spliced forms of TCR ζ mRNA that are less stable, and at the protein level where proteolysis of TCR ζ protein is mediated by increased caspase-3 expression and activity in SLE T cells.17-19 Treatment of SLE T cells with caspase-3 inhibitors has been shown to increase the

IMMUNOPATHOLOGY

C-reactive protein (CRP), mannose-binding lectin (MBL), cytokine genes such as tumor necrosis factor (TNF), and interferon-α (IFN-α) genes that may function by disrupting either the innate or adoptive arm of the immune response (reviewed in Nath et al.,9 Croker and Kimberly,10 and Shen and Tso11). While significant differences exist between human and murine lupus, animal models have served as valuable tools to evaluate the genetic basis of pathology of human SLE. The murine models of SLE such as the (NZB × NZW)F1, BXSB, and MRL mice are a few such examples. Chapters 17 and 18 categorize these mouse models into groups and discuss each model and the insights we have gained from them. In a nutshell, these models have helped in narrowing down the search for candidate genes to specific lupus susceptibility loci. It is expected that with the availability of powerful genetic and proteomic tools, the animal models will aid identification of disease susceptibility genes through positional cloning and in vivo complementation studies using bacterial artificial chromosome (BAC) transgenic technology.

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OVERVIEW OF THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

ab TCR ↑CD40L ↓CD45

CD28

CD4 CD3 edeg

FcRg

LAT

↑Vav ↑PLC-g1

Grb2 Sos

(+)

↓Lck

(+) (–)

(–)

(+)

ZAP-70

p21 Ras

↑Syk ↓

T cell activation

↑[Ca2+]i response

ERK

(–)

↑ PP2A

(–)

(–) pCREB

T cell activation

(+)

↑ CREM

(–)

(+) (+)

↑

CaMKIV

(–) IL-2 transcription

Fig. 6.1 Abnormal TCR-induced signaling in SLE. SLE T cells down-regulate TCR ζ chain, and instead up-regulate FcRγ chain that associates with TCR and preferentially recruits and activates Syk kinase. These events alter the kinetics of phosphorylation of proteins such as Vav and PLC-γ1, and induce their abnormal recruitment to lipid rafts to amplify signaling. Lck levels are reduced in SLE T cells, whereas co-stimulatory signals through CD28 are enhanced. Heightened and faster influx of intracytoplasmic calcium induced by TCR ligation activates CaMKIV, which enhances the activity of transcriptional repressor CREM leading to decreased IL-2 production. Decreased IL-2 transcription is also mediated by increased levels of PP2A that decreases phosphorylation of the enhancer of IL-2 transcription, pCREB.

58

expression of TCR ζ while simultaneously decreasing the expression of FcRγ chain.19 Similarly, forced expression of FcRγ into normal T cells resulted in down-regulation of the expression of TCR ζ.20 These observations suggest that the regulation of expression of TCR ζ and FcRγ is reciprocally linked. An interesting aspect about TCR signaling in SLE is that the heightened TCR-induced early response of SLE T cells does not translate into increased production of IL-2. The mechanisms underlying this observation involve defects in the transcriptional regulation of the IL-2 gene. First, SLE T cells express increased amounts of transcriptional repressor cAMP response element modulator (CREM) that binds to the IL-2 promoter and represses its activity,21,22 which may be induced by factors present within the serum of SLE patients. Indeed, it has been observed that anti-TCR/CD3 present in SLE serum could induce the expression of CREM in normal T cells treated with SLE serum.23 In addition, the level of calcium/calmodulin-dependent kinase IV (CaMKIV) is increased in the nucleus of SLE

T cells, and it increases the expression of CREM and its binding to the IL-2 promoter.23 The second mechanism of decreased IL-2 production by SLE T cells involves increased levels of the ser/thr phosphatase PP2A that causes dephosphorylation of phosphorylated cAMP response element-binding protein (pCREB), which is a transcriptional enhancer of IL-2 gene.24 Suppression of the expression of CREM or PP2A in SLE T cells by means of siRNA or antisense or dominant negative constructs leads to correction of IL-2 production, thus providing a target for defective molecular therapy of IL-2 production in SLE. Alterations in expression of co-stimulatory molecules and defective cytokine production by SLE T cells are considered in subsequent sections.

B-Cell Abnormalities and Antibodies Similar to T cells, B cells also display abnormalities at several levels such as phenotype, life span, function, and altered signal transduction. It has been shown that phenotypically, the naive B-cell compartment is

Location

Molecule

Expression Status

CD40 L

Increased

CD70

Increased

Cell surface

TCRζ chain

Decreased

FcRγ chain

Increased

Lck

Decreased

Syk

Increased

Vav

Increased

PLC-γ1

Increased

Lipid rafts

Cytoplasm/nucleus

Lck

Decreased

Syk

Increased

Vav

Increased

MAP kinase

Decreased

Protein kinase C

Decreased

NF-κB, p65 subunit

Decreased

Elf-1-p98

Decreased

PP2A

Increased

CREM

Increased

CaMKIV

Increased

replaced by activated plasma cells that arise from abnormal polyclonal activation/differentiation of B cells that are also long-lived. In terms of function, these cells are efficient presenters of autoantigens to T cells, are responsible for producing numerous autoantibodies, and modulate the activity of T cells to secrete cytokines that further contribute to the immune pathology (See Chapter 11). Autoantibody production by B cells in SLE has been demonstrated to be mediated by both T-cell– dependent and T-cell–independent mechanisms. Among the autoantibodies, the most important are anti-dsDNA, anti-ssDNA, anti-Ro, anti-poly ADP ribose, anti-Sm, anti-phospholipid antibodies, and antinucleosome antibodies, all involved in various pathologic outcomes as discussed in Chapters 22A through G. At the center of these B-cell abnormalities lies aberrant signal transduction across the B-cell receptor (BCR). Similar to T cells, ligation of the BCR results in earlier induction of intracellular tyrosine phosphorylation events and heightened intracytoplasmic calcium flux.25 These events occur in conjunction with abnormal signaling through other molecules that regulate BCR signaling such as the altered expression of complement receptor-2

(CR2, which is involved in regulating B-cell tolerance) and FcγRII receptors (discussed above). In addition, the expression of Lyn kinase, a cytoplasmic signaling molecule that is involved in negative regulation of B-cell signaling is also decreased in B cells in SLE, providing another rationale for increased B-cell responses.25 Another surface receptor that merits special mention is the B-cell–activating factor of the TNF family receptor (BAFF-R). The survival of B cells depends on whether B cells can compete for BAFF (BAFF–BAFF-R interaction) instead of a homologous protein A proliferation-inducing ligand (APRIL). BAFF can bind to three receptors—TACI, BCMA, and BAFF-R—whose expression varies throughout B-cell ontogeny, whereas APRIL binds only TACI and BCMA.26 In SLE, BAFF production by dendritic cells is increased in response to CpG DNA-inducible cytokine (IFN-α). The resulting activation of B cells can induce T-cell–independent IgG production. Thus, antagonism of BAFF and APRIL has been attempted as a means of treating SLE.26,27 The role of cytokines in modulating the effects of B cells and the effect of increased expression of co-stimulatory molecules by B cells on the functioning of T cells are discussed below.

IMMUNOPATHOLOGY

TABLE 6.1 ABNORMAL EXPRESSION OF PROTEINS IN SLE T CELLS

Role of Cytokines No discussion on the pathogenesis of SLE is complete without mentioning the role of cytokines. An attempt is often made to classify autoimmune diseases into Th1 or Th2 diseases on the basis of helper T-cell function and cytokine production. However, as discussed in Chapter 12, SLE fails to fulfill strict criteria of either one of these categories. As a result, the role of cytokines in SLE is largely decided by individual merit rather than as mediators of Th1 or Th2 phenotypes. On this front, a plethora of studies have examined a large number of cytokines and their possible contribution to SLE pathology. Significant among these cytokines with direct or indirect effect on mediating the abnormal immune processes in SLE are IL-2, IFN-γ, IFN-α, IL-4, IL-6, IL-10, TNF-α, and TGF-β (see Chapter 12). As observed earlier, IL-2 production is defective and occurs at least in part as a result of defective transcription.23 Defective IL-2 production would mean defective IL-2dependent functions of T cells such as proliferation, differentiation and activation-induced cell death (AICD) and efficient functioning of Tregs. Production of IFN-γ by SLE T cells in response to mitogens is diminished.28 (See Chapter 10.) A majority of SLE patients with high disease activity have shown increased expression of genes regulated by IFN in peripheral blood cells (“IFN signature”).29 In this group, IFN-α was found to be the major cytokine in SLE.30 Studies at the genetic level in both murine models and humans have confirmed an important role

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OVERVIEW OF THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

60

for IFN-α in the pathogenesis of SLE. IFN-α levels and activation of IFN-α pathway have been correlated with disease severity. High levels of IFN-α could augment the activity of antigen presenting cells and facilitate autoantibody production by aiding Ig class switching by B cells, or increase the rate of cellular apoptosis.30 Polymorphisms in the gene promoter and receptors have also been described for IL-4, but its role in SLE is not precisely clear. IL-6 is another cytokine that contributes to SLE pathogenesis predominantly through its action on B cells. Higher levels of serum IL-6 correlate with increased disease severity. B cells of SLE patients have been found to respond abnormally to IL-6 by virtue of spontaneous surface expression of IL-6 receptors.31 Blockade of IL-6 has been attempted in animal models with measurable success in terms of retardation of progression of the disease and holds promise for humans as well. Similarly, administration of antibodies against IL-10 also demonstrated retardation of the disease progression in murine models.5 Similar to IL-6, higher IL-10 levels also correlate with increased disease activity and is believed to influence the disease process via its effects on B-cell functions. Multiple studies have demonstrated an association of TNF-α gene polymorphisms with disease susceptibility. However, different ethnic groups display differential outcomes of these polymorphisms making it difficult to generalize their effects. Complicating the issue further is the observation in both human and animal models that have shown dual effects of TNF-α on SLE pathogenesis. Similarly, human studies have shown that administration of anti-TNF-α antibodies increases the titer of autoantibodies, and yet the patients display clinical improvement. Thus, further studies are awaited to precisely characterize the role of TNF-α in SLE. Diminished levels of TGF-β is also observed in SLE and may contribute to the autoimmune phenomenon via the resulting decrease in the number of Tregs that require TGF-β to differentiate from naive T cells. (See Chapter 13 for further discussion of immune abnormalities mediated by the abovementioned and other cytokines.)

have been discussed. Another defect inherent to SLE is defective clearance of immune complexes by phagocytes. Altered expression of at least two receptors could be responsible for this phenomenon: (1) allelic polymorphisms of FcgR gene that alter binding of FcγR to complexes containing different subtypes of IgG (discussed above), and (2) diminished expression of complement receptor (CR1) resulting from functional polymorphisms of CR1 gene that affect clearance of complexes containing C3 and C4 (reviewed in Mok and Lau5). In vitro studies have revealed abnormal stimulation and activation of B cells to produce IgG by T cells derived from SLE patients in the absence of extraneous agents such as antigens or mitogens, thus highlighting the importance of altered expression of co-stimulatory molecules on the surface of T cells. For example, CD40L that is expressed in high amounts on SLE T cells could provide co-stimulatory signals to B cells via CD40 expressed on its surface. Similarly, treatment of helper T cells with DNA methyltransferase inhibitors induces expression of high amounts of CD70, a co-stimulatory molecule that binds to CD27 expressed on B cells. These molecular defects could account for the autoantibody production, class switching, and somatic hypermutation displayed by B cells in SLE. Similarly, B cells are also involved in providing co-stimulation to activate T cells and dendritic cells (reviewed in Nagy et al.25). B cells in SLE also exhibit higher amounts of co-stimulatory molecules B7.1 and B7.2 that may further provide activation signals to the T cells. Polymorphisms of toll like receptor-9 (TLR9) have also been described in some populations. Autoimmune response against chromatin (CpG DNA) by B cells that occur independent of T cells has been shown to be mediated via cooperation between DNA containing immune complex–bound FcγRIIa and TLR9 that induce activation of plasmacytoid dendritic cells (pDC) (reviewed in Kyttaris et al.27). The role of BAFF–BAFF-R interaction is another mechanism by which T-cell– independent antibody response is elicited by B cells and has been considered in a previous section.

Consequence of Altered Expression of Cell Surface Receptors

Dendritic cells (DC) are powerful antigen-presenting cells that play a vital role in mediating peripheral tolerance. An immature DC binds both self as well as foreign antigens, but normally undergoes maturation to induce antigen-specific immunity only when it binds foreign antigens, thus acting as a check point for tolerance to self. Uncontrolled DC activation upon binding self-antigens can result in disruption of tolerance and development of autoimmunity. Several alterations to the DC homeostasis have been observed in SLE. A subset of DC bearing CD123 and negative for CD11c termed pDC is reduced in the

Since virtually all compartments of immune system function via ligation of cell surface receptors that participate in a multitude of functions such as antigen recognition, opsonization, complement fixation, signal transduction, and induction of apoptosis, in this chapter and in Chapter 14, we examine the pathology of SLE in terms of abnormalities in cell surface receptor expression/function. Possible alterations in antigen presentation/recognition as a result of abnormalities in the MHC molecules

Dendritic Cell Abnormalities

Environment

Genetic factors

IFN-α to induce maturation of monocytes to DC. The mature DC then captures autoantigens derived from apoptotic cells, undergo spurious maturation and present the autoantigens to self-reactive helper T cells. In this process, large numbers of plasma cells that are capable of producing autoantibodies are also perpetuated via activation of B cells by mature DC.32 The role of IFN-α is further considered in Chapters 12 and 13.

IMMUNOPATHOLOGY

peripheral blood of SLE patients, perhaps as a result of tissue migration. Instead, they are replaced by CD123-negative cells that possess the ability to secrete IFN-α. It has been shown that unlike normal peripheral blood monocytes that are inactive, monocytes derived from SLE patients function like dendritic cells. 32 A general model of DC-mediated pathogenesis of SLE favors viral-mediated activation of pDC, which in turn secretes large amounts of

Hormones

Critical threshold

Stochastic factors

Initiation of pathogenesis

Dendritic cell • IFN? secretion • T independent Ab production

Defective antigen presentation Defective priming

B cell Increased activity

T cell • Increased costimulation • Cytokine help Increased activity

Auto Ab production

• Abnormal complement fixation • Defective phagocyte activation • Circulating immune complexes

• Increased apoptosis • Defective AICD • Defective cytokine production

Tissue damage

Fig. 6.2 Pathogenesis of SLE. Environmental, hormonal, and stochastic factors act singly or in combination in genetically predisposed individuals to skew their disease liability to a critical threshold and initiate the pathogenesis of SLE. Abnormalities at the levels of antigen-presenting cells, B and T lymphocytes are observed. Production of autoantibodies and abnormal cytokine response by T cells result. The autoantigens are believed to be provided by massive apoptotic cells and debris that overwhelm the scavenging ability of phagocytic cells. Defective complement fixation also contributes to the pathogenesis. The circulating immune complexes and abnormal cytokines induce massive tissue inflammation, tissue destruction and eventually organ damage that are typically seen in SLE.

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OVERVIEW OF THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

Defects in Apoptosis Apoptosis or programmed cell death is an active, tightly regulated process normally used by the body to delete self-reactive lymphocytes. Another process of equal measure is the ability to rapidly clear apoptotic cells and fragments by phagocytic cells. Disruption of either of these processes can contribute to development of autoimmunity. For example, in SLE massive apoptotic rates of cells overwhelm the phagocytic cells and thereby possibly unmask cryptic potential autoantigens. Conversely, lower rates of apoptosis of autoreactive cells can also result in augmenting the autoimmune response. Gene knockout and transgenic mouse models have revealed the role of several molecules in disrupting either the apoptotic pathway or altering mechanisms involved in clearing the dead cells, cellular debris, and immune complexes. These include, among others, Fas/FasL, Bcl-2, members of complement cascade, and DNase.33 In SLE patients, circulating peripheral blood monocytes display increased rates of apoptosis, as do freshly isolated lymphocytes when cultured in vitro, and are believed to be a result of abnormal Fas-mediated signaling processes. The rate of apoptosis has also been shown to directly correlate with the disease activity. These abnormalities may stem from genetic polymorphisms or defects in the expression of Fas-FasL.34,35 Other mechanisms that have been shown to enhance apoptosis in peripheral blood lymphocytes in SLE are abnormally elevated mitochondrial transmembrane potential (Δψm) and increased baseline reactive oxygen intermediate (ROI).25 Interestingly, SLE T cells also display resistance to TCR-mediated AICD compared to normal T cells.

This defect might prolong survival of activated SLE T cells and contribute to increased activation of B cells to produce autoantibodies. Resistance to AICD is believed to be partly linked to diminished levels of TNF-α in T cells resulting from a gene polymorphism in SLE. The defective apoptotic process observed in SLE is a classic example of how both increased and decreased rates of apoptosis in different contexts can contribute to the similar autoimmune process. The overall pathogenesis of SLE is summarized in Fig. 6.2.

CONCLUSION Etiopathogenesis of SLE is highly complex and remains unclear. While genetic factors appear to be the strongest determinants of disease susceptibility, hormonal, environmental, and stochastic events also contribute toward triggering the onset of the disease process. Recent advances in the field of SLE biology have unraveled several mechanisms at the cellular and molecular levels that are involved in initiating inappropriate activation of the immune system and perpetuating the autoimmune process. However, considering the wide variation among individuals in the presentation of SLE, much work remains to be done before extrication and elucidation of the complex web of immune abnormalities would become possible, and a “molecular signature” be assigned to each patient to plan an individual-tailored therapeutic strategy. To take the bull by the horns requires a concerted effort of identification of biomarkers that can be used to design targeted molecular therapy. The advances in the fields of genetics and proteomics and the expected effective utilization of animal models are reassuring in this regard.

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8. Tsao BP. Update on human systemic lupus erythematosus genetics. Curr Opin Rheumatol 2004;16:513-521. 9. Nath SK, Kilpatrick J, Harley JB. Genetics of human systemic lupus erythematosus: the emerging picture. Curr Opin Immunol 2004;16:794-800. 10. Croker JA, Kimberly RP. Genetics of susceptibility and severity in systemic lupus erythematosus. Curr Opin Rheumatol 2005;17:529-537. 11. Shen N, Tsao BP. Current advances in the human lupus genetics. Curr Rheumatol Rep 2004; 6:391-398. 12. Lang TJ. Estrogen as an immunomodulator. Clin Immunol 2004;113:224-230. 13. Tsokos GC, Nambiar MP, Tenbrock K, Juang YT. Rewiring the T-cell: signaling defects and novel prospects for the treatment of SLE. Trends Immunol 2003;24:259-263. 14. Vratsanos GS, Jung S, Park YM, Craft J. CD4(+) T cells from lupusprone mice are hyperresponsive to T cell receptor engagement with low and high affinity peptide antigens: a model to explain spontaneous T cell activation in lupus. J Exp Med 2001;193: 329-337.

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15. Enyedy EJ, Nambiar MP, Liossis SN, Dennis G, Kammer GM, Tsokos GC. Fc epsilon receptor type I gamma chain replaces the deficient T cell receptor zeta chain in T cells of patients with systemic lupus erythematosus. Arthritis Rheum 2001;44:1114-1121. 16. Krishnan S, Nambiar MP, Warke VG, Fisher CU, Mitchell J, Delaney N, et al. Alterations in lipid raft composition and dynamics contribute to abnormal T cell responses in systemic lupus erythematosus. J Immunol 2004;172:7821-7831. 17. Juang YT, Tenbrock K, Nambiar MP, Gourley MF, Tsokos GC. Defective production of functional 98-kDa form of Elf-1 is responsible for the decreased expression of TCR zeta-chain in patients with systemic lupus erythematosus. J Immunol 2002;169:6048-6055. 18. Chowdhury B, Tsokos CG, Krishnan S, Robertson J, Fisher CU, Warke RG, et al. Decreased stability and translation of T cell receptor zeta mRNA with an alternatively spliced 3’-untranslated region contribute to zeta chain down-regulation in patients with systemic lupus erythematosus. J Biol Chem 2005;280:18959-18966. 19. Krishnan S, Kiang JG, Fisher CU, Nambiar MP, Nguyen HT, Kyttaris VC, et al. Increased caspase-3 expression and activity contribute to reduced CD3zeta expression in systemic lupus erythematosus T cells. J Immunol 2005;175:3417-3423. 20. Nambiar MP, Fisher CU, Kumar A, Tsokos CG, Warke VG, Tsokos GC. Forced expression of the Fc receptor gamma-chain renders human T cells hyperresponsive to TCR/CD3 stimulation. J Immunol 2003;170:2871-2876. 21. Solomou EE, Juang YT, Gourley MF, Kammer GM, Tsokos GC. Molecular basis of deficient IL-2 production in T cells from patients with systemic lupus erythematosus. J Immunol 2001;166:4216-4222. 22. Tenbrock K, Juang YT, Gourley MF, Nambiar MP, Tsokos GC. Antisense cyclic adenosine 5’-monophosphate response element modulator up-regulates IL-2 in T cells from patients with systemic lupus erythematosus. J Immunol 2002;169:4147-4152. 23. Juang YT, Wang Y, Solomou EE, Li Y, Mawrin C, Tenbrock K, et al. Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV. J Clin Invest 2005;115:996-1005. 24. Katsiari CG, Kyttaris VC, Juang YT, Tsokos GC. Protein phosphatase 2A is a negative regulator of IL-2 production in patients

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PATHOGENESIS

7

The Environment in the Pathogenesis of Systemic Lupus Erythematosus Amr H. Sawalha, MD and Bruce C. Richardson, MD, PhD

INTRODUCTION Human lupus is a chronic relapsing autoimmune disease characterized by autoantibodies to a host of cellular components and immune complex deposition in the kidney and other tissues. Although the etiology of lupus is unknown, both genetic and environmental factors are implicated in disease pathogenesis. Evidence for a genetic contribution comes from familial aggregation of autoimmunity in approximately 20% of lupus cases,1 a higher concordance rate in monozygotic (~25%) relative to dizygotic twins (2%),2 the evidence for linkage at multiple loci across the human genome as shown by genome-wide scans of subjects with familial lupus,3 and the discovery of various susceptibility genes for the disease.4 The observations that the majority of lupus is sporadic and that drugs such as procainamide, hydralazine, and others (as well as UV light) trigger lupus-like autoimmunity, and the lack of complete concordance in identical twins, indicate a prominent role for exogenous agents.5 How environmental agents interact with the various genetic loci to produce autoantibodies, the hallmark of lupus, is incompletely understood. Current models postulate that autoimmunity develops in genetically susceptible hosts exposed to appropriate environmental triggering factors. However, the nature of the interactions among various genetic elements and the environment is yet to be revealed. Herein, we discuss selected environmental factors associated with systemic lupus erythematosus (SLE), and review the evidence for their pathogenic role in the disease. The environmental factors discussed include drugs, UV light exposure, infectious agents, chemicals, heavy metals, and dietary factors.

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64

Drug-induced lupus provides the clearest example of an exogenous agent causing lupus-like autoimmunity. In the majority of cases, withholding the offending medication results in the disappearance of autoantibodies and resolution of disease signs and

symptoms, and cautious readministration causes disease recurrence,6 supporting cause and effect. More than 100 drugs have been implicated in causing lupus and lupus-like syndromes. Whereas some of the reports are anecdotal, others (such as hydralazine and procainamide) are well established by clinical studies.5 A recent controlled case study utilizing the General Practice Research Database in the United Kingdom has now estimated the relative risk for developing SLE in 1437 patients exposed to 13 of the known lupus-inducing drugs and 6963 matched controls. This study found supportive results for the majority, 7 providing additional epidemiological evidence for an association between specific drugs and lupus-like diseases (see Table 7.1). Because the inciting agent is known, drug-induced lupus provides a unique opportunity to identify disease mechanisms potentially relevant to idiopathic lupus. Mechanistic studies have often focused on hydralazine and procainamide, recognized as causing lupus-like autoimmunity since 1953 and 1962, respectively.8 Recent studies demonstrate that these drugs modify epigenetic mechanisms regulating T lymphocyte gene expression, with important implications for the pathogenesis of both drug-induced and idiopathic lupus. Common to both forms of lupus is dysregulation of DNA methylation, an epigenetic mechanism regulating gene expression.

DNA Methylation in Drug-Induced Lupus DNA Methylation DNA methylation in vertebrates refers to the postsynthetic methylation of cytosine bases in DNA to form methylcytosine. Methylcytosine is usually found in CG pairs, and most CG pairs in the mammalian genome are methylated. The exceptions are those in promoter sequences of transcriptionally active genes. Promoters of most active genes are hypomethylated, and methylation of the promoter sequences renders the genes transcriptionally inactive.9 DNA methylation suppresses gene expression by several mechanisms.

Medication odds ratio

95% Confidence interval

Acebutolol

8.2

1.4–46.7

Captopril

2.4

1.5–4.1

Carbamazepine

3.4

2.3–4.8

Chlorpromazine

2.7

1.4–5.2

Hydralazine

6.7

1.9–23.6

Isoniazide

20

2.2–178.9

Methyldopa

NA

Minocycline

4.3

3.1–6.0

Penicillamine

29.6

6.6–132.1

Procainamide

NA

Propylthiouracil

NA

Quinidine

5.0

0.7–35.5

Sulfasalazine

39.9

17.1–93.2

a. Odds ratio for developing lupus upon exposure is indicated where available.7 NA: Not available or the study was underpowered to detect any difference.

These include inhibiting binding of some transcription factors10,11 and attracting methylcytosine binding proteins that sterically inhibit transcription factor binding, as well as tether chromatin inactivation complexes to methylated sequences. The chromatin inactivation complexes promote localized chromatin condensation into an inactive configuration,12 and may be the most transcriptionally important mechanism. Methylation patterns are established during differentiation and serve to suppress genes unnecessary or detrimental to the function of each specific cell type. DNA methylation is also involved in female X chromosome inactivation, genomic imprinting, and suppressing parasitic DNA. The importance of DNA methylation is evidenced by studies demonstrating that homozygous deficiency of any of the methyltransferases results in death during embryogenesis or in the early postnatal period, and that changes in established methylation patterns causes changes in gene expression that contribute to aging and cancer.13 De novo methylation of previously unmethylated DNA, such as occurs during differentiation, is mediated by DNA methyltransferases Dnmt3a and Dnmt3b. Methylation patterns are then replicated during mitosis by the maintenance methyltransferase Dnmt1. Dnmt1 binds proliferating cell nuclear antigen (PCNA) at the replication fork, recognizing and methylating hemimethylated DNA that results from a methylated

parent strand and unmethylated daughter strand, while ignoring unmethylated DNA, thereby replicating methylation patterns.13 Dnmt1 expression is linked to the cell cycle, and is regulated in part via signaling through the extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) pathways.14,15

DRUGS

TABLE 7.1 DRUGS MOST COMMONLY IMPLICATED IN DRUG-INDUCED LUPUSa

Inducing DNA Demethylation DNA can be demethylated using DNA methyltransferase inhibitors such as 5-azacytidine. 5-azacytidine is a cytosine analogue that is incorporated into DNA during S phase, where it covalently binds DNA methyltransferases during maintenance DNA methylation.16 This depletes cellular DNA methyltransferases with subsequent genome-wide hypomethylation of the newly synthesized DNA and activation of those genes repressed by methylation and for which the cell expresses the necessary transcription factors.

Effects of 5-azacytidine on T-cell Function and Gene Expression 5-azacytidine has been used to characterize how DNA methylation regulates T-cell function. Treating human and murine cloned and polyclonal antigen-specific CD4+ T-cells with 5-azacytidine causes loss of the requirement for nominal antigen and the ability to respond to self class II MHC determinants without added antigen. The autoreactive cells also acquire the ability to promiscuously kill autologous or syngeneic macrophages and overstimulate B-cell antibody production.17 This autoreactive response resembles the response of semiallogeneic CD4 T-cells to host class II determinants in chronic graft-versus-host disease, which causes an autoimmune disease with features of lupus in mice.18 The similarities between 5-azacytidine-induced CD4+ T-cell autoreactivity and CD4+ alloreactive responses to class II MHC molecules is further supported by studies demonstrating that 5-azacytidine-treated CD4+ T-cells cause a lupus-like disease in syngeneic recipients. The mice develop autoantibodies to singleand double-stranded DNA, a lupus band test and nephritis, resembling chronic graft-versus-host disease.19 T-cell genes affected have been identified with mechanism-focused approaches and more recently via oligonucleotide arrays. Genes modified by 5-azacytidine and relevant to autoimmunity include CD11a (LFA-1a), perforin, CD70, and INF-γ,17,20 among others. CD11a is a subunit of LFA-1 (CD11a/CD18), an adhesion molecule expressed on most hematopoetic cells. 5-azacytidine demethylates a series of alu repeats 5′ to the CD11a promoter, increasing transcription.21 CD11a demethylation participates in the autoreactivity of the treated cells through increased LFA-1 expression. Increasing LFA-1 expression by transfection of human or murine CD4+ T-cells causes the same loss of restriction to

65

THE ENVIRONMENT IN THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

nominal antigen and responses to self class II MHC molecules without specific antigen (as seen following treatment with DNA methylation inhibitors).21 The autoreactivity may be due in part to overstabilization of the low-affinity interaction between the T-cell receptor and self class II MHC determinants without specific antigen, thereby decreasing the threshold for activation.23 Murine CD4+ T-cells made autoreactive by LFA-1 transfection cause a lupus-like disease in vivo resembling the autoimmunity caused by injecting demethylated LFA-1 overexpressing cells,24 further supporting the importance of autoreactivity caused by this mechanism (see Fig. 7.1). Demethylated autoreactive CD4+ T-cells also acquire the ability to kill autologous or syngeneic macrophages. Aberrant perforin expression contributes to this autoreactive cytotoxic response. Perforin is a cytotoxic molecule normally expressed by NK cells and cytotoxic CD8+ T-cells. Demethylating a conserved region linking the perforin promoter to an upstream enhancer induces perforin expression in CD4+ T-cells and increases perforin expression in CD8+ T-cells.25 Concanamycin, a perforin inhibitor, decreases autoreactive macrophage killing by hypomethylated CD4+ cells, suggesting that perforin contributes to this cytotoxic response.26 The macrophage killing may contribute to autoantibody formation in the demethylation model by depleting scavenger cells responsible for clearing apoptotic debris while simultaneously increasing the release of apoptotic material, both of which cause lupus-like autoimmunity in animal models.27,28 CD70 and IFN-γ overexpression in hypomethylated CD4+ T-cells may contribute to autoimmunity by increasing antibody production. CD70 is expressed on stimulated T-cells, and interacts with CD27 on Bcells to promote immunoglobulin synthesis and secretion. 5-azacytidine increases CD70 by demethylating a region just upstream of the active promoter.29 CD70 overexpression on CD4+ T-cells treated with 5-azacytidine causes B-cell overstimulation and increased IgG

CD41

APC

secretion30 resembling the polyclonal B-cell activation seen in the peripheral blood B-cells of patients with lupus.31 In addition, IFN-γ is suppressed in Th2 cells by promoter methylation and is demethylated with 5-azacytidine, inducing expression.32 IFN-γ similarly stimulates B-cell immunoglobulin secretion, and lupus B-cells secrete significantly greater amounts of IgG in response to IFN-γ than controls.33 The effects of DNA hypomethylation on T lymphocyte function and gene expression are summarized in Fig. 7.2. Finally, 5-azacytidine can reactivate endogenous retrovirus expression in T-cells34 and Epstein-Barr virus proteins as well as the Epstein-Barr viral lytic cycle in B-cells.35 EBV and endogenous retroviral genes are normally suppressed by DNA methylation, and their reactivation has been associated with the development of lupus-like autoimmunity.34,36 The possible role of EBV and retrovirus expression in lupus is discussed further in material following and elsewhere in this textbook.

DNA Demethylation by Lupus-Inducing Drugs The observation that inhibiting DNA methylation causes lupus-like autoimmunity suggests that drugs causing lupus-like autoimmunity might inhibit DNA methylation. Indeed, when CD4+ T-cells are treated during S phase with either hydralazine or procainamide the cells become autoreactive, similar to what is seen with 5-azacytidine.37 Low concentrations (10−7 M) of hydralazine and procainamide induce autoreactivity, which increases in a dose-dependent fashion. Interestingly, the concentrations of hydralazine and procainamide causing autoreactivity fall in the therapeutic concentrations of these drugs and are similar to the concentrations that induce lupus. Hydralazine and procainamide also increase LFA-1 and CD70 expression, similar to 5-azacytidine.30,38 Consistent with the LFA-1 overexpression and autoreactivity, hydralazine and procainamide also inhibit methylation of newly synthesized DNA in human T-cells.37 Procainamide is a competitive T-cell

CD41 DNA methylation inhibition, LFA-1 transfection

APC

66

CD41

Autoreactivity

CD41

LFA-1 overexpression

Fig. 7.1 The effect of DNA methylation inhibition on CD4+ cells. CD4+ T-cells treated with DNA methylation inhibitors overexpress LFA-1 and become autoreactive, similar to cells transfected with LFA-1.

Decreased ERK pathway signaling (Lupus, hydralazine, UV light)

DRUGS

Fig. 7.2 Hypothetical schema uniting proposed mechanisms by which drugs, viruses, and UV light may contribute to lupus-like autoimmunity.

DNA methyltransferase inhibitors (5-azacytidine, procainamide)

Decreased Dnmt1 activity

DNA hypomethylation

Increased expression of B cell EBV genes

Increased expression of T cell:

CD11a

Perforin

T cell autoreactivity

Cytotoxicity

CD70

IFN-γ

Increased B cell help

Mø killing

Increased apoptosis and decreased clearance of apoptotic debris

DNA methyltransferase inhibitor.39 In contrast, hydralazine inhibits the extracellular signal-regulated kinase (ERK) pathway (thereby decreasing DNA methyltransferase expression) but does not directly inhibit DNA methyltransferase enzyme activity.40 DNA methyltransferase 1 and 3a levels are also decreased in murine T-cells treated with the ERK pathway inhibitor U0126, and the treated cells have hypomethylated DNA (similar to human T-cells treated with hydralazine).40 To determine if inhibiting the ERK pathway in T-cells causes autoreactivity, D10 cells, (cloned conalbumin reactive Th2 line from AKR mice) were treated with the MEK inhibitor U1026 or hydralazine. U1026- and hydralazine-treated cells overexpressed LFA-1 and responded to syngeneic antigen-presenting cells in the absence of the antigen, thereby resembling T-cells treated with 5-azacytidine (Fig. 7.1). In vivo effects of cells made autoreactive with procainamide and hydralazine have been similarly determined in murine models. When polyclonal CD4+ T-cells from DBA/2 mice are treated with 5-azacytidine or procainamide and then injected into unirradiated syngeneic female mice, the mice develop anti-DNA and antihistone antibodies, a positive lupus band test, and an

Lupus autoantibodies

Keratinocyte apoptosis

UV light

immune complex glomerulonephritis.19 This experiment has been repeated using D10 cells treated with 5-azacytidine. The treated cells overexpress LFA-1, become autoreactive, and produce large amounts of IL-6.41 Adoptive transfer of the treated cells into female AKR mice induces autoantibodies directed against single-stranded and double-stranded DNA as well as antihistone antibodies. Histologic examination reveals that these mice develop immune complex glomerulonephritis, pulmonary alveolitis, central nervous system pathologies (including fibrinoid necrosis, karyorrhexis, and meningitis), and bile duct proliferation with periportal inflammation similar to primary biliary cirrhosis.41 The same model has been used to demonstrate that procainamide is more potent than N-acetylprocainamide in inducing LFA-1 overexpression and autoreactivity in vitro and autoimmunity in vivo, and that hydralazine is more potent than phthalazine (the parent compound) in the same assays.38 D10 cells treated with the ERK pathway inhibitor U0126 or hydralazine and then adoptively transferred into nonirradiated syngeneic female mice, also induce the production of anti-double-stranded DNA antibodies, confirming that ERK pathway inhibition can induce autoimmunity.40

67

THE ENVIRONMENT IN THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

Together, these studies indicate that T-cells demethylated by treatment with either hydralazine or procainamide are sufficient to induce a lupus-like disease in vivo. This suggests a mechanism that might contribute to the development of some forms of drug-induced lupus.

DNA Methylation in Idiopathic Lupus: What We Learned from Drug-Induced Lupus The studies on DNA methylation and drug-induced lupus prompted confirming studies in patients with idiopathic SLE. The results suggest that idiopathic lupus may be caused by mechanisms very similar to those by which procainamide and hydralazine cause lupus-like autoimmunity. Early studies demonstrated that T-cells from lupus patients have a genome-wide reduction in methylated DNA and decreased DNA methyltransferase enzymatic activity.42 Subsequent studies revealed that lupus T-cells have decreased levels of Dnmt1 transcripts, due to defective ERK pathway signaling, similar to hydralazine-treated T-cells.14 Examination of the previously identified methylationsensitive genes revealed that CD4+ lupus T-cells aberrantly overexpress CD11a, CD70, and perforin (similar to experimentally demethylated T-cells),26,30,43 that LFA-1 and perforin overexpression contribute to the spontaneous macrophage killing characteristic of lupus T-cells,26 and that CD70 overexpression contributes to overstimulation of autologous B-cell IgG production.30 Moreover, the same CD11a, CD70, and perforin promoter sequences demethylated by methylation inhibitors are demethylated in lupus T-cells.21,25,29 Together, these studies suggest that a yet unidentified environmental factor may contribute to the pathogenesis of idiopathic lupus through effects on DNA methylation similar to hydralazine or procainamide in drug-induced lupus. This unidentified environmental element may interfere with ERK signaling and DNA methylation in T-cells, resulting in autoimmunity. Host genetics may determine the outcome of the interaction between the host and the environment, and thus the likelihood that this interaction will result in an autoimmune phenotype in any given individual. It is also possible that genetic factors may influence T-cell signaling pathways.

Ultraviolet Light Exposure

68

Sun exposure triggers lupus flares. In one report, ultraviolet light (both UVA and UVB) induced skin lesions clinically and histologically compatible with lupus erythematosus in a significant number of 128 patients with either cutaneous or systemic lupus erythematosus and who underwent phototesting.44 Furthermore, the use of sun screens that efficiently protect against both UVA and UVB were very effective

in protecting against UV-induced lupus erythematosus.45 In addition, UV light exposure in a tanning device was linked to the development of lupus in a previously healthy patient.46 Finally, disease flares are least common in January among lupus patients living north of the artic circle.47 Lupus-prone mouse strains demonstrate increased susceptibility to DNA damage by UVA.48 Moreover, in NZB/W F1 mice with high titers of circulating antinuclear and anti-DNA antibodies whole-body UV irradiation results in in vivo binding of ANA to epidermal cell nuclei and induced circulating DNA/anti-DNA immune complexes.49 In lupus-prone BXSB male mice, UV exposure results in a significant increase in serum single-stranded DNA autoantibody production, splenic polyclonal B-cell activity, and glomerular inflammatory changes. In addition to the accelerated autoimmunity observed, UV exposure results in premature death in BXSB males.50

UV Light and DNA Methylation Although the mechanism by which UV light induces or exacerbates autoimmunity is not entirely clear, some evidence indicates that DNA demethylation (due to decreased ERK pathway signaling) may be involved by mechanisms similar to those found in idiopathic and hydralazine-induced lupus. Early work demonstrated that UV light is a potent DNA methylation inhibitor.51 Subsequent studies demonstrated that relatively small amounts (~30 Joules/m2) of UVB-enriched light demethylates CD4+ T-cell DNA, causes LFA-1 overexpression, and induces autoreactivity.52 A recent study has now shown that the same amount of UV light inhibits TCR-mediated ERK pathway signaling,53 with effects identical to those seen in hydralazine treated T-cells and in idiopathic lupus.40 This provides an attractive common mechanism for drug-induced, UV-light-induced, and idiopathic lupus, although UV-treated T-cells have yet to be shown to cause lupus-like autoimmunity in adoptive transfer studies (as has been done for procainamide and hydralazine).

Apoptosis A large body of literature suggests that UV-induced keratinocyte apoptosis could contribute to the development of lupus-like autoantibodies. UVB irradiation of cultured keratinocytes induces an enrichment of lupus autoantigens such as Ro, La, snRNP, and Sm in apoptotic blebs on the cell surface.54 Patients with SLE have a high proportion of apoptotic cells, and the expression of autoantigens on the surface within apoptotic blebs may be an important step in inducing autoimmunity in this disease.55 This hypothesis is supported by recent evidence demonstrating that dendritic cells,

Infections Epstein-Barr Virus and Lupus A number of infectious agents have been proposed as contributing to the pathogenesis of human SLE, with varying degrees of experimental support for an etiologic role. Of these, the association between EpsteinBarr virus infection and SLE is perhaps the best studied at this point, and is discussed in detail elsewhere in this text. In brief, lupus patients have serologic evidence of early Epstein-Barr viral infection,57,58 and there is evidence for antigenic cross-reactivity between Epstein-Barr nuclear antigen-1 (EBNA-1) and the Sm B/B′ component of the spliceosome59 (as well as an autoantigenic epitope on 60 Kd Ro).60,61 Immunization with the determinants in Freund’s adjuvant can be shown to lead to epitope spreading,60,61 suggesting a role for antigenic mimicry in the 30 to 40% of lupus patients with anti-Sm and anti-Ro antibodies.62 Interestingly, lupus patients have an EBV viral load ~40 times greater than controls that is not explained by disease activity or immunosuppressive agents.63 Although altered T-cell responses to EBV may contribute,63 defective DNA methylation may also lead to reactivation of the EBV genome.64 Perhaps increased expression of EBV proteins, together with increased accumulation of apoptotic debris and excessive B-cell help, breaks tolerance and thus permits generation of autoantibodies. This is illustrated in Fig. 7.2.

H. pylori and Lupus Also of interest is a recent report suggesting a lack of association between H. pylori infection and lupus in a group of African-American lupus patients. Of the 113 African-American female lupus patients in that study group, 43 were seropositive for H. pylori. Female African-American patients with lupus had a lower prevalence of H. pylori seropositivity compared to controls (38.1% versus 60.2%, odds ratio = 0.41, p = 0.0009, 95% CI 0.24-0.69).66 A difference in therapeutic experience did not explain the increased seronegative frequency for H. pylori observed in African-American female patients with SLE. Furthermore, the mean age of onset for lupus was older in the seropositive group (34.4 years) compared to the seronegative SLE patients (28.0 years) (t = 2.11, p = 0.039).66

DRUGS

important in initiating antinucleosomal antibody responses, preferentially phagocytose the apoptotic blebs through “eat me” signals as well as through FcγRmediated mechanisms.56 The mechanisms by which procainamide, hydralazine, and UV light may inhibit DNA methylation and contribute to autoimmunity are summarized in Fig. 7.2.

Chemicals and Heavy Metals A variety of chemical factors and heavy metals have been reported in association with SLE.65,67 Of these, the epidemiologic data supporting an association is most compelling for crystalline silica and mercury (summarized in material following; see also Table 7.2).

Crystalline Silica Exposure to silica has been associated with a number of autoimmune diseases, including lupus, Sjogren’s syndrome, scleroderma, rheumatoid arthritis, vasculitis, and undifferentiated connective tissue disorders.68,69 High-level occupational exposure to crystalline silica has been reported in construction (masonry, heavy construction, and painting), iron and steel foundries (casting), and in metal services (sandblasting, grinding, or buffing of metal parts).70 In a population-based study designed to examine the association between occupational silica exposure and SLE in the southeastern United States, 265 lupus patients

Retroviruses and Lupus Increased expression of endogenous retroviral genes has also been reported in lupus patients and is potentially implicated in lupus pathogenesis. Endogenous retroviruses encode a number of proteins, some of which have direct effects on immunocytes. Others demonstrate molecular mimicry between the viral proteins and autoantigens (reviewed in Perl34). Because retroviruses are also suppressed by DNA methylation, authors have proposed that DNA demethylation in lupus may explain reactivation of these latent elements, with the potential of contributing to disease pathogenesis through mechanisms similar to EBV.34 However, as for many proposed agents conclusive evidence for a direct linkage between human lupus and endogenous or exogenous retroviruses is lacking.65

TABLE 7.2 CHEMICALS AND HEAVY METALS REPORTED TO BE ASSOCIATED WITH AUTOIMMUNITY65,67 Gold Mercury Cadmium Pristane Vinyl chloride Industrial solvents Silica and silicone Pesticides Hydrazines Hair dyes

69

THE ENVIRONMENT IN THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

70

were compared to 355 matched controls.71 Silica exposure was determined through blinded assessment of job histories by three industrial hygienists. The lupus patients had a higher exposure to silica than their matched controls (19% versus 8%) (medium silica exposure odds ratio 2.1 [95% CI 1.1-4.0], high silica exposure odds ratio 4.6 [95% CI 1.4-15.4]).71 The mechanism by which silica induces autoimmunity is unclear. Studies in model systems suggest that silica may act as a nonspecific adjuvant that enhances immune responses.72 Furthermore, silica may induce apoptosis,73 potentially resulting in autoantigen exposure through apoptotic blebs and consequent autoantibody production. In addition, silica can induce the production of proinflammatory cytokines such as TNF-γ and IL-1.74

no evidence that permanent hair dye use, age at first use, frequency of use, or duration of use is associated with the development of SLE.85 It thus seems unlikely that hair dyes contribute to the pathogenesis of human lupus.

Mercury

Dietary Factors

Exposure to mercury has long been recognized as a public health concern, due to known toxic effects on the skin, kidneys, lungs, and nervous system.75 Sources for mercury exposure come from fish consumption, dental amalgams, vaccines, thermometers, and a variety of electrical products. Gold miners are also at increased risk for toxic exposure. A recent epidemiologic study of 265 lupus patients and 355 controls revealed an association between self-reported occupational mercury exposure and human lupus (odds ratio = 3.6, 95% CI 1.3-10.0).76 The immunotoxic effects of mercury have been studied in animal models. Animals treated with mercury develop autoantibodies to fibrillarin and immune-complex glomerulonephritis.77 Of interest, the level of mercury exposure that induces autoimmunity in animals falls within the mercury levels detected in human occupational exposure.78 In addition, mercury has been shown to accelerate autoimmunity in lupus-prone animal models.79,80 The susceptibility to mercury-induced autoimmunity in animal models appears to be genetically determined.81,82 Furthermore, studies have shown that female mice are more susceptible to the development of autoantibodies following mercury exposure.81

Ingestion of the amino acid L-canavanine has been associated with a lupus-like disease in both animals and humans. Monkeys fed alfalfa seeds develop antinuclear antibodies, anti-dsDNA antibodies, low complement levels, hemolytic anemia, deposition of complement and immunoglobulin in the skin, and immune-complex deposition in the kidneys.89 Further studies reveal that L-canavanine, which is present in high levels in alfalfa seeds, is responsible for the autoimmune phenotype observed.90 In humans, exacerbation of lupus activity with alfalfa ingestion has also been reported.91 Moreover, it has been demonstrated that L-canavanine has dose-related effects in vitro on human immunoregulatory cells, which could explain the induction or exacerbation of SLE by alfalfa.92,93 These effects include decreased mitogenic response to both phytohemagglutinin and concanavalin A and abrogation of concanavalin A-induced suppressor T cell function.92 However, the association between alfalfa consumption and lupus in humans remains controversial. In a Swedish case-control study of 85 female lupus patients compared to 205 controls, the proportion of women who reported consumption of alfalfa sprouts was similar among the two groups.94

Hair Dyes

CONCLUSIONS

Despite initial reports of an association between lupus and the use of hair dyes,83 subsequent studies failed to confirm such an association.84-87 In a case-control study, Petri and Allbritton reported no significant difference in exposure to hair dye in 218 lupus patients studied before the diagnosis of SLE as compared to controls.84 Furthermore, there was no difference in lupus disease activity among patients who used hair products after their diagnosis as compared to those who did not.84 In a subsequent report of 106,391 women enrolled in a prospective cohort study, Sanchez-Guerrero and colleagues reported

Other Agents A number of other chemical factors have been reported in association with SLE, such as pesticides and chemical solvents. However, the association of these factors with the disease remains largely controversial, and unconfirmed. Similarly, the reported association of SLE with vaccination and smoking needs further evaluation.88 Finally, persuasive epidemiologic studies supporting a role for hydrazines, aromatic amines, and environmental endocrine disrupters are lacking.65

Alfalfa

In summary, a number of exogenous factors including drugs, sunlight, pesticides, xenobiotics (including silica, solvents, heavy metals and hydrazines), diet, and infectious agents have been proposed as lupus-inducing agents. Of these, drug-induced lupus provides perhaps the clearest example of exogenous agents triggering lupus, with persuasive data supporting etiologic roles for drugs such as procainamide and hydralazine through effects on DNA methylation resembling those occurring in idiopathic lupus.

which these agents could induce lupus are unclear. Persuasive epidemiologic studies supporting a role for hydrazines, aromatic amines, and environmental endocrine disrupters are lacking. Finally, the dietary compound canavanine has been implicated in animal studies, but confirmation in humans is lacking.

REFERENCES

Epidemiologic data supports an association with minocycline and with some of the biologics (such as TNF antagonists and interferons), although the mechanisms involved are less clear. DNA demethylation with resultant effects on endogenous gene expression, as well as the expression of latent EBV genes, may also play a role. UV light is an accepted lupus trigger, and although the mechanisms involved are unclear, increased apoptosis and DNA demethylation through signaling inhibition similar to hydralazine and idiopathic lupus are possible. Epidemiologic studies support roles for silica and mercury exposure, but further confirmation is desirable and the mechanisms by

ACKNOWLEDGMENTS This chapter was made possible by NIH Grant Number P20-RR015577 from the National Center for Research Resources, PHS grant AR42525, and a Merit grant from the Department of Veterans Affairs.

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inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice. J Clin Invest 1993;92:38-53. Sawalha AH, Richardson BC. DNA methylation in the pathogenesis of systemic lupus erythematosus. Current Pharmacogenomics 2005;3:73-78. Lu Q, Kaplan M, Ray D, et al. Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus. Arthritis Rheum 2002;46:1282-1291. Richardson B. Effect of an inhibitor of DNA methylation on T cells. II. 5-azacytidine induces self-reactivity in antigen-specific T4+ cells. Human Immunol 1986;17:456. Kaplan MJ, Beretta L, Yung RL, et al. LFA-1 overexpression and T cell autoreactivity: mechanisms. Immunol Invest 2000;29:427-442. Yung R, Powers D, Johnson K, et al. Mechanisms of druginduced lupus. II. T cells overexpressing lymphocyte functionassociated antigen 1 become autoreactive and cause a lupuslike disease in syngeneic mice. J Clin Invest 1996;97:2866-2871. Lu Q, Wu A, Ray D, et al. DNA methylation and chromatin structure regulate T cell perforin gene expression. J Immunol 2003;170:5124-5132. Kaplan MJ, Lu Q, Wu A, et al. Demethylation of promoter regulatory elements contributes to perforin overexpression in CD4+ lupus T cells. J Immunol 2004;172:3652-3661. Mevorach D, Zhou JL, Song X, et al. Systemic exposure to irradiated apoptotic cells induces autoantibody production. J Exp Med 1998;188:387-392. Walport MJ. Lupus, DNase and defective disposal of cellular debris. Nat Genet 2000;25:135-136. Lu Q, Wu A, Richardson BC. Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs. J Immunol 2005;174:6212-6219. Oelke K, Lu Q, Richardson D, et al. Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors. Arthritis Rheum 2004;50:1850-1860. Fauci AS, Moutsopoulos HM. Polyclonally triggered B cells in the peripheral blood and bone marrow of normal individuals and in patients with systemic lupus erythematosus and primary Sjogren’s syndrome. Arthritis Rheum 1981;24:577-583. Young HA. Regulation of interferon-gamma gene expression. J Interferon Cytokine Res 1996;16:563-568. Golbus J, Salata M, Greenwood J, et al. Increased immunoglobulin response to gamma-interferon by lymphocytes from patients with systemic lupus erythematosus. Clin Immunol Immunopathol 1988;46:129-140. Perl A. Role of endogenous retroviruses in autoimmune diseases. Rheum Dis Clin North Am 2003;29:123-143. Tao Q, Robertson KD. Stealth technology: how Epstein-Barr virus utilizes DNA methylation to cloak itself from immune detection. Clin Immunol 2003;109:53-63.

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36. Kang I, Quan T, Nolasco H, et al. Defective control of latent Epstein-Barr virus infection in systemic lupus erythematosus. J Immunol 2004;172:1287-1294. 37. Cornacchia E, Golbus J, Maybaum J, et al. Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity. J Immunol 1988;140:2197-2200. 38. Yung R, Chang S, Hemati N, et al. Mechanisms of drug-induced lupus. IV. Comparison of procainamide and hydralazine with analogs in vitro and in vivo. Arthritis Rheum 1997;40:1436-1443. 39. Scheinbart LS, Johnson MA, Gross LA, et al. Procainamide inhibits DNA methyltransferase in a human T cell line. J Rheumatol 1991;18:530-534. 40. Deng C, Lu Q, Zhang Z, et al. Hydralazine may induce autoimmunity by inhibiting extracellular signal-regulated kinase pathway signaling. Arthritis Rheum 2003;48:746-756. 41. Yung RL, Quddus J, Chrisp CE, et al. Mechanism of drug-induced lupus. I. Cloned Th2 cells modified with DNA methylation inhibitors in vitro cause autoimmunity in vivo. J Immunol 1995;154:3025-3035. 42. Richardson B, Scheinbart L, Strahler J, et al. Evidence for impaired T cell DNA methylation in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Rheum 1990;33:1665-1673. 43. Richardson BC, Strahler JR, Pivirotto TS, et al. Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus. Arthritis Rheum 1992;35:647-662. 44. Lehmann P, Holzle E, Kind P, et al. Experimental reproduction of skin lesions in lupus erythematosus by UVA and UVB radiation. J Am Acad Dermatol 1990;22:181-187. 45. Herzinger T, Plewig G, Rocken M. Use of sunscreens to protect against ultraviolet-induced lupus erythematosus. Arthritis Rheum 2004;50:3045-3046. 46. Fruchter O, Edoute Y. First presentation of systemic lupus erythematosus following ultraviolet radiation exposure in an artificial tanning device. Rheumatology (Oxford) 2005;44:558-559. 47. Haga HJ, Brun JG, Rekvig OP, et al. Seasonal variations in activity of systemic lupus erythematosus in a subarctic region. Lupus 1999;8:269-273. 48. Golan DT, Borel Y. Increased photosensitivity to near-ultraviolet light in murine SLE. J Immunol 1984;132:705-710. 49. Natali PG, Mottolese M, Nicotra M. Immune complex formation in NZB/W mice after ultraviolet radiation. Clin Immunol Immunopathol 1978;10:414-419. 50. Ansel JC, Mountz J, Steinberg AD, et al. Effects of UV radiation on autoimmune strains of mice: increased mortality and accelerated autoimmunity in BXSB mice. J Invest Dermatol 1985;85:181-186. 51. Lieberman MW, Beach LR, Palmiter RD. Ultraviolet radiationinduced metallothionein-I gene activation is associated with extensive DNA demethylation. Cell 1983;35:207-214. 52. Richardson B, Powers D, Hooper F, et al. Lymphocyte functionassociated antigen 1 overexpression and T cell autoreactivity. Arthritis Rheum 1994;37:1363-372. 53. Li-Weber M, Treiber MK, Giaisi M, et al. Ultraviolet irradiation suppresses T cell activation via blocking TCR-mediated ERK and NF-κB signaling pathways. J Immunol 2005;175:2132-2143. 54. Casiola-Rosen LA, Anhalt G, Rosen A. Autoantigen targets in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 1994;179:1317-1330. 55. Furukawa F. Photosensitivity in cutaneous lupus erythematosus: Lessons from mice and men. J Dermatol Sci 2003;33:81-89. 56. Frisoni L, McPhie L, Colonna L, et al. Nuclear autoantigen translocation and autoantibody opsonization lead to increased dendritic cell phagocytosis and presentation of nuclear antigens: A novel pathogenic pathway for autoimmunity? J Immunol 2005;175:2692-2701. 57. James JA, Kaufman KM, Farris AD, et al. An increased prevalence of Epstein-Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus. J Clin Invest 1997;100:3019-3026. 58. James JA, Neas BR, Moser KL, et al. Systemic lupus erythematosus in adults is associated with previous Epstein-Barr virus exposure. Arthritis Rheum 2001;44:1122-1126. 59. James JA, Scofield RH, Harley JB. Lupus humoral autoimmunity after short peptide immunization. Ann N Y Acad Sci 1997;8 15:124-127.

60. James JA, Gross T, Scofield RH, et al. 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. 61. McClain MT, Heinlen LD, Dennis GJ, et al. Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry. Nat Med 2005;11:85-89. 62. Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol 2000;53:424-432. 63. Kang I, Quan T, Nolasco H, et al. Defective control of latent Epstein-Barr virus infection in systemic lupus erythematosus. J Immunol 2004;172:1287-1294. 64. Tao Q, Robertson KD. Stealth technology: how Epstein-Barr virus utilizes DNA methylation to cloak itself from immune detection. Clin Immunol 2003;109:53-63. 65. Cooper GS, Dooley MA, Treadwell EL, et al. Hormonal, environmental, and infectious risk factors for developing systemic lupus erythematosus. Arthritis Rheum 1998;41:1714-1724. 66. Sawalha AH, Schmid WR, Binder SR, et al. Association between systemic lupus erythematosus and Helicobacter pylori seronegativity. J Rheumatol 2004;31:1546-1550. 67. Hess EV. Environmental chemicals and autoimmune disease: Cause and effect. Toxicology 2002;181/182:65-70. 68. Sanchez-Roman J, Wichmann I, Salaberri J, et al. Multiple clinical and biological autoimmune manifestations in 50 workers after occupational exposure to silica. Ann Rheum Dis 1993;52: 534-538. 69. Conrad K, Melhorn J, Luthke K, Dorner T, Frank K-H. Systemic lupus erythematosus after heavy exposure to quartz dust in uranium mines: Clinical and serological characteristics. Lupus 1996;5:62-69. 70. Linch KD, Miller WE, Althouse RB, et al. Surveillance of respirable crystalline silica dust using OSHA compliance data (1979-1995). Am J Ind Med 1998;34:547-558. 71. Parks CG, Cooper GS, Nylander-French LA, et al. Occupational exposure to crystalline silica and risk of systemic lupus erythematosus: A population-based, case-control study in the southeastern United States. Arthritis Rheum 2002;46:1840-1850. 72. Pernis B, Paronetto F. Adjuvent effects of silica (tridymite) on antibody production. Proc Soc Exp Biol Med 1996;110:390-392. 73. Lim Y, Kim JH, Kim KA, et al. Silica-induced apoptosis in vitro and in vivo. Toxicol Lett 1999;108:335-339. 74. Davis GS, Pfeiffer LM, Hemenway DR. Persistent overexpression of interleukin-1beta and tumor necrosis factor-alpha in murine silicosis. J Environ Pathol Toxicol Oncol 1998;17:99-114. 75. Clarkson TW, Magos L, Myers GJ. The toxicology of mercury: Current exposures and clinical manifestations. N Engl J Med 2003;349:1731-1737. 76. Cooper GS, Parks CG, Treadwell EL, et al. Occupational risk factors for the development of systemic lupus erythematosus. J Rheumatol 2004;31:1928-1933. 77. Hultman P, Enestrom S. The induction of immune complex deposits in mice by peroral and parenteral administration of mercuric chloride: Strain dependent susceptibility. Clin Exp Immunol 1987;67:283-292. 78. Hultman P, Enestrom S. Dose-response studies in murine mercury-induced autoimmunity and immune-complex disease. Toxicol Appl Pharmacol 1992;113:199-208. 79. Pollard KM, Pearson DL, Hultman P, et al. Lupus-prone mice as models to study xenobiotic-induced acceleration of systemic autoimmunity. Environ Health Perspect 1999;107(Suppl 5): 729-735. 80. Pollard KM, Pearson DL, Hultman P, et al. Xenobiotic acceleration of idiopathic systemic autoimmunity in lupus-prone bxsb mice. Environ Health Perspect 2001;109:27-33. 81. Hultman P, Bell LJ, Enestrom S, et al. Murine susceptibility to mercury. I. Autoantibody profiles and systemic immune deposits in inbred, congenic, and intra-H-2 recombinant strains. Clin Immunol Immunopathol 1992;65:98-109. 82. Hultman P, Bell LJ, Enestrom S, et al. Murine susceptibility to mercury. II. autoantibody profiles and renal immune deposits in hybrid, backcross, and H-2d congenic mice. Clin Immunol Immunopathol 1993;68:9-20. 83. Freni-Titulaer LW, Kelley DB, Grow AG, et al. Connective tissue disease in southeastern Georgia: A case-control study of etiologic factors. Am J Epidemiol 1989;130:404-409.

90. Malinow MR, Bardana EJ Jr, Pirofsky B, et al. Systemic lupus erythematosus-like syndrome in monkeys fed alfalfa sprouts: Role of a nonprotein amino acid. Science 1982;216:415-417. 91. Roberts JL, Hayashi JA. Exacerbation of SLE associated with alfalfa ingestion. N Engl J Med 1983;308:1361. 92. Alcocer-Varela J, Iglesias A, Llorente L, et al. Effects of L-canavanine on T cells may explain the induction of systemic lupus erythematosus by alfalfa. Arthritis Rheum 1985;28:52-57. 93. Morimoto I. A study on immunological effects of L-canavanine. Kobe J Med Sci 1989;35:287-298. 94. Bengtsson AA, Rylander L, Hagmar L, et al. Risk factors for developing systemic lupus erythematosus: A case-control study in southern Sweden. Rheumatology (Oxford) 2002;41:563-571.

REFERENCES

84. Petri M, Allbritton J. Hair product use in systemic lupus erythematosus: A case-control study. Arthritis Rheum 1992;35: 625-629. 85. Sanchez-Guerrero J, Karlson EW, Colditz GA, et al. Hair dye use and the risk of developing systemic lupus erythematosus. Arthritis Rheum 1996;39:657-662. 86. Hardy CJ, Palmer BP, Muir KR, et al. Systemic lupus erythematosus (SLE) and hair treatment: A large community based case-control study. Lupus 1999;8:541-544. 87. Jimenez-Alonso J, Sabio JM, Perez-Alvarez F, et al. Hair dye treatment use and clinical course in patients with systemic lupus erythematosus and cutaneous lupus. Lupus 2002;11:430-434. 88. Sanchez-Guerrero J. Is cigarette smoking a risk factor for systemic lupus erythematosus? Comment on the article by Costenbader et al. Arthritis Rheum 2005;52:1340-1341. 89. Bardana EJ Jr, Malinow MR, Houghton DC, et al. Diet-induced systemic lupus erythematosus (SLE) in primates. Am J Kidney Dis 1982;1:345-352.

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8

The Genetics of Lupus Bahram Namjou, MD, Jennifer A. Kelly, MPH, and John B. Harley, MD, PhD

INTRODUCTION Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disorder with a troublesome complex genetic phenotype. SLE is characterized by an extreme breakdown of self-tolerance, which results in a wide range of immunologic abnormalities (including pathogenic autoantibody production and immune complex formation, T- and B-cell dysregulation, and defective clearance of apoptotic materials). SLE has a prevalence rate of approximately 1 in 2000 in the United States, and targets women of childbearing age and minority ethnic groups more frequently than men or European-derived peoples. Familial aggregation of SLE is an expected keystone observation if there is an underlying genetic cause. Studies in twins and multiple inbred strains of mice that spontaneously develop lupus-like phenotypes further support the importance of a genetic influence on SLE. Since the early 1970s, investigators have strived to identify the genes that cause SLE and now know that both genetic and allelic heterogeneity play roles in the initiation of this complex disease. Linkage analyses in both man and mice have identified many regions that are likely to harbor loci that increase the risk of disease or of particular subphenotype. Genes in the major histocompatability complex (MHC)—as well as complement components, Fc receptors, and cytokines—have been extensively investigated.

The higher the λs the easier it is to identify the gene(s) responsible for disease. Among twin studies, an increased concordance rate among monozygotic twins (24 to 69%) is almost 10 times higher than that seen in dizygotic twins (2 to 9%), providing further support for a genetic contribution to SLE.4,5 The lack of a 100% penetrance among monozygotic twins, however, demonstrates that environmental factors, genetic imprinting, X-chromosome inactivation, or random processes may also play a role in the disease.6 Grennan et al. found that 25% of monozygotic twins were concordant for SLE. However, none of 18 HLA identical same-sex siblings of SLE probands had definite SLE—suggesting that most of the genetic predisposition to SLE is attributable to genes outside the HLA region.7 Except for isolated SLE cases with complement deficiency, the pattern of inheritance in SLE does not seem to follow a simple Mendelian trait such as autosomal dominant, autosomal recessive, or sex-linked recessive. However, dominant models have been shown to fit well among multi-case SLE families.8,9 In one study, a segregation analysis applied to 19 multi-case families for SLE demonstrated a Mendelian dominant inheritance pattern (with penetrance of 92% in females and 49% in males) and a gene frequency of 0.10.8 In a separate study, Winchester and Nunez-Roldan calculated that a polygenic model with at least three or four dominant alleles fit best among their SLE families.9

GENETIC EPIDEMIOLOGY

74

Familial aggregation of SLE has been consistently observed in many studies, with a sibling recurrence risk ratio (λs) of 20 to 40 (depending on the population studied).1,2 The λs is a statistic that measures the ratio of the phenotype frequency in siblings of affected probands compared to the phenotype frequency in the general population and is a crude measure of the potential a phenotype has for a genetic explanation.3 As a comparison, the λs for a monogenic trait such as cystic fibrosis is about 10-fold higher (λs=500).

GENOMIC METHODS Association and linkage studies are the two major approaches to genetic dissection of complex traits.10 Association studies are performed on both single cases (in case-control studies) and family-based studies. In the population-based case-control association design, differences in relative allele or genotype frequencies at a single marker locus are evaluated between unrelated affecteds and healthy controls. To avoid population stratification, which may cause spurious associations

published association lupus results are based on case-control association studies, which are subject to artifacts derived from phenotypic variation and population stratifications. Of the many purported associations published in lupus, studies confirming the initial results have been performed in only a minority of cases. As has proven typical for virtually all human phenotypes, many purported genetic associations in SLE have not been confirmed. Consequently, a complex literature has been emerging.

THE HLA REGION

in such studies, family-based association tests have been developed in which the transmission of marker alleles from heterozygous parents to affected and unaffected offspring are compared in small nuclear families.11 Transmission from generation to generation occurs in sets of linked loci called haplotypes. The measure of this linkage effect in the population (linkage disequilibrium, LD) differs substantially from population to population and decays gradually due to recombination and mutation. Genome-wide linkage disequilibrium mapping has been recently applied to detect disease genes.12,13 However, the extent of LD in the human genome is not completely characterized. Estimates of up to 500,000 single-nucleotide polymorphism (SNP) markers may be needed to capture most of the information.12 With rapid advancements in genotyping technology, genome-wide association scans using hundreds of markers are underway and are expected to become standard in the near future. In such studies, however, large numbers of association tests are computed requiring correction of statistical significance for multiple testing. Linkage analysis is another approach to detect susceptibility loci in complex traits. Families containing two or more affected relatives are genotyped at DNA markers evenly distributed throughout the genome and the cosegregation of marker alleles and disease within these multi-case families are evaluated either with or without specified models using parametric or nonparametric methods, respectively. The goal is to identify whether co-segregation of two loci occur more often than expected when they are not physically close together. In this systematic approach, the previous knowledge of disease gene location is not necessary. However, there is limited power to detect genes of modest effects in complex genetic diseases compared to single-gene Mendelian traits. Consequently, large collections of multi-case families are required. Not only sample size but family structure, ethnic background, and phenotypic variations can affect the results. SLE has substantial intrafamilial subphenotype variability among siblings. The classification for study typically requires the patient expressing 4 of 11 American College of Rheumatology (ACR) criteria.14,15 This, in turn, further increases genetic heterogeneity. Many epidemiologic and environmental elements such as ethnicity, gender, hormonal exposure, UV radiation, pregnancy, smoking habits, and viral exposures are thought to influence these phenotypic variations. The interpretation of these statistical analyses is especially complicated. Overall, association studies seem to be more powerful than linkage for complex diseases.13 Association studies of the entire genome depend on a dense map of markers. They are unlikely to detect extremely rare variants. Currently, most of the

THE HLA REGION The human major histocompatibility (MHC) genomic region located at chromosome 6 (6p21) in humans (syntenic to chromosome 17 in the mouse) encodes the human leukocyte antigen (HLA) genes and many genes that are critical in the regulation of the immune system. This small segment of the human genome has been associated with many immune diseases, including SLE, rheumatoid arthritis, diabetes, asthma, psoriasis, and various other autoimmune disorders. In 1999, the human MHC Sequencing Consortium annotated 224 genes in this 3.6-Mb genomic segment.16 Today, there are 239 genes in the region.17 The presentation of antigens to T-cells is the important known function of the products of MHC genes, but the functions of many other genes in this region have yet to be characterized. There are three class I alpha-chain genes (HLA -A, -B, and -C) and three pairs of highly polymorphic MHC class II alpha and beta-chain genes (HLA-DR, DP, and DQ). HLA-DR, however, may have an extra beta-chain gene whose product could pair with the DR alpha chain. Hence, the three sets of genes could give rise to four types of MHC class II molecules. The genes encoding the alpha chains of MHC class I and the alpha and beta chains of MHC class II molecules are linked within the complex. There are more than 200 alleles of human MHC class I and II, and therefore most individuals are likely to be heterozygous at MHC loci with co-dominant expression of both alleles. The MHC class III region contains genes that encode complement component C4 (C4A and C4B), C2, and factor B (Bf gene); some that encode cytokines such as tumor necrosis factor-alpha (TNF-alpha, TNF gene); and some that encode lymphotoxins (LTA and LTB). The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Many suggest that selective evolutionary pressure from infectious agents maintains a wide variety of MHC molecules in the population.18,19 For example, the HLA-B53 allele has

75

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strong association with recovery from a lethal form of malaria. This allele is very common in West Africa, where malaria is endemic. The DR-B1 alleles DR2 and DR3 have shown consistent associations with SLE in Caucasian populations, with a two- to threefold increase in frequency.20-22 However, HLA associations in many non-Caucasian populations have not been very convincing. This could be due to different levels of admixture in AfricanAmerican populations, and therefore matching the ethnic ancestry of the cases and controls is important and challenging. Despite many case-control studies, few studies have applied family-based association in the HLA region,23,24 and in European-Americans only DRB1*1501 (DR2) seems to be associated in two different family-based populations (p=0.0007, p<0.05).23,24 This is an example of association studies showing different results with HLA-DR3 and DR2. The association with HLA-DR2 can be localized to this allele, whereas the association with DR3 cannot be reduced to less than an extended haplotype containing HLA-DR3 and many other genes.23 Because antigen recognition by T-cells is dependent on the presence of specific MHC molecules, HLA class II alleles may show stronger association with the autoantibody profiles observed in SLE than with disease expression or particular clinical manifestations. In fact, several studies suggest that the contribution of HLA class II genes in SLE is predominantly at the level of production of specific autoantibodies rather than with SLE itself.25-27 There are a number of studies that have found association of HLA alleles with various autoantibodies. These include a study of HLA DR3/DR2 with anti-Ro,26 which identifies haplotypes at HLA-DQ (which also appears to be associated with anti-Ro through gene complementation and perhaps with polymorphism of the T-cell receptors).28,29

COMPLEMENT COMPONENTS

76

Complement cascade plasma proteins, key components of the innate immune system, can be activated by three distinct pathways: classical, alternative, and lectin. Complements have important roles in host resistance to bacterial infection and in the clearance of immune complexes. Therefore, they prevent autoimmunity. In addition, complements have an important role in lymph node organization, B-cell maturation, differentiation and tolerance, and IgG isotype switching.30,31 C2, C4A, C4B, and factor B are complement components located in the MHC class III region. Different alleles of these three components are linked to particular HLA haplotypes and are inherited as extended MHC haplotypes or complotypes. Considerable differences in

complotype frequencies have been observed among various SLE ethnic groups. Complement deficiency is usually inheritied as a recessive trait except for C1 inhibitor, CR1, and properdin deficiency (which are autosomal dominant, codominant, and X linked, respectively). In Europeans, homozygous C4 or C2 deficiency is found in up to 1 in 10,000.32,33 However, C2 deficiency has not been identified in the AfricanAmerican population. More than 35 variants have been observed in the human C4A and C4B loci, including null C4 variants (C4AQ0, C4BQ0) that have arisen due to allelic deletion. Heterozygous deficiency of C4 probably is the most common form of complement deficiency among lupus families.34 However, there is no correlation between the number of C4 genes expressed and C4 concentration. Indeed, the increase in C4AQ0 in SLE patients could be due to other genes on the extended haplotype of HLA-B8;SC01;DR3.35 C2 deficiency in Caucasians is linked to another HLA region extended haplotype: HLA-A25,B18,DR2,DQB1.36 Associations of anticardiolipin antibodies with C4A or C4B null allotypes in the U.S. black population,37 or anti-Ro with homozygous C2 deficiency in Caucasians,38 could be due to these MHC extended haplotypes. In fact, mild alterations of serum complement levels in heterozygous C2 and C4 deficiency patients suggest that the primary factors for inducing lupus in these patients may be beyond serum complement activity. Homozygous C2 and C4 deficient patients usually have a mild disease with a low titer of anti-nuclear antibodies. In contrast, C1q deficiency (although much less frequent) has a higher predictive risk for lupus (>90%) and is associated with more severe disease and glomerulonephritis. About 20 families with C1 (C1q, C1r, C1s) deficiencies have been described in the literature, and heterozygous deficiencies are difficult to identify. Complement components C3,C5 through C9 deficiencies (which are usually associated with infection) have also been reported with SLE. The CR1 complement receptor (C3b/C4b receptor) is expressed on erythrocytes and is the structural basis of the Knops blood group. Its genetic control is under the influence of two alleles with codominant expression, which is based on the varying number of long homologous repeats (LHR).39 Erythrocyte CR1 is crucial for buffering immune complexes in circulation and transporting them to phagocyte Fc gamma and complement receptors located in the liver and spleen. Therefore, the number of functional erythrocytes expressing CR1 is important in this process. The severe anemia of malaria and hemolytic anemia influences CR1 expression, as does the serum erythropoietin level. In fact, this is reason to use recombinant erythropoietin in SLE patients.40 Although low levels of CR1

FCγ RECEPTORS The low-affinity Fcγ receptor (FcγR) genes are attractive candidates for SLE disease susceptibility. Numerous studies have demonstrated association with one or more FcγR alleles and SLE. Three distinct but closely related classes of FcγR have been identified in humans: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRs vary in their binding capacity for IgG, their preference for IgG subclasses, the cell types in which they are expressed, and the intracellular signals they elicit. They may be stimulatory (FcγRIIA, FcγRIIIA, FcγRIIIB, FcγRIIC) or inhibitory (FcγRIIB) to immune responses.45 Normal individuals with an HLA haplotype containing DR2 or DR3, both of which are found with increased frequency in SLE patients, are more likely to have prolonged Fc receptor-mediated clearance of IgG than normal control groups without these haplotypes.46 There are also allelic variations of FcγRIIA and FcγRIIIA that influence the ability to bind certain IgG subclasses and alter the responses of phagocytes to IgG-opsonized antigens.47,48 A meta-analysis comprising more than 1000 patients established an association with FcγRIIA-R131 and SLE, especially in African-American populations, and with FcγRIIIA-F176 and SLE in European-Americans and other ethnic groups.49 In addition, a potential dose-response relationship between the FcγRIIAR131 allele and the risk of SLE was also identified,

demonstrating a greater odds of having SLE if a patient had the 131R/R genotype compared to the 131R/H genotype (OR=1.23, 95% CI 1.03-1.46) or 131H/H genotype (OR=1.55, 95% CI 1.21-1.98).50 The FcγRIIIA polymorphism has an impact on the development of lupus nephritis. A comparison of 1154 lupus nephritis patients with 1261 non-nephritis SLE subjects revealed a significant overrepresentation of the low-binding F158 allele among patients who developed renal disease (OR=1.20, 95% CI=1.06 to 1.36, p=0.003).51 The 176F/F genotype had the highest risk of renal disease when compared to the 176V/V genotype (OR 1.47, 95% CI=1.11 to 1.93, p=0.006).51 FcγR genes are in a cluster of ~300 kb on chromosome 1q23. However, the potential role of linkage disequilibrium between the FcγR genes is not yet established. In a cohort of 46 Hispanic SLE patients with a high prevalence of lupus nephritis in which there was a selection for haplotypes containing FcγRIIA-R131 and FcγRIIIA-F176, no LD was detected between FcγRIIA and FcγRIIIA (35 Kb separation).52 In a study of Japanese SLE patients, significant LD was detected, however, between FcγRIIIA and FcγRIIIB but not between FcγRIIA and FcγRIIIA (nor between FcγRIIA and FcγRIIIB).53 Associations of SLE with other Fcγ receptors have also been reported. At least two studies have shown an association between FcγIIB-232T and SLE in Asians.54,55 FcγIIB is the only gene among the FcγR family that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and has the ability to transmit inhibitory signals in B-cells and myelomonocytic cells. In fact, in one study of SLE in a Thai population an association of FcγRIIIA-176F was identified, but this may have been attributable to its strong LD with FcγRIIB-232T and/or FcγRIIIB-NA2.56 Alternatively, in a Europeanderived sample, association of SLE with FcγRIIB has been identified in a variant of the 2B.4 promoter haplotype.57 This study also demonstrated a lack of LD between the FcγRIIB promoter haplotypes with FcγRIIA and FcγRIIIA polymorphisms in EuropeanAmericans. These variable LD and association results obtained in the different studies could be due to random variation, admixture effects, phenotypic variation, lack of appropriate controls and population stratification, or the confounding influence of other susceptibility loci.

TUMOR NECROSIS FACTORS

expression in SLE have been shown in different studies, the deficiency is at least in part reversible (and inherited CR1 deficiency does not seem to clearly predispose to SLE).41 Both functional and structural polymorphisms of CR1 have been reported with different results among lupus patients. A recent meta-analysis of these polymorphisms among 18 studies suggests an association of CR1-B (also called S allele) and SLE in European-Americans (OR=1.66).42 However, the CR1C allele (which in theory may better explain the lack of clearance of immune complexes in SLE) was not associated.42 In fact, only one study suggests an association of the CR1-C allele with lupus.43 Another structurally related complement receptor, CR2 (CD21), is expressed only on lymphocytes and dendritic cells and has been implicated in lupus susceptibility in both human and animal models. Moreover, it serves as the receptor for Epstein-Barr virus. Although some variations in the CR2 gene due to alternative splicing have been detected, in contrast to CR1, a specific LHR variation in human CR2 has not been identified44 and may be the reason for a lack of association with this gene (which is suspected to be important in SLE).

TUMOR NECROSIS FACTORS The increased expression of the tumor necrosis factor (TNF) alpha, a proinflammatory cytokine in MHC class III region, has been correlated with a variety of different autoimmune and infectious disorders. The -308 polymorphism is located in the promoter region.

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THE GENETICS OF LUPUS

It contains two alleles (TNF1 and TNF2) that regulate low or high expression. TNF2 (-308 A) has been associated with SLE either independently of HLA haplotypes (OR=5 in Caucasians58 and OR=2.72 in African-Americans59) or as a part of an extended MHC haplotype (HLA-A1-B8-DRB1*0301-DQ2).60 In fact, the combined HLA-DR3, TNF-alpha -308A, IL-1alpha -889C/C genotype produced an OR = 8.0 (p < 0.00001) in a Caucasian population.61 The TNF-alpha -308 A polymorphic association is related to autoimmunity in general rather than specific SLE, and there were also some negative results in lupus that could be due to random variation or population heterogeneity.62,63 Interestingly, because TNF2 seems to be a common susceptibility allele for various autoimmune rheumatic diseases and at the same time has a protective role for tuberculosis, it has recently been suggested that autoimmune diseases might be a consequence of natural selection for enhanced TB resistance.64 Polymorphism of other related genes in this family [such as TNF-B or TNF-R2 (p75), FAS, and FAS ligand] have also been considered as risk factors for lupus with various results.

It appears that this SNP alters a binding site for the runt-related transcription factor 1 (RUNX-1), which could lead to aberrant regulation of PD-1. Additional reports suggest that SNPs affecting RUNX1 binding site are associated with rheumatoid arthritis as well as psoriasis.71,72 Interestingly, PD-1 originally became the focus of interest after a linkage effect was identified at 2q37 in Northern European lupus families.73 However, it is not completely clear whether this intronic SNP is responsible for the entire linkage observed. Like the PD-1 pathway, the cytotoxic T lymphocyte antigen-4 (CTLA-4) B7 pathway leads to the downregulation of T-cell activity. It is possibly the most robust regulatory process controlling autoreactivity, which is illustrated by severe lymphoproliferative disorders with early lethality in CTLA-4-deficient mice.74,75 Many autoimmune disorders including SLE have been associated with variants of this gene, but the results have not been consistent. However, two recent meta-analyses for the exon 1 (A/G) polymorphism suggestively confirm the implication of this gene in SLE with an OR=1.24 for the G allele in an Asian population76 and OR=1.39 for the GG genotype when assuming all studies come from the same population.77

MANNOSE-BINDING LECTIN (MBL) The lectin pathway is responsible for an antibodyindependent pathway of complement activation that is initiated by binding of the mannose-binding lectin (MBL) to carbohydrates on the surface of pathogens. MBL activates complement through associated serine proteases. MBL deficiency is not uncommon in the general population (5 to 10%) and is associated with increased susceptibility to infection as well as enhanced progression to SLE and severe rheumatoid arthritis.65,66 According to a meta-analysis of more than eight published studies and two additional unpublished samples, MBL variant alleles confer a 1.6 times overall increased risk for SLE (67,68). In general, MBL variants have been considered a severity marker rather than a susceptibility marker for SLE.

PDCD1, CTLA-4

78

The CD28-B7 and immune co-stimulatory molecule ICOS pathways promote T-cell activation, whereas the cytotoxic T lymphocyte antigen-4 (CTLA-4) and PD-1 pathways lead to down-regulation of T-cell activity. PD-1 belongs to the CD28-B7 family that contains an immunoreceptor tyrosin-based inhibitory motif (ITIM) and serves as a negative regulator of immune responses. C57BL/6 knockout mice for PD-1 have been shown to develop lupus-like nephritis and arthritis.69 The PD1.3A intronic SNP has been associated with human SLE in both Europeans and Mexicans.70

PTPN22 The protein tyrosine phosphatase non-receptor type 22 gene is a likely risk factor for the development of humoral autoimmunity. This gene encodes the lymphoid protein tyrosine phosphatase (LYP), a suppressor of T-cell activation. The minor allele (1858T), which encodes the amino acid substitution (R620W), disrupts the P1 proline-rich motif that is important for interaction with the C-terminal Src tyrosine kinase (Csk), a negative regulatory kinase. This potentially alters the normal function of this protein as a negative regulator of T-cell activation. Association with this SNP was first identified in type 1 diabetes78 and later with rheumatoid arthritis, SLE, and Graves’ disease.79-81 However, neither case-control nor family-based associations have been significant when a large number of families with multiple sclerosis have been tested.82 This is thought to be the due to the fact that multiple sclerosis is primarily a T-cell disease.82 In addition, the identified associations have been primarily in European-American families and the risk allele seems to be more common in European-American than African-American healthy controls (9 versus 2%). Therefore, there is much less power to detect this modest effect in African populations and a large number of cases and controls will be necessary if the association exists at all. A recent family-based study conducted in 4 large independent Caucasian cohorts, however, did not find significance in their lupus pedigrees

Work underway using large collections of subjects (>500) are likely to clarify whether these associations are robust or not.

THE INTERFERON PATHWAY

GENETIC DISEASE OR ABNORMALITY

Interferon (IFN) is important in the defense against viral infections and appears to have a major role in lupus pathogenesis. Serum interferon alpha (IFN-α) levels have been correlated with both lupus disease activity and severity since the early 1980s.84-86 In addition, microarray data consistently support the dominance of an “IFN signature” on the up-regulated gene expression of the IFN responsive gene in the global gene expression profiles of lupus patients.87-89 The gene IFN cluster is located on chromosome 9p and contains at least 26 genes, including IFN-α, IFN-γ, and IFN-β1. Although there are only a few association studies with lupus and the IFN cluster, new evidence provides an association for other genes that responds to IFN [including tyrosine kinase 2 (TYK2) and IFN regulatory factor 5 (IRF5)].90 TYK2 (located at 19p13.2) belongs to the Janus kinase family, which is important in JAK-STAT signaling and interestingly, this chromosomal region has also been linked in Caucasian lupus families with positive anti-dsDNA.134 IRF5, located at 7q32, is important in innate immunity response and may be directly up-regulated by apoptosis-inducing genes such as p53.91 Another IFN-related gene, immune interferon gamma (IFNγ), has also been implicated in the protection against infection and autoimmunity. However, the correlation of IFNγ with SLE activity may not be as strong as IFN-α. IFNγ is produced by mitogen- or antigen-stimulated T lymphocytes. An intronic microsatellite CA repeat in the IFNγ gene has been a subject of interest in different autoimmune disorders, including SLE, although with no evidence of association.92 On the other hand, mutations that have been identified in IFNγ receptors (IFNγR1 and R2) have been considered risk factors for SLE in Japanese lupus patients, especially when the IFNγR1 Met14/Val14 and the IFNγR2 Gln64/Gln64 genotypes are combined (OR=9.6, p=0.04).93

Identifying lupus patients with coexistent known genetic diseases or abnormalities may provide important clues for the potential role of underlying genes in lupus. Apart from the complement deficiency mentioned previously, there have been other case reports of specific genetic diseases or abnormalities with lupus.

OTHER GENES There are more than 50 genes that have been reported to be associated with human SLE, and many of them await evidence of confirmation. These include IL-4 Ra, IL-6, IL-10, Poly ADP-ribose polymerase (PARP), acute phase reactants (CRP), Bcl-2, T-cell receptors (TCR), Toll-like receptors 9 and 5 (TLR9, TLR5), immunoglobulin heavy and light chain genes, vitamin D, prolactin and estrogen receptors, N-acetyl transferase NAT1 and NAT2, Fas and its ligand, and many more.

GENETIC DISEASE OR ABNORMALITY

but found an association between the minor 1858T allele and concurrent SLE and autoimmune thyroid disease.83

Klinefelter’s Syndrome Over the last three decades, many case reports have suggested an increased rate of lupus in XXY males, which has primarily been attributed to the increased level of estrogen in these males. Recently, it has been shown that patients with lupus (both male and female) have an altered metabolism of estrogens with altered levels of both the 16- and 2-hydroxylated metabolites (a very feminizing group of hormones). This phenomenon remains to be explained.94,95 Men with Klinefelter’s syndrome and SLE also have many of the same characteristics of women with SLE. Indeed, one group has shown that the prevalence of lupus in XXY males is similar to females, indicating a gene dose effect on chromosome X (Scofield H. et al., unpublished data).

Metabolic Enzyme Deficiency Two hereditary metabolic enzyme deficiencies, prolidase and mannosidase, have been associated with SLE. Prolidase deficiency is a rare autosomal recessive disease with only ~40 reported cases in the world. It is characterized by mild to severe skin lesions with massive excretions of iminodipeptides in the urine. Because of high levels of iminoacids in collagen, this enzyme plays an important role in collagen metabolism. Presently, three cases of SLE with prolidase deficiency have been reported.96,97 Alpha-mannosidosis is another autosomal recessive disorder caused by the deficiency of lysosomal alpha-mannosidase (LAMAN) and is characterized by progressive mental retardation, facial coarsening, dysostosis multiplex, immune defects, deafness, and hepatomegaly. A deficiency of the enzyme causes intralysosomal accumulation of mainly unbranched oligosaccharides. Recently, two sisters with SLE and alpha-mannosidase have been reported.98 Another gene related to LAMAN (MAN2A1), which regulates the hybrid to complex branching pattern of extracellular asparagine (N)-linked oligosaccharide chains (N-glycans), results in a systemic autoimmune disease similar to human

79

THE GENETICS OF LUPUS

SLE in mice.99 Both LAMAN and prolidase are mapped to 19p13.2-q13 in man.

Noonan Syndrome This syndrome, which causes multiple malformations including congenital heart disease, short stature, and unusual facial structure, is not uncommon worldwide, with a prevalence of 1 in 1000 to 1 in 2500. It is unusually associated with multiple autoimmune phenomena, including SLE, vasculitis, anterior uveitis, autoimmune thyroiditis, and vitiligo.100,101 The gene responsible for the autosomal dominant form of this disease, PTPN11 (12q24), is in the same family as PTPN22—a lupus-associated gene responsible for humoral autoimmunity (see previous material).

Aicardi-Goutiéres Syndrome This autosomal recessive syndrome is characterized by an early-onset progressive encephalopathy and acquired microcephaly, basal ganglia calcification, and chronic cerebrospinal fluid (CSF) lymphocytosis (with increased levels of IFN-α in the CSF). Dysregulated production of IFN-α have been suggested to play a fundamental role in both diseases.102 At least two unrelated families containing affected sibpairs have been reported to have both SLE and Aicardi-Goutiéres.103,104

Chondrodysplasia Punctata Chondrodysplasia punctata are a heterogeneous group of bone dysplasias mainly characterized by premature and ectopic calcification of cartilage leading to cataracts, ichtyiosis, and short stature. Many genetic (autosomal dominant, recessive, X linked) and non-genetic causes (e.g., anticoagulants) have been described. It has been suggested that chondrodysplasia punctata might be one of the

manifestation of neonatal lupus in mothers with SLE, especially those with positive anti-U1 RNP.105,106 It has also been suggested that maternal autoantibodies due to anticoagulant activity may be responsible.107

Retinitis Pigmentosa This is a genetic disease with progressive visual loss due to pigmentary changes in the retinal pigment epithelium. More than 40 chromosomal loci and 30 candidate genes have been identified, with a prevalence of 1 in 4000 worldwide. Both discoid and systemic lupus cases have been reported with this disease, and antibodies to retinal antigens have been found in lupus.108-110 One of the known genes associated with retinitis pigmentosa, mer tyrosine kinase protooncogene (Mertk), has also been shown to have a critical role for the engulfment and efficient clearance of apoptotic cells in Mer knockout mice, which suggests a possible role of this gene in systemic autoimmune diseases and SLE.111

GENETIC LINKAGE IN LUPUS Multiple genome-wide linkage studies have been performed in multi-case families around the world. A few of the current significant linkage results (LOD>3.3) that have been confirmed in independent samples112-125 also correspond to syntenic regions in murine lupus (Table 8.1 and Fig. 8.1). Quantitative linkage analyses in murine lupus have identified more than 50 susceptibility loci, some of which are linked to specific traits (i.e., autoantibody production, lymphoid hyperplasia, nephritis, or mortality). The data in mice further support lupus being multigenic and support the contention that different phenotypes result from the specific combinations of susceptibility loci.

TABLE 8.1 SIGNIFICANT LINKAGES ESTABLISHED (LOD>3.3) AND CONFIRMED IN INDEPENDENT HUMAN SAMPLES

80

Location

Marker

Possible Candidate Genes

Murine Syntenic Region

References

1q23

Fcγ, D1S1677

FcγRIIA, FcγRIIIA, FcγRIIB, CRP

Sle1ab, Nba2, Lbw7

112–116

1q41

D1S2869

PARP, TLR5

Sle1c

112, 115–120

2q37

D2S125

PDCD1

4p16

D4S2366

6p11-21

D6S426

12q24

D16S395

16q12

D16S415

MHC Haplotype

121 Sle6

122

sles1, Lbw1

114, 119, 123 124

OAZ

114, 119, 123, 125

4p16 5p15 6p21

GENETIC LINKAGE IN LUPUS

African-American European-American Hispanic

5q14

1q23

2q34

1q41

2q37 1

2

3

4

5

6

7

8

11p13

10q22

11q14

12q24 9

10

11

12

13

14

15

19

20

21

22

17p13

19p13 16q12 18q21

16

17

18

Fig. 8.1 Genome-wide linkage studies in human SLE: 1q23,112-116 1q41,112,115-120 2q34,132,133 2q37,121 4p16,122 5p15,128,129 5q14,135 6p21,114,119,123 10q22,132,133 11p13,138,139 11q14,130,131 12q24,124 16q12,114,119,123,125 17p13,136,137 18q21,134 and 19p13.134

Indeed, one of the first linkages in humans (1q41-43) was first demonstrated after its syntenic region in the mouse was targeted as a region containing potential SLE candidate genes.120 Although significant linkage to this region was only marginally confirmed in wholegenome scans, multiple suggestive linkages identified by many independent groups provide strong evidence

that a lupus candidate gene may lie in the 1q41-43 region (see Table 8.1).115-120 The 1q23 linkage, which is syntenic to linkages found in murine lupus models (Sle1a, Sle1b, Nba2, and Lbw7) and contains an important Fcγ receptor gene family was originally identified in a collection of 94 pedigrees (LOD=3.37).112 Subsequently, the linkage has been

81

THE GENETICS OF LUPUS

TABLE 8.2 SIGNIFICANT LUPUS LINKAGES IDENTIFIED BY A PEDIGREE STRATIFICATION APPROACH Stratifying Phenotype

Chromosomal Location

Ethnicity

Confirmed

Reference

Neuropsychiatric disorder

4p16

EA

No

127

Self-reported rheumatoid arthritis

5p15

EA

Yes

128, 129

Hemolytic anemia, nucleolar ANA

11q14

AA

Yes

130, 131

Renal disease

10q22 2q34

EA AA

Yes Yes

132, 133

Anti-dsDNA

19p13.2 18q21.1

EA AA

Yes No

134

Autoimmune thyroid disease

5q14

EA

Yes

135

Vitiligo

17p13

EA

Yes

136, 137

Thrombocytopenia, discoid lupus

11p13

AA

No

138, 139

confirmed by two independent studies.114,115 Later, evidence of both linkage and association in 126 pedigrees revealed suggestive multipoint NPL results for both FcγRIIIA (NPL=2.7, p=0.004) and FcγRIIA (NPL=2.6, p=0.006).126 In this study, family-based tests of association demonstrated an increased transmission of the low-affinity F176 allele at the locus FcγRIIIA (OR=2.18, p=0.0005) but not for FcγRIIA.126 Because of the extreme heterogeneity of the lupus phenotype, one approach has focused on performing linkage scans using pedigrees that contain at least one affected family member with a particular clinical or serological manifestation. The goal is to create a more genetically homogeneous sample. This approach has identified additional genomic loci linked to these particular subphenotypes and several have already been confirmed (Table 8.2127-139 and Fig. 8.1).

The chromosomal regions identified by linkage studies represent large genomic areas (usually 10 to 30 cM) and depend on map density. Usually, hundreds of genes may require evaluation in such large genomic interval. This remains a technically challenging task. Fine mapping of the region with additional markers may narrow the region of interest. After the region is narrowed to a <5 cM region, association studies are then performed using high-density arrays with pooled or individual DNA samples, linkage disequilibrium mapping, family-based transmission disequilibrium testing, and case-control study designs. With recent advances in high-throughput SNP genotyping, the analyses of complex diseases become more practical for both linkage and association studies. Gene identification will provide new insights into the pathologic pathways leading to autoimmunity and will influence subsequent work focused on therapeutic intervention.

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82

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55. Chu ZT, Tsuchiya N, Kyogoku C, Ohashi J, Qian YP, Xu SB, et al. Association of Fcgamma receptor IIb polymorphism with susceptibility to systemic lupus erythematosus in Chinese: A common susceptibility gene in the Asian populations. Tissue Antigens 2004;63:21-27. 56. Siriboonrit U, Tsuchiya N, Sirikong M, Kyogoku C, Bejrachandra S, Suthipinittharm P, et al. Association of Fcgamma receptor IIb and IIIb polymorphisms with susceptibility to systemic lupus erythematosus in Thais. Tissue Antigens 2003;61:374-383. 57. Su K, Wu J, Edberg JC, Li X, Ferguson P, Cooper GS, et al. A promoter haplotype of the immunoreceptor tyrosine-based inhibitory motif-bearing FcgammaRIIb alters receptor expression and associates with autoimmunity. I. Regulatory FCGR2B polymorphisms and their association with systemic lupus erythematosus. J Immunol 2004;172:7186-7191. 58. Rood MJ, van Krugten MV, Zanelli E, van der Linden MW, Keijsers V, Schreuder GM, et al. TNF-308A and HLA-DR3 alleles contribute independently to susceptibility to systemic lupus erythematosus. Arthritis Rheum 2000;43(1):129-134. 59. Sullivan KE, Wooten C, Schmeckpeper BJ, Goldman D, Petri MA. A promoter polymorphism of tumor necrosis factor alpha associated with systemic lupus erythematosus in African-Americans. Arthritis Rheum 1997;40(12):2207-2211. 60. Wilson AG, de Vries N, Pociot F, di Giovine FS, van der Putte LB, Duff GW. An allelic polymorphism within the human tumor necrosis factor alpha promoter region is strongly associated with HLA A1, B8, and DR3 alleles. J Exp Med 1993;177(2): 557-560. 61. Parks CG, Pandey JP, Dooley MA, Treadwell EL, St Clair EW, Gilkeson GS, et al. Genetic polymorphisms in tumor necrosis factor (TNF)-alpha and TNF-beta in a population-based study of systemic lupus erythematosus: Associations and interaction with the interleukin-1alpha-889 C/T polymorphism. Hum Immunol 2004;65(6):622-631. 62. Tobon GJ, Correa PA, Gomez LM, Anaya JM. Lack of association between TNF-308 polymorphism and the clinical and immunological characteristics of systemic lupus erythematosus and primary Sjogren’s syndrome. Clin Exp Rheumatol 2005;23(3):339-344. 63. Chen CJ, Yen JH, Tsai WC, Wu CS, Chiang W, Tsai JJ, Liu HW. The TNF2 allele does not contribute towards susceptibility to systemic lupus erythematosus. Immunol Lett 1997; 55(1):1-3. 64. Correa PA, Gomez LM, Cadena J, Anaya JM. Autoimmunity and tuberculosis: Opposite association with TNF polymorphism. J Rheumatol 2005;32(2):219-224. 65. Turner MW. Deficiency of mannan binding protein—a new complement deficiency syndrome. Clin Exp Immunol 1991;86(Suppl 1):53-56. 66. Saevarsdottir S, Vikingsdottir T, Vikingsson A, Manfredsdottir V, Geirsson AJ, Valdimarsson H. Low mannose binding lectin predicts poor prognosis in patients with early rheumatoid arthritis: A prospective study. J Rheumatol 2001;28(4):728-734. 67. Garred P, Voss A, Madsen HO, Junker P. Association of mannosebinding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients. Genes Immun 2001;2(8):442-450. 68. Lee YH, Witte T, Momot T, Schmidt RE, Kaufman KM, Harley JB, Sestak AL. The mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: Two case-control studies and a meta-analysis. Arthritis Rheum 2005 (in press). 69. H. Nishimura, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999; 11:141-151. 70. Prokunina L, Castillejo-Lopez C, Oberg F, Gunnarsson I, Berg L, Magnusson V, et al. A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat Genet 2002;32(4):666-669. 71. Tokuhiro S, Yamada R, Chang X, Suzuki A, Kochi Y, Sawada T, et al. An intronic SNP in a RUNX1 binding site of SLC22A4, encoding an organic cation transporter, is associated with rheumatoid arthritis. Nat Genet 2003;35(4):341-348.

72. Helms C, Cao L, Krueger JG, Wijsman EM, Chamian F, Gordon D, et al. A putative RUNX1 binding site variant between SLC9A3R1 and NAT9 is associated with susceptibility to psoriasis. Nat Genet 2003;35(4):349-356. 73. Lindqvist AK, Steinsson K, Johanneson B, Kristjansdottir H, Arnasson A, Grondal G, et al. A susceptibility locus for human systemic lupus erythematosus (hSLE1) on chromosome 2q. J Autoimmun 2000;14(2):169-178. 74. Waterhouse P, et al. Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science 1995; 270:985-988. 75. Tivol EA, et al. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 1995;3:541-547. 76. Lee YH, Harley JB, Nath SK. CTLA-4 polymorphisms and systemic lupus erythematosus (SLE): A meta-analysis. Hum Genet 2005:116(5):361-367. 77. Barreto M, Santos E, Ferreira R, Fesel C, Fontes MF, Pereira C, et al. Evidence for CTLA4 as a susceptibility gene for systemic lupus erythematosus. Eur J Hum Genet 2004;12(8):620-626. 78. Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, et al. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 2004;36(4):337-338. 79. Begovich AB, Carlton VE, Honigberg LA, Schrodi SJ, Chokkalingam AP, Alexander HC, et al. A missense singlenucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004;75(2):330-337. 80. Kyogoku C, Langefeld CD, Ortmann WA, Lee A, Selby S, Carlton VE, et al. Genetic association of the R620W polymorphism of protein tyrosine phosphatase PTPN22 with human SLE. Am J Hum Genet 2004;75(3):504-507. 81. Velaga MR, Wilson V, Jennings CE, Owen CJ, Herington S, Donaldson PT, et al. The codon 620 tryptophan allele of the lymphoid tyrosine phosphatase (LYP) gene is a major determinant of Graves’ disease. J Clin Endocrinol Metab 2004;89(11):5862-5865. 82. Begovich AB, Caillier SJ, Alexander HC, Penko JM, Hauser SL, Barcellos LF, et al. The R620W polymorphism of the protein tyrosine phosphatase PTPN22 is not associated with multiple sclerosis. Am J Hum Genet 2005;76(1):184-187. 83. Wu H, Cantor RM, Graham DS, Lingren CM, Farwell L, Jager PL, et al. Association analysis of the R620W polymorphism of protein tyrosine phosphatase PTPN22 in systemic lupus erythematosus families: Increased T allele frequency in systemic lupus erythematosus patients with autoimmune thyroid disease. Arthritis Rheum 2005;52(8):2396-2402. 84. Ytterberg SR, Schnitzer TJ. Serum interferon levels in patients with systemic lupus erythematosus. Arthritis Rheum 1982;25(4):401-406. 85. Kim T, Kanayama Y, Negoro N, Okamura M, Takeda T, Inoue T. Serum levels of interferons in patients with systemic lupus erythematosus. Clin Exp Immunol 1987;70(3):562-569. 86. Matei I, Ghyka G, Savi I, Tudor A. Correlation of serum interferon with some clinical and humoral signs of systemic lupus erythematosus. Med Interne 1990;28(4):289-294. 87. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci USA 2003;100(5):2610-2615. 88. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau J, Pascual V. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 2003;197(6):711-723. 89. Kirou KA, Lee C, George S, Louca K, Papagiannis IG, Peterson MG, et al. Coordinate overexpression of interferon-alpha-induced genes in systemic lupus erythematosus. Arthritis Rheum 2004;50(12):3958-3967. 90. Sigurdsson S, Nordmark G, Goring HH, Lindroos K, Wiman AC, Sturfelt G, et al. Polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associated with systemic lupus erythematosus. Am J Hum Genet 2005; 76(3):528-537.

114. Shai R, Quismorio FP Jr, Li L, et al. Genome-wide screen for systemic lupus erythematosus susceptibility genes in multiplex families. Hum Mol Genet 1999;8:639-644. 115. Tsao BP, Cantor RM, Grossman JM, Kim SK, Strong N, Lau CS, et al. Linkage and interaction of loci on 1q23 and 16q12 may contribute to susceptibility to systemic lupus erythematosus. Arthritis Rheum 2002;46(11):2928-2936. 116. Johanneson B, Lima G, Von Salome J, et al. A major susceptibility locus for systemic lupus erythematosus maps to chromosome 1q31. Am J Hum Genet 2002;71:1060-1071. 117. Moser KL, Gray-McGuire C, Kelly J, et al. Confirmation of genetic linkage between human systemic lupus erythematosus and chromosome 1q41. Arthritis Rheum 1999;42:1902-1907. 118. Graham RR, Langefeld CD, Gaffney PM, et al. Genetic linkage and transmission disequilibrium of marker haplotypes at chromosome 1q41 in human systemic lupus erythematosus. Arthritis Res 2001;3:299-305. 119. Gaffney PM, Kearns GM, Shark KB, et al. A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families. Proc Natl Acad Sci USA 1998;95:14875-14879. 120. Tsao BP, Cantor RM, Kalunian KC, Chen CJ, Badsha H, Singh R, et al. Evidence for linkage of a candidate chromosome 1 region to human systemic lupus erythematosus. J Clin Invest 1997;99(4):725-731. 121. Magnusson V, Lindqvist AK, Castillejo-Lopez C, et al. Fine mapping of the SLEB2 locus involved in susceptibility to systemic lupus erythematosus. Genomics 2000;70:307-314. 122. Gray-McGuire C, Moser KL, Gaffney PM, et al. Genome scan of human systemic lupus erythematosus by regression modeling: Evidence of linkage and epistasis at 4p16-15.2. Am J Hum Genet 2000;67:1460-1469. 123. Gaffney PM, Ortmann WA, Selby SA, et al. Genome screening in human systemic lupus erythematosus: Results from a second Minnesota cohort and combined analyses of 187 sib-pair families. Am J Hum Genet 2000;66:547-556. 124. Nath SK, Quintero-Del-Rio AI, Kilpatrick J, et al. Linkage at 12q24 with systemic lupus erythematosus (SLE) is established and confirmed in Hispanic and European American families. Am J Hum Genet 2004;74:73-82. 125. Nath SK, Namjou B, Hutchings D, Garriott CP, Pongratz C, Guthridge J, et al. Systemic lupus erythematosus (SLE) and chromosome 16: Confirmation of linkage to 16q12-13 and evidence for genetic heterogeneity. Eur J Hum Genet 2004;12(8):668-672. 126. Edberg JC, Langefeld CD, Wu J, Moser KL, Kaufman KM, Kelly J, et al. Genetic linkage and association of Fcgamma receptor IIIA (CD16A) on chromosome 1q23 with human systemic lupus erythematosus. Arthritis Rheum 2002;46(8):2132-2140. 127. Nath SK, Kelly JA, Reid J, et al. SLEB3 in systemic lupus erythematosus (SLE) is strongly related to SLE families ascertained through neuropsychiatric manifestations. Hum Genet 2002;111:54-58. 128. Namjou B, Nath SK, Kilpatrick J, et al. Stratification of pedigrees multiplex for systemic lupus erythematosus and for selfreported rheumatoid arthritis detects a systemic lupus erythematosus susceptibility gene (SLER1) at 5p15.3. Arthritis Rheum 2002;46:2937-2945. 129. Nath SK, Namjou B, Garriott CP, et al. Linkage analysis of SLE susceptibility: confirmation of SLER1 at 5p15.3. Genes Immun 2004;5:209-214. 130. Kelly JA, Thompson K, Kilpatrick J, et al. Evidence for a susceptibility gene (SLEH1) on chromosome 11q14 for systemic lupus erythematosus (SLE) families with hemolytic anemia. Proc Natl Acad Sci USA 2002;99:11766-11771. 131. Sawalha AH, Namjou B, Nath SK, et al. Genetic linkage of systemic lupus erythematosus with chromosome 11q14 (SLEH1) in African-American families stratified by a nucleolar antinuclear antibody pattern. Genes Immun 2002;3(Suppl 1):S31-S34. 132. Quintero-Del-Rio AI, Kelly JA, Kilpatrick J, et al. The genetics of systemic lupus erythematosus stratified by renal disease: linkage at 10q22.3 (SLEN1), 2q34-35 (SLEN2), and 11p15.6 (SLEN3). Genes Immun 2002;3(Suppl 1):S57-S62. 133. Quintero-del-Rio AI, Kelly JA, Garriott CP, Hutchings DC, Frank SG, Aston CE, et al. SLEN2 (2q34-35) and SLEN1 (10q22.3) replication in systemic lupus erythematosus stratified by nephritis. Am J Hum Genet 2004;75(2):346-348.

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91. Barnes BJ, Kellum MJ, Pinder KE, Frisancho JA, Pitha PM. Interferon regulatory factor 5, a novel mediator of cell cycle arrest and cell death. Cancer Res 2003;63:6424-6431. 92. Lee JY, Goldman D, Piliero LM, Petri M, Sullivan KE. Interferongamma polymorphisms in systemic lupus erythematosus. Genes Immun 2001;2(5):254-257. 93. Nakashima H, Inoue H, Akahoshi M, Tanaka Y, Yamaoka K, Ogami E, et al. The combination of polymorphisms within interferongamma receptor 1 and receptor 2 associated with the risk of systemic lupus erythematosus. FEBS Lett 1999;453(1/2):187-190. 94. Lahita RG. The connective tissue diseases and the overall influence of gender. Int J Fertil 1996;41:156-165. 95. Lahita RG. The influence of sex hormones on the disease systemic lupus erythematosus. Springer Semin Immunopathol 1986;9:305-314. 96. Bissonnette R, Friedmann D, Giroux JM, Dolenga M, Hechtman P, Der Kaloustian VM, et al. Prolidase deficiency: A multisystemic hereditary disorder. J Am Acad Dermatol 1993;29(5 Pt 2):818-821. 97. Shrinath M, Walter JH, Haeney M, Couriel JM, Lewis MA, Herrick AL. Prolidase deficiency and systemic lupus erythematosus. Arch Dis Child 1997;76(5):441-444. 98. Urushihara M, Kagami S, Yasutomo K, Ito M, Kondo S, Kitamura A, et al. Sisters with alpha-mannosidosis and systemic lupus erythematosus. Eur J Pediatr 2004;163(4/5):192-195. 99. Chui D, Sellakumar G, Green R, Sutton-Smith M, McQuistan T, Marek K, et al. Genetic remodeling of protein glycosylation in vivo induces autoimmune disease. Proc Natl Acad Sci USA 2001;98(3):1142-1147. 100. Alanay Y, Balci S, Ozen S. Noonan syndrome and systemic lupus erythematosus: Presentation in childhood. Clin Dysmorphol 2004;13(3):161-163. 101. Amoroso A, Garzia P, Vadacca M, Galluzzo S, Del Porto F, Mitterhofer AP, et al. The unusual association of three autoimmune diseases in a patient with Noonan syndrome. J Adolesc Health 2003;32(1):94-97. 102. Dale RC, Tang SP, Heckmatt JZ, Tatnall FM. Familial systemic lupus erythematosus and congenital infection-like syndrome. Neuropediatrics 2000;31(3):155-158. 103. Aicardi J, Goutieres F. Systemic lupus erythematosus or AicardiGoutieres syndrome? Neuropediatrics 2000;31(3):113. 104. Rasmussen M, Skullerud K, Bakke SJ, Lebon P, Jahnsen FL. Cerebral thrombotic microangiopathy and antiphospholipid antibodies in Aicardi-Goutieres syndrome: Report of two sisters. Neuropediatrics 2005;36(1):40-44. 105. Kelly TE, Alford BA, Greer KM. Chondrodysplasia punctata stemming from maternal lupus erythematosus. Am J Med Genet 1999;83(5):397-401. 106. Austin-Ward E, Castillo S, Cuchacovich M, Espinoza A, CofreBeca J, Gonzalez S, et al. Neonatal lupus syndrome: A case with chondrodysplasia punctata and other unusual manifestations. J Med Genet 1998;35(8):695-697. 107. Wessels MW, Den Hollander NJ, De Krijger RR, Nikkels PG, Brandenburg H, Hennekam R, et al. Fetus with an unusual form of nonrhizomelic chondrodysplasia punctata: Case report and review. Am J Med Genet A 2003;120(1):97-104. 108. Desatnik H, Ashkenazi I, Regenbogen L. Retinitis pigmentosa and discoid lupus erythematosus. Metab Pediatr Syst Ophthalmol 1992;15(1-3):9-11. 109. Rahi AH, Addison DJ. Autoimmunity and the outer retina. Trans Ophthalmol Soc UK 1983;103(Pt 4):428-437. 110. Sekimoto M, Hayasaka S, Noda S, Setogawa T. Pseudoretinitis pigmentosa in patients with systemic lupus erythematosus. Ann Ophthalmol 1993;25(7):264-266. 111. Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 2001;411:207-211. 112. Moser KL, Neas BR, Salmon JE, et al. Genome scan of human systemic lupus erythematosus: Evidence for linkage on chromosome 1q in African-American pedigrees. Proc Natl Acad Sci USA 1998;95:14869-14874. 113. Cantor RM, Yuan J, Napier S, et al. Systemic lupus erythematosus genome scan: Support at 1q23, 2q33, 16q12 and 17q21 and novel evidence at 3p24, 10q23, 13q32 and 18q23. Arthritis Rheum 2004;50(10):3203-3210.

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134. Namjou B, Nath SK, Kilpatrick J, et al. Genome scan stratified by the presence of anti-double-stranded DNA (dsDNA) autoantibody in pedigrees multiplex for systemic lupus erythematosus (SLE) establishes linkages at 19p13.2 (SLED1) and 18q21.1 (SLED2). Genes Immun 2002;3(Suppl 1):S35-S41. 135. Namjou B, Kelly JA, Kilpatrick J, Nath SK, Scofield RH, Harley JB. Linkage at 5q14.3-15 in pedigrees multiplex for systemic lupus erythematosus (SLE) stratified by autoimmune thyroid disease (AITD). Arthritis Rheum 2005;52(11):3646-3650. 136. Nath SK, Kelly JA, Namjou B, et al. Evidence for a susceptibility gene, SLEV1, on chromosome 17p13 in families with vitiligorelated systemic lupus erythematosus. Am J Hum Genet 2001;69:1401-1406.

137. Spritz RA, Gowan K, Bennett DC, Fain PR. Novel vitiligo susceptibility loci on chromosomes 7 (AIS2) and 8 (AIS3), confirmation of SLEV1 on chromosome 17, and their roles in an autoimmune diathesis. Am J Hum Genet 2004;74(1):188-191. 138. Scofield RH, Bruner GR, Kelly JA, et al. Thrombocytopenia identifies a severe familial phenotype of systemic lupus erythematosus and reveals genetic linkages at 1q22 and 11p13. Blood 2003;101:992-997. 139. Nath SK, Namjou B, Kilpatrick J, et al. A candidate region on 11p13 for systemic lupus erythematosus: A linkage identified in African-American families. J Investig Dermatol Symp Proc 2004;9:64-67.

PATHOGENESIS

9

Hormonal Influences in the Expression of Systemic Lupus Erythematosus Virginia Rider, PhD, Xiaolan Li, MD, and Nabih I. Abdou, MD

INTRODUCTION The ability of the immune system to distinguish self from non-self is essential to maintain nonresponsiveness to self. Although self-recognition occurs during development, checkpoints that operate in the adult are critical for maintaining and establishing tolerance to self-antigens that appear after maturity.1 The process of self-tolerance continues in adult life. In autoimmune diseases, it is postulated that self-reactive cells expressing antigen receptors escape clonal deletion.2 Moreover, somatic mutations can potentially generate autoreactive antigen receptors.3 Loss of self-recognition, or autoimmunity, is more prevalent in females than in males.4 The timing of disease onset for some autoimmune disorders, such as lupus, occurs after puberty—suggesting that sex steroids could influence the gender bias of disease onset.5 The correlation between sexual maturity and some autoimmune diseases is consistent with the view that sex steroids enhance or suppress molecules that regulate pathways important in maintaining tolerance to self-antigens. In general, estrogens have been shown to promote or stimulate the immune response, whereas androgens and progesterone are considered more suppressive6-8 (Table 9.1).

SEXUAL DIMORPHISM AND AUTOIMMUNITY Several autoimmune diseases (including SLE, Sjögren syndrome, multiple sclerosis, rheumatoid arthritis, Graves’ disease, and Hashimoto thyroiditis) are sexually dimorphic because they occur more frequently in women than in men.4 In murine models of autoimmune diseases such as autoimmune encephalomyelitis and collagen-induced arthritis, females typically respond more robustly than males. The factors contributing to the development of autoimmune disease appear to involve lymphocyte-mediated mechanisms

that control disease pathogenesis, activation, or regression. Although it is still not known how sex hormones influence cells in the immune system, emerging evidence suggests that hormones affect cellular differentiation, cytokine production, T helper (Th)1 and Th2 cell polarization, MHC class 2 expression, and antigenpresenting cell recruitment or function8-10 (Table 9.1). Females have more immunoglobulins in circulation than males. Estrogen increases in vitro production of IgG and IgM in isolated human peripheral blood mononuclear cells.9 Estrogen may also affect antigen presentation by stimulating the differentiation of dendritic cells and the proliferation of naive CD4+ cells.11 Dendritic cells from mice induced to develop experimental allergic encephalomyelitis show a reduced capacity to present specific antigen to T-cells when cultured in medium containing estradiol. These cells express less TNF-a, interferon gamma, and IL-12 (suggesting that estrogen stimulates a shift to the Th2 phenotype and reduces inflammation).12 We have shown that estrogen suppresses TNF-a production by lupus T-cells in vitro and that it reduces apoptosis.13

HORMONES AND THE IMMUNE SYSTEM Hormones are chemical messengers secreted into the blood stream that bind to specific receptor proteins in the nucleus (steroid hormones) or on the surface (peptide hormones) of target cells. Prolactin is a hormone that is synthesized and secreted from the anterior pituitary gland. Receptors for prolactin are expressed on T- and B-cells,14 and 20 to 30% of patients with SLE have increased levels of prolactin in circulation.15,16 Treatment of NZB/NZW F1 mice with the prolactin inhibitor bromocriptine improves survival of the mice,17 suggesting that prolactin stimulates disease progression in SLE. Prolactin exerts its effects, at least in part, on B-cells by rescuing autoreactive

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TABLE 9.1 HORMONES AND SOME OF THEIR EFFECTS ON THE IMMUNE SYSTEM Hormone

Role in Immune System

Gene Targets

Estrogen (estradiol 17-β)

Immunostimulatory, protective for some Bcl-2, calcineurin, CD154, autoimmune diseases, activates B- & T-cells, IL-10, IL-4, interferon-gamma, decrease/increase apoptosis Foxp3, CD22, vcam-1, shp-1 IL-2, IL-2 receptor, cyclin A, TNF-α

Progesterone

Immunoprotective, gamma/delta TCR+

Progesterone-induced blocking factor (PIBF) Th-2 switch? IL-12

Androgens

Immunoprotective, B and T lymphocytes, thymic atrophy, thymocyte apoptosis

TGF-β, macrophage migration inhibitory factor

Dehydroepiandrosterone (DHEA)

Stabilizes disease symptoms in SLE, androgen precursor, anabolic steroid

Prolactin

Stimulates disease activity in SLE, T-cell proliferation, B-cell maturation

Interferon regulatory factor-1, Bcl-2, CD40

Hormones alter the expression of a variety of genes that control the proliferation, differentiation, and apoptosis of lymphocytes, macrophages, and dendritic cells of the immune system. Hormones also regulate the production of cytokines and cell adhesion molecules. Changes in the expression of many of these gene targets have been associated with pathogenesis in autoimmune diseases but the mechanisms involved require clarification.

B-cells that would normally be targeted for apoptosis.18 Continuous administration of prolactin to mice transgenic for anti-DNA antibody heavy chain (R4A-IgG2b, BALB/c) over a period of four weeks led to a lupus-like phenotype.18 In these mice, prolactin increased B-cell survival, presumably by up-regulating Bcl-2 and protecting autoreactive cells from apoptosis. Interestingly, prolactin effects are influenced by the genotype of these mice because R4A-IgG2b C57BL/6 mice maintain tolerance with the same prolactin treatment that induced a lupus phenotype in the R4AIgG2b BALB/c mice. Prolactin levels in human females with SLE correlate with disease activity,16,19 and conventional treatment for SLE reduces disease activity and prolactin levels in these patients. Prolactin expression increases in response to estrogen, and the use of estrogen receptor (ER) selective agonists indicates that both ER-a and ER-β stimulate prolactin secretion in the rat.20 It is now important to test if hyperprolactinemia results from a defect in estrogen-dependent regulation of the prolactin gene or if increased secretion of prolactin in SLE patients is controlled by other factors. Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are the main adrenal androgens produced in humans.21 DHEA is unique compared with other adrenal steroids because the levels in circulation fluctuate from birth into advancing age.22 DHEA is a precursor for sex hormone synthesis and acts as an anabolic steroid.23 Pharmacologic studies suggest that DHEA therapy increases testosterone levels in women but not in men.22 SLE patients treated with prednisone have

low levels of DHEA.23 In SLE patients with mild disease, DHEA improves or stabilizes disease symptoms.24,25

ESTROGENS AND THE IMMUNE SYSTEM The action of estradiol in the classical target tissues of the uterus and the breast has been extensively studied.26-28 In classical target cells, estradiol increases protein synthesis, stimulates proliferation, induces the production of growth factors and their receptors, and recruits infiltration of macrophages into target tissues such as the uterus. We are just beginning to understand how estradiol action can stimulate the immune system.29-31 It seems likely that estradiol will affect processes in target cells of the immune system in a manner similar to classical endocrine cell types, although the target genes may be different. Recent studies of endocrine effects on cells of the immune system support the growth-promoting effects of estradiol through increased production of cytokines32,33 and immunoglobulins in circulation.34,35 Estradiol enhances the proliferation of T-cells36 and macrophages37 and affects apoptosis of autoreactive B-cells.29,31,38 There is also exciting new evidence that suggests activation of the ER can be compartmentalized to specific cell types of the immune system.39-41 Mice lacking functional ER-a by target mutagenesis were induced to develop experimental allergic encephalomyelitis.39 Inflammation of the central nervous system (CNS) was initiated by autoantigen-specific T lymphocytes located in the CNS compartment of

that results from a loss of self-recognition, and the resulting secretion of autoantibodies leads to a variety of pathologic outcomes.52,53 Because estrogen can both stimulate and inhibit gene expression, it is not surprising that for some autoimmune diseases estrogen may be protective. The clinical assessment of various diseases suggests that estrogen can be beneficial in autoimmune disorders, including multiple sclerosis, rheumatoid arthritis, uveitis, and thyroiditis. These autoimmune diseases improve during pregnancy most likely owing to estrogen influence on inducing a shift from Th1 to Th2 cytokine profiles during gestation.54 In experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis, estrogen decreased production of proinflammatory cytokines (including tumor necrosis factor, interferon gamma, and IL-2).55 The protective effect in these autoimmune mice was postulated to be due to down-regulation of proinflammatory cytokines and an estrogen-dependent increase in Th2 cytokines such as IL-5.55 Mice induced to develop EAE benefit by the administration of low levels of estrogen prior to induction of the disease.56 One of the mechanisms involved in this protective effect of estrogen pretreatment is postulated to be an increase in the expression of Foxp3 and the subsequent regulation of genes involved in the maintenance of tolerance to self-antigens.40,56 Foxp3 appears to be a master controller of regulatory T-cells because increased expression of the FOXP3 protein stimulates expansion of CD4+ CD25+ T-cells.40 This regulatory T-cell subset is thought to play an essential role in the maintenance of tolerance.40,57 Several observations suggest that FOXP3 may exert its effects by transcriptional repression because it decreases the expression of IL-2 and may down-regulate a variety of other cytokines, including IL-4, TNF, and granulocyte macrophage colony-stimulating factor.57 Additional studies are now warranted to investigate the estrogendependent regulation of Foxp3 in gender-biased autoimmune diseases such as lupus.

ESTROGEN RECEPTORS

these mice. The authors39 suggest that estrogen may play a role in activating the vascular endothelium of the blood/brain barrier and inhibit T-cell adhesion in the CNS. Taken together, the emerging evidence suggests that estrogen regulates genes that promote growth and activity of cells that function in the immune system. Because estrogen is a key regulator of molecules involved in inflammation, any organ system in which inflammation pathways are activated is a likely target for estrogen action.10,42,43 Estrogen enters target cells from the circulation, and once in the cell the hormone binds to high-affinity receptor proteins (Fig. 9.1). The estrogen-dependent changes in cell behavior occur in response to the binding of the ligand-receptor complex to specific DNA sites that induce or suppress gene transcription.26,44-46 At least some of the potential gene targets in cells comprising the immune system have been identified.29,30,47 Estrogen up-regulates the expression of genes that are markers for B-cell and T-cell activation. Estrogen directly stimulates the expression of genes in mouse B-cells, which allows some autoreactive cells to escape B-cell-mediated apoptosis.29 Estrogen action thus rescues a population of autoreactive B-cells that would be deleted from the repertoire in the absence of the hormone. We have shown that two markers of T-cell activation, calcineurin and CD154, are up-regulated by estrogen in SLE but not in normal T-cells.48,49 Abnormal T-cell regulation of B-cells results in antibody/autoantibody secretion.50,51 SLE is a disorder

ESTROGEN RECEPTORS

Fig. 9.1 Classical mechanism of nuclear receptor action. The hormone estradiol17-b (E) is carried through the blood stream to the target cell. Estrogen enters the T-cell and binds to specific estrogen receptors (ERs) located in the nucleus. The hormone-receptor complex undergoes a conformational change and binds to specific regulatory sites along the DNA of target genes. DNA binding stimulates the transcription of specific genes (including calcineurin, CD154, and perhaps others), leading to new protein synthesis and altered cell function.

Sex steroid receptors are expressed in most cells that comprise the immune system.30,58-60 However, their function in the immune system remains to be clearly established. Steroid receptors are part of the nuclear receptor gene family that contains a large group of homologous proteins, including the steroids, thyroid hormone, retinoic acid, and vitamin D.44-46 In the general scheme of nuclear hormone receptor action, the hormone or ligand binds to the receptor and this interaction stimulates a conformational change that leads to DNA binding at specific sequences along target genes (see Fig. 9.1).

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Evidence suggests that the interaction of activated receptors with target genes can both increase and decrease transcription and thereby alter cell function. Although this classical mechanism of sex steroid action is still considered a valid model, more recent evidence indicates that additional layers of complexity are required for appropriate signaling events activated by nuclear receptors. It is now evident that there are a host of cofactors that interact with the receptors and modulate gene transcription.61 Although this is a relatively unexplored area of research in cells comprising the immune system, it is logical to assume that cofactors will be major modulators of hormone-dependent gene regulation in all cells that express steroid receptors. In this chapter, we focus specifically on information relating to the steroid hormone estrogen and the ER because compelling evidence is emerging for estrogen effects on the development and pathogenesis of autoimmune diseases.30,31,62-64 Estrogen effects are exerted through two receptor proteins termed estrogen receptor-alpha (ER-α) and estrogen receptor-beta (ER-β). The receptors are coded for by separate genes, but the proteins exhibit regions of high homology in the DNA and ligand binding domains.27,28 ER subtypes are modular in structure, with functional domains characteristic of all nuclear receptors (see Fig. 9.2). The amino termini of the ERs have diverged and only share about 18% homology. This divergent region may be important for the differential action of ER-a and ER-β in cells that coexpress both receptors.65,66 There is evidence that the magnitude of an estrogenic response to ER-a and ER-β activation varies depending on promoter context and cell type.66 Evidence suggests that ER-a and ER-β

function is not redundant in cells in which receptor subytpes are coexpressed. It is now important to investigate how differential receptor subtype action influences target cell behavior and if altered function underlies the development of endocrine-dependent autoimmune disease.

MOLECULAR MECHANISMS OF ESTROGEN ACTION IN HUMAN SLE Over the last 10 years our laboratory and others have investigated whether estrogen contributes to the gender bias of some autoimmune diseases such as SLE. The accumulating evidence30,31,67-70 is compelling in both humans and mice that estrogen is an important modulator of the immune system in females with SLE. Calcineurin is phosphatase that stimulates T-cell activation. We found that calcineurin transcripts and phosphatase activity increase in SLE but not in normal T-cells cultured with estradiol.71 SLE is the autoimmune disease with the largest number of detectable autoantibodies,72 suggesting that estrogen-dependent T-cell activation is a molecular link among hormone activation of the T-cell, increased T-/B-cell interactions, and SLE pathogenesis. We tested this postulate by measuring CD154 expression in T-cells cultured with estradiol. Estradiol increased CD154 transcripts and the amount of CD154 on the surface of SLE but not on normal T-cells.49 This sensitivity of SLE T-cells to estrogen suggests that there are differences between female SLE T-cells and normal T-cells. Our initial idea was that the differential sensitivity of SLE T-cells might be due to the expression, or lack of expression, of one of the ER

Fig. 9.2 Comparison of the shared structural features of all nuclear receptors with the human estrogen receptors (ER-α, ER-β). The nuclear receptor gene family is comprised of proteins that are modular in nature and contain six functional domains (A—F), as depicted in the top of the diagram. The human estrogen receptors, ER-a and ER-β, are coded for by separate genes and share a high degree of homology. The percent of homology, shown by numbers in brackets, between ER-a and ER-β is highest in the DNA binding domain (97%) and most divergent in the A/B region (18%) of the two receptor subtypes. AF = activation function. Amino acid numbers are indicated by numbers within each domain. See text for additional details.

CONCLUSIONS Recent studies highlight the importance of sex steroids in regulating various functions of the immune system. In the classical model of estrogen action, estrogen

binds to the receptor and interacts with specific DNA sequences along target genes. It is becoming clear, however, that there are many variations on this mechanism. The identification of two ERs that do not share redundant functions in cells in which both receptor subtypes are coexpressed complicates our understanding of estrogen action in target cells. ERs can interact with other transcription factors such as SP1 and AP-1 and increase transcription without binding to the classical ERE. Additional complexities have appeared with the identification of non-genomic estrogen responses that suggest that receptors interact with other signaling molecules at the cell surface and activate a variety of cellular responses.81 Transcriptional coregulators interact with ERs and the transcriptional machinery, and these interactions may be central to promoter- and cell-specific responses. Moreover, corepressors bind to steroid receptors and ensure that gene transcription does not occur inappropriately. The absence or inactivation of corepressors could lead to promiscuous transcription in response to estrogen. We have shown that SLE T-cells respond to estrogen differently than normal T-cells.30,64 Although the molecular basis underlying this estrogen sensitivity is unknown, the potential mechanisms are currently under investigation in our laboratory (see Box 9.1). In this chapter, we attempted to clarify some of the controversial effects of hormones (in particular, estrogen) in autoimmune disease. It is important to keep in mind that there are numerous immunocompetent cells that may respond differently to estrogen with respect to their differentiation, proliferation, and cytokine production. It seems that the diverse and opposing effects of estrogen in some autoimmune diseases will

CONCLUSIONS

(ER-a /ER-β). We used RT-PCR and Southern blotting to identify ER transcripts in human T-cells.71 Somewhat to our surprise, transcripts for both receptor subtypes were present in male and female T-cells from patients and normal controls. Thus, the absence of ER-a or ER-β mRNA in normal versus SLE T-cells (and in female versus male T-cells) cannot explain the differential sensitivity of female SLE T-cells to estrogen. We also explored the possibility that alternative splicing of ER-a could explain the differential sensitivity of SLE T-cells to estrogen. The results from that study58 do not support the idea that alternative splicing of a particular ER variant was associated with SLE T-cells. Moreover, the binding activity of ER-a (assessed by Scatchard analysis)73 suggests that the primary structure and ligand binding to ER-a is similar between SLE and normal T-cells. Many of the critical genes regulating the immune response are controlled at the post-transcriptional level.74 Sex steroids, including estrogen, are known to stabilize the mRNA from several genes.75 Calcineurin mRNA stability was assessed in both resting and activated T-cells.76 The results suggest that the estrogen-dependent increase in calcineurin is not due to increased mRNA stability at the time points analyzed in that study. Estrogen effects on CD154 mRNA stability in SLE T-cells are currently under investigation in our laboratory (Li and colleagues, unpublished). Development of compounds that act as receptorspecific agonists provide the tools to selectively activate one receptor subtype and study the response of estrogen-regulated genes.77,78 In a recent study, we compared the amount of estrogen receptor subtypes in T-cells and then measured the ability of receptor agonistspecific ligands to activate marker gene expression.79 We cultured SLE T-cells with ER-a and ER-β agonists and found that SLE T-cells respond to both agonists by increased expression of T-cell activation marker genes. The ability of SLE T-cells to respond to an ER-a agonist suggests that the low level of ER-a in these cells does not necessarily indicate a lowered level of transcriptional activity. Continuous turnover of ER-a through the proteasome-mediated degradation pathway is linked to transcriptional activity.80 Therefore, low levels of ER-a in SLE T-cells could be due to ER-a activation in these cells. Current experiments are testing this possibility by blocking the proteasome degradation pathway and measuring T-cell activation target gene expression.

BOX 9-1 ●

●

●

●

●

●

●

The major function of estrogen, working through estrogen receptors (ER-α and ER-β), is to alter gene transcription in target cells. ER-α and ER-β bind to DNA as homodimers and in cells that coexpress both receptor subtypes as heterodimers. ERs interact with coactivators and corepressors to modulate gene activity. Inappropriate interactions could alter normal estrogen responsiveness. ERs bind to specific sequences along the DNA of target genes, or ERs can alter transcription through protein-protein interactions at SP1 and AP-1 sites. ERs can alter gene transcription by stimulating the MAPK signal transduction cascade. The significance of this pathway in normal cell function and pathogenesis is not clear. Skewed metabolism of estradiol can lead to the production of biologically active metabolites and can sustain estrogen effects on target cells. Transcriptional regulation may require appropriate turnover of ER-α via the proteasome degradation pathway.

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be clarified only after we more clearly understand the effects of this hormone on the various immune cell types, on the genes that are estrogen responsive, and the signal transduction pathways that are stimulated in response to estrogen action. Although the details of molecular action discussed in this chapter may seem a long way from the bedside, understanding differences in ER subtypes and their function should ultimately lead to highly specialized treatments. ERs are ligand-activated transcription factors that regulate estrogen-responsive genes. Evidence suggests that estrogen is a major modulator, perhaps even a master regulator, of the immune system. Clarification of the biological roles of ER-a and ER-β is essential to further evaluate estrogen effects, particularly in autoimmune diseases with strong gender bias such as SLE.

FUTURE DIRECTIONS As we seek to understand estrogen action, it is important to tie in other hormones that may contribute to autoimmunity. It is well established that estrogen-dependent feedback mechanisms regulate gonadotropin-releasing hormone and the production of follicle-stimulating and -luteinizing hormones. Moreover, prolactin expression can increase in response to estrogen. It is therefore important to investigate the interplay among these hormones and to identify how abnormalities in their regulation may contribute to human autoimmune diseases. Gene profiling of SLE immune cells stimulated with estrogen and with receptor-specific agonists and antagonists can now provide an overview of gene expression changes that contribute to autoimmune disease. This research approach seems of particular importance for translating basic research into clinical applications. For example, the identification of the ER subtypes involved in stimulating the immune response could permit a chemical blockade of single-receptor subtype and reduce potential side effects. ER-specific blockers such as Faslodex are currently available and may provide a safe treatment for some patients in whom estrogen contributes to disease progression.

Although animal models of autoimmune disease have contributed enormously to our understanding of these disorders, the mechanisms are complicated and diverse. It is our view that mechanistic studies are required in human autoimmune diseases because the information gleaned from animal models may not always be directly applicable to humans. Understanding the role of estrogen in human disease and whether the hormonal effects are global in nature or are restricted to the maintenance and survival of immunoreactive clones is an essential first step in this process. It is challenging and rewarding to unmask the mechanisms that play a role in the overexpression of the immune system that leads to the female preponderance to develop autoimmune disease. This challenge requires systematic studies that include clinical, serologic, cell, and molecular approaches. The future is bright, as more scientists and clinicians work together using available technology in well-defined cohorts of patients with autoimmune disorders followed during the various phases of disease. Understanding the role of estrogen and its receptors in the immune system is expected to facilitate the use of current drugs and to lead to the development of new pharmaceuticals for the treatment of estrogenassociated pathologies. New knowledge about the interplay among hormones and signal transduction pathways that are crucial in maintaining self-tolerance in the adult will present novel treatments for SLE patients and improve the quality of their lives.

ACKNOWLEDGMENTS The research discussed in this chapter has been supported by NIH grants AI-49272 and RR-1647 from the Idea Network of Biomedical Research Excellence (K-INBRE), by the Evans Memorial fund, and by private funds from the Center for Rheumatic Diseases. We thank our patients who volunteered in these research efforts. We are grateful for the assistance of Cindy Greenwell (research coordinator) and Hope Schreck (for secretarial help) in preparing the manuscript.

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PATHOGENESIS

10

T-Cells and Systemic Lupus Erythematosus Robert Hoffman, DO and Marcos E. Maldonado, MD

INTRODUCTION AND BACKGROUND The pathogenesis of systemic lupus erythematosus (SLE) is complex and appears to be influenced by environmental and genetic factors. Currently, there is substantial evidence that the adaptive and innate immune system both contribute to the development of SLE. The role of T-cells, B-cells, cytokines, cell surface receptors, and dendritic cells in pathogenesis are reviewed elsewhere in this book. The focus of this chapter is on the role of T-cells in SLE, with an emphasis on what has been learned directly from studies on T-cells from patients with SLE. T-cells appear to play a central role in the complex series of events that culminates in the development of SLE. In this chapter, we consider loss of immunologic tolerance, autoantigen reactive T-cells, abnormalities of T-cell signaling, autoantigen processing and presentation, abnormalities of T-cell co-stimulation, and the role of T regulatory cells in disease pathogenesis. A model of how these factors may influence disease pathogenesis is shown in Fig. 10.1. It is well established that T-cells are a central component of the adaptive immune system, and numerous observations support a role for T-cells in SLE pathogenesis.1-4 Early descriptive studies reported (and subsequent studies have repeatedly confirmed) that T-cells obtained from patients with active SLE display markers of activation (such as increased expression of HLA-DR on the cell surface2) and exhibit gene expression typical of cellular activation, including up-regulation of proto-oncogenes such as c-myc.5 Histologic studies have demonstrated close physical proximity of activated T-cells to sites of tissue injury. Studies have also documented infiltration of affected organs, such as the salivary glands and kidneys with T-cells.4,6-8 A recent example was a study using renal biopsy in SLE patients where it was found that infiltrating T-cells in the kidneys can be oligoclonal based on restricted T-cell receptor usage. These T-cells also exhibit evidence of recent activation.7

Indirect evidence of a role for T-cells in the pathogenesis of SLE has been suggested by immunogenetics studies where associations between select major histocompatibility complex (MHC) alleles and SLE have been reported.9 These studies have been interpreted as supporting a central role for T-cells in SLE pathogenesis, either through direct effects on T-cell-peptide-MHC antigen selection and presentation or through indirect effects on T-cell repertoire selection. Furthermore, in support of such a hypothesis antinuclear antibody-producing B-cells exhibit evidence of having undergone T-cell-derived cytokine-driven affinity maturation. Such B-cells are of the IgG isotype, and this IgG can be present in high levels in serum.8 Immunoglobulin heavy and light chain gene rearrangements in autoantibody-producing murine B-cells, and in the more limited number of instances where they have been examined in SLE patients, demonstrate nucleotide substitutions typical of an antigen-driven T-cell-dependent immune responses. These studies indirectly support a central role for T-cells in disease pathogenesis.

LOSS OF IMMUNOLOGIC TOLERANCE IN SLE To participate in the pathogenesis of SLE, T-cells presumably must escape normal immunologic tolerance. There are a number of possible mechanisms whereby immunologic tolerance may be lost by T-cells, leading to autoimmunity. Some of the proposed mechanisms are summarized in Table 10.1. It has been demonstrated that T-cells reactive with self-antigens can escape thymic selection and are detectable in the peripheral blood of both SLE patients and healthy individuals.10-16 The T-cell receptor on these T-cells has low avidity for self-antigens and this may in part be why they are able to escape deletion in the thymus. In healthy individuals, such self-reactive T-cells are presumably kept from expanding by regulatory mechanisms. For example, during T-cell activation

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Surface Ig, FcR or other receptors capturing apoptic debris including cleaved 70kD antigen Antigen presented in context of HLA-DR

Ag

Abnormal Ag processing?

T cell receptor

Abnormal activation threshold for cell signaling? Signal

Ag

Process Ag

Debris including DNA and RNA stimulating ACP through Toll-like receptor (TLR) signaling

CD4+ T cell

Cytokines

HLA synthesis

Accessory molecules Excess co-stimulation?

Antigen presenting cell Dendritic cell, B cell or macrophage

Tissue damage

T cell help? CD4, CD8, T cells, B cells, neutrohils, other cells

Autoantigen recognition B cell

Apoptotic debris

Autoantibody

Innate immunity activation (TLRs) Presentation and processing of autoantigens

Maturation and survival Tissue damage

Antigen presentation

Dendritic cell Autoantigen recognition

T cell

Fig. 10.1 The hypothetical points where T-cells interacting with B-cells, dendritic cells, macrophages, apoptotic debris or necrotic tissue products, CD8+ cells, neutropils, other cells and serum factors could lead to loss of immunologic tolerance. Ultimately, these result in tissue injury and the development of clinical manifestations of SLE. It appears that the innate and adaptive immune systems can contribute to the development of SLE. In this model, apoptotically modified or necrotic debris that is not rapidly cleared through noninflammatory mechanisms (including DNA, RNA, and proteins bound to these) provide proinflammatory innate immune signals and is opsinized by antigen-presenting cells (dendritic cells or B-cells). Through antigen-specific signals, MHC-bound autoantigenic peptides, and nonspecific proinflammatory second signals, T-cells become activated and mature into effector and memory T-cells. These in turn provide maturation and differentiation signals to autoantibody-producing cells, recruit other cells to sites of inflammation, and directly mediate tissue injury (inducing more cellular debris). Under these conditions, B-cells undergo cytokine-induced affinity maturation, produce high quantities of complement-fixing antibodies that also induce injury at sites of autoantigen recognition, or form circulation immune complexes. In this model, factors that contribute to prolonged exposure to apoptotic debris, resistance to activation-induced cell death of B- and T-cells, and ineffective generation of regulatory cells may promote autoimmunity.

96

Central tolerance: ● Defect(s) that allow escape of autoreactive T-cells into the periphery Peripheral tolerance: Failure of mechanisms of anergy resulting in expansion of autoreactive T-cells

●

●

Defect in activation-induced cell death with persistence of expanded autoreactive T-cells

●

Ineffective control or elimination of autoreactive T-cells by regulatory cells

in healthy individuals such autoreactive T-cells may become anergic, undergo activation-induced cell death, or be limited in their expansion by other regulatory cells. Under the pathologic conditions found in SLE, however, autoreactive T-cells persist and expand and thereby contribute to the development of disease. Theoretical mechanisms for the expansion of such pathogenic T-cells include stimulation by newly exposed cryptic T-cell epitopes on self-antigens during apoptosis or during antigen processing, inappropriate T-cell help, excess T-cell co-stimulation, abnormal activation thresholds for T-cell signaling pathways, and inadequate control of T-cell growth by regulatory cells. The evidence for each of these in SLE is considered in this chapter.

AUTOANTIGEN-REACTIVE T-CELLS Self-reactive T-cells may escape normal mechanisms of immunologic tolerance, expand, and be detectable in the peripheral blood of patients with SLE.10-16 T-cells reactive with a number of lupus nuclear autoantigens [including DNA; histones; the small nuclear ribonucleic proteins Sm-B, Sm-D, U1-70kD, and U1-A; and heterogeneous nuclear ribonucleoprotein (hnRNP) A2 protein] have been isolated from the peripheral blood of SLE patients and characterized. These are outlined in Table 10.2.10-12,17-32

TABLE 10.2 ANTIGEN-REACTIVE T-CELLS ●

DNA-histone-reactive T-cells

●

Small nuclear ribonucleoprotein-reactive T-cells ❍ Sm-B-reactive T-cells ❍ Sm-D-reactive T-cells ❍ U1-70kD-reactive T-cells ❍ U1-A-reactive T-cells

●

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2-reactive T-cells

Rajaogopalan et al. were the first to describe human T-cell lines reactive with double-stranded DNA isolated from patients with SLE.17 The activated T-cells they identified selectively augmented the production of pathogenic IgG anti-DNA antibodies ex vivo, supporting the conclusion that they might have a role in pathogenesis.17 Datta and colleagues subsequently characterized chromatin-reactive T-cells in SLE in detail and reported that these T-cells are typically CD4+, can provide help to anti-DNA and antihistone antibody-producing B-cells, and that they have restricted T-cell receptor CDR3 usage with characteristics of antigen selection by a limited number of cationically charged antigenic epitopes. They mapped the major T-cell epitopes present on the core nucleosomal histone protein complex to four regions: histone H2B amino acid residues 10 through 33, histone H3 residues 85 through 105, histone H4 residues 16 through 39, and histone H4 residues 71 through 94. They demonstrated that these autoantigenic peptides can be promiscuously presented by several HLA-DR alleles. Furthermore, they found that nucleosomereactive human T-cells produce substantial quantities of INF gamma. They found in parallel studies done in a murine model system that such nephrogenic complement-fixing antinucleosome autoantibodies belong to INF gamma–dependent IgG subclasses. They subsequently proposed that expansion of these low-affinity chromatin autoantigen-reactive T-cells is essential for sustaining anti-DNA/histone autoantibody-producing B-cells.17-19 In addition to DNA and nucleosomes, human T-cells reactive with various small nuclear ribonucleoprotein self-antigens (including Sm-B, Sm-D, U1-70kd, and U1A) have been identified and characterized. The characteristic features of autoantigen-reactive T-cells that have been described are outlined in Table 10.3.10-12,20,21,23-32 These small nuclear ribonucleoproteins are ubiquitous self-antigens that are components of the spliceosome complex, which physiologically functions to excise introns and generate messenger RNA transcripts lacking intervening RNA.33,34 Sm-reactive T-cell lines and T-cell clones reactive with the Sm-D or Sm-B small nuclear ribonucleoproteins were first described by Hoffman and colleagues from patients with SLE.10 U1 small nuclear ribonucleoprotein-reactive peripheral blood T-cells were first reported by O’Brien and colleagues from patients classified as SLE.20 T-cell clones from connective tissue disease patients reactive with the U1-70kD small nuclear ribonucleoproteins antigen were described by Hoffman and colleagues.23,26 Okubo and colleagues were the first to describe peripheral blood mononuclear-cell-derived CD4+ T-cells from SLE or MCTD patients that reacted to the U1-A small nuclear ribonucleoprotein.21 Subsequently, such various small nuclear ribonucleoprotein-reactive T-cell clones have

AUTOANTIGEN-REACTIVE T-CELLS

TABLE 10.1 IMMUNE TOLERANCE: PROPOSED DEFECTS IN CENTRAL OR PERIPHERAL TOLERANCE IN SLE

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98

TABLE 10.3 CHARACTERISTICS OF AUTOANTIGEN-REACTIVE T-CELLS ●

CD4 T cell phenotype typical but CD8 also described

●

Provide T-cell help to autoantibody-producing cells

●

Produce large amounts of interferon gamma and alpha by some

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Produce variable quantities of Th2-like cytokines, IL-2, and IL-4

●

T-cells recognize a very limited number of T-cell epitopes on the autoantigen

●

T-cell epitopes reside on RNA binding domains or Sm protein-protein motifs of antigens

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Limited diversity of T-cell receptors utilized; most use alpha/beta T-cell receptors

●

Persistence over time of clones expressing similar or identical T-cell receptor isolated from peripheral blood of patients

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HLA-DR used as restriction element by most autoantigen-reactive T-cells

been extensively characterized (see Table 10.3). Typically, they are CD4-positve T-cells that produce large amounts of IFN gamma, moderate quantities of IL-2, and variable quantities of IL-4 and IL-10.4,23-30 They recognize antigen in the context of HLA-DR. T- and B-cell responses are linked in SLE, and both small nuclear ribonucleoprotein and hnRNP-reactive T-cells can provide B-cell help for autoantibody production.31 T-cell epitope mapping studies of human T-cell clones reactive with the small nuclear ribonucleoproteins U1-70kD, Sm-B, and Sm-D have been done to determine the precise regions recognized on the autoantigen by T-cells. These studies have revealed that there are limited T-cell epitopes on these antigens. Interestingly, virtually all T-cell antigen recognition regions (or so-called T-cell epitopes) reside within functional regions of the protein—either within the Sm motifs for Sm-B and Sm-D or within the RNA binding domain for U1-70kD and hnRNP.27,29,34 T-cell clones have recently been identified and characterized from patients with SLE that are reactive with another nuclear ribonucleoprotein antigen known as hnRNP A2.31 Greidinger and colleagues cloned human T-cells reactive with hnRNP A2 from SLE patients and found that such hnRNP-reactive T-cells when cocultured in vitro with autologous B-cells could augment anti-hnRNP autoantibody production.31 Haffman and Steiner also identified and characterized hnRNPreactive T-cells and found that similar to the findings described previously for U1-70kD T-cell epitope mapping, hnRNP-reactive T-cells also recognize the RNA binding domain portion of the antigen.32 Collectively, these studies reveal a recurring theme: ribonucleoproteinreactive T-cells are directed against highly conserved

regions that function to bind their associated RNA (the RNA binding domain of U1-RNP and hnRNP) or their associated proteins (Sm protein-protein binding domains). Finally, a novel mechanism for autoantigen “cross reactivity” by T-cells in SLE has recently been reported. De Silva-Udawatta and colleagues reported that T-cell receptor usage by small nuclear ribonucleoproteinreactive T-cells can have significant flexibility or “plasticity”.30 For example, they found that a single T-cell receptor can recognize two distinct small nuclear ribonucleoprotein autoantigenic peptides that have no apparent sequence homology.30 This cross reactivity is limited to the U1-70kD and a Sm-B peptide. However, a series of other closely related small nuclear ribonucleoprotein-derived peptides did not cross stimulate the T-cell receptor. These studies indicate that there are now a number of distinct mechanisms for immunologic cross reactivity that may result in loss of tolerance in SLE, including cross reactivity occurring at the level of the T-cell receptor.

T-CELL SIGNALING IN SLE In the previous sections we have considered the findings that immunologic tolerance and the expansion of self-reactive T-cells may play an important role in the pathogenesis of SLE. We now consider a series of studies indicating that there are defects in T-cell signaling pathways in SLE that may also play a significant role in disease pathogenesis. A summary of some of the abnormalities of T-cell signaling that have been reported for T-cells from patients with SLE is provided in Table 10.4. The interaction of the T-cell receptor with its cognate antigenic ligand peptide (in this case an autoantigen) bound to an MHC molecule on the surface of an antigenpresenting cell results in a series of signaling events. Binding of an autoantigenic peptide-MHC complex to TABLE 10.4 T-CELL SIGNALING PATHWAY ABNORMALITIES IN SLE ●

Exaggerated intracellular calcium responses

●

Deficient IL-2 production

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Increased intracellular phosphorylation

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Decreased T-cell receptor/CD3 zeta chain complex expression

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Defective formation of lipid rafts/immunologic synapse

●

Mutations in type I protein kinase A (PKA) regulatory subunit alpha

●

Deficient mitogen-activated protein kinase (MAPK) activity

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Resistance to apoptosis; mediate via cyclo-oxygenase-2dependent pathway

●

Decreased activation-induced cell death

AUTOANTIGEN PROCESSING AND PRESENTATION Antigen-presenting cells, including B-cells and dendritic cells, are central to T-cells’ recognition of antigen and subsequent downstream immunologic events. B-cells and dendritic cells are reviewed in detail elsewhere in this book. It is important here to briefly consider the role of B-cells and dendritic cells in antigen presentation to T-cells in SLE. B-cells and dendritic cells can present autoantigen to T-cells, and dendritic cells may be of particular importance in their position at the interface of innate and adaptive immunity (see Fig. 10.1). There are a number of mechanisms where antigen presentation could influence T-cell response. MHC selection of antigen peptides during immunologic development could influence T-cell repertoire development and peripheral tolerance.

TABLE 10.5 ANTIGEN PROCESSING AND PRESENTATION ●

MHC may influence peptide binding and/or T-cell repertoire selection

●

Activation or “danger” signal may be triggered via innate immune Toll-like receptors (TLR) ❍ RNA can stimulate via TLR-3 and possibly TLR-7 ❍ DNA can stimulate via TLR-9

●

TLR-dependent or TLR-independent signaling may occur following internalization of RNA/DNA-containing complexes

●

Autoantigen modification by proteases, oxidation, or other processes reveals new epitope(s)

●

Defective clearance of apoptotic material may influence antigen-processing pathways and results in prolonged exposure of antigens to immune cells

If during antigen processing/presentation dendritic cells encounter a so-called “danger” or activation signal it may switch from noninflammatory disposal of cellular debris to inflammatory processing/ presentation of dying apoptotic or necrotic cells.46-48 These mechanisms are outlined in Table 10.5. Recent studies have identified the potential role of cell-derived DNA and RNA in triggering inflammatory responses. Chromatin containing CpG motif-rich DNA or small nuclear ribonucleoprotein antigenic complexes containing RNA could potentially trigger “danger” responses in the pathogenesis of SLE by providing accessory signals through TLR9 on human dendritic cells, macrophages, or B-cells, or through TLR3 on human dendritic cells.49-52 Recent studies suggesting a role for activation of TLR3 and TLR7, as well as TLR-independent signaling pathways, in SLE have been published. These studies indicate that TLR4, TLR9, and TLR3 can up-regulate MHC (and can influence antigen processing pathways), potentially linking antigen epitope selection to activation of TLRs via CpG DNA or RNA through the binding of TLR9 to CpG DNA-chromatin or TLR3 to RNA small nuclear ribonucleoprotein complexes, respectively.49-52 In addition to the recognition of the potential for DNA and RNA to activate cells through the TLR pathway, there is an emerging body of evidence demonstrating that during apoptosis autoantigens can be modified by protease cleavage (caspases or granzyme B), by oxidative cleavage, or by other modification mechanisms. There is also evidence that these modifications of selfantigens might reveal previously cryptic epitopes or “neoepitopes” to the immune system.53-58 In the T-cell epitope mapping studies previously reviewed, it has been found that there are limited numbers of T-cell epitopes on histones H2B, H3, and H4, and on the small nuclear ribonucleoproteins Sm-B, Sm-D, and U1-70kD. The presence of a limited number of T-cell epitopes could

AUTOANTIGEN PROCESSING AND PRESENTATION

the T-cell receptor in turn leads to the activation of a cascade of protein kinases, mobilization of calcium, and eventually the translocation of activated transcription factors to the nucleus that in turn up-regulate a variety of inducible genes. Following cell activation, activationinduced cell death through apoptosis or other death pathways may occur. A number of these events appear to be altered in SLE (see Table 10.4). At the cell surface T-cell receptor engagement with antigen results in a series of downstream signaling events. Abnormalities in several of these have been observed in SLE and they appear to be unrelated to disease activity, supporting the hypothesis that they may play a role in pathogenesis. Tsokos’ laboratory has reported and extensively characterized abnormal IL-2 production, exaggerated calcium responses, and increased signaling events that follow T-cell activation using mitogen or antigen activation of T-cells.35-41 They and others have extensively characterized the defects in subsequent signaling events found in T-cells from patients with SLE. Tsokos and colleagues have recently reviewed this topic.35 In related studies, Kammer’s laboratory has reported identification of an mRNA mutation of type I protein kinase A regulatory subunit alpha in T-cells from patients with SLE that may contribute to abnormal T-cell function in SLE.42,43 In additional studies on cell signaling, Cedeno and colleagues evaluated the Ras/Raf/mitogen-activated protein kinase (MAPK) cascade in patients with SLE and concluded that a deficiency in MAPK activity in activated SLE T-cells they observed was caused by aberrant transduction of a signal downstream of the T-cell receptor but proximal to the activation of Ras.44 Other studies in SLE have additionally reported abnormalities of cell activation, including resistance to apoptosis through a cylco-oxygenase-2-dependent pathway and decreased activation-induced cell death.45

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reflect selective antigen processing and presentation of cryptic epitopes by antigen-presenting cells to T-cells. In addition, defects in the clearance of apoptotic material have been reported in SLE, and it has been proposed that prolonged exposure to apoptotic material results in the prolonged stimulation of T-cells that could also be an important factor in breaking T-cell tolerance.48,58

ABNORMALITIES OF T-CELL CO-STIMULATION Another potential mechanism for the development of autoimmunity is excess co-stimulation during T-cell/antigen engagement. Co-stimulatory and adhesion molecules play an important role in forming the so-called “immunologic synapses” that ultimately lead to T-cell receptor signal transduction during antigen recognition. Abnormalities of these could lead to enhanced T-cell/antigen interactions and subsequent abnormal cellular signaling events (Table 10.6). Two groups have reported that there is abnormal expression of molecules involved in the co-stimulatory CD40-CD40 ligand pathway on T-cells from patients with SLE.59-62 They found that there are abnormalities of CD40 ligand (CD40L, also known as CD154) expression on lupus B- and T-cells and proposed that this leads to prolonged co-stimulation of T-cells. They postulated that hyperexpression of CD40L leads to triggering of lupus T-cells by subthreshold stimuli and the presentation of apoptotic material by APC (including dendritic cells, macrophages, and B-cells).47-62 Finally, they reported that T-cells from SLE patients exhibited regulatory defects in the Cb1 and mitogen-activated protein kinase pathway (which is key to prolonged overexpression of CD40L) and suggest that this may be the mechanism of abnormal CD40L expression.59 A series of adhesion molecules plays an important role in T-cell/APC interactions, as they serve to assist in stabilizing T-cell receptor-antigen interactions. In studies examining the role of adhesion molecules in SLE pathogenesis, Richardson and colleagues have observed that there can be overexpression of a key accessory adhesion molecule in SLE that appears to be important in pathogenesis. This is known as Leukocyte-Function-Associated antigen (LFA-1, also known as CD11a). LFA-1 is a member of the beta-2 integrin family that can interact

with intercellular adhesion molecule-1 (ICAM-1) during antigen-specific T-cell activation. Richardson and colleagues have reported that DNA hypomethylation of LFA-1 gene induces a lupus-like syndrome in vivo in an animal model, and they have linked DNA methylation status with the development SLE in patients.63-66 The mechanism of this remains to be fully defined. Finally, additional molecules may be involved in aberrant signaling in SLE. For example, polymorphism of the programmed cell death receptor gene (PDCD1) has been reported as a susceptibility factor for the development of SLE in some populations, although the mechanism of these associations also remains to be fully elucidated.67

T REGULATORY CELLS There is currently significant interest in understanding the role of regulatory cells in maintenance of peripheral tolerance68-70 (see Table 10.7). Cells expressing high levels of CD4 and CD25 (the alpha chain of the IL-2 receptor) molecules on their surface and expressing high intracellular levels of the gene for Fox P3 have been shown to have an important role in regulating activated T-cells and preventing the development of autoimmunity. In SLE, one small study has reported that there was a deficiency of circulating CD4-postive/CD25-postive T-cells of the T regulatory phenotype in SLE when compared to health controls.71 Deficiency of such cells in SLE could be a factor in the development of disease. In addition, natural killer (NK) and CD8-positive cells can also have regulatory functions, and deficiency or abnormalities of function of these could potentially contribute to the development of SLE.72 Additional studies of these regulatory cells in SLE pathogenesis are ongoing and will continue to advance the science.

CONCLUSIONS In summary, T-cells play a central role in the adaptive immune response and appear to be of central importance in the complex pathogenesis of SLE (see Fig. 10.1). It is widely accepted that defects in peripheral tolerance occur in SLE and that failure to normally control the expansion of autoreactive T-cells in the periphery can contribute to the development of autoimmunity (Tables 10.1 and 10.7). Currently, there is great interest

TABLE 10.6 CO-STIMULATION AND CELL ADHESION PATHWAY ABNORMALITIES IN SLE

100

●

Excess co-stimulation ❍ CD40L

●

Adhesion molecules ❍ LFA-1 methylation

●

Programmed cell death receptor 1 (PD1/PDCD1) gene polymorphisms associated with SLE in some populations

TABLE 10.7 REGULATORY CELLS THAT MAY BE IMPORTANT IN SLE ●

CD4-positive, CD25-positive, Fox P3 expressing T regulatory (Tregs)

●

CD8-positive regulatory cells?

●

Natural killer (NK) regulatory cells?

has reviewed the characteristics of autoreactive human T-cells that have been identified from patients with SLE, including those that are reactive with DNA, histones, small nuclear ribonucleoproteins, and heterogeneous nuclear ribonucleoprotein A2 (Tables 10.2 and 10.3). In addition, a series of abnormalities in SLE have been described for T-cell signaling (Table 10.4), antigen processing/presentation (Table 10.5), and co-stimulation (Table 10.6) that may also play key roles in the complex series of immunologic events that result in the development of SLE.

REFERENCES

in the role of regulatory cells in autoimmunity and it is hoped that it may be possible to manipulate T regulatory cells for treatment of SLE.72 Although defects in central tolerance has not been the favored theory for the development of systemic, recent studies on the Aire gene in humans and in animal models suggest that central defects in tolerance (such as those seen with Aire deficiency) may also contribute to autoimmunity and be independent of defects in peripheral tolerance or regulatory T cells.73 Detection of peripheral tolerance defects remains of great importance, and this chapter

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52. Hoffman RW, Gazitt T, Foecking MF, Ortmann RA, Misfeldt M, Jorgenson R, et al. U1-RNA induced innate immunity signaling. Arthritis Rheum 2004;50:2891-2896. 53. Greidinger EL, Foecking MF, Ranatunga S, Hoffman RW. Apoptotic U1-70kD is antigenically distinct from the intact form of the U1-70kD molecule. Arthritis Rheum 2002; 45:1264-1269. 54. Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK. Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum 2005;52:1491-1503. 55. Zhuang H, Narain S, Sobel E, Lee PY, Nacionales DC, Kelly KM, et al. Assiociation of anti-nucleoprotein autoantibodies with upregulation of Type 1 interferon-inducible gene transcripts and dendritic cell maturation in systemic lupus erythematosus. Clin Immunol 2005 (Epub ahead of print). 56. Casciola-Rosen LA, Anhalt G, Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apototic keratinocytes. J Exp Med 1994;179:1317-1330. 57. Andrade F, Casciola-Rosen L, Rosen A. Apoptosis in systemic lupus erythematosus: Clinical implications. Rheum Dis Clin North Am 2000;26:215-227. 58. Walport MJ. Complement and systemic lupus erythematosus. Arthritis Res 2002;4:S279-293. 59. Desai-Mehta A, Lu L, Ramsey-Goldman R, Datta SK. Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest 1996;97:2063-2071. 60. Yi Y, McNuerney M, Datta SK. Regulatory defects, in Cb1 and mitogen-activated protein kinase (extracellular signal-related kinase) pathways cause persistent hyperexpression of CD40 ligand in human lupus T cells. J Immunol 2000;165:6627-6634. 61. Vakkalanka RK, Woo C, Kirou KA, Koshy M, Berger D, Crow MK. Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis Rheum 1999;42:871-881. 62. Crow MK, Kirou KA. Regulation of CD40 ligand expression in systemic lupus erythematosus. Curr Opin Rheumatol 2001; 13:361-369. 63. Lu Q, Wu A, Richardson BC. Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus inducing drugs. J Immunol 2005;174:6212-6219. 64. Richardson BC, Powers D, Hooper F, Yung RL, Rourke KO. Lymphocyte function-associated antigen 1 overexpression and T cell autoreactivity. Arthritis Rheum 1994;37:1363-1372. 65. Yang J, Deng C, Hemati N, Hanash S, Richardson BC. Effect of mitogenic stimulation and DNA methylation on human T cell DNA methyltransferase expression and activity. J Immunol 1997;159:1303-1309. 66. Kammer GM, Perl A, Richardson BC, Tsokos GC. Abnormal T cell signal transduction in systemic lupus erythematosus. Arthritis Rheum 2002;46:1139-1154. 67. Prokunina L, Castillejo-Lopez C, Oberg F, Gunnarsson I, Berg L, Magnusson V, et al. A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat Genet 2002;32:666-669. 68. Shevach EM. Regulatory T cells in autoimmunity. Ann Rev Immunol 2000;18:432-449. 69. Powrie F, Maloy KJ. Regulating the regulators. Science 2003;299:1030-1031. 70. Shevach EM. Regulatory/Suppressor T Cells in health and disease. Arthritis Rheum 2004;50:2721-2724. 71. Crispin JC, Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun 2003;21:273-276. 72. Bisikirska B, Colgan J, Luban J, Bluestone JA, Herold KC. TCT stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs. J Clin Invest 2005;115:2904-2913. 73. Chen Z, Benoist C, Mathis D. How defects in central tolerance impinge on a deficiency in regulatory T cells. Proc Natl Acad Sci 2005;102:14735-14740.

PATHOGENESIS

11

B-cells in Human Systemic Lupus Erythematosus Stamatis-Nick C. Liossis, MD and George C. Tsokos, MD

Among the numerous immunologic abnormalities encountered in patients with SLE, the most striking is B-cell overactivity. B lymphocytes in SLE produce an array of autoantibodies (autoAb) against soluble and cellular constituents but most commonly and most characteristically against the macromolecular complexes of the cell nucleus, the antinuclear antibodies (ANA). Although the spectrum of autoAb specificity in SLE may seem unrestricted, only a handful of autoAB have been shown to contribute convincingly to disease-related tissue injury. The latter are best represented by the anti–blood-cell antibodies that activate complement and cause cytopenias and the cationic anti-dsDNA autoAb that are thought to contribute to the expression of nephritis.1 Apart from producing Ab, B-cells play a role in such things as the production of cytokines, antigen presentation, and immune regulation. Abnormalities of B-cell functions encountered in patients with SLE are discussed in this chapter.

THE ORIGIN OF AUTOREACTIVE B-CELLS In contrast to previous beliefs, it is currently understood that autoreactive B-cells do exist in normal individuals. Moreover, although the naturally autoreactive B-cell pool was thought to be small, novel studies have presented evidence that this is an underestimation. Under normal conditions, or in disease states ranging from viral infections to malignancies, normal autoreactive B-cells produce a variety of natural autoAb that have a short life span and do not cause autoimmune disease or tissue damage. Natural autoAb usually belong to the IgM isotype, do not undergo isotype switching and affinity maturation, and may help in removing dying or dead cells. Still, under conditions of hypoxic tissue damage they may bind to newly revealed antigens, activate complement, and confer extensive tissue damage.2 In contrast, autoAbs in SLE undergo isotype switching and affinity maturation. Their presence is not helpful to the host because they have the capacity to cause tissue damage and their production is continuous and their presence is long lived,

indicating that the relevant regulatory mechanisms are profoundly defective. Autoreactive antibodies arise from autoreactive B-cells, but the mechanisms involved in the preservation of autoreactive B-cells are unclear. If we consider that detection of autoAb witnesses the presence of autoreactive B-cells, we now know that such B-cells exist in patients with SLE several years before the development of clinically evident disease. It was recently reported that 88% of previously healthy individuals that subsequently developed SLE have detectable autoAb in their sera as early as 9.4 years (mean: 3.3 years) before diagnosis, and in some perhaps even earlier. AutoAb were detected in the sera of 3.8% of healthy control individuals.3 The exhaustive analysis of different early B-cell subpopulations in a few patients with juvenile-onset SLE disclosed that autoreactive B-cells in SLE arise early in B-cell ontogeny and that the tolerance checkpoints imposed during B-cell development are violated. Significant numbers of antigen-inexperienced naïve B-cells (25 to 50%) are capable of producing autoAb in patients with SLE, whereas in healthy individuals this percentage does not exceed 20%.4 Despite the striking B-cell overactivity encountered in patients with SLE, challenging patients with standard immunizations or stimulating SLE peripheral B-cells with polyclonal activators in vitro can result paradoxically in substantially decreased amounts of specific Ab production compared to the responses of B-cells obtained from normal individuals5 (Fig. 11.1).

B-CELL SUBPOPULATIONS Peripheral B-cells are phenotypically distinguished into the CD5+ B-1 cells (less than 20% of circulating B-cells) and the conventional B-cells (B-2 cells). B-1 cells may be involved in the pathophysiology of some human autoimmune diseases such as Sjögren’s syndrome and rheumatoid arthritis because they produce IgM rheumatoid factor. The potential pathogenic role of B-1 cells in human lupus is questionable principally

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Production of cytokines IL-6, IL-10 BLys Production of various autoantibodies Enhanced signaling

Antigen T cell BCR

VH4.34-producing B-cells) in patients with SLE and healthy individuals disclosed that in healthy donors the naive VH4.34 B-cells are numerous. However, following a sequence of selection steps in secondary lymphoid organs their progression into the final steps of differentiation is efficiently blocked. In contrast, naive VH4.34 B-cells in patients with SLE progress unopposed to the final steps of maturation into plasma cells. This suggests that tolerance checkpoints in patients with lupus are malfunctioning.11

CD40L Lupus B cell

B7

Bidirectional T-B cell interactions

Fig. 11.1 A central role for the B-cell in SLE. Under the influence of enhanced antigen receptor-mediated signaling, lupus B-cells overexpress surface co-stimulatory molecules and over-interact with autoreactive lupus T-cells. B-cells in SLE are also under the influence of increased concentrations of soluble stimulators via autocrine (IL-6, IL-10) and paracrine (BLyS) loops and produce a spectrum of autoantibodies.

because in patients with SLE both B-1 and B-2 cells contribute to the production of pathogenic autoAb such as anti-dsDNA.6 A detailed analysis of circulating B-cells in patients with SLE revealed major disturbances in the different peripheral B-cell compartments. A B-cell subpopulation representing immature B-cells (generated in the bone marrow but not having undergone final maturation steps in peripheral lymphoid organs) named type I transitional (T1) B-cells are reportedly increased in the circulation in patients with SLE.7 In patients with active (but not in those with inactive) SLE there was a marked reduction in the numbers of naive (CD19+CD27−) B-cells and an enhanced representation of the CD27highCD38+CD19dimsIglowCD20−CD138+ plasma cells in the periphery.8 In addition, increased numbers of CD27high plasma cells correlated with increased disease activity more accurately than a combination of clinical and serological parameters, suggesting that this may be of value in monitoring disease activity.9 Immunosuppressive therapy commonly used in SLE patients induces differential changes in the different B-cell subsets: it reduces significantly the numbers of CD27high plasma cells and of CD27- naive B-cells but does not affect the population of memory CD27+ B-cells.8 A similar reduction of both naïve and memory B-cells and a significant expansion of plasma-cell precursors is encountered in pediatric SLE patients. These B-cell subset alterations were independent of disease activity.10 Studying the development and fate of specific autoreactive B-cells (the natural autoAb

THE ABNORMAL IMMUNOREGULATORY ENVIRONMENT Isotype switching and affinity maturation (both features of lupus autoAb) indicate that the lupus autoAb response is a T-cell dependent (auto) antigen-driven immune process. But is the T-cell compartment responsible for B-cell hyperreactivity? If this is correct, B-cells should be under either decreased T-cell–mediated suppression or under excessive unopposed T-cell–derived help, or both. There is not much evidence supporting a decreased suppression, but there is evidence that increased T-cell–derived help is responsible for the increased production of antibody and autoAb in SLE. Increased nonspecific help to autoreactive B-cells can also lead to autoimmunity. A chronic graft-versus-host murine model supports this view12 and may provide an explanation for the increased occurrence of autoimmunity in patients undergoing bone marrow transplantations.13 The previously cited data conclude that B-cell overactivity and the production of autoAb are due to factors exogenous to the B-cell and lie within the T-cell compartment. Although the contribution of T-cells to the production of autoAb is clear, there are sufficient data challenging the view of an entirely T-cell–dependent process. Studies of murine and human lupus have produced direct or indirect evidence that B-cells in SLE are not innocent bystanders but that their role in disease initiation and perpetuation may be central.

INTRINSIC SLE B-CELL DEFECTS: FUNCTIONAL STUDIES A number of studies have concluded that the B-cell in lupus may contribute in a T-cell–independent manner to the production of autoAb. Polyclonal B-cell activation is commonly encountered but is not the principal cause of the production of autoAb.14 According to the “two-signal” hypothesis, contact of B-cells with (self) antigen in the absence of T-cell–derived help tolerizes B-cells15 but in the presence of T-cell–derived help at the same time results in B-cell tolerance breakdown. Nevertheless, experimental data support the contention that this rule can be violated and that B-cell

SLE B CELLS ARE EFFICIENT (AUTO) ANTIGEN-PRESENTING CELLS Other investigators taking into account the particularly efficient antigen-presenting capacity of the B-cell have produced data indicating that the B-cell in lupus may represent the central pathogenic cell that triggers other immune cells toward hyperactivity. In certain experimental lupus models, the presence of B-cells even in the total absence of autoAb was the one and only determinant for the appearance of the autoimmune murine syndrome.19 In the absence of B-cells such autoimmune mice had neither activated T-cells nor lupus.20 It is thus possible that under certain circumstances lupus B-cells are not restricted to the production of autoAb only but mediate abnormal T-cell activation.21 In patients with SLE treated with anti-CD20 monoclonal antibody, the clinical effect was associated with decreased numbers of circulating T-cells expressing the early activation marker CD40L,22 suggesting that (as in mice) in humans lupus B-cells are involved in the generation of activated T-cells.

CYTOKINES CONTRIBUTE TO B-CELL ACTIVATION IN SLE Human B-cells are capable of producing and secreting cytokines and lupus B-cells display an abnormally increased production of and response to IL-6 and IL-10. Both IL-6 and IL-10 are B-cell stimulatory cytokines, whereas IL-10 also inhibits type-1 cytokine responses.

Patients with active SLE have elevated serum levels of IL-6,23 increased numbers of IL-6-secreting cells, and increased IL-6 mRNA content in their peripheral blood mononuclear cells. B-cells from patients with SLE express constitutively cell-surface IL-6 receptors. IL-6 stimulates SLE B-cells in vitro to produce anti-DNA Ab. SLE patients have elevated serum levels of IL-10, increased numbers of IL-10-secreting cells, and increased IL-10 mRNA content in their peripheral blood mononuclear cells. Recombinant human interleukin-10 promotes (and an anti–IL-10 mAb inhibits) the in vitro production of autoantibodies from B-cells obtained from patients with SLE.24 Increased production of IL-10 is also found in B-cells from relatives of patients with SLE. It has been reported that circulating lupus CD5+ B-cells (B-1 cells) expressing the surface marker CD40L spontaneously produce IL-10.25

PHENOTYPIC CHANGES OF THE LUPUS B-CELL

tolerance can be broken without the support of T-cell-mediated help. Such data have been produced in lupus-prone mice genetically manipulated to have no TCRαβ+ (and no CD4+) T-cells or even no T-cells at all. The B-cells of such autoimmunity-prone animals were still able to produce significant amounts of pathogenic IgG autoantibodies16 and to develop immune-complex–mediated nephritis,17,18 making the role of increased T-cell–mediated help or decreased suppression of relative importance. Do overactive B-cells contribute to the expression of the disease directly, or through the production of autoAb? Lupus-prone MRL/lpr mice genetically manipulated to have B-cells incapable of secreting immunoglobulin still developed autoimmunity characterized by nephritis with cellular infiltrates, indicating that inherently autoimmune B-cells (without the production of autoAb and immune complexes) contribute directly to disease pathogenesis.19 The absolute dependence of the expression of SLE on either direct or indirect interactions of B-cells with either hyperactive or autoreactive T-cells is therefore uncertain under certain experimental conditions.

PHENOTYPIC CHANGES OF THE LUPUS B-CELL Functional CD40 ligand (CD40L, CD154) is expressed on the surface membrane of SLE B-cells,26 which increases further after stimulation in vitro. Because not only T-cells but B-cells from SLE patients express functional CD40L on their cell surface, and because both types of lymphocytes express CD40, a bidirectional cognate stimulatory loop may function between lupus T- and B-cells.27 The role of CD40L-expressing B-cells in the production of autoAb in patients with SLE was supported by limited observations that indicate that treatment of patients with anti-CD40L mAb resulted in decreased numbers of IgG- and IgG anti-DNA autoAbproducing cells.28 Expression of CD40L in mice results in autoimmune manifestations, including nephritis.29 In addition to CD40L, members of the B7 family of co-stimulatory molecules (CD80 and CD86) are also aberrantly expressed on the surface of lupus B-cells. Expression of B7-family molecules has been shown to be a prerequisite for the disruption of immune tolerance toward self-antigen. It has been recently suggested that both CD80 and CD86 (the latter is absent in normal B-cells) were significantly up-regulated on the surface of circulating B-cells from patients with SLE. An anti-CD40L mAb inhibited the expression of CD86 on B-cells from patients with lupus, and an antiCD86 (but not of anti-CD80) mAb inhibited the production of polyclonal immunoglobulin and of anti-single-stranded DNA, suggesting a functional role for the abnormally expressed CD86 molecule on B-cells from patients with lupus.30 The incompletely characterized B-cell surface molecule RP105 is underexpressed on the surface of B-cells from patients with SLE. Such RP105- lupus B-cells, but not RP105+ cells, were capable of producing in vitro anti-dsDNA autoAb31 (Table 11.1).

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TABLE 11.1 ABNORMAL PHENOTYPE OF B-CELLS IN SLE ●

Molecule abnormality function

●

CD40L increased co-stimulation

●

ICOS-L decreased co-stimulation

●

CD80 slightly increased co-stimulation

●

CD86 increased co-stimulation

●

RP105 decreased unknown

●

CR1 decreased complement receptor

●

CR2 decreased complement receptor

●

EBV receptor

ABNORMALITIES OF BCR-SIGNALING REGULATORY MOLECULES When B-cells encounter properly presented antigen, other B-cell surface molecules are engaged and some of them provide regulatory control over the intensity, duration, and fate of the biochemical signal generated by the interaction of BCR with the antigen. In this manner, the signaling-regulatory molecules eventually regulate the final outcome of the B-cell/antigen encounter. Engagement of signaling regulatory molecules triggers separate intracytoplasmic biochemical cascades. The net sum of these signals may result in an increased, attenuated, or qualitatively different BCR-initiated signal outcome.

COMPLEMENT RECEPTORS ON LUPUS B-CELLS ABERRANT B-CELL ANTIGEN RECEPTOR SIGNAL TRANSDUCTION Crucial aspects of lymphocyte function (such as activation, proliferation, cytokine production, effector functions, and apoptosis) are determined by the signaling biochemical pathway initiated following ligation of the surface antigen receptor. This biochemical cascade begins upon binding of the B-cell surface antigen receptor (BCR), the surface immunoglobulin, with the relevant ligand (antigen and/or autoantigen) and ends at the cell nucleus with the induction of transcription of specific genes. It may thus be possible that heterogeneous defects such as those described previously may have a common underlying central biochemical abnormality. Stimulation of circulating B-cells from patients with SLE through their surface IgM or IgD BCR produced significantly higher fluxes of free intracytoplasmic Ca2+ when compared to similarly induced responses of B-cells from patients with other systemic rheumatic diseases, or to the responses obtained from normal B-cells. Even though the rise of cytoplasmic Ca2+ concentration is an early signaling event, the production of tyrosyl phosphorylated proteins occurs even earlier. This earliest known signaling event is similarly increased in B-cells from patients with SLE following stimulation via their BCR.32 It is of interest that strikingly similar abnormalities of antigen-receptor signaling have previously been reported from the study of fresh T-cells, T-cell lines, and autoantigen-specific T-cells from patients with SLE, pointing toward a potentially unifying Ag-receptor-mediated signaling defect(s) in lupus lymphocytes. It has been proposed that the signaling abnormalities encountered in SLE lymphocytes may provide a biochemical and molecular background for such diverse functions as lymphocyte activation, anergy, and cell death.33

The most important signal-augmenting B-cell surface molecule is the complement receptor type 2 (CR2). It forms the hetero-oligomeric complex (the CR2 complex), which includes CD21 (CR2), CD19 (the signaling molecule of the complex), and CD81. Physiologically, CR2 binds iC3b, C3dg, or C3d. The cytoplasmic signal produced when antigens decorated with the aforementioned complement fractions bind to B-cells represents the sum of co-crosslinking BCR and CR2 and is several orders of magnitude higher when compared to the signal produced by the same antigens via the BCR alone. Despite a significant decrease in the expression of cell surface CR2, the cytoplasmic Ca2+ responses recorded were significantly enhanced compared to normal B-cells.34 Previous studies have shown that expression of both CR2 and of complement receptor type 1 (CR1, an alternatively spliced product of the CR2 gene) is decreased on the surface of B-cells obtained not only from patients with SLE but on B-cells from their healthy relatives, suggesting a potentially genetic alteration.35 Other studies have argued that decreased expression of CR2 on lupus B-cells correlates well with increased disease activity, suggesting an acquired defect.36 CR2 also represents the B-cell surface receptor for Epstein-Barr virus (EBV). Young patients with recentonset SLE have evidence of EBV infection more commonly than their healthy counterparts (99 versus 70%),37 and more EBV-infected blood cells are detected in patients with SLE, correlating with disease activity.38 Studies in vitro have shown that EBV infection of lupus B-cells cannot be contained by lupus T-cells, suggesting that defective EBV control in patients with SLE may be a T-cell defect.39 CR2 enhances the strength of the BCR-initiated signaling process and its deficiency on the surface of lupus B-cells does not help to explain the pathophysiology of the overactive lupus B-cell. Nevertheless, recent data

STIMULATORY B-CELL SURFACE MOLECULES B lymphocyte stimulator (BLyS),41 a novel member of the TNF superfamily, stimulates B-cells both in vitro and in vivo principally via the receptor for the B-cell activating factor belonging to the TNF superfamily (BAFFR). BLyS is a principal focus of research interest because it has clearly been shown that BLyS protein is vital and indispensable for normal B-cell development.42 Some patients with SLE have increased levels of circulating BLyS and the titers of BLyS correlated well with the titers of anti-dsDNA autoAb but not with disease activity.43 Nevertheless, there is a heterogeneity in BLyS levels among different lupus patients, and there are patients with SLE who have normal or even lower than normal levels of circulating serum BLyS. Based on the central role of BLyS in the B-cell immune response, different groups evaluated the effects of BLyS blockade in experimental lupus and such an approach was fruitful in delaying disease progression and improving animal survival. Anti-BLyS-based treatment is currently in phase II clinical trials in patients with SLE.

B-CELL SURFACE RECEPTORS THAT PROVIDE NEGATIVE REGULATION There are many B-cell surface signaling inhibitory receptors. Among them, the functions of CD5, CD22,

and FcγRIIB1 are best understood. The role of the B-cell surface inhibitory receptor for the Fc fraction of IgG type IIB1 (FcγRIIB1, CD32) is best characterized. When antigen bound to IgG is presented to the BCR, BCR and FcγRIIB1 are co-crosslinked and result in a net signal of smaller magnitude than the signal generated by antigen alone. The cytoplasmic Ca2+ fluxes are of shorter duration, and the resulting B-cell response is incomplete activation and proliferation. Previous studies have reported that the receptors for the Fc fraction of IgG malfunction in SLE and result in the production of excess antibodies and the accumulation of immune complexes.44 In addition, a single-nucleotide polymorphism (a G-C substitution at position -343) in the human FCGR2B promoter was recently identified, particularly in European-American patients with SLE. The -343 G/C polymorphism results in decreased transcription, providing thus a molecular explanation for the dysregulation of FcγRIIB1 in some patients with SLE (181). In other patients with SLE, the surface expression of FcγRIIB1 on B-cells was similar to controls but its inhibitory function was defective because it could not efficiently diminish BCR-mediated signaling.45 Another B-cell signaling molecule (the protein tyrosine kinase Lyn) has been reported decreased in the cytoplasm of B-cells from 2/3 of patients with SLE.46 According to recent studies, it appears that Lyn is crucial for the function of several other signaling inhibitory molecules of B-cells such as the surface receptors CD22 and FcγRIIB1, suggesting that decreased Lyn may contribute to lupus B-cell overactivity.47,48

REFERENCES

propose that complement receptor-mediated signaling maintains B-cell immune tolerance to self-antigens.40 Therefore, the low amounts of membrane complement receptors (due to genetic or acquired defects) found in SLE may explain why lupus B-cells display decreased immune self-recognition. It has been proposed that complement plays a role in the early stages of B-cell negative selection. According to this theory, circulating natural autoAb identify highly conserved self-antigens and activate the classical pathway of complement. Such complexes bind to the BCR and complement receptors of highly autoreactive immature B-cells arising daily in the bone marrow and facilitate their depletion in the normal host. In the case of early complement factor (or complement receptor) deficiencies, autoreactive immature B-cells are not efficiently removed, and lupus-like autoimmunity develops.

CONCLUSIONS Recent data support a central pathogenic role for the lupus B-cell because it functions as an independent contributor to the appearance of the disease, apart from the established contribution of potentially harmful autoAb production. Improving our understanding of the lupus B-cell physiology and pathophysiology will ultimately improve our understanding of the disease and may provide us with useful tools to deal with SLE more rationally. As discussed, B-cell depletion and inhibition of the function of factors that promote B-cell function may prove of therapeutic value in the treatment of patients with SLE.

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4. Yurasov S, Wardemann H, Hammersen J, Tsuiji M, Meffre E, Pascual V, et al. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med 2005;201:703-711. 5. Gottlieb AB, Lahita RG, Chiorazzi N, Kunkel HG. Immune function in systemic lupus erythematosus: Impairment of in virto T-cell proliferation and in vivo antibody response to exogenous antigen. J Clin Invest 1979;63:885.

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6. Suzuki N, Sakane T, Engleman EG. Anti-DNA antibodies production by CD5+ and CD5- B cells of patients with systemic lupus erythematosus. J Clin Invest 1990;85:2347-2348. 7. Sims GP, Ettinger R, Shirota Y, Yarboro CH, Illei GG, Lipsky PE. Identification and characterization of circulating human transitional B cells. Blood 2005;105:4390-4398. 8. Odendahl M, Jacobi A, Hansen A, Feist E, Hiepe F, Burmester GR, et al. Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus. J Immunol 2000;165:5970-5979. 9. Jacobi AM, Odendahl M, Reiter K, Bruns A, Burmester GR, Radbruch G, et al. Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus. Arthritis Rheum 2003;48:1332-1342. 10. Arce E, Jackson DG, Gill MA, Bennett LB, Banchereau J, Pascual V. Increased frequency of pre-germinal center b cells and plasma cell precursors in the blood of children with systemic lupus erythematosus. J Immunol 2001;167:2361-2369. 11. Pugh-Bernard AE, Silverman GJ, Cappione AJ, Villano ME, Ryan DH, Insel RA, et al. Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance. J Clin Invest 2001;108:1061-1070. 12. Feuerstein N, Chen F, Madaio M, Maldonado M, Eisenberg RA. Induction of autoimmunity in a transgenic model of B cell receptor peripheral tolerance: changes in coreceptors and B cell receptorinduced tyrosine-phosphoproteins. J Immunol 1999;163:5287-5297. 13. Sekiguchi DR, Jainandunsing SM, Fields ML, Maldonado MA, Madaio MP, Erikson J, et al. Chronic graft-versus-host in Ig knockin transgenic mice abrogates B cell tolerance in antidouble-stranded DNA B cells. J Immunol 2002;168:4142-4153. 14. Gharavi AE, Chu JL, Elkon KB. Autoantibodies to intracellular proteins in human systemic lupus erythematosus are not due to random polyclonal B cell activation. Arthritis Rheum 1988;31:1337-1345. 15. Bretscher P, Cohn M. A theory of self-nonself discrimination. Science 1970;169:1042-1049. 16. Wen L, Roberts SJ, Viney JL, Wong FS, Mallick C, Findly RC, et al. Immunoglobulin synthesis and generalized autoimmunity in mice congenitally deficient in alpha beta(+) T cells. Nature 1994;369:654-658. 17. Peng SL, Madaio MP, Hughes DP, Crispe IN, Owen MJ, Wen L, et al. Murine lupus in the absence of alpha beta T cells. J Immunol 1996;156:4041-4049. 18. Reininger L, Radaszkiewicz T, Kosco M, Melchers F, Rolink AG. Development of autoimmune disease in SCID mice populated with long-term “in vitro” proliferating (NZB x NZW)F1 pre-B cells. J Exp Med 1992;176:1343-1353. 19. Chan OT, Hannum LG, Haberman AM, Madaio MP, Shlomchik MJ. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J Exp Med 1999;189:1639-1648. 20. Chan O, Shlomchik MJ. A new role for B cells in systemic autoimmunity: B cells promote spontaneous T cell activation in MRL-lpr/lpr mice. J Immunol 1998;160:51-59. 21. Chan OT, Madaio MP, Shlomchik MJ. The central and multiple roles of B cells in lupus pathogenesis. Immunol Rev 1999;169:107-121. 22. Sfikakis PP, Boletis JN, Lionaki S, Vigklis V, Fragiadaki KG, Iniotaki A, et al. Remission of proliferative lupus nephritis following B cell depletion therapy is preceded by down-regulation of the T cell costimulatory molecule CD40 ligand: An open-label trial. Arthritis Rheum 2005;52:501-513. 23. Linker-Israeli M, Deans RJ, Wallace DJ, Prehn J, Ozeri-Chen T, Klinenberg JR. Elevated levels of endogenous IL-6 in systemic lupus erythematosus: A putative role in pathogenesis. J Immunol 1991;147:117-123. 24. Llorente L, Zou W, Levy Y, Richaud-Patin Y, Wijdenes J, AlcocerVarela J, et al. Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus. J Exp Med 1995;181:839-844. 25. Diaz-Alderete A, Crispin JC, Vargas-Rojas MI, Alcocer-Varela J. IL-10 production in B cells is confined to CD154+ cells in patients with systemic lupus erythematosus. J Autoimmun 2004;23:379-383. 26. Desai-Mehta A, Lu L, Ramsey-Goldman R, Datta SK. Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest 1996; 97:2063-2073.

27. Datta SK, Kalled SL. CD40-CD40 ligand interaction in autoimmune disease. Arthritis Rheum 1997;40:1735-1745. 28. Huang W, Sinha J, Newman J, Reddy B, Budhai L, Furie R, et al. The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum 2002;46:1554-1562. 29. Higuchi T, Aiba Y, Nomura T, Matsuda J, Mochida K, Suzuki M, et al. Cutting edge: Ectopic expression of CD40 ligand on B cells induces lupus-like autoimmune disease. J Immunol 2002;168:9-12. 30. Nagafuchi H, Shimoyama Y, Kashiwakura J, Takeno M, Sakane T, Suzuki N. Preferential expression of B7.2 (CD86), but not B7.1 (CD80), on B cells induced by CD40/CD40L interaction is essential for anti-DNA autoantibody production in patients with systemic lupus erythematosus. Clin Exp Rheumatol 2003;21:71-77. 31. Kikuchi Y, Koarada S, Tada Y, Ushiyama O, Morito F, Suzuki N, et al. RP105-lacking B cells from lupus patients are responsible for the production of immunoglobulins and autoantibodies. Arthritis Rheum 2002;46:3259-3265. 32. Liossis SN, Kovacs B, Dennis G, Kammer GM, Tsokos GC. B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events. J Clin Invest 1996;98:2549-2557. 33. Tsokos GC, Liossis SN. Immune cell signaling defects in lupus: activation, anergy and death. Immunol Today 1999;20:123-128. 34. Mitchell JP, Enyedy EJ, Nambiar MP, Lees A, Tsokos GC. Engagement of complement receptor 2 on the surface of B cells from patients with systemic lupus erythematosus contributes to the increased responsiveness to antigen stimulation. Lupus 2002;11:299-303. 35. Wilson JG, Ratnoff WD, Schur PH, Fearon DT. Decreased expression of the C3b/C4b receptor (CR1) and the C3d receptor (CR2) on B lymphocytes and of CR1 on neutrophils of patients with systemic lupus erythematosus. Arthritis Rheum 1986;29:739-747. 36. Marquart HV, Svendsen A, Rasmussen JM, Nielsen CH, Junker P, Svehag SE, et al. Complement receptor expression and activation of the complement cascade on B lymphocytes from patients with systemic lupus erythematosus (SLE). Clin Exp Immunol 1995;101:60-65. 37. James JA, Kaufman KM, Farris AD, Taylor-Albert E, Lehman TJ, Harley JB. An increased prevalence of Epstein-Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus. J Clin Invest 1997;100:3019-3026. 38. Gross AJ, Hochberg D, Rand WM, Thorley-Lawson DA. EBV and systemic lupus erythematosus: a new perspective. J Immunol 2005;174:6599-6607. 39. Tsokos GC, Magrath IT, Balow JE. EBV induces normal B cell responses but defective suppressor T cell responses in patients with SLE. J Immunol 1983;131:1797-1801. 40. Prodeus AP, Goerg S, Shen LM, Pozdnyakova OO, Chu L, Alicot EM, et al. A critical role for complement in maintenance of selftolerance. Immunity 1998;9:721-731. 41. Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, et al. BLyS: Member of the tumor necrosis factor family and B lymphocyte stimulator. Science 1999;285:260-263. 42. Yan M, Marsters SA, Grewal IS, Wang H, Ashkenazi A, Dixit VM. Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity. Nat Immunol 2000;1:37-41. 43. Cheema GS, Roschke V, Hilbert DM, Stohl W. Elevated serum B lymphocyte stimulator levels in patients with systemic immunebased rheumatic diseases. Arthritis Rheum 2001;44:1313-1319. 44. Frank MM, Hamburger MI, Lawley TJ, Kimberly RP, Plotz PH. Defective reticuloendothelial system Fc-receptor function in systemic lupus erythematosus. N Engl J Med 1979;300:518-523. 45. Enyedy EJ, Mitchell JP, Nambiar MP, Tsokos GC. Defective FcgammaRIIb1 signaling contributes to enhanced calcium response in B cells from patients with systemic lupus erythematosus. Clin Immunol 2001;101:130-135. 46. Liossis SN, Solomou EE, Dimopoulos MA, Panayiotidis P, Mavrikakis MM, Sfikakis PP. B-cell kinase lyn deficiency in patients with systemic lupus erythematosus. J Investig Med 2001;49:157-165. 47. Nishizumi H, Horikawa K, Mlinaric-Rascan I, Yamamoto T. A doubleedged kinase Lyn: A positive and negative regulator for antigen receptor-mediated signals. J Exp Med 1998;187:1343-1348. 48. Cornall RJ, Cyster JG, Hibbs ML, Dunn AR, Otipoby KL, Clark EA, et al. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity 1998;8:497-508.

PATHOGENESIS

12

Cytokines in Systemic Lupus Erythematosus Violeta Rus, MD and Charles S. Via, MD

INTRODUCTION

IL-2

Systemic lupus erythematosus is characterized by a loss of tolerance to nuclear antigens and is accompanied by profound dysregulation of the immune system. Cytokines have been extensively studied in both human and murine lupus. However, in many cases the results for a given cytokine have appeared conflicting due to variation in the type of specimen tested (cells versus serum), the method of detection (mRNA expression versus cytokine protein secretion in vitro), and variability among commercial kits. The reader is referred to several comprehensive reviews on cytokines in lupus.1-3 This chapter focuses on recent developments, and where possible on areas of emerging consensus.

The role of IL-2 in both murine and human lupus has been extensively investigated.2,3,8 IL-2 is made primarily by T-cells (especially CD4+ T-cells) and promotes T-cell growth. Defective in vitro production of IL-2 following restimulation is a long-standing and robust observation.9 Nevertheless, given the central role that T-cells play in SLE pathogenesis (and the central role of IL-2 in T-cell growth and differentiation, activation-induced cell death, the development of regulatory T-cells, and resistance to viral infection) the meaning of defective IL-2 production in SLE is clearly of interest. A number of postulated explanations include intrinsic T-cell defects, T-cell exhaustion, decreased co-stimulation by APC, and excessive suppressor cell function. Several intracellular signaling pathways that contribute to T-cell IL-2 production have been shown to be defective.10 Recent evidence indicates that defective IL-2 production is a consequence of defective promoter activivity.8,11 A major question has been whether these defects reflect a primary predisposing defect or instead reflect a secondary defect induced by the altered immunoregulation characteristic of lupus. Supporting a secondary induced defect are the following observations: in vitro IL-2 production is normal in very young MRL/lpr mice, declines rapidly with age-related disease progression, and is related to the presence of CD4+ T cells (which can suppress in vitro IL-2 production by T-cells from MRL/+ mice).12 Similarly, in the lupus-like disease induced in normal F1 mice following the transfer of homozygous parental strain CD4+ T-cells, recipient F1 mice exhibit defective in vitro IL-2 production in conjunction with the appearance of anti-ssDNA ab as early as two weeks after transfer.13 Last, in human SLE intracellular signaling defects resulting in impaired IL-2 production can be induced by IgG from SLE patients.14 Although these results do not exclude the existence of primary preexisting defects in IL-2 production in some SLE patients, they support the idea that defective in vitro T-cell production of IL-2 can be a consequence of disease.

TH1/TH2 PARADIGM As initially described, T-cell clones have been divided into two types: Th1 clones [which produce interferon gamma (IFN-g), interleukin (IL)-2, and lymphotoxin] and Th2 clones, which produce IL-4 and IL-5. It was postulated that Th1 cytokines mediate primarily cellmediated immunity and Th2 cytokines mediate primarily antibody responses.4 With the discovery of additional cytokines and the pleiotropic nature of many cytokines, the original concept has become problematic. For example, IFN-g (a prototypic Th1 cytokine) also promotes isotype switching and antibody formation.5 Recent evidence indicates that the primary role of IL-2 is in the proliferation and differentiation of Th precursor cells into effector cells rather than as an effector cytokine.6 In addition, tumor necrosis factor alpha (TNF-a, a pro-inflammatory cytokine) has a critical role in the induction of cytotoxic T lymphocytes (CTL) in vivo.7 As discussed in material following, the complexity and heterogeneity of immune dysregulation in lupus combined with the pleiotropic properties of cytokines [depending on the stage of disease (induction phase versus effector phase)] prevents the classification of lupus as simply a Th1 or Th2 disease.

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110

IFN-G (TYPE II IFN) IFN-g is a 20- to 25-kd glycoprotein secreted by T-cells and NK cells in response to a variety of stimuli. CD8+ T-cells and Th1 CD4+ cells produce IFN-g during viral infections.15 The IFN-g receptor consists of the alpha chain (known as IFN-g RI) that binds IFN-g and the associated beta chain (IFN-g RII) required for biologic activity. Binding of IFN-g to its receptor leads to activation of tyrosine kinases JAK1 and JAK2, which then phosphorylate the transcription factor STAT1 that dimerizes and translocates to the nucleus (where it binds and activates the gamma activation site in the promoter of IFN-g inducible genes). IFN-g has multiple and pleiotropic effects on immune and nonimmune cells, including the ability to inhibit the proliferation of Th2 cells and to induce Ig class switching (promoting IgG2a production).16 Production of IFN-g during an immune response results in preferential expansion of Th1 cells and promotes macrophage killing in response to intracellular microbes and parasites. IFN-g induces or up-regulates MHC class II expression on immune and nonimmune cells, an effect that may contribute to the pathogenesis of autoimmunity. IFN-g has a pathogenic, albeit complex, role in SLE. Administration of IFN-g to NZB/NZWF1 mice accelerates the development of glomerulonephritis, whereas treatment of these mice with neutralizing antibody to IFN-g or cDNA encoding INFgR/Fc results in renal remission.17-19 A complex dichotomous role of IFN-g was seen in MRL-lpr mice in that prophylactic IFN-g administration to preautoimmune mice was beneficial whereas a deleterious effect was seen with IFN-g administration to older mice.20 In the parent-into-F1 model of lupus, IFN-g is critical for up-regulation of Fas and Fas ligand (which prevents lupus development by promoting elimination of autoreactive B-cells by CTL). In the absence of IFN-g, however, mice do not develop lupus in the short term due to B-cell elimination by residual perforin-dependent CTL.21 Studies in humans have also been conflicting. Elevated levels of serum IFN-g are seen in lupus patients but may22 or may not be related to disease activity.23 Increased IFN-g mRNA in PBMC from lupus patients24 and increased intracellular levels in CD4 T-cells from patients with lupus nephritis have also been reported. A mechanism for the increased production of IFN-g in SLE is suggested by studies showing that native nucleosomes and some of their peptides induce a strong IFN-g response in lupus patient T-cells.25 In contrast to the previous, T-cells from lupus patients exhibit a diminished ability to produce IFN-g following in vitro stimulation by mitogens.26,27 In one study, the in vitro production of IFN-g at basal and

stimulated levels did not differ from normal controls overall.28 However, within lupus patients a correlation was seen with disease activity, suggesting that an increase in IFN-g production may occur during exacerbation. In contrast, peripheral blood from lupus patients with active disease contained fewer IFN-g-secreting cells compared to controls,29 and patients with lupus nephritis had lower IFN-g serum levels.30 Polymorphism within the genes for the two IFN-g receptor chains has been associated with the risk of developing SLE.31,32 A significant difference in the induction of HLA-DR by IFN-g stimulation and a shift to Th2 cytokines was noted in individuals bearing the variant receptor IFN-gR1 Met14/Val14. The greatest risk of development of SLE was detected in the individuals who had the combination of IFN-g R1 Met14/Val14 genotype and IFN-gR2 Gln64/Gln64 genotype. Aside from differences due to methodology, the pleiotropic effects of IFN-g may explain the conflicting results in human and animal models and this suggests that both decreased and increased IFN-g production contributes to disease pathogenesis at different stages. Higher levels of IFN-g secreted at disease onset may increase MHC class II expression, contributing to the breaking of tolerance to self-antigens and production of pathogenic autoantibodies. Later in the disease, lower production of IFN-g could permit the production of higher levels of Th2 cytokines that in turn permit greater B-cell activity. IFN-g may also amplify effector mechanisms responsible for end-organ damage.

IFN-ALPHA One of the first cytokine abnormalities to be documented in SLE was an increase in serum IFN-a levels in most SLE patients33-35 (reported more than 25 years ago). Increased IFN-a levels correlated with disease activity, disease severity, immune activation (as measured by anti-dsDNA titers and complement levels), and clinical features such as fever, rash, and lymphopenia.33-35 With the advent of IFN-a as a treatment of malignancies and hepatitis C infection 20 years ago, a possible causative link between lupus and IFN-a has been suggested by reports of exacerbation of preexisting autoimmune diseases and/or the occurrence of overt organ-specific and systemic autoimmune diseases with IFN-a therapy.36 Recent studies using microarray technology have demonstrated an IFN-a signature in PBMC from adult and pediatric lupus patients.37 Moreover, the level of IFN-a-induced gene expression correlated with disease activity (suggesting that activation of the IFN-a pathway defines a subgroup of SLE patients with severe disease).38 In addition to the IFN-a gene signature found in SLE patients, gene expression data in lupus-prone

IL-12 IL-12 is a heterodimeric cytokine of 70 kDa comprising two covalently linked subunits p35 and p40. The p40 chain is overproduced relative to the p35 chain and forms homodimers that bind to the IL-12 receptor, competing with the bioactive p70 heterodimer for receptor occupancy and thereby serving as a receptor antagonist. IL-12 is predominantly produced by dendritic cells, monocytes, and macrophages, and to a lesser extent by B-cells.48 IL-12 is widely accepted as an important regulator of Th1 responses. It also promotes the expansion and survival of activated T-cells and NK cells and modulates the cytotoxic activity of CTLs and NK cells. During the adaptive immune response, IL-12 primes antigen-specific T-cells for high IFN-g production (driving their differentiation toward the Th-1 pathway). IL-12 can also act as an adjuvant for humoral immunity by enhancing production of IgG2a and IgG2b antibodies, and it may enhance antibody production by B-cells.

Several groups have examined IL-12 in the pathogenesis of lupus. IL-12 has been reported as up-regulated in the kidneys of NZB/W F1 mice and MRL/lpr mice49 independently of T-cells or T-cell-produced IFN-g.50,51 Elevated levels of IL-12 in serum from lupus patients have been reported in one study,52 whereas impaired production of IL-12 by SLE monocytes or PBMC was observed in vitro (which correlated with disease activity).53-55 IL-12 production was positively correlated with IFN-g anti-dsDNA antibody and negatively correlated with IL-10.56 In that study, IL-10 inhibition or IFN-g addition enhanced IL-12 production (but only the latter restored it to normal levels). Addition of exogenous IL-12 to lymphocytes derived from lupus patients reduced spontaneous polyclonal production IgG production by B-cells and the number of anti-dsDNA-secreting cells.57 This effect was directly mediated by IL-12 and not by IFN-g upregulation or IL-10 down-regulation. In vivo administration of IL-12 to lupus-like chronic GVHD mice prevented autoantibody production by promoting CTL activity that eliminated activated autoreactive B-cells.58 This effect was not blocked by administration of neutralizing anti–IFN-g mAb, indicating a direct CTL-promoting effect of IL-12. However, deletion of IL-12 gene in lupus-prone mice did not significantly influence autoimmunity.59 Taken together, it is difficult to determine the exact role of IL-12 in lupus pathogenesis (i.e., whether the alterations in IL-12 cited previously are secondary to lupus-related altered immunoregulation, whether they reflect endogenous compensatory pathways attempting to normalize immune function, or some combination of both). At present, it does not appear that IL-12 defects or excess are central to disease induction. A role for IL-12 in therapy is speculative at present.

IL-4

mice further substantiated an important role for IFN-a in the pathogenesis of lupus. Results from animal models also suggest a key role for IFN-a in lupus pathogenesis. Homozygous IFNa/b receptor-deleted NZB mice demonstrated an ameliorated disease pattern,39 whereas administration of IFN-a induced an early lethal disease course in pre-autoimmune NZBWF1 mice but not in normal BALB/c mice.40 The major source of IFN-a and mechanisms involved in lupus pathogenesis are an area of active investigation. Immune complexes containing anti-dsDNA/dsDNA or apoptotic bodies induce IFN-a production by plasmacytoid dendritic cells (pDC).35,41 Interestingly, several studies have documented a decreased frequency of pDCs in the blood of SLE patients,42 perhaps reflecting an accelerated migration of pDC into tissues such as the skin.43 Regarding pathogenic mechanisms, IFN-a in SLE serum can induce the differentiation of monocytes into dendritic cells with autoantigen-presenting capacity.44 In the presence of IFN-a, self-antigens derived from apoptotic cells and expressed on the surface of activated antigen-presenting cells may trigger sustained activation of self-reactive T-cells (which could provide help for autoantibody production by autoreactive B-cells). IFN-a can also enhance B-cell responses and promote immunoglobulin class switching through stimulation of dendritic cells.45 Of note, IFN-a also promotes Th1 development and IFN-g production, which in turn promotes Fas ligand and TRAIL expression on NK and activated T-cells (augmenting their capacity to mediate target cell apoptosis).46,47 How the Th1 actions of IFN-a promote lupus, if at all, is unclear.

IL-4 IL-4 is a multifunctional cytokine with B-cell stimulatory and Th2-promoting properties. IL-4 can rescue B-cells from apoptosis, enhancing their survival,60 and is responsible for immunoglobulin isotype switching to IgG1 and IgE.61 A T-cell suppressor role for this cytokine has also been suggested.62 Conflicting data exists regarding the role of IL-4 in the development of lupus. Elevated levels of IL-4 have been found in the sera of some lupus patients,63 and purified B-cells from lupus patients have been reported to spontaneously produce a soluble factor with IL-4–like activity.64 In addition, ex vivo mitogen-induced production of IL-4 was increased in one recent study65 (and elevated levels of IL-4 mRNA were reported in another).66 In contrast, other studies have demonstrated normal IL-4 mRNA from PBMC of lupus patients,67,68

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and the number of IL-4-producing CD4+ T-cells was significantly decreased in lupus patients.69 Polymorphisms in the IL-4 promoter and IL-4 receptor genes have been associated with the development of SLE.70,71 The preponderance of data from animal models of lupus suggests a role for increased IL-4 production in the pathogenesis of the disease. However, some conflicting results have also been reported. Spleen cells from BXSB lupus-prone mice produce normal amounts of IL-4 following in vitro mitogen stimulation,72 whereas increased IL-4 production was reported for lymph node from cells from either C3H/lpr73 or MRL/lpr mice74 compared to control mice. Complicating interpretation is the observation that spleen cells from MRL/lpr mice expressed significantly less IL-4 mRNA and less protein upon mitogen stimulation than controls.72 Interleukin-4–positive cells were reported in kidney biopsies from patients with active lupus nephritis in one study,75 whereas the expression of IL-4 and IL-4R mRNA correlated negatively with the degree of glomerular injury in another study.76 Evidence for a more novel role for IL-4 in the development of lupus nephritis comes from recent studies in which blockade of IL-4 by antibody treatment or of its signaling by inactivation of the Stat6 gene ameliorates glomerulosclerosis and delays or even prevents the development of end-stage renal disease in NZM.2410 mice, despite the presence of high levels of IgG antidsDNA.77 Thus, IL-4 may serve multiple roles in the development of lupus: it may enhance autoantibody production via its direct B-cell effects, protect against autoimmunity via its T-cell suppressor effect, or perpetuate tissue damage via direct effects on target organs.

IL-5

112

A possible role for IL-5 in the pathogenesis of lupus has not been extensively evaluated. Elevated IL-5 mRNA has been reported in cutaneous lupus erythematosus,78 suggesting a role for Th2 cells in the development of skin involvement in SLE. In the periphery, however, the number of IL-5–secreting T-cells was not significantly different between SLE patients and controls.79 A number of studies have suggested that abnormally high levels of IL-5 may play an important role in the abnormal expansion of activated B-cells in lupus. Activation-induced B-cell apoptosis could be rescued with the addition of cytokines such as interleukin IL-5 (or IL-10) in vitro.80 NZBW mice congenic for IL-5 transgene exhibited a reduced incidence of lupus nephritis, a decrease in anti-DNA antibody production, and a progressively increased frequency of peripheral B-1 B-cells with age—suggesting that dysregulated continuous high expression of IL-5 in SLE-prone

mice may directly or indirectly promote expansion of autoreactive B-1 B-cells and the subsequent suppression of autoimmune disease.81 The potential relevance of these results to human lupus is unclear.

IL6 IL-6 is a cytokine with pleiotropic effects that shares pro-inflammatory effects with IL-1 and TNF-a and exerts immunoregulatory functions on B- and T-cells. It is produced primarily by monocytes, fibroblasts, and endothelial cells, but also by T-cells, B-cells, and mesangial cells.82 IL-6 promotes B-cell maturation to plasma cells and secretion of immunoglobulins. Increasing evidence suggests an important role of IL-6 in the B-cell hyperactivity and immunopathology of SLE. Patients with SLE have increased serum IL-6 levels that correlate with disease activity, with antiDNA levels,83,84 with anemia,85 and with an increased frequency of IL-6–secreting PBMCs.29 IL-6 production has been detected at the site of various organs involved in lupus: in the cerebrospinal fluid in patients with CNS involvement,86 in the urine87 or renal glomeruli of patients with lupus nephritis,88 and in affected skin.89 Serum levels of IL-6–have been correlated with disease activity,83 and urinary levels of IL-6 have been correlated with renal pathology score90 (although one study found that urinary but not serum levels correlated with disease activity).87 In addition, a rise in plasma IL-6 before lupus exacerbation has been reported by Spronk and colleagues to occur in a subgroup of patients with serositis but not with other manifestations.84 Last, with successful treatment of CNS lupus cerebrospinal fluid IL-6 levels decreased significantly.91 An intrinsic abnormality in lupus B-cell responsiveness to IL-6 has been reported. Cells from normal individuals do not spontaneously express IL-6 receptors. However, the majority of B-cells from lupus patients spontaneously express IL-6 receptors.92 Low-density B-cells from healthy subjects did not respond to IL-6, whereas those from patients with active SLE differentiated into immunoglobulin-secreting cells without an additional co-stimulatory signal.93 An association between lupus development and IL-6 has been reported in several murine models. Older autoimmune MRL/lpr mice have increased serum IL-6 and IL-6R in membrane-associated and soluble form.94-96 In old NZB/W mice, IL-6 blockade reduced (and exogenous IL-6 increased) the ex vivo production of IgG anti-dsDNA antibody.97 IL-6 is an early key cytokine in B6.Sle1 lupus, as demonstrated by increased production of IL-6 by splenic B lymphocytes and monocytes and in vitro suppression of ANA production by anti-IL-6 Ab.98 In NZB/W mice, administration of recombinant human IL-6 (rhIL-6) accelerated

IL-10 IL-10 is a potent stimulator of B lymphocyte survival, proliferation, and differentiation and promotes the production of anti-DNA autoantibodies. IL-10 also inhibits activation, cytokine production, and antigen presentation by macrophages and has important interactions in inducing and sustaining immune and inflammatory responses.102 The inhibitory effect of IL-10 on IL-1 and TNF-a production is crucial to its antiinflammatory activities. Several lines of evidence suggest that IL-10 may play a role in the pathogenesis of lupus. For example, low-level increases in splenic IL-10 mRNA expression are observed in the P◊F1 model of lupus.103 Elevated serum levels of IL-10 and increased spontaneous production of IL-10 in several newly diagnosed untreated patients with SLE (but not in normal controls) have also been reported.104 Elevated serum levels of IL-10 have been reported in roughly a third of patients with SLE studied, and levels correlated with disease activity as measured by SLEDAI.105 Disease severity measured by a visual analog scale was reported to correlate with an elevated ratio of IL-10: IFNg-secreting cells as measured by ELISPOT.29 Similarly, a correlation between changes in disease activity measured by SLEDAI and IL-10 levels over a period of 4 weeks of treatment has also been reported.106 Furthermore, neutralizing anti-IL-10 antibody delays the onset of proteinuria, glomerulonephritis, and anti-dsDNA antibody production; decreases mortality in NZB/W mice;107 and inhibits autoantibody production in SCID mice injected with PBMC from SLE patients.108 The protective effect of anti-IL-10 is due to up-regulation of TNF-a production, because treatment with neutralizing anti-TNF-a mAb resulted in rapid development of autoimmunity. Moreover, AS101 (an immunomodulator that reduces serum levels of IL-10) was beneficial in SCID mice transplanted with mononuclear cells from SLE patients and in NZB/W mice.109 In contrast, IL-10 had a protective effect in MRL/lpr mice, as evidenced by the fact that IL-10–deficient mice on this background developed more severe lupus and the administration

of rIL-10 reduced anti-dsDNA production in MRL/+ mice.110 Interestingly, Llorente and colleagues found high levels of in vitro spontaneous production of IL-10 measured at both protein level and mRNA in 83% of SLE patients and in 75% of healthy members of multiplex families (suggesting that high levels of IL-10 may predispose to disease and precede onset).111 Lastly, IL-10 plays an important role in the pathogenesis of immunoregulatory abnormalities in SLE. IL10 blockade can reverse the impaired allogeneic response in SLE patients.112 Of note, a defect in the responsiveness to IL-10–induced suppression has been reported for IL-6 monocyte production.113 Such a defect may explain the concomitant elevation of IL-10 and IL-6 in SLE and contribute to perpetuation of polyclonal B-cell activation that characterizes SLE. The effects of IL-10 on B-cell function have lent support for the therapeutic use of IL-10 antagonists in SLE. In a pilot study, treatment of 6 patients with SLE with rh-anti-IL10 antibody achieved a long-lasting reduction of most clinical parameters in 5/6 patients.114 Further studies will be required to evaluate the full potential of anti-IL-10 therapy.

IL-1

the progression of glomerulonephritis99 without changes in anti-dsDNA antibody levels. Chronic IL-6 blockade using anti-IL-6 or anti-IL-6R monoclonal antibody in pre-autoimmune NZB/W mice prevented increases in anti-dsDNA antibody levels, prevented progression of proteinuria, and resulted in increased survival.100,101 Based on these data, it is possible that IL-6 blockade in human SLE could lead to a decrease in antidsDNA antibody levels and interfere with the inflammatory autoimmune process both systemically and locally. A phase I clinical study is currently under way.

IL-1 The IL-1 gene family is composed of IL-1a, IL-1b, and IL-1receptor-antagonist (IL-1Ra), the last being the only known naturally occurring cytokine antagonist. IL-1 peforms multiple biologic activities and is a regulator of the host response to infection and injury. Spontaneous release of IL-1 from SLE monocytes has been reported as increased in many115,116 but not all studies.117 In one study, increased release of IL-1a and IL-1b correlated with anti-RNP autoantibodies.115 IL-1 concentrations are increased in both the serum and CSF of lupus patients.91,118 High levels of IL-1 production by unstimulated B-cells from lupus patients have also been reported64 and may result in autocrine promotion of sustained B-cell activity.64,116 In contrast, monocytes from some lupus patients exhibit decreased IL-1 production in vitro.119 Polymorphisms in the IL-1 and IL-1Ra genes have been associated with an increased risk for SLE in patients from southeastern United States and China. However, no association with clinical manifestations was found.120,121 Significantly higher in vitro basal production of IL-1Ra by PBMC from lupus patients and increased serum levels in patients with active disease with extrarenal manifestations have been reported.122,123 In contrast, Fc gamma-R–mediated production of IL-1Ra was deficient in patients with active disease.117 Given the pro-inflammatory properties of IL-1, IL-1 blockade has been tried in SLE patients. In one study,

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CYTOKINES IN SYSTEMIC LUPUS ERYTHEMATOSUS

114

a small number of lupus patients treated with the human receptor-antagonist IL-1Ra (Anakinra) for refractory arthritis and myositis were reported to exhibit transient efficacy on arthritis (but there was no effect on myositis).124,125

TNF-A TNF-a is a pro-inflammatory and immunoregulatory cytokine with pleiotropic effects on a variety of cells. It has been suggested that the strong association of both human and murine lupus with specific alleles of MHC gene products may actually be due to an association with the TNF-a gene, which is located within the MHC class II region in close proximity to HLA-DR. Mononuclear cells from patients who are DQw1 or DR2 positive (strongly associated with SLE) produce lower amounts of TNF-a upon stimulation than do DR3 or DR4 positive subjects.126 Polymorphic variants of the TNF-a promoter have been associated with increased production of TNF-a. A number of studies have reported the association of TNF-a gene polymorphism with susceptibility and clinical-immunological findings. For example, a significant association was reported between TNF-a-308A promoter polymorphism and a susceptibility to SLE for AfricanAmericans in the United States,127 and for Caucasians in the Netherlands,128 whereas TNF-a-238 polymorphism conferred susceptibility to SLE in Mexican Mestizos.129 In another study, neither TNF-238 nor TNF-308 promoter polymorphisms conferred susceptibility to SLE.130 Several TNF microsatellite alleles have been reported as being associated with SLE but not independent of HLA associations.131 In Japanese patients, TNFB*2/2 genotype132 and TNFR II polymorphism (196R allele) were reported to confer susceptibility to SLE. The latter was not confirmed in either Spanish or UK populations.133 There is substantial evidence in both humans and animal models of lupus that TNF-a can exert opposing effects on SLE. It may be either protective or accelerate development of SLE. Deguchi and Kishimoto have reported that in vivo treatment of MRL/lpr mice with anti–TNF-a antibodies inhibited the development of inflammatory pulmonary lesions.134 The effect of in vivo rTNF-a on disease progression in NZB/W mice depends on the dose and timing of the administration. Administration of high doses to pre-autoimmune mice improved survival and delayed renal disease, whereas low doses and administration to younger B/W F1 mice accelerated renal disease and mortality rate.135,136 In vivo neutralization of TNF-a induced a lupus-like disease in P◊F1 mice that would otherwise develop acute GVHD mice.7 This outcome was due to critical requirement

for TNF-a in the production of IFN-g and in the induction of CTL capable of eliminating activated autoreactive B-cells. Serum TNF-a levels have been reported to be normal or increased in SLE patients.137,138 In some studies, stimulated T-cells and monocytes contained less intracellular TNF-a and produced lower levels of TNF-a in culture supernatants.53,139 In other studies, increased TNF-a production in vitro was observed.140 Jacob and colleagues reported lower levels of TNF-a production in DR2-positive patients with lupus nephritis,126 whereas Studnicka-Berke and colleagues reported high levels of serum TNF-a correlating with disease activity,138 the highest levels being associated with active lupus nephritis. On the other hand, no correlation with disease activity was reported in three patients followed during 19 clinical periods of active or inactive disease.141 The experimental evidence from murine models suggests that administration of anti-TNF-a might predispose to the development of SLE in humans. TNF-a blocking agents are now in widespread use for the treatment of rheumatoid arthritis, Crohn’s disease, and other conditions. Antinuclear antibodies, antidsDNA, and anti-cardiolipin antibodies have been reported in 7 to 15% of patients who have received anti-TNF-a therapy.142 The majority of these patients develop IgM but not IgG anti-ds DNA antibodies, and only 0.2% develop clinical features of SLE. However, there are a number of well-documented cases of SLE,143 which are typically mild and remit when the drug is stopped. One potential mechanism by which TNF-a blockade may promote humoral autoimmunity is the complete inhibition of IFN-g and the induction of CTL that could suppress autoreactive B-cells.7 Another potential mechanism is suggested by the demonstration of increased transcription of IFN-a-regulated genes in patients on anti-TNF therapy and sustained in vitro IFN-a release by anti-TNF-treated PDC,144 indicating a reciprocal relationship between TNF-a and IFN-a. Nevertheless, TNF blockade may be a useful therapeutic adjunct in lupus patients. An open label study using infliximab along with immunosuppressants in 6 patients with SLE demonstrated significant improvement in clinical manifestations in all patients145 despite increased titers of anti-dsDNA antibodies (Table 12.1).

TRANSFORMING GROWTH FACTOR-BETA (TGF-B) TGF-b is a pleiotropic cytokine with potent proinflammatory and immunoregulatory properties. Among hematopoetic cells, TGF-b is produced by platelets, activated T- and B-cells, macrophages, and dendritic cells.146 TGF-b is one of the most important

Cytokine

Murine Models of SLE

IFN-g

●

●

●

IL-6

●

●

●

IFN-a

●

●

IL-10

●

Human SLE

IFN-g blockade results in renal remission or prevention of glomerulonephritis Dichotomous effect of exogenous administration with disease. Improvement in preautoimmune mice and worsening in older mice

●

Age-related increase in serum IL-6 and sIL-6R levels IL-6 blockade prevents increase in anti-dsDNA levels, proteinuria improves survival IL-6 increases autoantibody production and accelerates glomerulonephritis

●

Ameliorated disease pattern in IFNa/b receptor-deleted NZB mice Exogenous administration induces lethal disease course in pre-autoimmune NZBF1 but suppresses autoimmunity in MRL/lpr and B6.Sle2 congenic mice

●

IL-10 blockade delays onset of proteinuria, glomerulonephritis, and anti-dsDNA antibody and decreases mortality in NZB/W mice

●

● ●

● ●

● ●

●

● ●

TNF-a

●

Complex effects in vivo can accelerate or delay autoimmunity in mouse models, depending on dose and timing

●

●

●

Elevated or decreased serum levels Increased IFN-g mRNA expression Increased intracellular expression in CD4 T-cells from patients with lupus nephritis Decreased number of IFN-g-secreting cells Decreased production by mitogen stimulated T-cells Increased serum levels, number of IL-6-secreting PBMCs, and in vitro production by T- and B-cells Increased expression of IL-6R on low density B cells. Increased IL-6 expression in the kidneys of lupus nephritis and increased urinary IL-6 levels during acute lupus nephritis Increased serum levels correlating with disease activity and immune activation parameters IFN-a up-regulated genes in PBMC

TRANSFORMING GROWTH FACTOR-BETA (TGF-B)

TABLE 12.1 SUMMARY OF ACTIVITIES FOR SELECTED CYTOKINES IN SLE

IL-10 promotes production of anti-ds-DNA antibodies (increased spontaneous production in vitro) Contributes to impaired-cell-mediated immunity Enhances Fas-mediated apoptosis Normal or increased serum levels (lower levels of TNF-a production in patients with nephritis) Decreased intracellular expression and production by stimulated T-cells and monocytes Increased renal expression of TNF-a in mesangial cells

Cytokine polymorphism in SLE: Gene

Site of Polymorphism

Type of Polymorphism

IL-10

Promoter-1082 IL10.G IL10.R

Single-base G/A Microsatellite Microsatellite

IL-4

Promoter-590

Single-base C/T

IL-6

3’ AT-rich minisatellite

AT-rich repeat

IL-1a

Promoter-889

Single base C/C

IL-1Ra

Intron 2 IL1RN*1 Intron 2 IL1RN*2

86-bpVNTR 86-bpVNTR

TNF-a

TNFa Promoter-238 Promoter-308

Microsatellite Single-base A/G Single-base A/G

IFNR1

Val14Met nt 88

Single-base A/G

cytokines in immune regulation and serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of immune cells. TGF-b1 can suppress T- or B-cell functions and can enable CD8+ T-cells to develop suppressor activity.147 Most importantly, TGF- b1 can serve as a co-stimulatory factor in the development of regulatory T-cells.148,149

The actions of TGF-b are not only complex but context dependent (in that the variable activities reflect not only the cell type and its state of differentiation but the cytokine milieu).150 The three isoforms of TGF-b (TGF-b 1, 2, and 3) have similar but not identical targets. TGF-b can induce its own production and secretion, potentially perpetuating TGF-b production. Studies of

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CYTOKINES IN SYSTEMIC LUPUS ERYTHEMATOSUS

cytokine regulation of TGF-b have demonstrated that IL-2 and TNF-a increase the production of TGF-b, whereas IL-10 has the opposite effect. Within the immune network, TGF-b mediates a negative feedback effect that inhibits activation, proliferation, and effector functions. T lymphocytes are clearly influenced by TGF-b at all stages of development, and the effects of TGF-b are a function of their state of differentiation. Specifically, TGF-b inhibits T-cell proliferation in response to IL-1, IL-2, recall antigens, and mitogens,151 and decreases production of IL-2 and IL-2 receptor expression.152 CD4+ T-cells are predominantly affected, and the cytokine profile is shifted from a Th1 to a Th2 phenotype.153 TGF-b can also enhance the growth of T-cells (predominantly of the naive phenotype) to become regulatory T-cells rather than Th1 or Th2 effector cells.154 CD8+ T-cells activated in the presence of IL-2 and TGF-b can inhibit IgG production,147 and CD4+ T cells activated in the presence of TGF-b inhibit CD8+ T-cell responses to alloantigens.149 The phenotype of these regulatory T-cells is identical to the naturally occurring thymic-derived CD4+ CD25+ regulatory T-cells. The role of TGF-b in the development of regulatory cells is particularly relevant to lupus. Decreased in vitro production of stimulated total and active TGF-b was reported in SLE patients.155 Furthermore, production of total but not active TGF-b was inversely correlated with disease activity.156 In line with this observation is the finding that CD4+CD25+ regulatory T-cells are decreased in patients with clinically active SLE.157 Plasmin is the principal protease that converts the latent form of TGF-b to its active form. Levels of plasmin activator are decreased in serum from SLE patients, whereas levels of plasmin activator inhibitor are increased.158 The abnormal plasmin levels along with the decreased IL-2 and TNF-a (cytokines that can induce TGF-b production) and increased amounts of IL-10 (which inhibits TGF-b production) could all contribute to decreased TGF-b observed in SLE.

TGF-b typically inhibits B-cell proliferation, decreases secretion and synthesis of IgG and IgM, and may induce apoptosis of B-cells and plasma cells (therefore serving as an important regulatory feedback loop to limit expansion of an activated B-cell population).146 Many of the functional activities of TGF-b (such as inhibiton of MHC class II expression by activated macrophages, inhibition of T-cell secretion of IL-2, and proliferation and inhibition of mitogenstimulated B-cell and NK cell function) are reminiscent of the immune system abnormalities seen in SLE. Thus, TGF-b activity may represent an attempt by the immune system to down-regulate the chronic lymphocyte hyperactivity characteristic of SLE. This idea is supported by the phenotype of TGF-b knockout mice characterized by the development of an autoimmunelike phenotype, including enhanced expression of MHC class I and II antigens, circulating SLE-like IgG antibodies to nuclear antigens, and progressive infiltration of lymphocytes into multiple organs (leading to myocarditis, polymyositis, and Sjogren-like syndrome).159

CONCLUSIONS The disordered immunoregulation characteristic of SLE is accompanied by multiple abnormalities in a diverse array of cytokines. A complete mechanistic and unifying understanding of these complex and often contradictory abnormalities has yet to be achieved. The recent appreciation for the role of regulatory cells (and their relevant cytokines) in the immune network has added complexity to the interpretation of cytokine abnormalities in SLE and has resulted in a reappraisal of the roles of several cytokines. As the role of cytokines in regulatory cell function is separated out from their role in antigen-driven lymphocyte respones, it is anticipated that a clearer understanding of SLE immunoregulation will emerge.

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76. Furusu A, Miyazaki M, Koji T, et al. Involvement of IL-4 in human glomerulonephritis: An in situ hybridization study of IL-4 mRNA and IL-4 receptor mRNA. J Am Soc Nephrol 1997;8:730. 77. Singh RR, Saxena V, Zang S, et al. Differential contribution of IL-4 and STAT6 vs STAT4 to the development of lupus nephritis. J Immunol 2003;170:4818. 78. Stein LF, Saed GM, Fivenson DP. T-cell cytokine network in cutaneous lupus erythematosus. J Am Acad Dermatol 1997; 36:191. 79. Amel-Kashipaz MR, Huggins ML, Lanyon P, et al. Quantitative and qualitative analysis of the balance between type 1 and type 2 cytokine-producing CD8(-) and CD8(+) T cells in systemic lupus erythematosus. J Autoimmun 2001;17:155. 80. Chen YY, Suen JL, Wu WM, et al. The effect of cytokines on the activation-induced apoptosis of B cells in autoimmune NZB x NZW F1 mice. Scand J Immunol 2001;53:596. 81. Wen X, Zhang D, Kikuchi Y, et al. Transgene-mediated hyperexpression of IL-5 inhibits autoimmune disease but increases the risk of B cell chronic lymphocytic leukemia in a model of murine lupus. Eur J Immunol 2004;34:2740. 82. Naka T, Nishimoto N, Kishimoto T. The paradigm of IL-6: From basic science to medicine. Arthritis Res 4 Suppl 2002;3:S233. 83. Linker-Israeli M, Deans RJ, Wallace DJ, et al. Elevated levels of endogenous IL-6 in systemic lupus erythematosus: A putative role in pathogenesis. J Immunol 1991;147:117. 84. Spronk PE, ter Borg EJ, Limburg PC, et al. Plasma concentration of IL-6 in systemic lupus erythematosus: An indicator of disease activity? Clin Exp Immunol 1992;90:106. 85. Ripley BJ, Goncalves B, Isenberg DA, et al. Raised levels of interleukin 6 in systemic lupus erythematosus correlate with anaemia. Ann Rheum Dis 2005;64:849. 86. Hirohata S, Miyamoto T. Elevated levels of interleukin-6 in cerebrospinal fluid from patients with systemic lupus erythematosus and central nervous system involvement. Arthritis Rheum 1990;33:644. 87. Peterson E, Robertson AD, Emlen W. Serum and urinary interleukin-6 in systemic lupus erythematosus. Lupus 1996;5:571. 88. Horii Y, Iwano M, Hirata E, et al. Role of interleukin-6 in the progression of mesangial proliferative glomerulonephritis. Kidney Int Suppl 1993;39:S71. 89. Nurnberg W, Haas N, Schadendorf D, et al. Interleukin-6 expression in the skin of patients with lupus erythematosus. Exp Dermatol 1995;4:52. 90. Malide D, Russo P, Bendayan M. Presence of tumor necrosis factor alpha and interleukin-6 in renal mesangial cells of lupus nephritis patients. Hum Pathol 1995;26:558. 91. Alcocer-Varela J, Aleman-Hoey D, Alarcon-Segovia D. Interleukin-1 and interleukin-6 activities are increased in the cerebrospinal fluid of patients with CNS lupus erythematosus and correlate with local late T-cell activation markers. Lupus 1992;1:111. 92. Nagafuchi H, Suzuki N, Mizushima Y, et al. Constitutive expression of IL-6 receptors and their role in the excessive B cell function in patients with systemic lupus erythematosus. J Immunol 1993;151:6525. 93. Kitani A, Hara M, Hirose T, et al. Heterogeneity of B cell responsiveness to interleukin 4, interleukin 6 and low molecular weight B cell growth factor in discrete stages of B cell activation in patients with systemic lupus erythematosus. Clin Exp Immunol 1989;77:31. 94. Kobayashi I, Matsuda T, Saito T, et al. Abnormal distribution of IL-6 receptor in aged MRL/lpr mice: Elevated expression on B cells and absence on CD4+ cells. Int Immunol 1992; 4:1407. 95. Suzuki H, Yasukawa K, Saito T, et al. Serum soluble interleukin-6 receptor in MRL/lpr mice is elevated with age and mediates the interleukin-6 signal. Eur J Immunol 1993;23:1078. 96. Tang B, Matsuda T, Akira S, et al. Age-associated increase in interleukin 6 in MRL/lpr mice. Int Immunol 1991;3:273. 97. Mihara M, Ohsugi Y. Possible role of IL-6 in pathogenesis of immune complex-mediated glomerulonephritis in NZB/W F1 mice: Induction of IgG class anti-DNA autoantibody production. Int Arch Allergy Appl Immunol 1990;93:89. 98. Liu K, Liang C, Liang Z, et al. Sle1ab mediates the aberrant activation of STAT3 and Ras-ERK signaling pathways in B lymphocytes. J Immunol 2005;174:1630.

122. Scuderi F, Convertino R, Molino N, et al. Effect of proinflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus. Autoimmunity 2003;36:71. 123. Sturfelt G, Roux-Lombard P, Wollheim FA, et al. Low levels of interleukin-1 receptor antagonist coincide with kidney involvement in systemic lupus erythematosus. Br J Rheumatol 1997; 36:1283. 124. Moosig F, Zeuner R, Renk C, et al. IL-1RA in refractory systemic lupus erythematosus. Lupus 2004;13:605. 125. Ostendorf B, Iking-Konert C, Kurz K, et al. Preliminary results of safety and efficacy of the interleukin 1 receptor antagonist anakinra in patients with severe lupus arthritis. Ann Rheum Dis 2005;64:630. 126. Jacob CO, Fronek Z, Lewis GD, et al. Heritable major histocompatibility complex class II-associated differences in production of tumor necrosis factor alpha: Relevance to genetic predisposition to systemic lupus erythematosus. Proc Natl Acad Sci USA 1990;87:1233. 127. Sullivan KE, Wooten C, Schmeckpeper BJ, et al. A promoter polymorphism of tumor necrosis factor alpha associated with systemic lupus erythematosus in African-Americans. Arthritis Rheum 1997;40:2207. 128. van der Linden MW, van der Slik AR, Zanelli E, et al. Six microsatellite markers on the short arm of chromosome 6 in relation to HLA-DR3 and TNF-308A in systemic lupus erythematosus. Genes Immun 2001;2:373. 129. Zuniga J, Vargas-Alarcon G, Hernandez-Pacheco G, et al. Tumor necrosis factor-alpha promoter polymorphisms in Mexican patients with systemic lupus erythematosus (SLE). Genes Immun 2001;2:363. 130. Rudwaleit M, Tikly M, Khamashta M, et al. Interethnic differences in the association of tumor necrosis factor promoter polymorphisms with systemic lupus erythematosus. J Rheumatol 1996;23:1725. 131. Hajeer AH, Worthington J, Davies EJ, et al. TNF microsatellite a2, b3 and d2 alleles are associated with systemic lupus erythematosus. Tissue Antigens 1997;49:222. 132. Takeuchi F, Nabeta H, Hong GH, et al. The genetic contribution of the TNFa11 microsatellite allele and the TNFb + 252*2 allele in Japanese RA. Clin Exp Rheumatol 2005;23:494. 133. Al-Ansari AS, Ollier WE, Villarreal J, et al. Tumor necrosis factor receptor II (TNFRII) exon 6 polymorphism in systemic lupus erythematosus. Tissue Antigens 2000;55:97. 134. Deguchi Y, Kishimoto S. Tumour necrosis factor/cachectin plays a key role in autoimmune pulmonary inflammation in lupusprone mice. Clin Exp Immunol 1991;85:392. 135. Gordon C, Ranges GE, Greenspan JS, et al. Chronic therapy with recombinant tumor necrosis factor-alpha in autoimmune NZB/NZW F1 mice. Clin Immunol Immunopathol 1989; 52:421. 136. Brennan DC, Yui MA, Wuthrich RP, et al. Tumor necrosis factor and IL-1 in New Zealand Black/White mice: Enhanced gene expression and acceleration of renal injury. J Immunol 1989; 143:3470. 137. Maury CP, Teppo AM. Tumor necrosis factor in the serum of patients with systemic lupus erythematosus. Arthritis Rheum 1989;32:146. 138. Studnicka-Benke A, Steiner G, Petera P, et al. Tumour necrosis factor alpha and its soluble receptors parallel clinical disease and autoimmune activity in systemic lupus erythematosus. Br J Rheumatol 1996;35:1067. 139. Mitamura K, Kang H, Tomita Y, et al. Impaired tumour necrosis factor-alpha (TNF-alpha) production and abnormal B cell response to TNF-alpha in patients with systemic lupus erythematosus (SLE). Clin Exp Immunol 1991;85:386. 140. Jones BM, Liu T, Wong RW. Reduced in vitro production of interferon-gamma, interleukin-4 and interleukin-12 and increased production of interleukin-6, interleukin-10 and tumour necrosis factor-alpha in systemic lupus erythematosus. Weak correlations of cytokine production with disease activity. Autoimmunity 1999;31:117. 141. Meijer C, Huysen V, Smeenk RT, et al. Profiles of cytokines (TNF alpha and IL-6) and acute phase proteins (CRP and alpha 1AG) related to the disease course in patients with systemic lupus erythematosus. Lupus 1993;2:359.

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99. Ryffel B, Car BD, Gunn H, et al. Interleukin-6 exacerbates glomerulonephritis in (NZB x NZW)F1 mice. Am J Pathol 1994;144:927. 100. Finck BK, Chan B, Wofsy D. Interleukin 6 promotes murine lupus in NZB/NZW F1 mice. J Clin Invest 1994;94:585. 101. Mihara M, Takagi N, Takeda Y, et al. IL-6 receptor blockage inhibits the onset of autoimmune kidney disease in NZB/W F1 mice. Clin Exp Immunol 1998;112:397. 102. Moore KW, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683. 103. Rus V, Svetic A, Nguyen P, et al. Kinetics of Th1 and Th2 cytokine production during the early course of acute and chronic murine graft-versus-host disease. Regulatory role of donor CD8+ T cells. J Immunol 1995;155:2396. 104. Horwitz DA, Jacob CO. The cytokine network in the pathogenesis of systemic lupus erythematosus and possible therapeutic implications. Springer Semin Immunopathol 1994;16:181. 105. Houssiau FA, Lefebvre C, Vanden Berghe M, et al. Serum interleukin 10 titers in systemic lupus erythematosus reflect disease activity. Lupus 1995;4:393. 106. Park YB, Lee SK, Kim DS, et al. Elevated interleukin-10 levels correlated with disease activity in systemic lupus erythematosus. Clin Exp Rheumatol 1998;16:283. 107. Ishida H, Muchamuel T, Sakaguchi S, et al. Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J Exp Med 1994;179:305. 108. Llorente L, Zou W, Levy Y, et al. Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus. J Exp Med 1995;181:839. 109. Kalechman Y, Gafter U, Da JP, et al. Delay in the onset of systemic lupus erythematosus following treatment with the immunomodulator AS101: association with IL-10 inhibition and increase in TNF-alpha levels. J Immunol 1997;159:2658. 110. Yin Z, Bahtiyar G, Zhang N, et al. IL-10 regulates murine lupus. J Immunol 2002;169:2148. 111. Llorente L, Richaud-Patin Y, Couderc J, et al. Dysregulation of interleukin-10 production in relatives of patients with systemic lupus erythematosus. Arthritis Rheum 1997;40:1429. 112. Lauwerys BR, Garot N, Renauld JC, et al. Interleukin-10 blockade corrects impaired in vitro cellular immune responses of systemic lupus erythematosus patients. Arthritis Rheum 2000; 43:1976. 113. Mongan AE, Ramdahin S, Warrington RJ. Interleukin-10 response abnormalities in systemic lupus erythematosus. Scand J Immunol 1997;46:406. 114. Llorente L, Richaud-Patin Y, Garcia-Padilla C, et al. Clinical and biologic effects of anti-interleukin-10 monoclonal antibody administration in systemic lupus erythematosus. Arthritis Rheum 2000;43:1790. 115. Aotsuka S, Nakamura K, Nakano T, et al. Production of intracellular and extracellular interleukin-1 alpha and interleukin-1 beta by peripheral blood monocytes from patients with connective tissue diseases. Ann Rheum Dis 1991;50:27. 116. Jandl RC, George JL, Silberstein DS, et al. The effect of adherent cell-derived factors on immunoglobulin and anti-DNA synthesis in systemic lupus erythematosus. Clin Immunol Immunopathol 1987;42:344. 117. Andersen LS, Petersen J, Svenson M, et al. Production of IL1beta, IL-1 receptor antagonist and IL-10 by mononuclear cells from patients with SLE. Autoimmunity 1999;30:235. 118. Liou LB. Serum and in vitro production of IL-1 receptor antagonist correlate with C-reactive protein levels in newly diagnosed, untreated lupus patients. Clin Exp Rheumatol 2001;19:515. 119. Whicher JT, Gilbert AM, Westacott C, et al. Defective production of leucocytic endogenous mediator (interleukin 1) by peripheral blood leucocytes of patients with systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis and mixed connective tissue disease. Clin Exp Immunol 1986;65:80. 120. Parks CG, Cooper GS, Dooley MA, et al. Systemic lupus erythematosus and genetic variation in the interleukin 1 gene cluster: A population based study in the southeastern United States. Ann Rheum Dis 2004;63:91. 121. Huang CM, Wu MC, Wu JY, et al. Interleukin-1 receptor antagonist gene polymorphism in Chinese patients with systemic lupus erythematosus. Clin Rheumatol 2002;21:255.

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PATHOGENESIS

13

Biology of Dendritic Cells Virginia Pascual, MD, A. Karolina Palucka, MD, and Jacques Banchereau, MD

INTRODUCTION The primary role of the immune system is to protect the body against infections. Innate (non Ag-specific) and adaptive (Ag-specific) immunity cells act in concert with effector proteins such as cytokines, antimicrobial peptides, complement, and antibodies to eradicate pathogens.1-3 Lymphocytes (T cells, B cells, NK and NK T cells) and their products are under the control of dendritic cells (DCs).4-7 DCs are composed of multiple subsets with distinct functions. These cells populate peripheral tissues where they capture antigens (Fig. 13.1). Antigen-loaded DCs migrate from the tissues through the afferent lymphatics into the draining lymph nodes. There, they present processed protein and lipid Ags to T cells via both classical (MHC class I and class II) and nonclassical (CD1 family) antigen-presenting molecules.6 Immature (nonactivated) DCs present self-antigens to T cells8-10 and, in the absence of appropriate co-stimulation, induce tolerance. Mature, antigen-loaded DCs induce antigen-specific immunity11 characterized by T-cell proliferation and differentiation into helper and effector cells with unique function and cytokine profiles. DCs can also induce immune tolerance through T-cell deletion and through activation of regulatory T cells. How this complex balance is maintained in health and broken in disease, and how it is regulated through distinct DC subsets and their functional plasticity, is now starting to be understood. We will focus on recent progresses in our knowledge of the physiology of DCs, on the identification of distinct DC subsets that induce distinct types of immune response6,12,13 and how this information might impact our understanding of autoimmune diseases like SLE.

BIOLOGY OF DENDRITIC CELLS

DCs Capture and Present Antigens Immature DCs are remarkably efficient at Ag capture while mature DCs are remarkably efficient at antigen presentation. DCs use several pathways to capture antigen, including (1) macropinocytosis;

(2) receptor-mediated endocytosis via C-type lectins (e.g., mannose receptor, DEC-205, DC-SIGN)14-21 or Fcγ receptors type I (CD64) and type II (CD32) (uptake of immune complexes or opsonized particles)22; (3) phagocytosis of apoptotic and necrotic cells,8,9,23 viruses, and bacteria including mycobacteria,24,25 as well as intracellular parasites such as Leishmania major; and (4) internalization of heat shock proteins, hsp70 or gp96-peptide complexes, through multiple receptors including LOX-126 and TLR2/4.27 Captured antigens are processed in distinct intracellular compartments and loaded onto DCs antigen presenting molecules (reviewed in Trombetta and Mellman28). Protein antigens are presented by classical MHC class-I and class-II molecules, while lipid antigens are presented through nonclassical CD1 antigen-presenting molecules.6

Presentation via MHC Class II and Class I Captured antigens are presented by MHC class-II molecules,29 which upon DC maturation are transported from lysosomal compartments to the cell membrane.30,31 In fact, this translocation of peptide-MHC (pMHC) class-II complexes from intracellular compartments to cell membrane represents a hallmark of DC maturation. pMHC complexes are very stable on the cell membrane of mature DCs, thereby facilitating TCR recognition. Furthermore, as opposed to macrophages that favor antigen degradation, DCs display lower levels of lysosomal proteases, thereby permitting low rate of antigen degradation.32 This in turn allows antigen retention in lymphoid organs in vivo for extended periods that might favor antigen presentation.32 Thus, prolonged availability of antigen for generation of pMHC complexes and prolonged presentation of such complexes on the cell surface might explain the unique efficiency of DCs in triggering naive T-cell differentiation. MHC class-II molecules are under the control of a transcriptional coactivator, MHC class-II transactivator (CIITA).33 The expression of CIITA is regulated by three independent promoters, the activity of which quantitatively determines MHC class-II expression.34

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CD34+ progenitors Plasmacytoid

Plasmacytoid dendritic cells

Bone marrow

Myeloid

CD11C– pDCs Monocytes

CD11C+ mDCs

Langerhans cells

Blood Interstitial dendritic cells Tissue Macrophages

Distinct subsets of antigen-presenting cells utilize different promoters, that is, plasmacytoid DCs (pDCs) and B cells rely on promoter pIII while myeloid DCs (mDCs) and macrophages use pI.35 These differences may have fundamental impact on the antigen presentation on MHC class II by these cell types and on ensuing immune responses. MHC class-I molecules represent another antigenpresentation pathway exploited by DCs.28 This involves the classical presentation of endogenous peptides, originating from cellular and viral proteins, as well as the presentation of exogenous antigens via crosspriming/presentation. In fact, cross-priming/presentation might be the main pathway through which immunity to tumors and microbes that do not infect DCs directly is generated.8,36,37 The cellular mechanisms that DCs use to present exogenous antigens on MHC class I remain to be defined. Cross-priming might actually depend on the transfer of proteasome substrates rather than peptides.38 Furthermore, the loading compartment remains as yet an unresolved issue, that is, whether it is the endoplasmic reticulum (ER) or a mixed phagosome-ER compartment.39

Presentation via CD1 Family of Nonclassical MHC Molecules 122

CD1 proteins present lipid antigens to effector T cells.40,41 In the human, but not in the mouse, this family consists of four members, CD1a-c (group 1 molecules) and CD1d

Fig. 13.1 Subsets of human dendritic cells. DC progenitors originate from bone marrow CD34+FLT3+ hematopoietic progenitor cells (HPCs). A myeloid pathway generates both Langerhans cells (LCs), found in stratified epithelia such as the skin, and interstitial (int)DCs, found in all other tissues. Another pathway generates plasmacytoid DCs (pDCs), which secrete large amounts of IFN-α after viral infection. Until recently, monocytes and pDCs were considered to be major circulating DC precursor populations. Activated monocytes (e.g., via GM-CSF) yield DCs with different phenotype and function when exposed to different cytokines (IL4, IL15, IFNα/β, TSLP, and so on). Recent studies demonstrated that γ/δ T cells as well as NK cells can acquire DC phenotype and function under the environmental pressure. It remains to be determined how all these DCs relate to each other and which of them can prime naive T cells, a seminal DC function.

(group 2 molecules), with distinct expression patterns.40,41 Different CD1 molecules display distinct intracellular trafficking pathways that likely result in antigen delivery into distinct compartments.40,41 CD1d is involved in antigen presentation to natural killer (NK) T cells, a unique subset of T cells expressing a limited TCR repertoire mostly composed of Vα24Vβ11.42 These innate-like T cells contribute to immune responses to infections and malignancies. Recent studies have identified lysosomal glycosphingolipid, isoglobotrihexosylceramide (iGb3),43 as endogenous antigens, and bacterial glycosylceramides44 as exogenous antigens for presentation by CD1d to NKT cells. Interestingly, CD1d ligation on monocytes triggers translocation of NFκB and IL-12 secretion,45 thus providing a possible mechanism through which NKT cells modulate antigen presenting cells and immune responses.46

DCs Migrate and Orchestrate Migration of Other Cells During their life span, DCs migrate from the bone marrow through the blood to peripheral and lymphoid tissues. Both DC migration and their capacity to orchestrate the migration of immune effectors are fundamental for launching and coordination of immune responses. Blood immature/nonactivated DCs are attracted to tissues through MIP3-a (via CCR6) or MCP chemokines

DC Maturation DC migration is intimately linked with maturation, and has an important impact on T-cell immunity.

Maturation Signals DCs can receive maturation signals from (1) T cells through CD40 ligand,66 as well as from NK, NKT cells, and γ/δ T cells (reviewed in Munz et al.46); (2) proinflammatory molecules including IL-1β, TNF, IL-6, and PGE2,67 or a combination of IL-1β and TNF with typeI (IFN-α/β) and -II (IFN-γ) interferons68; and (3) DC surface molecules involved in pathogen recognition, including Toll receptors (TLRs) and C-type lectins (reviewed in Klechevsky et al.69). It is likely that in vivo DCs will be exposed to a combination of these signals, which then influence the net result of T-cell activation. Interestingly, TLRs are differentially expressed by distinct DC subsets. For example, TLR9 (a receptor for demethylated DNA) is expressed only by pDCs, while mDCs preferentially express TLR 2 and 4 (receptors for bacterial products such as peptidoglycan and

lipopolysaccharide, respectively).70 Similarly, distinct DC subsets express unique lectins,20 which display immunostimulatory (ITAM) or inhibitory (ITIM) motifs. Such differential expression may confer distinct maturation signals and yield a distinct type of immune responses.71 TLR-mediated signaling in other cells (i.e., stromal cells) will trigger expression of specific chemokines/cytokines and adhesion molecules, which in turn will indirectly modulate DC maturation.72

Maturation Phenotype

BIOLOGY OF DENDRITIC CELLS

(via CCR2), as demonstrated for Langerhans cells (LCs) in vitro47 and in vivo,48 respectively. Upon pathogen entry, epithelial cells secrete ligands that provide signals for enhanced DC influx. Indeed, DCs are the first cells to arrive at the site of pathogen entry, even preceding neutrophils.49-51 Little is known about how DCs enter and traffic through the lymphatic vessels. The chemokine receptor CC-chemokine receptor 7 (CCR7) appears to be fundamental in this process.52 Thus, distinct maturation/activation signals—for example, prostaglandin PGE253,54 that induce the preferential expression of CCR7 by DCs—might increase the capacity of DCs to respond to appropriate ligands such as CCL19 and CCL21 expressed in lymphatic vessels and secondary lymphoid organs.55 Distinct DC subsets might enter secondary lymphoid organs through distinct routes. Indeed, upon bacterial triggering, mDC precursors migrate to peripheral tissues and subsequently to draining lymph nodes, while pDC precursors directly enter the lymph nodes in a CXCL9 and E-selectin dependent manner.56 Differential migration of cutaneous DC subsets, that is, LCs and dermal DCs, has been demonstrated recently with the use of vital imaging.57 Thus, after skin immunization, dermal DCs arrived in lymph nodes first and colonized areas distinct from those colonized by the slower-migrating LCs.57 These results might shed light on the way an immune response develops upon cutaneous immunization, a classical route of vaccination. DCs not only migrate, but they orchestrate the migration of immune effectors through chemokines,58-64 and regulate their maturation and function through cell-cell contact and/or soluble factors.4-7,65

As DCs mature, a series of coordinated events take place such as (1) loss of endocytic/phagocytic receptors; (2) up-regulation of co-stimulatory molecules CD40, CD80, CD86, and several members of the TNF/TNF receptor family including CD70 (ligand for CD27), 4-1BB-L, and OX40-L, all of which can have co-stimulatory effects on T cells73; (3) changes in morphology that include the loss of adhesive structures, cytoskeleton disorganization, and acquisition of high cellular motility74; (4) shift in lysosomal compartments with down-regulation of CD68 and up-regulation of DC-LAMP75; (5) change in class-II MHC compartments; and (6) secretion of cytokines including IL-12 and IL-23, which are important for type-1 polarization of T-cell immunity. This basic process of DC maturation can be modulated by pathogens via interaction with TLRs expressed on DCs. For example, TLR ligands together with a T-cell-like signal delivered through CD40, may enhance DC function.76 Indeed, TLR-mediated signals are involved in the control of CD4+ T-cell activation,77 and may play a role in autoimmunity. For example, DCs loaded with a heart-specific self-peptide induce CD4+ T-cell-mediated myocarditis in nontransgenic mice if activated through both CD40 and TLRs.78 Functional DC maturation can also be modulated by C-type lectins. Dectin-1, a yeast binding C-type lectin, synergizes with TLR2 to induce TNF-α and IL-12 production.79 Yet, Dectin-1 can also promote synthesis of IL-2 and IL-10 through recruitment of Syk kinase. Accordingly, syk-/- DCs do not make IL-10 or IL-2 upon yeast stimulation, but produce IL-12, which indicates that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently.79 These results demonstrate that pathogens use several surface molecules to modulate DC function.

Dendritic Cells Determine Type of T-Cell Response DCs control lymphocyte priming and the type of induced T-cell immunity. This is intimately linked with several aspects of DC biology, including DC migration and maturation, and with distinct DC subsets.

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DC Maturation and Outcome of Interaction with T Cells It is currently thought that immature DCs are tolerogenic, while mature DCs are immunostimulatory.80 This has been formally demonstrated in vivo with the use of fusion proteins targeted to immature DCs that lead to the induction of antigen-specific tolerance.81 By contrast, concomitant activation of the DCs with a CD40specific antibody results in a potent immune response because DCs are induced to express a large number of co-stimulatory molecules.82 Thus, immature DCs in the steady state are thought to maintain peripheral tolerance through their ability to present tissue antigens without appropriate co-stimulation. However, mature LPSactivated DCs efficiently expand CD25+CD4+ regulatory T cells.83 Additionally, LCs reaching the lymph nodes under steady-state or inflammatory conditions have been found to express similar levels of MHC class II, CD40, and CD86.57 Thus, the outcome of tolerance induction or priming might not only be related to the maturation stage, but to specific maturation signals, the threshold of activation,84 and/or the activation of a unique set of inhibitory molecules on DCs.

DC Maturation and Regulatory/Suppressor T Cells

Two broad subsets of CD4+ T cells with regulatory function have been characterized,85-87 both of which can be activated/expanded by DCs at distinct maturation stages.

Naturally Occurring CD4+CD25+ T Cells These T cells are produced in the thymus and mediate their suppressive effects in a cell-contact-dependent, antigen-independent manner, without the requirement of IL-10 or TGF-β.88-91 These cells are naturally “anergic” and require stimulation via their TCR for optimal suppressive function. Mature DCs allow their expansion, which is partially dependent on the production of IL-2 by the T cells and B7 co-stimulation by the DCs.83

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T-regulatory (TR) cells derive from CD4+25- T cells and mediate their effects through the production of suppressive cytokines such as IL-10 and TGF-β.92-94 Two types have been described: TR1 cells produce large amounts of IL-10 and low to moderate levels of TGF-β,92 and Th3 cells produce preferentially TGF-β95 and provide help for IgA production.96 Immature DCs induce the differentiation of naive T cells into TR cells.92,97,98 Injection of immature DCs pulsed with influenzaderived peptide has been shown in two healthy adults to lead to antigen-specific silencing of effector T-cell function.99 Murine pulmonary DCs induce the

development of TR in an ICOS-ICOS-L-dependent fashion that leads to the production of IL-10 by DCs.100 Furthermore, a population of “semimature” CD45RBhigh CD11clow murine DCs located within the spleen and lymph nodes has been described. These cells secrete IL10 after activation with LPS or CpG oligonucleotides, but do not up-regulate MHC class II or co-stimulatory molecules under the same conditions. Most importantly, they are highly potent at inducing tolerance that is mediated through the differentiation of TR cells in vivo.98,100 The complexity of the lineage and/or the subpopulations of DCs that may be responsible for tolerance induction is further illustrated by the description of unconventional DCs101 that display phenotypic and functional properties of both natural killer (NK) and dendritic cells (DCs). These cells appear able to induce protection against virally induced type-1 diabetes in a mouse model.

DC Subsets and Type of Induced T-Cell Immunity Finally, distinct DC subsets differentially modulate T-cell immunity. In mice, splenic CD8α+ DCs prime naive CD4+ T cells to make Th1 cytokines in a process involving IL-12, whereas splenic CD8α− DCs prime naive CD4+ T cells to make Th2 cytokines.102,103 Furthermore, different signals can induce different T-cell polarization by the same DCs, as shown by the induction of IL-12 production and Th1-cell polarization when DCs are activated with Escherichia coli LPS, but no IL-12 production and Th2-cell polarization when DCs are exposed to LPS from Porphyromonas gingivalis.104 In humans, CD40-ligand (CD40-L)– activated, monocyte-derived DCs prime Th1 responses through an IL-12-dependent mechanism, whereas pDCs activated with IL-3 and CD40-L have been shown to secrete negligible amounts of IL-12 and prime Th2 responses.105 Furthermore, IL-3- and CD40-L–activated pDCs induce CD8+T cells with regulatory/suppressor function.106,107 Thus, both the type of DC subset and the activation signals to which DCs are exposed are important for T-cell polarization.

Dendritic Cells Are Composed of Subsets Classically, two main DC differentiation pathways are recognized.6,7 A myeloid pathway generates Langerhans cells (LCs), which are found in stratified epithelia such as the skin, and interstitial (int)DCs, which are found in all other tissues.108 Another pathway generates plasmacytoid DCs (pDCs),109 which secrete large amounts of IFN-α/β after viral infection.110-112

DC Progenitors and Precursors

DC progenitors reside within CD34+ hematopoietic progenitor cells (HPCs).113 Both lymphoid and common

Myeloid DC Diversity This concept of plasticity/flexibility of the DC system is even further exemplified by monocytes and their response to environmental signals. Thus, monocytes can differentiate into either macrophages, which act as scavengers, or DCs that induce specific immune responses.130,131 Different cytokines skew the in vitro differentiation of monocytes into DCs with different phenotypes and function. Thus, when activated (e.g., by GM-CSF) monocytes encounter IL-4, they will yield IL4-DCs.132-134 By contrast, after encountering IFN-α/β, TSLP, TNF, or IL-15, activated monocytes will differentiate into IFN-DCs,135-138 TSLP-DCs,139,140 TNFDCs,141 or IL15-DCs,142 respectively. Thus, rather than the classical distinction of LCs and intDCs, we should consider myeloid DCs as a gradient of immunostimulatory DCs. Thus, mDCs are polarized by other cells and their products including IFN-α from pDCs, IFNγ from γ/δ T cells and NK cells, IL-4 and TNF from mast cells, IL-15 and TSLP from stromal cells, and IL10 from lymphocytes. These distinct DCs will induce distinct types of T-cell immunity. The challenge for years to come will be to link these distinct DC phenotypes in vitro with a specific type of immune response and immune pathology in vivo as exemplified by TNF and IFN-α143,144 or by TSLP in allergic inflammation.139

Distinct DC Subsets Are Endowed with Distinct Functional Properties Each DC subset has common as well as unique biological functions determined by a unique combination of cell-surface molecules and cytokines. Thus, in vitro experiments showed that LCs and interstitial DCs generated in cultures of CD34+ hematopoietic progenitors differ in their capacity to activate lymphocytes: interstitial DCs induce the differentiation of naive B cells into immunoglobulin-secreting plasma cells,108,145 whereas LCs seem to be particularly efficient activators of cytotoxic CD8+ T cells. They also differ in the cytokines that they secrete (e.g., only interstitial DCs produce IL-10) and their enzymatic activity,108,145 which might be fundamental for the selection of peptides that will be presented to T cells. Indeed, different enzymes are likely to degrade a given antigen into different peptide repertoires, as recently shown for HIV nef protein.146 This will lead to different sets of pMHC complexes being presented and to distinct antigen-specific T-cell repertoires. DC subsets express unique lectins,20 which at least partially account for the biological differences. Thus, LCs express langerin, critical to the formation of Birbeck granules.147,148 The role of these structures is not yet understood. The intDCs express DC-SIGN, which is involved in the interactions with T cells and DC migration, but is also used by pathogens, such as HIV, to hijack the immune system.149-151 pDCs express yet another lectin BDCA2, which is absent on mDCs.152,153 TLRs are also differentially expressed, as only pDCs express the nucleic acid-recognizing TLR-7/8 and TLR9. Such differential expression may permit specific in vivo targeting of DC subsets for induction of a desired type of immune responses, as recently demonstrated in mice by targeting DEC-205.81,82 Importantly, we begin to understand the molecular pathways underlying differential responses of pDCs and mDCs to pathogens and/or pathogen derived factors. This can be best illustrated by studies on mechanisms regulating type-I interferon secretion. Thus, pDCs are recognized as a main source of type-I interferon produced in response to viral110 or CpG154,155 triggering. Recent studies demonstrated that IRF-7 is critical for IFNα/β secretion in response to both stimuli. However, IRF-7 activation in response to virus is MyD88 independent, while response to CpG is dependent on both IRF-7 and MyD88.157 It turns out that pDCs but not mDCs can direct CpG to endosomal compartments, thereby allowing MyD88/IRF-7 activation and IFNα/β secretion.157

BIOLOGY OF DENDRITIC CELLS

myeloid progenitors yield, at the clonal level, mDCs as well as cells with pDC phenotype and capacity to secrete large amounts of IFN-α.114 Interestingly, the progenitors of pDCs and mDCs can be found within FLT3+ HPCs..114,115 This is consistent with the wellestablished role of FLT3 ligand (FLT3-L) in DC differentiation/mobilization in vivo in both humans and mice.117-120 Accordingly, FLT3-L is essential in the generation of pDCs and myeloid DCs (mDCs),122-124 and FLT3-L deficient mice show a considerable decrease in numbers of DCs in both peripheral and lymphoid tissues.117 Thus, FLT3-L appears as a major factor governing DC homeostasis in the steady state. Given the role of GM-CSF in DC generation,113,125,126 activation and survival,127 it is possible that GM-CSF is actually a major factor governing DC homeostasis upon infection. Until recently monocytes, lineage− mDC precursors and pDCs have been considered as major circulating DC precursor populations (Fig. 13.1). However, recent studies demonstrated that γ/δ T cells can acquire DC phenotype and function.128 Furthermore, proinflammatory cytokines can endow human NK cells with the ability to acquire antigen and stimulate T cells.129 These observations suggest remarkable plasticity in the antigen-presenting cell system. Thus, monocytes may yield all myeloid DCs while pDCs, γ/δ T cells, and NK cells yield another set of cells with DC properties.

Subsets of pDCs pDCs display distinct functions at two distinct stages of differentiation: (1) precursor pDC secrete large amounts of IFN-a after viral infection,110-112 and (2) mature pDC

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activate and modulate T-cell responses.158 Indeed, depending on the type of activation pDCs give rise to unique T-cell responses: they induce T cells secreting IFN-γ and IL-10 upon viral triggering and type-2 T cells upon activation with IL-3 and CD40 ligand.158 Recently the existence of pDCs subsets has been demonstrated. Thus, in the mouse, expression of lymphoid-related genes (RAG1 and Ig rearrangement products158) or proteins (CD4159) distinguishes between two subsets of pDCs. While the functional consequence of RAG1 and Ig rearrangement product expression remains to be determined, CD4neg pDCs appear mainly responsible for migration to lymphoid tissue and IFN-α secretion upon exposure to CpG.159

DCs Interact with Other Cells of Immune System DCs also regulate naive145 and memory160 B cells, NK cells,161 and NKT cells.162

Interaction with B Cells As discussed above, myeloid DCs can prime naive B cells. Several molecules have been shown to be involved in this process, including IL-12, IL-66 and, more recently, BAFF/Blys,163-167 a molecule up-regulated by IFN-α. IFN-α and IL-6 are also important in the differentiation of activated B cells into efficient Ig-secreting plasma cells upon exposure to virus-triggered pDCs.160 Strikingly, the plasma cells generated under these conditions express very high levels of CD38, similar to that of plasma cells isolated from lymphoid tissues. In contrast, plasma cells generated by culturing activated B cells with the T-cell-derived cytokines IL-2 and IL-10, although efficient Ig secretors, do not express high levels of CD38.168 This suggests that IFNα may represent an important cytokine in the generation of plasma cells in tissues. Indeed, studies in the mouse also support that IFN-α is an excellent adjuvant for humoral immunity.169 However, myeloid DCs may also indirectly contribute to plasma cell differentiation.170-173 Finally, differential activation of CD4+T cells with B-cell helper function by distinct DC subsets might play an important role in the induction of protective humoral immunity.

Cross-talk with Innate Lymphocytes

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DCs also interact with natural killer (NK), NKT, and γ/δ T cells. Indeed, these innate immune effectors induce DC maturation through a combination of soluble and cell-mediated signals (reviewed in Munz et al.46). In turn, mature DCs stimulate NK,161,174,175 NKT,176 and γ/δ T cells.177,178 These reciprocal interactions occur largely in secondary lymphoid organs and are important for the amplification of type-1 T-cell immunity. Distinct DC subsets appear to differentially

interact with innate lymphocytes. Thus, intDCs derived either from monocytes or from CD34+HPCs directly stimulate NK-cell proliferation and cytotoxic function.179 On the contrary, LCs require exogenous cytokines to activate NK cells.179

DENDRITIC CELLS AND TOLERANCE DCs are now thought to play a pivotal role in the control of both central and peripheral tolerance.10,11,80,180

Central Tolerance The thymus steadily produces thymocytes expressing newly assembled TCR, some of which may be reactive with components of self. High-affinity autoreactive thymocytes are eliminated upon encountering selfMHC peptide.181-183 There is evidence that both thymic epithelial cells as well as mature DCs in the thymus may be involved in this process.184,185 However, autoreactive T cells that are not deleted in the thymus need to be controlled in the periphery to prevent immune responses to self; hence, the need for peripheral tolerance that occurs in lymphoid organs.

Peripheral Tolerance through DCs There is evidence that immature/steady-state DCs control peripheral tolerance (reviewed in Steinman et al.80). In the absence of inflammation, these DCs present tissue antigens to T cells in the absence of appropriate co-stimulation, leading to T-cell anergy or deletion,80 or the development of IL-10-producing, regulatory T cells as discussed earlier.97,99 By suppressing a mandatory T-cell help, DCs may also avoid self-Ag–specific and T-dependent B-cell activation. The molecular mechanisms underlying the tolerogenic properties of peripheral DCs might involve (1) lack of and/or inappropriate co-stimulation, (2) cell death induction by expression of indoleamine 2,3-dioxygenase (IDO), which induces the catabolism of tryptophan, or by Fas/Fas-L interaction, (3) secretion of IL-10/TGF-β, and (4) inhibitory receptors.

Inhibitory Receptors The effector function of immune cells is regulated by positive and negative signals provided through class-I recognition receptors.186-191 In humans, these are provided by KIR (killer-cell immunoglobulin receptor) and KLR (killer-cell lectin-like receptor) family members that interact specifically with certain HLA allotypes and class-I-related genes.192 A third class of receptors with both activating and inhibitory functions includes immunoglobulin-like molecules (LILR, for leukocyte immunoglobulin-like receptors, also referred to as ILT, for immunoglobulin-like transcript). ILT family members are expressed on a wide

DCs in SLE Monocytes cannot initiate primary immune responses unless they are triggered to differentiate into mDCs. However, monocytes from SLE patient blood act as DCs since they are able to induce proliferation of allogeneic CD4 T cells.138 These results suggest that SLE blood represents a DC-inducing environment. Indeed, exposure

of normal monocytes to SLE serum results in rapid generation of cells with DC morphology and function.138 As discussed above, several cytokines have been shown to allow the differentiation of precursor cells into DCs. In particular, IFN-α together with GM-CSF allow monocytes to become DCs.135-137 Accordingly, the DC-inducing property of SLE serum is mediated by IFN-α, because it can be inhibited by an anti-IFN-α antibody and reproduced by spiking normal sera with IFN-α. Finally, the DC-inducing capacity of SLE serum correlates with disease activity according to SLEDAI.138 These results point to IFN-α as a factor driving unabated DC differentiation/activation in SLE. IFN-α can be produced by numerous cell types.201 However, pDCs are remarkable by the amount of IFN-α that they can produce, and these cells represent a likely source of IFN-α in SLE patients. Yet, pDCs (lineage CD123+ Class II+ cells) numbers are reduced in SLE blood.138,202 However, these cells infiltrate inflamed lupus skin,203,204 suggesting that the reduction in SLE blood pDCs results from their accelerated migration to sites of inflammation, as demonstrated in allergen-challenged nasal mucosa.205 Even though the effects of IFN-α explain many of the features of SLE, not every SLE patient displays elevated serum IFN-α via ELISA. This raised the question as to whether IFN-α deregulation occurs in all or only in a subset of SLE patients. Gene expression signatures using oligonucleotide microarrays demonstrated IFNα-regulated genes in PBMCs of all active pediatric SLE patients206 and in the majority of adult SLE patient blood.207,208 What causes the unabated production of IFN-α in SLE remains to be determined. Apoptotic and/or necrotic cell-derived DNA and RNA give rise to interferogenic immune complexes in vitro.209,210 In fact, chromatin-containing immune complexes can be internalized via FcgRIIa, reach the endosomal compartment and activate TLR-7/9 to induce IFN-α production.211,212 Recently, it has been shown that specific, highly conserved RNA sequences within snRNPs can stimulate TLR 7 and 8 as well as activate pDCs, which respond by secreting high levels of type-I IFN. Furthermore, SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the FcgRII and efficiently stimulate pDCs to secrete type-I IFNs.213 A number of clinical observations confirm critical role of IFN in the development of SLE. IFN therapy in cancer and viral infections is frequently associated with induction of autoantibodies (4 to 19%),214-217 and a variety of SLE symptoms have been reported in these patients with a frequency of 0.15 to 0.7%.218 IFN-α has also been known to exacerbate the underlying predisposition to develop SLE of certain lupus-prone

DENDRITIC CELLS AND TOLERANCE

variety of cells of both lymphoid and myeloid origin, and can be divided into three groups according to function193: (1) inhibitory receptors with cytoplasmic ITIM motifs, (2) activating receptors that associate with the ITAM-containing FcR-gamma chain, and (3) a single member ILT6 that does not contain a transmembrane region and might be secreted. The two most extensively studied members are the inhibitory receptors ILT2 and ILT4. ILT2 is broadly expressed on all monocytes, DCs, most B cells, and subsets of T and NK cells.193 ILT4 is restricted to myeloid cells and expressed on all monocytes and some DC populations. Both ILT2 and ILT4 interact with multiple class-I alleles including HLA-G.194 Signaling through these molecules may be at least in part responsible for the immunosuppressive effects of HLA-G on antigenpresenting cell function. Ligation of ILT4 on DCs by tetrameric HLA-G attenuates maturation in response to CD40L and reduces DC alloproliferative capacity.195 Expression of inhibitory ILT receptors appears to be a general feature of tolerogenic DCs.196 DCs treated with IL-10 specifically up-regulate inhibitory ILT receptors,196 while blocking inhibitory ILT receptors lead to a restoration of the alloproliferative capacity in spite of reduced co-stimulatory molecule expression.197 This novel approach to understanding of DC immunoregulatory function will be important for unraveling how pathogens evade immunity and for targeting of these pathways for therapy and for vaccination. The importance of immunostimulatory and immunoregulatory signals has actually been already demonstrated in the Fc receptor (FcR) system.198 FcRs share the same ligands; therefore, the consequence of immune complex engagement on DC function depends on the balance between activating (ITAM) and inhibitory (ITIM) receptor types. In the steady state, this balance favors inhibition with the expression of ITIM-containing FcγRIIb.199 It is shifted, however, in the presence of proinflammatory cytokines such as IFN-γ and TNF-α with the up-regulation of FcγR3 and down-regulation of RIIb, permitting immune complex-mediated activation.199 Furthermore, the blockade of inhibitory FcγR leads to spontaneous DC maturation with secretion of IL-12 p70.200 Thus, inhibitory and stimulatory receptors expressed on DCs appear fundamental in determining tolerance or immunity.

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Mature DCs

Monocytes

T cells IFN

α/β

CTLs

B cells

Plasma cells

Plasmacytoid DCs Fig. 13.2 IFN-α and myeloid DCs at the center of SLE pathogenesis. The pleiotropic effects of type-I IFN include the maturation of myeloid DC precursors, the differentiation of mature B cells into the plasma cell lineage, and the expansion of cytotoxic CD8 T cells. This last effect can be driven directly by IFN-α or indirectly by mDC that have matured in the presence of IFN-α. Plasma cell differentiation and antibody secretion may lead to the generation of immune complexes that may in turn activate pDCs via FcgRIIa and TLR interaction with chromatin-containing ICs.

In humans, mutations in genes involved in the production of IFN-α/β, such as the recently described IRF-5 polymorphism,224 may predispose to SLE by increasing the ability of PDCs to release these cytokines upon activation. Deficiency in apoptotic cell removal may provide DCs with an excessive load of nuclear antigens to present to T cells. These and other genetic alterations may contribute to a progressive loss of tolerance to nuclear antigens, which has been shown to sequentially precede the development of clinically overt disease. Some of these autoantibodies may form complexes able to activate both B cells, through the co-engagement of BCR and TLRs, and pDCs through the co-engagement of FcgR and TLRs. pDC-derived IFN-α, together with IL-6, promotes the differentiation of mature B cells into plasma cells, which in turn may increase autoantibody secretion and immune complex formation, closing this self-perpetuating loop. IFN-α will contribute to enhance any of these loops and precipitate the development of clinical disease.225

ACKNOWLEDGMENTS mouse strains.219,220 Conversely, the cross of both NZB and B6 lpr/lpr mice with a type-I IFN receptor KO strain significantly decreases morbidity and prolongs the survival of these animals.221,222 Finally, B6.Nba2 congenic mice display an increased expression of Ifi202, an interferon-inducible gene located within the NZB genetic interval that may mediate some of the lupus-promoting effects of IFN-α.223

Supported by grants from Baylor Health Care Systems Foundation, the National Institutes of Health (PO1 CA84512, U19 AIO57234, CA78846, CA085540, and CA89440), and the Alliance for Lupus Research. JB holds the Caruth Chair for Transplant Immunology Research. AKP holds the Michael A. Ramsay Chair for Cancer Immunology Research.

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192. Middleton D, Curran M, Maxwell L. Natural killer cells and their receptors. Transpl Immunol 2002;10:147-164. 193. Allan DS, McMichael AJ, Braud VM. The ILT family of leukocyte receptors. Immunobiology 2000;202:34-41. 194. Colonna M, Nakajima H, Navarro F, Lopez-Botet M. A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells. J Leukoc Biol 1999;66:375-381. 195. Liang S, Horuzsko A. Mobilizing dendritic cells for tolerance by engagement of immune inhibitory receptors for HLA-G. Hum Immunol 2003;64:1025-1032. 196. Manavalan JS, Rossi PC, Vlad G, Piazza F, Yarilina A, Cortesini R, et al. High expression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells. Transpl Immunol 2003;11:245-258. 197. Beinhauer BG, McBride JM, Graf P, Pursch E, Bongers M, Rogy M, et al. Interleukin 10 regulates cell surface and soluble LIR-2 (CD85d) expression on dendritic cells resulting in T cell hyporesponsiveness in vitro. Eur J Immunol 2004;34:74-80. 198. Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol 2001;19:275-290. 199. Ravetch JV. A full complement of receptors in immune complex diseases. J Clin Invest 2002;110:1759-1761. 200. Dhodapkar KM, Kaufman JL, Ehlers M, Banerjee DK, Bonvini E, Koenig S, et al. Selective blockade of inhibitory Fcgamma receptor enables human dendritic cell maturation with IL12p70 production and immunity to antibody-coated tumor cells. Proc Natl Acad Sci U S A 2005;102:2910-2915. 201. Taniguchi T, Ogasawara K, Takaoka A, Tanaka N. IRF family of transcription factors as regulators of host defense. Annu Rev Immunol 2001;19:623-655. 202. Gill MA, Blanco P, Arce E, Pascual V, Banchereau J, Palucka AK. Blood dendritic cells and DC-poietins in systemic lupus erythematosus. Hum Immunol 2002;63:1172-1180. 203. Farkas L, Beiske K, Lund-Johansen F, Brandtzaeg P, Jahnsen FL. Plasmacytoid dendritic cells (natural interferon—alpha/betaproducing cells) accumulate in cutaneous lupus erythematosus lesions. Am J Pathol 2001;159:237-243. 204. Blomberg S, Eloranta ML, Cederblad B, Nordlin K, Alm GV, Ronnblom L. Presence of cutaneous interferon-alpha producing cells in patients with systemic lupus erythematosus. Lupus 2001;10:484-490. 205. Jahnsen FL, Lund-Johansen F, Dunne JF, Farkas L, Haye R, Brandtzaeg P. Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy. J Immunol 2000;165:4062-4068. 206. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau J, et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 2003;197:711-723. 207. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A 2003;100:2610-2615. 208. Crow MK, Kirou KA, Wohlgemuth J. Microarray analysis of interferon-regulated genes in SLE. Autoimmunity 2003;36:481-490. 209. Bave U, Alm GV, Ronnblom L. The combination of apoptotic U937 cells and lupus IgG is a potent IFN-alpha inducer. J Immunol 2000;165:3519-3526. 210. Lovgren T, Eloranta ML, Bave U, Alm GV, Ronnblom L. Induction of interferon-alpha production in plasmacytoid dendritic cells

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Apoptosis Erika Darrah, BS and Antony Rosen, MD

INTRODUCTION Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by a wide array of clinical manifestations involving multiple organ systems. Unraveling the mechanisms responsible for disease initiation and propagation has proven to be equally complex. By the time that patients present with clinical symptoms of disease, the autoimmune response is well established, making early pathways in disease initiation difficult to determine. While it is unlikely that one defect accounts for the complex disease phenotype, significant data now implicate pathways of apoptosis in the pathogenesis of SLE.

KINETICS OF AUTOANTIBODY APPEARANCE IN SLE HIGHLIGHT TWO-STEP PATHOGENIC MODEL

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A centrally important element in the pathogenesis of SLE has become apparent through kinetic studies on individuals during the evolution of autoantibodies and clinical SLE.1,2 The appearance of antiphospholipid and antinuclear antibodies can be detected several years prior to development of SLE, clearly separating initiation of autoimmunity from the propagation phase that generates the clinical phenotype. Coincident with or slightly prior to development of clinical SLE, the antibody profile of individuals broadens to include antibodies to ribonucleoproteins and components of the spliceosome. In contrast, additional autoantibody specificities tend not to appear after development of disease. The asymptomatic autoantibody-positive state can thus be viewed as representing the precursor of the amplified disease phenotype, with targeting of ribonucleoproteins playing a potentially important and direct role in disease amplification and clinical manifestations in the second phase. Importantly, while initiation can be viewed as the generation of the immune response by antigen, the amplifying character of the propagation phase is better viewed as the generation of antigen by the immune response that can further drive

the process. It is becoming increasingly evident that once self-antigen becomes the target of an active immune response, lymphoid, inflammatory, and target tissues may contribute to the self-sustaining loop underlying disease propagation through a cycle of proliferation and apoptosis. It is within this kinetic construct of SLE that the roles of abnormalities in apoptotic pathways will be explored. In particular, impaired tolerance induction by apoptotic cells, dysfunctional clearance of apoptotic material, and novel pro-immune forms of apoptotic death will be examined in terms of possible roles in initiation of the autoimmune response. The role of changes in the availability or context of self-antigen, ligation of Toll-like receptors by self-nucleic acid, and opsonization of apoptotic cells by autoantibodies will be emphasized as possible drivers of the amplification phase.

SLE AUTOANTIGENS CLUSTER INTO CELL SURFACE BLEBS DURING APOPTOSIS Apoptosis is a form of programmed cell death in which a highly specific and orderly set of biochemical changes underlie the unique morphologic changes and the ultimate disposition of the dying cell and its contents. Apoptotic cells undergo a striking, orderly fragmentation and disassembly, and are strong inducers of immune tolerance (see below). Several specific biochemical pathways form the apoptotic framework. In addition to a specialized signaling apparatus (which transduces proapoptotic signals from a variety of subcellular domains), apoptosis is mediated through the activation of a proteolytic caspase cascade, in which a restricted subset of cellular targets are cleaved after aspartic acid residues. Substrates include proteins whose fragments function directly in generating the apoptotic phenotype, as well as a variety of molecules in which cleavage abolishes critical, antiapoptotic functions.3 Reorganization of multiple cellular components occurs during apoptosis, with clustering of a variety of

APOPTOSIS INDUCES TOLERANCE Significant data demonstrate that when apoptosis results in the removal of dead cells, it is associated with induction of immune tolerance to the contained antigens.7-12 Although the mechanisms underlying tolerance induction are numerous and are not fully elucidated, signals at the apoptotic cell surface (e.g. phosphatidyl serine) and cytokine secretion patterns in phagocytic cells (increased secretion of IL-10 and TGF-β and decreased secretion of IL-12 and TNF-α) are clearly important in this regard.13-17 This pattern of cytokines is associated with induction of peripheral tolerance to antigens contained in apoptotic cells. Unlike engulfment of apoptotic cells, necrotic cells trigger phagocytic maturation, proinflammatory cytokine secretion, and up-regulation of co-stimulatory molecules.18,19 Apoptosis is vital in providing self-antigen to induce the central and peripheral tolerance of naive lymphocytes. During development and maturation, T cells20 and B cells,21 which have high affinity for self-antigens, are clonally deleted. The source of this antigen largely comes from the phagocytosed apoptotic bodies of dying lymphocytes present in the thymus,22 bone marrow,23,24 and lymph nodes.10,25,26 Recent data have shown that nucleosome-specific T cells undergo selection in the thymus, as evident by clonal deletion of autoreactive T cells in normal mice. In lupus-prone SNF1 mice, however, a defect exists in the ability of thymic dendritic cells to successfully present an array of endogenous nucleosome antigens to developing thymocytes, allowing the survival of potentially autoreactive T cells.27 Dendritic cells are also important in maintaining peripheral T-cell tolerance to apoptotic antigen by presenting phagocytosed apoptotic material on MHC class-II molecules and migrating to the lymph node. Defects in B-cell tolerance checkpoints in the bone marrow and periphery have been observed in human and mouse SLE, which allow for the production of autoantibodies.28-33 It has been proposed that defects in the tolerance-inducing clearance of apoptotic

material, with the resulting incomplete deletion of autoreactive lymphocytes, might result in susceptibility to initiation of autoimmunity to autoantigens normally presented from apoptotic cells.34,35 It is important to note that, under some circumstances, apoptotic death may not follow the typical biochemical and morphologic patterns generally observed, which may provide an opportunity for novel processing and presentation of self-antigens. For example, the organization of nuclear antigens during apoptosis can be altered under intense death stimuli such as exposure to high doses of UVB. While low doses of UVB cause translocation of nuclear antigens to the plasma membrane in keratinocytes, high-dose UVB causes release of antigen from the cell. This UV-induced release of nuclear autoantigen has been postulated to contribute to initiation of disease observed in some lupus patients after prolonged sun exposure.36

IMPAIRED CLEARANCE OF APOPTOTIC CELLS RENDERS INDIVIDUALS SUSCEPTIBLE TO SYSTEMIC AUTOIMMUNITY The first indication that defects in the clearance and antiinflammatory effects of apoptotic cells rendered individuals susceptible to systemic autoimmunity came from studies on C1q deficiency. C1q-deficient humans or mice have a striking propensity to develop systemic autoimmunity with lupus-like features.37-41 When Botto and colleagues41 examined the phenotype that developed in C1q null mice, they observed a striking accumulation of apoptotic bodies in the kidneys of affected animals. They subsequently noted that clearance of apoptotic cells from the peritoneal cavity was diminished in C1q-deficient animals, and could be corrected by C1q administration.40,41 Interestingly, C1q-deficient animals have a high prevalence of autoantibodies, but frequently have milder autoimmune phenotypes.41 In contrast, deficiencies in several other receptor–ligand pairs that are also associated with impaired clearance of apoptotic cells, and in which animals develop features of SLE, tend to be strongly associated with prominent autoimmune phenotypes. For example, mice deficient for the receptor tyrosine kinase Mer or milk fat globule epidermal growth factor 8 (MFG-E8) exhibit dramatically impaired phagocytosis of apoptotic cells and spontaneously develop autoantibodies. Mer tyrosine kinase appears to be important for the intracellular signaling necessary to trigger the uptake of apoptotic cells, but not bacteria or opsonized particles, by phagocytes.42,43 Mer deficiency causes a lupus-like phenotype in mice with development of rheumatoid factor, antichromatin, and anti-dsDNA antibodies.42 The glycoprotein MFG-E8 is secreted by tingible body macrophages in the spleen and lymph node, and mediates clearance of the apoptotic debris.44

IMPAIRED CLEARANCE OF APOPTOTIC CELLS RENDERS INDIVIDUALS SUSCEPTIBLE TO SYSTEMIC AUTOIMMUNITY

otherwise intracellular molecules in blebs at the surface of the apoptotic cell.4 Prior to nuclear fragmentation, DNA, RNA, ribosomes, and ER components such as Ro can be found clustered at the surface of apoptotic blebs.4-6 Redistribution of histones to the surface of apoptotic blebs and release from the nucleus early in apoptosis has also been documented.5,6 More than a decade ago, it was noted that many of these redistributed intracellular components are autoantigens in lupus and other systemic autoimmune diseases, suggesting that apoptotic blebs might be an important source of autoantigens for both tolerance induction and systemic autoimmunity.

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In germinal centers, tingible body macrophages play an important role in removing low-affinity B cells that have undergone apoptosis.45 Mice lacking MFG-E8 show accumulation of apoptotic material around germinal center macrophages and develop anti-dsDNA and antinuclear antibodies with age, leading to immune complex deposition and proteinuria.44,46 Interestingly, similar features have been observed in the lymph nodes of a small group of human SLE patients in which reduced numbers of germinal center macrophages, increased apoptotic cells, and apoptotic cell accumulation around follicular dendritic cells was observed.47 The mechanisms underlying reduced clearance of apoptotic cells in these patients remain unclear. While it is possible that deficiency in tingible body macrophage function may be genetic in some patients, it is also possible that the defect is acquired, potentially through antibodies that block relevant receptor–ligand interactions. This latter category of defect, where abnormalities in apoptotic cell clearance affect immune cells during processes of selection in an ongoing immune response, may be particularly relevant to disease amplification. Defining such defects and pathways in human SLE remains a high priority.

antigen receptors and TLRs, and that this may be important pathogenetically in the feed-forward loop of SLE.57 It is important to note that eukaryotic nucleic acids are poor ligands for the TLRs, suggesting that additional modifications of nucleic acids may be relevant for recruiting the amplifying properties of these autoantigens51 (see below).

DISEASE INITIATION: RECRUITMENT OF FEED-FORWARD LOOP INVOLVING TOLL-LIKE RECEPTORS

MODIFIED PROTEIN STRUCTURE DURING CELL DEATH

Although the mechanisms underlying the synchronized change in autoantigen targets and the onset of clinical symptoms in SLE remain unknown, this change in specificity strongly suggests recruitment of a feed-forward amplification loop focused on the splicing ribonucleoproteins. Several recent discoveries are particularly tantalizing in this regard. For example, there is now striking data demonstrating that Toll-like receptors (TLRs) recognize self-nucleic acid in the context of splicing ribonucleoproteins and nucleosomes, and induce proinflammatory signals in dendritic cells (DCs) and B cells.48-55 TLRs recognize invariant, repeating, pathogen-associated patterns, and trigger the innate immune response to microbial infections. Most TLRs, including TLR 7, 8, and 9, signal through the adapter molecule MyD88 to trigger proinflammatory cytokine secretion, up-regulation of co-stimulatory molecules, and increased antigen presentation. TLR 9 is known to bind hypomethylated CpG DNA found in bacteria and mammalian DNA promoter regions while TLRs 7 and 8 bind ssRNA (reviewed in Ishii et al.56). Recent data demonstrate that splicing RNAs are highly effective activators of TLR7 and possibly TLR8.55 The emerging consensus from these studies is that a group of frequently targeted autoantigens in SLE have the unusual capacity to co-ligate

DYING CELLS AND AUTOANTIGENS IN SLE Significant data now demonstrate that numerous lupus autoantigens undergo a variety of modifications during apoptotic cell death, which may be of relevance to processing and epitope selection of autoantigens, as well as potentially to the ability to ligate TLRs. We discuss below predominantly the modifications of the protein components of autoantigens observed during cell death. There are also data demonstrating that the nucleic acid components of autoantigens can be modified during cell death, but these studies are at a much earlier stage.58-61 In both cases though, the mechanistic relevance of modified autoantigen structure during various forms of cell death on immunogenicity remains to be directly demonstrated.

Many cellular proteins are altered during apoptosis by proteolytic cleavage and other posttranslational modifications including phosphorylation,62,63 transglutamination,64 oxidation,65,66 citrullination,67,68 and ubiquitination.69 Antigen processing by cellular proteases is highly dependent on protein structure, sequence, and post-translational modifications. While lymphocytes are exposed to the surface and contents of apoptotic cells in the bone marrow and thymus, it is likely that they only encounter forms of antigens that represent the “core” apoptotic process. Novel antigenic modifications not normally presented to lymphocytes during development and selection might be generated under some nonhomeostatic circumstances, exposing cryptic epitopes (reviewed in Hall et al.70). Although each of the modifications noted above may be relevant in terms of providing novel forms of antigens not previously seen and tolerized by the immune system, much attention has been paid to the apoptosis-specific proteolytic modifications of autoantigens. Two observations are most pertinent in this regard: (1) many autoantigens are unified by their susceptibility to cleavage by the caspases, which are themselves responsible for generating the apoptotic phenotype; and (2) the relevance of caspase cleavage to immunogenicity still remains unclear. It has been proposed that caspase-cleaved autoantigens are the

APOPTOSIS AND VIRUS INFECTION There is a large amount of evidence demonstrating that viral infection can induce and/or modify apoptotic death and signaling.84-88 Additionally, there is strong evidence that viral proteins co-cluster with SLE autoantigens at the surface of apoptotic blebs, potentially in direct complexes with viral RNA and other antigens. It has previously been proposed that such co-clustering could place modified self-antigen in a proimmune context and potentially trigger an autoimmune response during infection.89 The recent evidence that autoantigens that co-ligate TLR molecules might be central effectors of the propagation phase of systemic

autoimmunity is particularly relevant in this regard. Cells dying in the context of a viral infection likely contain viral associated nucleic acid, such as single and double stranded RNA, capable of ligating TLRs on phagocytes or B cells. Ligation of TLRs in macrophages and DCs may trigger the release of proinflammatory cytokines, up-regulation of co-stimulatory molecules, and presentation of antigen to T cells. Presenting apoptotic or modified self-antigen in this proinflammatory context, particularly in an individual with preexisting autoantibodies, might trigger an active immune response against self-antigens and contribute to disease propagation.

SLE-IgG, DYING CELLS AND IFN-α

predominant tolerance-inducing forms of these molecules, and that abnormalities in the execution of apoptotic death (e.g., antiapoptotic Bcl-2 family member expression) may allow uncleaved forms of the molecules to be presented with selection of different epitopes.66,71-73 The effects of caspase cleavage on processing and presentation of endogenous autoantigens remain to be directly determined. There are also data demonstrating novel proteolytic cleavage events affecting a large number of autoantigens during non–caspase-mediated cell death processes, including cytotoxic lymphocyte granule–mediated death71 or necrotic death.74,75 Of particular relevance in this regard is granzyme-induced death of target cells, which is not usually homeostatic or developmental in nature, and is generally focused on virus-infected or transformed cells. Autoantigens in systemic autoimmunity are frequently cleaved by granzyme B (GrB), generating unique fragments.71 GrB is a serine protease found in the cytolytic granules of CD8+ cytotoxic T lymphocytes (CTLs) and NK cells. These cells primarily kill virally infected or transformed cells by triggering apoptosis through cleavage of intracellular substrates, including caspases, at aspartate residues. Although both GrB and caspases cleave at aspartate residues, they prefer different consensus sequences and can generate different peptides from the same substrate.76,77 Furthermore, granzyme-induced apoptosis does not occur in the thymus and bone marrow, and thus peptides generated by granzyme cleavage are not likely presented to developing lymphocytes.78-81 Lastly, the granule pathway is able to kill target cells through caspase-independent pathways,82,83 thus increasing the chances of generating non–caspase-cleaved, novel autoantigen fragments. Taken together, the available data suggest that autoantigens are preferentially modified during specific forms of nonhomeostatic death, in which caspaseindependent structural modifications predominate. Such uniquely modified molecules may form the substrate for systemic autoimmunity.

DISEASE PROPAGATION IN SLE There is significant complexity surrounding apoptotic death and disease propagation in SLE. Although studies are in their early phases, there is significant support for the notion that serum components, dying cells and their contents, and interferon-α (IFN-α) play important roles in this feed-forward cycle. Although the role of ongoing B-cell death in germinal centers as a source of immunogen will not be addressed here, this is likely an important additional component of pathogenesis. Cycles of expansion and apoptosis of autoreactive cells as well as damage to target tissue could drive disease propagation by supplying a renewable source of antigen.33 Serum components could further modulate clearance defects and cytokine secretion in response to apoptotic material.

SLE-IgG, DYING CELLS, AND IFN-α Serum factors have been implicated in contributing to disease propagation and phenotype by modulating normal modes of phagocytic clearance and maturation. The proinflammatory cytokine IFN-α has long been implicated in disease pathogenesis since early observations of elevated serum IFN-α levels in patients with SLE compared to controls.90,91 Subsequent work from Blanco and colleagues92 demonstrated altered maturation of phagocytes in SLE. SLE serum, but not serum from controls or patients with other systemic autoimmune diseases, was shown to induce monocyte differentiation into myeloid dendritic cells (DCs) with up-regulation of class-II and co-stimulatory molecules. These DCs were able to capture and present antigen from dying allogenic cells and induce proliferation of autologous CD4+ T cells. IFN-α was shown to drive this differentiation because neutralizing antibodies to IFN-α, but not to CD40 ligand, IL-4, or GM-CSF, inhibited DC induction. DC induction also correlated with the amount of IFN-α present in the serum from

137

APOPTOSIS

138

SLE patients.92 While plasmacytoid dendritic cells (pDCs) produce the majority of IFN-α in SLE, their depletion from SLE PBMCs does not abrogate secretion as in controls, suggesting that other cell types may be involved. Secretion of IFN-α and IL-6 by pDCs has also been shown to drive the proliferation and differentiation of activated B cells into antibody-secreting plasma cells.93 These are highly significant observations, considering that high levels of IFN-α90,91 as well as high-titer, high-affinity autoantibodies are present in the serum of SLE patients. These observations are strongly supported by work from numerous other investigators, including work by Lovgren and colleagues and Baechler and colleagues,94 who have all emphasized a role for type I interferons in SLE activity and severity. In recent studies from Ronnblom’s group, the interaction of contents of dying cells and interferons was explored. Treatment of plasmacytoid dendritic cells (pDCs) with supernatant from apoptotic or necrotic monocytes in the presence of SLE IgG was noted to trigger IFN-α secretion.95,96 In contrast, no IFN-α was detected when pDCs were treated with supernatant alone or in the presence of control IgG. Similarly, experiments using autoreactive rheumatoid factor (RF+) B cells, chromatin-specific B-cell clones, and purified myeloid DCs showed proliferation in response to sera from autoimmune mice, but not wild-type controls.50 Immune complexes containing nucleic acid released by apoptotic and necrotic cells were implicated in triggering the response. This was supported by data that artificial nucleosome, chromatin, and RNA/antibody complexes were able to induce a similar proliferative response in B cells.50 The nature of the autoantigen/antibody complex appeared to be critical in eliciting an immune response, and suggested a role for endogenous nucleic acid in triggering the response to immune complexes since digestion with DNase or RNase dramatically reduced B-cell proliferation.50 Using engineered DNAs, it was demonstrated that sequence and methylation status of the nucleic acid were vital to initiating the response. Interestingly, only hypomethylated DNAs could induce proliferation, raising the possibility that changes in the methylation pattern of selfnucleic acids could contribute to recognition by TLRs. The proliferation observed in response to nucleic acid containing immune complexes was sensitive to chloroquine treatment, suggesting a role for endosomal TLRs. The response to CpG DNA and chromatin IgG complexes was dramatically reduced in the presence of inhibitory CpG DNAs, chloroquine, and in TLR9 knockout mice.48,49,51 Proliferation in response to RNA IgG complexes was also blocked by chloroquine and in TLR7 knockout mice, suggesting recognition of endogenous RNA by TLR 7.50 Aside from proliferation,

lupus B cells responded to CpG complexes by increasing expression of prosurvival IL-10, autoantibody production, and up-regulating co-stimulatory molecules, compared to controls.54 Immune complexes induced secretion of TNF-α by myeloid DCs and was dependent on the presence of FcγRIII,48 while IFN-α secretion by pDCs was dependent on the binding of surface FcγIIa to the Fc portion of IgG in immune complexes.95,96 These data suggest a cooperative effect between ligation of antigen-presenting cell surface receptors by a multivalent immune complex, subsequent endocytosis, and ligation of endosomal TLRs by endogenous nucleic acids as a trigger of the autoimmune response in SLE. Since IFN-α and TNF-α are powerful activators of the immune response, inappropriate release in response to self-apoptotic antigen/autoantibody complexes might contribute disease propagation.

ANTIBODY OPSONIZATION OF APOPTOTIC CELLS TRIGGERS RELEASE OF PROINFLAMMATORY CYTOKINES Under normal circumstances, phagocytosis of apoptotic cells triggers the release of anti-inflammatory factors including IL-10, TGF-β, PAF, and prostaglandin E2.15,16 Secretion of these factors is dependent on recognition of molecules on the surface of the dying cell by phagocytic receptors. Phosphatidyl serine (PS) is exposed in the outer leaflet of the plasma membrane during early stages of apoptosis, and is critical for the uptake of apoptotic bodies. Ligation of PS receptors on the surface of phagocytes triggers membrane ruffling and uptake of apoptotic cells.97 TGF-β secretion in response to apoptotic cells is dependent upon the presence of surface PS since cells not expressing PS do not induce TGF-β.13 The anti-inflammatory effect of apoptotic cells is illustrated by their ability to accelerate resolution of tissue inflammation by reducing proinflammatory chemokine secretion and the number of infiltrating cells. Opsonization has been shown to facilitate apoptotic cell uptake through ligation of Fc receptors on the phagocytic surface.13,97 Opsonization of apoptotic cells, independent of antibody specificity, interferes with PSdependent phagocytosis and abrogates TGF-β secretion while allowing secretion of proinflammatory cytokines such as IL-1, IL-8, and TNF-α.13 This phenomenon is particularly relevant in the case of SLE, given that autoantibodies have been shown to preferentially bind to the surface of apoptotic cells.98,99 It has been proposed that opsonization of apoptotic cells by autoantibody in SLE facilitates phagocytosis and triggers the secretion of proinflammatory cytokines in response to self-antigen.100,101 Autoantibodies recognizing antigens on the surface of dying cells (which may

SERUM FACTORS CONTRIBUTE TO FEED-FORWARD LOOP OF AUTOIMMUNITY IN SLE In addition to the role of autoantibodies and type-I interferons in the feed-forward loop in SLE, additional contributions from less well-defined serum components have been suggested. For example, macrophages isolated from the peripheral blood of pre-disease SLE prone mice have been reported to show abnormal cytokine secretion in response to apoptotic cells.102 This effect was dependent on the presence of lipid and protein serum factors and caused the reduction of cytokines including IL-1, IL-6, IL-12, and TNF-α.102 Similar observations have also been made in human SLE. Macrophages isolated from patients with SLE

A. INITIATION

exhibit increased cell death and reduced ability to phagocytose apoptotic material.103,104 Phagocytic ability was shown to negatively correlate with disease activity and anti-dsDNA titer.103,105 A decreased number of phagocytosis-competent macrophages in SLE and a reduction in the number of apoptotic bodies per cell have been observed, compared to controls.103 The addition of control serum to these cells was able to rescue the defect and restore phagocytic capability to normal levels.103,104 Interestingly, normal cells treated with SLE serum showed a dramatic reduction in their ability to phagocytose apoptotic cells, suggesting a primary role for serum factors in this effect.103,105 The ability of these serum factors to modulate phagocytosis and cytokine secretion could contribute to the feed-forward autoimmune loop seen in SLE by enhancing preexisting clearance defects.

CONCLUSIONS

be present preferentially in the prediseased state) might become pathogenic if large amounts of “pro-immune” apoptotic death occur, which upon opsonization, allow additional autoantigens inside apoptotic cell blebs to become targets of the immune response.

CONCLUSIONS This review highlights a two-step model of SLE pathogenesis (Fig. 14.1). Susceptibility factors contributing to initial development of autoantibodies include apoptotic

B. TRANSITION

C. PROPAGATION

B cell Δ ppp

T cell help to autoreactive B cells

2

Modulation of apoptotic cell clearance

3

1

APCs

T cells TLR

Asymptomatic

Proinflammatory cytokines

Clinical phenotype

Fig. 14.1 A, Survival of autoreactive cells and clearance defects initiate autoantibody production directed against apoptotic cells. During initiation, antigens clustered at the surface of apoptotic cells drive autoantibody production in asymptomatic predisease individuals. B, The transition from initiation to propagation occurs when self-antigen is presented in a proinflammatory context. Viral infection, TLR/Fc receptor co-ligation, or altered autoantigen processing can recruit a feed forward loop of disease propagation marked by autoantibodies targeting ribonucleoproteins, with development of clinical SLE. C, Apoptotic cells drive the immune response in SLE via ligation of TLRs (1 and 3), T cell (2), and B cell (3) receptors. Immune cells drive disease propagation by supplying antigen through cycles of proliferation and apoptosis as well as through target tissue death. Serum factors (such as proinflammatory cytokines and autoantibodies) contribute to disease propagation by modulating apoptotic cell clearance, antigen presenting cell (APC) maturation, and immune activation.

139

APOPTOSIS

cell-clearance defects mediated through defective phagocytic receptors and soluble factors as well as impaired tolerance induction of T and B cells. Presence of autoantibodies in prediseased individuals may express pathogenic potential when a superimposed apoptotic event places self-antigen in a proinflammatory context. The modification of antigen structure during cell death or viral infection and the acquisition of TLR-ligating function may be critical in this regard. An interesting feature of SLE is the self-sustaining nature of inflammation and autoimmunity, reflecting the recruitment of feed forward loops that comprise renewable sources of antigen and proinflammatory signals. Target tissue death, as well

as death of immune cells following clonal expansion might contribute to the renewable antigen source. Once the amplification cycle begins, serum factors including IFN-α, autoantibodies, and other factors may play important roles in sustaining and further amplifying disease propagation by modulating apoptotic cell clearance and proimmune presentation by antigen-presenting cells. In this model, disease propagation describes the stage in which the immune response itself generates additional antigen, providing a feed-forward loop of autoimmunity. Interdiction of pathways that provide antigen and enable TLR ligation may have significant therapeutic potential.

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PATHOGENESIS

15

Infection and Autoimmunity Brian D. Poole, PhD, and Judith A. James, MD, PhD

Infections are associated with both the etiology and morbidity of systemic lupus erythematosus (SLE). Mounting evidence suggests that viral infections could serve as a trigger for the production of autoantibodies and subsequently lupus. Several viruses have been identified as potential agents, based on association studies, cross-reactivity, or similarity of the symptoms of infection with SLE (Box 15.1). Viruses might trigger the development of autoimmunity through molecular mimicry, alteration of the immune system, the effects of tissue damage, or other mechanisms. After developing lupus, patients are more susceptible to infection with a variety of bacterial, fungal, and viral pathogens. Susceptibility to infection has been associated with both disease activity and the use of immunosuppressive therapies to treat lupus.

ETIOLOGY Understanding of the etiology of lupus has proven difficult. No single factor has ever been identified as solely causal, be it genetic, hormonal, or environmental. Therefore, most researchers believe that lupus has a multifactoral etiology, with genetics, environmental, and hormonal factors all being variably involved in lupus susceptibility. Twin concordance studies demonstrate the importance, and ironically also the insufficiency, of genetics in lupus causation. Monozygotic twins have a concordance rate for SLE of 25 to 40%, while dizygotic twins have a rate closer to 9%.1-3 The difference BOX 15-1 EVIDENCE SUGGESTING POTENTIAL ROLES FOR INFECTION IN SLE ●

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Lupus has a complex etiology, which likely includes genetic, environmental, and hormonal influences. Antibodies against lupus autoantigens cross-react with viral proteins. Select viral infections have been associated with SLE. Viral infection often results in transient autoantibody production. Interferon response genes are up-regulated in lupus.

between these rates indicates that genetics are indeed important, and genome screening analyses have identified at least 12 chromosomal regions that are linked to SLE.4,5 The low concordance rate even among the monozygotic twins, however, illustrates that other factors are extremely significant in the development of lupus. Hormonal and other noninfectious environmental factors will be discussed in other chapters. However, one prime candidate for this environmental component would be viral infection. Viruses have multiple properties that could contribute to their ability to initiate or sustain the abnormal immune process leading to lupus. The mere presence of a viral infection activates the immune system to the end of eliminating the infection, and this activation has the potential to either inappropriately target the wrong antigens and turn into an autoreactive response, or re-set activation levels to a point that autoimmunity could develop in susceptible individuals. Viral infections often mimic the symptoms of lupus, including the production of autoantibodies. Viral infection could potentially lead to autoimmunity through at least two mechanisms: cross-reactivity leading to molecular mimicry or activation of innate and then adaptive immunity.

Autoimmunity through Molecular Mimicry Many viruses produce proteins with potential epitopes that are very similar to self-antigens targeted in lupus (Table 15.1). Antibodies produced against these proteins have the potential to cross-react with cellular proteins. In the case of lupus, the self-antigens that are recognized by antiviral antibodies are often located in nucleic-acid binding complexes such as the spliceosome, specifically the Sm, and nRNP complexes, other RNA-binding particles such as the Ro/La complexes, and nucleosomes, which contain DNA.6 These cross-reactive antibodies have the potential to initiate an autoimmune response based on improper recognition of self-proteins by antigenpresenting cells.

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self-antigen through a cross-reactive B-cell antigen receptor instead of soluble antibody. B cells present peptides from their specific antigen with extremely high efficiency,7 and would be highly effective at maintaining and broadening an autoimmune response. Once T-cell tolerance is lost, autoreactive T cells could then provide help for autoreactive B cells, leading to maturation and diversification of the autoantibody response, and eventually culminating in the development of pathogenic autoantibodies and lupus disease.

TABLE 15.1 CROSS-REACTIVITY OF ANTIVIRAL ANTIBODIES WITH SELF-ANTIGENS Virus

Viral Antigen

Self-Antigen

Epstein-Barr virus

EBNA-1

Sm B/B’ Sm D1 Ro

EBNA-2

SmB/B’ Sm D

Cytomegalovirus

gB

U1 70K

Autoimmunity through Bystander Effects

HIV

P24

Sm B/B’

HRES-1

P28

U1 70K

In the course of antiviral immune activation, some normal safeguards on the immune system are discounted. Virus infection results in immunostimulatory cytokine production. For example, interferon-alpha production is a hallmark of viral infection.8 Lupus patients exhibit elevated levels of interferon-alpha in the blood.9 Microarray analysis of gene expression shows a marked increase in expression of genes that are responsive to interferon-alpha in lupus patients.10-12 Interferon-alpha stimulates dendritic cells to activate T-cell immunity, including autoreactivity.13 Similarly, the presence of viral products activates toll-like receptors on antigen presenting cells,14 causing these cells to mature and present antigen in a stimulatory, rather than tolerogenic, fashion15,16 (Fig. 15.2).

In a molecular mimicry–based model of autoantibody generation, cross-reactive antipathogen antibodies bind to self-proteins. These opsonized self-antigens can then be phagocytosed by dendritic cells, processed, and presented to T cells in the context of MHC class II, leading to a loss of T-cell tolerance. The presence of viral infection would increase the likelihood of the T cells being stimulated by, rather than tolerized, by antigen presentation (Fig. 15.1). Similarly, B cells could serve as the antigen-presenting cells, but bind the

Cross-reactive antibodies

Activated antigen-presenting cell Internalization, processing and presentation of antigen

T T cell help

T

Activated autoreactive T cell

144

B Loss of tolerance Help for autoreactive B cells Autoantibody production B-cell epitope spreading

Fig. 15.1 Cross-reactivity-based initiation of lupus. Cross-reactive anti-pathogen (red) antibodies can bind to self-proteins (green triangles). The antibodies could be either in soluble form or as part of the B-cell antigen receptor. Antibody-bound self-protein is internalized and processed by dendritic cells. The presence of antibody in the immune complex, viral infection, interferon-alpha, or toll-like receptor stimulation could contribute to activation of dendritic cells to stimulate T cells. The processed autoantigen-derived peptides are presented to T cells, breaking tolerance.

Viral infection may be able to induce autoimmunity through one or a combination of these two mechanisms, or through other, undefined processes. It is clear, however, that viral infection affects immune system homeostasis on a fundamental level. Indeed, autoreactive antibodies are produced as a result of several types of viral infections. Part of the genetic susceptibility to lupus may lie in the inability to suppress the production of these autoantibodies once the infection is cleared.

ETIOLOGY

Infection could lead to usage of B-variable gene sequences that are prone to react with autoantigens and are not common without the presence of an active immune system.17,18 Additionally, viruses fundamentally alter the cells that are infected in ways that would increase their antigenicity. Alterations in apoptosis or clearance of apoptotic cells have been implicated in SLE.19 Viral infections can lead to apoptosis or necrosis due to the cytopathic effect of infection on cells. These mechanisms of cell death alter cellular proteins, potentially unmasking cryptic antigens and making the self-proteins more antigenic.20 Similarly, apoptosis or necrosis leads to the exposure on the cell surface of nuclear antigens, which are the main targets of lupus autoantibodies.21 The increase in cell death because of virus infection may dramatically increase the amount of autoantigen available to activate an inappropriate antibody response. Viral infection can also increase the expression of autoantigens in living cells.22 Similarly, viral infection is controlled by cytotoxic T cells, which kill infected cells via the formation of pores in the cell membrane and the introduction of granzyme B into the target cell. Granzyme B itself cleaves cell proteins, again potentially altering the presentation of self-antigens.23

Desirable Characteristics of Viral Etiologic Agent Over the past several decades, various infectious agents have been considered in SLE etiology. However, based upon the near impossibility to prove Koch’s postulates with many viral infections due to the lack of animal models and the variability of the outcomes of viral infection, indirect evidence is often required. Several factors contribute to this complexity. First is the multiplicity of factors involved. The process of developing lupus is long, with autoantibodies often appearing years before any clinical signs.24 The virus, if any, involved in SLE pathogenesis, therefore, would not necessarily be present when the symptoms of lupus are noticed.

Virus or viral components Activation through cytokines, interferons Activation through toll-like receptors APC

Self-peptides

Costimulation T cell

Autoreactive T cell

T cell activation Cytokine production Help for B cells

Fig. 15.2 Activation of autoimmunity by bystander effects of viral infection. Toll-like receptor activation, interferons, and other cytokines can influence antigen-presenting cells to activate T cells, breaking tolerance.

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Of course, if the presence of the virus is required to perpetuate or expand the autoimmune response, the virus would be expected to persist for years if not decades. If the virus were rare, the association between infection and SLE would be easy to identify, especially since lupus would be constrained by the geographic or population constraints of the virus. The widespread, although sporadic, nature of SLE indicates that a virus that is involved in causation should be similarly ubiquitous. When considering that a rare disease may be caused by a ubiquitous infection, it is necessary to explain why the infection does not always cause the disease. Showing causation with a nearly ubiquitous virus, however, is even more difficult because a high sample number is necessary to obtain sufficient power to observe significant differences between cases and controls. In summary, essential characteristics of a virus that would be considered as a causative agent are that it should be capable of infecting the patient years before the onset of symptoms, be relatively ubiquitous, and be strongly influenced by genetic and immune factors. Such a virus would also ideally be persistent to allow increased opportunity for cross-reactivity and modulation of the immune system. The presence of viral proteins that cross-react with important lupus autoantigens would also be highly desirable. Several viruses meet these criteria and have been investigated as potential causal agents. Those discussed herein are Epstein-Barr virus (EBV), Cytomegalovirus (HCMV), parvovirus B19 (B19), and retroviruses.

Epstein-Barr Virus as Etiologic Agent

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Epstein-Barr virus is a member of the gamma herpes virus family. This virus primarily infects B cells, and EBV infection in vitro leads to transformation of infected cells. Infection with EBV is lifelong, as the virus is capable of maintaining latency in memory B cells, allowing very little immune surveillance.25 Reactivation from the latent phase occurs periodically.26 The primary viral protein expressed during in vivo latency is Epstein-Barr nuclear antigen 1 (EBNA-1), while the majority of viral genes are expressed during lytic infection.27,28 EBV possesses a multiplicity of characteristics that would make it an ideal initiating or perpetuating agent in SLE (Box 15.1). The lifelong nature of EBV infection, coupled with intermittent reactivation, provides a continual source of potentially cross-reactive antigenic stimulation. In addition, the natural host cells of EBV are B cells, which are responsible for the production of the autoantibodies that are central to SLE. Infection with EBV activates and/or immortalizes B cells, as well as interferes with the normal regulatory mechanisms that control antibody production.

In fact, in vitro EBV infection has been used to immortalize B cells producing anti-La and anti-Ro antibodies.29 A protein expressed during viral latency, LMP-1, mimics the signal from CD40,30 which would normally be stimulated by T-cell help. CD40 is a central signal in B-cell activation that provides co-stimulation and allows the B cell to proliferate and undergo antibody isotype class switching and affinity maturation.31 Expression of LMP-1 in mice leads to marked B-cell proliferation and increases in the serum levels of IL-6, suggesting that LMP-1 lowers the threshold for B-cell activation.30 Lowering of this threshold makes a response to inappropriate antigens much more likely. In addition to the general characteristics of EBV infection, EBV has specific properties that suggest that it may be involved in SLE. Lupus has been associated with alterations in apoptosis, the programmed death of cells. EBV contains a protein, BHRF1, which is homologous to the antiapoptotic protein Bcl-2, and acts to alter the apoptotic pathway in infected cells.32 Similarly, SLE patients demonstrate 7- to 10-fold increases in the levels of interleukin (IL)-10 when compared to controls.33 IL-10 is an immunomodulatory cytokine often expressed in antibody-favoring immune responses. EBV expresses a protein that is highly homologous to IL-10, and acts in a similar manner.34 Additionally, infection of B cells with EBV leads to increased production of IL-10.35 Interestingly, although IL-10 is usually thought of as immunosuppressive, pretreatment of cells with interferon-alpha imbues IL-10 exposure with proinflammatory characteristics.36 Therefore, EBV specifically affects the immune system in ways that are consistent with processes observed to be involved in SLE. The cross-reactivity of antibodies against EBNA antigens to Ro and Sm antigens is extremely significant in terms of SLE pathogenesis. Investigation into the development of autoantibodies before the onset of symptoms revealed that anti-Ro antibodies are among the first to appear. Anti-Sm antibodies, in addition to being extremely specific for lupus, appear close to diagnosis, suggesting that they are involved in the development of symptoms.34,37 Anti–60-kd Ro autoantibodies, when found alone, develop in a distinct sequence. In patients where the development of anti-Ro antibodies can be observed, the first sequence recognized usually contains amino acids 169 to 180 (TKYKQRNGWSHK). This sequence is crossreactive with a sequence on EBNA-1 that is bound by antibodies from lupus patients.38 In addition, anti–EBNA-1 antibodies always develop in these patients before anti-Ro antibodies, implicating anti–EBNA-1 immunity in the development of anti-Ro and not vice versa. Antibodies against the Sm complex are some of the most specific indicators of lupus. Anti-Sm B immunity

Association of EBV with SLE Because of the attractiveness of EBV as a candidate etiologic agent, attempts to determine if there is an association between EBV infection and SLE has had a long and controversial history. The major obstacles to a clear understanding of the relationship between EBV and SLE are the near ubiquity of EBV infection, the lack of a parallel animal model and the lack of sensitivity of the tests

used to detect infection. While seropositivity to EBV approaches 95% in adults in the United States,50 it is only recently that ELISA assays to examine antibody responses and PCR to probe for viral DNA have become sensitive enough to conclusively identify differences in prevalence of infection or to precisely quantify viral or immune differences between patients and controls. Initial experiments investigating the relationship between SLE and EBV infection focused on differences in antibody titers. The initial studies in this area utilized indirect immunofluorescence to titer anti-EBV nuclear antigen antibodies in two independent studies, and found that lupus patients had significantly higher titers than controls.51 Other studies using immunofluorescence have found no statistically significant difference between antibody titers to EBV between SLE patients and controls. However, of 10 studies examining this question before 1998, six have found significant increases in antibody titer to EBV in lupus patients, one found decreased titers in patients, and three were not able to show a significant difference.52-60 As can be inferred by the variability in the results, problems with these studies exist. Immunofluorescence uses serum antibody binding to cell nuclei as the readout for the assay. This is not an ideal way to titer sera from lupus patients, who nearly all have antibodies against nuclear antigens notwithstanding the presence of EBV antigens. These antibodies could result in either false positives or interfere with the assay conditions leading to false decreases in titer. Another problem with the study of anti-EBV titer to examine correlation is that patients with lupus have generalized hypergammaglobulinemia, and it has been suggested that increased antibody titers to EBV antigens may be a result of the general increase in antibodies in the patient. Similarly, although increased anti-EBV titers in lupus are suggestive, there is no definitive reason why increased titers would indicate EBV involvement in SLE. A more informative measure, especially when paired with antibody titer, is seroprevalence rate, which demonstrates past infection. Seroprevalence would examine whether there is a correlation between infection itself and SLE, while alterations in virus titer would show actual differences in the biology of EBV infection in SLE patients compared to controls. EBV infection clearly does not cause SLE in everyone who is infected, so if it is to be considered a causal agent, there must be some difference in the effect of EBV infection on patients that will go on to develop SLE. This difference could be in the viral life cycle, the immune response to EBV, or a combination of the two. As mentioned, the near ubiquity of EBV infection makes the detection of differences in infection rates difficult. To solve this problem, our group examined sera

ETIOLOGY

begins with binding to a single epitope, PPPGMRPP,39 and binding later spreads to encompass multiple epitopes. As is the case with Ro autoimmunity, this initial Sm B’ humoral epitope is highly cross-reactive with a sequence on EBNA-1 that is bound by serum from lupus patients.40 Not only is PPPGMRPP the first antigen bound by the anti-Sm B response, antibodies targeted against this sequence account for an overwhelming proportion of the anti-Sm antibody population, highlighting its critical role in anti-Sm autoimmunity.39 Immunization of animals with this peptide or the corresponding EBNA-1 peptide PPPGRRP leads to widespread autoantibody production and clinical symptoms suggestive of lupus.37,41,42 Similarly, rabbits immunized with the Ro peptide that cross-reacts with EBNA-1, or the corresponding EBNA-1 peptide, developed antibodies to multiple autoantigens, including Sm, nuclear RNP, and dsDNA. These rabbits also developed lupus-like symptoms, including leukopenia, serum creatinine increases, and thrombocytopenia.38 Whole EBNA-1 protein is also able to promote autoantibody generation. DNA vaccination of mice with EBNA-1 coding sequence leads to the development of anti-Sm and anti-dsDNA antibodies.43 During initial infection with EBV, a large panel of both anti-EBV and autoantibodies are generated. Antibodies against the PPPGRRP region of EBNA-1 are transiently present,44 as are lupus anticoagulant antibodies.45,46 Antiphospholipid antibodies, such as lupus anticoagulant, are among the earliest appearing autoantibodies in patients who will develop lupus.24 In normal patients, these antibodies disappear after a short time. However, it may be the case that in patients who will develop lupus, the regulation of the immune system that allows elimination of cross-reactive antibodies is altered, so these antibodies persist. Case reports of lupus developing subsequent to EBV infection, but not vice versa, detail cases where either the patient is hospitalized for EBV-induced mononucleosis and later develops SLE,47 or patients who are admitted for SLE-related symptoms and found to have indications of EBV infection, such as positive anti-EBV IgM or the presence of EBV antigens in affected tissues.48,49 The timeline of these reports is suggestive, since EBV infection is always determined to have occurred prior to or concomitant with the development of lupus, not the reverse.

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from a large population of pediatric patients with lupus and carefully matched controls. Because the prevalence of EBV infection is decreased in a younger population, it is possible to accumulate a sufficient number of pediatric samples to obtain adequate power for these studies. This study also used purified antigens as targets for antibody binding by enzyme-liked immunosorben assay (ELISA) instead of immunofluorescence. The results were that 99% of lupus patients were positive for EBV VCA antibodies, while only 70% of controls were positive.61 The increased prevalence of EBV infection in lupus patients was highly significant, with an odds ratio (OR) near 50.61 A second group of pediatric lupus patients was examined in a similar manner. In this cohort, 100% of patients were seropositive for EBV, while 68% of controls had been infected, again a statistically significant result with an OR of more than 14.62 Using these techniques, our group subsequently analyzed a large cohort of adult lupus patients for EBV infection. In this study, 99.5% of patients were seropositive for EBV, while 95% of the controls were positive. Because of the large sample size of 196 patients and 392 matched controls, the study had the power to determine that this result was significant with an OR of 9.35, a 95% confidence interval (CI) from 1.45 to infinity, and a p value of 0.014 when corrected for familial control issues. No significant difference was noticed in seroconversion to HCMV, herpes simplex virus types 1 and 2, or varicella-zoster virus, demonstrating that the differences in EBV infection rate in lupus are specific and not merely the result of nonspecific hypergammaglobulinemia.63 The persistent nature of EBV infection makes it possible to detect EBV DNA in the blood of infected persons years after infection. PCR detection of DNA provides an assay that is not affected by factors such as potential antibody cross-reactivity or non-specific increases in antibody levels. To confirm the results obtained by serologic analysis of pediatric patients, PCR was used to amplify EBV DNA. All 32 of the lupus patients tested positive, while only 23 of the 32 matched controls had detectable EBV DNA in their peripheral blood (OR>10, 95% CI=2.53–infinity, p<0.002).61 A similar study by another group also revealed that a significantly greater proportion of patients tested positive for EBV DNA than controls (81.6% of patients compared to 48.9% of controls, p<0.0001).64 The combined serology and DNA studies show that nearly 100% of lupus patients have evidence of past infection with EBV, while a significantly smaller number of normal controls have been exposed. This pattern suggests that EBV infection is involved in lupus. The association could be explained by EBV acting as a causal agent, or lupus patients being more susceptible to EBV infection. Longitudinal studies of

antibody development reveal that EBV antibodies always predate lupus autoantibodies, strongly suggesting that the second scenario is not correct.38

DIFFERENCES IN BIOLOGY OF INFECTION BETWEEN LUPUS PATIENTS AND CONTROLS

Viral Load Quantification of infection rates can show association, but not necessarily reveal the process through which EBV infection might bring about autoimmunity. Several techniques are available to investigate these processes. The use of PCR technology allows for the identification of EBV DNA, and can be modified to quantify viral load. Quantification of the amount of virus or virus-infected cells in patients as compared to controls allows the elucidation of differences in the process or control of EBV infection. Increased viral load could be the result of more frequent or a higher level of lytic cycle reactivation, or defective control of infection. Multiple studies have examined EBV viral loads in lupus patients. Real-time quantitative PCR showed a significant increase in the amount of EBV DNA in PBMCs from lupus patients compared to controls.64 EBV DNA levels in both mouthwash samples and blood have been examined using serial dilution of DNA samples. Although there was no difference in the amount of EBV in mouthwash samples, there was a 15-fold increase in EBV DNA concentration in the blood.65 A similar study found an even greater increase in viral load, with lupus patients exhibiting 30-fold higher levels of EBV DNA than controls in the peripheral blood.66 These studies examined only DNA levels, without being able to determine whether the increase in viral load was due to the presence of a larger number of latently infected cells or similar numbers of infected cells with a few cells undergoing lytic infection. During the lytic cycle, viral DNA replication would greatly increase the overall number of viral genomes, skewing the amount of DNA in the blood. To answer this question, limiting dilution was performed on isolated B cells from peripheral blood, and then quantitative PCR was used to quantify EBV viral load on a per cell basis. Using these techniques, it was found that lupus patients have a 10-fold increase in infected cells over controls. Interestingly, in this study patients who were undergoing lupus flares also had significantly increased numbers of EBV-infected cells than patients whose disease was under control.67 These studies, using very sensitive and quantitative methods, all agree that there is a much higher EBV burden in lupus patients than controls. Increased viral burden is important for many reasons. The presence of more infected cells means more chances for the

Immune Response: T Cells The immune response to EBV is altered in lupus, and is so in ways that strengthen the case for a role for EBV involvement in SLE. As part of their investigation into viral load, Kang and colleagues66 also examined the T-cell responses. The number of CD8+ T cells that produced interferon-gamma in response to EBV antigens was decreased in lupus patients, which may contribute to the increased viral load. However, the proportion of CD4+ T cells that responded to EBV antigens was significantly higher in lupus patients. This increase in CD4+ T cell responses could be compensatory for the decrease in CD8+ responses, or it may be a feature of lupus biology. CD4+ T cells are important drivers of autoantibody production in lupus. Hyper-responsiveness of these cells in response to EBV could be important, perhaps in providing help to autoantibody-secreting B cells.

Immune Response: B Cells Since autoantibody production is the predominant feature of SLE, the antibodies against EBV are of supreme interest. In addition to the aforementioned increase in serum antibody titers, the specificity of antibodies to EBV antigens is different in lupus patients and controls. Sera from lupus patients recognize a wider variety of EBV proteins than control sera. On Western blots, patient sera recognize multiple EBV-derived protein bands that are not recognized by control sera.68 Antibodies from lupus patient sera recognized all seven of the viral early diffuse antigens 61% of the time, while control patients with mixed connective tissue disease only recognized these antigens 5% of the time.69 In a similar study, 62% of lupus patient sera recognized the 42-kD envelope protein of EBV, while only 4% of controls recognized this peptide. However, control sera bound more frequently to the 100-kD envelope protein (92% of controls vs. 11% of patients), demonstrating that the differences in binding are not simply due to nonspecific increases in antibody production.70 The antibody response against the EBV nuclear antigens also differs in SLE. In one study, 70% of lupus patients developed antibodies against the nuclear antigens

EBNA-2 and EBNA-3, while less than 10% of controls developed these antibody specificities.71 Similarly, 36 of 36 lupus patients developed antibodies against EBNA-1, while only 25 out of 36 matched controls developed specificity to this antigen (McClain et al., unpublished, 2006). The pattern of EBV antigens recognized by SLE sera is similar to that seen in patients with chronic viral reactivation, in which increased levels of antibody to EBNA-1, 2, and 6 are prevalent.72 The serology correlates with the DNA findings that the Epstein-Barr viral load is increased in lupus patients, and demonstrates that the immune response to EBV is qualitatively different in lupus patients. In addition to the EBV antigens recognized by lupus patient antibodies, differences in antibody class are also informative, and again support the findings of greater reactivation of EBV in lupus. IgA is the antibody class most responsible for mucosal immunity, and IgA antibodies are indicative of reactivation of EBV infection. Two groups demonstrated an association between anti-EBV IgA production and lupus.73,74 Parks and colleagues found a significantly higher proportion of anti EBV-VCA IgA antibodies in AfricanAmerican lupus patients,73 while Chen and colleagues demonstrated association between IgA prevalence and lupus in Asian populations.74 Both of these studies were performed in adult patients, and so are likely to indicate reactivation rather than initial infection. The highest-expressed viral genes in EBV-infected cells are usually EBER-1 and EBER-2, which are small RNA molecules that do not code for proteins and are not well understood. However, sera from patients with SLE will immunoprecipitate these molecules in the form of protein-RNA complexes. The sera that precipitate the EBERs also precipitate the La antigen,75 which is known to be targeted in SLE and which forms a complex with Ro, one of the earliest antigens recognized by SLE sera.24 Whether the EBERs interact with La is currently not known. A clear increase in the number of EBV antigens recognized by lupus patients is evident, as well as increased usage of the IgA isotype. These findings are notable for several reasons. First, they corroborate the finding that there is increased lytic activity in lupus patients. Second, they demonstrate that the antibody response in lupus is broader than normal. Broadening of the immune response allows for the targeting of antigens which are cross-reactive or otherwise able to initiate autoimmunity. The broadening of the immune response is not limited to simply targeting of more proteins. Antibodies from lupus patients also recognize more epitopes of individual proteins than normal controls. Most of the research in this area has focused on the EBNA-1 protein. EBNA-1 is the only protein that is expressed in all

DIFFERENCES IN BIOLOGY OF INFECTION BETWEEN LUPUS PATIENTS AND CONTROLS

immune system to encounter viral proteins, which may lead to a stronger or altered response. Increased prevalence of infected cells may similarly keep the immune system at a more activated antiviral state, and a proinflammatory cytokine environment may make immune tolerance easier to break in susceptible individuals. In addition, increased prevalence of infected cells means an increased likelihood that one of the B cells immortalized by EBV infection may be a producer of autoreactive antibodies.

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forms of EBV latency, given that it is required to allow proper partitioning of the EBV genome.76 As such, it presents a constant target for the immune system even during latent infection. EBNA-1 contains three major regions: an N-terminal sequence, a large repeat of the amino acids glycine and alanine, and a C-terminal region. The antibody response to EBNA-1 in normal individuals is primarily targeted against the GA repeat, with 90 to 95 % of the total anti-EBNA-1 antibodies binding to this sequence.77 However, lupus patients have broader antibody specificity. Peptides derived from different sequences of EBNA-1, used to demonstrate that lupus sera, but not normal sera, bound to two epitopes outside of the GA repeat.78 This observation was greatly expanded by recent work in our group. Recombinant protein fragments representing the C-terminal, GA repeat, and N-terminal region of EBNA-1 were examined for antibody binding. Antibodies from normal controls bound primarily to the GA repeat region, as expected. However, antibodies from lupus patients showed high levels of binding to both the C-terminal (mean [OD]=0.451 vs. 0.268, p<0.001) and N-terminal fragments (mean OD =0.844 vs. 0.296, p<0.001), and decreased binding to the GA repeat (OD=0.396 vs. OD=0.779, p<0.001). Furthermore, we used solid-phase octapeptide mapping to discern exactly which epitopes were bound by each serum sample. The epitopes recognized by lupus patient and control sera were practically exclusive, with the lupus antibodies binding primarily to epitopes in the N-terminus and C-terminus, without significant binding to the GA repeat, while control sera bound almost exclusively to the GA repeat region. These observations were specific, since epitope mapping of CMV early antigen and EBV EBNA-2 revealed no differences between lupus patients and controls.79 The broadening of the immune response to EBNA-1 is especially significant when it is considered that the regions recognized by antibodies in lupus include several epitopes that are cross-reactive with epitopes on common lupus autoantigens. The N-terminal region of EBNA-1 contains a glycine-arginine–rich sequence that is highly homologous to the Sm D1 protein.78-80 Interestingly, even in controls who produce antibodies against the N-terminal region of EBNA-1, these antibodies are not cross-reactive with Sm D1, while in lupus patients there is a high degree of cross-reactivity.81 The C-terminal region of EBNA-1 contains a proline-rich sequence, PPPGRRP, which is cross-reactive with the major epitope of Sm B recognized in lupus, PPPGMRPP. Both of these sequences are bound by antibodies from lupus patients but not antibodies from controls.79 Similarly, EBNA-1 contains a sequence that cross-reacts with the Ro autoantigen, and this sequence is similarly bound by antibodies from lupus patients.38

In addition to cross-reactive antibodies to EBNA-1, lupus patients also generate antibodies to EBNA-2 that bind to SLE antigens. SLE patients make increased titers of antibodies against EBNA-2.45,71 Anti-EBNA-2 antibodies from lupus patients but not from controls will bind to a proline-rich sequence of EBNA-2 that is homologous to a region in Sm D.82 These findings provide strong support for the association of EBV with SLE. In patients who will go on to develop lupus, infection with EBV leads to an aberrant immune response because of genetic factors in the immune system or an altered viral life cycle. This aberrant response generates cross-reactive antibodies to multiple autoantigens, which can result in the development of the widespread autoimmunity seen in lupus.

Cytomegalovirus as Etiologic Agent Another herpesvirus that has been potentially implicated in lupus pathogenesis in a subset of patients is human cytomegalovirus (HCMV). HCMV shares many attributes with EBV. Infection with HCMV persists throughout the lifetime of the host. HCMV maintains latency in dendritic cells,83 myeloid progenitors,84 hematopoietic progenitors,85 and epithelial cells.86 Dendritic cells and myeloid cells are intimately involved in the regulation of the immune system. HCMV infection is extremely common with infection rates in adults, ranging from 42% in Germany87 to 95% in India.88 Like EBV, HCMV produces a protein that mimics the effects of IL-10.89 HCVM infection induces high levels of interferonalpha in lupus patients,90 which may be consistent with a role for HCMV as either an etiologic agent or as an exacerbating agent in established lupus. HCMV also interferes with apoptosis in infected cells. The viral proteins IE1 and IE2 are capable of blocking apoptosis in response to tumor necrosis factor (TNF) alpha.91 Interfering with apoptosis through induction by TNF-alpha is especially interesting in the context of autoimmunity, since TNF-alpha is a central cytokine involved in rheumatoid arthritis, and blocking TNF-alpha with antibodies can lead to a lupus-like syndrome.92 Several case studies of lupus have associated with HCMV infection. In these cases, active infection, as determined by either the presence of anti-HCMV IgM or viral DNA, has been seen at the same time as onset of symptoms93,94 or flare.95 Association studies using larger groups of patients have attempted to discern if the association seen in these case studies is generalizable to SLE as a whole. These studies, like those used to investigate association with EBV, take the form of both antibody tests and isolation of HCMV DNA. These experiments have been inconclusive, with some groups reporting positive associations96 and some reporting no association.61-63,97

Parvovirus B19 Infection in SLE Parvovirus B19 (B19) is a small, single-stranded DNA virus that, like the other viruses discussed above, is nearly ubiquitous.102 Unlike the herpesviruses EBV and HCMV, however, B19 infection is usually transient without a latent phase, although viral DNA can persist for years.103 Association of B19 with SLE is primarily considered because the symptoms of B19 infection are similar to those of SLE, and autoantibody production is common in B19 infection. B19 infection is also frequently observed to exacerbate lupus. Parvovirus B19 infection can present with malar rash, persistent arthralgia, fever, fatigue, and positive antinuclear antibodies. The antinuclear antibody present in B19 infection can include anti-SCL-70, Sm, RNP, Ro, La, phospholipids and DNA.104-106 The similarities between disease presentations can make diagnosis difficult, especially in juveniles.107

Several reports associate B19 infection with the initiation of a lupus flare108,109 although given the similarity between B19 infection and lupus, it may be difficult to distinguish actual lupus activation from the symptoms of B19 infection. Additionally, B19 infection has been associated with the onset of lupus in case reports.106,110 However, upon examination of a large population of lupus patients, no association between B19 infection and lupus was found.111 Infection with B19 often leads to the production of autoantibodies, especially antiphospholipid antibodies. The presence of antiphospholipid antibodies in pediatric patients with rheumatic disease shows a statistical association with past or current B19 infection.112 Furthermore, the isotype and specificity of antiphospholipid antibodies that are present in B19 infection are very similar to the antibodies found in lupus, binding primarily to cardiolipin and phosphatidyl serine.113,114 Although most likely not a causative agent for SLE, further study of parvovirus B19 infection may yield insights into lupus. Specifically, increased understanding of the mechanism of autoantibody generation, and the role of these autoantibodies in symptoms such as arthropathy could be found.

RETROVIRUSES AND SLE

The major rationale for considering HCMV important in SLE is the involvement of the virus in the generation of autoantibodies against the U1 small nuclear ribonucleoprotein (U1), an important target of lupus autoantibodies. Healthy carriers of HCMV have an increased prevalence of anti-U1 antibodies compared to uninfected individuals, with 46% of HCMV-seropositive individuals possessing U1-70k–reactive antibodies, while only 20% of uninfected individuals had antibodies reactive to U1-70k (p<0.01). The presence of anti-U1 antibodies in lupus patients also correlated with HCMV seropositivity (p<0.05).98 The most likely candidate molecule to explain this association is the gB protein. Immunization of mice with recombinant gB protein leads to the production of anti-U1 antibodies, and also low levels of anti-ds DNA.99 However, conflicting results were obtained upon vaccination of HCMV-seronegative human volunteers with gB protein for viral protection. None of these immunized individuals generated autoantibodies.100 A possible explanation for the discrepancy is that the production of autoantibodies in humans is the result of a combination of genetic and environmental factors, and normal volunteers, unlike lupus patients, do not have the genetic background to easily produce and sustain autoantibody responses. Alternately, the immune systems of the mouse and human are sufficiently different that humans simply do not make cross-reactive autoantibodies against gB. One additional way in which HCMV may contribute to the production of autoantibodies is through redistribution of lupus autoantigens. HCMV infection of keratinocytes redistributes the 60 KD/Ro antigen to the cell surface, where it is presumably accessible to B-cell receptors.101 Increased availability for binding may increase the antigenicity of these self-proteins, rendering them capable of being targeted by the immune system.

RETROVIRUSES AND SLE Retroviruses share many characteristics with herpesviruses. These viruses are persistent, latently integrating their genomes into the host cell for lifelong infection. Retroviruses can be either exogenous, like HIV and HTLV, or endogenous, which are the remnants of germline-integrated retroviral genomes. Retroviruses were identified as candidates for etiologic agents of SLE because approximately onethird of SLE patients have false-positive results on HIV tests using the viral protein p24 as the test antigen.115 These findings indicate that lupus patients make antibodies against proteins that are very similar to p24. Of course, it is very unlikely that HIV itself is a causal agent in SLE, a finding that has been borne out experimentally,116 but it is possible that other retroviruses, including the endogenous group, may contain similar proteins. In fact, a majority of lupus patients do make antibodies that react with the endogenous retroviral element–encoded nuclear protein HRES-1.117 The epitope in HIV p24 that reacts with lupus antibodies is a proline-rich sequence cross-reactive to the PPPGMRPP epitope of Sm.118 This sequence is also cross-reactive with EBNA-1. However, the antibodies that react to HRES-1 do not cross-react with Sm, but rather with the U1 RNP. In addition, the presence of antibodies to HRES-1 correlate with anti-U1 RNP antibodies, but each antigen is recognized by a subset of antibodies that do not recognize the other.

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Genetic susceptibility

Virus infection

Interferon-alpha Cross-reactive antibodies

Broadening of Virus autoantibody specificity reactivation/ persistence Pathogenic antibodies

Fig. 15.3 Pathway from infection to lupus. A virus, for example EBV, infects a genetically susceptible individual. Viral infection precipitates the production of inflammatory cytokines and autoantibodies. In the example of EBV, interferon alpha and potentially anti-Ro or anti-Sm antibodies would be made. Continual restimulation through viral persistence and reactivation leads to the broadening of the immune response to encompass multiple autoantigens, with the eventual production of pathogenic autoantibodies and the development of clinical lupus.

Although studies examining serology and the presence of viral genomes have ruled out HIV or HTLV as etiologic agents of lupus,119,120 the role of endogenous retroviruses is difficult to assess. Since they are ubiquitous and are actually self-antigens, serologic association studies provide little information. Antibody reactivity to endogenous retroviruses is associated with reactivity to other self-proteins; however, the degree of cross-reactivity makes it difficult to say which reactivity came first. To truly understand the role of endogenous retroviruses in SLE, studies examining sequence difference or differences in expression of these retroviral proteins still need to be undertaken.

INFECTION OF LUPUS PATIENTS

152

LUPUS

Patients afflicted with lupus have a high rate of infections, which are a cause of significant morbidity. The increased rates of infection may be the result of the immunosuppressive medication used to treat SLE, or as a result of the underlying immune dysregulation and tissue damage associated with the disease. The most common infections afflicting lupus patients are bacterial. Up to one-third of lupus patients admitted to the hospital will have infections, and approximately 15% of those will be major infections, mostly sepsis.121 The most common types of infection leading to hospitalization of lupus patients are lower respiratory tract infection, followed by urinary tract infections, skin infections, septicemia, and musculoskeletal infections.122 Viral and fungal infections are also common in lupus, including reactivation of herpes zoster,123,124 papilloma virus,125,126 candida infections,127 and Pneumocystis carinii infection.128,129 Infection in lupus patients is associated with immunosuppressive therapy. Of the drugs used to treat lupus, the one most associated with bacterial infection is steroid use.130,131 Cyclophosphamide treatment is also associated with increased risk of infection.127 Factors besides therapeutic agents are also important in the prevalence of infection. Disease activity as measured

by SLEDAI score positively correlates with the risk of infection.131 Nephritic disease correlates with an increased risk of infection.127 This may be a result of decreased kidney efficiency, leading to kidney infections, or it may be a result of the generally higher level of disease activity in patients with nephritis.

CONCLUSION Viral infection has great potential to be a precipitating event leading eventually to lupus in susceptible individuals (Fig. 15.3). Multiple viral infections have characteristics that could implicate them in the pathogenesis of lupus (Table 15.2). Some of these viruses have been associated with the presence or flare of lupus disease. Increased insight into the role of viral infection in lupus can suggest new treatment or prevention options, as well as an increased understanding of the mechanisms of autoimmunity. Further study of infection may also help in the early detection and aggressive treatment of infections in lupus patients.

TABLE 15.2 VIRUSES POTENTIALLY INVOLVED IN LUPUS CAUSATION Virus

Evidence for Etiological Role in Lupus

Epstein-Barr virus Increased seroprevalence in lupus patients Increased viral DNA load in lupus patients Cross-reactivity of viral proteins Case reports of lupus onset after infection Cytomegalovirus Cross-reactivity of viral proteins Case reports of infection associated with lupus onset or flare Serologic association with autoantibody subsets Parvovirus B19

Similarity of symptoms Autoantibody production

Retroviruses

Cross-reactivity of viral proteins

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112. Von Landenberg P, Lehmann HW, Knoll A, et al. Antiphospholipid antibodies in pediatric and adult patients with rheumatic disease are associated with parvovirus B19 infection. Arthritis Rheum 2003;48(7):1939-1947. 113. Yamazaki M, Asakura H, Kawamura Y, et al. Transient lupus anticoagulant induced by Epstein-Barr virus infection. Blood Coagul Fibrinolysis 1991;2(6):771-774. 114. Loizou S, Cazabon JK, Walport MJ, et al. Similarities of specificity and cofactor dependence in serum antiphospholipid antibodies from patients with human parvovirus B19 infection and from those with systemic lupus erythematosus. Arthritis Rheum 1997;40(1):103-108. 115. Deas JE, Liu LG, Thompson JJ, et al. Reactivity of sera from systemic lupus erythematosus and Sjogren’s syndrome patients with peptides derived from human immunodeficiency virus p24 capsid antigen. Clin Diagn Lab Immunol 1998;5(2):181-185. 116. Font J, Vidal J, Cervera R, et al. Lack of relationship between human immunodeficiency virus infection and systemic lupus erythematosus. Lupus 1995;4(1):47-49. 117. Perl A, Colombo E, Dai H, et al. Antibody reactivity to the HRES1 endogenous retroviral element identifies a subset of patients with systemic lupus erythematosus and overlap syndromes. Correlation with antinuclear antibodies and HLA class II alleles. Arthritis Rheum 1995;38(11):1660-1671. 118. Talal N, Flescher E, Dang H. Are endogenous retroviruses involved in human autoimmune disease? J Autoimmun 1992;5(Suppl A):61-66. 119. Nelson PN, Lever AM, Bruckner FE, et al. Polymerase chain reaction fails to incriminate exogenous retroviruses HTLV-I and HIV-1 in rheumatological diseases although a minority of sera cross react with retroviral antigens. Ann Rheum Dis 1994;53(11):749-754. 120. Bailer RT, Lazo A, Harisdangkul V, et al. Lack of evidence for human T cell lymphotrophic virus type I or II infection in patients with systemic lupus erythematosus or rheumatoid arthritis. J Rheumatol 1994;21(12):2217-2224. 121. Chen YS, Yang YH, Lin YT, et al. Risk of infection in hospitalised children with systemic lupus erythematosus: a 10-year followup. Clin Rheumatol 2004;23(3):235-238. 122. Wongchinsri J, Tantawichien T, Osiri M, et al. Infection in Thai patients with systemic lupus erythematosus: a review of hospitalized patients. J Med Assoc Thai 2002;85(Suppl 1):S34-S39. 123. Kahl LE. Herpes zoster infections in systemic lupus erythematosus: risk factors and outcome. J Rheumatol 1994;21(1):84-86. 124. Ishikawa O, Abe M, Miyachi Y. Herpes zoster in Japanese patients with systemic lupus erythematosus. Clin Exp Dermatol 1999;24(4):327-328. 125. Yell JA, Burge SM. Warts and lupus erythematosus. Lupus 1993;2(1):21-23. 126. Korkmaz C, Urer SM. Cutaneous warts in patients with lupus erythematosus. Rheumatol Int 2004;24(3):137-140. 127. Noel V, Lortholary O, Casassus P, et al. Risk factors and prognostic influence of infection in a single cohort of 87 adults with systemic lupus erythematosus. Ann Rheum Dis 2001;60(12):1141-1144. 128. Liam CK, Wang F. Pneumocystis carinii pneumonia in patients with systemic lupus erythematosus. Lupus 1992;1(6):379-385. 129. Kadoya A, Okada J, Iikuni Y, et al. Risk factors for Pneumocystis carinii pneumonia in patients with polymyositis/dermatomyositis or systemic lupus erythematosus. J Rheumatol 1996;23(7):1186-1188. 130. Gladman DD, Hussain F, Ibanez D, et al. The nature and outcome of infection in systemic lupus erythematosus. Lupus 2002;11(4):234-239. 131. Duffy KN, Duffy CM, Gladman DD. Infection and disease activity in systemic lupus erythematosus: a review of hospitalized patients. J Rheumatol 1991;18(8):1180-1184.

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91. Zhu H, Shen Y, Shenk T. Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol 1995;69(12):7960-7970. 92. De Bandt M, Sibilia J, Le Loet X, et al. Systemic lupus erythematosus induced by anti-tumour necrosis factor alpha therapy: a French national survey. Arthritis Res Ther 2005;7(3):R545-R551. 93. Hayashi T, Lee S, Ogasawara H, et al. Exacerbation of systemic lupus erythematosus related to cytomegalovirus infection. Lupus 1998;7(8):561-564. 94. Nawata M, Seta N, Yamada M, et al. Possible triggering effect of cytomegalovirus infection on systemic lupus erythematosus. Scand J Rheumatol 2001;30(6):360-362. 95. Vasquez V, Barzaga RA, Cunha BA. Cytomegalovirus-induced flare of systemic lupus erythematosus. Heart Lung 1992;21(4):407-408. 96. Rider JR, Ollier WE, Lock RJ, et al. Human cytomegalovirus infection and systemic lupus erythematosus. Clin Exp Rheumatol 1997;15(4):405-409. 97. Bendiksen S, Van Ghelue M, Rekvig OP, et al. A longitudinal study of human cytomegalovirus serology and viruria fails to detect active viral infection in 20 systemic lupus erythematosus patients. Lupus 2000;9(2):120-126. 98. Newkirk MM, van Venrooij WJ, Marshall GS. Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection. Arthritis Res 2001;3(4):253-258. 99. Curtis HA, Singh T, Newkirk MM. Recombinant cytomegalovirus glycoprotein gB (UL55) induces an autoantibody response to the U1-70 kDa small nuclear ribonucleoprotein. Eur J Immunol 1999;29(11):3643-3653. 100. Schleiss MR, Bernstein DI, Passo M, et al. Lack of induction of autoantibody responses following immunization with cytomegalovirus (CMV) glycoprotein B (gB) in healthy CMVseronegative subjects. Vaccine 2004;23(5):687-692. 101. Zhu J. Cytomegalovirus infection induces expression of 60 KD/Ro antigen on human keratinocytes. Lupus 1995;4(5):396-406. 102. Naides, SJ. Parvoviruses. In: Specter S, Lancz G, eds. Clinical Virology Manual. Essex: Elsevier Science Publishers, 1992:547-569. 103. Cassinotti P, Burtonboy G, Fopp M, et al. Evidence for persistence of human parvovirus B19 DNA in bone marrow. J Med Virol 1997;53(3):229-232. 104. Moore TL, Bandlamudi R, Alam SM, et al. Parvovirus infection mimicking systemic lupus erythematosus in a pediatric population. Semin Arthritis Rheum 1999;28(5):314-318. 105. Nesher G, Osborn TG, Moore TL. Parvovirus infection mimicking systemic lupus erythematosus. Semin Arthritis Rheum 1995;24(5):297-303. 106. Fawaz-Estrup F. Human parvovirus infection: rheumatic manifestations, angioedema, C1 esterase inhibitor deficiency, ANA positivity, and possible onset of systemic lupus erythematosus. J Rheumatol 1996;23(7):1180-1185. 107. Trapani S, Ermini M, Falcini F. Human parvovirus B19 infection: its relationship with systemic lupus erythematosus. Semin Arthritis Rheum 1999;28(5):319-325. 108. Hemauer A, Beckenlehner K, Wolf H, et al. Acute parvovirus B19 infection in connection with a flare of systemic lupus erythematodes in a female patient. J Clin Virol 1999;14(1):73-77. 109. Seve P, Ferry T, Koenig M, et al. Lupus-like presentation of parvovirus B19 infection. Semin Arthritis Rheum 2005;34(4):642-648. 110. Diaz F, Collazos J, Mendoza F, et al. Systemic lupus erythematosus associated with acute parvovirus B19 infection. Clin Microbiol Infect 2002;8(2):115-117. 111. Bengtsson A, Widell A, Elmstahl S, et al. No serological indications that systemic lupus erythematosus is linked with exposure to human parvovirus B19. Ann Rheum Dis 2000;59(1):64-66.

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Autoantibodies in Systemic Lupus Erythematosus David Isenberg, MD and Matthew Adler, MRCP

H

V

L

V H1

Hinge

C

L

C

1 CH

CL

156

Before discussing the origin of autoantibodies in SLE, the structure of immunoglobulins must be considered.

Variable region

Combining site VH

Basic Immunoglobulin Structure

N-terminal Combining site VH

CHO Constant region

CH2

Papain Pepsin

CH3

One of the hallmarks of systemic lupus erythematosus (SLE) is the presence of autoantibodies, over a hundred of which have been reported in the serum of these patients.1 The vast majority are found rarely in SLE, or indeed in other autoimmune diseases. Many are likely to be bystanders and represent epiphenomena, but good evidence suggests that some cause clinical disease. Autoantibodies found in more than 25% of patients with SLE include anti-ssDNA, anti-dsDNA, anti-Ro, anti-poly ADP ribose, and anti-histone/nucleosome antibodies and antiphospholipid antibodies. Antibodies to dsDNA are found in 50 to 70% of patients,2 but are very rarely found in healthy individuals and in relatives of patients with SLE.3 Interestingly, in one study, anti-dsDNA antibodies were detected in the sera for up to 9.4 years prior to developing symptomatic SLE.4 Anticardiolipin antibodies were estimated to occur in about 44% of patients with SLE, and 34% were found to have a lupus anticoagulant in one large meta-analysis.5 These results imply that not all anti-dsDNA or antiphospholipid antibodies are pathogenic. This observation will be discussed in some detail later in this chapter. Anti-Ro antibodies, while present in around a third of patients with SLE, are found in roughly twice as many patients with Sjögren’s syndrome. Anti-Sm antibodies are found less frequently (10% of white patients; 30% of black patients), but are highly specific for SLE. Antibodies that are likely to be pathogenic can be a useful warning marker of disease flare, in particular lupus nephritis, and the two most useful antibodies in this category are antibodies to dsDNA and antinucleosome antibodies.

CH 2

Overview of Autoantibodies in SLE

Antibodies, or immunoglobulins (Igs), play a pivotal role in the acquired immune response, and are produced by plasma cells in response to antigenic challenge. Igs consist of two heavy and two light chains, linked covalently by disulfide bonds (Fig. 16.1). The light chains may either be a kappa or lambda polypeptide and each of the two heavy chains contains four domains. It is the variable regions of the heavy and light chains at the N-terminal end that vary in amino acid sequence between antibody molecules and it is here that antigen binding occurs (Fab). The C-terminal end (Fc region) contains sites for complement fixation, and for example, binding to rheumatoid factors. Five classes of immunoglobulin are recognized on the basis of the Fc fragment of the heavy chain, and heavy-chain

CH3

INTRODUCTION

Cleavage sites

CHO

C-terminal Fig. 16.1 Representation of human IgG molecule. (Adapted from Morrow J, Nelson L, Watts R, Isenberg D. Autoimmune Rheumatic Disease. 2nd ed.. New York: Oxford University Press, 1999.)

Origin of Autoantibodies The origin of autoantibodies in SLE is largely unknown but there are a number of theories. First, environmental autoantigens may trigger the induction of autoantibodies. Second, it has been shown in the MRL/pr mice models that anti-dsDNA production may be antigen driven.6 Similar findings and conclusions supporting antigen-driven autoantibody production have also been documented by other groups.7-9 Third, polyclonal B-cell activation has been suggested as a way of producing a diverse population of autoantibodies, supporting the idea of an important role played by B cells in SLE.10 Fourth, there are a number of ways in which apoptosis, or programmed cell death, may be involved in autoantibody production in this disease. Cellular debris produced as a result of apoptosis may act as the trigger for the production of autoantibodies. Since complement is required to aid in the clearance of apoptotic material, models of complement-deficient animals have confirmed that these animals are more prone to developing lupus-like nephritis.11 Other mechanisms of tissue injury include impairment of apoptosis, which in itself can be multifactoral and has been described elsewhere,12,13 and autoantibody-driven apoptosis.14,15 Many researchers are convinced that the failure of macrophages to efficiently remove apoptotic cells is critical.16 Apoptotic cell fragments containing nuclear “debris,” including DNA, Ro, La, Sm, and so on, may be “presented” to T cells by “professional” antigen-presenting cells or by B cells acting as antigen presenters. The T cells in turn trigger other B cells to produce anti-DNA, Ro, La, and so forth. In this way lupus is considered by some to be a consequence of inadequate “waste disposal.” Other mechanisms, including disruption of the idiotype network, may also play a role17,18.

STRUCTURAL CONSIDERATIONS

Anti-dsDNA Antibodies Not all patients with these antibodies have overt clinical disease activity, suggesting that not all anti-dsDNA antibodies are pathogenic. This view has been confirmed in a number of animal studies whereby infusion of anti-dsDNA antibodies did not always result in disease.19-22 So what specific properties of anti-dsDNA antibodies make some but not others pathogenic? Furthermore, how and why do they develop and how do they cause tissue damage?

A considerable amount of work has been focused on attempting to answer these questions. For example, it has been established that IgG dsDNA antibodies tend to be the pathogenic isotype,23,24 which also tend to be positively charged and show high-affinity binding to dsDNA rather than ssDNA.2 The ability of some IgG anti-dsDNA antibodies to cross-react with a-actinin, which is found in the renal podocytes, may also be important.25

Role of Monoclonal Antibodies One method of determining whether an autoantibody is pathogenic is to study the effect of human monoclonal antibodies in severe combined immunodeficiency (SCID) mice. Despite the difficulties in producing these monoclonal antibodies, various groups have made use of molecular biological techniques to synthesize a number of monoclonal anti-dsDNA and antiphospholipid (APL) antibodies, and their isotype and binding properties have been characterized. Describing these in any great detail is beyond the scope of this chapter, but they have been reviewed by Ravirajan and colleagues.26 The renal effects of a number of IgG human monoclonal anti-dsDNA antibodies that have been produced in SCID mice are shown in Table 16.1. Again it is clear that some antibodies are more pathogenic than others. Monoclonal anti-dsDNA antibodies from SLE patients27 and from animal lupus models28 have been sequenced, and analysis of their structure has shown that the high-affinity antibodies of the IgG isotype contain a large number of somatic mutations in their heavy-chain variable regions (VH) and light-chain variable regions (VL). These mutations are thought to be driven by antigens by virtue of their distribution within the complementarity-determining regions (CDRs). This region forms the antigen-binding site. Specific amino acids

STRUCTURAL CONSIDERATIONS

classes are further divided into subclasses. Idiotypic determinants are unique sequences of amino acids located in the variable region. It is here that antigen binding occurs and the site that determines antibody specificity.

TABLE 16.1 IgG HUMAN MONOCLONAL ANTI-DNA ANTIBODIES Structure/function relationship EFFECTS IN SCID MICE Antibody

Proteinuria

Type of Deposition

B3

++→+++

In vivo ANA

D5

0/+

-

RH14

++→+++

b.m capillary loop and mesangial matrix

DIL-6

+

-

32.B9

+

-

33.C9

++→+++

Extracellular deposits in the capillary wall and mesangium

35.21

++→+++

In vivo ANA

C6.F7

Trace

-

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AUTOANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS

158

seem to be found more commonly in these CDRs as a result of somatic mutations, namely arginine, asparagine, and lysine. These amino acids are likely to be highly relevant by virtue of their ability to form electrostatic charges and hydrogen bonds with the negatively charged DNA phosphodiester backbone28,29 (see Fig. 16.1). Thus, antigen-driven clonal expansion is likely to result in the development of pathogenic antibodies, and it is this expansion that gives rise to the somatic mutations. To that end, dsDNA and histones are likely candidates in driving this process. The human monoclonal anti-dsDNA antibody B3 has been extensively analyzed and shown to be pathogenic in SCID mice.30 Computer models31 have demonstrated the importance of its arginine residue at position 27a in CDR1 of the Vγ gene 2a2. Substitution of this arginine by a serine both reduced the binding of the antibody to DNA and was associated with a significant reduction in proteinuria in the SCID model system.32 Many groups have looked at other autoantibodies and specific sequences in the heavy and light-chain variable regions and correlated this with DNA binding.33-35 Recent work has shown that particularly strong binding to dsDNA occurred when both light chains contained arginine residues (Arg 27a and Arg 92).35 They also showed changes in binding to DNA if arginine residues were positioned slightly differently. As reviewed elsewhere,36 most antibodies have more heavy-chain than light-chain contacts with DNA, especially in the heavy-chain third CDR. Perhaps there is a tendency for VH germline codons to mutate to arginine (or lysine), and a probability that B cells with arginine-containing CDR-VH domains will develop into anti-DNA antibody producers during clonal expansion.37 It is not merely affinity for dsDNA that determines pathogenicity. Cross-reactivity with cardiolipin, nucleosomes, laminin, a-actinin, heparan sulfate, and type-IV collagen may also be important. Kumar and colleagues38 investigated the human monoclonal autoantibodies B3, RH14 (anti-DNA), and UK4 (cardiolipin) in their IgG and cloned Fab formats, and looked to see whether they exhibited crossreactivity and whether this might contribute to their pathogenic potential. The fine binding characteristics of these three molecules have been elucidated and published elsewhere, and will not be discussed further.26,39,40 Both B3 and RH14 antibodies have been shown to induce proteinuria in SCID mice, and RH14 shows specific renal binding and induces early features of lupus nephritis under electron microscopy.26,30 Kumar and colleagues38 showed that B3 and RH14 possess high cross-reactivity against a DNA polymerase (PolIV) of bacterial origin and that some patients with

lupus have anti-PolIV activity in their sera. All three molecules bind to ssDNA and dsDNA, but RH14 is the predominant dsDNA binder and UK14 binds the most to cardiolipin. It is possible that changes in the physiologic milieu may modify the antigen-binding patterns of these antibodies, and thus modify their pathogenic potential, although this may be less true for anticardiolipin antibodies. Data from Kumar’s study also showed that the reactivity of RH14 for dsDNA involved high electrostatic charges, and phosphate interactions and B3 also significantly exhibited charge-independent cross-reactivity for cardiolipin.

Structural Consideration of Antiphospholipid Antibodies and Pathogenicity Pathogenic anticardiolipin antibodies are also generally of the IgG isotype,41,42 and bind the cofactor β2-glycoprotein I (β2GPI). Analogous to the range of anti-dsDNA antibodies, it is interesting to note that patients with antiphospholipid antibodies do not always have the antiphospholipid antibody syndrome, characterized by arterial and venous thromboses, recurrent spontaneous miscarriage, and thrombocytopenia. Antiphospholipid antibodies have been found in other infectious and neoplastic diseases, as well as in normal healthy individuals.43 In general they are of the IgM isotype and do not bind β2GPI. What seems to separate pathogenic from nonpathogenic antiphospholipid antibodies is the ability to bind neutral and negatively charged phospholipids in the presence of a co-factor, i.e., β2GPI.44 This co-factor is not the only one to adopt this “function,” and it has the ability to bind antiphospholipid antibodies in patients with the antiphospholipid syndrome even in the absence of phospholipids.45 Studies have shown similarities in the amino acid sequences of dsDNA antibodies and antiphospholipid antibodies, and arginine residues positioned at certain locations can influence the binding of cardiolipin.46 The molecular structure of β2GPI has been elucidated,47,48 and this has given insight into the way it binds to phospholipids via epitopes. The structure of β2GPI, which consists of five domains, has been examined in order to identify which contain epitopes that bind phospholipids. Domain V is not a major binding site in view of its hydrophobic loop that prevents binding to positively charged phospholipids.49,50 Current evidence points to domain I as the main binding site for phospholipid in patients with the syndrome,51-53 and that a mutation of arginine to glycine at position 43 ameliorated the binding in patients with disease.54 How antiphospholipid antibodies actually promote thrombosis is not fully understood, but they may in some way interfere with the endothelial cells, thereby reducing production of prostacyclin; they may also

Cardiolipin DXS

High

DNA Histones

Heparin sulfate GBM Crossreactivity

Nephritis

Fig. 16.2 Schematic diagram of DNA binding in the kidney.

affect other fibrinolytic mechanisms. Again, this may be mediated by binding to heparan sulfate (Fig. 16.2).

LINKS BETWEEN STRUCTURE AND PATHOLOGY The relation between the presence of anti-dsDNA antibodies and lupus nephritis has been known about for almost forty years.55-57 Early animal studies predicted a link between DNA antibodies and disease when they were identified in glomerular immune deposits in animals with active lupus nephritis. Furthermore, Ebling and Hahn demonstrated higher concentrations in the glomerular eluate than in the serum not only in mouse models but also in humans.58 When murine anti-dsDNA antibodies are administered to a healthy mouse, nephritis can develop.59,60 However even within the subset of “pathogenic” DNA antibodies, not all are nephritogenic as they have been identified in extra-renal sites.61 So how do immune deposits formed from autoantibodies find their way and bind in the kidney? A number of mechanisms have been proposed. Initially it was suggested that circulating immune complexes i.e., dsDNA and anti-dsDNA were deposited in the glomeruli.62 It proved difficult to confirm this idea when looking for deposition of immune complexes preformed in the circulation. The possibility that DNA might become “planted” onto the glomerular basement membrane63 also gained some support. It was proposed that antibodies might then bind to DNA, activating complement, and exciting inflammation. Berden and colleagues have proposed a more important role for nucleosomes in this context.64 Nucleosomes consist of DNA and histones and it was suggested

that positively charged histones might bind to the negatively charged proteoglycan components of glomerular membranes.65 The histones were thus in effect acting as a bridge between the anti-DNA/nucleosome antibodies and the kidney and this interaction is shown in Fig. 16. 2. Other proposed mechanisms include the ability of some anti-DNA antibodies to cross-react directly with renal membrane components.66,67 In further work by Mostoslavsky and colleagues,68 it was shown that the essential difference between some murine anti-dsDNA antibodies shown to be pathogenic in a mouse model was the ability to cross react with a-actinin (known to be an important constituent of the glomeruli). In follow-up studies by Mason and colleagues,69 affinitypurified anti-dsDNA antibodies from the sera of lupus patients was shown to be far more likely to have come from patients with active renal disease if they cross reacted with a-actinin. Another possible candidate for a glomerular cross-reaction is laminin,70 but this will not be discussed further. a-actinin is a 100-kD protein and is mainly located in the podocyte foot processes71 and mesangial cells68 and of the four known isoforms of the genes, it is the a-actinin 4 (a-ACTN4) that is present in the human kidney.72 Experiments with a-ACTN4 knockout mice have added weight to the hypothesis that this protein is important in maintaining normal podocyte cell function.73 Two groups have shown that only pathogenic murine anti-dsDNA antibodies bind directly to a-actinin.68,74 More recently, Mason and colleagues have investigated the ability of human pathogenic antidsDNA antibodies to bind to a-actinin.69 Their results confirmed that certain pathogenic human DNA antibodies (RH14, B3) bound strongly to a-actinin in patients with renal disease whilst non-pathogenic DIL6 bound very weakly. In a recent study using a panel of human anti-dsDNA and/or anti—a-actinin antibodies it was shown that binding of these immunoglobulins to glomeruli was not inhibited by DNAase treatment but was blocked by a-actinin.75 These data support the notion that anti-dsDNA antibodies can bind to glomerular a-actinin and may indeed be pathogenic. In this review we are focusing primarily on renal disease but the possibility remains that anti-DNA antibodies might also cross react with cell surface structures from other organs. Thus, as an example, DeGiorgio and colleagues showed that some anti-DNA antibodies could cross react with a DNA-mimicking pentapeptide sequence that occurs in the extra-cellular domain of the N-methyl-D-aspartate receptor in the brain causing the apoptotic death of neurons in vivo and in vitro.76 Antibodies with this notable activity were detected in the cerebrospinal fluid of a patient with lupus known to have CNS manifestations.

LINKS BETWEEN STRUCTURE AND PATHOLOGY

Anti-DNA avidity Low

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Another potential mechanism for inflicting renal damage was proposed by Yanase and colleagues who suggested that certain subsets of anti-dsDNA antibodies can penetrate living cells, enter the cell nucleus inducing apoptosis, and ultimately lead to glomerular damage as evidenced by the presence of increased proteinuria.77 However, for such deposited antibodies to induce glomerular damage other affected mechanisms need to be envisaged and one such possibility involves the activation of the Fc receptor. This claim is based upon the important early finding that complement activation is critical in immune complex deposition. To this end Clynes and colleagues demonstrated the essential part played by the FcR in a mouse model of lupus.78 In their study although the homozygous FcR gamma -/- mice produced DNA antibodies in immune complexes, they did not produce proteinuria or histological nephritis in the glomeruli. These data support the view that FcRs are crucial in immune activation and clinically evident disease. Supporting the argument that complement is important in mediating tissue damage is the observation that using an anti-C5 antibody to NXB/W mice can prevent the development of renal disease.79 In clinical practice, physicians take a rising antidsDNA level80-82 with a falling level of C383,84 as being indicative of a potential flare of lupus, although there are a group of patients with persistently very high levels of anti-dsDNA antibodies who remain clinically well.85,86 Paradoxically, some studies have shown little

correlation between rising dsDNA levels and disease flaring.87,88 Indeed some reports suggest that levels may actually fall before a flare82 of lupus nephritis for example probably because the DNA antibodies are being deposited in the kidney. Also, it has been suggested that patients with class III and IV lupus nephritis, rather than class V, are more likely to have higher levels of antibodies to dsDNA in the serum, but this is not always true.89 A strong piece of evidence in favor of the pathogenic role of DNA antibodies in lupus nephritis comes from a study by Linnik and colleagues who reported that reducing levels of anti-dsDNA antibodies using a novel dsDNA-based biconjugate (abetimus sodium, LJP394) gives rise to a reduced risk of lupus nephritis flares.90

CONCLUSIONS Autoantibodies are a hallmark of SLE although not all are pathogenic and cause tissue damage. Molecular biology is now being used to help determine, more precisely, the key features/binding characteristics of autoantibodies such as anti-dsDNA/phospholipids, which give them “pathogenic status.” The processes driving the production of autoantibodies are now clearer and the ways in which they bind to their targets in organs such as the kidney and cause damage are also being clarified. The close links between structure, function, and pathogenicity are increasingly evident.

REFERENCES

160

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9. Radic MZ, Cocca BA, Seal SN. Initiation of systemic autoimmunity and sequence specific anti-DNA autoantibodies. Crit Rev Immunol 1999;19:117-126. 10. Klinman DM, Steinberg AD. Systemic autoimmune disease arises from polyclonal B cell activation. J Exp Med 1987;165:1755-1760. 11. Botto M, Dell’Agnola C, Bygrave AE, et al. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 1998;19:56-59. 12. Greidinger EL. Apoptosis in lupus pathogenesis. Front Biosci 2001;6:1312-1325. 13. Vainshaw AK, Toubi E, Ohsako S, et al. The spectrum of apoptotic defects and clinical manifestations, including systemic lupus erythematosus, in human with CD95 (Fas/APO-1) mutations. Arthritis Rheum 1999;33-42. 14. Nakamura N, Ban T, Yamaji K, et al. Localization of the apoptosisinducing activity of lupus anticoagulant in an annexin V-binding antibody subset. J Clin Invest.1199;8:51-58. 15. Oshimi Y, Oda S, Honda Y, et al. Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells. J Immunol 1996;2909-2915. 16. Rosen A, Casciola-Rosen L. Autoantigens as substrates for apoptotic proteases: implications for the pathogenesis of systemic autoimmune disease. Cell Death Differ 1999;6(1):6-12. 17. Kalunian KC, Panosian-Sahakian N, Ebling FM, et al. Idiotypic characteristics of immunoglobulins associated with systemic lupus erythematosus. Studies of antibodies deposited in glomeruli of humans. Arthritis Rheum 1989;32:513-522.

42. Lynch A, Marlar R, Murphy J, et al. Antiphospholipid antibodies in predicting adverse pregnancy outcome. A prospective study. Ann Intern Med 1994;120:470-475. 43. Shah NM, Khamashta MA, Atsumi T, et al. Outcome of patients with anticardiolipn antibodies: a 10 year follow-up of 52 patients. Lupus 1998;7:3-6. 44. Hunt JE, McNeil HP, Morgan GJ, et al. A phospholipid-β2 glycoprotein I complex is an antigen for anticardiolipin antibodies occurring in autoimmune disease but not with infection. Lupus 1992;1:75-81. 45. Matsuura E, Igarashi Y, Yasuda T, et al. Anticardiolipin antibodies recognise β2 glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994;179:457-462. 46. Rahman A. Autoantibodies, lupus and the science of sabotage. Rheumatology 2004;43:1326-1336. 47. Bouma B, de Groot PG, van den Elsen JM, et al. Adhesion mechanism of human beta(2)-glycoprotein I to phospholipids based on its crystal structure. EMBO J 1999;18:5166-5174. 48. Schwarzenbacher R, Zeth K, Diederichs K, et al. Crystal structure of human beta2-glycoprotein I. implications for phospholipids binding and the antiphospholipid syndrome. EMBO J 1999;18:6228-6239. 49. Hunt J, Krilis S. The fifth domain of beta 2-glycoprotein I contains a phospolipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies. J Immunol 1994;152:653-659. 50. Wang MX, Kandiah DA, Ichikawa K, et al. Epitope specificity of monoclonal anti-beta 2- glycoprotein I antibodies derived from patients with the antiphospholipid syndrome. J Immunol neutrol: 1995:29-36. 51. Iverson GM, Victoria EJ, Marquis DM. Anti-beta2 glycoprotein I (beta2GPI) autoantibodies recognize an epitope on the first domain of beta2GPI. Proc Natl Acad Sci U S A 1998;95:15542-15546. 52. Igarashi M, Matsuura E, Igarashi Y, et al. Human beta2-glycoprotein I as an anticardiolipin cofactor determined using mutants expressed by a baculovirus system. Blood 1996;87:3262-3270. 53. George J, Gilburd B, Hojnik M, et al. Target recognition of beta2glycoprotein I (beta2GPI)-dependent anticardiolipin antibodies: evidence for involvement of the fourth domain of beta2GPI in antibody binding. J Immunol 1998;160:3917-3923. 54. Iverson GM, Matsuura E, Victoria EJ, et al. The orientation of beta2GPI on the plate is important for the binding of anti-beta2GPI autoantibodies by ELISA. J Autoimmun 2002;18:289-297. 55. Swaak AJ, Huysen V, Nossent JC, et al. Antinuclear antibody profiles in relation to specific disease manifestations of systemic lupus erythematosus. Clin Rheumatol 1990;9(Suppl 1):82-94. 56. Swaak T, Smeenk R. Clinical significance of antibodies to double stranded DNA (dsDNA) for systemic lupus erythematosus (SLE). Clin Rheumatol 1987;6(Suppl 1):56-73. 57. Clough JD, Couri J, Youssoufian H, et al. Antibodies against nuclear antigens: association with lupus nephritis. Cleve Clin Q 1986;53:259-265. 58. Ebling F, Hahn BH. Restricted subpopulations of DNA antibodies in kidneys of mice with systemic lupus. Comparison of antibodies in serum and renal eluates. Arthritis Rheum 1980;23: 392-403. 59. Vlahakos DV, Foster MH, Adams S, et al. Anti-DNA antibodies form immune deposits at distinct glomerular and vascular sites. Kidney Int 1992;41:1690-1700. 60. Winfield JB, Faiferman I, Koffler D. Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. Association of high avidity antinative DNA antibody with glomerulonephritis. J Clin Invest 1977; 59:90-96. 61. Sasaki T, Hatakeyama A, Shibata S, et al. Heterogeneity of immune complex-derived anti-DNA antibodies associated with lupus nephritis. Kidney Int 1991;39:746-753. 62. Dixon FJ, Oldstone MA, Tonietti G. Pathogenesis of immune complex glomerulonephritis of New Zealand mice. J Exp Med 1971;134:65s-71s. 63. Carlson JA, Hodder SR, Ucci AA, et al. Glomerular localization of circulating single-stranded DNA in mice. Dependence on the molecular weight of DNA. J Autoimmun 1988;1:231-241. 64. Berden JH, Grootscholten C, Jurgen WC, et al. Lupus nephritis: a nucleosome waste disposal defect? J Nephrol 2002; 15(Suppl 6):S1-S10. 65. Berden JH, Licht R, van Bruggen MC, et al. Role of nucleosomes for induction and glomerular binding of autoantibodies in lupus nephritis. Curr Opin Nephrol Hypertens 1999;8:299-306.

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PATHOGENESIS

17

What Do Mouse Models Teach Us about Human Systemic Lupus Erythematosus? Kui Liu, PhD and Chandra Mohan, MD, PhD

INTRODUCTION Systemic lupus erythematosus (SLE) is an autoimmune disease of complex etiology primarily characterized by the presence of high titers of autoantibodies directed to many nuclear as well as cytoplasmic antigens and end-organ damage. Antibody and immunocomplex–mediated inflammation in this disease can lead to the development of glomerulonephritis, dermatitis, serositis, and vasculitis. It is estimated to affect about 1 in 2000 people with a strong gender bias (female to male ratio is about 9:1). Furthermore, blacks and Hispanics are approximately two to four times more likely to develop this disease than whites. Both genetic and environmental factors contribute to its pathogenesis.1-4 Although deficiencies in some of the early components of the complement cascade, such as C1 and C4,2,5 can lead to the development of SLE, this disease is typically polygenic in origin.4 Over the past 30 years, mouse models have provided valuable insights concerning the pathogenesis of lupus. Based on how they were generated, these models can be divided into three groups: (1) spontaneous mouse lupus models, (2) congenic mouse models, and (3) engineered mouse models of lupus. The central goal of this review is to present the main features of these models and the lessons that we have learned from them.

SPONTANEOUS MURINE LUPUS MODELS REPRODUCE COMPLEXITY OF HUMAN SLE Spontaneous murine lupus models were the earliest to be reported (Table 17.1). Several inbred and hybrid strains were noted to develop elevated levels of antinuclear antibodies (ANA) with varying degree of lupuslike kidney disease. Studies performed with these models have provided important insights into lupus pathogenesis. Some of the best-characterized models include the hybrids of New Zealand black (NZB) and New Zealand white (NZW) mice, as well as the closely

related recombinant inbred NZM2410 strains.6-8 Numerous susceptibility loci have been mapped in these strains.4,9-14 The MRL/lpr and BXSB/Yaa strains also develop lupus.15-17 MRL/lpr mice carry the lpr mutation of Fas on the lupus-prone MRL background, while BXSB/Yaa mice bear the Y-linked autoimmune accelerator (Yaa) gene on the lupus-prone BXSB background. Besides these two loci, several additional loci have recently been identified in their background genomes.18-21 Likewise, (SWR x NZB)F1 (or SNF1) mice develop lupus nephritis that is clinically very similar (in onset, severity, female bias, and pathology) to the BWF1 model, as a consequence of multiple SWR and NZB loci.22-26 In these models, although disease susceptibility loci appear to be scattered randomly over all 19 autosomes, loci on four chromosomes appear to have been repeatedly mapped in several independent studies: chromosomes 1, 4, 7, and 17. Among these loci, the syntenic counterparts of the murine loci on chromosome 1 (Sle1, Nba2, etc.) and chromosome 17 (Sle4, Lbw1, etc.) have also been implicated in linkage analysis to be associated with human SLE.3,4 However, it is presently unknown if the same genes within these intervals are responsible for both the murine and human disease. It is also interesting to consider how well these mouse models of spontaneous lupus mimic the human disease. All of the mouse models exhibit high titers of IgG anti-dsDNA and antiglomerular antibodies, accompanied by severe glomerulonephritis, resembling human SLE. Beyond this commonality, the various models reflect certain aspects of the human disease fairly well. Thus, whereas the BWF1 and SNF1 models exhibit the female bias in disease prevalence (just as in human SLE), many of the other strains do not. In fact, the BXSB strain is rather unusual in afflicting males predominantly. Whereas the MRL/lpr and BXSB models are heavily dependent on single-gene accelerators, the other models (and human SLE) do not appear

163

WHAT DO MOUSE MODELS TEACH US ABOUT HUMAN SYSTEMIC LUPUS ERYTHEMATOSUS?

TABLE 17.1 SPONTANEOUS MOUSE MODELS FOR LUPUS Name

Genetic Background

(NZB × NZW)F1

Clinical Phenotypesa

Genderb

NZB, NZW

ANA, GN, lymphosplenomegaly, synovitis, Sjogren’s, CNS damage

F>M

38, 86

NZM2410

NZB, NZW

ANA, GN, lymphosplenomegaly

F>M

7, 8, 12, 87

NZM2328

NZB, NZW

ANA, GN, lymphosplenomegaly

F>M

88

MRL-Fas

MRL

ANA, GN, lymphosplenomegaly, synovitis, vasculitis, skin lesions, Sjogren’s, CNS damage

F>M

40-42, 89-91

MRL FasLgld

MRL

ANA, GN, lymphosplenomegaly, arteritis, arthritis, CNS damage

F>M

92, 93

BXSB/yaa

BXSB

ANA, GN, lymphosplenomegaly, CNS damage

M

(SWR x NZB)F1

SWR, NZB

ANA, GN, lymphosplenomegaly

F>M

lpr

40, 42, 89, 94 22, 23

a The clinical phenotypes shown here have been documented in the literature. However, it is unclear if all strains have been examined comprehensively for all potential phenotypes (e.g., skin and CNS involvement). b F>M means that females are predominantly affected. ANA, antinuclear autoantibodies; CNS, central nervous system; GN, glomerulonephritis.

to be so. Likewise, the massive degree of lymphoproliferation that characterizes the former models is also not a typical feature of lupus in other models or in humans. On the other hand, the joint and skin diseases exhibited by MRL/lpr mice represent features seen in human SLE but rarely noted in the other mouse models.27-30 More recently, it has become apparent that these mouse models may also be suitable for studying CNS lupus, a common feature of human SLE.31-43

CONGENIC MURINE LUPUS MODELS PROVIDE UNIQUE TOOLS FOR DISSECTING LUPUS PATHOGENESIS

164

References

Although linkage analyses have identified numerous loci that are associated with various disease phenotypes, they have not provided any mechanistic insights into how these loci actually contribute to these phenotypes. Congenic dissection is a strategy in which individual loci that contribute to a polygenic disease (such as lupus) can be segregated into a collection of unique substrains, each bearing an individual locus; one is thus allowed to study the component phenotypes contributed by each locus separately. Using this strategy, various lupus susceptibility loci have been successfully introgressed onto the genome of lupus-resistant strains, such as the C57BL/6 (B6) (Table 17.2). These newly generated congenic mouse strains constitute a unique and powerful tool for dissecting lupus pathogenesis. Since most lupus congenics derived to date have been generated on the B6 background, one can easily breed them to existing mouse tools such as transgenics and knockouts (involving various molecules of

immunologic importance), many of which are already on the B6 background. This would then allow the scientist to study the roles of specific cells or molecules in the context of different lupus susceptibility loci. Moreover, the same breeding approach used to create congenic strains can be repeatedly applied in order to further “narrow” the relevant congenic intervals. A good illustration of how lupus pathogenesis can be “dissected” using congenics stems from the genetic studies of the NZM2410 inbred model. In this model, lupus is contingent upon at least three non-MHC chromosomal intervals—Sle1, Sle2, and Sle3/5. Functional analyses of B6-based congenic strains bearing Sle1, Sle2, or Sle3/5 have demonstrated that each interval is responsible for very different component phenotypes.44-48 Among them, the breach of immune tolerance to nuclear antigens mediated by Sle1 appears to be essential for lupus development.12,49,50 However, Sle1 by itself does not lead to the development of fatal lupus but only modest serologic autoreactivity.49,50 In contrast, Sle1 mediates highly penetrant fatal glomerulonephritis in epistasis with Sle2, Sle3/5, Yaa, or lpr.49-53 Studies of the NZM2410-derived susceptibility intervals support a multi-step pathogenesis model, in which development of fatal lupus appears to be “initiated” by Sle1 and further exacerbated by additional susceptibility loci or genes.4,54 Congenic recombinant studies have identified that Sle1 is a cluster of four susceptibility subintervals: Sle1a, b, c, and d.55 Fine mapping of the Sle1b interval has already helped identify the association of extensive polymorphisms in the SLAM/CD150 gene cluster with the development of systemic autoimmunity.56 Likewise,

Genetic Background

Name

Chromosome

Clinical Phenotypesa

Genderb

References

Introgression of Disease Interval onto “Normal” Background

B10.Yaa.BXSB-Bxs1

C57BL/10

1, Y

GN

M

95

B10.Yaa.BXSB-Bxs1/4

C57BL/10

1, Y

ANA, GN

M

95

B10.Yaa.BXSB-Bxs1/2

C57BL/10

1, Y

ANA

M

95

B10.Yaa.BXSB-Bxs2/3

C57BL/10

1, Y

ANA, GN, lymphosplenomegaly

M

95

B6.Sle1

C57BL/6

1

ANA, lymphosplenomegaly

F>M

44

B6.Sle3/5

C57BL/6

7

ANA, GN

F=M

46

B6.Sle1.Sle2

C57BL/6

1, 4

ANA, GN, lymphosplenomegaly

F>M

50

B6.Sle1.Sle2.Sle3/5

C57BL/6

1, 4, 7

ANA, GN, lymphosplenomegaly

F>M

50

B6.Sle1.Sle3/5

C57BL/6

1, 7

ANA, GN, lymphosplenomegaly

F>M

49, 50

B6.Sle2.Sle3/5

C57BL/6

4, 7

ANA, GN

F>M

50

B6.Sle1.Yaa

C57BL/6

1, Y

ANA, GN, lymphosplenomegaly

F>M

50

B6.Sle1.Fasl

C57BL/6

1, 19

ANA, GN, lymphosplenomegaly

F>M

52

B6.Lmb3.Faslpr

C57BL/6

7, 19

ANA, GN, lymphosplenomegaly

F>M

96

pr

B6.Nba2

c

C57BL/6

1

ANA

F

58, 97

B6.Yaa. Nba2

C57BL/6

1, Y

ANA, GN

M

98

B6.Sgp3/2.Yaa

C57BL/6

13, Y

ANA

M

99

Fc

100

c

100

Introgression of “Normal” Interval onto Disease Background

NZM2328.Cgnz1C57L/C5 NZM2328.Adnz1

C57L/C5

NZM2328 NZM2328

1 4

GN ANA, GN

F

a

The clinical phenotypes shown here have been documented in the literature. The degree of lymphosplenomegaly has not been detailed in all strains. b F>M means that females are predominantly affected. c Only data for female mice were presented. ANA, antinuclear autoantibodies; GN, glomerulonephritis.

the culprit gene responsible for the Sle1c locus appears to be polymorphisms of the CR2 gene.57 In the NZB model, Ifi202 has been implicated as the responsible candidate gene within the Nba2 locus on chromosome 1.58 Besides these reports, the culprit genes responsible for the other lupus susceptibility loci still remain elusive. Congenic strains have also served as an excellent approach to investigate how various susceptibility loci may interact to mediate lupus development in terms of the cellular and molecular cascades involved. Thus, for example, whereas Sle2 appears to be responsible for the B-cell hyperactivity and the expanded B1 cells noted in lupus, Sle3 appears to be responsible for the hyperactive and proinflammatory antigen-presenting cells including dendritic cells (DCs) and macrophages.45,59-61 In contrast, Sle1 appears to be a gene that is very important in breaching B-cell and T-cell tolerance to self-antigens. However, the congenic dissection strategy still has its limitations. Although it is very powerful

CONGENIC MURINE LUPUS MODELS PROVIDE UNIQUE TOOLS FOR DISSECTING LUPUS PATHOGENESIS

TABLE 17.2 CONGENIC MOUSE MODELS FOR LUPUS

for segregating the disease susceptibility loci, it is much less so for further separating multiple disease genes that may be located within a small genomic interval. Identifying the phenotypic effect of each individual candidate gene within the small interval requires expensive genetic manipulation to generate engineered mouse models carrying individual candidate genes on a resistant-strain background in order to ascertain each gene’s contribution to disease. Nevertheless, this appears to be the most promising approach currently available to lupus researchers.

ENGINEERED MODELS CONSTITUTE POWERFUL TOOLS FOR STUDYING POTENTIAL ROLE OF INDIVIDUAL GENES IN LUPUS PATHOGENESIS Studies of various genes using the knockout technology have revealed that deficiency in many proteins can

165

WHAT DO MOUSE MODELS TEACH US ABOUT HUMAN SYSTEMIC LUPUS ERYTHEMATOSUS?

lead to pathology similar to SLE (Table 17.3). These proteins have diverse functions, and the occurrence of lupus-like disease cannot always be predicted from their known functions at the outset of the studies. The implicated molecules thus far have been in various pathways that regulate the immune response. One class of proteins implicated in lupus mediates the clearance of apoptotic cells. This clearance may be essential for removing potential autoantigens that might otherwise serve as triggers of ANA production. These include, among others, serum amyloid P component (SAP), Dnase I, C1q, and cmer. Among them, SAP specifically binds chromatin of apoptotic cells and nuclear debris, displaces H1 histones, and solubilizes native chromatin.62 Dnase I removes DNA from soluble or deposited autoantigenic nucleoprotein complexes.63 C1q also participates in the clearance of apoptotic cells.64 Disruption of the genes for these proteins apparently results in the impaired clearance of apoptotic cells. Consequently, the availability of excess autoantigens increases and apparently immune tolerance

TABLE 17.3 ENGINEERED MOUSE MODELS FOR LUPUS Chromosome Location

Clinical Phenotypesa

Gene Name

Genetic Background

Bcl2 (transgene)

(C57BL/6 x SJL), BALB/c

ANA, GN, lymphosplenomegaly

75, 76

BlyS (BAFF, transgene)

C57BL/6

ANA, GN, lymphosplenomegaly

77-79

C1q (−/−)

129 × C57BL/6

4

C4 (−/−)

129 × C57BL/6

17

CD22 (−/−)

129 × C57BL/6

7

ANA,

102

CD45 (E613R)

129 × C57BL/6

1

ANA, GN, lymphosplenomegaly

103

CTLA-4 (−/−)

C57BL/6

1

Lymphoproliferative syndrome

104

DNase I (−/−)

129 × C57BL/6

FcγRIIB (−/−)

C57BL/6

Fyn (−/−)

129 × C57BL/6

Gadd45a (−/−) Lyn (−/−) p21 (−/−)

129 × C57BL/6

PD-1 (−/−)

C57BL/6

1

ANA, GN, lymphosplenomegaly

110

PECAM-1(CD31) (−/−)

C57BL/6

11

ANA, GN

111

PKCδ (−/−)

C57BL/6

14

ANA, GN, lymphosplenomegaly

112, 113

PTEN

+/−

16

References

ANA, GN,

64

ANA, GN, lymphosplenomegaly

101

ANA, GN

63

ANA, GN, lymphosplenomegaly

105

10

ANA, GN

106

C57BL/6

6

ANA, GN

C57BL/6

4

ANA, GN, lymphosplenomegaly

107-109

17

ANA, GN, lymphosplenomegaly

80

1

81

C57BL6 x 129

19

ANA, GN, lymphosplenomegaly

114

Sap (−/−)

129 x C57BL/6

1

ANA, GN

62

TSAd (−/−)

C57BL/6

3

ANA, GN, lymphosplenomegaly

74

TGF-β (−/−)

C57BL/6

7

ANA, GN, massive inflammation

115-118

a

166

cannot be effectively maintained. Among these genes, C1q deficiency has been found to be associated with severe SLE in humans.65-68 Deficiencies in C2 and C4 also predispose to SLE.66,69-71 There is only one report on two SLE patients that show decreased DNAse-1 activity.72 Thus far, there have been no reports of deficiency of c-mer in human SLE. Similarly, C-reactive protein (CRP) appears to be genetically defective in some patients with SLE.73 Proteins in a second group control B- and T-cell activation and proliferation. Most of these proteins are involved in cell signaling. It can be expected that disruption of any of the molecules crucial in negative feedback regulation of lymphocyte signaling could potentially lead to uncontrolled lymphocyte activation and possibly autoimmunity. The molecules in this category include Lyn, Fyn, CD22, TGF-beta, CTLA-4, PD-1, FcγRIIB, Pten (+/−), PECAM-1/CD31, PKCδ, and so on (Table 17.3). Genetic manipulations that promote lymphocyte activation and proliferation also

The clinical phenotypes shown here have been documented in the literature. The extent to which a comprehensive search for all possible phenotypes was conducted varied widely from study to study. ANA, antinuclear autoantibodies; GN, glomerulonephritis.

when the targeted gene lies within a susceptibility interval, such as within distal chromosomes 1. Moreover, the knockout approach cannot provide much insight into elucidating the phenotypic consequences of subtle polymorphisms in candidate genes. In this regard, an ideal model would express the particular allele of the polymorphic gene on a resistant genetic background in order to help researchers fathom how subtle sequence differences can influence gene function.

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lead to autoimmunity. T-cell–specific adapter protein (TSAd) has been implicated in regulating IL-2 production and T-cell apoptosis; the disruption of this gene results in defective T-cell death and development of systemic autoimmunity.74 Bcl2 and BLyS promote the survival of lymphocytes, and their overexpression also leads to the development of lupus-like autoimmune phenotypes.75-79 Disruption of molecules that are involved in cell-cycle checkpoints, such as Gadd45α and P21, can also precipitate autoimmunity.80,81 Among these genes, the association of specific genetic polymorphisms in PD-1, FcγRIIB, and BLyS with human SLE has been documented.82-84 Gene knockout technology represents a very powerful tool for studying the roles of individual genes in the development of autoimmunity. However, when interpreting results from models generated by this approach, limitations must be recognized. First, most of these knockout mouse models have been generated on the 129-strain background, and accumulating evidence indicates that this genetic background possesses several autoimmunity-promoting loci.85 It is even more critical

CONCLUSION It is clear that all three types of mouse models are immensely instructive concerning the genetic origins, component pathogenic mechanisms, and contributing cells and molecules that act in concert to engender lupus. Using these leads, researchers can then ask if human lupus is also dictated by similar genes and molecules. Given the rapid pace of progress in genomics, transcriptomics, and proteomics, a large number of translational studies are likely in the near future, as the laboratory mouse helps us unravel the mysteries of lupus.

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113. Miyamoto A, K Nakayama, H Imaki, S Hirose, Y Jiang, M Abe, et al. Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cdelta. Nature 2002;416:865-869. 114. Di Cristofano A, P Kotsi, YF Peng, C Cordon-Cardo, KB Elkon, PP Pandolfi. Impaired Fas response and autoimmunity in Pten+/- mice. Science 1999;285:2122-2125. 115. Gorelik L, RA Flavell. Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 2000;12:171-181. 116. Kulkarni AB, CG Huh, D Becker, A Geiser, M Lyght, KC Flanders, et al. Transforming growth factor beta 1 null mutation in mice

causes excessive inflammatory response and early death. Proc Natl Acad Sci U S A 1993;90:770-774. 117. Shull MM, I Ormsby, AB Kier, S Pawlowski, RJ Diebold, M Yin, et al. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 1992;359:693-699. 118. Yaswen L, AB Kulkarni, T Fredrickson, B Mittleman, R Schiffman, S Payne, et al. Autoimmune manifestations in the transforming growth factor-beta 1 knockout mouse. Blood 1996;87:1439-1445.

PATHOGENESIS

18

Genes and Genetics of Murine Lupus Shozo Izui, MD

INTRODUCTION Systemic lupus erythematosus (SLE) is a disorder of systemic autoimmunity characterized by the formation of a variety of autoantibodies and subsequent development of immune complex (IC) glomerulonephritis, that is, lupus nephritis.1 The pathogenesis of SLE is a complex process in which major histocompatibility complex (MHC)–linked and multiple non–MHC-linked genetic factors contribute to the overall susceptibility and progression of the disease, along with contributions of hormonal and environmental factors. The involvement of genetic factors in SLE was initially suspected because of a familial tendency for SLE, leading to the concept of a special genetic background necessary for contracting SLE. However, in monozygotic twins the concordance rate was only between 30 to 50%, thus indicating that both genetic and environmental factors are critically involved in the pathogenesis of SLE. Because of the complex nature of the disease, a sophisticated genetic analysis of SLE is only possible by using animal models with well-defined backgrounds. Thus, the availability of several murine strains with distinct genetic backgrounds, such as (NZB x NZW)F1, MRL, and BXSB, which all spontaneously develop an autoimmune syndrome resembling human SLE, has offered an invaluable opportunity for elucidating the genetic basis underlying the etiopathogenesis of SLE. Indeed, genome-wide linkage analyses involving lupus-prone and nonautoimmune strains have helped identify multiple lupus susceptibility loci. Current efforts are focused on evaluation of candidate genes located within these susceptibility loci by analyzing autoimmune phenotypes in mice congenic for different susceptibility intervals.

MURINE MODELS OF SLE Mice of the (NZB x NZW)F1 hybrid strain, and MRL and BXSB strains have been extensively used as experimental models of human SLE.2 They are characterized

by a wide spectrum of autoimmune manifestations culminating in the development of IC-mediated lupus nephritis. The severity of kidney lesions is closely associated with the increase in serum titers of IgG autoantibodies directed against various nuclear antigens, such as DNA and chromatin, and of circulating retroviral gp70-anti-gp70 IC (gp70 IC). The NZB (H2d) and NZW (H2z) strains were developed in New Zealand from a murine stock of undefined background by selection on black and white color, respectively. NZB mice develop autoimmune hemolytic anemia, but neither NZB nor NZW mice develop a typical lupus-like syndrome. In contrast, (NZB x NZW)F1 hybrid mice develop a severe autoimmune disease resembling human SLE, which affects the females earlier than the males, and sex hormones have been shown to be responsible for the early development of disease in the females. These F1 mice have been used as the classical animal model of SLE. The MRL strain (H2k) is derived from a series of crosses involving four strains (LG/J, AKR/J, C3H/Di, and C57BL/6). The spontaneous and recessive lpr (lymphoproliferation) mutation in the MRL strain results in a generalized lymphadenopathy due to massive accumulation of a unique subset of T cells (TCRαβ+, CD4−, CD8−, B220+).3 The lpr mutation consists of an insertion of an endogenous retrovirus in the Fas gene that codes for a receptor implicated in apoptosis of lymphocytes.4 The presence of the Faslpr mutation markedly accelerates the progression of SLE-like autoimmune syndrome in MRL mice. Besides the SLE-like autoimmune syndrome, MRL-Faslpr mice produce high titers of IgM and IgG rheumatoid factors and develop arthritis-like joint lesions resembling human rheumatoid arthritis.5 The BXSB mouse (H2b) is a recombinant inbred strain derived from a cross between a C57BL/6 (B6) female and an SB/Le male. These mice spontaneously develop an SLE-like disease that affects male animals much earlier than females. The male-determined accelerated disease is independent of sex hormones, but due to the presence of an as yet unidentified mutant gene,

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GENES AND GENETICS OF MURINE LUPUS

1

Fcgr2b, SALM/CD2, Ifi202 Cr2

Nba2, Sle1, Lbw7, Bxs3

4

7 Sle3 Nba5, Lbw5, Lmb3

Cd22 Adnz1 Sle2 Nba1, Lbw2, Imh1, Sgp4

C1qa

13 Rsl

17 Sgp3, Bxs6

H2, Tnfa

Fig. 18.1 A schematic representation of five murine autosomes illustrating the locations of the major lupus susceptibility loci and selected candidate genes. The number of each chromosome is given above its centromere.

autoantibodies, development of lupus nephritis and production of nephritogenic gp70 autoantigen. Results from genome-wide linkage analyses in mice obtained through by intercrosses or back-crosses of different lupus-prone and nonautoimmune strains led to the identification of multiple autoimmune susceptibility regions distributed all over the murine genome.10-12 These analyses have shown that (1) lupus-like disease is controlled by sets of susceptibility loci that independently or additively contribute to the overall susceptibility and progression of the disease, (2) heterogeneous combinations of multiple disease-promoting genes operate in a threshold-dependent manner to achieve full expression of the disease, and (3) contributions are unlikely to be linked to “true” genetic mutations, but are rather due to polymorphic alleles with subtle functional differences, except for the Fas and Yaa mutations observed in MRL and BXSB mice, respectively.

SPONTANEOUS MUTATIONS PREDISPOSING TO SLE IN LUPUS MICE

Fas and Fas Ligand Gene designated Yaa (Y-linked autoimmune acceleration), present in the Y chromosome of BXSB mice, which is originally inherited from the SB/Le strain.6

MULTIGENIC FEATURES OF MURINE SLE

172

Since the development of an SLE-like syndrome was first reported in the F1 progeny of the NZB and NZW strains, the genetic basis for SLE in (NZB × NZW)F1 hybrids has been investigated in a number of laboratories. Since the major features of (NZB × NZW)F1 autoimmune disease are not present in the parental strains, it is clear that genes from each parent act in concert to produce the F1 phenotype. Early genetic studies on New Zealand mice have demonstrated that many individual autoimmune traits segregate independently of each other in (NZB × NZW)F1 × NZW back-cross mice,7 in (NZB × NZW)F2 mice8 and in recombinant inbred strains derived from the NZB strain through crosses with non-autoimmune strains of mice.9 This suggests that there is no common genetic defect causing overall autoimmune responses, but rather that each of the autoimmune traits is under the control of a separate genetic mechanism. Classic progeny studies have provided only limited information on the number, identity and chromosomal location of the lupus susceptibility genes. However, the availability of polymorphic microsatellite markers covering the entire mouse genome has permitted to map more precisely the genetic loci linked with a wide spectrum of autoimmune traits, that is, production of

The identification of defects in Fas mapped to chromosome 19, which is involved in apoptosis, in lupusprone MRL mice with the lpr phenotype, represented an important contribution to our understanding of the genetic basis of SLE.4 This mutation produces a massive enlargement of lymph nodes with the accumulation of a particular subset of T cells that are phenotypically TCRαβ+, CD4−, CD8−, but express the B220 molecule characteristic of B cells.3 Notably, the gld (generalized lymphoproliferative disease) mutation, discovered in a colony of the C3H/HeJ strain, induces marked lymphadenopathy phenotypically indistinguishable from that induced by the lpr mutation.13 As a matter of fact, gld was identified as a mutation of the gene encoding the Fas ligand (FasL), present in chromosome 1.14 These Faslpr and Faslgld mutations not only accelerate the progression of autoimmune disease in lupus-prone MRL mice, but also induce the production of a broad spectrum of autoantibodies in various strains of mice, including those not predisposed to SLE.2,15,16 Fas is highly expressed in activated B and T cells, while the expression of FasL is limited to activated T cells.3 However, the Fas apoptosis pathway does not appear to be essential for negative selection during T and B cell development in thymus and bone marrow, respectively.17,18 Therefore, it has been speculated that the abnormal regulation of the Fas apoptotic pathway could result in prevention of antigen-induced apoptotic death of autoreactive lymphocytes in the periphery, thereby promoting the development of lupus-like autoimmune responses. However, it should be stressed that the mutation of Fas or FasL alone is not sufficient

Yaa Mutation In contrast to the accelerated development of SLE in (NZB x NZW)F1 female mice, male BXSB mice develop disease much more rapidly than their female counterparts.2 This striking sexual dimorphism is not hormonally mediated, but results from a mutant gene, Yaa, present in the Y chromosome of the BXSB strain.25-27 The contribution of the Yaa mutation to lupus susceptibility remains limited without other background genes, since nonautoimmune strains, such as CBA/J and B6, were largely unaffected by the Yaa mutation. Notably, when B6.Yaa consomic males are mated with NZW females, which are phenotypically normal but have a genetic potential to develop SLE, F1 hybrid males bearing the Yaa mutation develop typical SLE.26 In addition, studies in B6 or C57BL/10 (B10) mice carrying different lupus susceptibility loci derived from either NZB, NZW or BXSB mice have shown that the combination of a single lupus susceptibility locus with Yaa can be sufficient to induce the development of lupus-like autoimmune syndrome, although the severity of the disease was variable, depending on the individual lupus susceptibility locus studied.28-30 These results indicate that the Yaa mutation by itself is unable to promote SLE in mice which are not predisposed to autoimmune diseases, but in combination with autosomal susceptibility alleles present in different lupus-prone strains, it can induce or accelerate the development of SLE. It is clear that the molecular characterization of Yaa would give valuable information on the general

mechanisms implicated in the development of SLE. Unfortunately, it is impossible to map this locus by conventional genetics because of the lack of homologous recombination of the Y chromosome. In an attempt to identify the cell types which express the Yaa mutation, we have produced Yaa plus non-Yaa double-bone-marrow chimeric mice, and analyzed the origin of autoantibodies produced in these mice. This analysis revealed that only B cells of Yaa origin participated in the production of IgG anti-DNA autoantibodies, and that they maintained this production even after selective depletion of T cells of Yaa origin.31,32 Thus, the Yaa abnormality is functionally expressed in B cells, but not in T cells. Based on this finding, it has been hypothesized that the action of Yaa may be to decrease the threshold for BCR-dependent stimulation, thereby promoting the activation of autoreactive B cells.33 This hypothesis is in agreement with the selective autoimmune enhancing effect of the Yaa mutation: the Yaa effect appears to be essential for the promotion of autoimmune responses in mice having only a limited activity of T-helper cells specific for a given autoantigen.34 It has been reported that lupus-like autoimmunity could be detected in genetically engineered mice lacking or overexpressing molecules implicated in the regulation of BCR signaling, which indicates that the deregulation of BCR signaling may be a critical element in the triggering and activation of potentially autoreactive B cells. According to this model, the Yaa mutation would have a direct enhancing effect on BCR signaling and thereby trigger the cascade of events initiating SLE. A unique cellular abnormality associated with the Yaa mutation is monocytosis.35 At 8 months of age, monocytes reached a frequency of more than 50% of peripheral blood mononuclear cells in BXSB Yaa male mice. The development of monocytosis was closely associated with the progression of SLE, since monocytosis was observed in (NZB × B6.Yaa)F1 male mice developing SLE, but not in B6.Yaa males, which fail to develop a lupus-like autoimmune syndrome.36 Furthermore, recent analysis of B6 × (NZB × B6.Yaa)F1 back-cross males bearing the Yaa mutation revealed a remarkable correlation of monocytosis with autoantibody production and subsequent development of lupus nephritis,37 indicating that monocytosis is a useful and predictive marker for severe SLE. Significantly, Yaa-mediated monocytosis resulted in selective expansion of a monocyte subset expressing the CD11c dendritic cell marker,36 which is therefore considered to be a potential source of tissue-resident dendritic cells. Thus, the association of monocytosis with the development of SLE could be explained by a possible expansion of dendritic cells, which would further promote the production of pathogenic autoantibodies. In addition, given the considerable role of infiltrating

SPONTANEOUS MUTATIONS PREDISPOSING TO SLE IN LUPUS MICE

to induce severe autoimmune disease in mice that are not predisposed to SLE,13,15,16 underlining the importance of other still undefined lupus susceptibility background genes in the development of full-blown SLE. Although Fas mutations have been identified in children with a rare autoimmune lymphoproliferative syndrome (ALPS),19,20 this was not really the case in SLE patients.21 Nevertheless, studies in mice bearing the lpr or gld mutation clearly indicate the importance of the genes regulating apoptosis in the development of SLE. It can be speculated that one of the defects in lupusprone mice may be the failure to efficiently eliminate autoreactive B cells upon interaction with autoantigens. In fact, the resistance of mature B cells to B-cell receptor (BCR)-mediated apoptosis has been reported in lupus-prone (NZB × NZW)F1 mice.22 This concept is consistent with the findings that transgenic overexpression of the antiapoptotic proto-oncogene bcl-2 in B cells led to spontaneous development of an SLE-like autoimmune syndrome in certain strains of mice,23 and that the constitutive expression of the bcl-2 gene is able to counteract the apoptotic death of autoreactive B cells upon interaction with autoantigens in the periphery.24

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GENES AND GENETICS OF MURINE LUPUS

macrophages in the progression of glomerular lesions, and given the implication of stimulatory IgG Fc receptor (FcγR) in murine lupus nephritis,38 monocytosis could promote glomerular inflammation and injury through increased secretion of proinflammatory cytokines, reactive oxygen species, and matrix-specific proteases, possibly as a result of IC-mediated, FcγRdependent activation of infiltrating macrophages. Although the precise cellular and molecular basis of the Yaa gene-linked development of monocytosis has not been defined, the analysis of Yaa plus non-Yaa mixedbone-marrow chimeras showed no overrepresentation of monocytes of Yaa origin over those of non-Yaa origin, indicating that the development of monocytosis is not due to an intrinsic abnormality in the growth potential of monocyte lineage cells from Yaa mice.36 Rather, Yaa-mediated monocytosis may result from an excessive production of monocyte-specific growth factor(s)—for example, by activated macrophages— possibly due to hyper-responsiveness of FcγR to autoimmune IC that arise during the course of the disease.

MHC ASSOCIATION OF MURINE SLE

Association of H2d/z Heterozygosity with Murine SLE

174

Extensive studies in New Zealand mice have demonstrated a strong association of H2d/z heterozygosity (vs. H2d/d or H2z/z) with the development of SLE, indicating a co-dominant contribution from each strain, i.e., H2d from NZB and H2z from NZW.39,40 However, it is still unknown how this H2 heterozygosity mechanistically contributes to murine SLE. It has been proposed that mixed haplotype class-II molecules produced by heterozygous pairing of an α-chain from one haplotype with a β-chain from the other haplotype might play a critical role in the development of SLE. However, the lack of disease enhancement by an Abz transgene introduced into H2d homozygous (NZB × NZW.H2d)F1 mice41 and by Ez or Az transgene in (B6 × NZB)F1 × NZB back-cross mice42,43 argue against this possibility. Significantly, a comparative serologic analysis of two different nephritogenic anti-DNA and antigp70 autoantibody productions in (NZB × NZW)F1 × NZW and (NZB × NZW)F1 × NZB back-cross mice revealed that in the F1 × NZW back-cross, H2d/z (compared with H2z/z) was associated preferentially with the production of anti-gp70 rather than anti-DNA autoantibodies, whereas the opposite influence was noted for H2d/z (compared with H2d/d) in the F1 × NZB backcross.44 These results suggest that enhancement of disease by H2d/z heterozygosity is related to separate contributions from H2d and H2z, thus providing one explanation as to why H2d/z heterozygosity is required for full expression of disease in (NZB × NZW)F1 mice.

Association of H2b with Murine SLE Another contribution of the MHC to the regulation of murine SLE, which is different from that seen in (NZB × NZW)F1 hybrid mice, has been well documented in BXSB and (NZB × BXSB)F1 mice, in which lupus susceptibility was more closely linked with the H2b haplotype than with the H2d and H2k haplotypes.45-47 However, this MHC effect was limited, as it was markedly influenced by other factors in the genetic background of individual lupus-prone mice. In the context of the BXSB background, in which the development of SLE is dependent on the Yaa mutation, the H2d or H2k haplotype almost completely prevented the development of autoimmune responses occurring in H2b-bearing conventional BXSB mice. In contrast, (NZB × BXSB)F1 female hybrids homozygous for H2d still developed typical SLE, although its development was markedly delayed as compared with mice homozygous for H2b. This indicates that the genetic complementation of NZB and BXSB genomes allows the development of spontaneous autoimmune responses in the context of H2d, even without the Yaa mutation. More strikingly, the Yaa mutation dramatically accelerated the progression of SLE in (NZB × BXSB)F1 H2d mice to an extent comparable with that observed in F1 H2b mice. Thus, no more MHC association was evident in these F1 hybrid males when they expressed the Yaa gene. Notably, similar results were observed in mice bearing the Faslpr mutation; the production of autoantibodies in B6 mice bearing the Faslpr mutation was highly dependent on H2b,48 while lupus-like disease was developed equally well in both H2k and H2b lupus-prone MRL-Faslpr mice.49 All these experiments indicate that the MHC class-II genes likely provide at least some of the genetic requirements for the predisposition to SLE, and that conventional MHC class-II molecules are sufficient in mice with an appropriate autoimmune genetic background. Most significantly, the MHC-linked autoimmune promoting effect is no longer apparent in mice which are highly predisposed to SLE, for example by powerful autoimmune accelerating genes, such as Yaa or Faslpr. The autoimmune inhibitory effect of the H2d and H2k haplotypes, as compared with H2b, can be related at least in part to the expression of I-E molecules, since mice bearing the H2b haplotype do not express I-E because of the deletion of the promoter region of the Ea gene. The development of SLE was almost completely prevented in BXSB (H2b) mice expressing two copies of an Ea transgene encoding I-E α-chains, which is the case of H2d and H2k BXSB mice. In addition, the expression of two functional Ea (one transgenic and the other endogenous) genes in either H2d/b (NZB × BXSB)F1 or H2k/b (MRL × BXSB)F1 mice provided protection from SLE at levels

claimed the presence of a lupus suppressor gene, Sles1 (SLE suppressor 1), within the H2 region of the NZW strain,57 although it remains to be confirmed whether the observed suppressive effect was due to this novel suppressor gene and not to MHC region polymorphisms. In addition, the Tnfa allele of the NZW strain, which is associated with down-regulated TNF-α synthesis, has been previously proposed as a candidate gene that may underlie the H2z contribution to lupus in (NZB × NZW)F1 mice.58,59

NON–MHC-LINKED LUPUS SUSCEPTIBILITY LOCI The results obtained with more than 20 genome-wide linkage analyses have helped to identify a number of non–MHC-linked lupus susceptibility loci, associated with autoantibody production and/or lupus nephritis and scattered all over the murine genome. However, it is important to note that several major loci identified in independent studies are co-localized in essentially identical chromosomal regions in different lupusprone mice. Among them, four non-MHC regions have been more extensively studied: Nba2 (New Zealand black autoimmunity 2); Sle1 (systemic lupus erythematosus 1), Lbw7 (lupus-NZB × NZW 7) and Bxs3 (BXSB 3) on chromosome 1; Nba1, Sle2, Lbw2, Imh1 (IgM hyper 1), Adnz1 (Anti-dsDNA antibody in NZM2328 locus 1), and Sgp4 (Serum gp70 production 4) on chromosome 4; Sle3, Nba5, Lbw5, and Lmb3 (lupus in [MRL-Faslpr × B6-Faslpr]F2 cross 3) on chromosome 7; and Sgp3 and Bxs6 on chromosome 13 (Table 18.1). Although they have not yet been well characterized, additional susceptibility loci have been mapped on other chromosomes, and some of them are apparently strain-specific. This implies that different clusters of genes confer lupus susceptibility in different strains of mice, though some loci are likely to be common to several murine models of SLE.

NON–MHC-LINKED LUPUS SUSCEPTIBILITY LOCI

comparable to those conferred by the H2d/d or H2k/k haplotype.47 These results suggest that the reduced susceptibility associated with the I-E+ H2d and H2k haplotypes (vs. the I-E− H2b haplotype) is largely, if not exclusively, contributed by the Ea gene. This idea is further supported by the recent demonstration that (NZB × NZW)F1 mice expressing I-Ad but lacking I-E molecules developed SLE as severe as that of wild-type H2d/z heterozygous (NZB × NZW)F1 mice.50 However, it should be stressed that since H2d/z (NZB × NZW)F1 mice express I-E, the unique autoimmune-promoting effect conferred by the H2d/z heterozygosity apparently overcomes the protective effect of I-E in this genetic background, as in the case of (NZB × BXSB)F1 mice expressing the Yaa mutation and MRL mice bearing the Faslpr mutation. The precise mechanism(s) responsible for the Ea gene-mediated protection from SLE remains to be elucidated. Studies of Ea transgenic and nontransgenic mixed-bone-marrow chimeras revealed that these chimeric mice developed a typical lupus-like autoimmune syndrome, in which anti-DNA autoantibody production was preferentially induced by nontransgenic B cells.51,52 These results suggested that B cells are the major target of Ea-mediated suppression of autoimmune responses, and that Ea gene expression may interfere with an efficient interaction between autoreactive T and B cells, possibly by modulating the presentation of pathogenic self-peptides by MHC class II molecules. This could occur as a result of increased formation of peptides derived from the I-E α-chains. In fact, one of the peptides, Eα52-68 peptide, has been identified as one of the major self-peptides presented by I-A molecules.53,54 The hypothesis that the protective action of the Ea gene is mediated through binding of its degradation products by MHC class-II molecules, thereby competing with potentially pathogenic self-peptides, was further supported by the demonstration that the protective effect of the Ea transgene was highly dependent on the host H2 haplotype.55 This idea was consistent with recent in vitro demonstration that the capacity of Ea transgenic B cells to activate T-helper cells by presenting I-A–restricted peptides of foreign antigens was substantially diminished, compared with that of nontransgenic B cells.56 The MHC class-II Ea gene apparently contributes to the reduced susceptibility to SLE by suppressing autoimmune responses in mice, but its protective effect is influenced by the host H2 haplotype. However, the Ea gene is not the only gene encoded within the MHC region that determines the genetic susceptibility to murine SLE, and the MHC region likely encodes additional lupus-associated genes, which can potentiate or suppress the development of SLE by acting at various levels of the disease process. Studies have

Lupus Susceptibility Loci Mapped to Chromosome 1 An NZB locus, Nba2, was initially mapped to the distal region of chromosome 1 by an analysis of (NZB × SM/J)F1 × NZB back-cross mice.12 Since this locus was found to be linked with the production of various autoantibodies, including anti-DNA, antichromatin, and anti-gp70, it apparently controls overall autoantibody production in SLE, and thereby the development of lupus nephritis.30,60 Nba2 is likely to be identical to Lbw7 of NZB origin, which was revealed by analysis of (NZB × NZW)F2 mice.11 The Sle1 locus, derived from the NZW strain, overlaps with the same region on chromosome 1, and was also linked to autoantibody production and lupus nephritis.10,61 B6 mice congenic

175

GENES AND GENETICS OF MURINE LUPUS

TABLE 18.1 MAJOR LUPUS SUSCEPTIBILITY LOCI IN MURINE SLE Chromosome

Locus

Origin

Locationa

Traitsb

1

Nba2 Lbw7 Sle1 Bxs3

NZB NZB NZW BXSB

153-193 157-184 145-195 150-189

ANA, Anti-gp70, LN ANA, LN ANA, LN ANA

4

Nba1 Lbw2 Imh1 Sgp4 Sle2 Adnz1

NZB NZB NZB NZB, NZW NZW NZW

122-140 128-134 133-140 133-150 60-123 74-109

LN LN IgM gp70 LN ANA

7

Sle3 Lbw5 Nba5 Lmb3

NZW NZW NZB MRL

10-75 25-66 20-73 20-73

ANA, LN LN Anti-gp70, LN ANA, LN

13

Sgp3 Bxs6

NZB, NZW, MRL BXSB

50-68 44-88

gp70, anti-gp70, LN gp70, anti-gp70, LN

17

H2

NZB/NZW, BXSB

31-36

ANA, Anti-gp70, LN

a

Approximate locations of lupus susceptibility loci indicated as Mb (megabases) from the centromere. ANA, antinuclear autoantibodies (including anti-DNA); LN, lupus nephritis.

b

176

for the Nba2 or Sle1 interval developed elevated titers of anti-DNA and antichromatin autoantibodies, but failed to develop lupus nephritis, while these congenic mice are able to develop severe lupus nephritis in the presence of the Yaa mutation.28,30 Cellular studies in B6.Sle1 congenic mice have claimed that Sle1 alone was associated with selective loss of tolerance to nuclear autoantigens.62 Significantly, the analysis of subinterval congenic mice carrying different portions of the Sle1 locus has revealed that three nonoverlapping loci within Sle1, termed Sle1a, Sle1b, and Sle1c, can independently cause loss of self tolerance.63 This indicates that Sle1 is represented by a cluster of functionally related lupus susceptibility genes. In addition to Nba2, Lbw7, and Sle1, a recent genetic analysis involving the BXSB and B10 strains has identified a lupus-susceptibility interval situated in chromosome 1, designated Bxs3, which overlaps directly with Nba2, Lbw7, and Sle1.64 Although the contribution of additional loci located in more centromeric region of BXSB chromosome 1 has been described in this analysis, studies in B10 congenic mice bearing each of the BXSB-derived susceptibility intervals confirmed that the Bxs3 locus provided a major contribution to spontaneous production of autoantibodies and subsequent development of lupus nephritis.29 Notably, the region of human chromosome 1 syntenic with Nba2/Lbw7/ Sle1/Bxs3 (1q41-q42) has been shown to be associated with SLE in humans.65,66 Thus, this region of

chromosome 1 is critical for the development of murine and human SLE. Analysis of sequence polymorphism has suggested that the Nba2/Lbw7/Sle1/Bxs3 interval likely contains several lupus susceptibility genes. The first important candidate gene is Fcgr2b encoding the inhibitory type II FcγR (FcγRIIB). The presence of promoter region polymorphism has been shown to result in defective expression of FcγRIIB in activated B cells in germinal centers of NZB mice.67,68 Since FcγRIIB is an inhibitory receptor containing an immunoreceptor tyrosinebased inhibitory motif (ITIM), its co-ligation to the BCR through IgG-containing IC prevents the activation of BCR signaling.69 FcγRIIB thus sets thresholds for the IC-mediated activation of B cells. However, FcγRIIB may play a limited role in the initial triggering of autoreactive B cells, since the major source of autoantigens implicated in SLE is unlikely to be in a form of IC. Therefore, the defective expression of FcγRIIB in B cells from lupus-prone mice may rather be involved in promoting sustained production of IgG autoantibodies through an enhanced activation of autoreactive B cells after the interaction with autoimmune IC. Furthermore, we have recently observed much lower levels of FcγRIIB expression on macrophages bearing the NZB-type Fcgr2b allele than on those bearing the B6-type allele.37 Since activating FcγR apparently plays a critical role in the development of lupus nephritis,38 the defective FcγRIIB

Lupus Susceptibility Loci Mapped to Chromosome 4 The Nba1 and Lbw2 loci, which are likely to be identical, were mapped to the mid-distal region of NZB chromosome 4 and identified as loci contributing to

the development of lupus nephritis, but not to the production of IgG antinuclear autoantibodies.11,77,78 Their contribution to lupus nephritis was further confirmed by the analysis of NZW.Nba1 congenic mice (Jørgensen TN and Kotzin BL, personal communication, 2006) and of (NZB × NZW)F1 mice with or without the NZB-derived Lbw2 locus.79 Notably, these loci overlap with Imh1 of NZB origin, which was found to be linked with IgM production.80 Another locus, Sle2 of NZW origin, was linked with lupus nephritis and mapped to a region more proximal.10 Significantly, B6.Sle2 congenic mice had increased serum levels of IgM in parallel to elevated number of B1 cells, which are known to be the major source of serum IgM.61,81 It is somehow difficult to understand how increased IgM production without the production of pathogenic IgG autoantibodies could promote the development of lupus nephritis. Thus, it is more likely that the Nba1/Lbw2 interval contains additional lupus-promoting gene(s) other than that regulating IgM production. In this regard, it is worth noting that we have identified a locus, designated Sgp4, in the distal region of the NZB chromosome 4 overlapping with the Nba1 locus, which was linked to the production of nephritogenic gp70 antigens.82,83 Therefore, the association of lupus nephritis with Nba1/Lbw2 could in part be a consequence of increased production of nephritogenic gp70 autoantigens, as in the case of the contribution of Sgp3 locus in chromosome 13 to lupus nephritis (see below). Additionally, the Nba1/Lbw2 genetic contribution may be operating distal to autoantibody production by affecting IC localization or inflammatory responses to deposited IC. In this regard, it is worth mentioning that the Nba1/Lbw2 interval contains the C1qa gene encoding the first component of complement C1q. Significantly, it has been shown that an insertion polymorphism in the NZB sequence upstream of C1qa appears to down-regulate the serum levels of C1q.84 This could result in an impairment of IC clearance, thereby promoting the deposition of IC and hence the development of lupus nephritis. It should also be stressed that C1q-deficient mice are able to develop lupus-like autoimmune syndrome in association with an accumulation of apoptotic bodies in tissues.85 Further studies revealed that C1q played a substantial role for the clearance of apoptotic bodies.86 Thus, it has been speculated that the failure of efficient elimination of apoptotic bodies in C1q-deficient mice may favor the development of autoimmune responses against nuclear antigens characteristic for SLE. This notion was further supported by the findings that other mice deficient in molecules implicated in clearance of apoptotic bodies developed antinuclear autoantibodies and that such a mutation also enhanced autoantibody production in lupus-prone mice.87,88

NON–MHC-LINKED LUPUS SUSCEPTIBILITY LOCI

expression in lupus-prone NZB, BXSB, and MRL mice, which all bear the NZB-type Fcgr2b allele,67,68 could additionally contribute to the effector phase of ICmediated lupus nephritis due to excessive activation of FcγR-bearing effector cells. Most significantly, it has recently been observed that the development of SLE was markedly prevented as a result of partial restoration of FcγRIIB levels70 or of congenic expression of the B6-type Fcgr2b allele on B cells in BXSB mice (Lin Q and colleagues, 2006, submitted for publication). These results, together with the finding that partial FcγRIIB deficiency, that is, heterozygous level of FcγRIIB expression, is sufficient to induce the production of autoantibodies and the development of lupus nephritis in B6 mice by the presence of the Yaa mutation,71 strongly support the contribution of Fcgr2b polymorphism to the development of lupus-like autoimmune syndrome. Second, lupus susceptibility has been shown to be associated with extensive polymorphisms of the signaling lymphocyte activation molecule (SLAM)/CD2 gene family (Cd244, Cd229, Cs1, Cd48, Cd150, Ly108, and Cd84), as all lupus-prone mice share the same SLAM haplotype, which is different from that of B6 mice.72 Since these genes encode cell surface molecules that play a role in the modulation of cellular activation and signaling in the immune system, they are also good candidates for promoting lupus-like autoimmune responses. Among the SLAM/CD2 gene family, the strongest candidate seems to be Ly108, the expression of which appears to be constitutively upregulated in B and T cells from B6.Sle1 congenic mice. Third, the Ifi202 (interferon-inducible p202) gene could also be implicated in SLE, since its expression is markedly increased in NZB mice, as compared with B6 mice.60 Given a remarkable protection from autoimmune syndrome in NZB mice deficient in the interferon (IFN)-α/β receptor,73 and given the role of type-I IFN in the differentiation of monocytes to immunostimulatory dendritic cells,74 it has been suggested that type-I IFN could play a critical role in the development of SLE. This idea is based on the hypothesis that uncontrolled, excessive activation of dendritic cells could divert self-antigen presentation from tolerance induction to autoimmunity. Finally, since the Cr2 gene of the NZW strain encodes the less functional complement receptor 2 (CR2),75 the NZW-type Cr2 allele could be an additional candidate allele of the Sle1 interval, in view of the possible role of CR2 in the induction of central B-cell tolerance in the bone marrow.76

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In an additional study using NZM2328 mice, one of the recombinant inbred NZM (New Zealand mixed) strains derived from a cross of NZB female and NZW male,89 NZB-derived locus, Adnz1, on the mid chromosome 4 was defined, which contributed to the production of anti-DNA autoantibodies, but not to lupus nephritis.90 Strikingly, NZM2328 mice bearing the B6type Adnz1 interval failed to produce antinuclear autoantibodies, but still developed severe lupus nephritis in kinetics comparable to those seen in wildtype NZM2328 mice.91 This raises a question concerning the pathogenic role of anti-DNA autoantibodies in lupus nephritis. In fact, it has repeatedly been shown that the development of murine lupus nephritis was associated with an increased production of retroviral gp70 IC much more than anti-DNA autoantibodies.6,8,27,78,82,92 In addition, one might also consider the involvement of other IC systems. For example, it has been claimed that anti-C1q autoantibodies were associated with the presence of human lupus nephritis, amplifying glomerular injury in SLE.93 Whatever the genetic mechanisms may be, the striking difference in autoimmune phenotypes conferred by Sle2 vs. Adnz1, both of which are derived from the same chromosomal region of NZW mice, further illustrate the complexity of lupus susceptibility.

Lupus Susceptibility Loci Mapped to Chromosome 7

178

The centromeric region of chromosome 7 contains lupus susceptibility genes regulating autoantibodies and lupus nephritis, which are the Sle3 and Lbw5 loci derived from the NZW strain,10,11 the Nba5 locus from the NZB strain,30 and the Lmb3 locus from the MRL strain.94 The contribution of these loci to murine SLE has been confirmed by the analysis of Sle3 or Nba5 congenic B6 mice30,61 and of Lmb3-congenic MRL mice (Santiago-Raber ML, Kono DH, Theofilopoulos AN, personal communication, 2006). One possible candidate gene present in this region is Cd22, which codes for a B–cell-restricted adhesion molecule that recognizes α2,6-linked sialic acid– bearing glycans and functions as a negative regulator of BCR signaling.95 The analysis of B6 × (NZW × B6.Yaa)F1 back-cross males has provided evidence that an NZW locus peaking at Cd22a was strongly linked with autoantibody production and lupus nephritis. 96 A link between dysregulated CD22 expression and lupus-like autoimmune disease has also been suggested by the findings that mice with a disrupted Cd22 gene developed increased serum titers of IgG anti-DNA autoantibodies97 and that partial CD22 deficiency, i.e., heterozygous level of CD22 expression, in B6 mice can result in an induction of IgG anti-DNA autoantibody production in the presence

of the Yaa mutation.98 Significantly, NZW and NZB mice carry the defective Cd22a allele: CD22 expression on Cd22a B cells is lower at steady state and less upregulated following B-cell activation than that of Cd22b B cells.98,99 It is also worth mentioning that B cells derived from BXSB mice bearing the Cd22c allele100 displayed the same defect as Cd22a B cells.98 Since CD22 functions primarily as a negative regulator of BCR-mediated signal transduction, a limited up-regulation of CD22 in activated B cells may have significant functional consequences in B-cell responses. In addition, Cd22a and Cd22c B cells appear to express aberrant forms of CD22, differing in the N-terminal sequences constituting the ligand-binding site, due to the synthesis of abnormally processed Cd22 mRNA as a result of the insertion of a short interspersed nucleotide element in the second intron.98 We have recently observed that CD22a molecules were less efficient in the binding to CD22 ligand (CD22L) than their CD22b counterparts and that Cd22a B cells displayed a phenotype reminiscent of constitutively activated B cells.99 In view of the importance of the CD22–CD22L interaction in the regulation of B-cell activation,101 these data support the idea that the expression of defective CD22a and CD22c could contribute to enhanced B-cell activation, and thus favor the development of autoimmune responses in combination with other susceptibility alleles present in lupus-prone mice. This notion is also supported by the finding that the spontaneous production of lupus autoantibodies could be induced in mice deficient in BCR negative regulators, such as Lyn kinase, SHP-1 phosphatase, FcγRIIB, and in mice overexpressing a positive BCR regulator, such as CD19, in which their B cells become abnormally hyper-responsive to antigenic stimulation. It should be stressed that unlike Sle3 and Lmb3, Nba5 was unable to promote the production of anti-DNA and antichromatin autoantibodies, and that its autoimmune-promoting effect was selective for nephritogenic gp70 autoantigens.30 Notably, the Nba5 locus, peaking at the D7Nds5 marker, is located more than 10 cM distal to Cd22. Thus, the candidate gene for Nba5 is most likely to be different from those that promote general hyperresponsiveness of B and/or T cells to enhance overall autoimmune responses. Since B6.Nba5 congenic mice did not have higher levels of serum gp70, it is improbable that this locus is implicated in overall production of serum gp70. However, it is possible that the Nba5 locus could regulate the expression of a subpopulation(s) of gp70 that is critically involved in anti-gp70 autoantibody responses and gp70 IC formation, given the possible heterogeneity of serum gp70 proteins.

Additional genes involved in the pathogenesis of SLE are those encoding nephritogenic autoantigens or regulating their expression. One of these autoantigens, which plays an important role in the development of murine lupus nephritis, is the endogenous retroviral envelope glycoprotein gp70.102 This is illustrated by the fact that the gp70 antigen is found in circulating IC and glomerular immune deposits within diseased kidneys of lupus mice.103 gp70 IC become apparent in the circulation close to the onset of disease, and their concentrations rise with the progression of lupus nephritis,6,8,27,78,82,92 thereby providing good evidence that gp70 IC are implicated in renal injury of lupus-prone mice. Interval mapping of back-cross progeny between lupus-prone mice (NZB, NZW, BXSB, and MRL) and B6 or B10 mice identified a major locus controlling gp70 production on the middle of chromosome 13, designated Sgp3 or Bxs630,92,104 (Kono DJ, Izui S, Theofilopoulos AN, unpublished data, 2006); the Bxs6 locus identified in BXSB mice is likely to be identical to Sgp3. In addition, we observed a significant linkage of the Sgp3 locus with anti-gp70 production and lupus nephritis, but not with anti-DNA production.30,96 Analysis in B6 or B10 mice congenic for the Sgp3 or Bxs6 locus derived from either the NZB, NZW or BXSB strain revealed that all three congenic mice had approximately 10-fold higher levels of gp70, as compared with B6 or B10 mice30,104 (Rankin J, Haywood MEK, Izui S, Morley BJ, unpublished data, 2006), suggesting that the underlying allele of Sgp3 is shared among these three different lupus-prone mice. However, serum concentrations of gp70 in Sgp3 congenic mice bearing the NZB- or NZW-derived Sgp3 locus were still lower than those seen in NZB and NZW mice, indicating the presence of other loci controlling the production of serum gp70. We have recently identified a second locus regulating the production of gp70 on distal chromosome 4 of both NZB and NZW mice.82,83 The presence of this locus, designated Sgp4, has been confirmed by the analysis of B6 mice bearing this NZB interval (Jørgensen TN, Kotzin BL, Izui S, unpublished data, 2006). These data indicate that Sgp3 and Sgp4 are critically involved in the regulation of gp70 production, and contribute to the development of lupus nephritis by favoring antigenic stimulation and gp70 IC formation. Notably, serum gp70 is secreted by liver cells and behaves as an acute phase protein; this acute phase response is also under genetic control, but different from that regulating the production of basal levels of gp70.104,105 It could be possible that with the onset of SLE and the corresponding systemic inflammation, the production of gp70 is boosted, thereby further accelerating lupus nephritis. It is worth noting that the Gv1

(Gross virus antigen 1) gene, which overlaps with the Sgp3 locus, controls the expression of thymic GIX gp70 antigen,106 that is closely correlated to serum levels of gp70.105 As Gv1 likely regulates in trans the expression of multiple endogenous retroviral transcripts in different tissues, including the liver,106,107 it is reasonable to assume that Gv1 and Sgp3 are identical or related genes regulating the expression of endogenous retrovirus. A recent study has identified two Rsl (regulator of sex limitation) genes in this region, Rsl1 and Rsl2, which encode KRAB (Krüppel-associated box) zinc-finger proteins and control male-dependent up-regulated gene expression in liver.108 In addition to the two identified Rsl genes, there exist in this region more than 20 Rsl candidate genes, the function of which has not yet been identified. Since serum gp70 is synthesized at higher levels in liver of male mice than female mice, it is possible that the expression of retroviral serum gp70 is regulated by one of the Rsl genes.

CONCLUDING REMARKS

Lupus Susceptibility Loci Mapped to Chromosome 13

CONCLUDING REMARKS Genetic analysis of SLE in several lupus-prone mice revealed that multiple, unlinked genes are responsible for the expression of various autoimmune manifestations, and that several, quite distinct genetic backgrounds are compatible with the development of this disease. Apparently, individual lupus-prone strains of mice have quite distinct genetic defects, but end up with similar immunopathologic abnormalities responsible for the development of lupus-like autoimmune disease. Although the nature of these genetic components has not been completely defined, it is becoming clear that certain classes of genes play a crucial role in the development of murine SLE, and that they can be classified into three categories (Box 18.1). The first group includes the genes, the products of which are implicated in the regulation of the production of nephritogenic serum gp70 antigens or in the pathways of waste disposal in the body. The former likely encodes a trans-activating factor that regulates the expression of endogenous retroviral gp70 in liver, and an example for the latter is the C1q protein, which helps to clear apoptotic cells. The second group of genes, which can confer susceptibility to SLE, encodes proteins that regulate the thresholds for activation of autoreactive B or T cells, such as FcγRIIB, CD22, SLAM/CD2, MHC class II (and the Yaa gene product). The genes encoding Fas, FasL, and CR2 can also be classified in this category, since these proteins can regulate the induction of tolerance in autoreactive B and T cells. Genes in the third category code for proteins that act in the effector phase of IC-mediated lupus nephritis. For example, C1q promotes clearance of circulating IC, and FcγRIIB modulates the IC-dependent activation of proinflammatory FcγR-bearing effector cells.

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BOX 18-1 MOLECULAR DEFECTS IMPLICATED IN MURINE SLE I. Proteins regulating expression and clearance of nephritogenic autoantigens Trans-activating factors regulating the production of serum gp70 in liver (Sgp3 and Sgp4) First component of complement, C1q (C1qa), involved in clearance of apoptotic bodies II. a. Proteins regulating thresholds for activation of autoreactive B and T cells FcγRIIB (Fcgr2b): BCR negative regulator CD22 (Cd22): BCR negative regulator Family of signaling lymphocyte activation molecules (SLAM/CD2) MHC class II (H2d/z, H2b) Yaa gene product II. b. Proteins regulating induction of tolerance of autoreactive B and T cells Fas apoptosis receptor and its ligand (Faslpr and Faslgld) Complement receptor 2 (Cr2) III. Proteins regulating clearance of IC and activation of inflammatory effector cells C1q (C1qa), involved in clearance of IC FcγRIIB (Fcgr2b): negative regulator of activating FcR on immune effector cells

Because different genetic components are likely to be involved at various levels of SLE disease progression, the absence of genetic abnormalities at a certain level of the disease process may well explain why mice without an appropriate SLE background fail to develop

full-blown SLE. Therefore, the variations of the onset and severity of SLE observed among various lupusprone mice could be interpreted as the result of different assortments of various genetic defects implicated in SLE. Obviously, further identification of the genetic defects critically involved in the development of murine SLE and of their interaction is of paramount importance for the understanding of the mechanism of human SLE, and is indeed a subject of extensive and active investigation. Concerning the Yaa mutation, it has recently been shown that Yaa-bearing mice express two copies of the TLR7 gene as a result of translocation from the telomeric end of X chromosome onto the Y chromosome (Pisitkun P, et al. Science 2006;312:1669; Subramanian S, et al. Proc Natl Acad Sci U S A 2006;103:9970). This suggests that the Yaa-induced acceleration of lupus-like diseases may be in part due to increased TLR7 signaling in B cells. However, further studies are awaited to define the precise role of the TLR7 gene duplication in the accelarated development of SLE in the context of the Yaa mutation.

ACKNOWLEDGMENTS I thank Dr. Thomas Moll for his critical reading of the manuscript. The studies from my laboratory discussed in this review were supported by the Swiss National Foundation for Scientific Research.

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67. Jiang Y, Hirose S, Sanokawa-Akakura R, et al. Genetically determined aberrant down-regulation of FcγRIIB1 in germinal center B cells associated with hyper-IgG and IgG autoantibodies in murine systemic lupus erythematosus. Int Immunol 1999;11:1685. 68. Pritchard NR, Cutler AJ, Uribe S, et al. Autoimmune-prone mice share a promoter haplotype associated with reduced expression and function of the Fc receptor FcγRII. Curr Biol 2000;10:227. 69. Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol 2001;19:275. 70. McGaha TL, Sorrentino B, Ravetch JV. Restoration of tolerance in lupus by targeted inhibitory receptor expression. Science 2005;307:590. 71. Moll T, Nitschke L, Carroll M, et al. A critical role for FcγRIIB in the induction of rheumatoid factors. J Immunol 2004;173:4724. 72. Wandstrat AE, Nguyen C, Limaye N, et al. Association of extensive polymorphisms in the SLAM/CD2 gene cluster with murine lupus. Immunity 2004;21:769. 73. Santiago-Raber ML, Baccala R, Haraldsson KM, et al. Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice. J Exp Med 2003;197:777. 74. Blanco P, Palucka AK, Gill M, et al. Induction of dendritic cell differentiation by IFN-α in systemic lupus erythematosus. Science 2001;294:1540. 75. Boackle SA, Holers VM, Chen X, et al. Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein. Immunity 2001;15:775. 76. Prodeus AP, Georg S, Shen LM, et al. A critical role for complement in maintenance of self-tolerance. Immunity 1998;9:721. 77. Drake CG, Babcock SK, Palmer E, et al. Genetic analysis of the NZB contribution to lupus-like autoimmune disease in (NZB x NZW)F1 mice. Proc Natl Acad Sci U S A 1994;91:4062. 78. Vyse TJ, Drake CG, Rozzo SJ, et al. Genetic linkage of IgG autoantibody production in relation to lupus nephritis in New Zealand hybrid mice. J Clin Invest 1996;98:1762. 79. Haraldsson MK, dela Paz NG, Kuan JG, et al. Autoimmune alterations induced by the New Zealand Black Lbw2 locus in BWF1 mice. J Immunol 2005;174:5065. 80. Hirose S, Tsurui H, Nishimura H, et al. Mapping of a gene for hypergammaglobulinemia to the distal region on chromosome 4 in NZB mice and its contribution to systemic lupus erythematosus in (NZB x NZW)F1 mice. Int Immunol 1994;6:1857. 81. Mohan C, Morel L, Yang P, et al. Genetic dissection of systemic lupus erythematosus pathogenesis: Sle2 on murine chromosome 4 leads to B cell hyperactivity. J Immunol 1997; 159:454. 82. Tucker RM, Vyse TJ, Rozzo S, et al. Genetic control of gp70 autoantigen production and its influence on immune complex levels and nephritis in murine lupus. J Immunol 2000; 165:1665. 83. Rigby RJ, Rozzo SJ, Gill H, et al. A novel locus regulates both retroviral glycoprotein 70 and anti-glycoprotein 70 antibody production in New Zealand mice when crossed with BALB/c. J Immunol 2004;172:5078. 84. Miura-Shimura Y, Nakamura K, Ohtsuji M, et al. C1q regulatory region polymorphism down-regulating murine C1q protein levels with linkage to lupus nephritis. J Immunol 2002;169:1334. 85. Botto M, Dell’Agnola C, Bygrave AE, et al. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 1998;19:56. 86. Taylor PR, Carugati A, Fadok VA, et al. A hierarchial role for complement pathway complement proteins in the clearance of apoptotic cells in vivo. J Exp Med 2000;192:359. 87. Scott RS, McMahon EJ, Pop SM, et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 2001;411:207. 88. Cohen PL, Caricchio R, Abraham V, et al. Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase. J Exp Med 2002; 196:135.

89. Rudofsky UH, Evans BD, Balaban SL, et al. Differences in expressison of lupus nephritis in New Zealand Mixed H-2z homozygous inbred strains of mice derived from New Zealand Black and New Zealand White mice: origins and initial characterization. Lab Invest 1993;68:419. 90. Waters ST, Fu SM, Gaskin F, et al. NZM2328: a new mouse model of systemic lupus erythematosus with unique genetic susceptibility loci. Clin Immunol 2001;100:372. 91. Waters ST, McDuffie M, Bagavant H, et al. Breaking tolerance to double stranded DNA, nucleosome, and other nuclear antigens is not required for the pathogenesis of lupus glomerulonephritis. J Exp Med 2004;199:255. 92. Haywood MEK, Vyse TJ, McDermott A, et al. Autoantigen glycoprotein 70 expression is regulated by a single locus, which acts as a checkpoint for pathogenic anti-glycoprotein 70 autoantibody production and hence for the corresponding development of severe nephritis, in lupus-prone BXSB mice. J Immunol 2001;167:1728. 93. Coremans IE, Spronk PE, Bootsma H, et al. Changes in antibodies to C1q predict renal relapses in systemic lupus erythematosus. Am J Kidney Dis 1995;26:595. 94. Vidal S, Kono DH, Theofilopoulos AN. Loci predisposing to autoimmunity in MRL-Faslpr and C57BL/6-Faslpr mice. J Clin Invest 1998;101:696. 95. Nitschke L, Tsubata L. Molecular interactions regulate BCR signal inhibition by CD22 and CD72. Trends Immunol 2004; 25:543. 96. Santiago ML, Mary C, Parzy D, et al. Linkage of a major quantitative trait locus to Yaa gene-induced lupus-like nephritis in (NZW x C57BL/6)F1 mice. Eur J Immunol 1998;28:4257. 97. O’Keefe TL, Williams GT, Davies SL, et al. Hyperresponsive B cells in CD22-deficient mice. Science 1996;274:798. 98. Mary C, Laporte C, Parzy D, et al. Dysregulated expression of the Cd22 gene as a result of a short interspersed nucleotide element insertion in Cd22a lupus-prone mice. J Immunol 2000;165:2987. 99. Nitschke L, Lajaunias F, Moll T, et al. Expression of aberrant forms of CD22 in murine B lymphocytes in Cd22a lupus-prone mice affects ligand-binding. Int Immunol 2006;18:59. 100. Lajaunias F, Ibnou-Zekri N, Fossati-Jimack L, et al. Polymorphisms in the Cd22 gene of inbred mouse strains. Immunogenetics 1999;49:991. 101. Poe JC, Fujimoto Y, Hasegawa M, et al. CD22 regulates B lymphocyte function in vivo through both ligand-dependent and ligand-independent mechanisms. Nat Immunol 2004;5:1078. 102. Yoshiki T, Mellors RC, Strand M, et al. The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J Exp Med 1974;140:1011. 103. Izui S, McConahey PJ, Theofilopoulos AN, et al. Association of circulating retroviral gp70-anti-gp70 immune complexes with murine systemic lupus erythematosus. J Exp Med 1979; 149:1099. 104. Laporte C, Ballester B, Mary C, et al. The Sgp3 locus on mouse chromosome 13 regulates nephritogenic gp70 autoantigen and predisposes to autoimmunity. J Immunol 2003; 171:3872. 105. Hara I, Izui S, Dixon FJ. Murine serum glycoprotein gp70 behaves as an acute phase reactant. J Exp Med 1982;155:345. 106. Oliver PL, Stoye JP. Genetic analysis of Gv1, a gene controlling transcription of endogenous murine polytropic proviruses. J Virol 1999;73:8227. 107. Levy DE, Lerner RA, Wilson MC. The Gv-1 locus coordinately regulates the expression of multiple endogenous murine retroviruses. Cell 1985;41:289. 108. Krebs CJ, Larkins LK, Price R, et al. Regulator of sex-limitation (Rsl) encodes a pair of KRAB zinc-finger genes that control sexually dimorphic liver gene expression. Genes Dev 2003; 17:2664.

PATHOGENESIS

19

Complement Deficiencies in Human Systemic Lupus Erythematosus (SLE) and SLE Nephritis: Epidemiology and Pathogenesis C. Yung Yu, DPhil, Georges Hauptmann, MD, PhD, Yan Yang, MD, PhD, Yee Ling Wu, BS, BA, Dan J. Birmingham, PhD, Brad H. Rovin, MD, and Lee A. Hebert, MD

SUMMARY Human SLE is triggered by multiple genetic and environmental risk factors, with each factor having a modest effect (low penetrance) on the disease pathogenesis. However, subjects with homozygous or total deficiency of complement component C1q or C4 are highly likely to have SLE or lupus-like disease. Other hereditary complement deficiencies associated with SLE include deficiencies of C1r, C1s, C2, mannan binding lectin (MBL), and C1 inhibitor. SLE patients with a primary deficiency of a complement protein tend to have severe cutaneous diseases with photosensitive skin rash, high titers of anti-Ro/SSA but low levels of antinuclear antibodies (ANA) and antidsDNA. A general feature of complement-deficient SLE patients is recurrent or invasive bacterial infections. The incidence of a primary complement deficiency in SLE is low. However, homozygous and heterozygous isotype deficiencies of complement C4A are very common (which may be present in about 40% of Caucasian SLE patients). Some ethnic groups have a deficiency of C4B instead. Acquired deficiencies of C1q, C3, C4, and erythrocyte complement receptor CR1 appear to be useful biomarkers for SLE disease activity (particularly of renal disease). Hypocomplementemia in SLE can be caused by immunecomplex (IC)-mediated complement activation/consumption or by the presence of autoantibodies against a specific complement protein. The complement system (when properly functioning) protects against SLE and SLE nephritis by providing defense against infections that may trigger flare, facilitates clearance of apoptotic debris that can induce autoimmunity, and prevents pathologic IC accumulation

in susceptible organs. If the complement system cannot perform these protective activities, because of genetic or acquired deficiencies, IC may accumulate in the kidneys and induce nephritis. Furthermore, excessive activation of complement generates products with pro-inflammatory and profibrotic activities that may directly damage the kidneys.

INTRODUCTION The disease initially described as lupus erythematosus (LE) represents the most clinically and serologically diverse form of autoimmune disease. LE was first recognized by its visible cutaneous manifestations before the introduction of more precise clinical and biological diagnostic criteria that established the multisystem nature and hence the designation as systemic lupus erythematosus (SLE). Indeed, the spectrum of disease manifestations among SLE patients is broad, ranging from various cutaneous manifestations [subacute, acute, or chronic cutaneous (discoid) lupus erythematosus (DLE)] to life-threatening involvement of vital organs/ systems that include the kidneys, the central nervous system, the cardiovascular system, and the lungs. The involvement of complement in the etiopathogenesis of SLE was revealed when the introduction of routine measurements of the hemolytic complement activities (CH50) and component protein levels (mainly C3 and C4) showed that serum complement protein levels are usually decreased in patients with SLE. However, complement abnormalities were not included in the 1997 revised criteria of the American College of Rheumatology for the diagnosis and classification of SLE, although they are among the most

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powerful markers for SLE disease susceptibility and disease activity.1 SLE is a complex disease that involves multiple genetic and environmental risk factors that initiate the disease.2,3 It is thought that each of numerous susceptibility genes contributes modestly to an increased risk of SLE in a subject. However, in a small subset of patients a homozygous single-gene defect can be the major driving force for disease onset. A total deficiency in one of the early components of the classical complement activation pathway represents such a single genetic factor that strongly predisposes a subject to SLE.

CHARACTERISTICS OF SLE ASSOCIATED WITH COMPLEMENT DEFICIENCY The earliest indication suggesting that human subjects with complement deficiencies are predisposed to immunopathogenic diseases came from reports in 1971 to 1974. In many cases of SLE, lupus-like syndromes, or glomerulonephritis patients were found to have genetic deficiencies of C1q, C1r, C1s, C4, C2, or C1inhibitor (Table 19.1). At present such deficiencies are the strongest disease susceptibility genes for the development of human SLE.4,5 It appears that the association of complement deficiency with SLE shows a hierarchy of prevalence and disease severity according to the position of the protein in the activation pathway.6 The highest frequency and the most severe disease is associated with a deficiency in one of the proteins of the C1 complex (C1q, C1r, and C1s) or with a total deficiency of complement C4 (i.e., C4A and C4B). Seventy-five to 90% of human subjects with a homozygous deficiency of C1 or C4 have SLE or lupus-like disease. Remarkably, the intrafamilial disease concordance rate for SLE in combination with C1 or C4 deficiency even exceeds that observed between siblings of monozygotic twins (24 to 69%). The disease associated with hereditary complement deficiency tends to be of early onset, and (unlike the

high female preponderance among the majority of SLE patients) the female-to-male ratio is approximately 1:1.7 By contrast, complement C2 deficiency (C2D) is associated with a lower prevalence of disease (estimated at approximately 10%) and a disease onset similar to regular SLE patients with a female predominance.8 In addition to a tendency to develop SLE, individuals with a homozygous deficiency of C1q/C1r/C1s, C4, or C2 often have a higher susceptibility to recurrent or invasive bacterial infections. SLE associated with homozygous complement deficiencies is characterized by a predominance of cutaneous manifestations, especially discoid lupus erythematosus (DLE) or subacute cutaneous LE with marked photosensitivity. It is marked by low titers or the absence of antinuclear antibodies (ANA) and antibodies to native DNA, but a frequent occurrence of anti-Ro/SSA antibodies.

DEFICIENCIES OF SUBCOMPONENT PROTEINS FOR THE C1 COMPLEX Deficiency in any of the three subcomponents of the C1 complex will lead to a loss of activation of the classical pathway.

C1q Deficiency The human A, B, and C genes of C1Q are closely linked on chromosome 1p34 through 1p36. Hereditary C1q deficiency is caused either by a failure to synthesize C1q (~60% of cases) or by the synthesis of a nonfunctional low-molecular-weight (LMW) C1q (~40%). Coding mutations have been identified that lead to the formation of a premature termination codon at amino acid residues 6 or 41, or frame-shift mutations from codon 43 together with a stop codon at residue 108 of the C-chain, a stop codon at residue 150 of the B-chain, and a stop codon at residue 186 of the A-chain.9-11 The vast majority of known human subjects with homozygous C1q deficiency (39 of 42, 93%) have

TABLE 19.1 SLE AND OTHER SYMPTOMS IN TOTAL DEFICIENCIES OF COMPLEMENT COMPONENTS OF THE CLASSICAL PATHWAY OF ACTIVATION* Components

184

Number of Cases

SLE and Other Disease Associations

C1q

42

SLE (30); GN (16); CNS disease (7); recurrent bacterial infections (13); with septicemia in early childhood (4); moniliasis (9)

C1r, C1s

14

Lupus-like syndrome (4); SLE (2) DLE (2); GN (2); infections (8); otitis media, gonococcal infection, tuberculosis, post-varicella encephalitis, virus-associated hemophagocytic syndrome

C4

28

SLE (17); SLE-like disease (5) GN (6); infections (7); dead (4); healthy (1)

C2

>150

SLE & SLE-like disease (34%); increased susceptibility to infections (25–30%); glomerulonephritis (~10%); cardiovascular disease (~15%)

CNS, central nervous system; DLE, discoid lupus erythematosus; GN, glomerulonephritis. *Number of cases in parenthesis.

C1QD

B

C4D

C

C2D

Fig. 19.1 Cutaneous lesions and pathology associated with complement-deficient SLE. LE- like syndrome and cutaneous infection in a male child with homozygous C1q deficiency (A, upper) and lesions of discoid LE with scarring lesions on the face in the same patient at the age of 22 years after 6 years of treatment with thalidomide 11 (A, lower). LE syndrome and osteomyelitis in a child with total C4 deficiency (B, upper) and butterfly rash and cheilitis at the age of 3 years and osteomyelitis of the femur at the age of 10 years (B, lower). The patient died at the age of 12 years from pulmonary infection and cardiovascular failure. (C) Anti-Ro/SS-A+ acute cutaneous LE in a young women with homozygous type I C2D (upper, typical butterfly rash; lower, photosensitive lesions on sun-exposed area).

developed a clinical syndrome related to SLE (Fig. 19.1) with skin rash (36 subjects, 86%), glomerulonephritis (GN; 16 subjects, 38%), and central nervous system (CNS) involvement (7 subjects, 17%). The disease is present equally in males and females and is typically of early onset, with a median age of 6 years (range: 6 months to 42 years). The prevalence of autoantibodies is slightly lower than that of regular SLE patients: ANA 24/34 (70.6%), ENA (extractable nuclear antigens Sm, RNP, Ro and/or La) 15/24 (62.5%), and anti-dsDNA 5/24 (20.8%). At least one-third of the C1q-deficient patients also suffered from recurrent bacterial infections, including otitis media, meningitides, and pneumonia. Four C1q-deficient patients died with septicemia in early childhood. Some patients developed diffuse monilia and aphtous lesions in the mouth and toenail deformity secondary to moniliasis9 (panel A, Fig. 19.1).

Low Levels of C1q Proteins In addition to coding mutations reported in patients with complete deficiency of C1q, a silent single-nucleotide polymorphism at codon 70 (GGG–>GGA) of the C1QA gene was found to be associated with decreased levels of C1q in patients with subacute cutaneous lupus erythematosus (SCLE).10 The cause for such a reduced level of C1q is not known. Hereditary C1q deficiencies should be distinguished from acquired C1q hypocomplementemia in SLE

DEFICIENCIES OF SUBCOMPONENT PROTEINS FOR THE C1 COMPLEX

A

patients or other autoimmune conditions, including hypocomplementary urticarial vasculitis syndrome (HUVS), cryoglobulinemia, and severe combined immunodeficiency syndromes. These are due to an increased protein consumption, especially under conditions related to the presence of autoantibodies against C1q. C1q autoantibodies are present in approximately one-third of SLE patients, who often have renal disease and higher clinical disease activities. 9,12,13

C1r and C1s Deficiencies The genes for C1r and C1s are in close linkage on chromosome 12p13. Typically, those individuals who have no C1r protein may also have reduced levels of C1s (∼20 to 40% of normal level).7 Deficiencies of C1r and C1s have been reported in 14 cases so far. Among them, 12 developed a lupus-like disease with skin rash, discoid lupus, or SLE. A majority of patients presented also had severe bacterial or viral infections. The female-to-male ratio was 1.7:1. Relatively lower prevalence of ANA (62.5%) was found. Molecular defects leading to C1s deficiency had been studied in three patients. Among them were two nonsense mutations in exon 12 at codon 53414 or codon 60815 and a 4-bp deletion in exon 10 that created frameshift mutations and formation of a premature stop codon.15,16 The molecular basis of C1r deficiency has not been determined.

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186

TOTAL AND ISOTYPE DEFICIENCIES OF COMPLEMENT COMPONENT C4

Total Deficiencies of C4A and C4B A remarkable feature of human complement C4 genetics is the variation in the gene copy number and gene size. One to five copies of long (21 kb) or short (14.6 kb) C4 genes can be present at the central region of the MHC on chromosome 6p21.3.17 To date, 2 to 7 copies of C4 genes in a diploid genome have been demonstrated to be frequently present among different healthy subjects. Each of those C4 genes may code for an acidic C4A protein or a basic C4B protein. The high copy number of C4 genes probably evolved to increase the diversity and reduce the possibility of a total deficiency of C4A and C4B proteins in an individual. To date, 28 individuals from 19 families with a complete deficiency of both C4A and C4B proteins have been firmly established.4,18,19 Among these C4D subjects, 17 were diagnosed with SLE according to the ACR criteria. Of the remaining 11 subjects, 5 had lupus-like disorders such as photosensitive skin lesions and/or discoid lupus and 6 had kidney disease such as mesangioproliferative glomerulonephritis, recurrent hematuria, membranous nephropathy, and Henoch-Schöenlein purpura with end-stage kidney failure. Repeated and invasive (and sometimes fatal) infections were reported in at least 7 individuals (bacterial meningitidis, osteomyelitis, otitis media, respiratory tract infections, and septicemia). Only one subject, age 21 at the time of report, remained relatively healthy. The age of SLE disease onset/diagnosis among C4D subjects varied from 2 to 41 years. The female-to-male ratio of affected patients was close to 1:1 (13 female/14 male). Four C4D patients died between 2 and 25 years of age. Common clinical manifestations of C4D are photosensitivity, severe skin lesions (sometimes with scarring atrophic lesions on the face and extremities), Raynaud’s phenomenon, infections, and renal disease (panel B, Fig. 19.1). Serologically, antinuclear antibodies are generally present at low titers or absent. Of special interests is the frequent presence of anti-Ro/SSA but the absence of anti-La/SSB antibodies. Anti-dsDNA antibody tests were negative in 9 of 11 patients studied.4,7,19 The molecular basis of total C4 deficiencies was elucidated in 12 Caucasian subjects with six different HLA haplotypes. The common cause of total C4 deficiency is due to the absence of a C4B gene and the presence of a single long C4A mutant gene in an MHC haplotype that has a mini-insertion or deletion (indel). There are also MHC haplotypes with two nonfunctional C4 genes in tandem. The most prevalent molecular defect leading to nonexpression of a C4 protein is a 2-bp insertion at codon 1213 in exon 29.20 Other deleterious mutations

include G–>A mutation at the donor site of intron 28 (of C4B), a 1-bp deletion at codon 497 (of C4B) or a 2-bp deletion at codon 522 (of C4A) in exon 13, and a 1-bp deletion at codon 811 of exon 20 (of C4A).18

C4A and C4B Isotype Deficiencies Gene copy number and gene size variations are the two major factors contributing to the quantitative diversity of complement C4 plasma protein levels among different individuals.21 They create a wide range of plasma protein levels for each isotype, including a homozygous lack of C4A or C4B. The initial observations in the early 1980s of an association between homozygous deficiency and partial deficiency of C4A (i.e., substantially lower expression level of C4A than C4B in a subject) and SLE22 have since been replicated and extended. Cumulative results from more than 35 different studies revealed that homozygous C4A deficiency is increased from less than 1% in the healthy populations to 3.5 to 5% in SLE patient cohorts, whereas partial or heterozygous deficiency of C4A increased from 21.7 to 24% in the healthy controls to 40.2 to 42.9% in SLE patient populations. Those study populations included Northern and Central Europeans, Anglo-Saxons, Caucasians in the United States, and East Asians. French SLE patients and controls showed relatively lower frequencies of C4AQ0, but the differences between the patient and control groups were statistically significant.18 Although C4AQ0 is significantly associated with SLE in many racial or ethnic groups, a difference in the C4BQ0 allelic frequencies between SLE patients and healthy controls is not observed in Northern and Central Europeans, African Americans, and most Orientals. However, the reverse situation was seen for Spanish, Mexican, and Australian Aborigine SLE patients, among whom a significant increase in frequency of C4BQ0 but not C4AQ0 has been demonstrated. Homozygous deficiency of C4B is associated with increased risk of IgA nephropathy and other autoimmune conditions such as insulin-dependent diabetes. Absence of at least one C4B gene that leads to low levels of plasma C4 protein has been associated with dermatological diseases, specifically DLE, angioedema, and urticaria. Such phenomena would suggest a delicate balance in the physiologic roles of C4A and C4B among different ethnic or genetic backgrounds, or differences in the influence of specific environmental factors on the requirement of C4A and C4B proteins. The clinical manifestations associated with homozygous C4A deficiency have been reported in several studies. In a cohort of 80 Swedish SLE patients, there were 13 homozygous C4A-deficient subjects, who had an increased incidence of photosensitivity but other clinical features were similar to non C4A-deficient individuals.

DEFICIENCY OF COMPLEMENT C2 C2 deficiency (C2D) is relatively common in Caucasians of European descent. Approximately 0.3 to 1.2 % of the white population in the United States and Europe is a carrier of a C2 “null” allele (C2*Q0). The association of homozygous C2D with SLE has been confirmed in more than 150 cases. SLE is thought to occur in up to 34% of C2D patients, with a female-to-male ratio of 8:1. However, actual prevalence of SLE among C2D individuals would be lower because many C2D subjects remain relatively healthy. The severity and prognosis of SLE associated with C2D is comparable to regular SLE patients. In contrast with C1q, C1r/C1s, and C4 deficiencies, SLE associated with C2D is less severe in disease activity and is exceptional before puberty. Discoid skin lesions and arthralgias are prominent, and pleuropericardial, neurologic, or renal involvement is absent or mild. Glomerular lesions in C2D patients with SLE vary in histologic types and include membranoproliferative, mesangial, membranous, and focal sclerosis. C2D patients usually have low or absent ANA titers and antibodies to dsDNA. In contrast, the prevalence of anti-Ro (SSA) antibodies in these patients is reported to be much higher than in non-C2D patients with SLE. Clinically and serologically the patients often resemble subsets of SLE such as ANA-negative SLE with photosensitivity, localized acute lupus erythematosus (ACLE) (panel C, Fig. 19.1), or SCLE. An increased susceptibility to bacterial infections may be present in up to 30% of C2D individuals, occurring mainly during infancy and childhood. These infections were related to concomitant immunoglobulin deficiencies (especially of IgG4, IgA, and IgD) or to coexistent abnormalities of the alternative pathway function. Cardiovascular diseases were noticed at a high rate in a cohort of 40 Swedish C2D patients. A total of 10 acute myocardial infarctions and 5 cerebrovascular episodes

in 6 patients were observed, indicating a possible role of C2D in the development of atherosclerosis.8,26,27 The C2 null phenotype is predominantly associated with a specific “ancestral” MHC haplotype HLA A*25, B*18, C2*Q0, BF*S, C4A*4, C4B*2, DRB1*1501 (DR2), and DQB1*0602. Such type I C2 deficiency is caused by a 28-base pair (bp) at the junction between exon 6 and intron 6.28 A less common type of C2D, referred to as the type II deficiency, is caused by missense mutations in a variety of HLA haplotypes that result in a failure to secrete the C2 protein.29,30 Whether there is an association of lupus disease with heterozygous C2 deficiency similar to that seen with partial C4 deficiency is controversial. There appears to be a slight increase in the prevalence of heterozygous C2 deficiency among SLE patients compared to controls. Compound heterozygous deficiencies of C2 and C4A or C2 and C4B remain usually unrecognized. Interestingly, 18 cases from 9 families have been reported and about 30% of them had SLE, DLE, lupus panniculitis, or another autoimmune disorder.29,31

DEFICIENCIES OF OTHER COMPLEMENT PROTEINS IN SLE

No differences were seen in the percentage of antidsDNA, Sm, RNP, Ro/SS-A, La/SS-B, rheumatoid factors, or anticardiolipin antibodies for these patients. In North American studies of Caucasian SLE, homozygous C4A-deficient patients were found to have a higher incidence of cutaneous disease but in general a milder disease course with lower incidence of renal involvement and proteinuria, lower incidence of seizures, and less common anti-dsDNA, anti-SM, anti-Ro, and anticardiolipin antibodies.23-25 On the contrary, East Asian SLE patients with homozygous C4A deficiency were associated with more severe disease activities (including higher incidence of renal disease and serositis and higher titers of anti-dsDNA) when compared with SLE patients without a homozygous C4A deficiency.

DEFICIENCIES OF OTHER COMPLEMENT PROTEINS IN SLE

Deficiency of Complement Component C3, C5, C6, C7, C8, or C9 The major disease association with homozygous deficiencies of components C3 through C9 is infection. However, cases of SLE and lupus-like disease were also found. Of the 27 reported cases with C3D, seven patients had SLE or lupus-like disease. The lupus-like patients had erythematous rash, photosensitivity, and arthralgia. Notably, six out of seven Japanese C3D patients developed lupus-like symptoms, suggesting that C3D in this ethnic group could be more susceptible to a breakdown of immunologic tolerance. Seven C3D patients had renal disorders such as proteinuria, mesangiocapillary glomerulonephritis, nephrotic syndrome, IgA nephropathy, or microhematuria.32 Only isolated cases of SLE were reported among patients with deficiencies in terminal pathway components (C5 to C9).7

Deficiency of the Mannose Binding Lectin (MBL) MBL is structurally and functionally analogous to C1q, and this has led to the hypothesis that individuals with MBL deficiency might be predisposed to the development of SLE. Five structural or promoter polymorphisms in the gene coding for MBL on chromosome 10 may result in reduced serum MBL levels and have been found to be associated with severe recurrent infections in children.33,34 Studies conducted in SLE patients of different ethnic backgrounds showed that both structural and promoter polymorphisms associated with low MBL

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COMPLEMENT DEFICIENCIES OF HUMAN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND SLE NEPHRITIS

were increased in patients with SLE. Furthermore, it was found in two studies that the combination of a C4 null allele and a dysfunctional MBL allele was more strongly associated with SLE than either allele alone.35,36

Deficiency of the C1-Inhibitor (C1-inh) Genetic and acquired forms of C1-inh deficiency are associated with angioedema. C4 and C2, the substrates of the C1 complex, are chronically depressed in most patients. This chronic depression of serum C4 and C2 levels may be similar to those seen in hereditary deficiency disorders. The incidence of SLE in individuals with hereditary C1-inh deficiency has been estimated at approximately 2%. Cases of SLE in the acquired form of C1-inh deficiency have also been reported. As with hereditary C2D and C4D, the lupus disease is characterized by a high incidence of skin rash, discoid, or atypical cutaneous lupus lesions. Tests for ANA are often negative or weakly positive. dsDNA tests negative.37

Deficiency of Erythrocyte Complement Receptor Type 1 (CR1) Humans express a complement receptor (known as CR1) on their erythrocytes that enables erythrocytes to bind complement-opsonized immune complexes (IC) in the circulation. This process aids in appropriate IC clearance, minimizing IC deposition in vulnerable tissue such as the kidney.38 Numerous studies have documented decreased levels of erythrocyte CR1 expression (detectable levels, binding function) in SLE patients. Although some of these studies have implicated genetic factors,39 acquired changes occur that account for the majority of this defect. Less certain are the mechanism and the consequence of this loss. Although commonly explained by a proteolytic removal of CR1 from the erythrocyte during removal of IC, there is evidence that acute regeneration of these levels occurs.40 This suggests that reversible conformation changes in CR1 account for some of this apparent CR1 expression deficiency. Interestingly, cycles of erythrocyte CR1 “loss” and regeneration appear to be the consequence of normal CR1 function that protects against kidney damage during an SLE flare.40

COMPLEMENT IN THE PATHOPHYSIOLOGY OF SLE AND SLE NEPHRITIS

188

Glomerular accumulation of immune complexes (IC) is the initial pathogenetic step in human SLE glomerulonephritis (GN). Indeed, the WHO classification of human SLE GN is based on the pattern of glomerular change initiated by the deposited IC.41 The connection to the complement system is the clear evidence that activation of each of its pathways [classical, alternative,

and mannose-binding lectin (MBL)] is involved in the development of the glomerular changes. However, in understanding complement’s role in SLE it is crucial to recognize that complement’s overarching role is to protect against IC-mediated injury. It is only when complement’s protective functions fail that its injurious functions take over.

Protection Against SLE Induction The complement system, properly functioning, protects against SLE induction and its exacerbations (flares). The protective action of complement takes two forms. ● Protection against infection: Interaction of the adaptive immune system with the complement and cellular components of the innate immune system are crucially important in protection against infection. The relevance to SLE is that infection may be a key environmental trigger for SLE induction or SLE reactivation (flare). For example, Epstein-Barr virus infection may induce SLE in genetically susceptible individuals. In addition, infections may inadvertently activate autoimmunity.42 As emphasized previously, the MBL pathway is an important defense against infection. Specific MBL polymorphisms and MBL deficiencies are strong and independent risk factors for SLE.36 Deficiencies or dysfunction of C1q, C3, C3, C4, factor H, factor I, and components of the terminal complement pathway are also significantly associated with infection and SLE activation, especially deficiency of the classical pathway. ● Protection against the inflammatory and immunizing effects of apoptotic cells: C1q and MBL are needed for effective clearance of apoptotic cells, which can be inflammatory and highly immunogenic, leading to induction of autoimmunity.43,44 C4 and decay-accelerating factor (DAF) are also needed to protect against autoimmunity.45,46

Protection Against Pathologic IC Accumulation When IC does form, a properly functioning complement system provides protection against pathologic IC accumulation. This protective function of complement, referred to collectively as IC processing, has three aspects.47 ● Inhibition of immune precipitation: This classical pathway function involves binding of C1q to IgM, IgG1, and IgG3 (the Ig that best activate the classical pathway), resulting in C3b and C4b intercalation into the IC matrix and disrupting the formation of large insoluble IC. Instead, smaller, less phlogistic, IC are formed that can be safely cleared without causing tissue injury.

●

Solubilization of deposited IC: This alternative pathway function involves C3b deposition within the matrix of large insoluble IC, causing disruption of Fc-Fc interactions and dissolution of the IC. This limits IC-induced tissue damages. Clearance of pathogenic IC that form in the circulation: In humans and other primates, this appears to be accomplished primarily by the erythrocyte immune complex-clearing mechanism, which is unique to humans and other primates. As discussed previously, this process of erythrocyte CR1 binding IC in the circulation appears to protect against deposition of IC in the kidney. When IC form at a rate that is too great for the clearing mechanism to handle, pathologic IC accumulation occurs (leading to multisystem injury that includes GN, as discussed in material following).

The Reno-Injurious Effects of the Complement System This regressive behavior of the complement system takes the following forms. ● Impaired IC processing: This permits excessive complement-activating effects of circulating and deposited IC. Acquired IC processing impairment can be the result of the following. a. Excessive activation and/or consumption of complement and its regulators because of an excessive IC load: This can create a vicious cycle in which the greater the IC load the greater the consumption of complement and its regulators.48 The most vulnerable complement component appears to be C3, which is the focal point of complement activation. Alternative pathway activation appears also to be critical to the induction of GN because MRL/lpr factor-Bor factor-D-deficient mice are protected from renal disease, as are mice in which C3 activation is blocked using a transgenic expression of soluble Crry (a murine C3 convertase inhibitor).49 b. Depletion or impairment of complement components or their regulators by autoantibodies: Autoantibodies relevant to SLE and its nephritis are antibodies to C1q, which are highly correlated with the presence of SLE GN.12,50 Other antibodies against complement components and its regulators that are possibly relevant to SLE are the antibodies that stabilize C3 convertases such as the C3NeF/C4 NeF and that interfere with CR1 function.50 c. Impaired production of the complement component or regulator: These mechanisms include anemia (decreased red cell CR1), malnutrition (decreased C3 production), and advanced liver disease (decreased C3 and C4 production).

●

●

●

Recruitment of other components of the innate immune system and the induction of other inflammatory systems: These mechanisms include the inflammatory effects of C3a and C5a and the chemotactic effects of C5a, much of which may be mediated by the alternative pathway amplification loop by IC. The effect of C5a, acting through its receptor, exacerbates inflammation in part by altering the ratio of the inhibitory and inflammatory FcγRs.51 Uncontrolled complement activation can broadly influence pro-fibrotic gene expression in resident kidney cells and infiltrating cells, cumulating in interstitial fibrosis.52 The inflammation induced by complement activation may induce C-reactive protein (CRP), which can activate the classical pathway and potentially contribute to tissue injury (although in some settings CRP may mitigate tissue injury, including proteinuria).53,54 Complement activation causes proteinuria, and proteinuria causes complement activation: Subepithelial IC deposits, characteristic of membranous nephropathy, require C5b-9 formation to induce proteinuria.55 The ensuing proteinuria includes all of the alternative pathway components. In addition, the kidney (or cells that infiltrate the kidney) are capable of synthesizing C2, C3, C4, and factor B.47,56,57 Complement activation in the tubular compartment by proteinuria is the major determinant of progressive renal interstitial inflammation and scarring in experimental models of renal disease.56,58 Complement activation by antiphospholipid antibodies: The pathogenicity of antiphospholipid antibodies appears to be related to their ability to activate the complement system.59 This may in part explain the common occurrence of low C3 and C4 in the antiphospholipid syndrome occurring in either SLE or non-SLE patients.60 Thrombotic glomerulopathy associated with antiphospholipid antibodies is a factor of all forms of SLE nephritis.61

COMPLEMENT IN THE PATHOPHYSIOLOGY OF SLE AND SLE NEPHRITIS

●

Diagnostic Significance of Complement Changes in SLE Nephritis SLE nephritic flares are usually associated with proportional decreases in both C3 and C4 (Table 19.2). Indeed, tight cross-sectional and serial associations of complement C4 and C3 serum or plasma protein levels have been observed in most SLE patients. Profiling of the C4 and C3 serum protein levels in a patient can reflect the fluctuation of SLE disease activities and the efficacy of medical treatment (Fig. 19.2).62 Renal SLE flares are more likely to cause low C3 and C4 than nonrenal SLE flares. In general, changes in C3 serum protein levels are diagnostically more sensitive than changes in C4.

189

SLE

C3 and C4 decreased proportionately

MPGN type 1

C3 decreased more than C4 (effect of C3 neF)

Cryoglobulinemia, Types I and III

C3 and C4 decreased proportionately

Cryoglobulinemia, Type II

C4 may be decreased more than C3

GN of chronic infection (e.g., endocarditis)

C3 and C4 decreased proportionately

Postinfectious (e.g., post-streptococcal GN)

C3 decreased much more than C4

Other conditions

a

Generally, C3 and C4 decreased proportionately

GN, glomerulonephritis; MPGN, membranoproliferative GN; NeF, nephritic factor. a Rheumatoid vasculitis, idiopathic vasculitis, repeated injection of foreign proteins, drug-induced SLE, hypersensitivity to drugs, chemotherapy for malignancy with immune-complex formation, thyroid disease and GN, jejunoileal bypass with vasculitis, B-cell lymphoproliferative disorder with immune-complex formation, cold agglutinin hemolytic anemia, catastrophic antiphospholipid syndrome.

30

L/S C4A3/C4B1 Mean [C4] = 8.3 ± 3.2 mg/dL Mean [C3] = 74.7 ± 6.8 mg/dL

25 20 15 10 5 0 09/16/01 10/21/02 11/25/03 04/04/02 05/09/03

A

200 180 160 140 120 100 80 60 40 20 0

40 Serum C4 conc. (mg/dL)

35

Serum C3 conc. (mg/dL)

40

35 30

LL/LS C4A3B1/C4A3B1 Mean [C4] = 13.5 ± 4.7 mg/dL Mean [C3] = 74.7 ± 17.2 mg/dL

25 20 15 10 5 0 01/05/98 04/19/01 08/01/04 08/28/99 12/10/02

B

Date

200 180 160 140 120 100 80 60 40 20 0

Serum C3 conc. (mg/dL)

PATIENT NO. 10

PATIENT NO. 23

Serum C4 conc. (mg/dL)

COMPLEMENT DEFICIENCIES OF HUMAN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND SLE NEPHRITIS

TABLE 19.2 RENAL DISEASES ASSOCIATED WITH HYPOCOMPLEMENTEMIA

Date

Serum C4 conc. (mg/dL)

40 35 30

LS/LS C4A3B2/C4A3B2 Mean [C4] = 19.1 ± 7.5 mg/dL Mean [C3] = 108.2 ± 23.2 mg/dL

25 20 15 10 5 0 10/26/01 06/18/03 02/07/05 08/22/02 04/13/04

C

190

200 180 160 140 120 100 80 60 40 20 0

Serum C3 conc. (mg/dL)

PATIENT NO. 16

Date

Fig. 19.2 Distinct patterns of serum complement C4 and C3 profiles in three representative SLE patients. In each panel, serum C4 concentrations are shown in solid lines (black) and serum C3 concentrations in dotted lines (grey). The X axis shows the dates of blood tests. The RCCX structures, C4A and C4B protein allotypes, the mean C4 protein concentration, and the mean C3 protein concentration of the patient are listed in each panel. Panel A shows the profile of a patient whose serum C4 concentrations were persistently low (mean C4 level: 8.3 ± 3.2 mg/dL). Panel B shows the profile of a patient whose serum C4 levels fluctuated above and below 10 mg/dL (mean C4 level: 13.5 ± 4.7 mg/dL). This particular profile is representative of SLE patients with high disease activities. Panel C shows the profile of a patient whose serum C4 levels were mostly within the normal range (mean C4 level: 19.1 ± 7.5 mg/dL).

Complement activation

Antigen

C3b, C3bi, C4b

Antibody

Erythrocyte Step 2: IC bind to erythrocyte CR1 and are shuffled through the circulation

Step 3: IC are transferred from the erythrocyte to the monocyte phagocytic system

COMPLEMENT IN THE PATHOPHYSIOLOGY OF SLE AND SLE NEPHRITIS

Step 1: IC are opsonized with complement

Fig. 19.3 The primate erythrocyte immune complex (IC) clearing mechanism. IC that formed in the circulation and activate complement become coated with covalently bound cleaved fragments of C3 and C4. Human erythrocytes, expressing CR1 with binding affinity for some of these fragments, bind and shuttle IC through the circulation until they pass through sites of fixed tissue phagocytes (such as the liver). At this point, the IC are removed from the erythrocyte by the phagocytes, and the erythrocytes return to the circulation, presumably capable of binding more circulating IC. Deficiencies in this pathway can lead to IC trapping at tissue sites such as the kidneys.38,68

With an SLE flare, it is more likely that the C3 level will fall below the normal range than the C4 level.63 This is because of the complex gene copy number and gene size variations in C4A and C4B, which lead to an overlapping range of C4 protein levels in healthy subjects and SLE patients. The low levels of C4 may be caused by low copy number of C4 genes and by complement consumption during the SLE disease process.62 This suggests that a revision of C4 protein normal range according to the C4 gene copy number is necessary (Fig. 19.3). Complement activation products can be markers for SLE activation, but they have not yet found an essential role in SLE management.47 However, the levels of split product C4d on reticulocytes, red blood cells, and platelets may become a reliable biomarker for the recent and past complement activations and SLE disease activities64,65 (a phenomenon similar to the application of glycosylation levels on

hemoglobin A1c as a marker for the control of sugar levels in diabetes).

Therapeutic Considerations There are theoretical reasons to suggest that inhibition of complement activation, at least temporarily, would be beneficial in acute severe SLE. A number of complement inhibitors are under consideration as therapeutic agents, including recombinant forms of naturally occurring complement regulators, antibodies against complement components, and peptide-based inhibitors.66 However, their role clinically is still unsettled. Controlling alternative complement activation in the renal tubular compartment is achievable with oral bicarbonate therapy to raise urine pH toward 7.4.67 This has been shown to reduce renal injury in models of proteinuric nephropathy and to reduce evidence of renal tubular compartment complement activation in proteinuric patients.47,56

191

COMPLEMENT DEFICIENCIES OF HUMAN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND SLE NEPHRITIS

192

REFERENCES 1. Petri M, Magder L. Classification criteria for systemic lupus erythematosus: A review. Lupus 2004;13:829. 2. Tsao BP. The genetics of human systemic lupus erythematosus. Trends Immunol 2003;24:595. 3. Croker JA, Kimberly RP. SLE: challenges and candidates in human disease. Trends Immunol 2005;26:580. 4. Hauptmann G, Tappeiner G, Schifferli J. Inherited deficiency of the fourth component of human complement. Immunodeficiency Rev 1988;1:3. 5. Sullivan KE. Complement deficiency and autoimmunity. Curr Opin Pediatr 1998;10:600. 6. Taylor PR, Carugati A, Fadok VA, et al. A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo. J Exp Med 2000;192:359. 7. Pickering MC, Botto M, Taylor PR, et al. Systemic lupus erythematosus, complement deficiency and apoptosis. Adv Immunol 2001;76:227. 8. Jonsson G, Truedsson L, Sturfelt G, et al. Hereditary C2 deficiency in Sweden: Frequent occurrence of invasive infection, atherosclerosis, and rheumatic disease. Medicine (Baltimore) 2005;84:23. 9. Walport MJ, Davies KA, Botto M. C1q and systemic lupus erythematosus. Immunobiology 1998;199:265. 10. Racila DM, Sontheimer CJ, Sheffield A, et al. Homozygous single nucleotide polymorphism of the complement C1QA gene is associated with decreased levels of C1q in patients with subacute cutaneous lupus erythematosus. Lupus 2003;12:124. 11. Petry F, Hauptmann G, Goetz J, et al. Molecular basis of a new type of C1q-deficiency associated with a non-functional low molecular weight (LMW) C1q: Parallels and differences to other known genetic C1q-defects. Immunopharmacology 1997;38:189. 12. Marto N, Bertolaccini ML, Calabuig E, et al. Anti-C1q antibodies in nephritis: Correlation between titres and renal disease activity and positive predictive value in systemic lupus erythematosus. Ann Rheum Dis 2005;64:444. 13. Holers VM. Anti-C1q autoantibodies amplify pathogenic complement activation in systemic lupus erythematosus. J Clin Invest 2004;114:616. 14. Dragon-Durey MA, Quartier P, Fremeaux-Bacchi V, et al. Molecular basis of a selective C1s deficiency associated with early onset multiple autoimmune diseases. J Immunol 2001; 166:7612. 15. Endo Y, Kanno K, Takahashi M, et al. Molecular basis of human complement C1s deficiency. J Immunol 1999;162:2180. 16. Inoue N, Saito T, Masuda R, et al. Selective complement C1s deficiency caused by homozygous four-base deletion in the C1s gene. Hum Genet 1998;103:415. 17. Yu CY, Chung EK, Yang Y, et al. Dancing with complement C4 and the RP-C4-CYP21-TNX (RCCX) modules of the major histocompatibility complex. Progr Nucl Acid Res Mol Biol 2003; 75:217. 18. Yang Y, Chung EK, Zhou B, et al. The intricate role of complement component C4 in human systemic lupus erythematosus. Curr Dir Autoimmun 2004;7:98. 19. Yang Y, Lhotta K, Chung EK, et al. Complete complement components C4A and C4B deficiencies in human kidney diseases and systemic lupus erythematosus. J Immunol 2004;173:2803. 20. Barba G, Rittner C, Schneider PM. Genetic basis of human complement C4A deficiency: Detection of a point mutation leading to nonexpression. J Clin Invest 1993;91:1681. 21. Yang Y, Chung EK, Zhou B, et al. Diversity in intrinsic strengths of the human complement system: Serum C4 protein concentrations correlate with C4 gene size and polygenic variations, hemolytic activities and body mass index. J Immunol 2003;171:2734. 22. Fielder AH, Walport MJ, Batchelor JR, et al. Family study of the major histocompatibility complex in patients with systemic lupus erythematosus: Importance of null alleles of C4A and C4B in determining disease susceptibility. Br Med J (Clin Res Ed) 1983;286:425. 23. Sturfelt G, Truedsson L, Johansen P, et al. Homozygous C4A deficiency in systemic lupus erythematosus: Analysis of patients from a defined population. Clin Genet 1990;38:427.

24. Welch TR, Brickman C, Bishof N, et al. The phenotype of SLE associated with complete deficiency of complement isotype C4A. J Clin Immunol 1998;18:48. 25. Petri M, Watson R, Winkelstein JA, et al. Clinical expression of systemic lupus erythematosus in patients with C4A deficiency. Medicine (Baltimore) 1993;72:236. 26. Alper CA, Xu J, Cosmopoulos K, et al. Immunoglobulin deficiencies and susceptibility to infection among homozygotes and heterozygotes for C2 deficiency. J Clin Immunol 2003;23:297. 27. Lipsker DM, Schreckenberg-Gilliot C, Uring-Lambert B, et al. Lupus erythematosus associated with genetically determined deficiency of the second component of the complement. Arch Dermatol 2000;136:1508. 28. Johnson CA, Densen P, Hurford RK Jr, et al. Type I human complement C2 deficiency: A 28-base pair gene deletion causes skipping of exon 6 during RNA splicing. J Biol Chem 1992; 267:9347. 29. Hartmann D, Fremeaux-Bacchi V, Weiss L, et al. Combined heterozygous deficiency of the classical complement pathway proteins C2 and C4. J Clin Immunol 1997;17:176. 30. Zhu ZB, Atkinson TP, Volanakis JE. A novel type II complement C2 deficiency allele in an African-American family. J Immunol 1998;161:578. 31. Boeckler P, Milea M, Meyer A, et al. The combination of complement deficiency and cigarette smoking as risk factor for cutaneous lupus erythematosus in men: A focus on combined C2/C4 deficiency. Br J Dermatol 2005;152:265. 32. Reis ES, Falcao DA, Isaac L. Clinical aspects and molecular basis of primary deficiencies of complement component C3 and its regulatory proteins factor I and factor H. Scand J Immunol 2006;63:155. 33. Nath SK, Kilpatrick J, Harley JB. Genetics of human systemic lupus erythematosus: The emerging picture. Curr Opin Immunol 2004;16:794. 34. Garred P, Madsen HO, Halberg P, et al. Mannose-binding lectin polymorphisms and susceptibility to infection in systemic lupus erythematosus. Arthritis Rheum 199;42:2145. 35. Tsutsumi A, Takahashi R, Sumida T. Mannose binding lectin: Genetics and autoimmune disease. Autoimmun Rev 2005;4:364. 36. Lee YH, Witte T, Momot T, et al. The mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: Two case-control studies and a meta-analysis. Arthritis Rheum 2005;52:3966. 37. Nettis E, Colanardi MC, Loria MP, et al. Acquired C1-inhibitor deficiency in a patient with systemic lupus erythematosus: A case report and review of the literature. Eur J Clin Invest 2005; 35:781. 38. Birmingham DJ, Hebert LA. CR1 and CR1-like: The primate immune adherence receptors. Immunol Rev 2001;180:100. 39. Wilson JG, Wong WW, Murphy EE3, et al. Deficiency of the C3b/C4b receptor (CR1) of erythrocytes in systemic lupus erythematosus: Analysis of the stability of the defect and of a restriction fragment length polymorphism of the CR1 gene. J Clin Invest 1982;69:900. 40. Birmingham DJ, Gavit KF, McCarty SM, et al. Consumption of erythrocyte CR1 (CD35) is associated with protection against systemic lupus erythematosus renal flare. Clin Exp Immunol 2006;143:274. 41. Weening JJ, D’Agati VD, Schwartz MM, et al. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 2004;65:521. 42. Zandman-Goddard G, Shoenfeld Y. Infections and SLE. Autoimmunity 2005;38:473. 43. Hebert LA, Agarwal G, Sedmak DD, et al. Proximal tubular epithelial hyperplasia in patients with chronic glomerular proteinuria. Kidney Int 2000;57:1962. 44. Seelen MA, van der Bijl EA, Trouw LA, et al. A role for mannosebinding lectin dysfunction in generation of autoantibodies in systemic lupus erythematosus. Rheumatology (Oxford) 2005;44:111. 45. Carroll MC. A protective role for innate immunity in systemic lupus erythematosus. Nat Rev Immunol 2004;4:825.

58. Sheerin NS, Sacks SH. Leaked protein and interstitial damage in the kidney: Is complement the missing link? Clin Exp Immunol 2002;130:1. 59. Shinzato MM, Bueno C, Trindade V, et al. Complement-fixing activity of anticardiolipin antibodies in patients with and without thrombosis. Lupus 2005;14:953. 60. Ramos-Casals M, Campoamor MT, Chamorro A, et al. Hypocomplementemia in systemic lupus erythematosus and primary antiphospholipid syndrome: Prevalence and clinical significance in 667 patients. Lupus 2004;13:777. 61. Frampton G, Hicks J, Cameron JS. Significance of anti-phospholipid antibodies in patients with lupus nephritis. Kidney Int 1991;39:1225. 62. Wu YL, Higgins GC, Rennebohm RM, et al. Three distinct profiles of serum complement C4 proteins in pediatric systemic lupus erythematosus (SLE) patients: Tight associations of complement C4 and C3 protein levels in SLE but not in healthy subjects. Adv Exp Med Biol 2006; 586:227. 63. Ricker DM, Hebert LA, Roche R, et al. Serum C3 levels are diagnostically more sensitive and specific for systemic lupus erythematosus activity than are serum C4 levels. Am J Kidney Dis 1991;18:678. 64. Manzi S, Navratil JS, Ruffing MJ, et al. Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus. Arthritis Rheum 2004;50:3596. 65. Liu CC, Manzi S, Kao AH, et al. Reticulocytes bearing C4d as biomarkers of disease activity for systemic lupus erythematosus. Arthritis Rheum 2005;52:3087. 66. Mollnes TE, Kirschfink M. Strategies of therapeutic complement inhibition. Mol Immunol 2006;43:107. 67. Peake PW, Pussell BA, Mackinnon B, et al. The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells. Nephrol Dial Transplant 2002;17:745. 68. Helmy KY, Katschke KJ Jr, Gorgani NN, et al. CRIg: A macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 2006;124:915.

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46. Miwa T, Maldonado MA, Zhou L, et al. Deletion of decay-accelerating factor (CD55) exacerbates autoimmune disease development in MRL/lpr mice. Am J Pathol 2002;161:1077. 47. Hebert LA, Cosio FG, Birmingham DJ. Complement and complement regulatory proteins. In Neilson E, Couser W (eds.), Immunologic Renal Diseases. Philadelphia: Lippincott Williams & Wilkins, 2001:367-393. 48. Hebert LA, Cosio FG, Birmingham DJ, et al. Experimental immune complex-mediated glomerulonephritis in the nonhuman primate. Kidney Int 1991;39:44. 49. Thurman JM, Holers VM. The central role of the alternative complement pathway in human disease. J Immunol 2006; 176:1305. 50. Trouw LA, Roos A, Daha MR. Autoantibodies to complement components. Mol Immunol 2001;38:199. 51. Ravetch JV. A full complement of receptors in immune complex diseases. J Clin Invest 2002;110:1759. 52. Bao L, Zhou J, Holers VM, et al. Excessive matrix accumulation in the kidneys of MRL/lpr lupus mice is dependent on complement activation. J Am Soc Nephrol 2003;14:2516. 53. Barnes EV, Narain S, Naranjo A, et al. High sensitivity C-reactive protein in systemic lupus erythematosus: Relation to disease activity, clinical presentation and implications for cardiovascular risk. Lupus 2005;14:576. 54. Rodriguez W, Mold C, Kataranovski M, et al. Reversal of ongoing proteinuria in autoimmune mice by treatment with C-reactive protein. Arthritis Rheum 2005;52:642. 55. Cunningham PN, Quigg RJ. Contrasting roles of complement activation and its regulation in membranous nephropathy. J Am Soc Nephrol 2005;16:1214. 56. Wilmer WA, Rovin BH, Hebert CJ, et al. Management of glomerular proteinuria: A commentary. J Am Soc Nephrol 2003;14:3217. 57. Welch TR, Beischel LS, Witte DP. Differential expression of complement C3 and C4 in the human kidney. J Clin Invest 1993; 92:1451.

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MECHANISMS OF TISSUE DAMAGE

20

Role of Complement in Systemic Lupus Erythematosus Pathogenesis Chau-Ching Liu, MD, PhD, Jeannine S. Navratil, MS, Amy H. Kao, MD, MPH, Susan Manzi, MD, MPH, and Joseph M. Ahearn, MD

INTRODUCTION

194

The complement system is intimately linked to SLE. Although paradoxical, this association has been recognized for decades. Two early observations formed the foundation for this conundrum. Vaughan and colleagues first assayed serum complement in four cases of SLE in 1951, and determined that diminished CH50 correlated with disease activity.1 This was confirmed by Elliott and Mathieson, who noted that complement depression was particularly associated with “albuminuria.”2 Lange and colleagues discovered that although low complement was characteristic of SLE whether or not there was renal involvement3 it was diminished in virtually all cases of acute, but not chronic, glomerulonephritis. Schur and Sandson found CH50 to be below 50% of normal in 46% of patients with renal involvement and 24% of patients with active SLE, but in only 4% of those with inactive disease—suggesting that complement levels were of particular value in following and evaluating patients with active SLE, especially those with nephritis.4 Thereafter, a large body of work demonstrated that complement activation (reflected by diminished serum levels of C3, C4, and CH50) plays a major role in the tissue inflammation and organ damage that result from lupus pathogenesis. Several other seemingly contradictory observations demonstrated a strong association between hereditary homozygous deficiency of the classical pathway components and development of SLE.5-7 In fact, inherited complement deficiency is recognized as conferring the greatest known risk for development of SLE. Thus, paradoxically, complement deficiency may be causative in SLE. However, activation of this same inflammatory cascade is detrimental in patients who already have the disease. This perplexing link between complement and SLE has been partially explained by discoveries made during the

past several years. Concomitant studies have identified potential strategies and opportunities for mining the complement system for lupus genes, biomarkers, and therapeutics. In this chapter we review the biology of the complement system in relation to SLE, summarize common methods for measurement of complement, discuss the utility of complement assays in clinical management of SLE, and consider the potential therapeutic interventions targeting the complement system.

COMPLEMENT BIOLOGY The complement system is composed of more than 30 plasma and membrane-bound proteins that form three distinct pathways and that protect against invading pathogens: classical, lectin, and alternative8-11 (Fig. 20.1). Functionally inactive complement pro-proteins exist in plasma until their activation is triggered. Once activated, the proteins undergo a cascade of sequential serine protease– mediated cleavage events within each pathway, thereby releasing biologically active fragments and selfassembling into multimolecular complexes. In general, activation of the complement system can be viewed as a two-stage process. In the first stage, unique to each of the three activation pathways, the early complement components are activated and lead to the formation of the C3 convertases. In the second stage, all three pathways converge to form a lytic complex, which consists of the terminal complement components (Fig. 20.1).

Complement Activation Pathways The classical pathway of complement activation plays an important role in SLE pathogenesis and is responsible for executing a major effector mechanism of antibodymediated immune responses. Five proteins are specific to the activation of the classical pathway: C1q, C1r, C1s, C4, and C2 (Fig 20.1). C1q sets into motion a sequential

cascade of events when it binds to the Fc portion of IgG (particularly IgG1 and IgG3) or IgM molecules that are bound to an antigen. This process then activates C1r (a serine protease), which in turn leads to activation of C1s (also a serine protease). When C1s enzymatically cleaves the other two classical pathway proteins, C4 and C2, two small soluble polypeptides (C4a and C2b) are generated and released. Concurrently, a surface-bound bimolecular complex (C4b2a, which functions as an enzyme and is referred to as the classical pathway C3 convertase) is formed. A variety of other molecules can also initiate activation of the classical pathway by interacting with C1q. These include C-reactive protein,12 amyloid P component,13 β-amyloid protein,14,15 and DNA.16 The lectin pathway shares several components with the classical pathway (Fig. 20.1). It is initiated when mannose-binding lectin [MBL; also known as mannose-binding protein (MBP)] binds to a variety of repetitive carbohydrate moieties such as mannose, N-acetyl-D-glucosamine, and N-acetyl-mannosamine, which are abundantly present on a variety of microorganisms.17,18 MBL, a plasma protein composed of a collagen-like region and a carbohydrate-binding domain, is structurally similar to C1q. MBL forms complexes in the plasma with other proteins such as mannose-binding protein-associated serine proteases (MASPs).19-21 Under physiologic conditions, MBL does not bind to mammalian cells, probably because these cells lack mannose residues on their surface. Once bound

COMPLEMENT BIOLOGY

Fig. 20.1 Schematic of the complement system and activation pathways.

to microbial pathogens, MASPs can cleave C4 and initiate the complement cascade. Alternatively, MBL (in place of C1q) may trigger the activation cascade by activating C1r and C1s. At this point, the lectin pathway intersects with the classical pathway and a C3 convertase (i.e., the C4b2a complex) is eventually generated. In contrast to the classical and lectin pathways, activation of the alternative pathway is not dependent on antibodies or other specific recognition molecules. Three plasma proteins (Factor B, Factor D, and properdin) are unique to the alternative pathway (Fig. 20.1). Normally, native C3 molecules undergo a so-called “C3 tickover” process—a continuous low-rate hydrolysis of the thioester group that generates iC3* (hydrolyzed C3) and subsequently C3b fragments.22 Via the formation of thioester bonds, some of these spontaneously generated C3b fragments may covalently attach to the surface of microbial pathogens and host cells. The bound C3b molecules are then capable of binding Factor B, which once bound is cleaved into Ba and Bb fragments by Factor D (a serine protease). The Bb fragment remains associated with C3b, whereas the small soluble Ba fragment diffuses away from the activation site. The surface-bound C3bBb complex serves as the alternative pathway C3 convertase, as the C4b2a complex does in the classical pathway. Self-damage of the host cells and tissue does not occur when the C3bBb complexes bind to mammalian cells because these complexes are rapidly degraded by several regulatory proteins. However, the C3bBb complexes associated with microbial pathogens, which do not generally express these regulatory proteins, will remain intact and can be further stabilized by the binding of properdin. C3, the central and most abundant component of the complement system, is proteolytically cleaved when C3 convertases are generated during the first stage of complement activation, giving rise to a smaller C3a fragment and a larger C3b fragment. The C3b molecules associate with C4bC2a or with C3bBb complexes to form the classical and alternative C5 convertases. The membrane attack complex (MAC, C5b-9) is formed by the activation and assembly of the terminal components C5, C6, C7, C8, and C9, which is initiated when the C5 convertases cleave C5 on the surface of foreign pathogens.

Effector Functions of Complement The complement system protects against invasion by four primary and basic biological functions: opsonization, activation of inflammation, clearance of immune complexes, and osmotic lysis of invading microorganisms.8-11 During SLE pathogenesis, self-antigens and autoimmune complexes (rather than foreign microbes) generate complement activation and its inflammatory consequences.

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ROLE OF COMPLEMENT IN SYSTEMIC LUPUS ERYTHEMATOSUS PATHOGENESIS

C3a, C4a, and C5a are soluble proteolytic fragments, which are highly potent and pro-inflammatory. By binding to specific receptors expressed on leukocytes (e.g., C5a receptor), they attract and activate them. The larger fragments (C3b, C4b) and their derivatives (e.g., iC3b and iC4b) can remain bound to the surface of microbial pathogens (or autoantigens) and facilitate recognition and uptake of opsonized particles by phagocytic cells via specific receptors (e.g., complement receptors 3 and 4, CR3 and CR4) expressed on the latter. When C3b and C4b bind to immune complexes, this binding prevents their aggregation into insoluble complexes and enhances their clearance. To mediate the clearance of C3b/C4b-opsonized immune complexes, erythrocytes that express CR1 transport these complexes to macrophages of the reticuloendothelial system in the spleen and liver.23,24 In addition, these

Regulators of Complement Activation and Complement Receptors In humans and other mammals, a redundant family of regulatory proteins controls complement activation

TABLE 20.1 COMPONENTS OF THE HUMAN COMPLEMENT SYSTEM Effector Protein

Mr (kD)

Function/Pathway Involved

C1q

450 (six-subunit bundle)

Recognition, binding/classical

C1r

85

Serine protease/classical

C1s

85

Serine protease/classical

205 (3-chain, αβγ, complex)

Serine protease (C4b); anaphylatoxin (C4a)/classical

C2

102

Serine protease (C2a); small fragment with kinin-activity (C2b)/classical

C3b

190 (2-chain, αβ, complex)

Membrane binding, opsonization (C3b); anaphylatoxin (C3a)/terminal

C5

190 (2-chain, αβ, complex)

MAC component (C5b), anaphylatoxin (C5a)/terminal

C6

110

MAC component/terminal

C7

100

MAC component/terminal

C8

150 (3-chain, αβγ, complex)

MAC component/terminal

C9

70

MAC component/terminal

Factor B

90

Serine protease/alternative

Factor D

24

Serine protease/alternative

Properdin

55 (monomers); 110, 165, 220, or higher (oligomers)

Stabilizing C3bBb Complexes/alternative

MBL

200-400 (2-4 subunits with three 32 KD chains each)

Recognition, binding/Lectin

MASP-1

100

Serine protease/Lectin

MASP-2

76

Serine protease/Lectin

C4

a

Membrane-Bound Mr (kD) Regulatory Protein c

CD35 (CR1 ) d

160-250 (4 isoforms)

Function Binding C3b and C4b; cofactor for Factor I

CD46 (MCP )

45-70 (different glycosylation forms) Promoting C3b and C4b inactivation by Factor I

CD55 (DAFe)

70

CD59 (protectin; H19)

196

complexes may bind to monocytes and neutrophils, which phagocytose them (often releasing lysosomal enzymes in the process). Finally, the C5b-9 MACs may perturb the osmotic equilibrium or disrupt the integrity of the surface membrane of target cells, thereby causing lysis of these cells. For several decades, the role of the complement system was thought to be limited to these four effector functions. Recently, however, many additional roles for complement (such as a link between innate and adaptive immunity) have been discovered.25,26 These novel functions are discussed in the material following.

Accelerating decay of the C3bBb and C4b2a complexes 18-20

Preventing C9 incorporation into the MAC in a homologous restriction manner

Soluble Regulatory Protein

Mr (kD)

Function

C1-inhibitor (C1-INH)

105

Removing activated C1r and C1s from the C1 complex

C4-binding protein (C4bp)

570 (7-subunit complex)

Displacing C2b in the C4bC2b complex; cofactor for Factor I

Factor H

160

Displacing Bb in the C3bBb complex; cofactor for Factor I

Factor I

88

Serine protease cleaving C3b and C4b

Clusterin

70

Preventing insertion of soluble C5b-7 complexes into cell membranes

S protein (vitronectin)

84

Preventing insertion of soluble C5b-7 complexes into cell membranes

Carboxypeptidase N

280 (multi-subunit complex)

Inactivating anaphylatoxins

Complement Receptor

Structure/Mr (kD)

Complement Ligand(s)f

CR1 (CD35)

Single chain, 190-280g

C3b, C4b, iC3b, C1q

CR2 (CD21)

Single chain, 140-145

C3dg/C3d, iC3b

CR3 (CD11b/CD18)

2-chain, α/β; 170/95

iC3b

CR4 (CD11c/CD18)

2-chain, α/β; 150/95

iC3b

cC1qR (calreticulin)

Single chain, 60

C1q (collagenous tail), MBL

gC1qR

Tetramer, 33/subunit

C1q (globular head)

C1qRP

Single chain, 126

C1q (collagenous tail)

C3a receptor

Single chain, 50?

C3a

C5a receptor (CD88)

Single chain, 50

C5a

COMPLEMENT BIOLOGY

TABLE 20.1 COMPONENTS OF THE HUMAN COMPLEMENT SYSTEM—cont’d

a. Serum concentration range considered normal: 20-50 mg/dL. b. Serum concentration range considered normal: 55-120 mg/dL. c. Complement receptor 1. d. Membrane cofactor protein. e. Decay accelerating factor. f. Noncomplement ligands (e.g., Epstin-Barr virus for CR2 and fibrinogen for CR3 and CR4) not listed. g. Four allotypes with different numbers of SCR and displaying distinct Mr under reducing condition: CR1-A (220 kD), CR1-B (250 kD), CR1-C (190 kD), and CR1-D (280 kD).

to ensure that host cells and tissues do not inappropriately activate this effective machinery (Table 20.1). Soluble or cell-surface regulatory molecules act at multiple steps of the activation pathways to control the potent consequences of complement activation. To do so, they use different mechanisms, functioning as proteolytic enzymes, cofactors for proteolytic enzymes, or competitive inhibitors of multimolecular convertases (Fig. 20.2). Receptors for proteolytic fragments of complement proteins (such as C3b, C4b, iC3b, and C3d or C1q) are expressed by a wide spectrum of cells and serve pivotal roles in executing many of the effector functions of complement described previously. Recent research

has led to the identification of at least four receptors for C1q: cC1qR (calreticulin, a collectin receptor), gC1qR, C1qRp, and CR1 (CD35).27-30 C1q receptors bind opsonized immune complexes to endothelial cells. This induces expression of adhesion molecules on these cells and enhances leukocyte binding/extravasation.31 On other cell types, C1q binding leads to enhanced phagocytosis, increased generation of reactive oxygen intermediates, and activation of platelets, presumably via distinct receptors.32-34 Two major receptors for C3- and C4-derived fragments, CR1 (CD35) and CR2 (CD21), belong to the “regulators of complement activation” (RCA) family.35,36 CR1 (which primarily binds C3b and C4b) is widely

197

ROLE OF COMPLEMENT IN SYSTEMIC LUPUS ERYTHEMATOSUS PATHOGENESIS

198

phagocytosis of C3-opsonized pathogens and adhesion of mononuclear phagocytes to endothelial cells.53-55

COMPLEMENT AND SLE

Fig. 20.2 Regulation of complement activation.

expressed by neutrophils, monocytes/macrophages, B lymphocytes, some T lymphocytes, glomerular podocytes, and erythrocytes,37-39 where it has the important function of binding and clearing immune complexes.23,24 CR1 also serves as a cofactor for Factor I, thereby playing a role in regulation of complement activation. Factor I cleaves C3b and C4b to iC3b and iC4b.40,41 CR2 (which is expressed mainly on B lymphocytes, activated T lymphocytes, and follicular dendritic cells) binds primarily iC3b, C3dg, and C3d.42-46 CR2, along with its cognate complement ligands, is a critical link between the innate and adaptive immune systems.26,47 For example, when CR2 (as part of the CD19/CD21/CD81 B-cell coreceptor complex) and B-cell receptors are co-ligated on the surface of B lymphocytes (which occurs via the binding of C3d-decorated immune complexes) B-cell activation, proliferation, and antibody production are enhanced.47-49 Furthermore, CR2 (which is expressed on follicular dendritic cells) can bind to antigens and immune complexes that when they have been opsonized by C3-derived fragments can be retained in the germinal centers of secondary lymphoid follicles.43,44 These retained antigen fragments provide essential signals for generation of memory B-cells, as well as for survival and affinity maturation of B-cells.50 CR3 and CR4 belong to the β2 integrin family and are composed of two subunits: a common β chain (CD18) and a specific α chain (CD11b for CR3 and CD11c for CR4). Phagocytic cells (e.g., neutrophils, monocytes, and macrophages), antigen-presenting cells (e.g., dendritic cells), and follicular dendritic cells express these receptors.51,52 CR3 and CR4 mediate

The role of complement in the etiopathogenesis of SLE is complex, intriguing, and paradoxical, and has been intensely studied over the past several decades. On the one hand, a hereditary deficiency of a component of the classical pathway (C1, C2, or C4) has been associated with the development of SLE.5-7,59-61 On the other hand, activation of the complement system [caused by tissue deposition of immune complexes (formed from autoantigens and autoantibodies)] results in tissue inflammation and damage.56-58 Studies performed over the past several years may reconcile seemingly discordant roles for complement in SLE. These studies have demonstrated that although the complement system plays a role in maintaining immune tolerance to prevent the development of SLE59,61,62 it also participates in tissue-destructive inflammatory processes once SLE is established in a patient.56,57

Immune Complex Abnormalities, Complement Activation, and SLE Immune complex abnormalities (e.g., decreased solubility and impaired disposal of immune complexes) and consequent complement activation are responsible for many of the clinical manifestations and pathology observed in patients with SLE. Decreased serum levels of C3 and C4 (due to genetic or acquired factors, or both) may prevent the formation of small soluble immune complexes63,64 by not permitting sufficient binding of C3 and C4 fragments to the antigen-antibody lattice. Furthermore, impaired binding, processing, and transporting of immune complexes to phagocytes of the reticuloendothelial system may be the result of reduced levels of CR1 on erythrocytes, which have been demonstrated in patients with SLE (see further discussion in material following). As a consequence, insoluble aggregates (which are formed from abnormally large quantities of immune complexes in the circulation) may eventually be deposited in various tissues. Deposited immune complexes, although they do not seem to cause tissue damage directly, do so indirectly by providing ample binding sites for complement components. The ensuing activation of the complement system causes the release of various mediators and promotes cellular infiltration and interaction, both of which culminate in tissue damage. This pathogenic sequence underlies the molecular basis for changes to the vascular endothelium and glomerular basement membrane, both of which are highly susceptible to this mode of inflammatory damage and lead to vasculitis

COMPLEMENT AND SLE

and glomerulonephritis (two hallmark manifestations of SLE).

Complement Deficiency and SLE Complement deficiency may occur as a hereditary or an acquired phenomenon. A deficiency of early complement components (i.e., C1, C4, and C2) is significantly associated with development of SLE. Such association is discussed elsewhere in this volume. The intriguing clinical association between complement deficiency and SLE is currently explained by three non mutually exclusive hypotheses. In the first hypothesis, impaired clearance of immune complexes due to deficiency of early complement components may trigger/augment the development of SLE. Interestingly, of the two isotopes of human C4, C4A has predominantly been implicated in binding and solubilizing immune complexes.65-67 Consequently, reports that the prevalence of C4A deficiency is higher in SLE patients than in the general population are not unexpected.68-70 Several studies showed impaired initial splenic processing of immune complexes in SLE patients,71-74 supporting the concept that impaired clearance of immune complexes may contribute to the development of SLE in the context of complement deficiency. The second hypothetical mechanism suggests that complement deficiency, through its capacity to determine activation thresholds of B and T lymphocytes, may alter the normal mechanism of negative selection of self-reactive lymphocytes.26,62,75-77 Co-ligation of CR2 and BCR augments B-cell activation by decreasing the threshold to antigenic stimulation.47 Therefore, selfantigens not opsonized by C4b or C3b are unlikely to trigger sufficient activation of self-reactive B-cells, and as a result these cells may escape negative selection. The escaped cells may breach self-tolerance to autoantigens once they become activated by encountering relevant autoantigens in the periphery. A third hypothetical mechanism also proposes to explain the link between complement deficiency and development of SLE.78,79 This hypothesis was first generated by the discovery that C1q can bind directly to apoptotic keratinocytes, and has been supported by subsequent observations. These studies demonstrated that endothelial cells and peripheral blood mononuclear cells undergoing apoptosis also bind C1q80 (Fig. 20.3), which subsequently triggers activation and deposition of C4 and C3 on these apoptotic cells.80,81 Thus, opsonized apoptotic cells and blebs can be effectively taken up by phagocytic cells via a complement receptor-mediated mechanism.81,82 During apoptosis, normally hidden intracellular constituents are often biochemically modified and redistributed/segregated into surface blebs of dying cells, resulting in the presence of “altered-self” constituents.83-85

Fig. 20.3 Binding of C1q to apoptotic endothelial cells and keratinocytes. Panels A-F: confocal analysis of C1q present on surface blebs of a human umbilical vein endothelial cell undergoing UVB-induced apoptosis. Shown are six consecutive cross sections through the cell stained for the presence of C1q by indirect immunofluorescence. The fluorescence intensity is represented by a color scale, with white being the highest intensity and black the lowest. Panels G-I: differential interference contrast images of panels D-F are shown to visualized morphology of the entire cells and apoptotic blebs. Panels J and K: Confocal analysis of bound C1q on apoptotic human keratinocytes. Shown are a merged phase and fluorescence image of a single plane of an apoptotic keratinocyte (J) and a stereo image of a Z-series of several planes though a single apoptotic keratinocyte (K). (Reproduced from Korb et al. J Immunol 1997;158:4525, and Navratil et al. J Immunol 2001;166:3231, copyrights 1997 and 2001, The American Association of Immunologists, Inc.)

When apoptotic cells are not promptly cleared due to complement deficiency, these constituents may persist, be recognized by the immune system, breach immune tolerance, and trigger autoimmune responses.86 Taken together, these studies suggest that complement plays a pivotal role in maintaining immune tolerance by facilitating the clearance of autoantigen-containing apoptotic bodies.7,59,61,87 Using a mouse model, Botto and colleagues were the first to report that mice deficient in C1q were defective in clearing apoptotic cells, as indicated by the accumulation of apoptotic bodies in the glomeruli of their kidneys. This led to autoimmune responses to nuclear autoantigens and glomerulonephritis.88 Subsequently, Taylor and colleagues reported similar but less severe defects in the

199

ROLE OF COMPLEMENT IN SYSTEMIC LUPUS ERYTHEMATOSUS PATHOGENESIS

clearance of apoptotic cells and spontaneous autoantibody production in C4-deficient mice.89 Results from these animal studies suggest that clearance of apoptotic cells is impaired or delayed in the absence of C1q, and less so in the absence of C4. Likewise, the persistence of apoptotic cells may lead to development of autoimmunity and tissue damage in humans. Reduced phagocytic activity of neutrophils, monocytes, and macrophages of SLE patients has previously been observed.90-92 Specifically, Hermann and colleagues reported a reduced capacity of SLE-derived macrophages to phagocytose apoptotic cells.93 Moreover, monocytederived macrophages prepared from SLE patients have shown impaired clearance of iC3b-opsonized apoptotic cells in vitro.94 Baumann and colleagues reported an abnormal accumulation of apoptotic cells accompanied by a significantly decreased number of tangible body macrophages (cells responsible for removing apoptotic nuclei) in the germinal centers of lymph nodes in a small subset of SLE patients, demonstrating in vivo evidence for impaired clearance of apoptotic cells in human SLE.95 Collectively, data from both animal and human studies not only substantiate the observed hierarchic role for complement components of the classical pathway and the risks of developing SLE but provide a strong mechanistic basis linking complement deficiency and SLE.

CR1 Deficiency in SLE

200

Reduced levels of CR1 expressed on erythrocytes of patients with SLE have been reported by several investigators.96-108 Studies performed in the 1980s led some investigators to conclude that erythrocyte CR1 deficiency is inherited,96,98,99,102 whereas later studies using SLE patients of various ethnic backgrounds suggested that erythrocyte CR1 deficiency is acquired.97,100,101,103-110 Some studies showed that reduced E-CR1 levels correlated with disease activity.100,104,107,108 Differing experimental methods and ethnic populations may account for these conflicting results. Wilson and colleagues examined E-CR1 levels in SLE patients and their relatives to elucidate the molecular basis of the observed differential erythrocyte CR1 expression in SLE patients versus healthy individuals.98 They found that a significant number of patients’ relatives had reduced E-CR1 levels. Three patterns of E-CR1 levels (high, intermediate, and low) could be identified in both SLE patients and family members used as controls. These studies indicated that E-CR1 levels were genetically regulated in a bialleleic co-dominant manner. Later, the same investigators demonstrated the presence of two HindIII-digested CR1 gene fragments, 7.4 kb and 6.9 kb in length, by using DNA probes for the CR1 gene and restriction fragment length polymorphism (RFLP) studies.111 They showed that the 6.9-kb

fragment (L allele) was associated with low E-CR1 levels, a heterozygous 7.4-kb/6.9-kb (H/L) pattern correlated with intermediate E-CR1 levels, and the 7.4-kb fragment (H allele) correlated with high E-CR1 levels. These results are consistent with the originally proposed bialleleic co-dominant fashion of inherited E-CR1 expression.98 However, it should be pointed out that E-CR1 levels were considerably lower in SLE patients than in healthy individuals with the matched HindIII RFLP pattern, suggesting that additional genetic or nongenetic factors may influence E-CR1 expression in SLE patients.102 Although these researchers initially postulated that increased prevalence of the HindIII L/L genotype may be associated with SLE, subsequent studies have not supported their theory. Reports from several studies do not indicate a significant difference in gene frequencies of HindIII H and L alleles between SLE patients and normal controls.101,103,106-108 This suggests that the L/L genotype does not increase susceptibility to SLE. Two other studies support the notion that low E-CR1 levels in SLE patients are acquired. A 1987 study demonstrated that normal erythrocytes transfused into SLE patients lost significant amounts of CR1 within a few days after transfusion.112 A 1999 study found that CR1 levels on the youngest form of erythrocytes (reticulocytes) of SLE patients were equivalent to those on reticulocytes of normal individuals and that SLE patients lost significantly greater amounts of E-CR1 in the peripheral circulation than did normal controls.113 In summary, both genetic and acquired factors are likely to contribute to the observed deficiency of erythrocyte CR1 in SLE patients, as experimental and clinical studies strongly indicate. However, the precise nature of these factors remains to be elucidated.

Abnormal Expression of CR2 and CR3 in SLE It has been reported that expression of CR2 on lymphocytes of patients with SLE was approximately 50 to 60% lower than those of healthy controls.114-116 Whether or not decreased CR2 expression correlates with increased activity of SLE (and if so, whether it is a cause or a result) is unclear. Nonetheless, animal studies using MRL/lpr mice have shown that decreases in CR1/CR2 expression preceded the development of clinically apparent disease,117 suggesting that decreased CR1/CR2 expression may be involved in the initiation or progression of autoimmune disease. The role of CR2 in the development and pathogenesis of SLE has been investigated using gene-knockout mice. In mice, CR2 and CR1 are encoded by the same gene through differential splicing of the RNA transcripts.118 Therefore, CR2-deficient (Cr2−/−) mice generated by gene-targeting techniques are also deficient in CR1.

MEASURING COMPLEMENT During flares of SLE, complement proteins would presumably be consumed at a rate proportional to activity of the disease. Measuring complement activation may

therefore be useful for diagnosing disease, assessing its activity, and determining response to therapy. Measuring complement activity and its individual component levels is also essential for detecting and diagnosing complement deficiency. Conventionally, the complement system is measured by one of two types of assays: functional and immunochemical. Complement-mediated hemolytic activity such as CH50 (indicative of the activity of the classical pathway) and APH50 (indicative of the activity of the alternative pathway) are measured by functional assays. Immunochemical assays measure serum concentrations of complement split products (or activation products) and individual complement components.

MEASURING COMPLEMENT

Initial studies have shown that Cr2−/−mice on the 129/B6 hybrid background did not develop an autoimmune phenotype.119,120 However, when back-crossed with B6/lpr mice (a mouse model of SLE) Cr2−/−/lpr mice on the 129/B6 hybrid background exhibited more aggressive disease featuring marked splenomegaly and lymhadenopathy, increased ANA and anti-dsDNA titers, and increased immune complex deposition in renal glomeruli at earlier time points than did B6/lpr mice.75 In comparison, Cr2−/−/lpr mice on the B6 background developed autoantibodies but not other manifestations.121 Recent studies of SLE susceptibility genes using the NZM2410 mouse model further support an important role of CR2 in the development of autoimmune phenotypes.122 Boackle and colleagues recently identified Cr2 as a candidate gene within the NZM2410 Sle1c locus.123,124 These investigators showed that B6 mice congenic for the Sle1c locus expressed dysfunctional CR1 and CR2 proteins, which are encoded by a defective Cr2 gene with a single nucleotide mutation. Although these mice developed autoantibodies to chromatin, they did not develop glomerulonephritis. Collectively, these results indicate that the Cr2 gene alone is insufficient to induce expression of a full spectrum of autoimmune phenotype/disease in lupus-prone mice, which apparently requires the contribution of additional SLE susceptibility genes. Reduced expression of CR3 on lymphocytes of patients with active SLE has also previously been reported.125,126 Immune vasculitis and panniculitis, arthritis, serositis, nephritis, and associated prominent cutaneous changes are some of the clinical manifestations seen in these patients. Although leukocyte adhesion deficiency, which is a congenital deficiency of CR3, is associated with increased susceptibility to pyogenic bacterial infections127,128 the pathophysiologic significance of decreased CR3 expression in SLE remains to be elucidated. Seemingly to the contrary, one study has even shown increased levels of CR3 on neutrophils of patients with SLE.129 Paradoxically, the highest neutrophil CR3 levels were detected in patients with the most severe disease, and in some of these patients the increased CR3 levels on neutrophils returned to normal when disease flares subsided. Researchers thus postulated that neutrophils, in response to complement activation, increase surface expression of CR3 in patients with active SLE. These activated neutrophils may contribute to endothelial injury in SLE.129

Measuring Complement Functional Activity Two simple quantitative tests for functional complement components in serum and other body fluids are the CH50 and APH50 assays, which measure complementmediated hemolysis. It is common to check CH50 in patients with SLE, in that complement activation in SLE is triggered predominantly by immune complexes that activate the classical pathway. Complement activity is quantified in the following way: the dilution of a serum (or other body fluid sample) required to lyse 50% of sheep erythrocytes sensitized with anti-sheep IgM (for CH50 assays) or unsensitized rabbit erythrocytes (for APH50 assays) is determined under standard conditions. Complement activity is represented by the reciprocal of this dilution in units per millileter (U/ml) of serum. For instance, complement activity would be reported as 160 CH50 U/ml in a sample in which a 1:160 dilution lyses 50% of erythrocytes. Serum samples, if not used immediately, should be stored at −70°C because some complement components are heat labile. This serves to optimize the preservation of complement proteins in functionally active forms.

Measuring Complement Proteins Measurement of serum levels of individual complement components is commonly used to identify deficiencies of specific complement proteins and to diagnose and assess disease activity in SLE. Traditionally, serum is used for complement measurements. (As cautioned previously, serum samples should be handled promptly and carefully to minimize possible degradation of complement proteins.) Immunochemical methods are generally based on the reactivity between complement components in the test sample and anticomplement antibodies added to the assays. Selecting a proper method depends on several factors: the level of sensitivity required, the availability of specific antibody, and the number and types of samples.

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ROLE OF COMPLEMENT IN SYSTEMIC LUPUS ERYTHEMATOSUS PATHOGENESIS

202

Nephelometry is routinely used to measure complement components present at relatively high concentrations in the serum (e.g., C3 and C4). Radial immunodiffusion (RID) or enzyme-linked immunosorbant assay (ELISA) can be used to measure other components that are usually present at low concentrations (e.g., C1, C2, C5 through C9, Factor B, Factor D, properdin, and so on). When C3 and C4 concentrations are too low to be measured accurately by nephelometry (i.e., < 20 mg/dl and < 10 mg/dl, respectively), RID is the alternative method of choice. ELISA is the most practical method to use for other body fluids or cell culture supernatants, in which the levels of complement components may be very low.

Measuring Complement Split Products Complement components constantly undergo synthesis, activation, and catabolism, and thus measurement of their serum concentrations is essentially a static appraisal of an extremely dynamic process. Because complement split products are generated only when complement activation occurs, direct determination of complement split products is thought to reflect more precisely the activation process of complement in vivo and hence the disease activity. Many clinical immunology laboratories currently measure complement split products in the plasma, yielded from activation of the classical pathway (C1rs-C1 inhibitor complex, C4a, and C4d), alternative pathway (Bb and C3bBbP), lectin pathway (C4a and C4d), and terminal pathway (C3a, iC3b, C3d, C5a, and sC5b-9). Plasma, instead of serum, should be used to measure complement split products. Using EDTA-anticoagulated plasma will avoid generating complement split products in vitro. ELISA and EIA are the most practical methods for the measurement of split products, in that only low levels of them may be present in the circulation even after significantly increased complement activation. Complement split products have commonly short, but different, half-lives in the plasma. Some split products that have very short half-lives (such as C3a, C4a, and C5a) are quickly converted to more stable and less active forms (such as C3a-desArg, C4a-desArg, and C5a-desArg). In contrast, some complement products (such as those that form multimolecular complexes) usually have a relatively long half-life in the circulation. Examples of these include products of classical pathway activation such as C1rs-C1 inhibitor complexes, products of alternative pathway (e.g., C3bBbP complexes), and sC5b-9 (the ultimate product of complement activation). C5b, C6, C7, C8, poly-C9, and the solubilizing protein (protein S) constitute sC5b-9, which is the soluble form of MAC.

COMPLEMENT AS A SOURCE OF BIOMARKERS FOR SLE DIAGNOSIS AND MONITORING

Complement measurement and SLE Activity In clinical practice, patients with SLE are often monitored by measures of serum C3, C4, and complement hemolytic activity (Table 20.2). Studies to evaluate the utility of these assays in the diagnosis and monitoring of SLE, with their noteworthy data and precautions, are succinctly reviewed in material following. First, there is no consensus regarding the actual value of complement measures in SLE monitoring, in spite of the conventional notion that decreased levels of complement components reflect activation of the classical and alternative pathway and correlate with clinical disease activity. Some investigators have found CH50 as well as serum C4 and C3 levels to be valuable as markers of SLE activity (Table 20.3) for the following reasons. First, patients with increased SLE disease activity, manifested by active nephritis and extra-renal involvement, have demonstrated significantly decreased levels of CH50 and serum C3 and C4.4,130-135 Second, clinical exacerbation has been preceded by a decrease in serum C4 levels.131,134,136 Third, remission/relapse of lupus nephritis has coincided significantly with an increase/decrease in serum C3 levels.133-135 Fourth, an impending flare of SLE may be heralded by a progressive fall of serum C3 or C4 levels.137 Fifth, serum C3 and C4 levels have normalized upon resolution of disease flares.131 On the contrary, the following observations argue against the usefulness of conventional complement measurement. First, the extent of changes in serum C3 and C4 levels do not correlate quantitatively with disease severity.138-140 Second, serum C4 and C3 levels during disease flares have been found to remain normal in some patients.141,142 Third, persistently low C4 levels have been detected in SLE patients with inactive disease.131,132,143,144 Fourth, increases in the split products (e.g., C3a, C3d, and C4d) have not always accompanied decreases in C3 and C4.

TABLE 20.2 COMMON PROFILES OF COMPLEMENT MEASUREMENT IN SLE AND OTHER INFLAMMATORY CONDITIONS Pathway(s) Involved

C3

C4

Factor B

Classical

↓

↓

N

↓

N

Alternative

↓

N

↓

N

↓

Classical/Alternative

↓

↓

↓

↓

↓

CH50 APH50

Complement Component(s)

Study Design

Results/Conclusions

C5b-9

Falk et al.156 108 serial plasma samples from 14 SLE patients

C5b-9 levels significantly elevated in SLE patients, and positively correlated with disease activity

Bb, C4d, C5b-9

Manzi et al.154 21 SLE patients prospectively followed for 1 year

C4d and Bb sensitive indicator of moderate-to-severe disease activity; C4d and Bb sensitive at predicting increasing disease activity

Ba, Bb, C4d, C5b-9

Buyon et al.153 380 serial plasma samples from 86 SLE patients with inactive, stable/moderate, or severe disease

Ba levels significantly elevated and positively correlated with disease activity; elevated C4 and increased Bb predictive of subsequent flares

C4d, C3d

Senaldi et al.146 Plasma samples of 48 SLE patients (11 inactive, 23 mildly active, 14 moderately/severely active)

Elevated C4d levels correlated with disease activity in a linear fashion; C3d levels elevated but not linearly correlated with disease activity

C3a, C4a, iC3b, C5b-9

Porcel et al.150 Plasma samples of 61 SLE patients (22 inactive disease; 39 active disease; defined by SLEDAI)

C3a, C4a, and C5b-9 significantly elevated in patients with active disease, with a positive correlation with disease activity scores; C5b-9 most sensitive and specific; iC3b not correlated with disease activity

C3a, C5a

Hopkins et al.148 Serial plasma samples from 23 SLE patients (7 pregnant; 5 CNS involvement)

C3a levels significantly higher in patients with a flare than in those with stable disease; rising C3a levels predictive of disease flares; highly elevated C3a and C5a in patients with CNS involvement; C3a levels elevated in most pregnant patients

C3a, C5a

Belmont et al.147 Plasma samples of 76 SLE patients with severe, moderate, or inactive disease

C3a significantly elevated in patients with severe or moderate disease activity and quantitatively correlated with disease severity; C5a significantly elevated in patients with severe disease activity

C1rs-C1inh, C3(Bb)P, C5b-9

Nagy et al.159 Plasma samples obtained from healthy controls and 65 SLE patients with active and inactive disease

All 3 activation products elevated in SLE patients with inactive disease compared to healthy controls; C3(Bb)P and C5b-9 but not C1rs-C1inh, distinguishing active disease from inactive disease

C1rs-C1inh, C3d

Sturfelt et al.145 Serial (at 6- to 8-wk intervals) samples from 33 SLE patients

Increased C1rs-C1inh consistently found during flares; Increased C1rs-C1inh detected before flares, especially extra-renal flares; Increased C3d associated with severe disease flares

C3, C4

Esdaile et al.137,209 Retrospective analysis of serum samples collected from 202 patients; serological tests: C3, C4, anti-dsDNA, immune complexes

All serological parameters tested poor predictor of SLE flares; Some values of patient-based serial measures in association with specific types of flares (e.g., decreased C3 and renal involvement)

CH50, C3, C4

Morrow et al.139 Prospective follow up of 35 SLE patients; serological tests: CH50, C3, C4, anti-dsDNA, immune complexes

None of the serological tests reliably distinguished the three clinical groups

C3

Abrass et al.138 Prospective study following 48 SLE patients; serological tests: C3, anti-dsDNA, immune complexes

C3 and anti-dsDNA neither associated with nor predictive of changes in disease activity

COMPLEMENT AS A SOURCE OF BIOMARKERS FOR SLE DIAGNOSIS AND MONITORING

TABLE 20.3 SELECT STUDIES OF COMPLEMENT MEASURES IN SLEa

203 Continued

ROLE OF COMPLEMENT IN SYSTEMIC LUPUS ERYTHEMATOSUS PATHOGENESIS

TABLE 20.3 SELECT STUDIES OF COMPLEMENT MEASURES IN SLEa—cont’d Complement Component(s)

Study Design

Results/Conclusions

C1q, C3, C4, C5, C9

Swakk et al.210 Prospective study of 143 patients; serological tests: C1q, C3, C4, C5, C9, anti-dsDNA

Decreased C4, C1q, and C3, in a sequential order, detected in patients with renal exacerbation; Decreasing C4 detectable 20-25 weeks before the flare; Decreased C1q and C3 detected during but not before the flare

C3, C4

Ricker et al.133 Retrospective study using serial serum samples obtained from 12 SLE patients with severe nephritis; serological tests: C3, C4

Normal C3 levels observed during disease remission; Abnormal C3 levels more frequently detected during flares than C4; Higher specificity and sensitivity of C3 than C4 in monitoring SLE disease activity

CH50, C1q, C3, C4

Valentijn et al.132 Retrospective study using serial serum samples obtained from 33 SLE patients; serological tests: CH50, C3, C4, immune complexes (C1q binding)

Significant correlation between overall disease activity, decreased C3/CH50, and increased immune complex levels; Low sensitivity, specificity, and predictive value; Correlation between C3/C4 levels and organ involvement in subgroups of patients

CH50, C1q, C3, C4

Lloyd and Schur131 Prospective study following 27 SLE patients through 47 cycles of clinical activity; serological tests: CH50, C1q, C3, C4, anti-dsDNA, and immune complex (C1q binding activity) levels

CH50, C3, C4 levels lower in patients with active renal disease than in patients with extra-renal involvement; Decreasing C4 levels preceded disease flares

a. Adapted from ref. 211. (Liu et al. Mining the complement system for lupus biomarkers. Clin Appl Immunol 2005;5:185-206, with permission from Elsevier).

Although direct determination of complement split products compared to conventional complement measurement should theoretically reflect more precisely the activation process of complement in vivo and thus more specifically clinically active disease, controversy regarding the utility of these assays still remains. Studies have generally shown that plasma concentrations of complement split products (including C1-C1 inh complex, C3a, C4a, C5a, C3d, C4d, C5b-9, Ba, and Bb) increased before or during clinical exacerbation,141,145-157 and in some cases the plasma levels correlated strongly with SLE disease activity scores.146,147,149-152 These studies would support the direct measurement of split complement products. On the contrary, elevated levels of split products such as C1-inh and C3d have been reported not only in almost all clinically ill patients but in a significant fraction of patients with quiescent disease, suggesting that plasma C1-inh and C3d levels bear little relationship to clinical activity and arguing against their use.139,158-161 Furthermore, inconsistent results have been reported for the utility of plasma levels of a given complement split product in distinguishing patients with different levels of disease activity and severity.150,158

Drawbacks and Problems Associated with Complement Measurement 204

Several factors that particularly confound measurement of C3 and C4 in disease may explain the discrepant reports

regarding the value of measuring serum C4 and C3 to monitor disease activity of chronic inflammatory diseases such as SLE. First, traditional concentration measurements reflect the presence of C3 and C4 protein entities irrespective of their functional integrity. Second, there is wide variation in serum C3 and C4 levels among healthy individuals. This range overlaps with that observed in patients with different diseases. Third, genetic variations such as partial deficiency of C4 (commonly present in the general population and in patients with autoimmune diseases70,162) may result in lower than normal serum C4 levels in some patients because of decreased synthesis rather than increased complement consumption during disease flares. Fourth, acute phase responses during inflammation may lead to an increase in C4 and C3 synthesis,163,164 which can counterbalance the consumption of these proteins during activation. Fifth, enhanced catabolism165-167 as well as altered synthesis of C3 and C4168,169 have been reported to occur in patients with SLE, which clearly can interfere with static measures of serum C3 and C4 levels. Sixth, tissue deposition of immune complexes may result in complement activation at local sites in patients with certain diseases. Therefore, the levels of complement products in the systemic circulation may not faithfully reflect such activity. Additional concerns should be raised regarding the measurements of complement

Complement Measurement and Organ-Specific Involvement in SLE Given the numerous confounding factors outlined here, it is not surprising that irreconcilable results regarding complement measurement have prevailed in the research arena of complement and SLE disease activity. However, if complement measurements are performed chronologically in the same patient and interpretation is based on the specific genetic and clinical characteristics of the patient they may still be informative. Measurement in the blood or other body fluid may also be valuable in assessing disease activity or predicting outcome under particular conditions, such as pregnancy and specific organ involvement. Lupus nephritis is one of the most serious clinical manifestations of SLE, and nephritic flare has been shown to be a predictor of a poor long-term outcome in SLE patients.170 Measurements of complement components and activation products in the plasma or in the urine may be useful for evaluating the extent of active inflammation in the kidneys. SLE patients with renal involvement were found to have markedly reduced serum levels of C3 and C4 more frequently than patients with only extra-renal involvement.131,132 Correspondingly, SLE patients with normal C3 and C4 levels were rarely found to have active nephritis.131,132 Therefore, the absence of a low C3 or C4 level in a patient with SLE may serve to exclude the possibility of ongoing renal disease. Because low C3 levels have been reported to be predictive of persistently active glomerular disease171 and have been associated with end-stage renal disease,172 low C3 and C4 levels may also be helpful in predicting long-term outcome in patients with SLE. In addition to low C3 and C4, very low levels of serum C1q were detected in SLE patients with active renal disease but not in those without it.131 Persistently low C1q levels, before and after intense treatment for lupus nephritis, have been shown to be indicative of continuously progressive damage to the kidneys and are thus associated with a poor outcome.173 Measurement of C3d in the urine has been pursued as a test for specific and accurate estimation of inflammation in the kidney because it is likely that C3d generated in the kidney at sites of immune complex deposition would pass into the urine. Kelly and colleagues174 and Manzi and colleagues154 have reported the detection of

C3d in the urine of SLE patients with acute nephritis, but also in that of patients without evidence of renal involvement. Thus, urinary C3d may also come from nonrenal origins and therefore may not be viewed as a specific marker of acute nephritis or a prognostic indicator of renal disease. Nevertheless, in the same study by Manzi and colleagues154 urinary C3d was better than serum C3, plasma C4d, Bb, and C5b-9 in distinguishing patients with acute lupus nephritis from those without such disease activity. Recently, Negi and colleagues reported that C3d levels were elevated in the urine of patients with active disease. Patients with active lupus nephritis had values as high as 0.87 AU/ml, whereas levels were measured at 0.31 AU/ml in those with active extra-renal disease and at 0.06 AU/ml in patients with inactive lupus nephritis.175 Collectively, these results suggest that increased levels of urinary C3d may reflect active SLE, particularly active lupus nephritis. In addition to renal disease, hematologic manifestations have been associated with low serum C3 and C4 levels. Ho and colleagues reported that decreases in C3 were correlated with concurrent decreases in platelet and white blood cell counts, as well as in hematocrit.176 However, these researchers did not find that decreased complement levels were consistently associated with SLE flares. Investigations have also explored the relationship between CNS disease in SLE and complement levels. One study showed that plasma C3a levels increased in SLE patients, and were particularly high in five patients who had acute CNS dysfunction. In addition, 4 of these 5 patients had significantly elevated plasma C5a levels as well.148 Similarly, Rother and colleagues155 found significantly higher levels of plasma C3d in SLE patients with CNS involvement than in those without such involvement. Finally, several studies have examined levels of complement proteins in other body fluids. Such investigations have included analyses of synovial fluid,177 pleural fluid,178,179 pericardial fluid,180,181 and cerebrospinal fluid,182-184 but these studies have been so few that little can be concluded about the clinical utility of such analyses.

COMPLEMENT AS A SOURCE OF BIOMARKERS FOR SLE DIAGNOSIS AND MONITORING

split products. As mentioned previously, complement activation can easily occur in vitro after blood sampling, and many of the split products have undefined (most likely short) half-lives both in vivo and in vitro. In combination, these factors may hamper accurate measures of soluble activation products that are derived from complement activation occurring in patients.

Measurement of Cell-Bound Complement Activation Products Recent studies have explored the hypothesis that cell-bound complement activation products (CB-CAPs) may serve as biomarkers for SLE diagnosis and monitoring.185,186 This hypothesis was based on the following rationale. First, as described previously serum C3 and C4 levels have no diagnostic utility and limited monitoring utility. Second, measurement of soluble complement activation products does have utility in certain clinical situations, but these assays have yet to replace measurement of serum C3 and C4

205

ROLE OF COMPLEMENT IN SYSTEMIC LUPUS ERYTHEMATOSUS PATHOGENESIS

in clinical practice. Third, cell surface receptors for complement activation products, present on all circulating cells, may confound accurate and reliable measurement of the soluble activation products. Fourth, C3- and C4-derived complement activation products are capable of covalent attachment to cell surfaces via thioester bonds, and this property may increase longevity in the circulation (making CB-CAPs more likely candidates for use in clinical practice than soluble CAPs). Fifth, C4-derived complement activation products are known to be present on surfaces of normal erythrocytes, although the physiologic significance of this phenomenon is unknown.187,188 Sixth, CB-CAPS on specific cell types with unique cellular properties (such as erythrocytes and reticulocytes) might provide additional clues to disease diagnosis, activity, and pathogenesis.

Erythrocyte-Bound C4d as a Biomarker for SLE Diagnosis and Monitoring Manzi and colleagues conducted a cross-sectional flow cytometric study to examine erythrocyte-bound C4d (E-C4d) levels in patients with SLE, those with other inflammatory and immune-mediated diseases, and healthy controls.185 In light of the previous reported association of low E-CR1 levels in SLE, erythrocyte-CR1 (E-CR1) was determined simultaneously. This study demonstrated that patients with SLE have significantly higher levels of E-C4d than those with other diseases and healthy individuals and that there is a pattern of abnormally high E-C4d levels in conjunction with abnormally low E-CR1 levels [which has high diagnostic sensitivity (81%) and specificity (91%) for SLE] as compared with healthy individuals, and with patients with other inflammatory diseases (72% sensitive, 79% specific).185 During this study, these investigators also observed significant longitudinal fluctuation of E-C4d for individual patients, which suggested that E-C4d levels might correlate with disease activity in SLE.

Reticulocyte-Bound C4d as a Biomarker for Disease Activity in SLE

206

These studies then took advantage of the knowledge that erythrocytes develop from hematopoietic stem cells in the bone marrow and emerge as reticulocytes, which then maintain distinct phenotypic features for 1 to 2 days before fully maturing into erythrocytes. Reticulocytes, if released into the peripheral circulation during an active disease state, may immediately be exposed to and bind C4-derived fragments generated from activation of the complement system. Because erythrocytes, which have a life span of approximately 120 days, may bind and retain activation-derived C4d throughout their lifetimes, an E-C4d level is likely to be the cumulative result of complement activation and

disease activity over a 120-day period. In contrast, due to the brief life span of reticulocytes the levels of C4d attached to these cells (R-C4d) might more likely originate from ongoing activation rather than being the result of past events. Therefore, it was hypothesized that R-C4d levels (as opposed to E-C4d levels) may more effectively and precisely reflect the current disease activity in a given SLE patient at a specific point in time. In this manner, R-C4d levels may serve as “instant messengers” of SLE disease activity. Initial studies indicated that R-C4d varies widely in SLE patients but not in patients with other diseases and healthy controls, that the mean R-C4d level of SLE patients is significantly higher than that of patients with other diseases or healthy controls, and that R-C4d levels fluctuate and correlate with clinical disease activity as measured by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and Systemic Lupus Activity Measurement (SLAM) indices.186 These findings suggest that C4d-bearing reticulocytes may provide clues to current and perhaps impending disease flares, thereby serving as biomarkers for SLE disease activity.

Platelet-Bound C4d as a Diagnostic Assay for SLE and Biomarker for SLE-Associated Cerebrovascular Disease Subsequent studies also explored the potential of platelet-bound C4d (P-C4d) as a source of complementbased biomarkers.189 Cross-sectional determination of P-C4d in SLE patients, patients with other diseases, and healthy controls demonstrated significant levels of C4d on platelets from 18% of SLE patients, 1.7% of patients with other diseases, and 0% of healthy controls. Accordingly, detection of C4d on platelet surfaces is 100% specific in distinguishing SLE patients from healthy controls, and 98% specific in distinguishing SLE patients from patients with other diseases. These results demonstrate great potential for P-C4d measurement as a confirmative diagnostic assay for SLE. These studies also suggest that deposition of C4d on platelets may reflect and/or contribute to vasculopathy in SLE patients. Indeed, preliminary results have demonstrated that the presence of C4d on platelets from SLE patients is significantly correlated with a history of a neurologic event and the presence of antiphospholipid antibodies. These data suggest that detection of P-C4d may identify SLE patients at increased risk for cerebrovascular disease and perhaps for other vascular complications of lupus.

ANTI-COMPLEMENT THERAPEUTICS FOR SLE The complement system has naturally been targeted for therapeutic intervention because of the fundamental

Alternative pathway

Classical pathway

Lectin pathway

C3b

C1q MBL

C4

C2

MASP C1r

B

C1s

C3 convertase

D Ba

C4 C2

C3 C4a Propendin

C2b

C5 convertase C3a

ANTI-COMPLEMENT THERAPEUTICS FOR SLE

Fig 20.4 Anticomplement therapeutics and potential target molecules.

C5 Anti-C5 mAb C5aR antagonists

C6 C5a C7

sCR1 Heparin Compostatin Protease inhibitors

C8 C9

Membrane attack complex (C5b-9)

role that complement activation plays in SLE pathogenesis. To date, a variety of reagents that inhibit or modulate complement activation at different steps of the cascade have been developed.190,191 Inhibitors of the early steps of complement activation and inhibitors of the terminal pathway constitute the two broad categories of these reagents190,191 (Fig. 20.4). Examples of the early inhibitors include soluble CR1 (sCR1, capable of regulating the generation of C3/C4 fragments and C3 convertases),192-194 heparin (a polyanionic glycosamine capable of binding/inhibiting C1, inhibiting binding of C1q to immune complexes, blocking C3 convertase formation, and interfering with MAC assembly),195-198 compostatin (a synthetic peptide capable of binding C3 and preventing its proteolytic cleavage), and protease inhibitors.199 Inhibitors of the terminal pathway include anti-C5 monoclonal antibodies (mAbs) that can bind C5, block its cleavage and formation of C5a, and abrogate MAC assembly.200-204 Synthetic antagonists of C5a receptors are also inhibitors of the terminal pathway, and have been exploited to block the anaphylactic and chemotactic effects of C5a.205-208 Considering that C3b opsonization

of pathogens and immune complexes is crucial for host defense and for prevention of immune complexassociated adverse reactions, it is reasonable to postulate that inhibitors of complement activation at a downstream step (such as C5 cleavage) will have therapeutic effects for patients with inflammatory diseases but may not increase the risk of infection in these patients. Eculizumab, a humanized anti-C5 mAb, has recently been studied in the NZB/W F1 mouse model of SLE and has been shown to improve significantly renal disease and increase survival of treated mice.202 A Phase I clinical trial of Eculizumab in patients with SLE concluded that it was safe and well tolerated, without significant adverse effects.202,203 Heparin, traditionally used as an anticoagulant and known to inhibit complement activation, has recently been demonstrated to prevent antiphospholipid antibody/complement-induced fetal loss in a murine model.198 This seminal observation suggests that heparin at “subtherapeutic” (non-anticoagulating) doses may be beneficial in pathological situations in which excess complement activation is unfavorable, such as ischemia/ reperfusion injury, antiphopholipid antibody syndrome, and lupus nephritis.

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208

CONCLUSIONS

ACKNOWLEDGMENTS

Recent genetic, biochemical, immunologic, and molecular biological studies not only have yielded further support for the well-accepted role of complement in mediating pathologic damage in SLE but have unraveled many previously unrecognized functions of complement in preventing the development of SLE. These newly discovered intricate roles have made the complement system the “Renaissance man” in the clinical and research arena of SLE. It is now time not only to revisit the issue of complement as a source of lupus biomarkers but to further explore the utility of anticomplement reagents as novel therapeutics for SLE. With a coordinated effort of investigators and physicians to conduct large-scale multicenter trials, these potentials of the complement system will undoubtedly be realized in a foreseeable future.

We thank our colleagues in the Lupus Center of Excellence and Division of Rheumatology and Clinical Immunology for providing clinical samples, helpful discussion, and skilled technical as well as administrative support. We also thank Diane Lattanzio for expert editorial assistance. Experimental work from the authors’ laboratory are supported by grants from the National Institutes of Health (RO1 AR-4676402, RO1 HL074335, RO1 AR-46588, NCRR/GCRC MO1-RR-00056, K24 AR02213, K23 AR051044, and P30 AR47372); the Lupus Foundation of Pennsylvania; the Alliance for Lupus Research; the Lupus Foundation of America, Southeastern Pennsylvania Chapter; and the Arthritis Foundation.

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162. Tsukamoto H, et al. Molecular analysis of a novel hereditary C3 deficiency with systemic lupus erythematosus. Biochem Biophys Res Commun 2005;330:298-304. 163. Sturfelt G, Sjoholm AG. Complement components, complement activation, and acute phase response in systemic lupus erythematosus. Int Arch Allergy Appl Immunol 1984;75: 75-83. 164. Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999;340: 448-454. 165. Alper CA, Rosen FS. Studies of the in vivo behavior of human C3 in normal subjects and patients. J Clin Invest 1967;46: 2021-2034. 166. Hunsicker LG, et al. Metabolism of third complement component (C3) in nephritis: Involvement of the classic and alternate (properdin) pathways for complement activation. N Engl J Med 1972;287:835-840. 167. Charlesworth, JA, et al. Hypercatabolism of C3 and C4 in active and inactive systemic lupus erythematosus. Annals of Rheumatic Diseases 1989;48:153-159. 168. Sliwinski JA, Zvaifler NJ. Decreased synthesis of the third component (C3) in hypocomplementemic systemic lupus erythematosus. Clin Exp Immunol 1972;11:21-29. 169. Tsukamoto H, et al. Increased production of the third component of complement (C3) by monocytes from patients with systemic lupus erythematosus. Clin Exp Immunol 1990;82: 257-261. 170. Ponticelli C, Moroni G. Lupus nephritis. J Nephrol 2000;13: 385-399. 171. Pillemer SR, et al. Lupus nephritis: Association between serology and renal biopsy measures. J Rheumatol 1988;15: 284-288. 172. Manger K, et al. Definition of risk factors for death, end stage renal disease, and thromboembolic events in a monocentric cohort of 338 patients with systemic lupus erythematosus. Ann Rheum Dis 2002;61:1065-1070. 173. Gunnarsson I, et al. Repeated renal biopsy in proliferative lupus nephritis: Predictive role of serum C1q and albuminuria. J Rheumatol 2002;29:693-699. 174. Kelly RH, et al. Complement C3 fragments in urine detection in systemic lupus erythematosus patients by Western Blotting. Appl Theor Electroph 1993;3:265-269. 175. Negi VS, et al. Complement degradation product C3d in urine: Marker of lupus nephritis. J Rheumatol 2000;27: 380-383. 176. Ho A, et al. A decrease in complement is associated with increased renal and hematologic activity in patients with systemic lupus erythematosus. Arthritis Rheum 2001;44: 2350-2357. 177. Pekin TJ Jr, Zvaifler NJ. Synovial fluid findings in systemic lupus erythematosus (SLE). Arthritis Rheum 1970;13: 777-785. 178. Hunder GG, McDuffie FC, Hepper NGG. Pleural fluid complement in systemic lupus erythematosus and rheumatoid arthritis. Ann Intern Med 1972;76:357-363. 179. Kinney E, et al. Pericardial-fluid complement. Am J Clin Pathol 1979;72:972-974. 180. Goldenberg DL, Leff G, Grayzel AI. Pericardial tamponade in systemic lupus erythematosus with absent hemolytic complement activity in pericardial fluid. NY State J Med 1975;75: 910-912. 181. Hunder GG, Mullen BJ, McDuffie FC. Complement in pericardial fluid of lupus erythematosus. Ann Intern Med 1974;80: 453-458. 182. Petz LD, et al. Serum and cerebrospinal fluid complement and serum autoantibodies in systemic lupus erythematosus. Medicine 1971;50:259-275. 183. Sanders ME, et al. Detection of activated terminal complement (C5b-9) in cerebraospinal fluid from patients with central nervous system involvement of primary Sjogren’s syndrome or systemic lupus erythematosus. J Immunol 1987;138: 2095-2099. 184. Jongen PJH, et al. Cerebrospinal fluid C3 and C4 indexes in immunological disorders of the central nervous system. Acta Neurol Scand 2000;101:116-121.

185. Manzi S, et al. Measurement of erythrocyte C4d and complement receptor 1 in the diagnosis of systemic lupus erythematosus. Arthritis Rheum 2004;50:3596-3604. 186. Liu CC, et al. Reticulocytes bearing C4d as biomarkers of disease activity for systemic lupus erythematosus. Arthritis Rheum 2005:52:3087-3099. 187. Tieley CA, Romans DG, Crookston MC. Localization of Chido and Todgers determinants to the C4d fragment of human C4. Nature 1978;276:713-715. 188. Atkinson JP, et al. Origin of the fourth component of complement related Chido and Rodgers blood group antigens. Complement 1988;5:65-76. 189. Navratil JS, Manzi S, Kao AH, Krishnaswami S, Liu C-C, Ruffing MR, et al. Platelet-C4d is highly specific for systemic lupus erythematosus. Arthriti Rheum 2002;54:670-674. 190. Morgan BP, Harris CL. Complement therapeutics: History and current progress. Mol Immunol 2003;40:159-170. 191. Sahu A, Lambris JD. Complement inhibitors: a resurgent concept in anti-inflammatory therapeutics. Immunopharmacology 2000;49:133-148. 192. Hill J, et al. Soluble complement receptor type 1 ameliorates the local and remote organ injury after intestinal ischemiareperfusion in the rat. J Immunol 1992;149:1723-1728. 193. Couser WG, et al. The effects of soluble recombinant complement receptor 1 on complement-mediated experimental glomerulonephritis. J Am Soc Nephrol 1995;5: 1888-1894. 194. Weisman HF, et al. Soluble human complement receptor type 1: In vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. Science 1990;249: 146-151. 195. Weiler JM, et al. Modulation of the formation of the amplification convertase of complement, C3b, Bb, by native and commercial heparin. J Exp Med 1978;147:409-421. 196. Weiler JM, et al. Heparin and modified heparin inhibit complement activation in vivo. J Immunol 1992;148: 3210-3215. 197. Mulligan MS, et al. Endothelial targeting and enhanced antiinflammatory effects of complement inhibitors possessing sialyl Lewis X moieties. J Immunol 1999;162:4952-4959. 198. Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nature Medicine 2004;10:1222-1226. 199. Sahu A, Morikis D, Lambris JD. Compstatin, a peptide inhibitor of complement, exhibits species-specific binding to complement component C3. Mol Immunol 2003;39:557-566. 200. Thomas TC, et al. Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv. Mol Immunol 1996;33: 1389-1401. 201. Fitch JC, et al. Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. Circulation 1999;100: 2499-2506. 202. Kaplan M. Eculizumab (Alexion). Curr Opin Invest Drugs 2002;3: 1017-1023. 203. Whiss PA. Pexelizumab Alexion. Curr Opin Invest Drugs 2002;3: 870-877. 204. Wang Y, et al. Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5. Proc Natl Acad Sci USA 1996;93:8563-8568. 205. Riley RD, et al. Recombinant human complement C5a receptor antagonist reduces infarct size after surgical revascularization. J Thorac Cardiovas Surg 2000;120:350-358. 206. Arumugam TV, et al. A small molecule C5a receptor antagonist protects kidneys from ischemia/reperfusion injury in rats. Kidney International 2003;63:134-142. 207. Sumichika H. C5a receptor antagonists for the treatment of inflammation. Curr Opin Invest Drugs 2004;5: 505-510. 208. Rother RP, Mojcik CF, McCroskery EW. Inhibition of terminal complement: A novel therapeutic approach for the treatment of systemic lupus erythematosus. Lupus 2004;13: 328-334.

products (C3d) in patients with systemic lupus erythematosus. Rheumatol Int 1985;5:215-220. 211. Liu CC, Denchenko N, Navratil JS, Nilson SE, Manzi S, Ahearn JM. Mining the complement system for lupus biomarkers. Clin Appl Immunol Rev 2005;5:185-206.

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MECHANISMS OF TISSUE DAMAGE

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Immune Complexes in Systemic Lupus Erythematosus Mark H. Wener, MD

INTRODUCTION Antigen-antibody complexes mediate much of the inflammation and tissue dysfunction associated with SLE. Deposition of ICs within tissues is responsible for glomerulonephritis and vasculitis and probably also for arthritis and some forms of cutaneous lupus. This chapter discusses the role of immune complexes in SLE.

BASIC IMMUNOCHEMISTRY OF IMMUNE COMPLEXES: THE PRECIPITIN CURVE

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The immunochemistry of immune complexes has been investigated over many decades.1 The classic precipitin curve demonstrates the importance of antigen/antibody ratios in determining the lattice formed by immune complexes in a typical antigen-antibody interaction. Adding increasing amounts of antigen to a constant amount of antibody demonstrates a curve with three general regions: the zone of antibody excess (“pro-zone”), the zone of equivalence, and the zone of antigen excess (“post-zone”). In some antigen-antibody systems, the pro-zone shows an extended region without precipitation. Immune complexes formed in the zone of far antigen or antibody excess are soluble. Large-lattice immune complexes containing IgG, formed at antigenantibody ratios close to the zone of equivalence, have multiple IgG Fc-regions available for interaction with C1q complement proteins and therefore activate complement efficiently. Immune complex lattice structure can be altered if there is interaction with complement proteins, because covalently bound complement peptides sterically inhibit immune complex interaction and extended lattice formation. Once immune precipitates are formed, their size can be reduced, leading to solubilization of preformed immunoprecipitates via activation of the alternative pathway of complement.2 Activation of the classical pathway of complement can inhibit immune complex growth by preventing extended lattice formation.3

Thus, in the presence of complement the precipitin curve can be more properly considered a precipitin “surface” in which either higher concentrations of complement components or antigen or antibody excess lead to smaller immune complexes or reduced immune precipitation (Fig. 21.1). Complement activation thus serves as a negative regulator of immune complex lattice extension. Hypocomplementemic sera from patients with SLE, in comparison with normal sera, fail to prevent formation of immune precipitates. This defective complementdependent prevention of immune precipitation is seen in early cases of SLE. Prevention of immune precipitation

Fig. 21.1 Modification of precipitin curve or precipitin “surface.” In the presence of complement, antigens and antibodies within immune complexes bind complement fragments (C’), preventing extension of nascent lattice formation or disrupting lattices, leading to smaller immune complexes. Y = antibody; 0 = antigen.

DISEASE ASSOCIATIONS

Immune Complexes and SLE Active renal SLE is associated with high serum concentrations of antidsDNA antibodies and enrichment of antiDNA within glomerular eluates of patients with SLE, supporting the role of antiDNA in the pathogenesis of SLE. DNA-antiDNA immune complexes are thought to be a central contributor to immune complex nephritis in SLE.6, 7 Several investigators have found evidence for circulating DNA-antiDNA immune complexes and other immune complexes in SLE patients and experimental models.8 Antibodies to the collagen-like region of C1q (anti-C1q) were also found to be concentrated within glomerular basement fragments isolated from kidneys of lupus patients.9 Together with data demonstrating a strong association between lupus nephritis and serum levels of anti-C1q, these data strongly implicate anti-C1q in the pathogenesis of lupus nephritis. Anti-C1q tends to be present if there are multiple autoantibodies (including antidsDNA, antiSSA, antiSm, or others) present and enriched in glomerular basement membrane fragments from kidneys of patients with SLE, suggesting a role for anti-C1q in promoting aggregation of immune complexes in the basement membrane.10 Assays for circulating immune complexes have been used to monitor SLE activity. Numerous studies have suggested that immune complex assays based on C1q binding or C3 content are positive in patients with SLE and can be helpful in assessing disease activity in patients with SLE.8 Assays for antidsDNA and complement components are more widely available than immune complex assays, and are similarly used to monitor disease activity and assist in the diagnosis of SLE. Therefore, measurement of immune complexes is not widely used clinically in comparison with those other measurements.

Immune Complexes and Vasculitis Using direct immunofluorescence microscopy, immunoglobulins and complement components can frequently be detected in vessels affected by some forms of vasculitis. In clinical situations, the antibody specificity of the deposited immunoglobulins is rarely determined, and therefore there is rarely direct evidence to conclude the source of the antigens recognized by those antibodies. The pattern of immunoglobulin deposition is most commonly used to infer the mechanism for antibody deposition. Immunoglobulins deposited in a smooth, linear, ribbon-like pattern are presumptively directed against high-density

continuous epitopes that are constitutive in the tissues (e.g., glomerular basement membrane collagen in patients with Goodpasture’s syndrome), whereas immunoglobulins with a discontinuous, discrete, granular distribution are presumed to be caused by immune complexes. The granular pattern is seen in SLE, and vasculitis contributes to the pathologic lesions in SLE.

PATHOPHYSIOLOGIC MECHANISMS

Immune Complexes as Initiators and Regulator of the Autoimmune Response Immune complexes have traditionally been thought of as having a critical role in causing tissue damage associated with SLE. The potential importance of immune complexes to initiate and enhance the immune dysregulation observed in SLE is also recognized. Immune complexes play a central role by harnessing the specificity of the acquired immune system (high-affinity autoantibodies) to augment the potent but less specific inflammatory response of the innate immune response (Fig. 21.2). Dysfunctional overactivity of the type I interferon system is considered a central factor in SLE.11-13 The cell that is the most potent producer of type I interferons is the plasmacytoid dendritic cell (pDC), and the trigger for type I interferon production by pDCs is typically viral DNA and/or RNA in response to infection. Immune complexes composed of nucleic acids and IgG from SLE patients are potent inducers of type I interferon production by pDCs.14 The nucleic acids responsible for interferon production can be released from apoptotic or necrotic cells. Efficient production of type I interferons by SLE IgG requires Fcγ receptors and intact IgG, whereas F(ab) or F(ab′)2 fragments of IgG are not sufficient.15 Thus, a key mechanism leading to sustained production of interferon in SLE is thought to be immune complexes containing nucleic acids, including most prominently DNA and antiDNA and possibly RNA and antibodies to RNA-protein complexes. Internalization of the immune complexes is mediated by Fcγ receptors, and the internalized nucleic acid then binds to toll-like receptors (TLRs), including TLR 9. Binding of characteristic CpG motifs to TLR9 induces cellular signaling pathways leading to enhanced interferon mRNA transcription and protein release. ICs binding to germinal center follicular dendritic cells (FDCs) facilitates antigen presentation by FDCs and thereby promotes the ability of FDCs to interact with B-cells and cause affinity maturation and class switching. It has been proposed that immune-complex-bearing FDCs may be required for development of high-affinity IgG antibodies, including antiDNA.16 Thus, nucleic acid containing immune complexes augments the autoimmune response in SLE by leading to sustained

PATHOPHYSIOLOGIC MECHANISMS

correlates positively with levels of C4A4 and inversely with the present of antibodies to C1q.5

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IMMUNE COMPLEXES IN SYSTEMIC LUPUS ERYTHEMATOSUS Fig. 21.2 Role of immune complexes in augmenting immune response and causing tissue damage in SLE. Immune complexes deposit in target organs and tissues, activate complement, and lead to tissue damage (including release of DNA, nuclear material, and cell debris as source of antigens). Immune complexes can be formed by the apoptotic and necrotic cell and nuclear antigens combining with antibodies directed against those antigens, which can include high-CpG islands, modified areas of mammalian DNA, and other potential pDC activators. Anti–nucleic-acid antibodies also combine with DNA and RNA from viruses and other exogenous sources of activating DNA. Facilitated by Fc γ receptor binding of immune complexes, activating DNA and RNA bind to TLRs and markedly up-regulate production of type I interferon by pDCs (considered a central feature of the altered inflammatory and immune milieu is SLE). Type I pDCs secrete cytokines that activate B-cells and T-cells, increasing the immune response. In germinal centers, immune complexes interact with FDCs, also augmenting the response of T-cells and B-cells (green arrows). Activated B and T lymphocytes together lead to production of inflammatory cytokines, high levels of high-affinity autoantibodies, and generation of a mature autoimmune response.

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overproduction of type I interferon and affinity maturation and continued production of antiDNA and related autoantibodies. Depressed clearance of immune complexes, as discussed in material following, may magnify this response by leading to higher levels of circulating immune complexes and/or higher binding of immune complexes to dendritic cells. The type I interferon expression profile correlates with SLE disease activity, in general, with particularly close correlation with the presence and levels of autoantibodies to nucleic acids.17,18 These correlations have been used to support the notion that type I interferon overexpression leads to SLE. Conversely, this association also lends support to the hypothesis that immune complexes containing autoantibodies to nucleic acids lead to the SLE phenotype by causing enhanced type I interferon production. Whether the type I interferon profile is caused by antibodies to nucleic acids or vice versa, the association between the two types of abnormalities suggests an active role of both in the immune and inflammatory dysregulation found in SLE.

Immune Complex Clearance The mononuclear phagocyte system plays the central role in removing immune complexes from the circulation, with clearance mediated by families of Fc and complement receptors on mononuclear phagocytes, neutrophils, and other cells. The presence of C3 receptors on primate erythrocytes but not erythrocytes from other species suggests a trafficking mechanism applicable to humans but not to non-primate experimental animals.19,20 Immune complexes that had activated complement and bound C3 in the circulation could bind to the complement receptor CR1 on the erythrocyte would be transported to the liver and spleen while bound to the red cell, and those immune complexes would be phagocytized by cells of the mononuclear phagocyte system (primarily via Fc receptors). In the liver, Kuppfer cells serve this phagocytic role. In the spleens of humans and some other species (but not mouse, rat, guinea pig, or rabbit), splenic filtering and immune complex trapping may be carried out at least in part in splenic ellipsoids, which are structures

indicating that complement modulates immune complex clearance by other mechanisms. Davies and colleagues27 administered murine IgG and human antimouse IgG to study immune complexes formed in vivo, an experiment that might be considered most representative of natural physiology. Patients with ovarian carcinoma were given 131I-murine monoclonal antitumor antibodies and subsequently 125 I-human antimouse IgG. Immune complexes were large but of a size possibly to be encountered physiologically. Soluble immune complexes formed within 5 minutes, activated complement, and were cleared with a half-life of 11 minutes in the liver and without a detectable increase in radioactivity over the spleen. Between 8 and 11% of the total available immune complexes bound to the erythrocyte, and at the time of peak red cell binding erythrocyte-bound immune complexes constituted approximately 20% of total circulating complexes. The majority of soluble immune complexes were cleared by mechanisms largely independent of red cells, and the site of clearance of these soluble complexes in the liver differed substantially from the splenic clearance of sensitized erythrocytes previously reported.28 In SLE patients, several studies have shown that the clearance of antibody-sensitized erythrocytes is slower than the clearance in normal controls, and slower in patients with active renal disease than in those without.29,30 Investigators at Leiden have administered radioiodinated aggregated human IgG (123I-AHG) to SLE patients to explore the fate of circulating soluble immune complexes in patients with SLE. The investigators described an initial rapid clearance and later slower clearance of immune complexes from the circulation (both reported in terms of the time to removal of 50% of the maximum material, T1/2). In their first study, the authors reported that the initial phase T1/2 was not significantly different between SLE patients and controls, whereas the second phase T1/2 was prolonged in the patient group.31 In the second study, SLE patients erythrocytes were observed to have a decreased number of CR1, which was associated with less binding of AHG to red blood cells and with a faster initial rate of clearance of AHG (mean half-time to removal 5.2 ±0.2 minutes in patients versus 6.6 ±0.2 minutes in controls, p = 0.01). The later phase of AHG clearance was similar in patients and controls (T1/2 148 ±18 versus 154 ±20 minutes). Both the maximum liver uptake and time required to reach the maximum liver uptake were similar in SLE patients and controls. Of interest, the feature most predictive of the rate of AHG clearance in SLE patients was the serum IgG concentration, which was inversely correlated (r = -0.66) with the rate of clearance. The authors speculated that the concentration of serum IgG in SLE

PATHOPHYSIOLOGIC MECHANISMS

consisting of specialized capillary segments surrounded by macrophages.21 A variety of probes have been employed to determine the kinetics and sites of immune complex clearance experimentally in humans. Investigators have used erythrocytes coated with IgG antibodies, aggregated IgG, preformed immune complexes, and antigens infused into preimmunized subjects. Davies and colleagues have performed studies using several different soluble immune complexes as probes, including tetanus/antitetanus, hepatitis B surface antigen/ antibodies, and murine IgG/human anti-mouse IgG.22 The former two types of immune complexes were formed in vitro and then injected into subjects. When soluble immune complexes of hepatitis surface antigen and antibody are made intentionally “small” (such as not to fix complement efficiently and which therefore do not bind to complement receptors on red cells), >90% are cleared by the liver [with a median clearance half-time of approximately 3 minutes (range 1 to 6 minutes)].23 The clearance half-time did not differ between normal individuals and subjects with SLE. Approximately 2 to 6% of these non-complementfixing immune complexes were cleared in the spleen, with no difference observed between SLE patients and normal individuals. In contrast to the normal removal of immune complexes observed in SLE patients, the fate of immune complexes in the liver was observed to be abnormal. The radiolabeled immune complexes were removed from the liver faster in SLE patients than in normal individuals, and in SLE patients there were significantly more intact IgG-containing immune complexes at later time points (after 1 and 4 hours), indicating release of immune complexes from the liver. These data suggest that retention and catabolism of immune complexes within the liver was impaired in SLE, leading to recirculation of intact immune complexes after release from the liver. In other studies, complement depletion led to accelerated clearance of immune complexes by the liver and spleen and might have been associated with increased tissue deposition of immune complexes,24 which suggested to the authors that red cell binding of immune complexes could have role in “buffering” excessive loads of immune complexes until they are removed by mononuclear phagocytes. Others have suggested that erythrocyte binding of immune complexes could have a role in immune complex processing or degradation while on the erythrocyte.25 However, C1q-deficient mice also demonstrate an initial accelerated hepatic clearance of immune complexes and reduced splenic clearance.26 Because mice lack erythrocyte complement receptors, the accelerated hepatic uptake in C1qdeficient mice is not likely to depend on erythrocytes,

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patients was a primary determinant of the proportion of Fc receptors occupied, and thereby governed the rate of clearance of AHG.32 The importance of the rapid, very early removal of immune complexes from the circulation was shown by Schifferli and colleagues, who examined the clearance of immune complexes composed of tetanus toxin and anti-tetanus in 4 patients with SLE, as well as 11 other patients and 9 normal subjects.33 The authors reported that the removal of these large complexes from the circulation occurred in two phases: a very rapid “trapping” phase that occurred within the first minute and a monoexponential later phase. In 1 of 9 normal individuals and 11 of 15 patients, over 8% of the injected immune complexes were removed from the circulation (“trapped”) within the first minute after administration, a time point and amount removed that could not be attributed to clearance by the liver and spleen and therefore trapping presumably resulted in deposition of immune complexes in peripheral tissues. This initial trapping was seen in patients with serum complement deficiencies, and was associated with lower levels of CR1 on erythrocytes. The later phase of immune complex clearance was exponential over the 60 minutes of measurement, with between 9.9 and 18.7% removed per minute in normals and 8.6 to 32.2% per minute removed in patients. When opsonized immune complexes bound in vitro to erythrocytes via CR1 were injected into patients there was release of 10 to 81% of the immune complexes from the erythrocytes within 1 minute of injection. The extent of this release was inversely correlated with CR1 number/cell. Together, these studies of clearance of soluble immune complexes in SLE patients argue that the hepatic clearance of immune complexes (which governs the late-phase removal of soluble immune complexes) is probably normal in SLE patients. Low CR1 numbers on erythrocytes or profound hypocomplementemia can permit deposition of immune complexes within tissues during the early phase of immune complex clearance. Reduction in CR1 numbers is an acquired abnormality associated with active SLE.34 It is unclear the degree to which the abnormalities in immune complex clearance mechanisms observed in these experiments contributes to immune complex deposition at sites of tissue injury. More recently, investigations have explored the implications of polymorphisms in various Fcγ receptors with regard to their potential role in clearing immune complexes from the circulation and causing a predisposition to SLE. Lack of the H131 allele of the FcγRIIA, which is responsible for efficient clearance of IgG2-containing immune complexes, has been associated with lupus nephritis in American blacks.35 A report

has implicated a functionally important genetic polymorphism of FcγRIIIA as a risk factor for SLE in a genetically diverse group of patients.36

Factors Governing Immune Complex Localization: Physicochemical Composition and Site of Formation Exploration of non-primate animal models of serum sickness demonstrated that a critical characteristic of circulating immune complexes that governed their clearance and deposition in tissues was their size or the extent of lattice formation. The lattice of an immune complex, defined as the number of antigen and antibody molecules in a given immune complex, governs the number and density of Fc regions in an immune complex (and thereby its ability to interact with Fc receptors and/or activate Fc-dependent functions). Large-lattice soluble immune complexes (>Ag2Ab2) tended to be cleared rapidly by the mononuclear phagocyte system, primarily by Fc receptors on the Kuppfer cells in the liver. If the mononuclear phagocyte system was saturated or blocked, these immune complexes would deposit in tissues (e.g., in the mesangial and subendothelial regions of the glomerular basement membrane). In comparison, small-lattice complexes (Ag2Ab2 or smaller) tended to have more prolonged time in the circulation. However, they had a lower tendency to deposit in tissues. Activation of complement proteins was also known to be size dependent, with complement activation occurring much more efficiently with larger-lattice immune complexes. In these rodent experimental systems, complement receptors play a role in removing immune complexes only if the immune complexes are very large. In experimental models, administration of preformed immune complexes results in mesangial and subendothelial localization of immune complexes within renal glomeruli. Studies in the 1980s using the Heymann model of nephritis and studies on isolated perfused kidneys emphasized that antibodies and antigens could deposit sequentially in the kidney, with the result that the immune complexes form in situ and tend to localize in the subepithelial region of glomeruli rather than being deposited from circulation.37 Formation of complexes in situ can occur because of direct binding of antigens or antibodies, initially because of interaction between the circulating molecule and structures within the kidney. This initial interaction can be relatively weak and/or nonspecific (e.g., because of charge-charge interactions). Electrical charge on either the antigen or the antibody within the immune complex governs interaction with fixed negative charges on proteoglycans in the basement membrane or in other structures and influences

IMMUNE COMPLEX REARRANGEMENT AND PERSISTENCE Circulating immune complexes are probably forming frequently in normal individuals by absorption of antigens from the gut with binding to serum antibodies. Only a minority of immune complexes escape uptake by the mononuclear phagocyte system and deposit in tissues, and only a minority of the immune complexes in tissues cause identifiable disease. Many immune complexes that deposit in target organs, such as the kidney, are present only for a few hours and then cleared. To develop immune deposits that are more persistent and visible as typical subendothelial or subepithelial electron dense deposits within the kidney, immune deposits of small or intermediate size that might deposit from the circulation into tissues must coalesce or rearrange to form larger immune deposits.51 This rearrangement may not occur between immune complexes composed of different antigens or antibodies that do not cross-react, in that they would not form a large-lattice immune deposit. Rearrangement of immune complexes may be associated with movement of deposits within the kidney. In an experimental rat immune complex model using immune deposits that could be localized by electron microscopy, it could be shown that immune deposits initially were formed and coalesced in subendothelial locations, and then moved to subepithelial locations, where they again underwent rearrangement.52 The solubilization of these complexes was associated with binding of C3, and the authors suggested that C3 solubilization of precipitates within the basement membranes facilitated the immune complex rearrangement. A hallmark of SLE is the wide variety of antigenantibody systems within a single individual. Although ample evidence indicates that DNA-antiDNA comprise the major antigen-antibody system in SLE, other antibodies may be present and enriched within the glomeruli. For example, glomerular enrichment of antibodies to the SSA/Ro antigen has been described,53 and multiple other antibodies are also present.10 Immune complexes and aggregated IgG may bind nonspecifically to histones,54 thus augmenting the formation of larger and more diverse immune deposits once histones are present. Additional factors contribute to persistence of immune deposits in tissues. Covalent cross-linking of antigens and antibodies to each other or to tissue antigens in kidneys and articular cartilage has been described,55,56 potentially leading to persistence of antigens and chronic inflammatory mechanisms at those sites. A mechanism has been proposed to explain immune complex covalent cross-linking.55,56 Activation of neutrophils leads to formation of reactive oxygen species by neutrophils,

IMMUNE COMPLEX REARRANGEMENT AND PERSISTENCE

both the deposition and persistence of antigens, antibodies, and immune complexes in tissues. In experimental systems, even a small proportion of positively charged (cationic) antibodies enhance binding and persistence of immune complexes in renal glomeruli.38 Deposition of antigens or antibodies could be augmented or facilitated also by antigen-specific receptors within the tissues. Particularly relevant for the study of SLE, Emlen and Burdick demonstrated that immune complexes containing DNA may be removed in part by DNA receptors.39 In experimental animals, the clearance of immune complexes containing glycosylated antigens is governed in part by specific carbohydrate receptors on hepatocytes.40 A serum carbohydrate binding protein, mannose-binding protein (MBP), may have an important role in clearing immune complexes containing antigens with selected carbohydrate residues. A member of the collagen motif-containing collectin family of proteins, MBP binds terminal mannose, fucose, glucose, fucose, or N-acetylglucosamine residues; can activate the classical or alternative pathways of complement;41 can activate macrophages via the C1q receptor;42 and can serve as an opsonin.43 Genetic polymorphisms responsible for depressed function and serum levels of MBP are associated with SLE in African Americans44 and other groups.45,46 Furthermore, certain ribonucleoprotein autoantigens (including the U1-specific 68kD and A proteins and the U2-specific B′ protein) are glycoproteins, with mannose, glucose, and N-acetylglucosamine detected on the 68kD protein.47 Thus, it is conceivable that the clearance of glycoprotein antigens or immune complexes containing such antigens (including the U1-RNP particle) could be influenced by MBP polymorphisms. These considerations suggest that MBP polymorphisms could participate in the pathogenesis of SLE by influencing immune complex clearance, analogous to the role of polymorphisms in complement components and FcR. Features on the antibodies within the immune complex can influence the physiology of immune complexes. The isotype of antibodies influences immune complex handling, in that the ability to activate complement influences both the ability to bind to complement receptors as well as to activate inflammatory cascades. AntidsDNA in SLE patients tend to be of subclasses IgG1, IgG2, and IgG3, and tend to be efficient in activation of complement.48 Experimental studies with murine monoclonal IgG3 immunoglobulins have demonstrated that deposition of cryoprecipitating or other self-associating immunoglobulin aggregates, a feature of certain immunoglobulin molecules, may cause glomerulonephritis.49 IgA-containing immune complexes may be cleared by distinct IgA receptors.50

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and activation of chrondrocytes leads to the formation of highly reactive nitric oxide by chondrocytes. These highly reactive molecules were shown to cause covalent cross-linking of antigen-antibody complexes on plastic surfaces. Similar mechanisms could be present in tissues.

Autoantibodies to the Collagen-Like Region of C1q Antibodies to C1q also augment aggregation of immune complexes in tissues. AntiC1q antibodies are found in association with lupus nephritis, and are less commonly demonstrable in serum of patients with nonrenal lupus.57,58 Rising concentrations of IgG antiC1q are generally associated with flares of lupus nephritis, and high levels of antiC1q are associated with proliferative forms of lupus glomerulonephritis. Antibodies to the collagen-like region of C1q are present and enriched in the glomeruli of many patients with lupus whose kidneys were examined at autopsy, and were associated with proliferative lupus nephritis.9 The fact that these antibodies were released under acid conditions suggests that they were present in the form of immune complexes. Release by DNAse suggests that the immune deposits also contained immune complexes composed of DNA and antiDNA, which then bound C1q and in turn antiC1q. Together, the clinical associations of antiC1q with active lupus nephritis and the data demonstrating that antiC1q is present and enriched in glomeruli strongly argue that antibodies to C1q play a pathogenic role in the proliferative forms of lupus nephritis. By binding to different molecules of C1q that have bound to immune complexes composed of different antigen-antibody systems, antibodies to C1q could promote aggregation of those different types of immune complexes, leading to larger, more persistent, and more pathogenic immune deposits. AntiC1q tends to be found in lupus kidneys when multiple autoantibodies are identified in the kidney, supporting the idea that antiC1q could be promoting the aggregation of different antigen-antibody systems.10 A murine model employing monoclonal anti-murine C1q has demonstrated that antiC1q contributes to the pathogenesis of experimental glomerulonephritis only if C1q-containing immune complexes have already deposited in the kidney.59 Thus, antiC1q augments pathogenic complement activation in the kidney but by itself does not appear to be sufficient to cause glomerulonephritis.

Tissue Effects of Immune Complexes

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Once deposited in tissues, immune complexes cause inflammation. Complement-mediated injury has been considered the dominant mechanism responsible. Clinically and experimentally, activation of complement can be demonstrated in serum, at tissue sites, and in urine. The well-known pro-inflammatory chemotactic

role of complement fragments is believed to lead to recruitment of inflammatory cells into the lesion. Larger lattice immune complexes, with a higher density of Fc regions, activate complement more efficiently. Release of pro-inflammatory cytokines from inflammatory cells is also greater for large-lattice immune complexes in synovial fluids than in smaller complexes.60 Nephrotoxic serum nephritis, a form of experimental nephritis probably caused by immune complexes that form in situ, could be improved substantially by administration of soluble complement receptor-1related gene/protein y (Crry), a potent complement inhibitor.61 Overexpression of the Crry protein in a transgenic mouse model also reduced this form of nephritis.62 Depletion of C5a function by antibody63 or through knockout of the C5a receptor gene64 demonstrated dependence of pulmonary immune complex disease on C5a. IgG Fc receptors (FcγR) have an important role in mediating immune complex disease. These data have been reported in a series of papers, largely using mice generated by Ravetch that lack the transmembrane signal-transducing gamma chain found on IgG FcRI and FcR III. The susceptibility to murine lupus nephritis65 and to collagen-induced arthritis was found to be altered in FcR-/- mice,66 indicating that similar mechanisms were important in these diseases. In a murine model of immune complex peritonitis, neutrophil migration was attenuated after complement depletion but totally abolished in mice lacking the FcR gamma chain. Additional data suggested that engagement of FcRIII did not lead to neutrophil recruitment and that engagement of FcRI was most important in causing inflammatory exudates.67 It has been proposed that local microenvironments within different tissues could influence expression of FcR on macrophages at different sites, thus modulating the local inflammation and other tissue effects of circulating or deposited immune complexes.68 Immune complexes themselves have a variety of other immunomodulatory effects. For example, binding of immune complexes to Fc receptors leads to aggregation of those receptors, triggering intracellular signaling pathways.69 Immune complexes augment the responsiveness of both B-cells and T-cells to antigen stimulation.70 Chromatin-containing immune complexes can augment activation of B-cells by engagement of Toll-like receptors such as TLR9.71 Cell activation by chromatin-containing immune complexes involves other mechanisms.72

Development of Therapies Based on the Immune Complex Model The immune complex model for the cause of tissue damage in SLE has been the dominant paradigm for several decades, and it remains so. Therapeutic approaches

Importance of Antigen within Circulating Immune Complexes Whereas in the classic serum sickness immune complex model the antigen in the immune complex (often heterologous serum albumin in experimental systems) bears little relevance to the resultant pathology, in SLE and other human immune complex diseases the antigen constituents within the immune complex could influence the pattern of clinical sequelae and the risk for cardiovascular disease. For example, antiphospholipid antibodies were found to be enriched in circulating immune complexes in patients with the antiphospholipid syndrome, with or without coexistent SLE.74 In these studies, aliquots of sera that were unfractionated or were fractionated by gel filtration or sucrose density gradients were analyzed for the presence of antiphospholipid antibodies. The relative concentration of anticardiolipin antibodies was up to 125 times higher in high-molecular-weight (HMW) fractions, compared with the antibody activity in unfractionated serum. Furthermore, in some sera minimal levels of antiphospholipid antibodies were detectable in the unfractionated serum, whereas high levels of antibodies to negatively charged phospholipids were found in the HMW fractions. The binding avidity of antiphospholipid antibodies was substantially higher in the immune complex fractions compared with the unfractionated sera, as assessed by binding curves and elution studies. Different types of immune complexes differ in their ability to bind to and activate platelets.75 Thus, antiphospholipid-containing immune complexes could augment the tendency of antiphospholipid antibodies to cause thrombosis and enhance vascular disease. Because antiphospholipid antibodies bind to other families of lipids, including oxidized low-density lipoproteins (LDLs),76 it is possible that the HMW antiphospholipid antibodies were part of immune complexes comprised of lipoproteins. Immune complexes containing antibodies to lipoproteins known to be associated with atherogenesis could play a role in development of coronary artery disease. Hasunama and colleagues found that the anticardiolipin cofactor β2-glycoprotein I (β2-GPI) bound preferentially to oxidized plasma lipoproteins [i.e., oxidized (ox)VLDL, oxLDL, and oxHDL] in comparison with the native forms of the lipoproteins.77 Antibodies to β2-GPI bound to the β2-glycoprotein I-oxLDL complex. Whereas binding of β2-GPI to oxLDL inhibited the uptake of oxLDL by macrophages, the uptake was

enhanced in the presence of immune complexes containing antiβ2-GPI and β2-GPI-oxLDL complexes. Uptake of oxLDL by macrophages predisposes to the formation of foam cells, leading to intimal disease and atherosclerosis. Thus, the enhanced uptake caused by lipoprotein-containing immune complexes could contribute to accelerated atherosclerosis78 and to immune complex disease. Given the growing importance of coronary disease and of the antiphospholipid syndrome in SLE, the role of immune complexes in those manifestations bears further investigation. As discussed previously, immune complexes containing nucleic acids have an important role in inducing production of type I interferon by dendritic cells, and thus may play a central role in the pathogenesis of SLE.14 Immune complexes containing DNA and nucleosomes can also bind to histones, which in turn can bind to negatively charged proteoglycans in the glomerular basement membrane, promoting deposition of DNAor nucleosome-containing immune complexes or in situ formation of those immune complexes.79-81 DNA and nucleosomes and/or immune complexes with those constituents are present in basement membranes of glomeruli82 and skin83 of patients with lupus, providing support for the importance of these nucleosome and DNA-containing immune complexes in SLE. In addition, immune complexes can bind nonspecifically to histones.

CLINICAL ASSAYS FOR CIRCULATING IMMUNE COMPLEXES

based on this paradigm, however, have been relatively disappointing. For example, whereas plasmapheresis for the treatment of SLE originally met with great enthusiasm, a controlled clinical trial of plasmapheresis in patients with lupus nephritis was unsuccessful.73

CLINICAL ASSAYS FOR CIRCULATING IMMUNE COMPLEXES A variety of assays for circulating immune complexes have been developed.84 The most commonly used assays include those based purely on the physical chemistry of immune complexes (e.g., tests depending on polyethylene glycol-induced precipitation of tests for cryoglobulins; i.e., cold-precipitating immunoglobulins), those dependent on binding to C1q, and those detecting the presence of IgG-C3 complexes either by employing cellular C3 receptors (Raji cell assay) or antigenic recognition. A problem with interpretation of results of immune complex assays is that they can give positive results when antibodies directed against the recognition moieties bind to those moieties as specific antibodies rather than in antigen-nonspecific immune complex interactions. For example, with the Raji cell assay antilymphocyte antibodies from some patients with systemic lupus erythematosus could give a positive result, recognizing antigens on the Raji cell as targets. Use of the C1q solid-phase assay allows autoantibodies directed against C1q to bind and give positive results, even in the absence of immune complexes. Because circulating immune complexes and antiC1q are both detected in standard C1q solid-phase binding assays, data associating positive test results with either immune complexes or antiC1q must be interpreted

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with caution.85 Similarly, autoantibodies to C3 components are frequently present in the sera of patients with SLE and related disorders, and could lead to positive results in immune complex assays based on recognition of C3. In the antiC3 assay, serum antibodies directed against the F(ab′)2 fragments of antiC3 used to detect the C3-bearing immune complexes have been reported to cause positive assay results. In these examples, the positive results are “false positive” in that the results are not caused by immune complexes, yet clinically useful results may be obtained (see material following). However, because the assays may become positive because of the presence of pathologic substances in addition to immune complexes investigators employing these assays and using them to make conclusions about circulating immune complexes should confirm that immune complexes are responsible for the positive results observed.85 One of the challenges of using the clinical assays for circulating immune complexes is that the lack of concordance between the methods has made interpretation of results difficult.86 Because of differences in the immunochemical properties of immune complexes and differences in principles of detection with the different methods, these differences may not be unexpected. Furthermore, pathogenically important immune complexes may not be present in the serum specimens usually analyzed, but may be deposited in peripheral tissues, carried on erythrocytes via their CR1 during their transit through the circulation, or lost during specimen handling. Immune complexes bound to circulating erythrocytes may be released into plasma during incubation, whereas they remain on the erythrocytes if serum is the specimen to be analyzed. Furthermore, even attempts to directly quantify erythrocyte-bound IgG, an approach that has the potential to measure immune complexes bound to cells, may be problematic because of the fact that CR1-bound IgG is relatively inaccessible to a variety of probes. Immune complexes contained within cryoglobulins are well recognized as potentially being diminished if the specimen is allowed to cool. Preanalytical factors (i.e., handling of clinical specimens prior to actual assay) and choices of specimen (serum, plasma, or erythrocyte) may substantially influence the results reported from any given patient, and these factors are often not carefully addressed.

Detection of Immune Complexes: Technical Issues for the Clinician

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Analyzing tissue biopsies using direct immunofluorescence microscopy, immunoglobulins, and complement components are routinely identified within vessels affected by some forms of vasculitis. The detection of these immune deposits depends in part on technical issues. Biopsy material ideally should be obtained from

new “fresh” lesions because immune deposits are transient and may be undetectable in older lesions. A portion of tissue biopsies should be snap-frozen to prevent degradation of immune deposits. For some biopsies, such as small punch biopsies of skin, one specimen may be obtained for routine histology and a second obtained for freezing and immunofluorescence studies. Specimens should be sent to an experienced laboratory because background staining, specificity of antibodies, and other factors can influence interpretation of results. Circulating immune complexes have been measured by a multitude of techniques, few of which are available to most clinicians.86 For the clinician, probably the most important immune complex assay is the assay for cryoglobulins. The test for cryoglobulins is frequently inaccurate because of problems with specimen handling, in that blood should be allowed to remain at 37° C while it clots and should be kept warm while the clot is centrifuged. Phlebotomists and laboratory personnel should be alerted to the possible presence of cryoglobulins, and should be reminded of the special handling requirements.

CONCLUSIONS Inflammation caused by immune complexes in tissues remains the single most important mechanism for clinical manifestations of SLE. Although substantial progress is being made investigating genetic contributions to clearance mechanisms of immune complexes, questions remain about the site and mechanism of immune complex formation and about factors that influence localization and pathogenicity at different sites. Mechanisms responsible for rearrangement and condensation (the process by which transient, probably nonpathogenic, immune complexes become sustained and pathogenic in SLE) also remain largely unexplored. Although the role of antiDNA as a contributor to lupus immune complex disease has been studied, the role of other antibodies (such as those directed to nucleoprotein complexes, C1q, and phospholipids) as constituents of immune complexes remains another relatively unexplored area of investigation. The relative role of complement and Fc receptor activation in the pathogenesis of immune complex disease is controversial. Although immune complexes are one of the fundamental causes of inflammation in autoimmune rheumatic diseases, many mysteries remain concerning their pathophysiology.

ACKNOWLEDGMENTS The author gratefully acknowledges informative and helpful discussions with Mart Mannik and numerous other colleagues concerning this topic. Fig. 21.2 was expertly prepared by Grace Klein.

1. Day E. Immune complexes. In Advanced Immunochemistry, Second Edition. New York: Wiley-Liss 1990:397-467. 2. Czop J, Nussenzweig V. Studies on the mechanism of solubilization of immune precipitates by serum. J Exp Med 1976; 143:615-630. 3. Johnson A, Harkins S, Steward MW, Whaley K. The effets of immunoglobulin isotype and antibody affinity on complementmediated inhibition of immune precipitation and solubilization. Mol Immunol 1987;24:1211-1217. 4. Arason GJ, Steinsson K, Kolka R, Vikingsdottir T, D’Ambrogio MS, Valdimarsson H. Patients with systemic lupus erythematosus are deficient in complement-dependent prevention of immune precipitation. Rheumatology (Oxford) 2004;43:783-789. 5. Arason GJ, Kolka R, Hreidarsson AB, et al. Defective prevention of immune precipitation in autoimmune diseases is independent of C4A*Q0. Clin Exp Immunol 2005;140:572-579. 6. Koffler D, Agnello V, Thoburn R, Kunkel HG. Systemic lupus erythematosus: Prototype of immune complex nephritis in man. J Exp Med 1971;134:169s-179s. 7. Hahn BH. Antibodies to DNA. N Engl J Med 1998;338:1359-1368. 8. Wener MH. Immune complexes and autoantibodies to C1q. In Kammer G, Tsokos G (eds.), Lupus Molecular and Cellular Pathogenesis. Totowa, NJ: Humana Press 1999:574-598. 9. Mannik M, Wener MH. Deposition of antibodies to the collagenlike region of C1q in renal glomeruli of patients with proliferative lupus glomerulonephritis. Arthritis Rheum 1997;40:1504-1511. 10. Mannik M, Merrill CE, Stamps LD, Wener MH. Multiple autoantibodies form the glomerular immune deposits in patients with systemic lupus erythematosus. J Rheumatol 2003;30:1495-1504. 11. Baechler EC, Gregersen PK, Behrens TW. The emerging role of interferon in human systemic lupus erythematosus. Current Opinion Immunol 2004;16:801-807. 12. Bennett L, Palucka AK, Arce E, et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 2003;197:711-723. 13. Rönnblom L, Alm GV. Systemic lupus erythematosus and the type I interferon system. Arthritis Res Ther 2003;5:68-75. 14. Lovgren T, Eloranta ML, Bave U, Alm GV, Ronnblom L. Induction of interferon-alpha production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG. Arthritis Rheum 2004;50:1861-1872. 15. Bave U, Magnusson M, Eloranta ML, Perers A, Alm GV, Ronnblom L. Fc gamma RIIa is expressed on natural IFN-alpha-producing cells (plasmacytoid dendritic cells) and is required for the IFN-alpha production induced by apoptotic cells combined with lupus IgG. J Immunol 2003;171:3296-3302. 16. Aydar Y, Sukumar S, Szakal AK, Tew JG. The influence of immune complex-bearing follicular dendritic cells on the IgM response, Ig class switching, and produciton of high affinity IgG. J Immunol 2005;174:5358-5366. 17. Dall’era MC, Cardarelli PM, Preston BT, Witte A, Davis JC Jr. Type I interferon correlates with serological and clinical manifestations of SLE. Ann Rheum Dis 2005;64:1692-1697. 18. Kirou KA, Lee C, George S, et al. Coordinate overexpression of interferon-alpha-induced genes in systemic lupus erythematosus. Arthritis Rheum 2004;50:3958-3967. 19. Cornacoff JB, Hebert LA, Smead WL. Primate erythrocyte immune complex clearing mechanism. J Clin Invest 1983;71:236-247. 20. Kimberly RP, Edberg JC, Merriam LT, Clarkson SB, Unkeless JC, Taylor RP. In vivo handling of soluble complement fixing Ab/dsDNA immune complexes in chimpanzees. J Clin Invest 1989;84:962-970. 21. Sørby R, Wien TN, Husby G, Espenes A, Landsverk T. Filter function and immune complex trapping in splenic ellipsoids. J Comp Path 2005;132:313-321. 22. Davies KA. Michael Mason Prize Essay 1995. Complement, immune complexes and systemic lupus erythematosus. Br J Rheumatol 1996;35:5-23. 23. Davies KA, Robson MG, Peters AM, Norsworthy P, Nash JT, Walport MJ. Defective Fc-dependent processing of immune complexes in patients with systemic lupus erythematosus. Arthritis Rheum 2002;46:1028-1038.

24. Waxman FJ, Hebert LA, Cornacoff JG, et al. Complement depletion accelerates the clearance of immune complexes from the circulation of primates. J Clin Invest 1984;67:1329-1340. 25. Medof ME, Prince GM. Immune complex alterations occur in the human red blood cell membrane. Immunology 1983;50:11-18. 26. Nash JT, Taylor PR, Botto M, Norsworthy PJ, Davies KA, Walport MJ. Immune complex processing in C1q-deficient mice. Clin Exp Immunol 2001;123:196-202. 27. Davies KA, Hird V, Stewart S, et al. A study of in vivo immune complex formation and clearance in man. J Immunol 1990; 144:4613-4620. 28. Frank MM, Lawley TJ, Hamburger MI, Brown E. Immunoglobulin G Fc receptor-mediated clearance in autoimmune disease. Ann Intern Med 1983;98:206-218. 29. Kimberly RP, Parris TM, Inman RD, McDougal JS. Dynamics of mononuclear phagocytes system Fc receptor function in systemic lupus erythematosus: Relation to disease activity and circulating immune complexes. Clin Exp Immunol 1983;51:261-268. 30. Van der Woude F, Van der Giessen M, Kallenberg G, et al. Reticuloendothelial Fc receptor function in SLE patients. I. Primary HLA linked defect or acquired dysfunction secondary to disease activity. Clin Exp Immunol 1984;55:473-480. 31. Lobatto S, Daha M, Breedveld F, et al. Abnormal clearance of soluble aggregates of human immunoglobulin G in patients with systemic lupus erythematosus. Clin Exp Immunol 1988;72:55-59. 32. Halma C, Breedveld F, Daha M, et al. Elimination of soluble 123I-labeled aggregates of IgG in patients with systemic lupus erythematosus: Effect of serum IgG and number of erythrocyte complement receptor type I. Arthritis Rheum 1991;34:442-452. 33. Schifferli JA, Ng YC, Paccaud J-P, Walport MJ. The role of hypocomplementemia and low erythrocyte complement receptor type 1 numbers in determining abnormal immune complex clearance in humans. Clin Exp Immunol 1989;75:329-335. 34. Walport M, Ross G, Mackworth-Young C, Watson J, Hogg N, Lachmann P. Family studies of erythrocyte complement receptor type 1 levels: Reduced levels in patients with SLE are acquired, not inherited. Clin Exp Immunol 1985;307:981-986. 35. Salmon JE, Millard S, Schachter LA, et al. Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans. J Clin Invest 1996;97:1348-1354. 36. Wu J, Bansal V, Redecha P, Salmon J, Edberg J, Kimberly R. A novel polymorphism of FcgRIIIA, which alters function, associates with the SLE phenotype (abstr). J Invest Medicine 1997;45:200A. 37. Nangaku M, Couser WG. Mechanisms of immune-deposit formation and the mediation of immune renal injury. Clin Exp Nephrol 2005;9:183-191. 38. Gauthier VJ, Mannik M. A small proportion of cationic antibodies in immune complexes is sufficient to mediate their deposition in glomeruli. J Immunol 1990;145:3348-3352. 39. Emlen W, Burdick G. Clearance and organ localization of small DNA anti-DNA immune complexes in mice. J Immunol 1988; 140:1816-1822. 40. Finbloom D, Magilavy D, Hartford J, et al. The influence of antigen on immune complex behavior in mice. J Clin Invest 1981;68:214. 41. Matsushita M, Fujita T. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J Exp Med 1992;176: 1497-1502. 42. Tenner A, Robinson S, Ezekowitz R. Mannose binding protein enhances mononuclear phagocytic function via a receptor that contains the 126,000 Mr component of the C1q receptor. Immunity 1995;3:485-493. 43. Kawasaki M, Kawasaki T, Yamashura I. Isolation and characterization of a mannose-binding protein from human serum. J Biochem 1983;94:937-942. 44. Sullivan KE, Wooten C, Goldman D, Petri M. Mannose-binding protein genetic polymorphisms in black patients with systemic lupus erythematosus. Arthritis Rheum 1996;39:2046-2051. 45. Davies EJ, Snowden N, Hillarby MC, et al. Mannose-binding protein gene polymorphism in systemic lupus erythematosus. Arthritis Rheum 1995;38:110-114.

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67. Heller T, Gessner J, Schmidt R, Klos A, Gautsch W, Kohl J. Cutting edge: Fc receptor type I for IgG on macrophages and complement mediate the inflammatory response in immune complex peritonitis. J Immunol 1999;162:5657-5661. 68. Bhatia A, Blades S, Cambridge G, Edwards JC. Differential distribution of Fc gamma RIIIa in normal human tissues and co-localization with DAF and fibrillin-1: Implications for immunological microenvironments. Immunology 1998;94:56-63. 69. Daëron M. Fc receptor biology. Annu Rev Immunol 1997;15: 203-234. 70. Marusic-Galesic S, Pavelic K, Pokric B. Cellular immune response to antigen administered as an immune complex. Immunology 1991;72:526. 71. Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, Marshak-Rothstein A. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 2002;416:603-607. 72. Boule MW, Broughton C, Mackay F, Akira S, Marshak-Rothstein A, Rifkin IR. Toll-like receptor 9-dependent and -independent dendritic cell activation by chromatin-IgG complexes. J Exp Med 2004;199:1631-1640. 73. Lewis EJ, Hunsicker LG, Lan SP, Rohde RD, Lachin JM. A controlled trial of plasmapheresis therapy in severe lupus nephritis: The Lupus Nephritis Collaborative Study Group. N Engl J Med 1992;326:1373-1379. 74. Årfors L, Lefvert AK. Enrichment of antibodies against phospholipids in circulating immune complexes (CIC) in the anti-phospholipid syndrome (APLS). Clin Exp Immunol 1997; 108:47-51. 75. Pfueller SL, Luscher EF. Review: The effects of immune complexes on blood platelets and their relationship to complement activation. Immunochem 1972;9:1151-1165. 76. Vaarala O, Alfthan G, Jauhiainen M, Leirisalo-Repo M, Aho K, Palosuo T. Crossreaction between antibodies to oxidized low-density lipoprotein and to cardiolipin in systemic lupus erythematosus. Lancet 1993;341:923-925. 77. Hasunuma Y, Matsuura E, Makita Z, Katahira T, Nishi S, Koike T. Involvement of b2-glycoprotein I and anticardiolipin antibodies in oxidatively modified low-density lipoprotein uptake by macrophages. Clin Exp Immunol 1997;107:569-573. 78. Puurunen M, Manttari M, Manninen V, et al. Antibodies against oxidized low density lipoprotein predicting myocardial infarction. Arch Intern Med 1994;154:2605-2609. 79. Kramers C, Hylkema MN, van Bruggen MC, et al. Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo. J Clin Invest 1994;94:568-577. 80. Morioka T, Woitas R, Fujigaki Y, Batsford SR, Vogt A. Histone mediates glomerular deposition of small size DNA anti-DNA complex. Kidney Int 1994;45:991-997. 81. Schmiedeke TM, Stockl FW, Weber R, Sugisaki Y, Batsford SR, Vogt A. Histones have high affinity for the glomerular basement membrane: Relevance for immune complex formation in lupus nephritis. J Exp Med 1989;169:1879-1894. 82. van Bruggen MC, Kramers C, Walgreen B, et al. Nucleosomes and histones are present in glomerular deposits in human lupus nephritis. Nephrol Dial Transplant 1997;12:57-66. 83. Grootscholten C, van Bruggen MC, van der Pijl JW, et al. Deposition of nucleosomal antigens (histones and DNA) in the epidermal basement membrane in human lupus nephritis. Arthritis Rheum 2003;48:1355-1362. 84. Wener M. Immune Complex Assays. In Rose N, et al. (eds.), Manual of Clinical Laboratory Immunology, Fifth Edition. Washington, DC: American Society of Microbiology 1997. 85. Kohro-Kawata J, Wener MH, Mannik M. The effect of high salt concentration on detection of serum immune complexes and autoantibodies to C1q in patients with systemic lupus erythematosus. J Rheumatol 2002;29:84-89. 86. Lambert PH, Dixon FJ, Zubler RH, et al. A WHO collaborative study for the evaluation of eighteen methods for detecting immune complexes in serum. J Clin Lab Immunol 1978; 1:1-15.

MECHANISMS OF TISSUE DAMAGE

22A

Antibodies to DNA David S. Pisetsky, MD, PhD

INTRODUCTION Antibodies to DNA (anti-DNA) are prototypic autoantibodies that are the serologic hallmark of systemic lupus erythematosus (SLE). These antibodies are virtually synonymous with autoimmunity and have been extensively characterized in patients as well as animal models to elucidate fundamental events in disease pathogenesis. In the clinical setting, anti-DNA antibodies remain a mainstay in patient evaluation and provide important information for both diagnosis and prognosis. As such, these antibodies bridge the realms of clinical care and basic research, making them probably the most studied of all autoantibodies in medicine.1,2 Although the anti-DNA response has been investigated for almost 50 years, research begun in the 1980s has revolutionized the conceptualization of this system and the activity of DNA on the immune system. At the heart of this research is the recognition that DNA can potently modulate immune responses and serve as an immunogen in both normal and aberrant immunity. Delineation of the immune activities of DNA came slowly, reflecting in part the dogma that a response to DNA occurs only in SLE and thereby reflects a major disturbance in immune regulation.3 With the understanding that DNA has rich and diverse immune properties, ideas on disease pathogenesis are evolving and suggest novel therapeutic approaches to block anti-DNA production or attenuate its consequences. This chapter reviews these ideas.

ASSAY OF ANTI-DNA ANTIBODIES DNA is a large polymeric macromolecule and theoretically presents a multitude of antigenic determinants that reflect sequence, backbone structure, and conformation. Despite the potential diversity of epitope structure, DNA was long dichotomized into two antigenic forms: single-stranded (ss) and double-stranded (ds) DNA. This dichotomy reflected clinical studies indicating that although antibodies to dsDNA occur essentially only in patients with SLE anti-ssDNA antibodies have broader expression among clinical diagnoses and

therefore have less specificity as markers. Because the diagnosis of SLE has important implications with respect to patient care, assays with high diagnostic specificity have been emphasized for clinical use.4,5

Assay Formats The assay of anti-dsDNA has shown continuous refinement, resulting from innovations in the form of the DNA used as substrate as well as the method for antibody detection. The following assays have been used in the clinical setting: complement fixation, Farr-type immunoprecipitation, Crithidia luciliae immunofluorescence, filter binding, solid-phase radiobinding assay, and enzyme-linked immunoabsorbent assay (ELISA). These assays differ in the source of DNA used as antigen as well as the spectrum of antibodies that can be detected.4,6 For example, a Farr-type immunoprecipitation assay involves the formation of an immune complex with a radiolabeled DNA that can be precipitated by ammonium sulfate. This complex must have sufficient avidity to remain intact in high salt. As such, a Farr assay likely detects a more limited subset of high-avidity antibodies than an ELISA (which can detect lower-avidity antibodies due to high antigen density at the solid-phase surface and the potential for cross-linking). (See Table 22A.1.)

TABLE 22A.1 PROPERTIES OF ANTI-DNA ANTIBODIES IN NORMAL SUBJECTS AND PATIENTS WITH SLE Anti-DNA Assays Normal

SLE

Species specificity Bacterial DNA

Bacterial and mammalian DNA

Epitope

Conserved

Nonconserved

Strand specificity ss and ds DNA

ss and ds DNA

Isotype

IgG2

IgG1, IgG3

Light chain

κ predominance κ and λ

Pathogenicity

None

Subset of nephritogenic antibodies

225

ANTIBODIES TO DNA

In general, anti-DNA assays provide useful information for diagnosis as well as prognosis, although results of various assays of individual sera can differ based on the immunochemical properties of the antibodies present. Among assays, an ELISA likely detects the broadest number of specificities because it measures low- as well as high-avidity antibodies. In a mature antigen-driven response, the significance of low-avidity specificities is uncertain. An ELISA does not entail the use of radioactivity, however, and facilitates high throughput screening because of the use of a multiwell plate platform. These features make an ELISA an attractive choice for the clinical laboratory despite the detection of lower-avidity specificities.7-11 There are several features of the routine testing of anti-DNA antibodies that bear note. The first concerns the dichotomy of anti-ss versus anti-dsDNA antibodies. Although antibodies to ssDNA can occur in patients with diagnoses other than SLE, sera from patients with SLE in general bind both antigenic forms. Indeed, as shown by cross-inhibition studies as well as the characterization of monoclonal antibodies, many antibodies bind ss and dsDNA. This pattern of specificity likely reflects antibody interaction with a determinant on the phosphodiester backbone that can be presented irrespective of strandedness.12 Sera with exclusive specificity for dsDNA are in fact uncommon among SLE patients, and in patient sera anti-ssDNA occur more frequently than anti-dsDNA. Anti-ssDNA antibodies are also technically easier to measure and provide more sensitive assays.

Antibody Avidity and Specificity

226

Another issue regarding anti-DNA assays centers on avidity. As an antigen, DNA presents a repeating structure that allows a single antibody to contact epitopes on an extended polynucleotide chain via each Fab site of an IgG molecule. This type of binding, termed monogamous (or bivalent) interaction, leads to a dramatic increase in antibody avidity because of cross-linking. Thus, although each Fab site can contact only a few nucleotides, most sera require much larger pieces of DNA for binding. These pieces are generally at least 35 to 40 nucleotides in length, a span that covers the distance between each Fab site. Furthermore, some antibodies require DNA pieces hundreds of bases long for binding, likely because of low concentration of each epitope on a DNA molecule or the need for a conformational change in the DNA chain for the juxtaposition of each epitope.13-15 For DNA, like other multivalent antigens, the term avidity is relative. (See Box 22A.1.) Finally, although antibodies to DNA can be readily detected in patient sera, DNA (both inside and outside the cell) exists in the form of nucleosomes, the basic structure of DNA in chromatin. In this structure, DNA

BOX 22A-1 PATHOGENICITY OF ANTI-DNA ● ● ● ● ●

Deposition of circulating immune complexes In situ immune complex formation Direct binding to glomerular antigens Penetration into cells Cytoxine induction by immune complexes via TLR9

is wrapped around a histone core and binds tightly to proteins. As an antigen, therefore, DNA can be considered a component or epitope of chromatin (with many antibodies to chromatin showing sufficient interaction with free DNA to allow detection in the absence of the protein components).16,17 Although chromatin preparations may mimic more closely the antigenic form of DNA in vivo, they are less well defined antigenically, leading to preference in the use of a purified DNA for serologic assays.

CLINICAL EXPRESSION OF ANTI-DNA

Anti-DNA Expression in SLE In the context of SLE, dsDNA in the B conformation is the relevant antigenic determinant. This structure is widely expressed on DNA independently of species origin and synthetic dsDNA molecules (depending on sequence). With dsDNA as an antigen, anti-DNA expression is highly specific for SLE and occurs rarely in patients with other clinical diagnoses. These antibodies are expressed in approximately 50% of patients at some time during the course of their illness.4,5 Frequently, antibodies to DNA are expressed concomitantly with antibodies to histones and other components of chromatin. Such expression, called linkage, likely reflects the role of nucleosomes in driving autoantibody production in SLE. The association is not invariable, however, because antibodies to histones can occur in the absence of antibodies to DNA in drug-induced lupus.5,16,17 Anti-DNA (in contrast to other antinuclear antibodies in SLE) shows highly variable levels of expression, leading to its utility in assessing prognosis and disease activity as well as diagnosis. In longitudinal studies, anti-DNA expression in individual sera can range from undetectable levels to striking amounts in terms of titers. Frequently, high levels of anti-DNA expression are associated with an intensification of disease activity, in particular of glomerulonephritis. In the clinical setting, a depression in complement levels often accompanies increased anti-DNA levels, pointing to a role of immune complexes that deposit in the kidney in the immunopathogenesis of nephritis.4-6 Whereas an elevation of anti-DNA levels can mark the worsening of renal disease, serologic and clinical disease activity can be discordant. Thus, patients with

Anti-DNA Expression in Normal Immunity As a molecule, DNA is structurally enormously diverse because of sequence microheterogeneity. Although this molecular diversity has been extensively characterized in the context of gene regulation, its potential role in immunology was long neglected. This neglect resulted from several factors: (1) the focus on clinically useful assays that facilitate the detection of antibodies to dsDNA in the B conformation, (2) the seemingly exclusive expression of anti-DNA antibodies in SLE, implying responses to DNA only in conditions of aberrant immunity, and (3) the prevailing belief that with respect to the immune system DNA is structurally simple and uniform, with single- and double-stranded conformations the only relevant antigenic forms. Because anti-DNA assays uniformly support the high association of anti-DNA with SLE, there was little reason to question the prevailing dogma on the antigenicity of DNA. Studies begun in the 1980s have redefined the antigenic properties of DNA, and the expression of anti-DNA in normal and aberrant immunity. These studies, which have explored the antigenicity of base sequence as well

as conformation, originated in a survey of the binding of sera from patients with SLE and normal human subjects (NHS) with a panel of naturally occurring DNA antigens that differed in species origin and included both bacterial and mammalian molecules. The rationale for assaying DNA from various species was to enlarge the spectrum of sequential determinants for testing.23 The results of these studies were remarkable and refuted the notion that anti-DNA expression is specific for SLE. Thus, these studies showed that sera from NHS contain antibodies to dsDNA from certain bacterial species.23-28 As shown by various immunochemical approaches, these antibodies differ significantly in immunochemical properties from antibodies found in SLE patients in their specificity, avidity, and pattern of light-chain and isotype expression (Table 22A.1). Importantly, these antibodies are highly specific in their binding to DNA from a given bacteria and do not cross-react with either mammalian DNA or DNA from another bacteria. This reactivity indicates interaction with a nonconserved determinant. In contrast, antibodies in SLE sera show broad binding to DNA, and as expected for antibodies binding to a conserved determinant (i.e., B DNA), show cross-reactivity with both bacterial and mammalian DNA. In general, levels of antibodies to bacterial DNA in NHS antibodies are similar to those of patients with SLE when measured with the same antigen. In their properties and expression, antibodies to bacterial DNA in normal human sera resemble antibodies to bacterial carbohydrates. These findings suggest that bacterial DNA can serve as an immunogen in normal immunity and drive the production of specific antibodies in ordinary encounters with microorganisms during infection or colonization. Because bacterial DNA differs from mammalian DNA in sequence, an obvious basis for antigenic recognition is present. The generation of antibodies to bacterial DNA is selective, however, and sera from NHS lack appreciable amounts of antibodies to DNA from many common sources, including E. coli. The reason antibodies are generated against only certain bacterial DNA is unknown.

IMMUNE PROPERTIES OF DNA

serologic activity (i.e., increased anti-DNA levels) may lack nephritis and patients with active nephritis may have only low levels of anti-DNA. Both situations can be readily explained. Thus, although anti-DNA production may be pathologic, only a subset of these antibodies may be pathogenic or nephritogenic. The properties conferring nephritogenicity are not well defined, although they likely depend on avidity, charge, and fine specificity for DNA. Existing assays do not distinguish those specificities that cause renal disease. The reverse situation (i.e., active nephritis without anti-DNA) may result from a failure of a particular assay to measure anti-DNA or from the role played by another autoantibody system in nephritis.18-22 The utility of anti-DNA measurements in staging other disease manifestations is much less clear. Thus, despite the value of anti-DNA determinations in nephritis, this antibody should not be viewed as a general measure of disease activity. Other issues concerning the role of anti-DNA as a marker involve the magnitude of change viewed as clinically significant and the timing with respect to flares. These issues have increased in relevance in the context of drug development with agents that can specifically lower anti-DNA production. At present, anti-DNA cannot be considered a surrogate marker for disease that can guide the development of new agents or the utilization of existing agents in routine care. For those patients in whom anti-DNA expression correlates with activity of nephritis this antibody system is nevertheless of a very useful laboratory test.

IMMUNE PROPERTIES OF DNA The recognition that normal individuals produce antibodies to bacterial DNA indicates that DNA is immunologically heterogenous, with bacterial DNA having features that can confer antigenicity and immunogenicity. Although these features could simply be nonconserved sequence motifs that serve as epitopes, studies conducted in vivo and in vitro have shown that bacterial and mammalian DNA have immunomodulatory activities that can affect antibody induction.

227

ANTIBODIES TO DNA

228

In particular, bacterial DNA can function as a PAMP (pathogen-associated molecular pattern) to stimulate a toll-like receptor (TLR) to induce cellular responses with adjuvant activities. These activities include activation of B cells, dendritic cells, and macrophages (among other cell types) and production of cytokines and chemokines. These responses result from the activation of TLR9, which functions on the inside of cells and contacts DNA after endocytosis.29-31 The immune activities of DNA were first defined in the murine system using naturally occurring bacterial DNA as well as synthetic oligonucleotides to reveal the structural features causing immune stimulation.32-34 These studies led to the identification of the CpG motif, a 6-base sequence in which unmethylated CpG dinucleotide is flanked by two 5’ purines and two 3’ pyrimidines. Such sequences can form palindromes, a feature emphasized in some descriptions of this motif. The CpG motif has enormous appeal as a PAMP structure because sequences of this type occur much more commonly in bacterial DNA than in mammalian DNA. Differences in cytosine methylation and CpG suppression in the mammalian genome determine the difference in the quantitative representation of the motif. Because short synthetic ODN can reproduce the immunostimulatory activity of bacterial DNA, the role of CpG motifs in immune activities appears certain. Although the CpG motif provides a simple and elegant model for immune stimulation by bacterial DNA, subsequent studies have added complexity to the picture. Thus, the sequences involved in the stimulation of murine and human cells show differences (with the sequences for activation also varying depending on DNA backbone structure and cell type activated).35 Rather than a single motif with broad activity, it appears that a variety of sequence motifs can trigger cells. Furthermore, studies indicate that DNA contains inhibitory as well as stimulatory sequences, with sequences rich in guanosine in particular able to suppress immune responses induced by CpG DNA and possibly other stimuli.36 The immune activity of any given DNA may therefore be a composite of its stimulatory and inhibitory sequences. A further complexity to this picture came with studies showing that immune activation is dependent on context as well as structure. Thus, although free bacterial DNA is stimulatory and free mammalian DNA is inhibitory, a complex of DNA with either antibody or a cytofectin agent can create a stimulatory moiety.37-39 Cytofectins are lipid-based agents commonly used to promote the uptake of DNA into cells for gene transfection. With both immune complexes and cytofectins, it appears that DNA can enter a cellular compartment where the stringency for sequence is reduced and essentially any DNA can become active.

These considerations suggest that immune activation by DNA can arise in at least two settings: (1) infection during which foreign DNA is released by bacteria or introduced into cells following phagocytosis and (2) autoimmunity during which anti-DNA antibodies bind circulating DNA to form immune complexes that can stimulate dendritic cells for cytokine production to promote immune responses. The existence of these activities provides the basis for a coherent mechanism for anti-DNA induction that can incorporate serologic findings from both normal individuals and patients with SLE.

INDUCTION OF ANTI-DNA IN SLE

The Role of Antigen Drive As shown by studies on both murine and human monoclonal antibodies, the anti-DNA response has features of an antigen-driven process in which DNA is the selecting antigen. These features include clonality and pattern of somatic mutation, with the introduction of charged residues into the variable region leading to an increase in antibody avidity.40-41 The coexistence of anti-DNA with antibodies to histones and other components of the chromatin further suggests that the nucleosome drives this response and elicits an array of specificities that in turn promote disease pathogenesis, including nephritis. Although the operation of an antigen-driven mechanism appears likely, antibody responses to DNA have been difficult to induce experimentally in normal animals by immunization with mammalian DNA in the B conformation (even in the presence of complete Freund’s adjuvant).42-43 These findings have suggested that SLE requires a perturbed immune system to allow anti-DNA responses. This perturbation may result from an intrinsic abnormality in the B-cell compartment that impairs the ordinary processes by which B cells are tolerized and allows retention in the repertoire of precursors with Ig receptors for DNA. Studies on mice with anti-DNA transgenes have shown that transgene expression differs depending on strain, with mice from autoimmune backgrounds allowing the expression of a transgene that in normal mice would be anergized or deleted.44-45 Multiple genes appear to contribute to this background effect. Added to a genetic backbone predisposed to autoimmunity must be a source of DNA antigen to drive the production of class-switched anti-DNA antibodies. This DNA may have two functions: to serve as an immunogen to which antibodies will be directed and to serve as an adjuvant to promote antibody production because of the induction of cytokines. Both bacterial and mammalian DNA can serve this function, although for mammalian DNA the presence of anti-DNA in the

Models for Anti-DNA Induction Given the different immune properties of bacterial and mammalian DNA, it appears reasonable to propose a model of autoantibody induction in which both DNA sources play a role, albeit at different times. According to this model, in patients with SLE as well as normal subjects, bacterial DNA induces the production of antibodies, although in patients with SLE these antibodies display cross-reactivity with mammalian DNA. This distortion of specificity and deviation from a highly specific recognition of nonconserved sites on bacterial DNA results from tolerance defects that create a repertoire replete with autoantibody precursors.46 In this response, bacterial DNA displays adjuvant activity to enhance antibody production in a way not possible with mammalian DNA alone. Indeed, in experimental models bacterial DNA can induce cross-reactive anti-DNA production in autoimmune NZB/NZW mice and a highly immunostimulatory CpG oligonucleotide can serve as an adjuvant for the induction of cross-reactive anti-DNA antibodies in normal BALB/c animals. Thus far, in animals anti-DNA antibody production occurs readily only with either bacterial DNA as an immunogen or a CpG ODN as an adjuvant.43,47,48 Once cross-reactive anti-DNA antibodies enter the system, endogenous DNA can replace bacterial DNA as the driving antigen because with immune complex formation mammalian DNA can exert adjuvant activity via its effects on dendritic cell cytokine production. At this point in the process the immune response can spread to other components of nucleosomes, leading to the linked responses. Although endogenous DNA can supplant exogenous DNA for this purpose, exogenous DNA may nevertheless induce a subsequent response. Thus, during flare anti-DNA levels can rise strikingly, implying either a major change in the overall immune poise of the host or the introduction of new antigen into the system. Because infection can bring antigen into the system, bacterial DNA may have an intermittent role in retriggering the response and initiating a cycle of autoantibody production.

PATHOGENICITY OF ANTI-DNA

Renal Disease The role of anti-DNA in the pathogenesis of lupus nephritis has been demonstrated by the correlation between serologic and clinical disease activity, the isolation of anti-DNA in enriched form from the glomeruli of affected patients, and the induction of nephritis in

normal mice by the administration of monoclonal anti-DNA antibodies. In the induction of nephritis, immune complexes appear to play a prominent role, with the charge of the antibody or bound antigen (i.e., DNA or nucleosome) leading to preferential localization at the glomerular basement membrane. The biopsies of patients clearly demonstrate the presence of complexes by both immunofluorescence and electron microscopy.18,21,49 Although immune complexes can form in the circulation by the union of DNA and anti-DNA, this process may also occur in the kidney by a mechanism termed in situ immune complex formation. According to this mechanism, DNA antigen localizes to the basement membrane, where it is bound or “planted” by chargecharge interactions. Once planted, DNA can trap anti-DNA circulating in the blood to assemble the complexes. Whether the complexes form in the circulation or in the kidney, once present they can activate complement and incite local inflammation. Anti-DNA antibodies may also contribute to renal disease by direct binding to sites on the basement membrane as well as penetration into cells.18,21,50

PATHOGENICITY OF ANTI-DNA

system would be required for the adjuvant properties to be manifest. Unless this antibody resulted from a nonspecific immune activation, the ability of self-DNA to initiate anti-DNA production appears limited.

Cytokine Production In addition to inducing nephritis, DNA/anti-DNA immune complexes may promote pathogenesis in SLE via effects on cytokine production. As shown in studies in vitro, immune complexes containing DNA as well as other nuclear antigens can induce the production of IFN-α by plasmacytoid dendritic cells. With DNA, stimulation appears to require both TLR9 and the Fc receptor, with cross-linking by IgG delivering a signal in addition to that resulting from the interaction of internalized DNA with TLR9.37,51-55 The resulting IFN-α can have generalized pro-inflammatory and immunostimulatory activities that promote various facets of SLE. In this regard, microarray analysis of RNA from peripheral blood cells of SLE patients demonstrates an “interferon signature” in many patients, pointing to the importance of this cytokine in immune regulation in this disease (see Figure 22A.1.).56,57

Immune Complex Formation For both of the roles of immune complexes (i.e., nephritis and cytokine production), the presence of DNA antigen is essential, with its release into the extracellular milieu a key step in pathogenesis. Extracellular DNA may play a role in anti-DNA induction as the inciting antigen, although it is possible that DNA displayed on the surface of apoptotic cells may serve this role. The release of DNA appears to result from cell death because high levels of DNA occur clinically in settings of trauma, infarction, and malignancy— all events associated with apoptosis or necrosis.

229

ANTIBODIES TO DNA

NHS

1.6

1.6

1.4

1.4

1.2

1.2

1

OD380

OD380

SLE

0.8

1 0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

Fig. 22A.1 Antibody responses to bacterial and mammalian DNA. Sera of patients with SLE as well as normal human subjects (NHS) were assaged for binding to the following mammalian and bacterial DNA antigens: calf thymus (CT), human placenta (HP), Micrococcus lysodeikticus (MC), Pseudomonas aeruginosa (PA), Staphylococcus epidermidis (SE), and Escherichia coli (EC). Results presented are the average for 12 sera in an ELISA using a dilution of 1:100. (Data are reproduced with modification from reference 23.)

0 CT HP MC PA SE EC

CT HP MC PA SE EC

DNA antigen

DNA antigen

Furthermore, the release of DNA into the circulation in animals can be modeled by the infusion of either apoptotic or necrotic cells as well as the administration of agents such as anti-Fas antibodies that elicit extensive apoptosis.58-62 The release process may not simply reflect cell death, however, because in mouse models macrophages are required. Thus, mice in which macrophages have been eliminated by treatment with clodronate fail to display blood DNA after infusion of dead cells.61 These findings suggest that release of blood DNA is an active process and/or requires intervention of other cell types. Furthermore, the release of DNA in mouse models is sensitive to the effects of glucocorticoids, which can modulate the macrophage activity. In mice, treatment with dexamethasone prior to the administration of dead cells or administration of a monoclonal anti-Fas antibody abrogates the subsequent rise in blood DNA levels.62 The effects of glucorticoids are notable because these agents can profoundly lower anti-DNA levels, causing decreases disproportionate to changes in other autoantibodies or total immunoglobulin levels. It is possible

that the actions of glucocorticoids reflect a decrease in available self-antigen to drive antibody production and to decrease the amount of immune complexes that induce cytokine and augment autoantibody production. Another agent that may impact on these responses is hydroxychoroloquine. This agent can affect endosomal acidification and the stimulation of TLR9 by DNA. Together, these observations suggest that drugs currently used to treat SLE are impacting on key steps that affect the induction of anti-DNA antibodies as well as their downstream effects.

CONCLUSIONS Antibodies to DNA are prototypic autoantibodies central to the pathogenesis of SLE. These antibodies arise by an antigen-driven process in an individual susceptible to autoimmunity because of genetic factors. By forming immune complexes that contain potentially immunomodulatory molecules, anti-DNA can elicit both tissue injury and functional disturbances of B and T cells. With the recognition that TLR9 mediates immune activation by DNA, new approaches to therapy will be possible.

REFERENCES

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1. Hahn BH. Antibodies to DNA. New Engl J Med 1998;338:1359. 2. Pisetsky DS. Antibody responses to DNA in normal immunity and aberrant immunity. Clin Diagn Lab Immunol 1998;5:1. 3. Pisetsky DS. Immune activation by bacterial DNA: A new genetic code. Immunity 1996;5:303. 4. Von Mühlen CA, Tan EM. Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum 1995;24:323. 5. Jang YJ, Stollar BD. Anti-DNA antibodies: Aspects of structure and pathogenicity. Cell Mol Life Sci 2003;60:309.

6. Kavanaugh AF, Solomon DH. The American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines: Guidelines for immunologic laboratory testing on the rheumatic diseases: anti-DNA antibody tests. Arthritis Care Res 2002;47:546. 7. Ward MM, Pisetsky DS, Christenson VD. Antidouble stranded DNA antibody assays in systemic lupus erythematosus: Correlations of longitudinal antibody measurements. J Rheumatol 1989; 16:609.

33. Krieg AM, Yi A-K, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 2002;374:546. 34. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Ann Rev Immunol 2002;20:709. 35. Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nature Rev Immunol 2004;4:1. 36. Gursel I, Gursel M, Yamada H, et al. Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J Immunol 2003;171:1393. 37. Vallin H, Perers A, Alm GV, et al. Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-α inducer in systemic lupus erythematosus. J Immunol 1999;163:6306. 38. Zhu FG, Reich CF, Pisetsky DS. Effect of cytofectins on the immune response of murine macrophages to mammalian DNA. Immunology 2003;109:255. 39. Jiang W, Reich CF III, Pisetsky DS. Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA. Cell Immunol 2004;229:31. 40. Marion TN, Bothwell ALM, Briles DE, et al. IgG anti-DNA autoantibodies within an individual autoimmune mouse are the products of clonal selection. J Immunol 1989;142:4269. 41. Schlomchik M, Mascelli M, Shan H, et al. Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation. J Exp Med 1990;171:265. 42. Pyun EH, Pisetsky DS, Gilkeson GS. The fine specificity of monoclonal anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. J Autoimmun 1993;1:11. 43. Tran TT, Reich CF III, Alam M, et al. Specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with mammalian DNA with a CpG oligonucleotide as adjuvant. Clin Immunol 2003;109:278. 44. Yachimovich-Cohen N, Fischel R, Bachar N, et al. Autoimmune NZB/NZW F1 mice utilize B cell receptor editing for generating high-affinity anti-dsDNA autoantibodies from low-affinity precursors. Eur J Immunol 2003;33:2469. 45. Seo S-J, Fields ML, Buckler JL, et al. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 2002;16:535. 46. Yurasov S, Wardemann H, Hammersen J, et al. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med 2005;201:703. 47. Gilkeson GS, Pippen AMM, Pisetsky DS. Induction of crossreactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA. J Clin Invest 1995;95:1398. 48. Wloch MK, Alexander AL, Pippen AMM, et al. Molecular properties of anti-DNA induced in preautoimmune NZB/W mice by immunization with bacterial DNA. J Immunol 1997;158:4500. 49. Fournieré GJ. DNA and lupus nephritis. Kidney Int 1988; 33:487. 50. Yanase K, Madaio MP. Nuclear localizing anti-DNA antibodies enter cells via caveoli and modulate expression of caveolin and p53. J Autoimmun 2005;24:145. 51. Båve U, Alm GV, Rönnblom L. The combination of apoptotic U937 cells and lupus IgG is a potent IFN-α inducer. J Immunol 2000;165:3519. 52. Magnusson M, Magnusson S, Vallin H, et al. Importance of CpG dinucleotides in activation of natural IFN-α producing cells by a Lupus-related oligodeoxynucleotide. Scand J Immunol 2001; 54:543. 53. Boulé MW, Broughton C, Mackay F, et al. Toll-like Receptor 9-dependent and -independent dendritic cell activation by chromatin-immunoglobulin G complexes. J Exp Med 2004; 199:1631. 54. Lövgren T, Eloranta M-L, Båve U, et al. Induction of Interferon-a production in plasmacytoid dendritic cells by immune complexes: Containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG. Arthritis Rheum 2004;50:1861. 55. Means TK, Latz E, Hayashi F, et al. Human lupus autoantibodyDNA complexes activate DCs through cooperation of CD32 and TLR9. J Cl Invest 2005;115:407. 56. Baechler EC, Batliwalla FM, Karypis G, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci USA 2003; 100:2610.

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8. Isenberg D, Smeenk R. Clinical laboratory assays for measuring anti-dsDNA antibodies. Where are we now? Lupus 2002;11:797. 9. Rahman A, Hiepe F. Anti-DNA antibodies, overview of assays and clinical correlations. Lupus 2002;11:770. 10. Haugbro K, Nossent JC, Winkler T, et al. Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 2004; 63:386. 11. Reveille JD. Predictive value of autoantibodies for activity of systemic lupus erythematosus. Lupus 2004;13:290. 12. Stollar BD, Papalian M. Secondary structure in denatured DNA is responsible for its reaction with antinative DNA antibodies of systemic lupus erythematosus. J Clin Invest 1980;66:210. 13. Papalian M, Lafer E, Wong R, et al. Reaction of systemic lupus erythematosus antinative DNA antibodies with native DNA fragments from 20 to 1,200 base pairs. J Clin Invest 1980;65:469. 14. Ali R, Dersimonian H, Stollar BD. Binding of monoclonal anti-native DNA autoantibodies to DNA of varying size and conformation. Mol Immunol 1985;22:1415. 15. Pisetsky DS, Reich CF. The influence of DNA size on binding of anti-DNA antibodies in the solid and fluid phase. Clin Immunol Immunopathol 1994;72:350. 16. Monestier M. Autoantibodies to nucleosomes and histone-DNA complexes. Methods 1997;11:36. 17. Amoura Z, Chabre H, Bach J-F, Koutouzov S. Antinucleosome antibodies and systemic lupus erythematosus. Adv Nephrol 1997;26:303. 18. Foster MH, Cizman B, Madaio MP. Nephritogenic autoantibodies in systemic lupus erythematosus: Immunochemical properties, mechanisms of immune deposition, and genetic origins. Lab Invest 1993;69:494. 19. Ohnishi K, Ebling FM, Mitchel B, et al. Comparison of pathogenic and non-pathogenic murine antibodies to DNA: Antigen binding and structural characteristics. Int Imunol 1993;6:817. 20. Suzuki N, Harada T, Mizushima Y, et al. Possible pathogenic role of cationic anti-DNA autoantibodies in the development of nephritis in patients with systemic lupus erythematosus. J Immunol 1993;151:1128. 21. Lefkowith JB, Gilkeson GS. Nephritogenic autoantibodies in lupus. Arthritis Rheum 1996;39:894. 22. Van Bruggen MCJ, Walgreen B, Rijke TPM, et al. Antigen specificity of anti-nuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo. Eur J Immunol 1997;27:1564. 23. Karounos DG, Grudier JP, Pisetsky DS. Spontaneous expression of antibodies to DNA of various species origin in sera of normal subjects and patients with systemic lupus erythematosus. J Immunol 1988;140:451. 24. Robertson CR, Gilkeson GS, Ward MM, et al. Patterns of heavy and light chain utilization in the antibody response to single-stranded bacterial DNA in normal human subjects and patients with systemic lupus erythematosus. Clin Immunol Immunopathol 1992;62:25. 25. Wu ZQ, Drayton D, Pisetsky DS. Specificity and immunochemical properties of antibodies to bacterial DNA in sera of normal human subjects and patients with systemic lupus erythematosus. Clin Exp Immunol 1997;109:27. 26. Pisetsky D, Drayton D, Wu ZQ. Specificity of antibodies to bacterial DNA in sera of healthy human subjects and patients with systemic lupus erythematosus. J Rheumatol 1999;26:1934. 27. Pisetsky DS. The antigenic properties of bacterial DNA in normal and aberrant immunity. Springer Semin Immunopathol 2000; 22:153. 28. Pisetsky DS, Drayton DM. Deficient expression of antibodies specific for bacterial DNA by patient with systemic lupus erythematosus. Proc Assoc Am Phys 1997;109:237. 29. Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nature Immunol 2004; 5:975. 30. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nature Immunol 2004;5:987. 31. Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol 2004;25:381. 32. Messina JP, Gilkeson GS, Pisetsky DS. Stimulation of in vitro murine lymphocyte proliferation by ceterial DNA. J Immunol 1991;147:1759.

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57. Kirou KA, Lee C, George S, et al. Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum 2005; 52:1491. 58. Sidransky D. Circulating DNA. What we know and what we need to learn. Ann NY Acad Sci 2000;906:1. 59. Pisetsky DS. DNA as a marker of cell death in systemic lupus erythematosus. Rheum Dis Clinics NAmer 2004;30:575.

60. Choi JJ, Reich CF III, Pisetsky DS. Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro. Scand J Immunol 2004;60:159. 61. Jiang N, Reich CF III, Pisetsky DS. Role of macrophages in the generation of circulating blood nucleosomes from dead and dying cells. Blood 2003;102:2243. 62. Jiang N, Pisetsky DS. The effect of dexamethasone on the generation of plasma DNA from dead and dying cells. Am J Pathol 2004;164:1751.

MECHANISMS OF TISSUE DAMAGE

22B

Antibodies and their Antigenic Targets in the Antiphospholipid Syndrome Bill Giannakopoulos, MB, BS, FRACP, Xiaokai Yan, PhD, and Steven A. Krilis, MB, BS, PhD

INTRODUCTION The antiphospholipid syndrome manifests as either venous or arterial thrombosis, and in females recurrent fetal loss. Antiphospholipid antibodies are an essential diagnostic feature of the syndrome. Antiphospholipid antibodies represent a heterogenous group of antibodies, targeting protein antigens that bind to anionic phospholipids and anionic phospholipids per se.1 In this chapter we review the methodology used to detect antiphospholipid antibodies, which can be detected by two methods: using the enzyme linked immunosorbent assay (ELISA) for the detection of a heterogenous group of antibodies that fall under the umbrella term anticardiolipin antibodies (aCL) and clotting assays for determination of the lupus anticoagulant (LA).1 We also examine the dominant antibodies in APS, which are those targeting β2GPI2-4 and prothrombin.5 The nature of the dominant antigenic targets is also examined within this context. Pathogenic mechanisms proposed for thrombosis and fetal loss, based on experimental evidence, are reviewed along with mechanisms responsible for the generation of autoantibodies.

DEFINITION OF THE ANTIPHOSPHOLIPID SYNDROME Diagnosis of the antiphospholipid syndrome requires at least one laboratory criterion and one clinical criterion.6 The laboratory criterion involves the detection of moderate to high titers of IgM or IgG anticardiolipin antibodies 12 weeks apart. Alternatively, the detection of the lupus anticoagulant on two separate occasions 12 weeks apart is required. The presence of anti-β2GPI Abs (either IgG or IgM) 12 weeks apart also constitutes positive serology for the syndrome, and represents a modification of the Sapporo

criteria undertaken in Sydney in 2004 during the Eleventh International Congress on antiphospholipid antibodies.6 The clinical criteria include the occurrence of venous, arterial, or small-vessel thrombosis anywhere in the circulation. The obstetric criteria include three recurrent miscarriages, in the absence of an alternate explanation, before 10 weeks gestation, or a miscarriage after or including the 10th week of gestation or delivery of a child before the 34th week of gestation due to preeclampsia, eclampsia, or intrauterine growth retardation.6 A number of clinical features have been noted to be associated with the syndrome, although they do not constitute a clinical criterion on their own. These include valvular heart disease, livedo reticularis, thrombocytopaenia, transverse myelopathy, and renal small artery vasculopathy.6 The catastrophic antiphospholipid syndrome is characterized by widespread intravascular thrombosis.7

ANTICARDIOLIPIN ELISA The aCL ELISA involves microtitre polystyrene plates coated with cardiolipin in ethanol and then dried by evaporation by leaving the ELISA plate open to air.8 The plate is subsequently washed with phosphate buffered saline (PBS), and blocked with either fetal or bovine serum or bovine albumin.8 The plate is then washed with PBS, and the patient’s samples are applied at a dilution of 1/50 to 1/100.8 After incubation and washing of the plates, the labeled secondary antibody (directed against either IgG or IgM) is applied. The aCL levels are then calculated from a standard curve created from a sufficient number of standards.8 It was initially believed that the antibodies from patients with APS were binding directly to cardiolipin in this test, and hence the name. It was subsequently

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ANTIBODIES AND THEIR ANTIGENIC TARGETS IN THE ANTIPHOSPHOLIPID SYNDROME

234

found that the antibodies were binding to anionic phospholipid binding proteins (which bound and became immobilized on the cardiolipin-coated plate). These proteins were contained within the patient’s sample and within the blocking buffer.9 The dominant antigenic target in patients with APS was subsequently found to be β2GPI, an abundant plasma protein.2-4 Other protein antigenic targets have been described, including annexin V10 and protein S11. The detection of these latter antibodies in APS has been noted in case reports in the literature, and their utility in the clinical diagnosis of APS or as mediators of pathogenesis remains unknown. Anti-prothrombin antibodies are not detected using this method.8

TESTS FOR THE LUPUS ANTICOAGULANT Four sequential steps have been determined to be important in the performance of lupus anticoagulant. They are prolongation of a phospholipid dependent clotting time, absence of correction of the prolonged clotting time on mixing the sample obtained from the patient with normal plasma (demonstrating that the prolongation is not due to a coagulation factor deficiency), evidence of phospholipid dependence (demonstrated by reversal of the prolonged clotting time by the addition of excess phospholipid), and exclusion of specific inhibition of any one coagulation factor.8 Two tests are usually performed to detect lupus anticoagulant, as no one test can detect them all. The tests are usually the dilute Russell Viper Venom time (DRVVT) and either an activated partial thromboplastin time (aPTT) or a Kaolin Clotting Time (KCT).9 The predominant antibodies responsible for the LA effect are anti-β2GPI Abs12 and anti-prothrombin antibodies.5 Recently it has been demonstrated that anti-β2GPI Abs with the lupus anticoagulant effect strongly correlate with thrombosis.13 Furthermore, anti-β2GPI Abs with LA activity appear to recognize the epitope Gly-40/Arg-43 in domain I of the β2GPI molecule.14 Anti-prothrombin antibodies with LA activity appear to be less specific for the diagnosis of APS.13 The LA is an in vitro phenomenon that does not correlate with in vivo activity. Otherwise, it would be associated with a bleeding diathesis rather than with the prothrombotic tendency associated with APS. The LA phenomenon can be explained mechanistically as follows. Clotting in vitro requires that an anionic phospholipid surface be present to allow for the prothrombinase complex to form, which leads to thrombin generation, which then leads to the generation of fibrin and hence clotting. Anti-β2GPI Abs and anti-prothrombin Abs in complex with their respective

antigens (β2GPI and prothrombin) bind with very high affinity to anionic phospholipids, thereby competitively inhibiting the binding and formation of the prothrombinase complex and leading to prolongation of clotting.9 The addition of excess phospholipids ensures that there are adequate binding sites for the prothrombinase complex to form, thereby removing the competitive inhibition.9

ANTI-b 2GPI ELISA The finding that patients with APS require β2GPI for binding in the aCL ELISA led to the testing of the β2GPI ELISA system without cardiolipin.15 It has been found that for the β2GPI ELISA to work, β2GPI has to be coated onto a negatively charged surface. This surface may be a preirradiated polystyrene microtitre plate.15 The reasons for this requirement have been closely studied, and the different explanations suggested are not necessarily mutually exclusive. One explanation is that the negatively charged surface allows β2GPI to bind via domain V.16 This may lead to a conformational change of the molecule that leads to exposure of a cryptic epitope on domain I, thus allowing antibodies to bind.16 Another explanation is that the negatively charged surface allows the β2GPI molecules to cluster closely together, and this has been shown to enhance the avidity of the antibodies for the antigenic target.17 Anti-β2GPI Abs tend to be low affinity and do not tend to form antibody antigen complexes in plasma.18 The β2GPI molecule is composed of five domains.9 Studies have been performed to determine the epitope specificity of the antibodies associated with the syndrome. It has been found that in APS the dominant antigenic target is in domain I.19 The fine epitope specificity is at Gly-40/Arg-43.20,21 In the majority of infections, anti-β2GPI Abs are not detected.22 In leprosy infection and atopic dermatitis associated with childhood, anti-β2GPI Abs have been detected. However, in these clinical situations the dominant antigenic target lies in domain V.23,24 It has been proposed that developing a diagnostic test that can detect anti-β2GPI Abs directed against the epitopes in domain I may further improve the specificity of testing for the antiphospholipid syndrome.14

b2GPI β2GPI is found in human plasma at a mean concentration of 4 μM.25 It is conserved among mammals, there being 60 to 80% amino acid sequence identity among the human, bovine, canine, and murine proteins.9 It is composed of 326 amino acids.26 In crystallographic analysis, its structure is J shaped.26 It is divided into five domains, each domain being typical of the complement

role in thrombus propagation.37 FXI-deficient mice display poorly formed and friable thrombus formation on ferric chloride-induced carotid artery injury, in contrast to complete blockage in the wild type.38 In primates, FXI plays an important role in thrombus propagation.39 It is interesting to note that β2GPI binding to FXI inhibits its activation by FXIIa and thrombin.28 When β2GPI is clipped by plasmin or FXIa, even though it retains its ability to bind FXI it loses its ability to inhibit its activation.35 This suggests that in vivo β2GPI may regulate FXI activation.35 It has been demonstrated that clipped β2GPI, which can be generated either by plasmin27 or activated FXI,28 can bind to plasminogen and inhibit its conversion to plasmin by tissue plasminogen activator.40 This set of results suggests that β2GPI may provide a regulatory link between the FXI activation pathway and fibrinolysis.

PROTHROMBIN

control protein module.26 The first four domains are composed of 60 amino acids.26 Each domain contains cysteine moieties, which allow the formation of disulfide bridges by linking the first with the third and the second with the fourth cysteine residues.26 The third and fourth domains are heavily glycosylated.26 The fifth domain contains an extra 20 amino acids at the C-terminus.26 The C-terminus is unusual in that it terminates with a cysteine moiety, which forms a disulfide bridge with a cysteine found between the standard second and third cysteine residue positions in the fifth domain.26 The fifth domain, between amino acids 281 and 288, contains a region that contains multiple positively charged lysine residues and is critical for phospholipid binding.26 Certain proteases [such as plasmin and activated factor XI (FXIa)] have been found to be able to cleave β2GPI between Lys 317 and Thr 318, abolishing its ability to bind to anionic phospholipids.27,28 β2GPI is predominantly generated in liver.9 The physiologic function of β2GPI is not clearly delineated as yet, although progress has been made in this field. β2GPI knockout mice have been generated. The hemostatic system has been studied in these mice, as has the reproductive system. In the hemostatic system it has been demonstrated that in vitro plasma from β2GPI knockout mice displayed an impairement of thrombin generation compared to plasma from wild-type mice.29 With regard to reproduction, β2GPI knockout mice produce litters that are smaller than wild-type mice. Furthermore, they display mild placental abnormalities.30 In the past, a number of functions were attributed to β2GPI based on its ability to bind the anionic phospholipid phosphatidylserine. Phosphatidylserine is expressed on apoptotic cells.31 It is also expressed by activated platelets and activated endothelial cells.1 It was suggested that β2GPI may bind to apoptotic cells, thereby allowing for their clearance.31 Its binding to activated platelets has been suggested to enable it to play an anticoagulant role by inhibiting ADP-induced aggregation of washed platelets32 and inhibition of the initiation of the contact pathway by competitively inhibiting FXII binding to the negatively charged surface.33 The ability of β2GPI to bind to phosphatidylserine on cell surfaces in conditions that mimic the in vivo milieu has recently been called into question by the finding that monomeric β2GPI in fluid phase displays negligible binding, suggesting that any physiologic function is unlikely to be mediated by binding to phosphatidylserine in the absence of cofactors yet to be defined.34 β2GPI has recently been found to directly bind factor XI.35 Factor XI plays an important role in the amplification of thrombin generation.36 It can be activated either by FXIIa or thrombin.36 In vivo evidence is accumulating that FXI activation may play an important

PROTHROMBIN Prothrombin is generated and secreted by the liver. It contains 579 amino acids. The mean concentration in the plasma is approximately 1 to 2 μM.41 It is a vitaminK-dependent glycoprotein. It is the zymogen precursor of thrombin.42 Unlike β2GPI, its physiologic function is well characterized. It represents a key point in the coagulation cascade. Thrombin is responsible for local fibrin formation, and upon binding to thrombomodulin for activating protein C (which serves to limit clot propagation in the region of intact and healthy endothelium).43 Thrombin also binds to the platelet surface via the GPIb-IX-V receptor, and by cleaving the proteaseactivated receptors 1 and 4 (PAR1 and PAR4).43 During its biosynthesis in the liver, prothrombin undergoes γ-carboxylation.42 These γ-carboxyglutamic residues (known as the Gla domain, and located on fragment 1 of the prothrombin molecule) are essential for the calcium-dependent binding of prothrombin to phosphatidylserine.9 The prothrombinase complex (Xa/Va/Ca2+/ phospholipid) activates prothrombin to thrombin by cleavage at several sites. The Gla domain is removed during this process, and hence thrombin does not have a phospholipid binding site.9

AUTOANTIBODY PRODUCTION MECHANISMS

Apoptosis Apoptosis is the term given to the process of programmed cell death the body uses to dispose of cells no longer required. In SLE it has been proposed that there

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may be impaired clearance of apoptotic cells, which may predispose a genetically susceptible individual to mount an immune response against the molecules bound to the apoptotic blebs, leading to autoantibody production against these antigens.44 Similarly, in APS it has been suggested that β2GPI31,45 and prothrombin46 may bind to apoptotic blebs, and in a manner analogous to what may be happening in SLE patients, if there is impairment in apoptotic cell clearance, this may predispose an individual to developing antibodies directed against β2GPI and prothrombin. Bevers and colleagues have suggested that β2GPI may not be able to bind the exposed phosphatidylserine surface on apoptotic cells in vivo, whereas prothrombin can.34 It may be that β2GPI can bind to the surface of apoptotic blebs via other exposed cofactors such as viral peptides or surface exposed chromatin. This is on the basis that β2GPI can bind to DNA1 and some viral particles (e.g., hepatitis B surface antigen).47

T-Cells and B-Cells T-cells may be involved in the generation of autoantibodies in APS.48 Antibodies of the IgG and IgA isotype have been identified.48 Furthermore, an analysis of B-cell hybridomas derived from patients with β2-GPI antibodies has demonstrated that the majority of IgG Abs carry somatic mutations.49 The analysis of mutations according to their impact on the amino acids translated (replacement/substitution ratio) suggests that B-cells have undergone affinity maturation to produce long-lasting class-switched antibodies consistent with T-dependent immune responses.49 Autoreactive T-cells against β2-GPI have been identified in patients with APS and in healthy individuals.48 Developing an understanding of the mechanisms by which this autoreactive population of T-cells may be differentially activated in patients who develop APS compared to healthy individuals may provide insights into how tolerance is able to be broken, and autoimmunity induced in APS.48 Autoreactive CD4+ and HLA class-II-restricted T-cells directed against β2-GPI were shown to become activated in vitro by antigen-presenting cells when native β2-GPI was complexed to phospholipids.50 They did not become activated when presented with native β2-GPI alone or phospholipid alone.50 The intracellular processing of β2-GPI may differ when it is complexed to phospholipid, compared to when it is on its own.50 Interleukin 6 and CD40-CD40L interaction mediate an important role in anti-β2-GPI Abs production by self-reactive B-cells.51

Dendritic Cells 236

Oxidised β2-GPI is able to bind to dendritic cells (DCs) and induce them to mature, subsequently priming naive

T-cells and causing Th1 polarization.52 DC activation leads to nuclear factor kappa B translocation. Oxidized β2-GPI-activated interleukin-1 receptor-associated kinase (IRAK), which is the first kinase recruited by the toll-like interleukin-1 receptor family in the MyD88 pathway.52 These results suggest that oxidized β2-GPI may bind to a toll-like receptor (TLR).52 This process may lead to enhanced presentation of β2-GPI to autoreactive T-cells, and thus initiate or sustain the autoimmune response against β2-GPI.52

Molecular Mimicry It has been suggested that anti-β2GPI Abs may be generated as a result of molecular mimicry between human β2GPI and molecules similar to β2GPI in invading organisms.53 The evidence for this concept arose from the demonstration that mice immunized with Haemophilus influenzae or Neisseria gonorrhoea or tetanus toxoid appear to develop anti-β2GPI Abs, which when extracted and passively transferred to pregnant mice are able to induce fetal loss.53 Anti-β2GPI Abs are not commonly generated as a result of bacterial infections.22 They have been described in humans in association with leprosy,23 but in this situation they were directed against an epitope in domain V and not domain I (as occurs in APS).19-21 Hence, further work is needed to delineate the possible contribution of molecular mimicry to disease pathogenesis in patients with APS.

THROMBOSIS AND FETAL LOSS: EFFECTOR MECHANISMS

Complement Complement activation may be an important mediator in thrombosis associated with APS.54 The study that suggests this involved the transfer of polyclonal aPL Abs from patients with APS to rats.54 It was noted during these experiments that it was the β2-GPI-directed antibodies that were responsible for inducing thrombosis.54 When the β2-GPI targeting subgroup was removed, pathology was not induced.54 Furthermore, the histology of the mesenteric microvessels revealed the deposition of IgG, and complement components C3 and C9.54 The contribution to pathology was confirmed with the findings that complement-C6–deficient rats or those treated with an antibody that blocks C5 activation did not develop thrombus.54 The passive transfer of aPL Abs, derived from humans or mice, to pregnant mice has been used to study the potential mechanisms responsible for fetal loss in APS.55-57 On the basis of this model, it has been suggested that the activation of complement may be an important effector mechanism.55,56,58 Pregnant mice that had the C5a receptors knocked out did not display

Disruption of Anticoagulant Pathways Phosphatidyserine (PS) is an anionic phospholipid that plays an important role in blood coagulation.59 PS on the endothelial or platelet surface allows for procoagulant and anticoagulant complexes to be generated.59 β2-GPI binds with low affinity to the activated platelet surface.34 However, its binding capacity for PS increases 1000-fold in the presence of anti-β2-GPI Abs.34 A number of studies have proposed that the anti-β2-GPI Ab/ β2-GPI complex may disrupt the functioning of anticoagulant proteins on the PS surface, which may predispose to thrombosis. Activated protein C (APC) serves an anticoagulant role by binding and inactivating the procoagulant factors Va (FVa) and VIIIa.60 Anti-β2-GPI Abs in the presence of β2-GPI have been shown to be able to disturb the inactivation of FVa in vitro, with the mechanistic explanation for why this occurs being that anti-β2-GPI Abs in complex with β2-GPI may compete with the APC complex for limited phospholipid binding sites.61 Anti-prothrombin Abs have also been shown to be able to disturb the inactivation of FVa in vitro via a similar mechanism.62 In the presence of oxidized phospholipids and phosphatidylethanolamine (PE), the β2-GPI-antibody complex may preferentially disturb APC function.61 This may explain why the anticoagulant pathway may be disturbed relative to the procoagulant pathways.61 Protein Z (PZ) is a vitamin-K-dependent naturally occurring anticoagulant that binds to factor Xa, in association with phospholipid, and serves as a cofactor for FXa inactivation by the protein-Z–dependent protease inhibitor (ZPI).63 The PZ/ ZPI complex leads to the inhibition of further thrombin generation.63 Anti β2-GPI Abs in complex with β2-GPI appear to inhibit the anticoagulant function of the PZ/ ZPI complex, by competing for phosphatidylserine binding sites.63 Annexin A5 is a protein that binds anionic phospholipids with high affinity, and it has been suggested that it may form a protective anticoagulant shield on vascular cells.64 aPL Abs in complex with their antigens may disturb the shield, and hence predispose to thrombosis.64,65 However, a study by a different group was unable to confirm enhanced displacement of annexin A5 from endothelium by aPL Abs.66 Another important issue that needs to be clarified with this hypothesis is whether the annexin A5 shield exists in vivo. Annexin A5 knockout mice did not appear to have problems with conception, nor thrombosis.67

Activation of Cells Involved in Thrombosis and Placental Development Anti-β2-GPI Abs in complex with β2-GPI are able to activate cells involved in the hemostatic response to induce a procoagulant phenotype,68 and hence create an enviroment in vivo that has an increased predisposition to thrombus formation.69 In vitro experiments demonstrate that platelets can be activated to express thromboxane A2.70-72 Endothelial cells can be activated to express various adhesion molecules, such as E-selectin, intercellular adhesion molecule1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and the major initiator of the extrinsic coagulation system, tissue factor (TF).68 Monocytes can be activated to express TF and the proinflammatory cytokines tumour necrosis factor-a (TNF-a) and interleukin 1 (IL-1).73,74 A number of in vitro studies looking at endothelial cells,75,76 platelets,77 and monocytes74 have suggested that involvement of the Fc receptor is not a necessary requisite for cellular activation to occur. This is consistent with an in vivo model that showed that the F(ab′)2 fragments of anti-β2-GPI Abs are able to induce platelet-rich thrombus to the same extent as the whole molecule.69 Specific receptors on platelets72,77 and endothelial cells76 have been identified, which appear to directly interact with the anti-β2-GPI Ab/ β2-GPI complex to mediate activation (see section on platelet and endothelial cell receptors). β2-GPI is able to bind trophoblasts via domain V.78 This enables anti-β2-GPI Abs to inhibit trophoblast gonadotropin secretion and invasiveness.79 It also leads to impairment of extravillous trophoblast differentiation.80 The question of whether a specific surface receptor is involved awaits to be determined, although preliminary evidence suggests that binding of the complex may occur on the exposed phosphatidylserine surface on trophoblasts.79 The mechanism for how this leads to the aforementioned effects in terms of intracellular pathways involved awaits to be established.

THROMBOSIS AND FETAL LOSS: EFFECTOR MECHANISMS

fetal resorption or fetal growth retardation.56 Activation of the C3 component of complement,55 neutrophils,56 and TNF-a57 also appeared to be important intermediates in the process.

Platelet Receptors Blockage of the apolipoprotein ER2′ (ApoER2′) receptor was noted to result in the inhibition of the increased collagen adhesive ability caused by the anti-β2-GPI Ab/ β2-GPI complex on platelets.77 These findings suggest that the ApoER2′ receptor mediates a role in the activation of platelets by the anti-β2-GPI Ab/β2-GPI complex. It was demonstrated that the ApoER2′ receptor is able to coprecipitate with dimerised β2-GPI, providing evidence for a direct interaction between β2-GPI and the receptor.77 β2-GPI is also able to bind the platelet adhesion receptor GP Ib-IX-V.72 Domains II through V of the β2-GPI molecule appear to be involved.72 β2-GPI binding to GPIba on the platelet surface enables

237

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238

anti-β2-GPI Abs directed against domain I to activate platelets leading to the generation of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 (TXA2).72 This may have occurred as a result of receptor cross-linking.72 One of the intracellular pathways involved in platelet activation involves p38 mitogen-activated protein kinase (p38MAPK) and phospholipase A2.71 This is consistent with the notion of activation downstream of the GPIba receptor81 and the ApoER2′ receptor.82 The phosphoinositide 3-kinase (PI3K)/Akt pathway is also activated by the anti-β2-GPI Ab/β2-GPI complex.72 This pathway is involved in the activation of the adhesion receptor aIIbβ3,83 and may play a role in the activation of aIIbβ3 by antibodies from patients with APS.84

Endothelial Cell Receptors

β2-GPI is able to bind annexin II.76 This was found to permit anti-β2-GPI Abs to activate endothelium, leading to the expression of a procoagulant phenotype.76 The annexin II receptor does not have a transmembrane region, and it is not yet clear as to how it leads to endothelial cell activation.76 It has been

suggested that annexin II in complex with β2-GPI may bind to other cell surface receptors that do contain a transmembrane region, allowing anti-β2-GPI Abs to induce activation via receptor clustering and crosslinking.76 A candidate receptor on endothelium is tolllike receptor 4 (TLR4), based on the involvement of intracellular enzymes that are characteristically downstream of this receptor.75 Similarly, based on the intracellular pathways activated it has been suggested that toll-like receptors (TLRs) may be involved in monocyte activation by the anti-β2-GPI Ab/β2-GPI complex.73

CONCLUSIONS Progress is steadily being made in understanding the mechanisms responsible for generating the pathogenic antibodies associated with APS, and the effector mechanisms by which they lead to pathology. The importance of continuing to pursue a complete understanding of all the facets of this disease process is that it may open up new vistas which lead to improved treatments for our patients.

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MECHANISMS OF TISSUE DAMAGE

22C

Antineuronal Antibodies John G. Hanly, MD

INTRODUCTION Autoantibody production is a cardinal feature in the pathogenesis of systemic lupus erythematosus (SLE) and is associated with most of the clinical manifestations of the disease. Antineuronal antibodies have been the subject of extensive research since they were first described in SLE patients in 1978,1 although their exact role in the pathogenesis and diagnosis of neuropsychiatric (NP) lupus is still unclear. Here we review the methodology for the detection of antineuronal antibodies, and explore their prevalence and association with NP-SLE and their potential role in the pathogenesis of the disease.

DETECTION OF ANTINEURONAL ANTIBODIES Antineuronal antibodies have been measured by a variety of assays using different substrates as the source

of neuronal antigens (Table 22C.1). These have included cultured viable and fixed neuroblastoma cell lines1-7 and brain sections of human origin.8 Complement-dependent cytotoxicity,1 indirect immunofluorescence,2-4,7 mixed hemadsorption,5 radioimmunoassay,7,9 and whole-cell ELISA6 assays have been some of the techniques used. Due in part to the wide heterogeneity of assay techniques, the prevalence of antineuronal antibodies in SLE patients has varied considerably (Table 22C.1). In general, IgG antibodies have been less frequent than the IgM isotype and the prevalence has varied between 13 and 82% and 26 and 75%, respectively. Virtually all studies included healthy and disease control groups, and it is noteworthy that antineuronal antibodies have also been reported in other neurologic disease states of both an inflammatory and degenerative nature10-15 and in rheumatoid arthritis.8

TABLE 22C.1 MEASUREMENT AND PREVALENCE OF ANTINEURONAL ANTIBODIES IN SLE Substrate

Assay

Neuroblastoma cell line SK-NSH (human)

Complement dependent cytotoxicity

Human brain sections

Patients____

IgG___

IgM___

Reference_______

40

17%

75%

Bluestein (1978) (1)

Indirect immunofluorescence

22

64%

(“minority”)

Bresnihan (1979) (8)

Neuroblastoma cell line SK-NSH (human)

Indirect immunofluorescence

40

25%

50%

Wilson (1979) (4)

Neuroblastoma cell line Neuro-2a (mouse)

Indirect immunofluorescence

41

82%

27%

Toh (1981) (3)

Neuroblastoma cell lines SKNM-C, NMB-7, IMR-6 (human)

Mixed hemadsorption assay

54

46%

__

How (1985) (5)

Neuroblastoma cell line SK-N- SH (human)

Radio immunoassay

16

38%

63%

Papero (1990) (7)

Neuroblastoma cell lines IMR-6, SK-N-SH (human)

Indirect immunofluorescence

70

13%

26%

Hanly (1993) (2)

Neuroblastoma cell line SK-N-SH (human)

Whole cell ELISA

87

NA

NA

Isshi (1998) (6)

241

ANTINEURONAL ANTIBODIES

CLINICAL ASSOCIATIONS WITH ANTINEURONAL ANTIBODIES Some groups have reported an association between circulating antineuronal antibodies and clinically overt NP manifestations of SLE (Table 22C.2).4,5,8 The correlation is strongest between antibodies of the IgG isotype and diffuse NP manifestations such as psychosis. For example, Wilson and colleagues4 demonstrated serum IgG reactivity to surface antigens on the human neuroblastoma cell line SK-N-SH in 45% of SLE patients with NP disease compared to 5% of SLE patients without NP manifestations. Similarly, 63% of patients with diffuse NP disease demonstrated antineuronal reactivity compared to none of the patients with focal disease.4 A similar but less striking correlation was reported by How and colleagues.5 However, others have not confirmed this clinical-serologic correlation.2,6,7,9,15 In particular, the association between circulating antineuronal antibodies and cognitive impairment has been inconsistent in cross-sectional studies. There is one report of a positive correlation between the presence of circulating antineuronal antibodies and cognitive impairment in SLE patients,16 but this was not confirmed in subsequent independent studies.2,7 Intuitively, the presence of antineuronal antibodies in a location where they would have ready access to brain tissue would support a pathogenic role. Thus, it is of interest that Bluestein and colleagues reported a striking association between the presence of antibodies in CSF of SLE patients with NP disease,9 an observation supported by two subsequent studies.6,15 Information is limited on the association between serial changes in antineuronal antibody reactivity with NP disease. Wilson and colleagues4 (8 patients) and Bresnihan and colleagues8 (2 patients) reported a close temporal association between serum IgG reactivity and clinical disease. Hanly and colleagues17 demonstrated a

similar although less striking correlation between cognitive function and serum IgG antineuronal antibodies in a group of 20 SLE patients evaluated retrospectively over a 2-year period. In this study, changes in antineuronal antibody levels were frequently associated with concurrent changes in anti-DNA antibodies and overall lupus disease activity. When neuropsychiatric disease or cognitive dysfunction was present, their course showed a close correlation with changes in antineuronal antibody levels. However, the concordance with anti-DNA antibodies limits the clinical relevance of these findings.

ANTIGENIC SPECIFICITY OF ANTINEURONAL ANTIBODIES Further insight into the pathogenic role and the diagnostic value of antineuronal antibodies in SLE patients might be derived from characterization of their antigenic specificities (Table 22C.3). Such information could permit the distinction between pathogenic and nonpathogenic antibodies and correlation between specific autoantibodies and subsets of nervous system lupus. Reactivity to a variety of surface neuronal antigens by SLE sera was first suggested by Bluestein,18 who demonstrated at least six different antibody specificities in immunoabsorption experiments using a panel of human neuroblastoma cell lines. Subsequent studies demonstrated cross-reactivity between antineuronal antibodies with surface proteins on lymphocytes,19-21 red blood cells,8 and glial cells22 and with mycobacterial antigens.23 Gangliosides are members of a family of glycolipids predominantly located on neuronal and myelin membranes in the central and peripheral nervous systems. Antiganglioside antibodies have been found in both the serum24-27 and CSF28 of SLE patients and other autoimmune inflammatory neurological disorders such as multiple sclerosis.29,30 Hanly and colleagues31 identified reactivity to a 97-Kd surface protein on two

TABLE 22C.2 ASSOCIATION BETWEEN IgG ANTINEURONAL ANTIBODY AND NP-SLE Serum

CSF

NP-SLE+

NP-SLE−

NP-SLE+

NP-SLE−

Reference

11/22 (92%)

2/10 (20%)

-

-

Bresnihan (1979) (8)

10/20 (50)%

1/20 (5%)

-

-

Wilson (1979) (4)

20/27 (74%)

2/18 (11%)

Bluestein (1981) (9)

-

-

How (1985) (5)

No group difference 14/33 (42%)

3/21 (14%)

-

-

No group difference 2/15 (13%)

242

No group difference

7/55 (13%)

4/24 (17%)

1/12 (8%)

Kelly (1987) (15)

-

-

Papero (1990) (7)

-

-

Hanly (1993) (2)

39/41 (95%)

Not available

Isshi (1998) (6)

Antigenic Specificity

Source of Antigen

Reference

Gangliosides

Neuronal and myelin membranes

Hirano (1980) (24)

97-Kd plasma membrane protein

Human neuroblastoma cell line IMR-6

Hanly (1988) (31)

50-Kd synaptic terminal membrane protein

Adult bovine and fetal human brain

Hanson (1992) (32)

Brain synaptosomes

Rat brain

Hanly (1993) (33)

Brain integral membrane proteins

Human and rat brain

Hanly (1993) (34)

NR2 glutamate receptor

Mouse brain

DeGiorgio (2001) (42)

human neuroblastoma cell lines, IMR-6 and NMB-7. This protein was not identified on non-neuronal cell lines and was not precipitated by sera from control subjects. Hanson and colleagues32 described antibodies to a 50-Kd neuronal membrane protein isolated from bovine brain synaptic plasma membrane. Affinity purified antibodies from SLE sera bound to the surface of cultured rat neuroblastoma cells, and upon Western blotting identified a protein of comparable size in human fetal brain. Additional antibody specificities have been described against proteins of different size derived from synaptosomes33 and integral membrane proteins from homogenized brain tissue.34 Although these studies clearly indicate that SLE patients have autoantibodies that target multiple neuronal and brain antigens, a reproducible and convincing association with clinical or subclinical nervous system disease has not so far been demonstrated. Most recently, attention has been focused on antiNR2 glutamate receptor antibodies as a potentially novel system that could explain some of the complexities of NP-SLE and provide a useful diagnostic tool. The NMDA (N-methyl-D-aspartate) receptors NR2a and NR2b bind the neurotransmitter glutamate and are present on neurons throughout the forebrain.35-37 The hippocampus, which is the anatomical structure closely linked to learning and memory, has the highest density of brain NMDA receptors.36 In addition to their putative role in learning and memory,38 these receptors display altered expression in major psychoses39 and if engaged by receptor antagonists cause hallucinations and paranoia.40

Studies have shown that a subset of anti-DNA antibodies, derived from both murine models of SLE and from a limited number of human subjects with the disease, cross-react with a pentapeptide consensus sequence41,42 that is present in the extracellular ligand binding domain of NR2 receptors. Moreover, these antibodies were present in the CSF of one SLE patient with progressive cognitive decline. Although of considerable interest, these findings are largely derived from animal studies and require confirmation in human subjects with NP-SLE. To date, the studies in human lupus examining the association between this subset of antineuronal antibodies and cognitive impairment have yielded conflicting results.43-45

OTHER AUTOANTIBODIES WITH BRAIN/NEURONAL CROSS-REACTIVITY

TABLE 22C.3 ANTIGENIC SPECIFICITY OF ANTINEURONAL ANTIBODIES

OTHER AUTOANTIBODIES WITH BRAIN/NEURONAL CROSS-REACTIVITY There are a number of lupus autoantibodies that crossreact with brain/neuronal antigens and thus may also be involved in the pathogenesis of nervous system disease Table 22C.4.

Lymphocytotoxic Antibodies Two classes of such antibodies have been demonstrated in SLE patients.46 IgM antibodies demonstrate maximal binding at 4°C and are cytotoxic for resting peripheral blood lymphocytes (hence the term cold-reactive lymphocytotoxic antibodies). In contrast, IgG antibodies demonstrate maximum binding at 37°C, preferentially react with activated lymphocytes, and result in target cell death by antibody-dependent

TABLE 22C.4 AUTOANTIBODIES THAT CROSS-REACT WITH NEURONAL CELLS Autoantibodies

Neuronal Cross-Reactivity

Reference

Lymphocytotoxic antibodies

Human brain homogenates

Bluestein (1976) (21)

Antiribosomal P antibodies

38 Kd surface human neuroblastoma cell line

Koren (1992) (65)

Antiphospholipid antibodies

Rat brain

Kent (2000) (69)

243

ANTINEURONAL ANTIBODIES

cell-mediated cytotoxicity in the absence of complement. Lymphocytotoxic antibodies have been demonstrated in serum and CSF of SLE patients and have been associated with nervous system manifestations, including cognitive dysfunction in some1,8,21,47 but not all studies.2,48 In patients with NP-SLE, lymphocytotoxic reactivity was frequently removed by preabsorbing with brain homogenate,21 suggesting the presence of cross-reactivity between lymphocytes and neuronal cells. Eluates with lymphocytotoxicity from brain homogenates were of both IgM and IgG isotypes.49-51 The antigenic specificity of IgM and IgG lymphocytotoxic antibodies has been studied. Cold-reactive IgM lymphocytotoxic antibodies demonstrate preferential binding to CD4+ T lymphocytes and the CD4+ 2H4+ subset.52,53 Using normal human peripheral blood lymphocytes and lymphocyte cell lines, reactivity to proteins of 55-, 70-, and 105- to 110-Kd proteins have been identified by Western blotting.54 Another study demonstrated IgM reactivity to 46- and 200-Kd glycoproteins isolated by lectin affinity chromatography from normal lymphocytes and lymphocyte cell lines.55 IgG anti-lymphocyte reactivity is directed predominantly against two proteins of 90- and 55-Kd molecular weight.56 Reactivity to a shared 52-Kd protein present on both lymphocytes (CD4+ and HUT-78 cell line) and neruoblastoma cells (SK-N-SH and IMR-6) has also been described,57 and the same group have reported an association with cognitive impairment in SLE.49

Antiribosomal P Antibodies

244

Anti-ribosomal P (anti-P) antibodies were first described in SLE patients in 1985 and are quite specific for SLE, with a prevalence of 13 to 20% depending on the ethnic group.58 Ribosomal P0, P1, and P2 proteins of 38, 19, and 17 Kd (respectively) are located on the 60s subunit of eukaryotic ribosomes. Anti-P antibodies recognize a number of epitopes, including a linear antigenic determinant of 22 amino acids at the carboxyl terminus (which is common to these three ribosomal proteins). In 1987, these autoantibodies were first linked to NP-SLE (in particular to psychosis).59 Subsequent work either supported,60 refuted,58,61 or extended this initial observation to include depression.60 Potential explanations for the differences in study outcomes include variability in diagnostic criteria for psychiatric disease, variance in the temporal relationship between clinical events and serologic testing, and differences in assay technique (particularly antigen preparation and purity). One of the largest human studies62 examined 394 SLE patients, 63 (16%) of whom had anti-P antibodies. There was a significant association with psychosis and depression, with odds ratios between 4 and 10. In contrast, a more recent study of 149 patients63 (12% of whom had anti-P antibodies) did

not find an association with any of the NP syndromes as defined by the ACR classification criteria.64 A potential link between antineuronal and anti-P antibodies has been described. Koren and colleagues65 reported an association between anti-P and antineuronal antibodies in SLE patients and furthermore demonstrated that anti-P antibodies bind a 38-Kd surface protein on human neuroblastoma and hepatoma cells (and to a lesser extent on human fibroblasts) that is closely related or identical to P0 ribosomal protein.65 In another study of 87 SLE patients6 there was a significant elevation in circulating anti-P antibodies in 34 patients with lupus psychosis, but there was no increase in the level of serum antineuronal antibodies. In contrast, examination of the CSF from the same patients revealed a significant elevation in antineuronal antibodies but not in anti-P antibody levels. These data suggest potential interaction between these two families of autoantibodies in the pathogenesis of NP-SLE and emphasize the importance of local versus systemic autoantibody production in the causality of an NP event.

Antiphospholipid Antibodies Autoimmune antiphospholipid antibodies, which are directed against phospholipid-binding proteins such as β2-glycoprotein I and prothrombin,66 are associated with predominantly focal manifestations of NP-SLE. The most common neurologic disorders are those of vascular origin (such as transient cerebral ischemia or stroke), but other associations include seizures, chorea, transverse myelitis, and cognitive dysfunction.67 In a review of over 1000 SLE patients, NP manifestations occurred in 38% of patients with lupus anticoagulant compared to 21% of patients without these antiphospholipid antibodies.68 The favored pathogenic mechanism for this subset of autoantibodies in NP-SLE is thrombosis within vessels of different caliber and subsequent cerebral ischemia. A procoagulant state may be induced through acquired resistance to protein C and protein S, platelet aggregation, and direct activation of endothelial cells.66 However, the in vitro evidence indicating antibody binding to brain tissue69 raises the possibility of an alternative and more direct immunopathogenic assault on the nervous system.

FUNCTIONAL SIGNIFICANCE OF ANTINEURONAL ANTIBODIES IN SLE The data from human studies supporting a role for antineuronal antibodies in the pathogenesis of NP-SLE is largely circumstantial. This includes the temporal relationship between clinical events and serologic findings,25 the presence of autoantibodies in the cerebrospinal fluid,9 and to a very limited extent their

Additional evidence supporting a pathogenic role for antineuronal antibodies is derived from animal studies in which alterations in behavior and neuropathologic changes have been demonstrated when antibodies have direct access to the nervous system. For example, the introduction of antisynaptosomal antibodies directly into the CSF of a rat model led to a particular subset of memory impairment,76 and the injection into the brain of antibodies against brain gangliosides induced seizures and neuropathologic changes.77

REFERENCES

identification in neuronal tissues from patients succumbing to the disease.22,70 Additional evidence in support of this disease model is the occurrence of antineuronal antibodies in other nervous system diseases, such as peripheral neuropathy,10 paraneoplastic syndromes,11 and multiple sclerosis.12 Autoantibodies gain access to the CSF of SLE patients by means of passive transfer from the circulation through a permeabilized blood/brain barrier,71,72 and independently by direct intrathecal production.25,71 Moreover, in animal models examining mechanisms of antineuronal injury, an enhanced permeability of the blood/brain barrier is a critical factor in allowing circulating autoantibodies to enter the CSF and gain access to neuronal cells.41 Although the original description of antineuronal antibodies used a cytotoxicity assay,1 most of the studies have not demonstrated a functional consequence either in vitro or in vivo of antibody binding to neuronal cells. In contrast, the recently described anti-NR2 antibodies have been shown to induce apoptotic cell death of neurons in vitro and in vivo, leading to neuronal injury in a manner similar to that seen in excitatory amino-acid toxicity.73 This effect is mediated via the antigen-binding portion of the antibody73 and is specifically inhibited by memantine, an NMDA receptor antagonist.41 Other studies indicating modulation of neuronal cell function74 and inhibitory effects on cultured rat brain astrocytes75 by antiphospholipid antibodies provide further evidence that antineuronal antibodies in SLE patients may have a functional impact on the nervous system.

CONCLUSIONS Autoantibody production is a hallmark of SLE and plays an important role in the pathogenesis of the disease. There is ample evidence for the presence of antineuronal antibodies in the circulation and CSF of some patients with SLE, although a reproducible and robust association with clinical manifestations of nervous system disease is lacking. The occurrence of shared epitopes between neuronal and non-neuronal cells provides a potential mechanism for initiation and perpetuation of autoantibody production. Although autoantibody may be produced de novo within the intrathecal space, increased permeability of the blood/brain barrier is critical for circulating antineuronal antibodies to gain access to the nervous system. A better understanding of the antibody specificity of antineuronal antibodies and their impact on neuronal viability and function should eventually lead to an improvement in the understanding, diagnosis, and therapy of nervous system lupus.

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56. Minota S, Winfield JB. IgG anti-lymphocyte antibodies in systemic lupus erythematosus react with surface molecules shared by peripheral T cells and a primitive T cell line. J Immunol 1987;138(6):1750-1756. 57. Denburg JA, Behmann SA. Lymphocyte and neuronal antigens in neuropsychiatric lupus: Presence of an elutable, immunoprecipitable lymphocyte/neuronal 52 kd reactivity. Ann Rheum Dis 1994;53(5):304-308. 58. Teh LS, Isenberg DA. Antiribosomal P protein antibodies in systemic lupus erythematosus. A reappraisal. Arthritis Rheum 1994;37(3):307-315. 59. Bonfa E, Golombek SJ, Kaufman LD, Skelly S, Weissbach H, Brot N, et al. Association between lupus psychosis and anti-ribosomal P protein antibodies. N Engl J Med 1987;317(5):265-271. 60. Schneebaum AB, Singleton JD, West SG, Blodgett JK, Allen LG, Cheronis JC, et al. Association of psychiatric manifestations with antibodies to ribosomal P proteins in systemic lupus erythematosus. Am J Med 1991;90(1):54-62. 61. Iverson GL. Are antibodies to ribosomal P proteins a clinically useful predictor of neuropsychiatric manifestations in patients with systemic lupus erythematosus? [letter; comment]. Lupus 1996;5(6):634-635. 62. Arnett FC, Reveille JD, Moutsopoulos HM, Georgescu L, Elkon KB. Ribosomal P autoantibodies in systemic lupus erythematosus. Frequencies in different ethnic groups and clinical and immunogenetic associations. Arthritis Rheum 1996;39(11):1833-1839. 63. Gerli R, Caponi L, Tincani A, Scorza R, Sabbadini MG, Danieli MG, et al. Clinical and serological associations of ribosomal P autoantibodies in systemic lupus erythematosus: Prospective evaluation in a large cohort of Italian patients. Rheumatology (Oxford) 2002;41(12):1357-1366. 64. The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes. Arthritis Rheum 1999;42(4):599-608. 65. Koren E, Reichlin MW, Koscec M, Fugate RD, Reichlin M. Autoantibodies to the ribosomal P proteins react with a plasma membrane-related target on human cells. J Clin Invest 1992; 89(4):1236-1241.

247

MECHANISMS OF TISSUE DAMAGE

22E

Anti-Histone Antibodies in Systemic Lupus Erythematosus Georg Schett, MD, Günter Steiner, MD, and Josef S. Smolen, MD

INTRODUCTION The autoimmune response to chromatin is one of the most typical pathologic features of systemic lupus. Detection and quantification of antibodies against various components of chromatin have not only facilitated the diagnosis of SLE but extended our disease monitoring repertoire. Although autoantibodies against chromatin components have been known for more than 50 years, the mechanism that drives the human immune system to attack well-hidden structures localized in the cell nucleus is still elusive. In this chapter we discuss the various forms of anti-chromatin antibodies and their value for the diagnosis and monitoring of SLE.

proteins, the most prevalent of which are HMG proteins. Among the five different forms of histones (H1, H2A, H2B, H3, and H4), four (except H1) form octamers that consist of two molecules of H2A, H2B, H3, and H4. These octamers constitute the core particle of the nucleosome, which has a size of 10 nm and where 200 base pairs of DNA are wrapped around twice (Fig. 22E.1). Outside the core particle, a single molecule of histone H1 is localized at the so-called linker region of DNA, which spans between two core particles. Histone H1 is important for stabilizing the highly organized tertiary structure of the nucleosome. Although of highly complex structure, chromatin is characterized by a limited number of antigenic targets that allow us to dissect the various patterns of anti-chromatin antibodies.

STRUCTURE OF CHROMATIN Chromatin is the highly organized storage form of DNA that consists of equal amounts of nucleic acid and histones, each of which attributes to 40% of total chromatin. The rest consists of other non-histone

HISTORY OF ANTI-HISTONE ANTIBODIES The first description of an immune phenomenon linked to autoantibodies against structures within chromatin goes back almost 60 years and was the detection

Fig. 22E.1 Structure of the nucleosome. DNA (black line) is wrapped around an octamer of core histones, consisting of each two molecules of H2A, H2B, H3, and H4. This structure is the nucleosome core. Histone H1 (green) is located outside the core particle at the linker region, which spans between two core particles.

Oktameric histone complex (H2A, H2B, H3, H4)

DNA

10 nm

258

H1 histone

Nucleosome

Granulocyte cytoplasm

Amorphous DNAcontaining material

Plasmamembrane Fig. 22E.2 Lupus erythematosus cell. Blood smear of a patient with SLE showing a lupus erythematosus cell (LEC). The LEC is a polymorphonuclear granulocyte, having engulfed the nucleus of a dead cell. LEC are highly specific for SLE and are based on anti-histone antibodies.

of the lupus erythematosus (LE) cell phenomenon by Hargraves and colleagues.1 The LE cell was exclusively found in bone marrow aspirates of patients with SLE and constitutes a polymorphonuclear granulocyte having engulfed the nucleus of a previously dead cell (Fig. 22E.2). Later studies have shown that the formation of LE cells depends on the presence of antibodies, which bind complement and react against self-structures defined by chromatin. Later this structure was defined as histone H1.2,3 Their typical morphology, their high

H1 H2a H2b H3 H4

DNA

specificity, and the fact that LE cells could also be detected in blood samples from SLE patients qualified them as a highly useful laboratory tool to diagnose SLE. The introduction of direct analysis of autoantibodies by specific immunoassays replaced the more complicated LE cell test in the routine diagnosis in later years. A second breakthrough in the research on the immune response against chromatin was the detection of antibodies against double-stranded DNA by Deicher and colleagues in 1959. Thus, measuring antibodies against double-stranded DNA has been established and has become a standard procedure in the diagnosis and monitoring of SLE patients.4-7 SLE patients commonly develop reactivity to several components of chromatin.8 Apart from the well-known antibodies against single- and double-stranded DNA, histones and the complex structures formed by the assembly of histones with DNA are major antigenic structures in SLE (Fig. 22E.3). Thus, antibodies against individual histones, histones complexes, complexes of histones with DNA, and nucleosomes can be differentiated and all constitute antigenic targets. Many SLE patients develop autoantibody responses against several different components of chromatin, but in several patients also highly selective immune response against single components can be observed.

ANTIBODIES AGAINST SINGLE HISTONES

Granulocyte nucleus

ANTIBODIES AGAINST SINGLE HISTONES Antibodies to histones were first described in 1957 and constitute a typical feature of spontaneous SLE and drug-induced LE.8,9 Up to 75% of SLE patients develop anti-histone immune responses.10-12 These antibodies

Single histones and DNA

Histonecomplexes

Histone-DNAcomplexes

Fig. 22E.3 Targets of anti-chromatin antibodies in SLE. Autoantibodies target different molecular structures of chromatin in SLE. They can bind individual histones or DNA. Other antibodies target complexes of histones, such as dimers of H2A and H2B or H3 and H4. In addition, there are antibodies specifically detecting complexes of histones and DNA, such as (H2A-H2B)-DANN, H1-DANN, and (H3-H4)2-DNA. Complex conformational epitopes on the nucleosome or on core particles lacking the assembly by H1 consitute antigenic targets in SLE.

Nucleosomes/ chromatin

259

ANTI-HISTONE ANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS

260

Fig. 22E.4 Antibodies against individual histones in SLE. Left panel: Immunoblot with sera from SLE patients binding to individual histones. Right panel: Absorption with individual histones leads to selective depletion of the antibody reactivity against the specific histone.

H1

Core histones

H1 H2A H2B

H3

H4

–

H1

H2A H2B

can specifically target each individual histone with almost no cross-reactivity to other histones (Fig. 22E.4). Among the four core histones and the one linker histone (H1), strongest autoantibody responses are found against histone H1 and the core histones H2A and B. Antibodies against H1 are found in 50 to 60% of SLE patients9,10,13-15 in cross-sectional analyses. The anti-H1 immune response is essential for the LE cell phenomenon (as discussed previously).3 Like most antibodies against chromatin structures, they belong to the IgG class.10 Autoantibody formation against histone H1, which compared to the core histones is phylogenetically less conserved, is predominantly directed against the trypsin-sensitive regions located within the amino- and carboxy-terminus of the molecule.15-19 A more detailed study using H1-peptides located the site of major immune reactivity to amino acid residues 204 to 218 (a lysine-rich region at the C-terminal end of histone H1), whereas minor epitopes were found within the amino-terminal region.21 Interestingly, the carboxy-terminal end of histone H1 is the most conserved region of the protein and is essential for its interaction with DNA. High-titer antibodies against H1 are highly specific for SLE. Rarely they are found in juvenile and adult rheumatoid arthritis if extra-articular manifestations are present in systemic sclerosis or in primary biliary cirrhosis. Their reactivity is usually low in these conditions.21-24 Importantly, hydralazin- and procainamid- induced LE can display an anti-H1 immune response.25 A crosssectional study with more than 400 sera of patients with rheumatic diseases has shown a similar specificity (98%) of anti-H1 as with antibodies against dsDNA or Sm.26 Interestingly, there is a strong concordance between H1 and dsDNA antibodies. Approximately two-thirds of anti-H1 positive samples contained anti-dsDNA antibodies and over 80% of anti-dsDNA positive sera showing anti-H1 reactivity, suggesting that these antibodies are immunopathologically linked. Longitudinal analyses of anti-H1 reactivity in SLE patients revealed dynamic changes of antibody titers over time, which was closely related to changes in

H3 H4

disease activity. Anti-H1 antibodies correlated with disease activity in a fashion similar to that of antibodies against double-stranded DNA. This is well in line with cross-sectional analyses showing higher disease activity and a higher frequency of severe organ involvement in SLE patients positively tested for the presence of LE cells or anti-H1 antibodies.13,27,28 Immune reactivity to the core histones (H2A, H2B, H3, and H4) is predominantly directed to H2B and somewhat less frequently to H2A. In contrast, autoantibodies to H3 and especially H4 are formed less frequently.29,30 Immune reactivity to individual core histones is closely linked to each other but much lesser to the appearance of anti-H1. As longitudinal analyses have shown, variation of anti-H2B response is also high over time in individual patients, but they reflect changes in disease activity less reliably than anti-H1 or anti-double-stranded DNA antibodies. An analysis of heavy and light change usage of B cells producing anti-H2A and anti-H2B antibodies has been performed by isolating four human monoclonal antibodies by a phage-display library. There was no clonal relation, as indicated by different heavy and kappa light chain groups as well different D and J gene arrangements among the antibodies.31 Due to the fact that the frequency and specificity of anti-histone antibodies as well as their linkage to disease activity varies among different histone components, the value of determining anti–total-histone reactivity seems to be rather limited. This is especially reflected in a number of conflicting data on the relationship of antibodies to total histone with SLE disease activity, showing a good, partial, or poor relation.9,10,32-36

ANTIBODIES AGAINST HISTONE-HISTONE AND HISTONE-DNA COMPLEXES Conformational antigens emerging from the dimerization and tetramerization of core histones as well as

In chlorpromazine- and hydralazine-induced lupus the immune response to (H2A-H2B)-DNA and (H3-H4)2-DNA complexes cannot be fully absorbed by intact nucleosomes, suggesting that buried motifs inside the nucleosome are recognized by these antibodies.37

ANTIBODIES AGAINST NUCLEOSOMES Finally, the complex structure the intact nucleosome provides conformational structures that represent a target for the autoantibody response in SLE. Dependent on their preparation, slightly different antigenic structures are used to detect anti-nucleosome antibodies. Thus, it is of relevance whether histone H1 is removed or not because the absence of H1 from chromatin opens its quaternary assembly and allows better access to the nucleosome core particle. Importantly, however, the antibody response to nucleosomes is the strongest among anti-chromatin antibodies in SLE, suggesting that many antigenic structures on chromatin are conformational.13,29 Antibodies to nucleosomes are highly specific for SLE. They are virtually absent in rheumatic disease other than SLE, such as in rheumatoid arthritis (Fig. 22E.5). Autoimmune type I (lupoid) hepatitis is

ANTIBODIES AGAINST NUCLEOSOMES

antigens (which result from conformational changes if DNA is bound to these complexes) are also targeted by the immune response in SLE. The most abundant antigenic structures are complexes of dimers of H2A and H2B with DNA (H2A-H2B-DNA) as well as complexes of H1 with DNA (H1-DNA).13,29 Antibodies against these complexes are found in up to 70% of SLE patients in cross-sectional analysis. Somewhat less frequently, dimers of H2A and H2B without DNA are identified as antigenic structures. Complexes consisting of H3 and H4, such as tetramers of these two histones with or without DNA, are only infrequently targeted by the autoantibody response in SLE.29 Strong antibody responses against H2A-H2B-DNA are also found in SLE patients negative for dsDNA antibodies.29 Antibodies against (H2A-H2B)-DNA also correlate with clinical signs of disease (especially proteinuria) in cross-sectional analyses, suggesting that testing for H2A-H2B-DNA could serve as a monitoring parameter for disease activity.29 Antibody reactivity to H2A-H2B-DNA can be almost fully abolished following absorption by intact nucleosomes, suggesting that the antigenic structures are located at the outer surface of the nucleosome.

RA

A

Mild SLE

B

Severe SLE Denat DNA

Native DNA

H1–DNA

NUC (+H1)

NUC (–H1)

H2A/B–DNA

H2A–H2B

H4

H3

H2b

H2a

H1

C

Fig. 22E.5 Antibodies against chromatin components in SLE. Autoantibody pattern against various chromatin antigens in the sera of patients with rheumatoid arthritis (A), mild systemic lupus (B), and severe systemic lupus (C). Antibodies are directed against the following antigens: individual histones H1, H2A, H2B, H3, and H4; complexes of H2A and H2B, and complexes of H2A and H2B with DNA; H1-stripped nucleosomes and whole nucleosomes; complexes of H1 with DNA; and native and denatured DNA.

261

ANTI-HISTONE ANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS

TABLE 22E.1 SPECIFICITY AND SENSITIVITY OF ANTI-NUCLEOSOME ANTIBODIES IN SLE Study Karsh 1982 (39)

N

Sensitivity

Specificity

Nephritis

145

86%

86%

ND

Burlingame 1994 (29)

40

78%

ND

Yes

Chabre 1995 (40)

40

48%

ND

Noa

Cacoub 1997 (41)

68

ND

100%

ND

Amoura 2000 (42)

496

72%

90%

Yes

Bruns 2000 (43)

445

56%

97%

Yes

Kiss 2001 (44)

109

39%

ND

Yes

Horak 2001 (45)

52

60%

ND

Noa

Ravirajan 2001 (46)

33

73%

ND

Noa

Hmida 2002 (47)

88

81%

98%

ND

Min 2002 (48)

129

76%

ND

Yes

Schett 2002 (26)

410

45%

95%

Noa

Cervera 2003 (49)

340

69%

92%

Yes

Benucci 2003 (50)

48

38%

ND

Yes

515

58%

99%

ND

63%

95%

Suer 2004 (51) Total

a. Association with clinical disease activity of SLE.

262

one of the few diseases for which anti-nucleosome antibodies have been additionally described.38 Importantly, more than a dozen studies have investigated antinucleosome antibodies in SLE and revealed a specificity of more than 90%26,27,39-51 (Table 22E.1). Roughly twothirds of SLE patients develop anti-nucleosome antibodies in cross-sectional studies, and their fraction may by even higher if longitudinal analyses are used because anti-nucleosome reactivity can strongly vary during the course of disease.26,27,39-51 Moreover, they are also found in SLE patients who lack double-stranded DNA antibodies, suggesting that testing for these antibodies could facilitate the diagnosis of SLE in subjects lacking antibodies against double-stranded DNA.52 Appearance of anti-nucleosome antibodies is also linked to increased disease activity of SLE. Most studies have either shown a link to disease activity scores such as ECLAM or SLEDAI or have unraveled a link to nephritis (Table 22E.1). This is reflected by the stronger anti-nucleosome antibody response in patients with severe SLE compared to mild SLE (Fig. 22E.5). The link of antibodies against components of the nucleosome with nephritis is interesting and may be based on the pathogenic role of anti-nucleosome antibodies in lupus nephritis. Kidney involvement in SLE is based on glomerular disease, where inflammation leads to progressive loss of its filter function resulting in proteinuria. Deposition of immune complexes and complement activation in the glomerular basement

membrane represent mechanisms in the expression of kidney pathology. Immune complexes deposited in the kidneys of patients with SLE contain antibodies against histones and nucleosomes.53-56 This suggests that antibodies to nucleosomes may precipitate glomerulonephritis by inducing the formation of immune complexes in the glomerular membrane. Their ability to activate the complement allows inflammation and tissue damage, which finally results in clinical apparent disease. Due to the fact that free nucleosomes circulate in the serum of SLE patients, the immune system in SLE patients is continuously challenged by self-antigens (which facilitates the formation of nucleosome- anti-nucleosome antibody immune complexes and their deposition in the kidneys).57

CONCLUSIONS Chromatin and its components are a central target of the autoimmune response in SLE. Whereas the antibody response against double-stranded DNA is widely known, many other components of the nucleosome constitute important antigenic structures that are highly specific for SLE and are linked to disease activity. The most important antigens are histone H1, core histones H2A, and H2B alone or complexed with DNA and the intact nucleosome. Detection of these antibodies can help establish the diagnosis of SLE and is useful in monitoring disease activity.

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44. Kiss E, Lakos G, Nemeth J, Sipka S, Szegedi G. Significance of anti-nucleosome (anti-chromatin) auto-antibodies in systemic lupus erythematosus. Orv Hetil 2001;142:1731-1736. 45. Horak P, Scudla V, Hermanovo Z, Pospisil Z, Faltynek L, Budikova M, et al. Clinical utility of selected disease activity markers in patients with systemic lupus erythematosus. Clin Rheumatol 2001;20:337-344. 46. Ravirajan CT, Rowse L, MacGowan JR, Isenberg DA. An analysis of clinical disease activity and nephritis-associated serum autoantibody profiles in patients with systemic lupus erythematosus: A cross-sectional study. Rheumatology (Oxford) 2001;40:1405-1412. 47. Hmida Y, Schmit P, Gilson G, Humbel RL. Failure to detect antinucleosome antibodies in scleroderma. Arthritis Rheum 2002;46:280-282. 48. Min DJ, Kim SJ, Park SH, Seo YI, Kang HJ, Kim WU, et al. Anti-nucleosome antibody: Significance in lupus patients lacking anti-double-stranded DNA antibody. Clin Exp Rheumatol 2002;20(1):13-18. 49. Cervera R, Vinas O, Ramos-Casals M, Font J, Garcia-Carrasco M, Siso A, et al. Anti-chromatin antibodies in systemic lupus erythematosus: A useful marker for lupus nephropathy. Ann Rheum Dis 2003;62:431-434. 50. Benucci M, Gobbi FL, Del Rosso A, Cesaretti S, Niccoli L, Cantini F. Disease activity and antinucleosome antibodies in systemic lupus erythematosus. Scand J Rheumatol 2003;32:42-45. 51. Suer W, Dahnrich C, Schlumberger W, Stocker W. Autoantibodies in SLE but not in scleroderma react with protein-stripped nucleosomes. J Autoimmun 2004;22(4):325-334.

52. Ghillani-Dalbin P, Amoura Z, Cacoub P, Charuel JL, Diemert MC, Piette JC, et al. Testing for anti-nucleosome antibodies in daily practice: A monocentric evaluation in 1696 patients. Lupus 2003;12:833-837. 53. Lefkowitz JB, Kiehl M, Rubenstein J, DiValerio R, Bernstein K, Kahl L, et al. Heterogeneity and clinical significance of glomerular-binding antibodies in systemic lupus erythematosus. J Clin Invest 1996; 98:1373-1380. 54. van Bruggen MC, Kramers C, Walgreen B, Elema JD, Kallenberg CG, van den Born J, et al. Nucleosomes and histones are present in glomerular deposits in human lupus nephritis. Nephrol Dial Transplant 1997;12:57-66. 55. Kramers C, Hylkema MN, van Bruggen MC, van de Lagemaat R, Dijkman HB, Assmann KJ, et al. Anti-nucleosomal antibodies complexed to nucleosomal antigens show anti-dsDNA reactivity and bind to rat glomerular basement membrae in vivo. J Clin Invest 1994;94:568-577. 56. Elouaai F, Lule L, Benoist H, Appolinaire-Pilipenko S, Atanassov C, Muller S, et al. Anti-histone antibodies are concentrated in glomerular eluates of lupus mice. Nephrol Dialysis Transpl 1994;9:362-366. 57. Amoura Z, Piette JC, Chabre H, Cacoub P, Papo T, Wechsler B, et al. Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: correlation with serum nucleosome antibody titers and absence of clear association with disease activity. Arthritis Rheum 1997;40: 2217-2225.

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22D

Antibodies to SSA/Ro and SSB/La: Potential Mechanisms of Tissue Injury in Neonatal Lupus-Congenital Heart Block Jill P. Buyon, MD and Robert M. Clancy, PhD

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Antibodies to SSA/Ro ribonucleoproteins in the maternal sera, often in association with SSB/La, have been almost universally demonstrated when congenital heart block (CHB) develops in utero in the absence of structural abnormalities.1-4 This model of passively acquired autoimmunity offers an exceptional opportunity to examine the effector arm of immunity and to define the pathogenicity of an autoantibody in mediating tissue injury. A molecular scenario in which maternal anti-SSA/Ro -SSB/La antibodies convincingly contribute to the pathogenesis of cardiac scarring has yet to be formulated. One difficulty in identification of a pathogenic effect of an autoantibody is accounting for the heterogeneity of that effect. CHB is a stellar example in that not only is the injury seemingly rare but the degree of injury varied, with the spectrum inclusive of clinically inconsequential first-degree block as well as third-degree block and an associated cardiomyopathy that is often fatal. The necessity of anti-SSA/Ro -SSB/La antibodies is supported by their presence in >85% of mothers whose fetuses are identified with conduction abnormalities in a structurally normal heart.3 However, when Brucato and colleagues5 prospectively evaluated 118 pregnancies in 100 patients with anti-SSA/Ro antibodies, the frequency of CHB in a fetus was only 1.7%. Although recurrence rates exceed the approximate 2% risk for a mother who has never had an affected child by five- to tenfold, the risk is not 100%. Moreover, the concordance rate in genetically identical twins is also not 100%. Accordingly, it is likely that antibody specificity alone cannot account for cardiac injury and that fetal factor(s) and/or the in utero environment must amplify the effects of the antibody, which may be necessary but insufficient to cause disease. Notably, one

mother in the series reported by Brucato and colleagues5 (who gave birth to two healthy children) developed complete heart block herself, raising the possibility that her heart had acquired the amplifying “fetal factors.” Clearly, this is a unique situation and one that needs to be further studied because it is likely to contribute important clues to pathogenesis. A direct pathologic consequence to cells by inhibiting function (as in neonatal myasthenia gravis,6 or Type II cytotoxic reactivity, as in hemolytic disease of the newborn7) would also predict a much higher recurrence rate of CHB in subsequent pregnancies than the observed recurrence rates of 18 to 20%.8,9 Another challenging aspect of the pathogenicity of this disease is that the candidate target antigens are normally sequestered intracellularly. This suggests several possibilities. First, the proposed target is not correct. Second, there is a cross-reactivity of the true target with an antigen normally found on the cardiac surface. Third, the target becomes available to maternal antibody following a change in the cell (which results in translocation to the membrane). The sections that follow address several of the challenges posed by trying to fit the clinical clues with pathogenesis. The majority of the clinical information and patient samples are derived from the Research Registry for Neonatal Lupus (RRNL), established in September of 1994.8

TARGET AUTOANTIGENS OF THE SSA/Ro-SSB/La SYSTEM: A RECENT EMPHASIS ON Ro52 Antibodies to the 52kD SSA/Ro protein are found in >75% of mothers whose children have CHB.10-13 Initial epitope mapping of this response revealed an immunodominant region spanning aa169-291 containing the

with anti-Ro52, additional factors are necessary to convert risk to disease expression. Eftekhari and colleagues recently reported that antibodies reactive with the serotoninergic 5-hydroxytryptamine (5-HT)4A receptor, cloned from human adult atrium, also bind 52kD SSA/Ro.17 Moreover, affinity-purified 5-HT4 antibodies antagonized the serotonin-induced L-type Ca channel activation in human atrial cells. Two peptides in the C terminus of 52kD SSA/Ro, aa365-382, and aa380-396 were identified that shared some similarity with the 5-HT4 receptor. The former was recognized by sera from mothers of children with NLS, and was reported to be cross-reactive with peptide aa165-185, derived from the second extracellular loop of the 5-HT4 receptor. These findings are of particular importance because >75% of sera from mothers whose children have CHB contain antibodies to 52kD SSA/Ro as detected by ELISA, immunoblot, and immunoprecipitation.12,18 Given the intriguing possibility that antibodies to the 5-HT4 receptor might represent the hitherto elusive reactivity directly contributing to AV block, we examined whether the 5-HT4 receptor is a target of the immune response in these mothers.19 Initial experiments demonstrated mRNA expression of the 5-HT4 receptor in the human fetal atrium. Electrophysiologic studies established that human fetal atrial cells express functional 5-HT4 receptors. Sera from 116 mothers enrolled in the RRNL, whose children have CHB, were evaluated: 99 (85%) contained antibodies to SSA/Ro, 84% of which were reactive with the 52kD SSA/Ro component by immunoblot. In sum, none of the 116 sera were reactive with the peptide spanning aa165-185 of the serotoninergic receptor. Rabbit antisera that recognized this peptide did not react with 52kD SSA/Ro. Accordingly, although 5-HT4 receptors are present and functional in the human fetal heart maternal antibodies to the 5-HT4 receptor are not necessary for the development of CHB. Most recently, Eftekhari’s group and ours jointly assessed the role of anti-5HT4 antibodies.20 Sera from 101 anti-SSA/Ro52-positive mothers (of whom 74 had children with CHB and 27 had children without heart block), 8 anti-Ro52-negative mothers who had other anti-Ro/La reactivity and children without heart block, and 18 healthy anti-Ro/La-negative donors were assessed in a single blind test using an ELISA coated with a 5-HT4 receptor-derived peptide. Also tested were 12 anti-Ro/La-negative mothers, of whom 1 had a child with CHB, 5 had children with structural heart block, and 6 had children who developed heart block after birth. Discrepancies between previous observations in our two groups could be ascribed to small differences in the setup of the assay. Of the 74 sera from Ro52+ mothers of children with CHB, 11 were reactive with the 5-HT4 peptide. Sera from

TARGET AUTOANTIGENS OF THE SSA/Ro-SSB/La SYSTEM

leucine zipper (which was recognized by the majority of the CHB-sera), frequently in the context of HLA-DRB1*0301, DQA1*0501, and DQB1*0201.11 The finer specificity of the anti-Ro52 response has been confirmed and extended with current focus on aa200-239 (p200).13 In an initial evaluation consisting of 9 CHB mothers and 26 anti-SSA/Ro positive mothers of healthy children, antibodies to p200 predicted CHB with greater certainty than currently available testing for either 60kD or 52kD SSA/Ro.13 Recent studies integrating an in vivo rodent model and in vitro culturing system suggest that anti-p200 antibodies bind neonatal rodent cardiocytes and alter calcium homeostasis.14 To address both the clinical necessity and sufficiency of this newly identified p200 reactivity in the development of CHB, as well as the reduced risk of CHB reportedly associated with aa176-196 (p176) and aa197-232 (p197),13 maternal sera were evaluated from The Research Registry for Neonatal Lupus (RRNL)8 and from the prospective study PR Interval and Dexamethasone Evaluation (PRIDE) in CHB.15 In addition, the PRIDE study provided the opportunity to address whether the level of anti-p200 antibodies positively correlated with length of the Doppler mechanical PR interval (>150 msec corresponds to first-degree block). The majority of the 156 Ro52-positive sera tested were reactive with p200 (>3 SD above control), irrespective of clinical status of the child.16 Mean OD values of p200 did not differ significantly among mothers of children with CHB (0.187 ± 0.363 SD), rash (0.176 ± 0.356 SD), or no manifestation of NL (0.229 ± 0.315 SD). p200 reactivity was found in 80/104 (77%) CHB mothers, 24/30 (80%) rash mothers, and 21/22 (95%) mothers who delivered healthy children and had no previous children with NL (P = NS for all comparisons). Sera from 4 CHB mothers with varied p200 titers (range OD 0.025 to 1.818) bound the surface of nonpermeabilized apoptotic but not healthy human fetal cardiocytes. These observations suggested that antibodies to p200, equivalent to antibodies to full-length SSA/Ro, do not bind the surface of fetal cardiocytes unless those cells become apoptotic (p200 is translocated to the membrane). For 32 Ro52-positive women completing the PRIDE study (22 no previous child with NL, 7 previous child with CHB, 3 previous child with rash) in whom p200 levels were determined during pregnancy, the correlation between level of p200 (OD range 0.000 to 1.170) and maximal fetal PR interval (range 115 to 168 msec) was not significant (Spearman R = 0.107, P = 0.58). Our interpretation of these data is that reactivity to p200 is a dominant but not uniform anti-Ro52 response in women whose children have CHB. Because exposure to this antibody specificity was observed with a similar frequency in children without CHB born to mothers

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the Ro52− mother of a child with CHB, one of 6 Ro52− mothers of children with structural HB, 3 of 35 Ro/La+ mothers of unaffected children, and 2 of 18 Ro52− controls were also 5-HT4-positive. Although 5-HT4 receptor autoantibodies do not have the predictive value of anti-Ro52 autoantibodies, the presence of these antibodies in a minor subset of mothers whose children have CHB suggests an additional risk factor that may contribute to the pathogenesis of disease.

CARDIAC HISTOPATHOLOGY Histopathologic studies constitute a major basis for formulating hypotheses regarding the pathogenesis of CHB. It appears logical to assume that the time of death relative to initial immune attack may influence the pathologic findings. Evidence of a cellular infiltrate might be present if death occurs close to the time a bradyarrhythmia is first detected, but calcifications and fibrosis may be the sole pathologic finding if death has occurred months later. However, based on data generated in our laboratory (see material following) the inflammatory phase seems to be rather evanescent (in that we have seen extensive fibrosis in a fetus electively terminated almost immediately following the in utero diagnosis).21 Moreover, based on serial echocardiograms a fetus can progress from normal sinus rhythm to complete block in a week.22 Although published literature on serial echocardiograms in mothers at high risk of a pregnancy complicated by CHB is limited (currently being addressed by the PRIDE study,15 discussed previously), it has been the general experience that the first clinically apparent abnormality in cardiac function is bradycardia, and only very rarely myocarditis (i.e., effusions, ventricular dysfunction, and so on). This implies that early inflammation is not clinically detectable and/or that atrioventricular (AV) nodal injury occurs independently of an inflammatory pancarditis. Specific vulnerability of the conducting system is unexplained.23 Ho and colleagues described the histopathology of seven hearts with CHB and associated maternal antibodies to the SSA/Ro polypeptide. In all of these hearts there was atrial-axis discontinuity: the AV node was replaced by varying degrees of fibrosis or fatty tissue.23 The distribution of the distal conducting system was normal. The diffuse fibroelastosis reported in some of these affected babies is considered to result from dilatation of the cardiac chambers secondary to the compensatory increased stroke volume present in CHB.24 However, Nield and colleagues25 have recently reported 13 CHB patients with endocardial fibroelastosis (EFE)—6 diagnosed in utero and 7 in the

postnatal period—despite presumed adequate ventricular pacing of all but one infant. EFE is associated with significant mortality and morbidity: 9 (70%) of these 13 patients died, and 2 (15%) required heart transplants. Given the importance of histologic data to infer pathogenic mechanisms, medical records of all families enrolled in the RRNL were reviewed to determine the incidence and timing of death, with emphasis on the pathologic findings in the affected fetal hearts.26 Complete autopsy reports were available in 11 cases. The mean time from detection of CHB to autopsy was 11 weeks. Although in three cases there were various lesions of the tricuspid valve, the pathologic descriptions were more suggestive of an imposed injury than a true developmental defect. These included nodularity, dysplasia, hypoplasia and fusion of valve leaflets, and fibrosis. The pulmonary valve was abnormal in two, one of which was described as stenotic dysplastic and the other as nodular and dysplastic. Aortic valve insufficiency and stenosis and hypoplasia of the mitral valve leaflet were observed in one. Endocardial fibroelastosis of the right and left ventricles (RV, LV), with or without calcification, was present in 7. Chronic changes in the myocardium were documented in 10, and included biventricular hypertrophy and increased RV and LV walls, thickened but hypoplastic RV, and hyperchromatic nuclei of the myocytes. Abnormalities of the AV node or vicinity were noted in 8 with involution, fibrosis, fatty infiltration, or calcification. However, in 2 the AV node per se appeared normal: in one there was calcification in adjacent tissue, and in another there was an atrophic His bundle with replacement by dense focally calcified fibrous tissue and scarring of the left and right bundle branches. Although previously unappreciated, autopsies obtained from the RRNL revealed a high incidence of valvular abnormalities. Although there were sufficient changes in the AV node to account for CHB in most cases, clinical conduction abnormalities may have been secondary to a functional exit block in a normal-appearing node. SA nodal disease expands the spectrum of conduction dysfunction. However, from a clinical standpoint sinus bradycardia is almost never seen. In the rare instances it has been observed, it has not been sustained.22 The absence of sinus bradycardia is probably due to the presence of other atrial pacers. These studies leave little doubt that the signature lesion of autoantibody-associated CHB is fibrosis, which can clearly extend beyond the conduction system. Consequently, the cascade leading to fibrosis is a major focus of investigation.

Apoptosis has traditionally been conceptualized from an immunologic point of view as either a means of maintaining B- and T-cell tolerance27,28 or as a mechanism for providing accessibility of intracellular antigens to induce an immune response.29 Casciola-Rosen and colleagues have demonstrated that autoantigens are clustered in two distinct populations of surface blebs on keratinocytes.29 The larger blebs, so-called apoptotic bodies (derived from the apoptotic nucleus), contain both SSA/Ro and SSB/La proteins with SSB/La detected at the cell surface surrounding large blebs in the later stages of apoptosis. The 52kD protein was not specifically identified but rather deduced because evaluation was done with a patient serum considered “monospecific” for 52kD SSA/Ro antibodies. The smaller blebs, arising from fragmented rough endoplasmic reticulum and ribosomes, contain SSA/Ro (presumably of cytoplasmic origin). SSB/La was not contained in these blebs. Apoptosis may be relevant in the pathogenesis of NLS. It is a selective process of physiologic cell deletion in embryogenesis and normal tissue turnover and plays an important role in shaping morphologic and functional maturity.30 Apoptosis is a process that affects scattered single cells rather than tracts of contiguous cells. In the normal adult myocardium, apoptosis has been observed only rarely.31,32 In contrast, apoptosis does occur during the development of the heart. In the 1970s, Pexeider extensively characterized the temporal and spatial distribution of cell death in the hearts of chicken, rat, and human embryos.33 Major foci included the AV cushions and their zones of fusion, the bulbar cushions and their zones of fusion, and the aortic and pulmonary valves. Albeit much of the cell death was noted in non-myocytes, a focus of myocyte death was apparent in the muscular interventricular septum as it grew toward the AV cushions in mid-gestation. Takeda and colleagues demonstrated apoptosis in mid-gestational rat hearts using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL), an in situ technique that detects DNA strand breaks in tissue sections.34 Although not coincident with the precise timing of CHB, it has also been suggested that apoptosis contributes to the postnatal morphogenesis of the SA node, AV node, and His bundle.35 Perhaps a novel view of apoptosis is that it facilitates the placing of cardiac target autoantigens in a location accessible to previously generated maternal autoantibodies. Tissue damage might be a consequence of being in the right place at the wrong time.

Apoptosis in electively terminated human abortuses aged 18 to 24 weeks has been recently assessed by our group.21 In fact, there was little detectable apoptosis, but that seen was most prominent in the septal region. We hypothesize that under conditions of physiologic remodeling apoptotic cardiocytes are rapidly cleared, thus accounting for the limited detection. To investigate the hypothesis that apoptosis indeed facilitates accessibility of SSA/Ro and SSB/La to circulating maternal autoantibodies, cultured human fetal cardiac myocytes were incubated with staurosporine or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ).36 By phase contrast microscopy, morphologic signs of early apoptosis were observed in 40% of the cardiocytes after approximately 4 hours and increased to 95% after 7 hours. The cellular topology of SSA/Ro and SSB/La was evaluated with confocal microscopy and determined in non-apoptotic and apoptotic cardiocytes by indirect immunofluorescence using two previously characterized antisera: one “monospecific” anti-SSB/La and the other recognizing both 52kD and 60kD SSA/Ro with goat anti-human IgG-FITC as secondary antibody. In non-apoptotic cardiocytes, SSA/Ro was predominantly nuclear with minor cytoplasmic staining and SSB/La was confined to the nucleus. In early apoptotic cardiocytes, condensation of the SSA/Ro- or SSB/Lastained nucleus was observed accompanied in some cells by a “ring” of bright green fluorescence around the periphery. In the later stages of apoptosis, the nuclear SSA/Ro and SSB/La staining became weaker. Blebs could then be seen emerging from the cell surface, stained with both SSA/Ro and SSB/La. Scanning electron microscopy unambiguously confirmed the surface expression of SSA/Ro and SSB/La (as assessed by gold particle labeling of autoantibodies) on cultured human fetal cardiocytes rendered apoptotic. These earlier published studies have now been extended to include a more in-depth evaluation of structure/function of extrinsic and intrinsic apoptosis pathways in human fetal and adult hearts, and surface accessibility of SSA/Ro-SSB/La Ag to maternal antibodies.37 High levels of Fas-associated death domain protein (FADD) and TNFR-associated death domain protein (TRADD), key components in the apoptotic machinery, were observed in CHB but not normal cardiac tissues. Fetal cardiocytes readily became apoptotic following stimulation with either anti-Fas or TNFα when plated on poly(2-) hydroxyethylmethacrylate (pHEMA), a nonadherent condition. However, these same stimuli did not induce apoptosis in adherent cells. Thus, in fetal cardiocytes adhesion to substrate was pivotal to escaping extrinsic pathway activation, whereas adult cardiocytes did not undergo apoptosis via the extrinsic pathway even in the absence of anchorage. However, adult cardiocytes

ACCOUNTING FOR ACCESSIBILITY OF INTRACELLULAR ANTIGENS TO CIRCULATING MATERNAL AUTOANTIBODIES: APOPTOSIS MAY BE THE CRITICAL LINK

ACCOUNTING FOR ACCESSIBILITY OF INTRACELLULAR ANTIGENS TO CIRCULATING MATERNAL AUTOANTIBODIES: APOPTOSIS MAY BE THE CRITICAL LINK

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treated with staurosporine underwent apoptosis, suggesting that these cells do have the machinery to execute apoptosis via the intrinsic pathway. Utilizing monoclonal antibodies generated from a chicken phage display library, it was demonstrated that Ro52, Ro60, and La48 are surface accessible on fetal cardiocytes regardless of the method used to induce apoptosis. Accessibility appeared to be restricted to select domains because not all antibodies that stained permeabilized cells were reactive with intact apoptotic cells. These studies support extrinsic activation of apoptosis, differentially operative in fetal compared to adult human cardiocytes, as a mechanism linking autoantibody to subsequent injury. In vivo studies have confirmed the observations made in vitro. Tran and colleagues have demonstrated the translocation of SSB/La in apoptotic cardiocytes in the conduction system of the unmanipulated mouse fetal heart.38 Clustering of SSB/La near the surface of apoptotic bodies occurs in vivo under physiologic conditions. To assess proof of concept and examine whether SSB/La and/or SSA/Ro epitopes on apoptotic cells are accessible for binding by antibodies in vivo, these same investigators have exploited a murine passive transfer model in which the fate of human autoantibodies actively transported across the placenta could be traced in fetal tissues known to have high rates of apoptosis.39 Specifically, BALB/c pregnant mice were injected with human anti-SSA/Ro-SSB/La serum, monospecific anti-Ro60 serum, affinity-purified anti-SSB/La, antidsDNA, or normal human serum. Apoptotic cells identified in the fetal conduction tissue (present under normal physiologic conditions of remodeling) showed redistribution of SSB/La from the nucleus to the surface of apoptotic bodies. Fetuses from anti-SSA/ Ro-SSB/La Ab-injected mothers showed a striking co-localization of human IgG with apoptotic cells in the atrium, AV node, liver, skin (with particulate epidermal deposition), and newly forming bone. The IgG-apoptotic cell complexes were organ specific and not detected in thymus, lung, or gut. No IgG deposits were identified in fetuses from mothers injected with anti-dsDNA, anti-Ro60, or normal sera. Experiments with affinity-purified anti-SSB/La and anti-SSA/Ro-SSB/La Abs absorbed with SSB/La confirmed the specificity of deposited IgG as anti-SSB/La. That Ro60 was not identified on the apoptotic cells remains puzzling.

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Four hearts were examined for histologic evidence of apoptosis,21 obtained from a 22-week female fetus

diagnosed at 22 weeks with CHB and electively terminated; a 20-week female fetus diagnosed at 18 weeks with third-degree block and hydrops who died within 2 weeks despite several days of maternal oral dexamethasone at 4 mg/day; a 34-week female fetus who died suddenly at 34 weeks, in which autopsy unexpectedly revealed pancarditis (absent heart block or infection); and a male newborn (40-week gestation) diagnosed with an enlarged right ventricle (RV) at 19 weeks and third-degree block at 24 weeks, who died 2 hours post-delivery. Normal age-matched controls included hearts obtained following elective termination of three fetuses (22, 23, and 24 weeks gestation) in which there was no known cardiac disease, and from a term newborn dying of noncardiac causes. As assessed by TUNEL (FITC and immunoperoxidase detection), apoptosis was increased in sections (including septal tissue, RV, and LV) from the 20-, 22- and 34-week fetuses with CHB/myocarditis, compared to the neonate with CHB dying at birth and 22-/23-week control hearts. Notably, apoptotic cardiocytes were not present in contiguous tracts but were diffusely scattered between nonapoptotic cells. In the 22-week CHB heart, the apoptotic index (AI), a quantitative measure of apoptosis [expressed as (TUNEL-positive nuclei/total nuclei) × 100, where the total number of nuclei is the number of nonapoptotic nuclei plus the apoptotic (TUNEL-positive) nuclei] in the septal tissue was 34%, compared to 8% for RV and 2% for LV. In contrast, tissue from all anatomic regions of the 23-week control heart revealed only scant TUNEL-positive cells consistent with physiologic apoptosis (AI <1% for septum, RV and LV) and in keeping with the hypothesis that apoptotic cardiocytes are rapidly cleared. Although IgG deposition was more limited in distribution than apoptosis, it too was greater in the 20-, 22-, and 34-week affected fetuses compared to the CHB neonate dying at birth. Specifically, IgG staining of the 20- and 22-week CHB fetal hearts was evident in areas proximal to the AV groove and in the LV of the 34-week fetus with myocarditis. In contrast, IgG was not found in the septum of the normal 23-week fetal heart or LV of the normal 22-week heart. Although apoptosis had not been previously examined, other investigative teams have reported deposition of IgG in CHB-hearts (29 and 30 weeks gestation) in several regions, including the conduction system.40,41 In one publication it was noted that in some areas “IgG appeared to outline cells.”40 Thus, the bulk of in vitro and in vivo evidence supports exaggerated apoptosis in the pathogenesis of CHB, and it remains to be explained whether this is secondary to an increase in the number of apoptotic cells and/or failure to clear these cells. In either case, the consequence is translocated intracellular antigens

MURINE MODEL OF CHB

that become accessible to transplacentally passaged maternal autoantibodies.

THE NEXT STEP: ACCOUNTING FOR FIBROSIS To address the functional consequences of opsonization of apoptotic cardiocytes, we designed coincubation experiments with human macrophages.42 TNFα was chosen as a readout of inflammation. Basal production of TNFα by the macrophages was 9.7 ± 0.9 SEM pg/ml and decreased to 3.3 ± 0.3 SEM pg/ml after coincubation with apoptotic cells, which was not observed in initial experiments using cardiocytes rendered necrotic after hypotonic lysis. Apoptotic cardiocytes preincubated with normal human IgG acted functionally as nontreated apoptotic cells. TNFα production by the macrophages was 5.7 ± 0.9 SEM pg/ml. In contrast, when macrophages were cocultured with apoptotic cardiocytes incubated with affinity-purified antibodies to each of the components of the Ro/La complex, TNFα production was increased threefold to fivefold over basal levels and 10- to 14-fold over that secreted after culture with apoptotic cells alone. Non-apoptotic cardiocytes incubated with medium alone or with serum containing antibodies reactive with 48kD SSB/La, 52kD SSA/Ro, and 60kD Ro did not modify the basal production of TNFα by the macrophages. A potential role for TGFβ in fibrosis of the AV node was supported by additional experiments in which we have demonstrated that macrophage-derived factors induce phenotypic changes in cardiac fibroblasts supportive of scarring.43 Human fetal cardiac fibroblasts exposed to supernatants obtained from macrophages incubated with opsonized apoptotic cardiocytes markedly increased expression of the myofibroblast marker, smooth muscle actin (SMAc), associated with scarring. This effect was blocked by anti-TGFβ antibodies.43 Immunohistology of the heart from a term male infant (diagnosed with AV block at 24 weeks of gestation and dying shortly after birth) supported macrophage crosstalk despite the 2-month lag time from detection to death.43 The ventricular tissue revealed microcalcification in which a predominant SMAc-positive infiltrate could be readily observed. Macrophages were also seen in areas of scar tissue. Notably, the fibrosis was not bland but involved an infiltrate of activated myofibroblasts months after the initial insult. Two other hearts from fetuses with CHB (20- and 22-week fetal deaths) were examined for myofibroblasts, which were found in all specimens, supporting the persistence of the myofibroblast phenotype in that this collection encompassed a spectrum of disease severity and timing of death relative to clinical detection.21

Fig. 22D.1 A proposed pathologic cascade leading from inflammation to fibrosis whereby maternal antibodies initiate events that lead to a persistent myofibroblast phenotype associated with scarring. We have previously reported that apoptosis of cardiocytes results in the surface expression of SSA/Ro and SSB/La components, subsequent opsonization by cognate antibodies, and the secretion by macrophages of cytokines such as TGFβ (which transdifferentiate fibroblasts into scar-promoting myofibroblasts). PS = phosphatidylserine. PSR = phosphatidylserine receptor.

To evaluate the extent of fibrosis, cardiac sections were stained with picrosirius for detection of collagen. In both the 20- and 22-week CHB hearts there was extensive fibrosis in the inferior portion of the atrial wall, where the AV node is likely to reside. Collagen deposition was absent in the septal tissue of a normal (fetal-age-matched) control heart. In the 20- and 22-week CHB hearts, TGFβ immunoreactivity was seen in the conduction tissue. In several sections, intense TGFβ staining was present in the extracellular fibrous matrix between SMAc-positive myofibroblasts concentrated in the adjacent subendocardium and infiltrating CD68-positive macrophages. Double labeling revealed colocalization of TGFβ in the cytoplasm of macrophages, including multinucleate giant cells. No fibrosis or TGFβ immunostaining was seen in conduction tissue or ventricles of control hearts from 22- or 23-week abortuses.21 A summary of our working hypothesis, highlighting apoptosis as the pivotal component that links antibody with the subsequent cascade to fibrosis, is depicted in Fig. 22D.1.

MURINE MODEL OF CHB Although clinical data leave little doubt regarding the association of anti-SSA/Ro and/or SSB/La antibodies with the development of CHB, and experimental data are beginning to suggest pathogenicity, efforts to establish an animal model have been limited. Kalush and colleagues reported that offspring of BALB/c mice immunized with the monoclonal anti-DNA idiotype 16/6 had conduction abnormalities.44 Of 31 pups born

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to mothers with experimental SLE, 8 had first-degree heart block, 2 had second-degree heart block, 2 had complete block, 10 had bradycardia, and 8 demonstrated widening of the QRS complex. None of these disorders could be detected in the 20 offspring of healthy control mice. One of the difficulties in interpreting these findings is that the immunized mothers synthesized a variety of autoantibodies, including antibodies reactive with 16/6 Id, ss/dsDNA, Sm, RNP, cardiolipin, SSA/Ro, and SSB/La. Accordingly, it was not possible to segregate out which specific antibody might be responsible for the arrhythmias detected in these pups. The electrocardiographic data are provocative. However, no histologic data were provided to assess the status of the SA or AV node, or the presence of myocarditis. To further establish an antibody-specific murine model to correlate arrhythmogenic effects of maternal autoantibodies with the in vivo genesis of CHB we have immunized female BALB/c mice with 100 μg of one of the following 6×His human recombinant proteins purified by Ni2+ affinity chromatography: 48kD SSB/La, 60kD SSA/Ro, 52kD SSA/Ro (52α full-length), and 52β.45 Control animals were given the same injections with a Ni2+ affinity-purified polypeptide encoded by pET-28 alone. Following primary immunization in complete Freund’s adjuvant and 2 boosters (50 μg) in incomplete Freund’s adjuvant, high-titer immune responses to the respective antigens were established by ELISA and immunoblot of recombinant antigens, and immunoprecipitation of [35S]-methionine-labeled in vitro translation products. Sera from mice immunized with either 52α or 52β immunoprecipitated radiolabeled murine 52kD SSA/Ro, confirming that these mice were specifically reactive with the murine homologue. Moreover, immunoblot of a newborn murine heart demonstrated the presence of 52kD SSA/Ro. Mice were mated and boosters continued every 3 weeks to ensure continued high-titer antibody responses. EKGs were performed on all pups using standard limb leads at birth or within 2 days postpartum. Maternal antibodies to the primary immunogens were detected by ELISA in the pups. Of 54 pups born to 6 fertile mice immunized with 60kD SSA/Ro, none had CHB. Of 27 pups born to 3 fertile mice immunized with 48kD SSB/La, none had CHB. In contrast, of 78 pups born to 5 fertile mice immunized with 52α and 86 pups born to 5 fertile mice immunized with 52β, 1 and 5 pups (respectively) had complete AV block. Accordingly, this antibodyspecific animal model provides strong preliminary evidence for a pathogenic role of antibodies reactive with 52kD SSA/Ro, particularly the 52β form, in the development of CHB. Moreover, analogous to the

frequency of 1 to 5% given for women with SLE who have anti-SSA/Ro and/or SSB/La antibodies4,5 this model suggests that additional factors must promote disease expression.

FETAL GENETIC FACTORS Because the pathogenesis of CHB involves a cascade from inflammation to subsequent scarring, polymorphisms of the TNFα promoter region and codons 10 and 25 of the TGFβ gene were evaluated in children with cardiac and cutaneous manifestations of NL. DNA was isolated from and genotyped in 137 children (59 CHB, 23 with rash, and 55 unaffected siblings) and 74 mothers of these children (>80% Caucasian) enrolled in the RRNL.46 Although an increased frequency of the -308A allele of TNFα (associated with higher production) was observed in children with CHB compared to controls, a clear association with disease could not be established because children with rash, unaffected children, and the mothers all had a significantly higher genotypic and allelic frequency of -308A compared to controls. In contrast to the results for TNFα, the TGFβ polymorphism Leu10 (associated with increased organ fibrosis) was distributed significantly differently in the children with CHB. There were no significant differences in genotypic or allelic frequencies between children with rash, unaffected children, or mothers and the published controls. However, the Leu10 polymorphism was significantly higher in CHB children (genotypic frequency 60%, allelic frequency 78%) than in unaffected children (genotypic frequency 29%, p = 0.016; allelic frequency 56%, p = 0.011), and controls. Cimaz and co-workers47 recently confirmed a high prevalence of the Leu10 polymorphism in two familes in which at least 1 child in each family was affected by CHB . The profibrotic TGFβ1 genotype was detected in a twin with CHB but not in its healthy twin, whereas each of 3 triplets (1 third-degree HB, 1 first-degree HB, 1 transient liver abnormality48) displayed Leu.10 Tissue from a 20-week fetus who died of CHB was available for isolation of DNA. This fetus was homozygous for the Leu10 polymorphism. Notably, the genotype of an unaffected older sibling at codon 10 was Leu/Pro. The mother had a genotype identical to the fetus with CHB. Thus, it appears that children with CHB have a higher frequency of a genetic polymorphism in TGFβ (which could lead to its exaggerated secretion) compared to unaffected anti-SSA/Ro exposed children, which fits well with the histologic observations. Amplification of antibody-induced injury secondary to a genetic polymorphism that inherently leads to

POTENTIAL IN UTERO ENVIRONMENTAL FACTORS Discordance of CHB in identical twins9 supports the contribution of an environmental influence that could critically amplify the pathologic cascade to scarring at the level of antibody access to antigen (initiating event) and/or subsequent fibrosis (progression) yet be operative in one fetus and not another despite all else being equal. Accordingly, our laboratory explored the novel hypothesis that in utero hypoxia amplifies one or several steps leading to cardiac fibrosis.49 The approach integrated an in vitro model of CHB, using separately isolated cardiocytes and fibroblasts from 16- to 24-week abortuses, with in vivo evaluation of hearts from fetuses that died of CHB. Initial experiments examined the effects of hypoxia on the downstream end of the cascade. Exposure of human fetal cardiac fibroblasts to hypoxia (<0.01% O2) for 24 and 48 hours had no effect on viability (FACS stain). Hypoxic fibroblasts expressed hypoxia-inducible factor 1α (HIF1α), which was not present in normoxia (RT-PCR, immunoblot). The fetal fibroblasts clearly displayed a myofibroblast phenotype, based on expression of the TGFβ downstream effectors plasminogen activation inhibitor 1 (PAI-1; blocks fibrin degradation; detected by RT-PCR) and smooth muscle actin (SMAc; detected by RT-PCR, immunofluorescence). Specifically, expression of PAI-1 increased twofold and SMAc by 4.5-fold (hypoxia versus normoxia), wheras GAPDH was the same under both conditions. Protein analysis of supernatants generated from hypoxic fibroblasts revealed increased release of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), PAI-1, and growth-related oncogene (GRO): 6.3-fold, 2.0-fold, 1.7-fold, and 2-fold, respectively (hypoxia versus normoxia). Three fetal CHB hearts were interrogated for expression of HIF1α, RANTES, and PAI-1: in the conduction tissue, prominent intracellular fibroblast expression

of HIF1α was seen in the region of fibrosis, whereas expression of RANTES (extracellular staining) and PAI-1 (both intracellular and extracellular) was seen in areas of calcification. An age-matched fetal heart (normal abortus) expressed none of these factors. Cell supernatants were analyzed for lactate dehydrogenase and cells were analyzed for apoptosis and human IgG binding, which was assessed by a FACS method after staining the cells using Annexin V, anti-active caspase-3 antibody, maternal anti-Ro/La IgG, rabbit IgG, or healthy control IgG. For each treatment, annexin V or IgG binding was reported as % positive cells (>95th percentile of isotype control). Fetal cardiocytes readily undergo apoptosis when plated under hypoxia (Annexin V staining, 23.9 ± 1.9 versus 9.3 ± 2, hypoxia versus normoxia, P = 0.01). Furthermore, anti-active caspase 3 and maternal anti-Ro/La IgG each tended to bind the cardiocytes rendered apoptotic following hypoxia. Negative controls stained appropriately (IgG healthy control, rabbit IgG). Confirmation that the cells had not undergone necrosis with loss of membrane integrity was supported by absence of significant LDH release (<6% total, not shown). In sum, the data support hypoxia as an environmental stress that potentially serves to amplify the proximal and distal components of the pathologic cascade to scarring, and provide a potential explanation for the progression to permanent injury in one fetus and not another despite identical genes and maternal antibody exposure.

SUMMARY

increased TGFβ production could be a factor relating to susceptibility. It is acknowledged that further polymorphisms need be considered, not only in the TGFβ genes but in those encoding molecules involved in the activation of latent TGFβ. Of relevance here may be the still unexplained clinical spectrum of disease, in which some fetuses develop fibrosis in an extraordinarily short time frame and others with incomplete blocks that progress postnatally even up to years after the antibodies have been cleared from the circulation.

SUMMARY The development of CHB is clearly a rare event, and we hypothesize that this rarity is due to the contribution of at least three components, as depicted schematically in Fig. 22D.2. The maternal component, antibodies reactive with SSA/Ro (frequently in association with anti-SSB/La), is necessary but insufficient. Apoptosis is considered a central event that links the maternal component to subsequent inflammation and ultimately scarring. The fetal component is likely to comprise factors that exaggerate apoptosis and fibrosis. These factors may be genetic. The third component must account for the discordance of disease in identical twins. One plausible candidate is environmental (i.e., intrauterine) stress, specifically hypoxia. Both fetal and environmental components serve to amplify the effect of maternal antibodies accounting for the spectrum as well as the rarity of this complex disease.

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Fig. 22D.2 Anti-SSA/Ro-SSB/La antibodies associate strongly with CHB, yet this injury to the developing heart remains a rare event. We hypothesize that this rarity is due to the contribution of at least three components. First, the maternal component—antibodies reactive with SSA/Ro (frequently in association with anti-SSB/La)—is necessary but insufficient. Apoptosis is considered a central event that links the maternal component to subsequent inflammation and ultimately scarring. Second, the fetal component is likely to comprise factors that exaggerate apoptosis and fibrosis. These factors may be genetic. A third component must account for the discordance of disease in identical twins. One plausible candidate is environmental (i.e., intrauterine) stress, specifically hypoxia. Both fetal and environmental components serve to amplify the effect of maternal antibodies accounting for the spectrum as well as the rarity of this complex disease. NSR = normal sinus rhythm.

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maternal autoantibodies in apoptotic human fetal cardiac myocytes. J Immunol 1998;161:5061-5069. Tran HB, Ohlsson M, Beroukas D, Hiscock J, Bradley J, Buyon JP, et al. Subcellular redistribution of La(SS-B) autoantigen during physiologic apoptosis in the fetal mouse heart and conduction system: A clue to the pathogenesis of congenital heart block. Arthritis Rheum 2002;46:202-208. Tran HB, Macardle PJ, Hiscock J, Cavill D, Bradley J, Buyon JP, et al. Anti-La (SS-B) antibodies transported across the placenta bind apoptotic cells in fetal organs targeted in neonatal lupus. Arthritis Rheum 2002;46:1572-1579. Miranda-Carús ME, Dinu Askanase A, Clancy RM, Di Donato F, Chou TM, Libera MR, et al. Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of tumor necrosis factor α by macrophages. J Immunol 2000;165:5345-5351. Lee LA, Coulter S, Erner S, Chu H. Cardiac immunoglobulin deposition in congenital heart block associated with maternal anti-Ro antibody. Am J Med 1987;83:793-796. Litsey SE, Noonan JA, O’Connor WN, Cottrill CM, Mitchell B. Maternal connective tissue disease and congenital heart block. Demonstration of immunoglobulin in cardiac tissue. New Engl J Med 1985;312:98-100. Clancy R, Askanase AD, Chiopelas E, Azar N, Miranda ME, Buyon JP. Pivotal role of human fetal cardiac fibroblasts in the pathogenesis of autoantibody-associated congenital heart block [abstract]. Arthritis Rheum 2001;44:S160. Clancy RM, Askanase AD, Kapur RP, Chiopelas E, Azar N, Miranda-Carus ME, et al. Transdifferentiation of cardiac fibroblasts, a fetal factor in anti-SSA/Ro-SSB/La antibody-mediated congenital heart block. J Immunol 2002;169:2156-2163. Kalush F, Rimon E, Keller A, Mozes E. Neonatal lupus erythematosus with cardiac involvement in offspring of mothers with experimental systemic lupus erythematosus. J Clin Immunol 1994;14:314-322. Miranda-Carús ME, Boutjdir M, Tseng CE, DiDonato F, Chan EKL, Buyon JP. Induction of antibodies reactive with SSA/Ro-SSB/La and development of congenital heart block in a murine model. J Immunol 1998;161:5886-5892. Clancy RM, Backer CB, Kapur RP, Yin X, Molad Y, Buyon JP. Cytokine polymorphisms and histologic expression in autopsy studies: Contribution of TNFα and TGFβ1 to the pathogenesis of autoimmune-associated CHB. J Immunol 2003;171:3253-3261. Cimaz R, Borghi MO, Gerosa M, Biggioggero M, Raschi E, Meroni PL. Transforming growth factor β1 in the pathogenesis of autoimmune congenital complete heart block: Lesson from twins and triplets discordant for the disease. Arthritis Rheum 2006;54:356-359. Fesslova V, Mannarino S, Salice P, Boschetto C, Trespidi L, Acaia B, Mosca R, Cimaz R, Meroni PL. Neonatal lupus: fetal myocarditis progressing to atrioventricular block in triplets. Lupus 2003;12:775-778 Clancy R, Zheng P, Gardner L, Buyon JP. Hypoxia, an environmental stress, in initiation and progression of the pathologic cascade to congenital heart block. Arthritis Rheum 2005;52: S303.

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20. Kamel R, Eftekhari P, Clancy R, Buyon JP, Hoebeke J. Autoantibodies against the serotoninergic 5-HT4 receptor and congenital heart block: a reassessment. J Autoimmun 2005;25:72-76. 21. Clancy RM, Kapur RP, Molad Y, Askanase AD, Buyon JP. Immunohistologic evidence supports apoptosis, IgG deposition, and novel macrophage/fibroblast crosstalk in the pathologic cascade leading to congenital heart block. Arthritis Rheum 2004;50:173-182. 22. Askanase AD, Friedman DM, Copel J, Dische MR, Dubin A, Starc TJ, et al. Spectrum and progression of conduction abnormalities in infants born to mothers with anti-Ro/La antibodies. Lupus 2002;11:145-151. 23. Ho YS, Esscher E, Anderson RH, Michaelsson M. Anatomy of congenital complete heart block and relation to maternal anti-Ro antibodies. Am J Cardiol 1986;58:291-294. 24. Hogg GR. Congenital acute lupus erythematosus associated with subendocardial fibroelastosis: Report of a case. Am J Clin Pathol 1957;28:648-654. 25. Nield LE, Silverman ED, Taylor CP, Smallhorn JF, Mullen JB, Silverman NH, et al. Maternal anti-Ro and anti-La antibodyassociated endocardial fibroelastosis. Circulation 2002;105: 843-848. 26. Tseng C, Friedman D, Buyon JP. Spectrum of cardiac histopathology in cases of autoimmune-associated congenital heart block (CHB) obtained from the Research Registry for Neonatal Lupus [abstract]. Arthritis Rheum 1997;40:S333. 27. Watanabe-Fukunaga R, Brannan CL, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1993; 356:314-317. 28. Bretscher P. The two-signal model of lymphocyte activation twenty-one years later. Immunol Today 1992;13:74-76. 29. Casciola-Rosen LA, Anhalt G, Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 1994;179:1317-1330. 30. Ucker DS. Death by suicide: One way to go in mammalian cellular development? New Biol 1991;3:103-109. 31. Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 1996;74:86-107. 32. Cheng W, Li B, Kajstura J, Li P, Wolin MS, Sonnenblick EH, et al. Stretch-induced programmed myocyte cell death. J Clin Invest 1995;96:2247-2259. 33. Pexeider T. Cell death in the morphogenesis and teratogenesis of the heart. Adv Anat Embryo Cell Bio 1975;51:1-100. 34. Takeda K, Yu ZX, Nishikawa T, Tanaka M, Hosoda S, Ferrans VJ, et al. Apoptosis and DNA fragmentation in the bulbus cordis of the developing rat heart. J Mol Cell Cardiol 1996;28:209-215. 35. James TN. Normal and abnormal consequences of apoptosis in the human heart: From postnatal morphogenesis to paroxysmal arrhythmias. Circulation 1994;90:556-573. 36. Miranda-Carus ME, Tseng CE, Rashbaum W, Ochs RL, Casiano CA, DiDonato F, et al. Accessibility of SSA/Ro and SSB/La antigens to

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Standardization of Autoantibody Testing in Systemic Rheumatic Diseases Marvin J. Fritzler, MD, PhD and Alan Wiik, MD, PhD

INTRODUCTION Autoantibody testing has become a powerful tool for clinicians in the management of patients with systemic rheumatic diseases, but these tests are currently not being used optimally due to lack of widely used standardization of kit manufacturing, assay performance, workplace conditions, post-marketing surveillance, and test interpretation. Regrettably, because of significant logistical challenges there have been very few prospective, unbiased, and multicenter studies. Thus, the clinical accuracy of certain laboratory diagnostic approaches is still unknown. It is clear that standardization of kits, reagents, and protocols could markedly improve these discrepancies. New serologic technologies and assays are being developed at a very rapid pace and may eventually be accompanied by acceptance of global standards of practice. To achieve this goal, the accuracy of such testing must be demonstrated in studies involving sufficiently large patient cohorts before new assays are approved or accepted for clinical use. This chapter provides an overview of the challenges involved in and proposed solutions put forward for the standardization of autoantibody testing. It is most important to appreciate that the primary goal of standardization is to improve the quality of patient care through the correct use and interpretation of autoantibody testing.

AUTOANTIBODY ASSAY KITS AND PROCEDURES For over half a century, the detection of human autoantibodies has become an increasingly important approach to the diagnosis and management of patients with a variety of autoimmune conditions. Although some diagnostic laboratories still use assays that are developed in-house, there is growing and widespread use of commercial diagnostic kits from a large number

of manufacturers and distributors.1 These commercial kits are cost effective, are easy to use, and for the most part have been tested and validated according to established protocols and criteria. Most kits come ready to use and provide all of the necessary reagents and clearly written protocols for performance of the assay. Commercial autoantibody assay kits employ a variety of technological platforms that include indirect immunofluorescence (IIF), immunodiffusion (ID), immunoblotting (IB), enzyme-linked immunoassays (ELISA), LINE assays, addressable laser bead immunoassays (ALBIA), and more recently antigen arrays in a number of formats.2,3 One of the current popular technology platforms is based on the ELISA because ELISAs are available through a global network of manufacturers and distributors, and they offer sensitivity, high throughput, relatively low cost, and modest laboratory equipment needed to perform the assay. Unfortunately, there has been little done to standardize these kits,4,5 and post-marketing surveillance and quality assurance is largely left to the manufacturers. It might be assumed that assays that have been in use for several decades (such as IIF) have achieved a high level of inter-laboratory consistency, but this is not the case. A number of studies have evaluated the performance characteristics of IIF ANA6-17 and ANCA18 kits. In addition, studies that compared ELISA kits from different manufacturers to conventional assays such as IIF and ID concluded that there was significant discordance between these conventional assays and ELISA.7,9 This lack of test result correlation is compounded by variability of the performance of diagnostic kits from different manufacturers.8,19 However, at least one study that used a cross section of serum referred to a rheumatology laboratory found moderate to good agreement between ANA-IIF and anti-DNA results with two commercial ELISA kits.10 Analysis of the design of some studies suggests that lack of agreement between

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ELISA and conventional assays may depend on the diagnosis and/or the selection bias of the patients under study.6,7,8,10 A study of the Serology Committee of the International Union of Immunological Societies and the Arthritis Foundation focused on the ELISA kits themselves and deficiencies in intrinsic properties of the kits (sensitivities and specificities) were identified as a specific concern.19 A more recent study by the same committee focused on academic clinical laboratories and found surprising lack of consistent inter-laboratory results even when highly characterized sera were distributed and tested.16 As in other studies,4 it was suggested that quality control procedures for daily performance of tests in the clinical laboratory setting should be adhered to and that a minimal performance target of correlation values in ELISAs should be established.16

MANUFACTURING AND ADOPTION OF DIAGNOSTIC KITS BY THE CLINICAL LABORATORY In many countries, the delivery of health care has been “streamlined” through consolidation of services, including those provided by diagnostic laboratories. This has often translated into higher workloads and pressure to improve the interval between when the test is requested to the time the test result is reported to the ordering physician (turnaround time). To meet these market demands, manufacturers have developed a wide variety of easy-to-use and relatively inexpensive diagnostic kits designed for high throughput and the detection of a spectrum of more autoantibodies. Unfortunately, the demand for prepackaged diagnostic kits has led to a fast-track approach in the development, validation, and approval of commercial kits. Regulatory agencies such as the Food and Drug Administration (FDA) in the USA have attempted to maintain a reasonable level of quality before allowing the release and marketing of diagnostic kits by manufacturers. It is in the context of this activity that some problems with kit performance occur because kit validation at this level may only be discovered during post-marketing performance. Validation studies would be significantly facilitated by a centralized collection of highly characterized normal and disease cohort sera that could be used in evaluation of kit performance and establishing appropriate levels of positive and negative boundaries. Currently, validation is often performed on state-ofthe-art equipment, using the freshest reagents and kits just off the assembly line. A second level of validation involves providing the kits to laboratories that are willing and able to “beta test” the kit. If the results from the external beta tests are in agreement with internal data, the kit is submitted for approval by

regulatory agencies. Upon approval, marketing and manufacturing rapidly begins. Another source of variation is that manufacturers tend to purchase kit components from a wide variety of suppliers and not all kit manufacturers use the same supplier. The decision to produce or purchase critical components such as purified antigens and secondary antibodies is based on a number of factors that include cost and performance. When a reagent satisfies these features, manufacturers tend to purchase large lots of these reagents to minimize variability between production lots. After the commercial kit has been developed, validated by the manufacturer, and marketed, it is then left to the clinical laboratories to evaluate the products available and make an “informed” decision about which kit is suited to that laboratory’s particular environment so that cost/performance issues are adequately addressed. The continuous development and adoption of new technologies provides yet another challenge. New technologies commonly focus on achieving high diagnostic (nosographic) sensitivity but less emphasis on high diagnostic specificity. Unfortunately, in practice diagnostic specificity generally decreases when a certain assay achieves high sensitivity. Therefore, when a new test is introduced, laboratories and clinicians may overlook the loss of diagnostic specificity and focus on the sensitivity of the assay. Because new assays are frequently released before the ability of the assay to accurately predict a specific diagnosis is fully known, it is imperative that testing sera from local control patients with inflammatory rheumatic diseases be used to measure the predictive performance of a new assay. This clinical evaluation must be performed by expert laboratories in close collaboration with experienced clinicians who strive for an accurate diagnosis. Defined patient samples provided by the experienced clinicians tend to produce superior coefficients of variation and receiver operator analyses that are used to determine performance characteristics of new assays and kits.5

AUTOANTIBODY TESTING IN THE CLINICAL LABORATORY The clinical diagnostic laboratory is usually dependent on the integrated and optimal performance of a number of individuals that perform different tasks, ranging from the laboratory manager, to the clinical laboratory specialist (usually a clinical laboratory immunologist with a PhD or MD degree and/or other certification), to the technologist and support staff that handle the specimens and perform the assay. As noted previously, the decision to adopt a particular kit is often based on fiscal matters and is left to the discretion of the manager in consultation with the laboratory specialist.

(epifluorescence) light. In addition, the quality of microscope objective lenses (numerical aperture) varies from manufacturer to manufacturer and the choice of objectives is often dictated by cost. Some laboratories use oil immersion objectives and others insist that dry objectives are adequate. Some laboratories use their own mounting medium and cover slips even when these are supplied as components of kits. ELISA and dot blot protocols are often performed on equipment with varying capacity and photonics that bear little resemblance to the equipment used by manufacturers. In addition, equipment is becoming more sophisticated and that generally results in assays becoming more sensitive. As that occurs, the diagnostic specificity decreases and cut-off points must be adjusted. For years it was thought that the cut-off and appropriate screening dilution for ANA on HEp-2 substrates was 1/40 or 1/80. However, after a multicenter study showed that 32% of normal sera were positive at 1/40 it was recommended that a cut-off of 1/160 is more appropriate.22 The impact of variations in equipment on assay performance is compounded when the test requires a subjective assessment by the technologist or laboratory immunologist. Unless there are stringent in-house validation procedures, this can lead to a wide range of results. A typical example of this variation is the discrepant results that may occur when a different technologist in the same lab performs and interprets the results one day and another performs and interprets the results on weekends. It might be concluded that this results in a complete breakdown of inter-laboratory and inter-test performance. Thankfully, that is not always the case because despite these variables performance characteristics might be considered remarkably high. This in large part is due to quality assurance and accreditation programs, discussed in material following.

INTERPRETATION OF THE AUTOANTIBODY RESULT

In a modern clinical laboratory where knowledge and highly specialized technologies are rapidly expanding and specialized products are being produced, the clinical laboratory specialist is expected to be an expert. These laboratory clinicians are doing their best to maintain and advance their level of competence, while carrying out burdensome but necessary quality assurance and accreditation standards. Meanwhile, industry is advancing quickly to adapt, modify, and advance diagnostic technologies as well as offer new diagnostic kits to make the laboratory clinician’s life more manageable. It is in this dynamic that further challenges to standardization of autoantibody testing arise. The characteristics of diagnostic laboratories that perform well are not clearly understood. Even though manufacturers provide clear directions on how to optimally use their kit and perform a certain assay, some laboratories adopt kits but use their own variation of the protocols.16 One study showed that when the laboratories are a part of or affiliated with academic institutions they might perform better, but this is not always the case.16 It is clear that when laboratories perform well, they invariably engage in a process of standardization and internal validation of new kits using clinically defined serum samples before they are adopted. As noted previously, even in ideal manufacturing circumstances (after internal evaluations and external beta testing) the capability of the assay to accurately predict a specific diagnosis is not fully known. Therefore, it is incumbent on the clinical diagnostic laboratory to evaluate each new kit with test sera from local patients with inflammatory rheumatic diseases. This process needs to be attended by close collaboration of diagnostic laboratories, experienced clinicians who strive for an accurate diagnosis and patients that willingly donate their blood for testing and research purposes. In the very early phase of diagnostics of inflammatory rheumatic diseases, the finding of a particular autoantibody has a great impact on the setting of diagnosis and estimation of prognosis in a patient.20 Therefore, serum samples taken at the very early onset of inflammatory disease symptoms should be stored together with clinical data seen at that time so that later follow-up and final diagnosis can help us focus on the most relevant tests to order in early phases of disease.21 There are a number of other factors that impact assay performance. The first is the equipment used to perform the test, but of pivotal importance are microscopes used for IIF and spectrophotometers and plate readers used for ELISA. These instruments vary in performance, not only with respect to intra-laboratory configurations but inter-laboratory configurations. A key element related to microscopy is the use of a transmitted UV light source rather than incident

INTERPRETATION OF THE AUTOANTIBODY RESULT The next significant problem leading to poor laboratory standardization is the content and design of autoantibody test reports. Clinicians must be able to understand the reported results without having to pour over the report. If the report is not easily understood, it can be erroneously interpreted. In some instances, the clinician may take unnecessary time to contact the laboratory to request an interpretation or (even worse) file the report as useless information. The results must be clearly expressed and an indication of whether it represents a high, moderate, low, borderline, or negative result. In most cases, there is little clinical value in reporting numerical results such as the OD values for ELISA.

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Clinicians usually want to know if the result is positive or not, if it is highly abnormal or borderline, and a general interpretation of the result. Some laboratories also provide current literature references to support the result so that clinicians can inform themselves further if they desire. When an autoantibody is found, the positive result is communicated to the clinician as a printed report or a secured digital report sent directly to the doctors. Other general information to aid in the interpretation, such as the sensitivity and specificity of a positive result, should be tabulated in a printed or an Internet-based guide or printed handbook. It is clear that a uniform internationally approved autoantibody reporting format would be desirable. Even if the result is accurately and clearly reported, other factors contribute to how a test is interpreted. Of utmost importance, the result must be interpreted in the context of the patient’s symptoms and/or clinical findings. An autoantibody test on its own rarely establishes a diagnosis because systemic rheumatic diseases involve multiple organ systems and, particularly early in the disease course, there is rarely a pathognomic feature. Therefore, multiple criteria have been developed and must be fulfilled to confirm a particular diagnosis. Importantly, each disease is associated with different autoantibody profiles and specificities,23-28 and there are ongoing efforts to develop improved criteria for particular groups of patients by reevaluating or adding new autoantibody markers.29,30 The goal is to increase the likelihood that a diagnosis or tentative classification of the disease is correct. The more knowledgeable the clinician is with regard to the clinical and laboratory characteristics of diseases the greater the chance a diagnosis will be correct. The use and revision of disease classification criteria is one example of progress in the area of standardization. Although there is progress in the evaluation and updating of widely used classification criteria there is a growing need for practical evidence-based and reasoned approaches for diagnostic testing. Hence, clinical practice guidelines (CPGs) are needed for a rational, judicious, and cost-effective use of serologic testing.31-35 A large number of variables must be considered before CPGs are developed. First, CPGs should take into full account evidence-based research. Second, CPGs should be based on consensus of experts in the field. The conclusions from some studies are dependent on the quality of the data, and it is necessary to develop inclusion and exclusion criteria to evaluate a large body of data.31 The value and application of conclusions based on grouped literature review in the setting of rapidly changing technology is open to debate. There seems to be limited clinical value in developing CPGs when high-performance laboratories are grouped with

poor performance laboratories. Third, the application of CPGs is difficult to apply in all settings. In small laboratories and clinical service environments it is easier to achieve consensus on testing strategies than it is for large laboratories that provide service to more extensive populations. A CPG may include a guide to clinicians suggesting which screening tests should be used for a particular patient with a certain tentative diagnosis and those tests that are useful to monitor disease activity or progression (Table 22F.1). This guide could also provide a flow diagram of tests necessary in support of a diagnosis and estimate prognosis. A positive screening test may lead to the referral of a patient to a specialist, who will then initiate further serological testing. It is important to realize that the pre-test probability of detecting a useful diagnostic laboratory result increases with each clinical feature that has been incorporated into the tentative diagnosis.36,37 These issues emphasize the importance of developing CPGs that are current and in tune with conventional autoantibody testing. Concerns arise that widespread and “inappropriate” use of autoantibody testing can lead to referral of patients to subspecialists for evaluation of a positive autoantibody test when clinical correlates are lacking or insufficient to lead to an immediate diagnosis. Although such concerns abound, it is important to appreciate a number of issues before decrying such practice. First, presumably the autoantibody test was ordered by a primary care clinician with some degree of justification or clinical suspicion of disease. Although ongoing physician education is of paramount importance, it is inappropriate to condemn this practice simply because a consultant is unable to make a firm diagnosis. It is well known that most of the systemic rheumatic diseases can present with protean symptoms and signs that are related to maladies that range from minor illnesses to major life-threatening diseases. In addition, it also known that the interval from first symptom to the definitive diagnosis for diseases such as SLE and SSc can range from 1 to 25 years. The clinical impact of this feature of disease latency is even more heightened when it is appreciated that the presence of a number of autoantibodies precedes the clinical diagnosis and the presence of ANA in asymptomatic individuals increases the risk of developing SLE by as much as 20-fold.38,39 A question that should naturally follow all of this evidence is, “What is the cost of not performing an autoantibody test and allowing a patient to progress to full-blown disease and endorgan damage or failure before the diagnosis is made?” Unfortunately, the focus of virtually all studies to date has been the cost effectiveness of an assay based on pre-test predictability without taking these factors into consideration.

Tentative Diagnosis

Primary Screen Test

Secondary Tests

Other Tests

Systemic lupus erythematosus

ANA

ds-DNA Sm Rib-P CL

U1RNP B2GP1

Primary Sjuögren’s syndrome

ANA

Ro/SS-A La/SS-B

IgM RF

Scleroderma

ANA

Scl-70 (topo I) U1RNP CENP

PM/Scl Fibrillarin

Mixed connective tissue disease

ANA

U1RNP

IgM RF

Poly-Dermatomyositis

ANA

U1RNP Jo-1

PM/Scl Ku

Anti-phospholipid syndrome

CL LAC ANA

B2GP1

Prothrombin

Primary small-vessel vasculitis

ANCA

PR3-ANCA MPO-ANCA

BPI-ANCA Elastase-ANCA

Rheumatoid arthritis

RF CCP

IgA RF

Autoimmune rheumatic disease

ANA ANCA

Many different, as above

ANA, antinuclear antibody; ANCA, anti-neutrophil cytoplasmic antibody; B2GP1, beta 2 glycoprotien 1; BPI, bactericidal/permeability-increasing protein; CCP, cyclic citrullinated peptide; CENP, centromere protein; CL, cardiolipin; LAC, lupus anti-coagulant; MPO, myeloperoxidase; PR3, proteinase 3; RF, rheumatoid factor; Rib-P, ribosomal protein; RNP, ribonucleoprotein; Sm, Smith antigen.

STANDARDIZED REFERENCE SERA The ongoing demands of quality control and quality assurance are highly dependent on the availability of prototype sera and the participation of patients who are willing to donate blood. The serum from patients with monospecific and polyspecific autoantibody reactivity is critical in research and development of new diagnostics products. Equally important is the long-term participation of patients in clinical studies so that the accurate and reliable correlations of diagnostic markers to clinical outcome can be studied and validated. The difficulties in obtaining and exchanging human sera have been increased in many jurisdictions because of reluctant human ethics research boards. A number of agencies provide carefully characterized reference sera for autoantibody testing. A notable example is the reference sera evaluated and established by the Serology Subcommittee of the International Union of Immunology Societies (IUIS) found at http://www.autoab.org (Table 22F.2). Both the industry and diagnostic laboratories have free access to and utilize these standardized sera that are made available through the Centers for Disease Control in Atlanta.40

IMPROVED STANDARDIZATION THROUGH REGULATORY AND QUALITY ASSURANCE AGENCIES

TABLE 22F.1 AN APPROACH TO AUTOANTIBODY TESTING

The reference sera available through this program are continuously monitored and plans are to expand this list to include antibodies to RNA polymerase I/III, PR3-ANCA, CCAP, and MPO-ANCA. In addition, sera used as the standard for a particular methodology need reevaluation from time to time as exemplified by the reevaluation of AF/CDC reference sera for immunoblotting purposes.40 At this time, standardized secondary antibodies are not widely available.

IMPROVED STANDARDIZATION THROUGH REGULATORY AND QUALITY ASSURANCE AGENCIES Many laboratories are required to participate in a number of improvement and quality assurance programs, such as the one administered by the College of American Pathologists (www.cap.org). In addition, the Clinical Laboratory Improvement Amendments of 1988 set standards for all laboratories engaged in clinical testing. These standards include requirements for trained and competent supervisory and testing personnel, record keeping, instrument maintenance, daily quality

269

STANDARDIZATION OF AUTOANTIBODY TESTING IN SYSTEMIC RHEUMATIC DISEASES

TABLE 22F.2 REFERENCE SERA AVAILABLE FROM THE CENTERS FOR DISEASE CONTROLa Antibody Designation

Patternb

ANA 1

Homogeneous

Native DNA

ANA 2

Speckled

SS-B/La

ANA 3

Speckled

—

ANA 4

Coarse speckled

U1-RNP

ANA 5

Coarse speckled

Sm

ANA 6

Clumpy nucleolar

Fibrillarin (U3-RNP)

ANA 7

Speckled

SS-A/Ro

ANA 8

Interphase nuclear discrete speckles and discrete speckles on metaphase chromosomes

Centromere

ANA 9

Nucleolar + grainy nuclear

Topoisomerase I (Scl-70)

ANA 10

None

Jo-1 (histidyl tRNA synthetase)

ANA 11

Nucleolar/speckled nuclear

PM/Scl

ANA 12

None

Ribosomal P

ANA 13

None

cardiolipin

a. Postal address: Biological Reference Reagents, Mailstop C-21, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333 U.S.A. Tel: (404) 639-3354, Fax: (404) 639-3086, e-mail: [email protected]. b. Pattern dependent on substrate.

control practices, result reporting, and laboratory inspection and maintenance. It is not clear that these standards are being met in routine practice in many laboratories performing autoantibody testing. In Europe, several standards under the EU ISO standard program can be used in clinical immunology laboratories (e.g., the ISO 17025 general rules for diagnostic performance supplemented with more targeted demands and strategies formulated in subspecialty standard documents).

A PARADIGM OF STANDARDIZATION

270

Specificity

An example of a recent test that has achieved high inter- and intra-laboratory reliability is the anti-cyclic citrullinated peptides (a-CCP) ELISA. This new assay has been constructed to reveal a majority of autoantibodies specifically directed to citrullinated proteins, antigens that are found in chronically inflamed synovial membranes of patients with many forms of arthritides.41 The reason only RA patients react with anti-citrulline antibody production seems to be explained by presence of a shared epitope allele of the arthritisassociated HLA-DRB1 class II molecule in most patients with seropositive RA.42 Although this specific surrogate marker antibody population would be expected to cover only part of the existing specific anti-citrulline antibodies, this seems not to be the case.43 The second generation of an a-CCP ELISA (a-CCP 2) is reported to be 98% specific for the

diagnosis of RA among inflammatory disease control patients. The first version of the ELISA for a-CCP had a sensitivity of about 70.44 Further studies have indicated a sensitivity of 50 to 70% in patients with early RA, where the presence of a-CCP at the onset of disease has been shown to be associated with more severe radiologic damage during follow-up.45 The finding of anti-CCP in patients with early undifferentiated polyarthritis has a strong predictive value for later development of RA.46 Several studies on seemingly healthy donors who later developed RA have appeared the last few years, indicating that anti-CCP may be detected up to a decade before the onset of clinical symptoms of RA.47 Factors that contribute to the high reliability of the a-CCP test include the fact that the principal target CCP antigen is derived from a single source and most ELISA kits sold by different manufacturers have been coated in a single commercial laboratory following a prescribed set of conditions and protocols. In addition, large post-marketing testing was performed in several centers before the test was made available to routine customers. This has resulted in an assay that produces comparable results irrespective of the laboratory in which the anti-CCP 2 test has been performed. Perhaps there are lessons in this example where other autoantibody kits are concerned. Another area in which some standardization of tests has been undertaken is the development of tests for

better ELISAs exist in the field of ANCA determination than in several other fields of autoantibody testing.

CONCLUSIONS

CONCLUSIONS

anti-neutrophil cytoplasm antibodies. A European multicenter study supported by the European Commission started by reviewing and testing what techniques were used in some European expert laboratories and then standardizing these assays. Results of this study were published.48 The further step of this study then focused on the best ways to isolate proteinase 3 (PR3), one of the main ANCA antigens, to preserve optimal reactivity. The final study involved the standardized assays and sera from 169 newly diagnosed and 189 historical patients with idiopathic small-vessel vasculitis (systemic or renal limited) using 184 sera from inflammatory disease controls and 740 healthy donors as controls.49 A high diagnostic specificity of the solid phase assays was obtained by setting cut-off values so as to attain about 95% specificity toward the disease controls. After most of the known data on ANCA testing in ANCA-associated vasculitides in the literature was reviewed, an international consensus statement was published to help scientists obtain an overview of minimal and optimal testing procedures and details on the best way to report positive results.50 Although the steps mentioned previously may seem to be very time consuming and laborious, it is the impression that

The principal goal of a clinician is to obtain an early and accurate diagnosis of patients that can present with a wide variety of symptoms and signs during development, progression, or regression of systemic rheumatic and other autoimmune conditions. Over the past two decades, commercial autoantibody assay kits have had a tremendous impact on achieving this goal. However, there remain significant concerns with the accuracy (sensitivity, specificity), reliability, quality, and interpretation of the results derived from commercial autoantibody testing kits. Table 22F.3 summarizes some of the principal issues and positive resolutions discussed in this chapter. To achieve a higher level of effectiveness will require a commitment and coordination of all groups and individuals involved in the network of autoantibody testing. An ongoing challenge is to identify and secure the commitment of those groups that will take responsibility for this area of utmost importance to evidence-based and cost-effective diagnosis and treatment of autoimmune conditions.

TABLE 22F.3 RECOMMENDATIONS FOR IMPROVED PERFORMANCE AND STANDARDIZATION OF AUTOANTIBODY TESTINGa Principal

Problem

Recommendations

Variable reagents and analytes Variable secondary antibodies Pre-marketing, beta testing, and selling kits Quality control and kit performance

Standardized or common sources of reagents and analytes. Standardized or common sources of secondary antibodies. Wider testing in clinical laboratories before marketing and selling.

Equipment Protocols and SOPs

Upgrading and use of equipment to standards. Follow manufacturer’s protocols. Utilize international reference sera. Utilize local sera from clinically defined and normal controls to assess performance of kit before adopting into practice. Technologist required to demonstrate competence and participate in appropriate educational forums. Design and adopt a universal format. Should be clear and concise. Provide clear algorithm of tests provided and approach to use. Report graded positive results when appropriate or give clinically meaningful cut-off. Staff with specific skills serve as primary physician liaison to provide advice and assist with test interpretation.

Manufacturer

Willingness and ability to adjust and improve post-marketing surveillance.

Clinical Laboratory

Training and Maintenance of competence Test requisitions and reports

Physician liaison

Continued

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STANDARDIZATION OF AUTOANTIBODY TESTING IN SYSTEMIC RHEUMATIC DISEASES

TABLE 22F.3 RECOMMENDATIONS FOR IMPROVED PERFORMANCE AND STANDARDIZATION OF AUTOANTIBODY TESTINGa—cont’d Principal

Problem

Recommendations

Ordering tests

Aware of clinical laboratory capabilities. Utilize graded approach to ordering tests. In cooperation with ethics boards move to digital or electronic receipt of test results while maintaining patient confidentiality. If result not understood contact physician liaison at laboratory. Timely action after report received because diseases can progress rapidly. If appropriate, enquire about patient’s willingness to participate in research. Seek informed consent. Promote and lobby for research and development of standardization of laboratory testing through professional organizations (i.e., ACR, PANLAR).

Physician

Receiving tests Interpreting result Communication of results Patient advocate and educator

Quality assurance

Regulatory Bodies Quality assurance

Attention to quality of data submitted for laboratory diagnostic kit approval. Attention to quality of samples provided for ongoing QA programs. Utilize international standards and reference sera.

Budgeting

Ensure that budget keeps pace with appropriate advances in serology. Support the use of tests that have been proven to have an impact on patient care and outcome. Ensure that laboratory services are optimized. Consider impacts of laboratory consolidation on quality of care.

Health Care Underwriters

Alignment of services Disease Specific Lay Groups Limited involvement in support or advocacy of standardization

Representation to quality control agencies. Support of research in quality assurance of laboratory testing.

a. Adapted with permission from Fritzler et al.1 Abbreviations: ACR, American College of Rheumatology; PANLAR, Pan-American League Against Rheumatism, QA, quality assurance.

REFERENCES

272

1. Fritzler MJ, Wiik A, Fritzler ML, et al. The use and abuse of commercial kits used to detect autoantibodies. Arthritis Res Ther 2003;5:192-201. 2. Fritzler MJ. New technologies in the detection of autoantibodies. In Conrad K, Fritzler MJ, Meurer M, et al. (eds.) Autoantigens, Autoantibodies, Autoimmunity. Lengerich: Pabst Scientific Publishers 2002:50-63. 3. Robinson WH, DiGennaro C, Hueber W, et al. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nature Med 2002;8:295-301. 4. Feltkamp TEW. Antinuclear antibody determination in a routine laboratory. Ann Rheum Dis 1996;55:723-727. 5. Wiik AS, Gordon TP, Kavanaugh AF, et al. Cutting edge diagnostics in rheumatology: On the role of patients, clinicians, and laboratory scientists in optimizing the use of autoimmune serology. Arthritis Care Res 2004;51:291-298. 6. Jaskowski TD, Schroder C, Martins TB, et al. Comparison of three commercially available enzyme immunoassays for the screening

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of autoantibodies to extractable nuclear antigens. J Clin Lab Anal 1995;9:166-172. Jaskowski TD, Schroder C, Martins TB, et al. Screening for antinuclear antibodies by enzyme immunoassay. Am J Clin Pathol 1996;105:468-473. Emlen W, O’Neill L. Clinical significance of antinuclear antibodies: Comparison of detection with immunofluorescence and ELISA. Arthritis Rheum 1997;40: 1612-1618. Fawcett PT, Rose CD, Gibney KM, et al. Use of ELISA to measure antinuclear antibodies in children with juvenile rheumatoid arthritis. J Rheumatol 1999;26:1822-1826. Ulvestad E, Kansetrom A, Madland TM, et al. Evaluation of diagnostic tests for antinuclear antibodies in rheumatological practice. Scand J Immunol 2000;52:309-315. Bossuyt X. Evaluation of two automated enzyme immunoassays for detection of antinuclear antibodies. Clin Chem Lab Med 2000;38:1033-1037.

33. Kavanaugh A. The utility of immunologic laboratory tests in patients with rheumatic diseases. Arthritis Rheum 2001;44: 2221-2223. 34. Smolen JS, Steiner G, Tan EM. Standards of care: The value and importance of standardization. Arthritis Rheum 1997;40: 410-412. 35. Tozzoli R, Bizzaro N, Tonutti E, et al. Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases. Am J Clin Pathol 2002; 117:316-324. 36. Keren DF, Nakamura RM. Progress and controversies in autoimmune disease testing. Clin Lab Med 1997;17:483-497. 37. Bizzaro N, Wiik A. Appropriateness in anti-nuclear antibody testing: From clinical request to strategic laboratory practice. Clin Exp Rheumatol 2004;22:349-355. 38. Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med 2003;349:1526-1533. 39. McClain MT, Arbuckle MR, Heinlen LD, et al. The prevalence, onset, and clinical significance of antiphospholipid antibodies prior to diagnosis of systemic lupus erythematosus. Arthritis Rheum 2004;50:1226-1232. 40. Smolen JS, Butcher B, Fritzler MJ, et al. Reference sera for antinuclear antibodies. II. Further definition of antibody specificities in international antinuclear antibody reference sera by immunofluorescence and Western immunoblotting. Arthritis Rheum 1997;40:413-418. 41. Vossenaar ER, Smeets TJ, Kraan MC, et al. The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum 2004;50:3485-3494. 42. Hill JA, Southwood S, Sette A, et al. The conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule. J Immunol 2003;171:538-541. 43. Nijenhuis S, Zendman AJ, Vossenaar ER, et al. Autoantibodies to citrullinated proteins in rheumatoid arthritis: Clinical performance and biochemical aspects of an RA-specific marker. Clin Chim Acta 2004;350:17-34. 44. Schellekens GA, Visser H, De Jong BAW, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 2000;43: 155-163. 45. Kroot EJJA, De Jong BAW, Van Leeuwen MA, et al. The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis. Arthritis Rheum 2000;43: 1831-1835. 46. Van Gaalen FA, Linn-Rasker SP, Van Venrooij WJ, et al. Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis: A prospective cohort study. Arthritis Rheum 2004; 50:709-715. 47. Rantapää-Dahlqvist S, De Jong BAW, Berglin E, et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003;48:2741-2749. 48. Hagen EC, Andrassy K, Csernok E, et al. Development and standardization of solid phase assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). A report on the second phase of an international cooperative study on the standardization of ANCA assays. J Immunol Methods 1996; 196:1-15. 49. Hagen EC, Daha MR, Hermans J, et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis: EC/BCR Project for ANCA Assay Standardization. Kidney Int 1998;53:743-753. 50. Savige J, Gillis D, Benson E, et al. International consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA). Am J Clin Pathol 1999;111:507-513.

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12. Jansen EM, Deng J, Beutner EH, et al. Comparison of commercial kits for the detection of anti-nDNA antibodies using Crithidia lucilae. Am J Clin Pathol 1987;87:461-469. 13. Avina-Zubieta JA, Galindo-Rodriguez G, Kwan-Yeung L, et al. Clinical evaluation of various selected ELISA kits for the detection of anti-DNA antibodies. Lupus 1995;6:370-374. 14. Cordiali FP, D’Agosto G, Ameglio F, et al. Determination of antibodies to extractable nuclear antigens by commercial kits: a multicenter study. Int J Clin Lab Res 1998;28:29-33. 15. Kern P, Kron M, Hiesche K. Measurement of antinuclear antibodies: Assessment of different test systems. Clin Diagn Lab Immunol 2000;7:72-78. 16. Fritzler MJ, Wiik A, Tan EM, et al. A critical evaluation of enzyme immunoassay kits for detection of antinuclear antibodies of defined specificities. III. Comparative performance characteristics of academic and manufacturers’ laboratories. J Rheumatol 2003;30:2374-2381. 17. Bizzaro N, Tozzoli R, Tonutti E, et al. Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: A collaborative study with the biomedical industry. J Immunol Methods 1998;219:99-107. 18. Csernok E, Ahlquist D, Ullrich S, et al. A critical evaluation of commercial immunoassays for antineutrophil cytoplasmic antibodies directed against proteinase 3 and myeloperoxidase in Wegener’s granulomatosis and microscopic polyangiitis. Rheumatology (Oxford) 2002;41:1313-1317. 19. Tan EM, Smolen J, McDougal JS, et al. A critical evaluation of enzyme immunoassays for the detection of antinuclear antibodies of defined specificities. I. Precision, sensitivity and specificity. Arthritis Rheum 1999;42:455-464. 20. Wiik AS. Anti-nuclear autoantibodies: clinical utility for diagnosis, prognosis, monitoring, and planning of treatment strategy in systemic immunoinflammatory diseases. Scand J Rheumatol 2005; 34:260-268. 21. Fenger M, Wiik A, Hoier-Madsen M, et al. Detection of antinuclear antibodies by solid-phase immunoassays and immunofluorescence analysis. Clin Chem 2004;50:2141-2147. 22. Tan EM, Feltkamp TEW, Smolen JS, et al. Range of antinuclear antibodies in “healthy” individuals. Arthritis Rheum 1997;40: 1601-1611. 23. Targoff IN. Laboratory testing in the diagnosis and management of idiopathic inflammatory myopathies. Rheum Dis Clin North Am 2002;28:859-890. 24. Ho KT, Reveille JD. The clinical relevance of autoantibodies in scleroderma. Arthritis Res Ther 2003;5:80-93. 25. Cervera R, Khamashta MA, Font J, et al. Systemic lupus erythematosus: Clinical and immunologic patterns of disease expression in a cohort of 1,000 patients. Medicine (Baltimore) 1993;72:113-124. 26. Wiik A. Clinical use of serological tests for antineutrophil cytoplasmic antibodies: What do the studies say? Rheum Dis Clin NA 2003;27:799-813. 27. Permin H, Hørbov S, Wiik A, et al. Antinuclear antibodies in juvenile chronic arthritis. Acta Paediatr Scand 1978;67:181-185. 28. Rosenberg AM. The clinical associations of antinuclear antibodies in juvenile rheumatoid arthritis. Clin Immunol Immunopathol 1988;49:19-27. 29. Nadashkevish O, Davis P, Fritzler MJ. A proposal of criteria for the classification of systemic sclerosis. Medical Science Monitor 2004;10:615-621. 30. Albrecht J, Berlin JA, Braverman IM, et al. Dermatology position paper on the revision of the 1982 ACR criteria for systemic lupus erythematosus. Lupus 2004;13:839-849. 31. Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: Antinuclear antibody testing. Arthritis Rheum 2002;47:434-444. 32. Kavanaugh AF, Solomon DH. Guidelines for immunologic laboratory testing in the rheumatic diseases: Anti-DNA antibody tests. Arthritis Rheum 2002;47:546-555.

273

MECHANISMS OF TISSUE DAMAGE

22G

Anti-Spliceosomal Autoantibodies Edward K.L. Chan, PhD, Carol Peebles, MS, MT, Marvin J. Fritzler, MD, PhD, and Minoru Satoh, MD, PhD

INTRODUCTION Anti-Sm was one of the first autoantibodies to non-histone proteins described in systemic autoimmune rheumatic diseases.1 Antibodies to the Sm antigen are highly diagnostic of systemic lupus erythematosus (SLE) and are present in 10 to 30% of unselected SLE populations.2,3 The identification of Sm antigen as well-defined proteins bound to U-rich small nuclear RNAs (snRNAs) has been considered a significant advance in biology, and these autoantibodies have served as useful probes to help investigate the important components of the spliceosome [which is the multiprotein complex responsible for pre-mRNA splicing of heterogeneous nuclear RNA to mature messenger RNA (mRNA)].4 In this chapter we discuss the major classes of anti-spliceosome antibodies [including anti-Sm, anti-U1 RNP (also known as anti-nRNP in older terminology), and anti-U2 RNP] and briefly review other minor autoantibodies to other classes of U RNPs, such as LSm (Like Sm) proteins.

SPLICEOSOMAL AUTOANTIGENS

274

Small nuclear ribonucleoproteins (snRNPs) are the major autoantigens in the spliceosome. They are classified by association with specific U-rich snRNAs, including the most abundant U1, U2, U4, U5, and U6 RNAs (Fig. 22G.1). These snRNPs associate with pre-mRNA in a sequential manner to assemble the spliceosome into a functional complex, which can catalyze the splicing reaction. Fig. 22G.1A illustrates the composition of the major snRNPs: U1, U2, U4/U6, and U5 snRNP sharing the Sm core proteins B or B′ (27/28kDa), D1/D2/D3 (14 kDa), E (12 kDa), F (11 kDa), and G (9 kDa), which are organized as seven-member ring structures (Sm ring). Analysis of individual snRNPs has disclosed that, in addition to the shared peptides, some proteins are specifically associated with certain snRNPs. U1 snRNPs contain three distinct proteins designated 70k (68/70 kDa), A (32 kDa), and C (22 kDa), shown in blue in Fig. 22G.1A. U2 snRNPs have two unique proteins: A’ (31 kDa) and B’’ (29 kDa, shown in green).

U4 is always associated with U6 and is thus often designed U4/U6 snRNP. There are several other proteins associated with U5 or U4/U6 snRNPs3 but only the 200 kDa doublet of U5snRNPs is shown in Fig. 22G.1A because this can be used to differentiate sera with anti-U1 RNP alone versus anti-U1 RNP plus anti-Sm (Fig. 22G.1C).5,6 U6 snRNP uses one of the LSm rings, which is structurally similar to the Sm ring, as its core complex (Fig. 22G.1D). Other snRNPs (such as U3, U8 and U13) are primarily localized to the nucleolus and are not part of the spliceosomal complex. U11 and U12 snRNPs occur in relatively lower abundance and are required for U2-snRNP-independent splicing. Other components of the spliceosome include SR proteins, which are serine-arginine–rich proteins involved in the mRNA splicing reaction.

ANTI-SM AUTOANTIBODIES Anti-Sm antibodies recognize the Sm ring core protein complex (Fig. 22G.1D) associated with the snRNA U1, U2, U4/U6, and U5 (Fig. 22G.1A). The Sm core proteins are the B/B′/N, D1, D2, D3, E, F, and G proteins described previously. Sm B and B′ are products of alternative splicing from a single gene, whereas Sm N shares 93% amino acid homology with B′ and is derived from a different gene with its expression restricted to certain cell types and stages of development. Earlier work of the crystal structures of two Sm protein complexes, D3/B and D1/D2, suggested that the seven Sm core proteins could form a closed ring and the snRNAs may be bound in the positively charged central hole. Because these core proteins are common to U1, U2, U4/U6, and U5 snRNPs, a wider range of snRNAs are immunoprecipitated by anti-Sm antibodies than are precipitated by anti-U1 RNP antibodies (Fig. 22G.1B). Anti-Sm produces a nuclear speckled staining pattern on HEp-2 cell nuclei by conventional indirect immunofluorescence (IIF) (Fig. 22G.2). However, this staining pattern is practically indistinguishable from that of anti-U1 RNP by this technique. Double immunodiffusion (DID), Western blotting (WB), line immunoassay

ANTI-SM AUTOANTIBODIES

Fig. 22G.1 Spliceosomal components. (A) Composition of U1, U2, U4, and U5 snRNPs illustrated as protein bands immunoprecipitated using anti-Sm or anti-U1 snRNP (nRNP) autoantibodies. The four snRNPs share the Sm core complex shown in orange. U1-snRNP-specific proteins are shown in blue, U2-snRNP-specific in green, and U5-snRNP–specific in black. Anti–Sm-positive sera immunoprecipitate all four snRNPs and associated proteins, whereas anti-nRNP–specific sera will only bring down U1 RNP. (B) snRNAs immunoprecipitated from a cell lysates using human anti-Sm or anti-nRNP, analyzed in a urea polyacrylamide gel, and detected by silver staining of RNA bands. (C) snRNP-associated proteins immunoprecipitated from lysates of cells metabolically labeled with [35S]-methionine using human anti-Sm or anti-nRNP, fractionated in SDS-PAGE, and detected by autoradiography. (D) Seven-member Sm and LSm rings are illustrated showing structural similarity among these doughnut-shaped structures, with the center of the doughnut involved in binding of single-stranded RNA.

Fig. 22G.2 Immunofluorescence of human anti-Sm using the CDC prototype serum on HEp-2 cells. Speckles are restricted primarily to nuclei and sparing nucleoli. snRNP complexes are concentrated in nuclear bodies known as Cajal bodies (arrows), which are often seen adjacent to nucleoli. M = mitotic cell. Anti-U1 RNP staining is practically identical. (See Color Plate 1.)

(LIA), analysis of precipitated RNAs (Fig. 22G.1B), enzyme-linked immunoassays (ELISA), or addressable laser bead immunoassays are preferred to definitively identify this autoantibody. Advances in cloning and epitope mapping of Sm-related peptides has led to the use of synthetic peptides in clinical studies. However, the clinical usefulness of these anti-peptide antibodies is unclear. Because most lupus sera bind to the glycine-arginine repeat of the Sm peptide regardless of the presence of anti-Sm antibodies under conventional methods such as DID or Western blot (reactivity with the D protein),7,8 the significance of reactivity with the Sm peptide needs to be carefully evaluated. The techniques required to demonstrate antibody to Sm necessitate the use of standard reference sera that are available through the Centers for Disease Control in Atlanta, Georgia.9 Antibodies to both Sm and U1 RNP, which commonly coexist in the same serum,2 are primarily detected in SLE patients. In SLE it is common to detect antibodies to U1 RNP alone or antibodies to both

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U1 RNP and Sm, but antibodies to Sm alone are rarely found.2,6 Over the years, it has been repeatedly confirmed that this antibody is present almost exclusively in SLE and is considered a diagnostic marker for that disease to the extent it is included in the American College of Rheumatology (ACR) criteria for the classification of the SLE.10 Although anti-Sm is present in only 10 to 30% of patients with SLE,2 it is a highly specific disease marker. It has only rarely been detected in normal sera or in patients with other systemic rheumatic diseases such as Sjögren’s syndrome (SjS), scleroderma, polymyositis, or drug-induced lupus. However, it is important to note that lupus-related autoantibodies (including anti-Sm) have been observed prior to the development of full clinical features or diagnosis.11 Therefore, anti-Sm can be detected occasionally in a patient who does not have SLE but may develop SLE in the future. Some reports suggested that the titers of anti-Sm antibodies correlate with the disease activity.3,12 Anti-Sm–positive SLE were associated with milder renal and central nervous involvement or late-onset renal disease13 in some reports, but this is controversial.3 The reactivity of anti-Sm antibodies has been refined and further based on the understanding that the B/B′ and D polypeptides are considered the major target autoantigens (although anti-U1 RNP antibodies often react with B/B′ in Western blot testing.5,13,14 It is known that SmB/B′ share cross-reactive epitopes (PPPGMRPP) with U1-specific RNPs, which are more frequently targeted by antibodies present in patients with mixed connective tissue disease (MCTD). Thus, the SmD polypeptides are regarded as the more disease-specific Sm antigens. Recently, it was shown that the polypeptides D1 and D3 contain the modified amino acid symmetrical dimethylarginine within a major autoepitope of the SmD1 and SmD3 C-terminus.15,16 A synthetic peptide of SmD3 containing symmetrical dimethylarginine at position 112 represents a promising tool for the detection of a highly specific subpopulation of anti-Sm antibodies.17

ANTI-U1 RNP (NUCLEAR RNP, NRNP) AUTOANTIBODIES

276

Autoantibodies to U1 RNP were first defined using DID as anti-Mo that recognize soluble nuclear ribonucleoprotein (nRNP).18 In the same year, Sharp and colleagues described a group of patients associated with high levels of antibodies to saline extractable nuclear antigen (ENA) detected by passive hemagglutination testing.19 This group of patients was described as MCTD, which is characterized by overlapping symptoms and signs of SLE, scleroderma, and polymyositis.19,20 The main clinical features of MCTD are a high prevalence of

Raynaud’s phenomenon, edema of the fingers, arthritis/arthralgias, myositis, serositis, and a relative absence of renal disease. The characteristic serologic feature of MCTD is the presence of high titers of RNase-sensitive anti-ENA antibodies, which were later confirmed as anti-U1 RNP. This was distinguished from RNaseresistant anti-ENA antibodies, which is anti-Sm antibodies, or anti-dsDNA antibodies.19-21 When anti-U1 RNP antibodies are present alone and in high titer, they are often associated with MCTD. Anti-U1 RNP antibodies are also detected, usually at a lower titer, in other systemic rheumatic diseases (including 30 to 40% of SLE) and in much lower frequency in rheumatoid arthritis, systemic sclerosis, Sjögren’s syndrome, polymyositis, discoid lupus, and (rarely) in drug-induced lupus.3,22 Because of the clinical relevance of the titers observed in systemic rheumatic diseases, it became necessary not only to detect but quantify the level of anti-U1 RNP antibodies in patient’s sera. It is important to appreciate that some SLE and MCTD sera bind most of the snRNP polypeptides whereas others bind to little, if any, 70k or C polypeptides. In one study, only 8% of SLE sera containing U1 RNP antibodies bound to the 70k antigen, but 76% of MCTD sera bound this protein.23 The observed higher frequency of anti-70k antibodies in MCTD has been supported in several studies and has been reported to be as high as 95% in MCTD, whereas the range of reactivity in SLE is 20 to 50%. The frequency of anti-U1 RNP is highly dependent on the assay employed, as evidenced by the fact that when recombinant 70k protein was used in an ELISA, up to 85% of SLE patients showed elevated antibody levels. It has been suggested that the presence of anti-70k is correlated with the presence of Raynaud’s phenomenon, esophageal dysmotility and myositis, and a negative indicator for the presence of renal disease.23-25 Taken together, the data suggest that antibodies to the 70k protein may be more characteristic of patients with classic features of MCTD. These may be important observations for the clinician who attempts to identify patients at high risk of developing end-organ disease when they present with only a few features (e.g., Raynaud’s phenomenon or myositis) of other systemic rheumatic diseases. Although antibodies to the 70k protein quantitatively vary during the disease course, there is little evidence that they correlate with disease activity or that they are involved in disease pathogenesis.24,25 There has been interest in the possibility that anti-U1 RNP antibodies have a unique property of being able to “penetrate” living cells.26 Although the pathogenic significance of such observations are not clear, this property may account for other occasional observations

not seen in 141 SLE patients but were identified in 8 patients with an overlap syndrome that most commonly included features of myositis.39 Two of these patients had features of SLE and one of the SLE patients also had an erosive arthritis and features of scleroderma.

ANTI-SNRNPS RECOGNIZING CONFORMATIONAL STRUCTURES In contrast to short linear epitopes for T cells, a classic characteristic of autoimmune B-cell epitopes is that they are discontinuously conformational (as indicated by epitope mapping studies using WB).2 Several reports have described autoantibodies that recognize the conformational structure of the multiprotein snRP complexes. Luhrmann and associates have reported that SLE sera have a unique and prevalent reactivity to a complex of Sm E, F, and G proteins.40 In this study, the individual Sm proteins E, F, and G were synthesized and then allowed to form complexes. Although the individual proteins were not recognized in WB, the EFG complex was preferentially immunoprecipitated by all anti-Sm sera but not sera from other diseases. These observations suggest that many anti-Sm sera recognize unique conformational epitopes in the EFG complex. It was noted that nearly all anti-U1 RNP-positive sera react with the native U1-C protein, in contrast to low frequency of recognition of the SDS-denatured U1-C by Western blot testing.5 One study showed that all anti-U1 RNP sera had autoantibodies that stabilize the U1-C and the Sm core proteins (some also stabilize U1-A-Sm), possibly by recognizing the quaternary structure.6,41 In contrast, the Sm core particle itself was recognized by anti-Sm antibodies but not by most of anti-U1 RNP sera. The stabilizing autoantibodies appear to be a significant component in anti-U1 RNP autoimmune response versus antibodies of individual components.41

ANTI-LSm4 AND LSm COMPLEX

that anti-U1 RNP antibodies appear in nuclei of patients’ tissue specimens. However, whether this is in fact an in vivo phenomenon or an in vitro artifact is still controversial. Antibodies to the A and C proteins are found in approximately 25% of unselected SLE cohorts and in 75% of SLE patients preselected for antibodies to U1 RNP.24,27,28 Like antibodies to the 70k protein, the titers may vary during the course of the disease but there is no clear evidence that they predict disease activity or that they are involved in the pathogenesis.24 U1 RNA has been described as acting as an endogenous adjuvant for the development of a pro-inflammatory state via activation of toll-like receptor 3 (TLR3) signaling.29 More recent studies have suggested that U1 RNA may induce innate immunity through other cellular RNA sensors, including TLRs 7 and 8 and RNA-dependent protein kinase.30-32 This may explain why RNA-protein complexes are frequent targets of lupus autoantibodies. Anti-U1 RNP antibodies are thought to preferentially recognize the protein components of the U1 RNP. However, several reports showed that coexistence of autoantibodies to U1 RNA are quite common.33-36 van Venrooij and colleagues reported that 38% of anti-snRNP sera were positive for antibodies to U1 RNA and that they were always accompanied by antiU1 RNP but not by anti-Sm.36 Hoet and colleagues35 reported that 9 patients with SLE overlap syndrome demonstrated changes in titers of anti-U1 RNA antibodies that were correlated with disease activity. More recently, Asano and colleagues34 suggested that anti-U1 RNA antibodies are a serologic indicator for pulmonary fibrosis in systemic sclerosis patients with anti-U1 RNP antibodies. As discussed by Greidinger and Hoffman,30 assays for anti-U1 RNA are poorly standardized and of limited availability to clinicians in most areas, making this observation primarily of research relevance.

ANTI-U2 RNP U2 snRNPs contain unique proteins A′ (31 kDa) and B′′ (29 kDa) in addition to the Sm core proteins (Fig. 22G.1A).37,38 Anti-U2 snRNP autoantibodies were first identified by a Ya prototype serum that mainly immunoprecipitated U2 snRNPs by recognizing U2 snRNP unique protein A′.37 In that U1-A and U2-B′′ proteins share a homologous domain, a group of patients who had autoantibodies that cross-reacted with U1-A and U2-B′′ (designated anti-U1/U2 snRNP antibodies) were described subsequently.39 Antibodies that preferentially immunoprecipitate U2 snRNPs via recognition of U2-specific A′ protein appeared to be uncommon. In one study, antibodies to U2 RNP were

ANTI-LSm4 AND LSm COMPLEX Recent studies have identified macromolecular complexes composed of Sm-like proteins that form distinct complexes in the form of an Sm ring (Fig. 22G.1D). They have been named like-Sm proteins, or LSm proteins (LSm1 to LSm8) because they each bear sequence homology with one of the seven Sm core proteins.42 For example, LSm4 was found to have the highest sequence similarity to SmD3, whereas hLSm1 and LSm8 have the highest similarity to the core domain of B/B′.43 Likewise, D1, D2, E, F, and G are most similar (respectively) to LSm2, LSm3, LSm5, LSm6, and LSm7. The complexes they form with each other have

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the seven-member ring doughnut configuration that resembles the Sm protein complexes.44 The LSm rings exist in two configurations: the LSm2-8 complex bound to U6 snRNA (involved in pre-mRNA splicing) and the LSm1-7 complex (involved in mRNA decapping and 5′-3′ mRNA degradation).43 Autoantibodies to LSm4 were detected in ~80% of all anti-Sm sera when analyzed by immunoprecipitation of in–vitro–translated LSm4.45 A small fraction [7.2% (28/391)] of the same group of anti-Sm sera immunoprecipitated LSm4 together with the LSm complex from HeLa cell extracts. These findings document that IgG autoantibodies to LSm4 are detected in a large overlapping subset of anti-Sm-positive SLE. LSm4 was immunoprecipitated from a few lupus sera that contained little or no co-precipitating antibodies to the Sm core proteins.45 The clinical significance of anti-LSm antibodies remains to be determined.

OTHER SPLICEOSOMAL ANTIBODIES Autoantibodies from a variety of systemic rheumatic diseases bind proteins that are components of U4/U6,46 U5, U7, and U11 snRNPs. These autoantibodies appear to be rare in SLE and tend to immunoprecipitate specific snRNPs because of their unique reactivity with snRNP particles. For the non-snRNP proteins involved in pre-mRNA splicing, autoantibodies to serine-arginine-rich (SR) proteins were reported in 50% of SLE patients and the epitopes were shown to be phosphorylated residues abundant in the protein members.47

CONSIDERATIONS IN THE DETECTION OF AUTOANTIBODIES

278

Anti-nRNP and anti-Sm were first identified as two independent precipitin lines in Ouchterlony DID.1,18 It was noted early on that when a patient serum with antibodies to Sm is placed next to a patient serum with antibodies to nRNP in DID a spur developed that would point in the direction of the nRNP well, suggesting the common constituents in Sm and nRNP antigens. The components in anti-nRNP and anti-Sm precipitin lines or the mechanism of spur formation are not known. Counterimmunoelectrophoresis (CIE) was also used to increase the sensitivity and reliability while reducing time for the reaction.48 The common sources of antigens were soluble extracts of either calf or rabbit thymus (CTE, RTE) or tissue culture cells. DID has been used for more than 50 years and is still used in some clinical laboratories because it is inexpensive and specific. However, it lacks sensitivity and can take up to 48 hours before precipitin lines are interpretable. DID and CIE generally favor high-titer sera and

sometimes cannot discriminate multiple autoantibody responses that are characteristic of systemic rheumatic disease sera. Although identification of the antibodies was achieved by DID, it was not considered the method of choice for quantitation. As mentioned earlier, two important properties of the nRNP antigen that allow for differentiation and subsequent quantitation of nRNP and Sm are that the antigenic reactivity of nRNP is destroyed by either treatment with RNase or by heat inactivation at 56°C for 30 minutes whereas that of Sm is not. Utilizing these differences, two methods for the quantitation of anti-Sm and -nRNP antibodies were developed. One method is the passive hemagglutination assay (PHA), in which both Sm and nRNP antigens are adsorbed on to tanned sheep red blood cells (anti-ENA antibodies) and then the nRNP reactivity is selectively removed by treatment with RNase (RNase-resistant anti-ENA antibodies).19-21 and the other method is CIE, in which a portion of the cell extract is heat inactivated to destroy the nRNP reactivity. In both of the assays the treated and untreated antigens are titered in parallel and the endpoint titers reported. Because PHA and CIE differ in the types of antibodies preferentially measured, titers will vary between the methods. Although RNase treatment removes antinRNP reactivity from RTE or CTE by DID or from ENA coated red blood cells used in PHA, the effects of RNase on intact snRNPs from cell lysate tested by immunoprecipitation are quite different.6 RNase treatment dissociates U1 snRNPs into the Sm core proteins plus U1-70K, A, and C. Anti-nRNP sera immunoprecipitate U1-70k, A, and C, whereas anti-Sm immunoprecipitate the Sm core particle.6,41 This is further complicated by the fact that anti-nRNP antibodies retain the Sm core particle plus U1-C if they bind prior to RNase digestion.6 With the application of WB, the difference between the protein antigens that were recognized by anti-Sm and anti-nRNP could be determined. It was demonstrated that most anti-Sm antibodies reacted with the B/B′ and the D proteins and occasionally with the E protein.2,3 Anti-nRNP antibodies, on the other hand, reacted with the U1-specific proteins U1-70k, A, and C proteins. However, many or most of anti-U1 RNPalone sera also recognize B/B′ proteins by Western blot analysis.5,13,14 This apparent discrepancy may be explained by the inaccessibility or absence of the Western blot–specific B/B′ epitopes on the native molecules6 or the presence of B/B′ epitope uniquely expressed in U1 snRNP but not on other snRNPs.14 It has been shown that anti-B/B′ antibodies from anti-U1 RNP sera are specific for B/B′, whereas anti-B/B′ antibodies from anti-Sm sera cross-react with B/B′ and D.13 In many clinical diagnostic laboratories, ELISA has become the method of choice for identification of

antigens of differing components. This obviously leads to different antigens being available for reactivity and to variable results. This must always be taken into consideration in comparing discrepant results between kits. The ANA Standardization Subcommittee of the Standardization Committee of the International Union of Immunological Societies conducted a critical evaluation of ENA ELISAs that compared ELISA kits from nine companies.51 The samples tested were prepared and coded at the Centers for Disease Control (CDC) using the CDC reference sera.9,52 The antibodies tested include nDNA, ssDNA, histone, Sm, Ul-RNP, SS-A/Ro, SS-B/La, Scl-70, centromere, and Jo-1. Although not all nine companies had assays for all of the antigens, most performed quite well when they used their own kits to detect antibodies to SS-A/Ro, SS-B/La, Scl-70, and Jo-1. In contrast, there was considerable variability in detection of antibodies to Sm and nDNA.51

REFERENCES

anti-Sm/nRNP. The procedure is usually based on purified antigens that are adsorbed on a solid surface of 96 well-microtiter plates. An alternative method is to apply the purified antigens on a nitrocellulose surface in a format referred to as LIA. ELISA has rapidly advanced, but highly specific, sensitive, and reliable assays that use highly purified or recombinant proteins can be expensive and are limited by inter-manufacturer and inter-laboratory variation.49 A recent report suggested that ELISA using a mixture of recombinant U1-70k, A, and C proteins along with U1 RNA helps to improve ELISA detection of anti-nRNP antibodies.50 In ELISA or LIA, the nature and integrity of the antigen will determine which epitopes are available for antibody binding. An understanding of the differences in antigens will lead to a better understanding of assay results. From the previous discussion, knowing that Sm and nRNP are part of an RNA and protein complex and that antibodies in patients react with numerous epitopes on the specific proteins one can appreciate the fact that different assays sometimes give different results. In commercially available ELISA kits, some manufacturers coat the microtiter plates with the Sm/RNP complex and free Sm using biochemically separated antigens. Others coat the plates with cloned

ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grants AI47859, AI39645 AR40391, and M01R00082.

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10. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271-1277. 11. Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med 2003;349: 1526-1533. 12. Boey ML, Peebles CL, Tsay GJ, et al. Clinical and autoantibody correlations in Orientals with systemic lupus erythematosus. Ann Rheum Dis 1988;47:918-923. 13. Homma M, Mimori T, Takeda Y, et al. Autoantibodies to the Sm antigen: Immunological approach to clinical aspects of systemic lupus erythematosus. J Rheumatol 1987;13:188-193. 14. Ohosone Y, Mimori T, Fujii T, et al. Autoantigenic epitopes of the B polypeptide of Sm small nuclear RNP particles: Identification of regions accessible only within the U1 small nuclear RNP. Arthritis Rheum 1992;35:960-966. 15. Mahler M, Fritzler MJ, Bluthner M. Identification of a SmD3 epitope with a single symmetrical dimethylation of an arginine residue as a specific target of a subpopulation of antiSm antibodies. Arthritis Res Ther 2005;7:R19-R29. 16. Brahms H, Raymackers J, Union A, et al. The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies. J Biol Chem 2000; 275:17122-17129. 17. Mahler M, Stinton LM, Fritzler MJ. Improved serological differentiation between systemic lupus erythematosus and mixed connective tissue disease by use of an SmD3 peptide-based immunoassay. Clin Diagn Lab Immunol 2005; 12:107-113.

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18. Mattioli M, Reichlin M. Characterization of a soluble nuclear ribonucleoprotein antigen reactive with SLE sera. J Immunol 1971;107:1281-1290. 19. Sharp GC, Irvin WS, LaRoque RL, et al. Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest 1971;50:350-359. 20. Sharp GC, Irvin WS, Tan EM, et al. Mixed connective tissue disease: An apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 1972;52:148-159. 21. Sharp GC, Irvin WS, May CM, et al. Association of antibodies to ribonucleoprotein and Sm antigens with mixed connectivetissue disease, systematic lupus erythematosus and other rheumatic diseases. N Engl J Med 1976;295:1149-1154. 22. Tan EM. Interactions between autoimmunity and molecular and cell biology: Bridges between clinical and basic sciences. J Clin Invest 1989;84:1-6. 23. Pettersson I, Wang G, Smith EI, et al. The use of immunoblotting and immunoprecipitation of (U) small nuclear ribonucleoproteins in the analysis of sera of patients with mixed connective tissue disease and systemic lupus erythematosus: A cross-sectional, longitudinal study. Arthritis Rheum 1986;29:986-996. 24. Takeda Y, Wang GS, Wang RJ, et al. Enzyme-linked immunosorbent assay using isolated (U) small nuclear ribonucleoprotein polypeptides as antigens to investigate the clinical significance of autoantibodies to these polypeptides. Clin Immunol Immunopathol 1989;50:213-230. 25. St Clair EW, Query CC, Bentley R, et al. Expression of autoantibodies to recombinant (U1) RNP-associated 70K antigen in systemic lupus erythematosus. Clin Immunol Immunopathol 1990;54:266-280. 26. Alarcon-Segovia D, Ruiz-Arguelles A, Llorente L. Broken dogma: Penetration of autoantibodies into living cells. Immunol Today 1996;17:163-164. 27. Pettersson I, Hinterberger M, Mimori T, et al. The structure of mammalian small nuclear ribonucleoproteins Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies. J Biol Chem 1984;259: 5907-5914. 28. Habets WJ, Hoet MH, van Venrooij WJ. Epitope patterns of anti-RNP antibodies in rheumatic diseases: Evidence for an antigen-driven autoimmune response. Arthritis Rheum 1990; 33:834-841. 29. Hoffman RW, Gazitt T, Foecking MF, et al. U1 RNA induces innate immunity signaling. Arthritis Rheum 2004;50:2891-2896. 30. Greidinger EL, Hoffman RW. Autoantibodies in the pathogenesis of mixed connective tissue disease. Rheum Dis Clin North Am 2005;31:437-50. 31. Vollmer J, Tluk S, Schmitz C, et al. Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8. J Exp Med 2005;202: 1575-1585. 32. Lau CM, Broughton C, Tabor AS, et al. RNA-associated autoantigens activate B cells by combined B cell antigen receptor/ Toll-like receptor 7 engagement. J Exp Med 2005;202: 1171-1177. 33. Wilusz J, Keene JD. Autoantibodies specific for U1 RNA and initiator methionine tRNA. J Biol Chem 1986;261: 5467-5472. 34. Asano Y, Ihn H, Yamane K, et al. The prevalence and clinical significance of anti-U1 RNA antibodies in patients with systemic sclerosis. J Invest Dermatol 2003;120:204-210. 35. Hoet RM, Koornneef I, de Rooij DJ, et al. Changes in anti-U1 RNA antibody levels correlate with disease activity in patients with

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systemic lupus erythematosus overlap syndrome. Arthritis Rheum 1992;35:1202-1210. van Venrooij WJ, Hoet R, Castrop J, et al. Anti-(U1) small nuclear RNA antibodies in anti-small nuclear ribonucleoprotein sera from patients with connective tissue diseases. J Clin Invest 1990; 86:2154-2160. Mimori T, Hinterberger M, Pettersson I, et al. Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome. J Biol Chem 1984; 259:560-565. Habets W, Hoet M, Bringmann P, et al. Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA. EMBO J 1985;4:1545-1550. Craft J, Mimori T, Olsen TL, et al. The U2 small nuclear ribonucleoprotein particle as an autoantigen: Analysis with sera from patients with overlap syndromes. J Clin Invest 1988;81: 1716-1724. Brahms H, Raker VA, van Venrooij WJ, et al. A major, novel systemic lupus erythematosus autoantibody class recognizes the E, F, and G Sm snRNP proteins as an E-F-G complex but not in their denatured states. Arthritis Rheum 1997;40:672-682. Satoh M, Akaogi J, Kuroda Y, et al. Autoantibodies that stabilize U1snRNP are a significant component of human autoantibodies to snRNP and delay proteolysis of Sm antigens in vitro. J Rheumatol 2004;31:2382-2389. Salgado-Garrido J, Bragado-Nilsson E, Kandels-Lewis S, et al. Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin. EMBO J 1999;18:3451-3462. He W, Parker R. Functions of LSm proteins in mRNA degradation and splicing. Curr Opin Cell Biol 2000;12:346-350. Kambach C, Walke S, Young R, et al. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 1999;96:375-387. Eystathioy T, Peebles CL, Hamel JC, et al. Autoantibody to hLSm4 and the heptameric LSm complex in anti-Sm sera. Arthritis Rheum 2002;46:726-734. Okano Y, Medsger TA Jr. Newly identified U4/U6 snRNP-binding proteins by serum autoantibodies from a patient with systemic sclerosis. J Immunol 1991;146:535-542. Neugebauer KM, Merrill JT, Wener MH, et al. SR proteins are autoantigens in patients with systemic lupus erythematosus: Importance of phosphoepitopes. Arthritis Rheum 2000;43: 1768-1778. Kurata N, Tan EM. Identification of antibodies to nuclear acidic antigens by counterimmunoelectrophoresis. Arthritis Rheum 1976;19:574-580. Tan EM, Smolen JS, McDougal JS, et al. A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. II. Potential for quantitation of antibody content. J Rheumatol 2002;29:68-74. Murakami A, Kojima K, Ohya K, et al. A new conformational epitope generated by the binding of recombinant 70-kd protein and U1 RNA to anti-U1 RNP autoantibodies in sera from patients with mixed connective tissue disease. Arthritis Rheum 2002; 46:3273-3282. Tan EM, Smolen JS, McDougal JS, et al. A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity. Arthritis Rheum 1999;42:455-464. Smolen JS, Butcher B, Fritzler MJ, et al. Reference sera for antinuclear antibodies. II. Further definition of antibody specificities in international antinuclear antibody reference sera by immunofluorescence and western blotting. Arthritis Rheum 1997;40: 413-418.

MECHANISMS OF TISSUE DAMAGE

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Neutrophils (Polymorphonuclear Leukocytes) in Systemic Lupus Erythematosus Yair Molad, MD and Bruce Cronstein, MD

INTRODUCTION Neutrophils are the most abundant leukocyte in the peripheral blood, and these cells are the first to arrive at inflamed sites. Although most investigators consider the involvement of neutrophils in the pathogenesis of SLE a relatively minor active role, this function of neutrophils in SLE was among the earliest experimental observations in patients with this disease. LE cells (neutrophils), neutropenia, and a role for neutrophils in the pathogenesis of immune complex-mediated vasculitis have been appreciated for many years. Intravascular neutrophil activation also plays a role in some cases of CNS lupus and pulmonary abnormalities observed in these patients. More recently, as the role for diminished clearance of immune reactants in the pathogenesis of SLE has been more widely appreciated, defective neutrophil-mediated clearance of apoptotic cells and immune complexes is thought to play a more basic role in the pathogenesis of SLE.

LE CELLS: A NEUTROPHIL-MEDIATED PHENOMENON The discovery of the lupus erythematosus (LE) cell phenomenon by Hangraves and colleagues in 19481 was a pivotal milestone not only from a diagnostic aspect but from the standpoint of its pathophysiologic significance. As recent studies show, the LE cell represents a hallmark stage in the initiation and perpetuation of the autoimmune process that underlies the role of apoptosis (programmed cell death) in the pathogenesis of systemic lupus erythematosus (SLE). It has recently been shown that the LE cell results from phagocytosis of apoptotic bodies by neutrophils [polymorphonuclear cells (PMN)] induced by anti-double-stranded (ds) DNA autoantibodies.2 Studies using flow cytometric analysis (FACS) of LE cells demonstrated the neutrophil as the phagocyte that engulfs apoptotic cells.3 Anti-dsDNA antibodies are a serologic hallmark as well as a crucial factor in the pathogenesis of immune complex–mediated tissue damage in SLE.4

The mechanism suggested to be involved in the phagocytic uptake of nuclei from apoptotic cells by neutrophils is induced by anti-dsDNA antibodymediated inhibition of enzymatic cleavage of the nuclei.5 The elucidation of the mechanism of production of the LE cells sheds light on the relevance and possible active role neutrophils play in the relationship between innate immune reaction presented by neutrophils and the specific immune response presented by antinuclear antibodies that underlie the pathogenesis of SLE.

NEUTROPHILS: THE ROLE OF INNATE IMMUNITY IN THE PATHOGENESIS OF SLE Neutrophils (PMNs) are a key component of the primary host defense line against noxious pathogens (together with macrophages and dendritic cells). This host defense line comprises the cellular arm of the innate immunity. Impaired clearance of apoptotic cells has been suggested as a crucial factor in the pathogenesis of SLE.6 To succeed with their physiologic reactions (such as chemotaxis, degranulation and phagocytosis) neutrophils become activated by various stimuli, including bacterial products, complement split products (such as C5a), immune complexes (ICs), chemokines, and cytokines [such as interleukin (IL)-1 and -8]. PMN activation is initiated upon recognition of antibodyor complement-opsonized particles as well as directly by microbial products via the toll-like receptors.7,8 Table 23.1 summarizes the main neutrophil surface receptors and their expression regulation in SLE. Neutrophils constitutively express surface complement receptors (CR), CR1 (CD35), and CR3 (β2-integrin, CD11b/CD18, Mac-1), which recognize C3b/iC3b split products of the complement system. IC are abundantly present in the serum as well as deposited in target organs affected in SLE. Neutrophil interaction with IC is mediated through binding to CR and Fcγ receptors (FcγR) present on these cells.

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NEUTROPHILS (POLYMORPHONUCLEAR LEUKOCYTES) IN SLE

TABLE 23.1 NEUTROPHIL SURFACE RECEPTORS AND THEIR EXPRESSION IN SLE Surface Receptor

Counter Ligand

Surface Expression in SLE

Complement receptor-1 (CD35)

C1q, C3b, iC3b, mannose binding lectin

Normal

Complement receptor-3 (CD11b/CD18)

ICb3, intercellular adhesion molecule-1 (ICAM-1), E-selectin, junctional adhesion molecule C (JAM-C), fibrinogen, heparin, elastase, neutrophil inhibitory factor, complement factor-H, glycoprotein Iba, urokinase receptor (uPAR), laminin, collagen, vitronectin, connective tissue growth factor

Increased

L-selectin (CD62L)

Sialomucins (CD34 family)

Decreased

Fc gamma receptor I (CD64)

Immunoglobulins, immunocomplexes

NPD

Fc gamma receptor II (CD16)

Immunoglobulins, immunocomplexes

NPD

Fc gamma receptor III (CD32)

Immunoglobulins, immunocomplexes

NPD

C5a receptor

C5a

Normal

Interleukin-8 receptor

Interleukin-8

NPD

Interleukin-1 receptor

Interleukin-1

NPD

Interferon I receptor

Interferons

NPD

Tumor necrosis factor-a-receptor (TNFa)

Tumor necrosis factor-α

NPD

TNFa-related apoptosis—inducing ligand (TRAIL) receptor-3

TNFα-related apoptosis-inducing ligand (TRAIL)

Normal/decreased in patients with neutropenia

Glycoprotein Fas (CD95)

Fas-ligand

Increased

Lipopolysaccharide receptor (CD14, toll-like receptor-4)

Lipopolysaccharide

NPD

Toll-like receptor-9

CpG motifs of bacterilal DNA

NPD

Granulocyte-colony stimulating factor (G-CSF)

G-CSF

NPD

Granulocyte and macrophage-CSF

GM-CSF

NPD

Glycoprotein receptor (CD44)

Hyaluronan, fibronectin, collagen, fibrin

Decreased

NPD, no published data

282

Neutrophil activation in patients with SLE is mediated by IC binding to FcγR, as reflected by a unique cell adhesion expression. Neutrophils constitutively express β2 integrins (CR3, CD11b/CD18) and L-selectin (LS, CD62L) on their surface, which play a role in the adhesion of neutrophils to endothelium and their egress to an extravascular site of inflammation. Upon neutrophil activation by chemoattractants [such as C5a, IL-8, and formylmethionyl-leucyl phenylalanine (FMLP)] there is an up-regulation of β2-integrin with a concomitant shedding (down-regulation) of LS. By contrast, in patients with active SLE there is a marked up-regulation of β2 integrin surface molecules with no change in the expression of LS.9 This discrepancy between the lack of LS shedding despite an

up-regulation of β2-integrin was shown to be related to FcγR activation by IC.9,10 Other studies confirmed that neutrophils are activated in the circulation of patients with active SLE, as reflected by increased expression of CD11b/CD18 on their surface with no decrease in LS expression.11 Moreover, the expression of β2-integrin on the surface of circulating neutrophils correlated positively with activity scores in patients with SLE.11,12 In contrast, there was no correlation between LS expression and disease activity.11 Interaction between FcR expressed on phagocytic cells and antibodies plays a critical role in innate immune response. Neutrophils (the major type of phagocytic cell in the blood) express two types of FcR [FcγR-IIA (CD32) and FcγR-IIIB (CD16)], both of

target tissues. Both increased neutrophil apoptosis and delayed removal of apoptotic neutrophils from the circulation have been described in lupus.21,22 Increased neutrophil apoptosis was correlated with disease activity and could serve as a source for dsDNA, as suggested by a positive correlation between antibodies to dsDNA and increased neutrophil apoptosis found in lupus patients.21 Moreover, delayed clearance of apoptotic neutrophils was observed in lupus and suggested as a major step in the pathogenesis of SLE.22 A decreased ability of macrophages to ingest apoptotic neutrophils was found in SLE and inversely correlated with activity scores.22 Taken together, these data indicate that increased neutrophil apoptosis together with decreased macrophage clearance of apoptotic cells yield a large nuclear-derived autoantigen burden that may induce or facilitate the formation of nucleosomeimmunoglobulin complexes. Various mechanisms have been suggested to explain the delayed clearance of apoptotic cells in lupus, such as complement deficiency and low pentraxin levels. With regard to delayed clearance of apoptotic neutrophils in SLE, reduced expression of CD44 and resistance to the apoptosis-inhibiting effects of granulocyte-colony stimulation factors (G-CSFs) were suggested as possible underlying mechanisms.23,24 In conclusion, cumulative data suggest that neutrophil apoptosis plays a major role in the pathogenesis of SLE by serving as the largest source of nucleosomes and nuclear debris for autoantigens resulting in production of anti-dsDNA and other antinuclear antibodies in lupus.

LUPUS NEUTROPENIA

which bind effectively to IC. IC binding to FcR results in neutrophil activation.13 In addition, FcR expressed on phagocytic cells are important in the process of IC clearance from the circulation. There is evidence suggesting that Fc-mediated clearance of IC is defective in patients with SLE.14 Allelic variants of FcγR are common within the general population. However, certain polymorphism patterns have been linked to increased susceptibility to SLE.15 Several studies addressing the association of FcγR-IIA and IIIA polymorphism and susceptibility for SLE in various ethnic groups yielded inconclusive results. In a meta-analysis comprising 17 studies, the homozygosity for the R131 allele of the FcγR-IIA was associated with a 1.3-fold greater risk for developing SLE but not for lupus nephritis.16 The evidence for an SLE susceptibility was not conclusive for patients of European descent compared to those of Asian and African descent. Another meta-analysis looking for the association of V/F158 genotype of FcγR IIIA demonstrated that the F158 allele poses a 20% greater risk for the development of lupus nephritis.17 FF homozygotes were more prone to renal damage as compared to VV homozygotes (odds ratio 1.47). These polymorphisms are associated with low-affinity FcγR, which have a lower capacity to clear ICs. The half-life of IgG-coated erythrocytes in the blood was prolonged in lupus patients expressing the FcγR-IIAR/R131 genotype, whereas the homozygous genotype FcγR-IIIA-F (F158) was associated with arthritis and/or serositis in these patients.18 This association of FcγR polymorphism with an increased risk of SLE and/or certain manifestations of the disease emphasizes the importance of neutrophils in the process of IC clearance, in that a delay in clearance results in greater IC deposition in tissues and organs such as the kidneys and blood vessels. FcγRs play another role in the pathogenesis of SLE; namely, clearance of apoptotic cells. The half-life of circulating neutrophils is about 7 hours, and following egress to an extravascular tissue they die within 1 to 2 days. Under normal conditions, neutrophils are removed from the circulation as well as from inflamed tissue by apoptosis, genetically programmed cell death.19 Delayed neutrophil apoptosis has been associated with autoimmune disorders, especially so in SLE where nuclear materials serve as autoantigens for the production of an array of antinuclear antibodies.20 Nucleosomes are known as major autoantigens in SLE and are exposed at the cell surface in apoptosis. Thus, increased apoptosis and/or delayed clearance of apoptotic cells could contribute to an overload of autoantigens in the circulation that will eventually lead to an increased nucleosome-containing IC deposition in

LUPUS NEUTROPENIA Neutropenia is one of the hematologic features of patients with SLE and is found in up to half of these patients.25 Several mechanisms have been suggested for the pathogenesis of lupus-associated neutropenia (Table 23.2). As mentioned earlier, apoptosis of circulating neutrophils is increased in patients with SLE.21,22

TABLE 23.2 THE PATHOGENESIS OF LUPUS-ASSOCIATED NEUTROPENIA ●

Increased neutrophil apoptosis

●

Increased TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis

●

Anti Ro/SS-A and La/SS-B antibody-associated apoptosis

●

Diminished granulopoiesis

●

Bone marrow hypocellularity

●

Anti–G-CSF mediated

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NEUTROPHILS (POLYMORPHONUCLEAR LEUKOCYTES) IN SLE

Regarding neutrophil apoptosis, Fas and tumor necrosis factor (TNF)-a induce neutrophil death. TNF-related apoptosis-inducing ligand (TRAIL) can induce neutrophil apoptosis. A recent study presented data that suggest that TRAIL can accelerate the apoptosis of neutrophils in patients with SLE.26 The serum TRAIL level was significantly higher in those patients with neutropenia than those with normal neutrophil count. The expression of TRAIL receptor-3 was significantly lower in SLE patients with neutropenia than in patients without neutropenia or healthy controls. G-CSF is an essential growth factor for the normal physiologic process of granulopoiesis. G-CSF stimulates the proliferation, differentiation, and maturation of neutrophil precursors in the bone marrow. In one study, IgG antibodies against G-CSF were detected in 73% of lupus patients.27 The serum level of G-CSF is increased in some but not all patients with lupus-associated neutropenia and no significant correlation could be found between G-CSF level and neutrophil count.21,27 Nonetheless, diminished granulopoiesis and bone marrow hypocellularity compatible with myelofibrosis or aplasia have been reported in patients with SLE.28 Another mechanism for the induction of lupusrelated neutropenia is anti-neutrophil antibodies. Anti-Ro/SS-A and anti-La/SS-B antibodies are commonly found in SLE and have been associated with leucopenia.29 Anti-Ro/SS-A antibody was associated with neutropenia in patients with SLE,30 as was antiLa/SS-B antibody.31 Moreover, anti-La/SS-B antibody is capable of exerting PMN apoptosis and activation as well as decreased PMN phagocytosis.31

NEUTROPHIL FUNCTION IN SLE

284

Increased susceptibility to infections is a leading cause of morbidity and mortality in SLE.32 Neutropenia and impaired PMN function are crucial predisposing factors for the increased tendency to incur infections in patients with SLE. Chemotaxis, recognition of microorganisms, phagocytosis, and oxidative metabolism are abnormal in SLE.33-35 Defective neutrophil chemotaxis in SLE has been associated with low complement serum level and positively correlated with serious infections in those patients with a lower chemotactic index.33 Neutrophil phagocytosis and TNFainduced phagocytosis are also significantly defective in patients with SLE.34 Others have reported a reduction in spontaneous and lipopolysaccharide (LPS)-induced production of IL-8 by PMN that correlates with disease activity.35 All of these various defects in neutrophil function likely contribute, among other factors, to the increased susceptibility for infections found in SLE patients.

LEUKOCYTOCLASTIC VASCULITIS One of the nonspecific cutaneous manifestations of SLE is small-vessel vasculitis.36 Leukocytoclastic vasculitis (LCV) is a neutrophil and FcR-dependent vasculitis of the post-capillary venules initiated by IC deposition and subsequent leukocyte recruitment, which results in neutrophilic inflammation in the blood vessel wall with a characteristic histologic finding of neutrophil nuclear debris (leukocytoclasis) and fibrinoid necrosis.37 In one study, LCV was found in 13.7% of patients with SLE who had nonspecific cutaneous manifestations, usually in the active phase of the disease.38 Upon circulating IC formation in excess and their deposition in blood vessel walls, there is an up-regulation of endothelial adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), as well as secretion of endothelial-derived chemokines (such as IL-8, which is a potent chemoattractant for neutrophils).39 As a result of IC-deposition, the complement classical pathway is activated. This results in elevation of C5a, which also acts as a neutrophil chemoattractant.40 This IC-mediated endothelial and neutrophil activation leads to increased neutrophil β2-integrin and endothelial ICAM-1-dependent neutrophil adhesiveness to the endothelium, as was demonstrated in animal models of IC-mediated vasculitis.41-44 Subsequently to IC and C5a-induced neutrophil activation, there is an increased production and release of neutrophil proteolytic enzymes along with free oxygen radicals that damage the vessel walls and the surrounding tissues.45-47 Apoptotoc cell death mediated by the Fas/Bc12 system probably leads to the characteristic feature of leukocytoclasis.37 Although not a specific feature of SLE, LCV is a prototypic example of an IC-induced and neutrophil-mediated inflammation that demonstrates the pivotal role neutrophils play in vascular and tissue damage in IC-mediated diseases.

CONCLUSIONS Neutrophils play a central role in the pathogenesis of some manifestations of SLE. As discussed previously, inappropriate neutrophil activation by immune reactants, diminished neutrophil-mediated clearance of immunogens and immune complexes, lower numbers of neutrophils in the peripheral circulation, and enhanced apoptosis of these cells all contribute to the pathogenesis of SLE. It is that much more ironic then that diminished neutrophil function contributes to the inadequate responses of SLE patients to microorganisms and increased morbidity and mortality from infections in patients with SLE.

1. Hargroves MM, Richmond H, Morton R. Presentation of two bone marrow elements: The “tart” cell and the LE cell. Proc Staff Mayo Clin 1948;23:25-28. 2. Schmidt-Acevedo S, Perez-Romano B, Ruiz-Arguelles A. “LE cells” result from phagocytosis of apoptotic bodies induced by antinuclear antibodies. J Autoimmun 2000; 15:15-20. 3. Böhm I. Flow cytometric analysis of the LE cell phenomenon. Autoimmunity 2004;37:37-44. 4. Linnik MD, Hu JZ, Heibrunn KR, Strand V, Hurley FL, Joh T. LJP 394 Investigator Consortium. Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus. Arthritis Rheum 2005;52:1129-1137. 5. Böhn I. LE cell phenomenon: Nuclear IgG deposits inhibit enzymatic cleavage of the nucleus of damaged cells and support its phagocytic clearance by PMN. Biomed Pharmacother 2004; 58:196-201. 6. Herrmann M, Voll RE, Kalden JR. Etiopathogenesis of systemic lupus erythematosus. Immunol Today 2000;21:424-426. 7. Greenberg S, Grinstein S. Phagocytosis and innate immunity. Curr Opin Immunol 2002;14:136-145. 8. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388:394-397. 9. Molad Y, Haines KA, Anderson DC, Buyon JP, and Cronstein BN. Immunocomplexes stimulate different signalling events to chemoattractants in the neutrophil and regulate L-selectin and beta 2-integrin expression differently. Biochem J 1994; 299:881-887. 10. Kocher M, Siegel ME, Edberg JC, Kimberly RP. Cross-linking of Fcγ receptor IIa and Fcγ receptor IIIb induces different proadhesive phenotypes on human neutrophils. J Immunol 1997; 159:3940-3948. 11. Molad Y, Buyon J, Anderson DC, Abramson SB. Cronstein BN. Intravascular neutrophil activation in systemic lupus erythematosus (SLE): Dissociation between increased expression of CD11b/CD18 and diminished expression of L-selectin on neutrophils from patients with active SLE. Immunol Immunopathol 1994;71:281-286. 12. Buyon JP, Shadick N, Berkman R, Hopkins P, Dalton J, Weissmann G, et al. Surface expression of Gp 165/95, the complement receptor CR3, as a marker of disease activity in systemic lupus erythematosus. Clin Immunol Immunopathol 1988; 46:141-149. 13. Ji H, Ohmura K, Mahmood U, Lee DM, Hofhuis FM, Boackle SA, et al. Arthritis critically dependent on innate immune system players. Immunity 2002;16:157-168. 14. Davies KA, Robson MG, Peters M, Norsworthy P, Nash JT, Walport MJ. Defective Fc-dependent processing of immune complexes in patients with systemic lupus erythematosus. Arthritis Rheum 2002;46:1028-1048. 15. Karassa FB, Trikalinos TA, Ioannidis JP. The role of FcgammaRIIA and IIIA polymorphisms in autoimmune diseases. Biomed Pharmacother 2004;58:286-291. 16. Karassa FB, Trikalinos TA, Ioannidis JP. Fc gamma RIIA-SLE meta-analysis investigators. Role of the Fc gamma receptor IIa polymorphism in susceptibility to systemic lupus erythematosus and lupus nephritis: a meta-analysis. Arthritis Rheum 2002; 24:1563-1571. 17. Karassa FB, Trikalinos TA, Ioannidis JP. Fc gamma RIIIA-SLE metaanalysis investigators. The Fc gamma RIIIA-F158 allele is a risk factor for the development of lupus nephritis: a meta-analysis. Kidney Int 2003;63:1475-1482. 18. Dijstelbloem HM, Bijl M, Fijnheer R, Scheepers RH, Oost WW, Jansen MD, et al. Fc gamma receptor polymorphisms in systemic lupus erythematosus: association with disease and in vivo clearance of immune complexes. Arthritis Rheum 2000; 43:2793-800. 19. Maianski NA, Maianski AN, Kuijpers TW, and Roos D. Apoptosis of neutrophils. Acta Haematol 2004,111:56-66. 20. Kalden J. Apoptosis in systemic autoimmunity. Autoimmun Rev 2004,3 Suppl 1:S9-10.

21. Courtney PA, Crockard AD, Williamson K, Irvine AE, Kennedy RJ, Bell AL. Increased apoptotic peripheral blood neutrophils in systemic lupus erythematosus: relations with disease activity, antibodies to double stranded DNA, and neutropenia. Ann Rheum Dis 1999;58:309-314. 22. Ren Y, Tang J, Mok MY, Chan AW, Wu A, Lau CS. Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus. Arthritis Rheum 2003;48:2888-2897. 23. Cairns AP, Crockard AD, McConnell JR, Courtney PA, Bell AL. Reduced expression of CD44 on monocytes and neutrophils in systemic lupus erythematosus: relations with apoptotic neutrophils and disease activity. Ann Rheum Dis 2001;60:950-955. 24. Armstrong DJ, Whitehead EM, Crockard AD, Bell AL. Distinctive effects of G-CSF, GM-CSF and TNFalpha on neutrophil apoptosis in systemic lupus erythematosus. Clin Exp Rheumatol 2005; 23:152-158. 25. Nossent JC, Swaak AJ. Prevalence and significance of haematological abnormalities in patients with systemic lupus erythematosus. Q J Med 1991;80:605-612. 26. Matsuyama W, Yamamoto M, Higashimoto I. Oonakahara K, Watanabe M, Machida K, et al. TNF-related apoptosis-inducing ligand is involved in neutropenia of systemic lupus erythematosus. Blood 2004;104:184-191. 27. Hellmich B, Csernok E, Schatz H, Gross WL, Schnabel A. Autoantibodies against granulocyte colony-stimulating factor in Felty’s syndrome and neutropenic systemic lupus erythematosus. Arthritis Rheum 2002;46:2384-2391. 28. Rosenthal NS, Farhi DC. Bone marrow findings in connective tissue disease. Am J Clin Pathol 1989;92:650-654. 29. de Rooij OJ, Van de Putte LB, Habets WJ, Verbeek AL, van Venrooij WJ. The use of immunoblotting to detect antibodies to nuclear and cytoplasmic antigens. Clinical and serologic associations in rheumatic diseass. Scand J Rhematol 1988;17:353-364. 30. Kurien BT, Newland J, Pacakowski C, Moore KL, Scofield RH. Association of neutropenia in systemic lupus erythematosus (SLE) with anti-Ro and binding of an immunologically crossreactive neutrophil membrane antigen. Clin Exp Immunol 2000;120:209-217. 31. Hsieh SC, Yu HS, Lin WW, Sun KH, Tsai CY, Huang DF, Stai YY, Yu CL. Anti-SS-B/La is one of the antineutrophil autoantibodies responsible for neutropenia and functional impairment of polymorphonuclear neutrophils in patients with systemic lupus erythematosus. Clin Exp Immunol 2003;131:506-516. 32. Juárez M, Nisischia R, Alarcón GS. Infections in systemic connective tissue diseases: systemic lupus erythematosus, scleroderma, and polymyositos/drmatomyositis. Rheum Dis Clin North Am 2003;29:163-184. 33. Alvarez I, Vazquez JJ, Fontan G, Gil A, Barbado J, Ojeda JA. Neutrophil chemotaxis and serum chemotactic activity in systemic lupus erythematosus. Scand J Rheumatol 1978;7:69-74. 34. Yu CL, Chang KL, Chiu CC, Chiang BN, Han SH, Wang SR. Defective phagocytosis, decreased tumor necrosis factor-alfa production and lymphocyte hyporesponsiveness predispose patients with systemic lupus erythematosus to infections. Scand J Rheumatol 1989;18:97-105. 35. Hsieh SC, Tsai CY, Sun KH, Yu HS, Tsai ST, Wang JC, et al. Decreased spontaneous and lipopolysaccharide stimulated production of interleukin-8 by polymorphonuclear neutrophils of patients with active systemic lupus erythematosus. Clin Exp Rheumatol 1994;12:627-633. 36. Calamia KT, Balabanova M. Vasculitis in systemic lupus erythematosus. Clin Dermatol 2004;22:148-156. 37. Claudy A. Pathogenesis of leukocytoclastic vasculitis. Eur J Dermatol 1998;8:75-79. 38. Cardinali C, Caproni M, Bernacchi E, Amato L, Fabbri P. The spectrum of cutaneous manifestations in systemic lupus eythematosus - the Italian experience. Lupus 2000;9:417-423. 39. Lentsch AB, Ward PA. Regulation of inflammatory vascular damage. J Pathol 2000;190:343-348. 40. Abramson S, Belmont HM, Hopkins P, Buyon J, Winchester R, Weissmann G. Complement activation and vascular injury in systemic lupus erythematosus, J Reumatol 1987;13:43-46.

REFERENCES

REFERENCES

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41. Fletcher DS, Osinga D, Bonney RJ. Role of polymorphonuclear leukocytes in connective tissue breakdown during the reverse passive Arthus reaction. Biochem Pharmacol 1986; 35:2601-2606. 42. Rote WE, Dempsey E, Maki S, Vlasuk GP, Moyle M. The role of CD11/CD18 integrins in the reverse passive Arthus reaction in rat dermal tissue. J Leukoc Biol 1996;59:254-261. 43. Smith RJ, Chosay JG, Dunn CJ, Manning AM, Justen JM. ICAM-1 mediates leukocyte-endothelium adhesive interactions in the reversed passive Arthrus reaction. J Leukoc Biol 1996; 59:333-340.

44. Kaburagi Y, Hasegawa M, Nagaoka T, Shimada Y, Hamaguchi Y, Komura K,et al. The cutaneous reverse Arthus reaction requires intercellular adhesion molecule 1 and L-selectin expression. J Immunol 2002;168:2970-2978. 45. Cochrane CG. Role of granulocytes in immune complex-induced tissue injuries. Inflammation 1977;2:319-333. 46. Fantone JC, Ward PA. Polymorphonuclear leukocyte-mediated cell and tissue injury: oxygen metabolites and their relations to human disease. Hum Pathol 1985;16:973-978. 47. Jancar S, Sanchez Crespo M. Immune complex-mediated tissue injury: a multistep paradigm. Trends Imunol 2005;26:48-55.

MECHANISMS OF TISSUE DAMAGE

24

Renal Damage in Systemic Lupus Erythematosus Steven G. Dimitriou, DO and Michael P. Madaio, MD

Renal damage in systemic lupus erythematous (SLE) involves a complex interaction among immune and inflammatory reactants that affect disease severity, clinical course, and response to therapy. In general, there is a breakdown in immunologic tolerance to chromatin that leads to production of autoreactive cells that either through direct infiltration (e.g., T cells, macrophages) and/or through their secretory products (e.g., autoantibodies, cytokines) initiate and perpetuate inflammation. Along with immune deposition and cellular infiltration, there is a sophisticated interplay among cytokines, chemokines, and complement along with the degree to which affected renal cells, respond to the aforementioned inflammatory milieu, that determines disease expression. Both the propagation and amplification of inflammation and fibrosis are influenced by genetic predisposition.1-13 Disease concordance in monozygotic twins, familial clustering, and increased incidence in selected ethnic backgrounds support a role for genetic background in lupus pathogenesis. In this context, one of the most intriguing aspects of SLE is that there is variable expression of disease among patients. For example, not everyone develops nephritis, and among patients with nephritis disease expression varies considerably. This is in part due to differential pathogenic characteristics among autoantibodies, and variability in the quantity and distribution of immune deposits. Furthermore, the cellular response to the immune reactant is variable. The key participants in lupus nephritis are discussed in material following. This is divided according to the players that initiate nephritis, and an emphasis is placed on the interplay leading to variability in expression of nephritis. In particular, the ensuing discussion focuses on autoantibodies, B cells, and T cells. Although inflammatory cells (mediators of inflammation and the renal cellular response) are crucial to disease activity, the plethora and redundancy of mediators and variable expression in individual patients over the course of disease limits targeting one mediator as therapy.

They are discussed elsewhere.14-29 Furthermore, the reader should also be aware that the events leading to autoimmunity are in general complex and are reviewed elsewhere.21,30,31

NEPHRITOGENIC AUTOANTIBODIES Although multiple factors influence the variability of disease expression, autoantibodies are commonly involved in initiating disease activity.30,32-34 Anti-dsDNA antibodies have been the most extensively studied.30,35,36 In general, there is correlation of serum anti-dsDNA antibody levels with nephritis, a temporal association of rising titers and increased disease activity, and antidsDNA antibodies are enriched in glomerular immune deposits.37-41 Furthermore, administration of some anti-dsDNA antibodies to normal mice can induce nephritis. However, it is also established that not all autoantibodies are pathogenic.31,36,42 Moreover, among those that are nephritogenic their pathogenic profiles differ considerably. For example, after administration of anti-dsDNA antibodies to normal mice there was variability in location of deposits (e.g., subendothelial, mesangial) and the type of nephritis that resulted also differed.32,33,36 These observations are consistent with clinical observations of patients with variable expression of nephritis. Pathogenic insights are also provided by elution studies.43-45 Antibodies eluted from human or murine lupus nephritis kidneys are typically of IgG isotype. They are distinguished from serum autoantibodies by their antigen-binding properties and their capacity to engage FcR and fix complement. Nephritogenic antibodies are highly cross-reactive and react with multiple autoantigens when compared to nonpathogenic serum autoantibodies, that prefer one antigen (e.g., DNA). These observations raised the possibility that nephritogenic antibodies cross-react with glomerular antigens. Further insights were derived from administration of monoclonal lupus autoantibodies to normal mice. Consistent with the aforementioned clinical

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observations in lupus patients, not all autoantibodies were nephritogenic (i.e., form immune deposits). However, cross-reactive autoantibodies appear to be more pathogenic than those that react with only DNA. Furthermore, there are subsets of autoantibodies that produce distinguishable histologic and clinical patterns of nephritis. One explanation for these observations is due to differences in both autoantibody binding properties and the mechanism by which they form immune deposits.40,46,47 In this context, glomerular immune deposits can potentially form by direct binding of the autoantibody to glomerular antigens, by immune complex formation where circulating autoantigens become complexed to glomerular antigens (and thereby serve as planted antigens for circulating autoantibodies), or by the deposition of circulating preformed immune complexes. Most evidence supports the initiation of immune deposition by in situ mechanisms, with either antibody binding to intrinsic glomerular antigens [e.g., cell surface antigens (cross-reactive theory) or circulating autoantigens that initially localize within glomeruli (planted antigen theory)].35,39,47-49 Passive trapping of preformed immune complexes within glomeruli is more likely to accentuate disease by activating resident glomerular cells or cells that have infiltrated the kidney (e.g., through FcR interaction). In this regard, transfer of preformed immune complexes has not convincingly been observed to recapitulate nephritis. Nevertheless, immune complexes have been shown to transiently localize in glomeruli, typically in the mesangium. In culture, immune complexes stimulate mesangial cells, and in kidneys of lupus patients this may lower their threshold for activation, cause an accentuation of cytokine release, and/or increase in matrix production. This situation may be amplified in lupus patients because clearance of immune complexes by phagocytic cells is often impaired. Thus, although immune complex deposition may not initiate inflammation it likely plays an important role. Further support for direct binding to glomeruli comes from experiments in which antibodies eluted from both human and murine lupus kidneys bound to glomerular extracts.4,45,50,51 Subsequently, multiple autoantibody-glomerular cell surface and matrix antigen interactions have been described. For example, anti-DNA antibodies have been shown to react with glomerular cell surface antigens, laminin, heparan sulfate, and α-actinin.48,52-56 Differences in cross-reactivity among autoantibody populations likely contribute to the diversity of disease observed in lupus patients. For example, different autoantibody specificities that predominate in a given individual influence where the deposits form and how disease is expressed. Evidence also supports the “planted antigen” mechanism of immune deposition in lupus. Intracellular antigens released into the circulation after cell death

(e.g., nucleosomes) may deposit in various sites within the glomerulus, where they can then serve as a nidus for binding of circulating autoantibodies. In support of a central role for nucleosomes, anti-DNA, anti-histone, and anti-nucleosome antibodies have all been shown to bind to nucleosomes previously localized within glomeruli. In this case, the nucleosome-glomerular interaction is facilitated by the relatively cationic charge of the histones with the relative negative charge of the GBM. Studies in both murine and human lupus sera have identified negatively charged heparan-sulfated glycosaminoglycan as the candidate ligand for this initial nucleosome binding. The deposition then presumably exposes the DNA within the nucleosome, which serves as a planted antigen for circulating anti-DNA antibodies. In most instances, deposition of antibody alone is insufficient for nephritis. For inflammation to develop, activation of effector mechanisms is required, and this is largely influenced by the Fc region of the complexed antibody and the location of the deposits. For many years, the accepted model of immune-mediated injury (based on studies done in the 1950s and 1960s involving the Arthus reaction) held that complement activation was responsible for all effector responses and consequences of immune complex deposition.57 More recently, an essential role for complement independent mechanisms were derived from observations in mice with targeted disruptions of complement components C3 or C4.58 They still mounted an Arthus reaction and other inflammatory responses. Interestingly, mice that are deficient in the activating FcγR receptors (FcγRIII) did not develop inflammation. In elegant studies directly related to lupus nephritis, Ravetch and colleagues demonstrated the obligatory function of FcR by back-crossing lupus-prone (NZB/NZW F1) mice with a congenic strain that had a homozygous deletion for the activating Fc receptor [FcRγ (−/−)].58-62 The FcRγ (−/−)-deficient NZB/NZW mice developed circulating autoantibodies and glomerular deposits of IgG and complement. However, they did not develop proteinuria or nephritis. The results support the conclusions that deposited immune complexes and C3 are insufficient to trigger effector cell activation and that functional activator FcRs play a pivotal role in mediating inflammation in autoimmune nephritis. The role of FcRs in autoimmunity is not limited to its triggering of effector cells. The inhibitory FcγRIIB suppresses B-cell activation and proliferation by opposing activation through the B-cell receptor (BCR).62 By contrast, activation of B cells and exaggerated antibody responses occur if FcγRIIB expression is decreased or signaling is impaired. This can result in loss of tolerance and the development of autoimmunity. In this regard, inactivation of FcγRIIb in normal mice

and anti-La) appeared years prior to the diagnosis of SLE whereas others (anti-Sm and anti-nuclear ribonucleoprotein antibodies) appeared only during the periclinical stages. These observations are consistent with the notions that there are specific properties of nephritogenic antibodies that initiate disease and/or that other factors (e.g., activation of B cells) are required.

ROLE OF CELLS

(C57BL/6) leads to a spontaneous lupus-like disease with anti-dsDNA autoantibodies, glomerular immune complexes, and severe glomerulonephritis.59 Some autoimmune-prone mice (such as NZB and MRL) show reduced surface expression of Fc γRIIB, which has been attributed to polymorphisms in the promoter region of this receptor gene.63 Furthermore, genetic polymorphisms of Fc receptors have also been described in humans. Some allelic variants alter the binding of the FcR Ig and thereby affect immune complex clearance. In some populations, the low-affinity binding polymorphisms confer a higher risk for development of lupus nephritis in comparison to the higher binding variants. Thus, altering FcR expression is a rational therapeutic approach for study. Nevertheless, in lupus nephritis (like other forms of nephritis) the anatomic location of the immune deposits influences both the predominant effector mechanism and the ultimate clinical and histologic manifestations of the disease.33,36,40,48 For example, if immune deposits form in the subepithelial area (as in membranous lupus) the presence of the GBM prevents inflammatory cell recruitment to the site, and the resultant pathology is nonexudative. This presumably occurs because FcR on circulating cells are not engaged. However, in this setting the membrane attack complex (C5b-9, generated from complement activation) mediates sublytic injury to cells. Consequently there is effacement of podocytes. This leads to altered glomerular barrier function and heavy proteinuria. Subsequently, there may be podocyte loss that may be exacerbated by the formation of very large deposits, contributing to overproduction of extracellular matrix components and leading to scarring. By contrast, if the immune deposits are accessible to the vascular space (such as in the subendothelial and mesangial regions) effector cells are recruited and inflammatory lesions dominate. In the latter situation, activation of resident glomerular cells through FcR (largely) and complement cascade contribute to the lesions described previously. Although the relationship of autoantibodies to lupus and other autoimmune diseases is well accepted,36-38,43,48,53,64 recent data suggest that other factors are required.65-68 In this regard, Arbuckle and colleagues evaluated serum samples obtained from the Department of Defense Serum Repository in order to investigate the onset of autoantibody development prior to the clinical well diagnosis of lupus.65-67 The investigators determined that autoantibodies were present well before a diagnosis of SLE in 88% of patients studied. An important finding was that there was a temporal pattern of autoantibody specificity with respect to the appearance of clinical manifestations/diagnosis of SLE. Specifically, certain autoantibodies (ANA, anti-Ro,

GENETIC FACTORS IN THE PATHOGENESIS OF LUPUS NEPHRITIS Genetic deficiency of complement factor(s) is a wellestablished human gene polymorphism associated with a pathogenetic role in SLE.57,69 Nevertheless, this is a relatively uncommon cause of lupus, and many of these patients do not develop nephritis. Nonetheless, there is a complex polygenic influence on susceptibility and disease expression, and there are numerous candidate loci. Animal models have provided insight into the influence of genetic contributions. Lupus-prone NZM2328 mice have unique genetic susceptibility loci: Cgnz1 on chromosome 1 is linked to chronic glomerulonephritis, Agnz1 on chromosome 1 is suggestively linked to acute glomerulonephritis, and Adnz1 on chromosome 4 is suggestively linked to elevated levels of ANA and anti-dsDNA.3,41,70 Waters and colleagues studied NZM2328 mice back-crossed with C57L/J mice (a nonlupus-prone strain) to generate two congenic strains in which the chromosomal segments containing Cgnz1 and Agnz1 (NZM.C57Lc1) or Adnz1 (NZM.C57Lc4) were replaced accordingly.41 The strain lacking the nephritic loci (NZM.C57Lc1) had reduced incidence of glomerulonephritis and proteinuria. The strain with the nephritic loci intact but lacking the autoantibody locus (NZM.C57Lc4) had chronic glomerulonephritis (severe proteinuria) despite the low levels of circulating ANA, anti-dsDNA, or anti-nucleosome antibody. Interestingly, the NZM.C57Lc4 kidneys had immune complex deposition. However, kidney eluates did not contain ANA, anti-dsDNA, or anti-nucleosome antibodies. The results suggest that breaking tolerance to nuclear antigens may not be the sine qua non of SLE pathogenesis, and that the development of glomerulonephritis is not contingent on autoantibody prevalence alone.

ROLE OF CELLS As implied previously, the involvement of B cells has emerged as a critical part of lupus pathogenesis beyond antibody secretion. Evidence supports a prominent role of autoreactive B cells as specific antigen-presenting cells in lupus at multiple levels.31,42,44,71-89 It has been shown that breaking T-cell tolerance to self-antigens

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was dependent on the ability of autoreactive B cells to process and present lupus autoantigens.83 Using immunization of normal mice with cross-reactive variants of self-proteins, Lin and colleagues82 and Mamula and colleagues83 have suggested a key role for B cells in initiating T-cell autoreactivity, which in turn leads to cell interactions that lead to B-cell autoreactivity. In separate studies, Shlomchik and co-workers further elucidated the role of B cells in MRL-lpr/lpr mice. They created novel strains of mice by crossing mice homozygous for a mutation (JhD) that prevents development of mature B cells.89 In the absence of B cells, lpr/Ipr mice did not develop glomerulonephritis, vasculitis, or interstitial nephritis. In contrast, all of the lpr/lpr littermate mice with B cells developed severe nephritis. Aside from its effect on nephritis, a lack of B cells was associated with reduced accumulation of T cells. Subsequent work by Chan and Shlomchik demonstrated that B cells are necessary for spontaneous activation and expansion of T cells in MRL-lpr/lpr mice involving CD4+ and CD8+ T cells.73,77 Lack of B cells correlated with the absence of cellular infiltrates in the kidney and skin, supporting an APC role for B cells. The presence of B cells within interstitial and perivascular infiltrates within the kidney also suggests that they serve as antigen-presenting cells for autoreactive T cells at these locations. Based on these observations, new therapeutic targets and strategies have emerged for lupus patients. Specifically, this has involved B-cell depletion using antibodies to CD20, a cell surface molecule specific to B cells. To evaluate the efficacy of the anti-CD20 monoclonal antibody, Sfikakis and colleagues evaluated the effect of rituximab infusions and oral prednisolone in patients with active proliferative lupus nephritis.88 In uncontrolled studies, the investigators found clinical remission of lupus nephritis, also associated with a decrease in B cells. (This was also associated with a reduction in T helper cell activation.) The percentage

of CD4+ T cells that express CD69 and HLA-DR were significantly lower in patients with clinical signs of partial remission of nephritis, and expression of the CD40L was also suppressed. These findings confirm the aforementioned findings in murine lupus, and support the role of B cells as APCs that then foster the activation of T helper cells in human lupus nephritis.

CONCLUSIONS Multiple cells and soluble factors participate in the initiation and perpetuation of lupus nephritis. Variable disease expression, responsiveness to therapy, and disease progression commonly observed among patients is typical. This is both genetically determined and under the influence of environmental and exogenous factors. In human lupus, T-cell-dependent autoantibody production leads to antibody deposition, and this is associated with cellular infiltration and activation/ proliferation of endogenous renal cells. Nevertheless, B cells play a crucial role as both antigen-presenting cells for T cells and for nephritogenic autoantibody production. Disease severity is determined by the nature and intensity of the autoimmune response and the intensity of renal inflammation. The latter is influenced by systemic and local (within the kidney) factors. Although not documented in this review, it is also evident that renal cells participate in augmenting inflammation and fibrosis. This is likely determined by the intensity of the systemic inflammatory response and by genetic factors. In lupus patients, disease progression is determined by the effectiveness with which therapy modulates systemic autoimmune nephritogenic responses and renal inflammatory and fibrogenic events. Thus, successful therapeutic regimens should be directed at suppressing both the systemic autoimmune response leading to nephritis and the renal inflammatory process resulting in fibrosis.

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MECHANISMS OF TISSUE DAMAGE

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Mitochondrial Dysfunction and Oxidative Stress in T Cells of Patients with Systemic Lupus Erythematosus Andras Perl, MD, PhD

SUMMARY Activation, proliferation, and cell death pathway selection of T lymphocytes depend on reactive oxygen intermediate (ROI) production and ATP synthesis, which are tightly regulated via the mitochondrial transmembrane potential (Δψm). Mitochondrial hyperpolarization (MHP) and ATP depletion represent early and reversible steps in T-cell activation and apoptosis. By contrast, T lymphocytes of systemic lupus erythematosus (SLE) patients exhibit persistent MHP, cytoplasmic alkalinization, increased ROI production, and ATP depletion that mediate enhanced spontaneous and diminished activation-induced apoptosis and sensitize lupus T cells to necrosis. Necrotic, but not apoptotic, cell lysates activate dendritic cells and may account for increased interferon-a production and inflammation in lupus patients. Persistent MHP is associated with increased mitochondrial mass and increased mitochondrial and cytoplasmic Ca2+ content in T cells of SLE patients. Nitric oxide (NO) has recently been recognized as a key signal of MHP and mitochondrial biogenesis in human T lymphocytes. NO-induced mitochondrial biogenesis in normal T cells regenerated the Ca2+ signaling profile of lupus T cells. Mitochondria constitute major Ca2+ stores, and NO-dependent mitochondrial biogenesis may account for altered Ca2+ handling by lupus T cells. MHP is proposed as a key mechanism of T-cell dysfunction and target of pharmacologic intervention in SLE.

INTRODUCTION SLE is a chronic inflammatory disease characterized by T- and B-cell dysfunction and production of antinuclear antibodies. Potentially autoreactive T and B lymphocytes during development1 and after completion of an immune response are removed by apoptosis.2 Abnormal T-cell activation and cell death underlie the

pathology of SLE.3,4 Paradoxically, lupus T cells exhibit both enhanced spontaneous apoptosis5 and defective activation-induced cell death.6-10 Increased spontaneous apoptosis of PBL has been linked to chronic lymphopenia5 and compartmentalized release of nuclear autoantigens in patients with SLE.11 By contrast, defective CD3-mediated cell death may be responsible for persistence of autoreactive cells.6 Both cell proliferation and apoptosis are energydependent processes. Energy in the form of ATP is provided through glycolysis and oxidative phosphorylation.12 The synthesis of ATP is driven by an electrochemical gradient across the inner mitochondrial membrane maintained by an electron transport chain and the membrane potential (Δψm, negative inside and positive outside). A small fraction of electrons react directly with oxygen and form ROI. Disruption of Δψm has been proposed as the point of no return in apoptotic signaling.13-15 Mitochondrial membrane permeability is subject to regulation by an oxidation-reduction equilibrium of ROI, pyridine nucleotides (NADH/NAD + NADPH/NADP), and GSH levels.16 Regeneration of GSH by glutathione reductase from its oxidized form GSSG depends on NADPH produced by the pentose phosphate pathway (PPP).17-19 Metabolic fluxes between glycolysis and the PPP are particularly relevant for balancing cellular requirements for energy and ROI production.20 Although ROI have been considered toxic by-products of aerobic existence, evidence is now accumulating that controlled levels of ROI modulate various aspects of cellular function and are necessary for signal-transduction pathways (including those mediating T-cell activation and apoptosis).20 Elevation of Δψm or MHP was discovered in our laboratory as an early event preceding caspase activation, phosphatidylserine (PS) externalization, and disruption of Δψm in Fas-(19) and H2O2-induced apoptosis of Jurkat human leukemia T cells and normal human PBL,21

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as well as in HIV-1-induced apoptosis.22 These observations were confirmed and extended to p53;23 tumor necrosis factor a;24 and staurosporin,25 camptothecin,26 and NO-induced apoptosis.27 MHP is also triggered by activation of the CD3/CD28 complex7 via ROI- and Ca2+-dependent production of NO28 or stimulation with Con A19 or galectin 1,29 IL-10, IL-3, IFN-?, and TGFβ.8 Thus, elevation of Δψm or MHP represents an early but reversible switch not exclusively associated with apoptosis. With ??m hyperpolarization and extrusion of H+ from the mitochondrial matrix, the cytochromes within the electron transport chain become more reduced (which favors generation of ROI).30 Therefore, MHP is a likely cause of increased ROI production and may be ultimately responsible for increased susceptibility to apoptosis following T-cell activation.20 MHP and ATP depletion play key roles in abnormal T-cell death (which shift susceptibility from apoptosis to necrosis) in patients with SLE.7 In turn, increased necrosis rates could play a central role in enhanced inflammatory responses in SLE. Δψm and ROI levels (as well as cytoplamic pH) are elevated in patients with SLE in comparison to healthy or RA controls.7,8 Baseline MHP and ROI levels correlated with diminished GSH levels, suggesting increased utilization of reducing equivalents in patients with SLE. It is presently unclear whether de novo synthesis of GSH or its regeneration from the oxidized form (GSSG) is deficient in lupus patients. GSH is required for interleukin-2-dependent T-cell proliferation31 as well as CD2- and CD3-mediated T-cell activation.32 Thus, low GSH content may also inhibit CD3-induced H2O2 production. Moreover, GSH deficiency predisposes cells for ROI-induced cell death.18,21,33 Diminished H2O2-induced apoptosis of cells with low baseline GSH levels indicates a severe dysfunction of redox signaling in patients with SLE. Increased ROI production may lead to skewed expression of redox-sensitive surface receptors and lymphokines. As examples, ROIs regulate gene transcription and release of TNFa and interleukin-10,34 both of which are elevated in sera35,36 and freshly isolated PBL of SLE patients.37,38 Elevated serum levels of type I interferon (IFN) raised the possibility of a viral etiology in lupus.39 Recently, overexpression of genes regulated by IFN were noted by microarray analysis of lupus PBL.40,41 Interestingly, expression of type I IFN is enhanced by ROI.42-45 Microarray studies also indicated overexpression of cytokines, cytokine receptors,46 bcl-2, superoxide dismutase,41 apoptosis mediators TNFR6 (Fas/CD95), TRAIL,40 TRAIL DR3, and TRAIL DR4,47 all consistent with oxidative stress in SLE. Interestingly, NADH dehydrogenase/complex-I of the electron transport chain was found to be downregulated in SLE.48

Expression of TCR? chain is sensitive to oxidative stress49 and thus increased ROI levels may explain, at least in part, low TCR? chain expression in lupus T cells.50 Cell surface expression of the Fas receptor51-53 and Fas ligand is also redox sensitive.54 Thus, higher ROI levels may lead to increased IL-10 production, release of FasL, and overexpression of the FasR in SLE.55-57 Mitochondrial ROI production and ??m are early checkpoints in Fas-19 and H2O2-induced apoptosis.21 Increased ROI levels confer sensitivity to H2O2-, NO-, TNFa-, or Fas-induced cell death.18 Enhanced NO production may also contribute to increased spontaneous apoptosis.58,59 Therefore, elevated baseline ROI and ??m levels may have key roles in abnormal T-cell activation and apoptosis in patients with SLE. Indeed, MHP could represent a novel target of pharmacological intervention in patients with SLE.60-62

REDOX CONTROL OF T-CELL ACTIVATION AND APOPTOSIS SIGNAL PROCESSING ROI modulate T-cell activation, cytokine production, and proliferation at multiple levels.32 The antigenbinding αβ or γδTCR is associated with a multimeric receptor module comprised of the CD3γδe and TCRζ chains. The cytoplasmic domains of CD3 and δ chains contain a common motif termed the immunoglobulin receptor family tyrosine-based activation motif (ITAM), which is crucial for coupling of intracellular tyrosine kinases.63 Expression of TCRζ chain is suppressed by ROI.49 Binding of p56lck to CD4 or CD8 attracts this kinase to the TCRζ-CD3 complex, leading to phosphorylation of ITAM. Phosphorylation of both tyrosines of each ITAM is required for SH-2-mediated binding by zeta-associated protein-70 (ZAP-70) or the related SYK. ZAP-70 is activated through phosphorylation by p56lck. Activated ZAP-70 and SYK target the two key adaptor proteins LAT and SLP-76.63 Oxidative stress reduces phosphorylation and displacement of LAT from the cell membrane, causing T-cell hyporesponsiveness.64 Phosphorylated LAT binds directly to phospholipase C-γ1, which controls hydrolysis of phosphatydilinositol-4,5-biphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Phosphorylation of inositol lipid second messengers is mediated by phosphatidylinositol 3’hydroxyl kinase (PI3K). The stimulatory effect of the TCR alone on PI3K activity is small. Concurrent triggering of the CD28 co-stimulatory molecule by its ligands CD80 or CD86 is required for optimal PI3K activation. IP3 binds to its receptors in the endoplasmic reticulum, opening Ca2+ channels that release Ca2+ to the cytosol. Increased cytosolic Ca2+ concentration activates the phosphatase calcineurin, which dephosphorylates the transcription factor NFAT. Dephosphorylated NFAT can translocate to the nucleus, where it promotes

triggered by activation of the CD3-CD28 complex7 or stimulation with Con A,19 IL-10, IL-3, IFN-γ, or TGFβ.8 Therefore, MHP represents an early but reversible switch not exclusively associated with apoptosis. MHP is a likely cause of increased ROI production12 and may be ultimately responsible for increased susceptibility to apoptosis following T-cell activation.7 MHP in T lymphocytes is associated with a dramatic increase, more than sixfold, of NO production lasting 24 hours after CD3-CD28 co-stimulation. Molecular ordering of T-cell activation-induced NO production revealed critical roles for ROI production and cytoplasmic and mitochondrial Ca2+ influx.28 CD3-CD28 co-stimulation-induced ROI production (similar to H2O2) enhances expression of NOS isoforms eNOS and nNOS, which require elevated Ca2+ levels for enzymatic activity. These results suggest that T-cell activation-induced ROI and Ca2+ signals contribute to NO production, with the latter representing a final and dominant step in MHP. Proteins of the Bcl-2 family are localized to membranes of distinct organelles, including mitochondria.71 Both the proapoptotic (Bax, Bad) and antiapoptotic (Bcl-2, Bcl-XL) members of the family can form ionconducting channels in lipid membranes.70 Bax can create a channel in the outer mitochondrial membrane, thus releasing cytochrome c and other caspase-activating moieties into the cytosol. Bcl-2 and Bcl-XL inhibit this process through dimerization with Bad or Bax. Bcl-2 expression appears to be unaltered in lupus PBL.72 The mitochondrion is the site of ATP synthesis via oxidative phosphorylation. The synthesis of ATP is driven by an electrochemical gradient across the inner mitochondrial membrane maintained by an electron transport chain and the membrane potential. Activity of caspases require ATP to the extent that depletion of ATP by inhibition of F0F1-ATPase with oligomycin73 or exhaustion of intracellular ATP stores by prior apoptosis signals, Fas stimulation,73 or H2O2 pretreatment leads to necrosis.74 Thus, intracellular ATP concentration is a key switch in the cell’s decision to die via apoptosis or necrosis.73

MITOCHONDRIAL CHECKPOINTS OF CELL DEATH PATHWAY SELECTION: ΔψM, ATP SYNTHESIS, ROI AND NO PRODUCTION, CA2+ FLUXING, AND REDUCING CAPACITY PROVIDED BY GSH AND NADPH

Coordinate MHP and ATP depletion play key roles in abnormal T-cell death in lupus patients.7 Δψm and ROI levels as well as cytoplamic pH are elevated in patients with SLE in comparison to healthy or rheumatoid arthritis controls.7,8 Baseline MHP and ROI levels correlated with diminished GSH levels, suggesting increased utilization of reducing equivalents in patients with SLE. It is presently unclear whether synthesis of

MHP appears to be the earliest change associated with several apoptosis pathways.10,20 Elevation of Δψm is also

MHP, INCREASED MITOCHONDRIAL MASS, ROI PRODUCTION, CYTOPLASMIC ALKALINIZATION, AND ATP DEPLETION IN LUPUS T CELLS

transcription of IL-2 in concert with AP-1, NF?B, and Oct-1. Whereas activities of AP-1 and NF?B are increased by oxidative stress,65 both thiol insufficiency and H2O2 treatment suppress calcineurin-mediated activation of NFAT.66 Thus, expression of cytokines [i.e., IL-2 (with AP-1 and NFAT motif-containing promoter) and IL-4 (with AP-1-less NFAT enhancer)] can be selectively regulated by oxidative stress (depending on the relative expression level of transcription factors involved).10 Programmed cell death (PCD) or apoptosis is a physiologic mechanism for elimination of autoreactive lymphocytes during development. Signaling through the Fas or structurally related TNF family of cell surface death receptors has emerged as a major pathway in elimination of unwanted cells under physiologic and disease conditions.67 Fas and TNF receptors mediate cell death via cytoplasmic death domains (DD) shared by both receptors.68 They trigger sequential activation of caspases, resulting in cleavage of cellular substrates and the characteristic morphologic and biochemical changes of apoptosis.69 Disruption of the mitochondrial membrane potential (Δψm) has been proposed as the point of no return in apoptotic signaling that leads to caspase activation and disassembly of the cell.14 Interestingly, MHP and ROI production precede disruption of Δψm, activation of caspases, and phosphatidylserine (PS) externalization in Fas-,19 TNFa-24 and H2O2-induced apoptosis of Jurkat human leukemia T cells and normal human peripheral blood lymphocytes.21 Elevation of Δψm is independent of activation of caspases and represents an early event in apoptosis.19 Pretreatment with caspase inhibitors completely abrogated Fas-induced PS externalization, indicating that activation of caspase-3, caspase-8, and related cysteine proteases were absolutely required for cell death.19 ROI levels were partially inhibited in caspase inhibitor-treated Jurkat cells, suggesting that caspase-3 activation (perhaps through damage of mitochondrial membrane integrity) contributes to ROI production and serves as a positive feedback loop at later stages of the apoptotic process. Cleavage of cytosolic Bid by caspase-8 generates a p15 carboxyterminal fragment that translocates to mitochondria. This may increase mitochondrial membrane permeability and lead to secondary elevation of ROI levels in the Fas and TNF pathway.70

MHP, INCREASED MITOCHONDRIAL MASS, ROI PRODUCTION, CYTOPLASMIC ALKALINIZATION, AND ATP DEPLETION IN LUPUS T CELLS

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TABLE 25.1 REDOX SIGNALING ABNORMALITIES IN T CELLS OF PATIENTS WITH SLE Signal

Effect

Reference

Δψm ↑

ROI ↑, ATP ↓

(7)

ROI ↑

Spontaneous apoptosis ↑, IL-10 production ↑

(7, 8)

GSH ↓

ROI ↑, spontaneous apoptosis ↑

(7, 18)

Spontaneous apoptosis ↑

Compartmentalized autoantigen release, disease activity ↑

(5, 7, 11)

H2O2

Apoptosis ↓, necrosis ↑

(7)

CD3 and CD28

AICD ↓, necrosis ↑

(8)

ATP ↓

Predisposes for necrosis

(7, 73)

Necrosis ↑

Inflammation ↑

(7, 80)

AICD ↓

Persistence of autoreactive cells

(6, 8)

FasR ↑

Spontaneous apoptosis ↑

(57)

FasL ↑

Spontaneous apoptosis ↑

(57)

IL-10 ↑

Selective induction of apoptosis in SLE

NO ↑

2+

(8, 57, 93)

Mitochondrial biogenesis, altered Ca fluxing

(58, 92)

IL-10 blockade

Spontaneous apoptosis ↓, ROI ↓

(8, 57)

IL-12

Spontaneous apoptosis ↓, ROI ↓

(8)

↑ = increase; ↓ = decrease.

GSH or its regeneration from its oxidized form is deficient in lupus patients. GSH is also required for interleukin-2-dependent T-cell proliferation31 as well as CD2- and CD3-mediated T-cell activation.32 Thus, low GSH content may also inhibit CD3-induced H2O2 production. Nevertheless, GSH deficiency predisposes for ROI-induced cell death.18,21 Diminished H2O2induced apoptosis of cells with low baseline GSH levels indicate a severe dysfunction of redox signaling in patients with SLE.7 Increased ROI production may lead to skewed expression of redox-sensitive surface receptors and lymphokines in SLE (Table 25.1). As examples, ROIs regulate gene transcription and release of TNFa and interleukin-10,34 both of which are elevated in sera35 and freshly isolated PBL of SLE patients.37 Expression of TCR? chain is sensitive to oxidative stress,49 and thus increased ROI levels may in part explain low TCR? chain expression in lupus T cells.50 Cell surface expression of the Fas receptor51 and ligand is also redox sensitive.54 Increased ROI levels may be related to increased IL-10 production, release of FasL, and overexpression of the FasR in SLE.57 Elevated NO production may also contribute to increased spontaneous apoptosis.59 Increased ROI levels confer sensitivity to H2O2, NO, TNFa, and Fas-induced cell death.18 Therefore, persistent MHP [causing increased ROI production (a trigger of apoptosis) and depletion of ATP (required for AICD)] may be responsible for the

paradox of increased spontaneos apoptosis and diminished AICD in SLE.

MHP AND ATP DEPLETION PREDISPOSE LUPUS T CELLS TO NECROSIS In response to treatment with exogenous H2O2, a precursor of ROI, lupus T cells failed to undergo apoptosis and cell death preferentially occurred via necrosis. Endogenous H2O2 is generated by superoxide dismutase from ROIs, O2-, or OH- in mitochondria.75 In turn, H2O2 is scavenged by catalase and glutathione peroxidase.17 Although H2O2 is freely diffusible, it has no unpaired electrons and by itself is not an ROI.75 Induction of apoptosis by H2O2 requires mitochondrial transformation into an ROI (e.g., OH-) through the Fenton reaction.12,75 As previously noted,21 H2O2 triggered a rapid increase of Δψm and ROI production that was followed by apoptosis of PBL in healthy subjects. By contrast, H2O2 failed to elevate Δψm, ROI production, and apoptosis but rather elicited necrosis of lupus T cells. Both CD3-CD28-induced H2O2 production and H2O2-induced apoptosis require mitochondrial ROI production. Therefore, diminished CD3-CD28-induced H2O2 production and H2O2-induced apoptosis together with deficient elevation of Δψm and ROI levels reveal deviations of key biochemical checkpoints in mitochondria of patients with SLE.

Indeed, lymphocyte necrosis occurs in the bone marrow79 and lymph nodes of lupus patients and may significantly contribute to the inflammatory process.80

INCREASED NECROSIS PROMOTES PRO-INFLAMMATORY STATE IN PATIENTS WITH SLE Swollen lymph nodes of patients with SLE harbor increased numbers of necrotic T lymphocytes and dendritic cells (DCs).81 Necrotic, but not apoptotic, cell death generates inflammatory signals necessary for the activation and maturation of DCs, the most potent antigen-presenting cells.82-84 High-mobility group B1 (HMGB1) protein, an abundant DNA-binding protein, remains immobilized on chromatin of apoptotic bodies. However, it is released from necrotic cells.85 HMGB1 stimulates human monocytes to release TNFa, IL-1a, IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1a, and MIP-1β (but not IL-10 or IL-12)86 and induces arthritis.87 Necrotic but not apoptotic cells also release heat shock proteins (HSPs), HSPgp96, hsp90, hsp70, and calreticulin. HSPs stimulate macrophages to secrete cytokines and to induce expression of antigen-presenting and co-stimulatory molecules on the DCs.83 Mature DCs express high levels of the DC-restricted markers, CD83 and lysosome-associated membrane glycoprotein (DC-LAMP), and the co-stimulatory molecules CD40 and CD86,84 which may contribute to altered intercellular signaling in SLE.10 CD14+ monocytes isolated from the blood of lupus patients, but not those from healthy individuals, act as DCs.88 Their activation is driven by circulating interferon-a (IFN-a) that may come from one of the DC subsets [i.e., plasmacytoid dendritic cells (PDC) that infiltrate lupus skin lesions]. Tissue lesions89,90 and blood of patients with SLE harbor activated PDC, which may be responsible for increased production of IFN-a in SLE.88,91

MITOCHONDRIAL TARGETS OF THERAPEUTIC INTERVENTION IN SLE

The mitochondrial transmembrane potential (Δψm, negative inside and positive outside) is dependent on the electron transport chain transferring electrons from NADH to molecular oxygen and on proton transport mediated by the F0F1-ATPase complex.12 During oxidative phosphorylation, the F0F1-ATPase converts ADP to ATP utilizing the energy stored in the electrochemical gradient. Alternatively, using the energy of ATP hydrolysis F0F1-ATPase can pump protons out of the mitochondrial matrix into the intermembrane space (causing Δψm elevation). Thus, MHP may occur in several ways. First, deficiency of cellular ADP could cause diminished utilization of the electrochemical gradient, ATP depletion, and MHP. However, ADP levels were not diminished but slightly elevated in lupus PBL.7 This suggested that ATP depletion and Δψm hyperpolarization were not due to a lack of ADP in patients with SLE. Second, MHP may occur through calciumactivated dephosphorylation of cytochrome c oxidase.76 Phosphorylation of cytochrome c oxidase is mediated by protein kinase A (PKA). Thus, deficiency of PKA may also contribute to MHP in SLE.77 Third, inhibition of the enzymatic activity of F0F1-ATPase would decrease utilization of the electrochemical gradient and cause Δψm hyperpolarization, ATP depletion, and ADP accumulation. Because blocking of F0F1-ATPase by oligomycin led to Δψm hyperpolarization and elevated ROI production, prevented H2O2- or CD3-CD28-induced elevation of Δψm in normal PBL, and sensitized to H2O2-induced necrosis, a similar mechanism may also be operational in patients with SLE.7 With Δψm hyperpolarization and extrusion of H+ ions from the mitochondrial matrix, the cytochromes within the electron transport chain become more reduced (which favors generation of ROI).12 Thus, MHP is a likely cause of increased ROI production and may be ultimately responsible for increased spontaneous apoptosis in patients with SLE. A 28 to 32% increase of the -200 mV Δψm may have a tremendous impact on mitochondrial energy coupling and ATP synthesis.12 Both T-cell activation and apoptosis require the energy provided by ATP.78 Intracellular ATP concentration is a key switch in the cell’s decision to die via apoptosis or necrosis,73 and therefore depletion of ATP may be responsible for defective H2O2-induced apoptosis and a shift to necrosis in patients with SLE. Apoptosis is a physiologic process that results in nuclear condensation and breakup of the cell into membrane-enclosed apoptotic bodies suitable for phagocytosis by macrophages (thus preventing inflammation). By contrast, necrosis is a pathologic process that results in cellular swelling, followed by lysis and release of proteases, oxidizing molecules, and other pro-inflammatory and chemotactic factors resulting in inflammation and tissue damage.78

MITOCHONDRIAL TARGETS OF THERAPEUTIC INTERVENTION IN SLE MHP represents an early but reversible checkpoint associated with activation and apoptosis of human T lymphocytes. Although Ca2+-, ROI-, and NADPHdependent production of NO appears to be a dominant factor in T-cell activation-induced MHP, relative impact and hierarchy of the metabolic and redox signaling pathways involved requires further study. The chemical composition of ROI (i.e., OH-, O2-, ONOO-, and ONOOH) and their compartmentalization during T-cell activation and cell death are unexplored. Selective targeting of ROI may prove valuable in regulating T helper cell differentiation and cytokine

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production, activation of cytotoxic T cells, and cell death pathway selection. Although MHP was not affected, IL-10 antibody or IL-12 normalized ROI production and intracellular alkalinization in lupus PBL.8 Therefore, IL-10 antagonists may partially correct redox signaling dysfunction in lupus. Bz-423, an experimetal drug binding to benzodiazepine receptor in mitochondria, was found to reduce Δψm, induce selective death of autoreactive lymphocytes, and improve clinical outcome of lupus in two different murine models.60,61 NO production is increased in patients with SLE,58 and it may play a key

role in persistent MHP, increased mitochondrial biogenesis, and enhanced mitochondrial Ca2+ fluxing and T-cell dysfunction.92 Precise delineation of the mechanism of MHP and ATP depletion may identify novel targets for pharmacologic intervention in patients with SLE.

ACKNOWLEDGMENTS This work was supported in part by grants RO1 AI 48079 and AI 61066 from the National Institutes of Health and the Central New York Community Foundation.

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49. Otsuji M, Kimura Y, Aoe T, Okamoto Y, Saito T. 1996. Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 zeta chain of T-cell receptor complex and antigen-specific T-cell responses. Proc Natl Acad Sci USA 1996;93:13119-13124. 50. Liossis SN, Ding XZ, Dennis GJ, Tsokos GC. Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus. Deficient expression of the T cell receptor zeta chain. J Clin Invest 1998;101:1448-1457. 51. Li D, Yang B, Mehta JL. Ox-LDL induces apoptosis in human coronary artery endothelial cells: Role of PKC, PTK, bcl-2, and Fas. Am J Physiol 1998;275:H568-H576. 52. Orlinick JR, Vaishnaw A, Elkon KB, Chao MV. Requirement of cysteine-rich repeats of the Fas receptor for binding of the Fas ligand. J Biol Chem 1997;272:28889-28894. 53. Bennett M, Macdonald K, Chan S-W, Luzio JP, Simari R, Weissberg P. Cell surface trafficking of Fas: A rapid mechanism of p53-induced apoptosis. Science 1998;282:290-293. 54. Kasibhatla S, Genestier L, Green DR. Regulation of Fas ligand expression during activation-induced cell death in T lymphocytes. J Biol Chem 1999;274:987-992. 55. He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR. Human immunodeficiency virus type 1 viral protein R (vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 1995;69:6705-6711. 56. Lorenz HM, Grunke M, Hieronymus T, Herrmann M, Kuhnel A, Manger B. In vitro apoptosis and expression of apoptosisrelated molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases. Arth Rheum 1997;40:306-317. 57. Georgescu L, Vakkalanka RK, Elkon KB, Crow MK. Interleukin-10 promotes activation-induced cell death of SLE lymphocytes mediated by Fas ligand. J Clin Invest 1997;100:2622-2633. 58. Oates JC, Christensen EF, Reilly CM, Self SE, Gilkeson GS. Prospective measure of serum 3-nitrotyrosine levels in systemic lupus erythematosus: Correlation with disease activity. Proc Assoc Am Phys 1999;111:611-621. 59. Cooper GS, Dooley MA, Treadwell EL, St. Clair EW, Parks CG, Gilkeson GS. Hormonal, environmental, and infectious risk factors for developing systemic lupus erythematosus. Arth Rheum 1998;41:1714-1724. 60. Blatt NB, Bednarski JJ, Warner RE, Leonetti F, Johnson KM, Boitano A, et al. Benzodiazepine-induced superoxide signals B cell apoptosis: Mechanistic insight and potential therapeutic utility. J Clin Invest 2002;110:1123-1132. 61. Bednarski JJ, Warner RE, Rao T, Leonetti F, Yung R, Richardson BC, et al. Attenuation of autoimmune disease in Fas-deficient mice by treatment with a cytotoxic benzodiazepine. Arth Rheum 2003;48:757-766. 62. Habeck M. Benzodiazepine library yields lupus target. Drug Discovery Today 2002;7:1193-1194. 63. Koretzky GA, Boerth NJ. The role of adapter proteins in T cell activation. Cell Mol Life Sci 1999;56:1048-1060. 64. Gringhuis SI, Leow A, Papendrecht-Van Der Voort EA, Remans PH, Breedveld FC, Verweij CL. Displacement of linker for activation of T cells from the plasma membrane due to redox balance alterations results in hyporesponsiveness of synovial fluid T lymphocytes in rheumatoid arthritis. J Immunol 2000; 164:2170-2179. 65. Beiqing L, Chen M, Whisler RL. Sublethal levels of oxidative stress stimulate transcriptional activation of c-jun and suppress IL-2 promoter activation in Jurkat T cells. J Immunol 1996; 157:160-169. 66. Furuke K, Shiraishi M, Mostowski HS, Bloom ET. Fas ligand induction in human NK cells is regulated by redox through a calcineurinnuclear factors of activated T cell-dependent pathway. J Immunol 1999;162:1988-1993. 67. Nagata S. Apoptosis by death factor. Cell 1997;88:355-365. 68. Itoh N, Nagata S. A novel protein domain required for apoptosis: Mutational analysis of human Fas antigen. J Biol Chem 1993; 268:10932-10937. 69. Martin SJ, Green DR. Protease activation during apoptosis: Death by a thousand cuts. Cell 1995;82:349-352. 70. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13:1899-1911.

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71. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-1312. 72. Rose LM, Latchman DS, Isenberg DA. Bcl-2 expression is unaltered in unfractionated peripheral blood mononuclear cells in patients with systemic lupus erythematosus. Brit J Rheumatol 1995;34:316-320. 73. Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P. 1997. Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis. J Exp Med 185:1481-1486. 74. Lee Y, Shacter E. Oxidative stress inhibits apoptosis in human lymphoma cells. J Biol Chem 1999;274:19792-19798. 75. Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Meth Enzymol 1990;186:1-85. 76. Kadenbach B. Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochimi Biophys Acta 2003;1604: 77-94. 77. Kammer GM, Perl A, Richardson BC, Tsokos GC. Abnormal T-cell signal transduction in systemic lupus erythematosus. Arth Rheum 2002;46:1139-1154. 78. Fiers W, Beyaert R, Declercq W, Vandenabeele P. More than one way to die: Apoptosis, necrosis and reactive oxygen damage. Oncogene 1999;18:7719-7730. 79. Lorand-Metze I, Carvalho MA, Costallat LT. Morphology of bone marrow in systemic lupus erythematosus. Pathologe 1994;15: 292-296. 80. Eisner MD, Amory J, Mullaney B, Tierney L Jr., Browner WS. Necrotizing lymphadenitis associated with systemic lupus erythematosus. Semin Arth Rheum 1996;26:477-482. 81. Kojima M, S. Nakamura S, Y. Morishita Y, H. Itoh H, K. Yoshida K, Y. Ohno Y, et al. Reactive follicular hyperplasia in the lymph node lesions from systemic lupus erythematosus patients: A clinicopathological and immunohistological study of 21 cases. Pathol Internat 2000;50:304-312. 82. Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: Endogenous activators of dendritic cells. Nature Med 1999; 5:1249-1255. 83. Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK. Necrotic but not apoptotic cell death releases heat shock proteins, which

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MECHANISMS OF TISSUE DAMAGE

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Nitric Oxide in Systemic Lupus Erythematosus James C. Oates, MD and Gary S. Gilkeson, MD

INTRODUCTION Whereas acquired autoimmunity is the sine qua non of lupus, the innate immune system plays an integral role in promulgating inflammatory responses and tissue destruction. An integral part of that innate immune response is the production of reactive nitrogen and oxygen intermediates (RNI and ROI). One of the most widely studied RNI, nitric oxide (NO), is overproduced in the setting of lupus activity. Its pathogenic potential in lupus or any other disease lies largely in the extent of its production and the proximity of its synthesis to ROI, such as superoxide (SO). NO and SO react to form peroxynitrite (ONOO1), a much more reactive and potentially pathogenic molecule. There is convincing evidence in murine lupus nephritis that inducible nitric oxide synthase (iNOS) activity increases with the progression of disease and leads to glomerular, joint, and dermal pathology. In addition, ONOO1-mediated modifications of proteins and DNA may increase the immunogenicity of these self-antigens, leading to a break in immune tolerance. In humans, there are observational data suggesting that overexpression of iNOS and increased production of ONOO1 leads to glomerular and vascular pathology. Therapies designed to target iNOS activity or scavenge ROI/RNI have not been tested in humans in part due to concerns over the specificity of many available compounds for their targets. However, several new compounds are in development that offer promise for human trials in the near future.

REACTIVE SPECIES IN INNATE IMMUNITY Free radicals are highly reactive molecules with one or more unpaired electrons that are important to the innate immune response. In systemic lupus erythematosus (SLE), there is evidence to support the notion that overproduction of free radicals in the absence of infection leads to a break in immune tolerance, increased tissue damage, and altered enzyme function. For the purposes of this chapter, the discussion of reactive species is confined to a select group of reactive oxygen

and nitrogen free radicals. Examples of ROI include SO, hydrogen peroxide, and hydroxyl radicals, whereas NO and ONOO1 are the RNI to be discussed. Reactive oxygen and nitrogen intermediates (RONI) play an important role in cellular signaling processes when produced at low levels. At higher levels, these molecules can cause direct toxicity to cells and induce modifications to lipids, amino acids, RNA, and DNA. NO is a membrane-permeable free radical molecule derived from arginine through the catalytic activity of nitric oxide synthase (NOS). There are three isoforms of NOS that are transcribed from three separate genes. All isoforms require dimerization of identical monomers to become active. Each monomer contains a reductase and oxygenase domain. The reductase domain, with assistance from calmodulin, catalyzes the transfer of two electrons from the electron donor nicotinamide adenine dinucleotide phosphate (NADPH), through flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), to heme iron in the oxygenase domain. Heme and tetrahydrobiopterin (BH4) interact in the oxygenase domain to catalyze a reaction of O2 and L-arginine to form NO and citrulline (Fig. 26.1). Two isoforms (endothelial or eNOS and neuronal or nNOS) are generally constitutively expressed and are dependent on sufficient concentrations of calcium for activity. In the vascular system, NO (once termed endothelium-derived relaxing factor, or EDRF) produced by eNOS is a potent vasodilator and regulator of vascular tone in response to shear stress. Nitroglycerin mimics the activity of eNOS by acting as a donor of NO.1 The beneficial effect of NO produced by the constitutively expressed NOS isoforms is blunted when NO is produced in or near cells producing high levels of ROI (as discussed later in the chapter). A third NOS gene (NOS2) produces an inducible isoform (termed iNOS) that is primarily found in immune cells, most notably macrophages and macrophage-derived cells. iNOS is expressed in response to inflammatory stimuli that are well characterized in murine cells. iNOS is expressed during pathologic states in human endothelial cells,

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NITRIC OXIDE IN SYSTEMIC LUPUS ERYTHEMATOSUS

NADP+ + H+

NADPH

e– FAD and FMN

Reductase

e–

Calmodulin calcium

Oxygenase

Heme Fe

BH4

e–

Arginine + O2

Citrulline + NO

Fig. 26.1 NO synthesis from arginine by iNOS and cofactors. Electrons (e-) are donated by NADPH to FAD and FMN in the reductase domain. This step requires Ca2+ (much higher levels for eNOS and nNOS than for iNOS) and calmodulin. Two cycles of electrons are then transferred by these carriers to heme iron in the oxygenase domain of the adjacent dimmer. This reaction is similar to that in P450 enzymes. The role of tetrahydrobiopterin (BH4) in this process is unclear, but it may assist in the coupling of NADPH oxidation and NO formation, thus preventing SO formation. With arginine and O2 as subtrates, donated electrons then catalyze two reaction steps, the formation of Nω-hydroxyL-arginine (NHA) followed by conversion of NHA to NO and citrulline.1

302

synovial fibroblasts, polymorphonuclear cells, lymphocytes, and natural killer cells.2 In normal human tissue, expression is strong in myocytes, skeletal muscle, and Purkinje cells.3 iNOS produces log-fold higher amounts of NO than the constitutively expressed isoforms and produces SO via the reductase domain when arginine substrate is less abundant.4 NO (when combined with SO) forms peroxynitrite (ONOO1), a more reactive and toxic molecule than NO itself. ONOO1 produced by immune cells is capable of killing intracellular pathogens and tumors cells. Glutathione peroxidase, catalase, superoxide dismutase, and antioxidants serve to protect host cells during inflammatory states by reducing the total free radical burden.5 iNOS aids in microbial defense during certain types of infection. Mice that lack the iNOS (NOS2) gene were less capable of inhibiting the growth of pathogens such as Mycobacterium tuberculosis6 and developed more significant clinical sequelae of infection when challenged with coxsackievirus.7 iNOS inhibitor therapy increased the clinical severity of infections in mice challenged with pathogens such as HSV-18 and Plasmodium chabaudi.9

Certain polymorphisms of the NOS2 promoter were studied in young West African Ghanian children with Plasmodium falciparum infection. These polymorphisms were associated with high plasma levels of NO metabolites, lower levels of parasitemia, and a milder disease course in this population.10 One of these polymorphisms has been associated with lower rates of malarial attacks in patients from Gabon and higher levels of iNOS activity in vitro,11 an effect not seen in East African Tanzanian subjects.12 Whereas chronic expression of ONOO1 may lead to tumor formation by inducing chronic DNA damage, NO and ONOO1 also have acute cytostatic, cytotoxic and pro-apoptotic effects on tumor cells.13 Whether similar NOS2 polymorphisms lead to suppression of tumor growth is not known. NO has the potential to induce both physiologic and pathologic effects, a dichotomy that pervades the literature. The ability of NO to induce cellular pathology is largely dependent on its conversion to more reactive nitrogen species such as ONOO1. In turn, the production of ONOO1 is dependent on levels of SO in the cellular microenvironment in which NO is released. The following will serve as an example of this concept. When NO is produced in proximity to mitochondria in high redox states, it can react with SO to form ONOO−. This molecule can induce apoptosis of that cell via cytochrome c-mediated caspase activation. Mitochondria in this state can be found in activated T cells of lupus subjects more frequently than in healthy controls.14 Alternatively, if NO is produced in low levels in the absence of ROI it promotes cell survival.15 Thus, the fate of NO and its ultimate pathogenicity depends on levels of oxygen versus SO in its immediate cellular milieu (Fig. 26.2).

NITRIC OXIDE BIOLOGY IN MURINE MODELS OF LUPUS

Observational Studies Although iNOS activity may have beneficial effects in the setting of parasitemia or tumor growth, its overexpression in the setting of lupus disease activity appears to lead to organ damage and an altered immune response. Several studies involving murine models of lupus support this concept. Both MRL/MpJ-Faslpr/J (MRL/lpr) and (New Zealand Black × New Zealand White)F1 (NZB/W) mice develop spontaneous proliferative lupus nephritis. MRL/lpr mice developed increasing levels of urine NO metabolites (nitrate + nitrite or NOX) in parallel with clinical expression of glomerulonephritis.16 This increase in iNOS activity was associated with post-translational modifications of proteins, specifically nitration of tyrosines (Tyr) to form 3-nitrotyrosine (3NTyr). Such modifications

NO

O2 –

NO

Intracellular redox gradient

NO2

ONOO –

O2 NO3 Fig. 26.2 Cellular microenvironment dictates the fate of NO. After synthesis by iNOS, eNOS, or nNOS, NO freely diffuses across membranes, forming a concentration gradient. Within this microenvironment also exists a redox gradient (represented by the bacl rectangle (formed by the presence of oxidant/reductant-coupled species. The redox state thus determines whether NO ultimately forms what are usually benign vs. pathogenix reactive nitrogen species (RNS). For instance, when formed in the presence of O2, NO can oxidize NO2 and NO3. Wherease when formed in the presence of superoxide (SO or O2−), NO oxidizes to form peroxynitrite (ONOO−).15

reduced the activity of catalase in the MRL/lpr kidney. Because catalase removes superoxide, its inactivation may expose cells to increased oxidative stress and accelerate tissue damage or modification.17 Immune complex formation and tissue deposition appear to be proximal to increased iNOS activity in murine lupus. Supporting this hypothesis is the observation that iNOS inhibitor therapy, although improving renal histopathology, had no effect on glomerular immune complex deposition in MRL/lpr mice.16 Autoantibodies increase markers of iNOS activity (3NTyr formation) in other antibody-mediated autoimmune diseases as well. For example, serum 3NTyr levels were increased after implantation of human β2-glycoprotein I antibody-producing hybridomas into mice with severe combined immunodeficiency syndrome.18 A similar link between autoantibody deposition and 3NTyr formation has been observed in anti-glomerular basement membrane (GBM) and myeloperoxidase (MPO) antibody models of glomerulonephritis. In both models, autoantibodies were passively transferred to disease-free mice. This intervention was followed by up-regulation of iNOS protein and formation of 3NTyr in glomerular tissue.19-21

Manipulation of iNOS in Murine Lupus Several inhibitor studies suggest that iNOS activity is pathogenic in murine lupus. Blocking iNOS activity in MRL/lpr mice before disease onset with the nonspecific arginine analog L-NG-monomethyl-L-arginine (L-NMMA) reduced 3NTyr formation in the kidney,

partially restored renal catalase activity, and inhibited cellular proliferation and necrosis within the glomerulus.16,17,22 This effect occurred in the absence of a change in immunoglobulin or complement deposition in the glomerulus, suggesting that increased iNOS expression occurred after immune complex deposition and complement activation.16 These results were confirmed using the partially selective iNOS inhibitor l-N6(1-iminoethyl)lysine (L-NIL) to treat mice prior to disease onset. In the L-NIL-treated mice, glomerular histopathology was significantly improved over controls and slightly better than in L-NMMA-treated mice. However, proteinuria was only partially inhibited in the L-NIL-treated mice, whereas L-NMMAtreated mice developed no significant proteinuria. L-NMMA therapy in NZB/W mice that were already expressing clinical nephritis had a similar but less profound effect on proteinuria and renal histopathology than did preventative therapy. However, L-NMMA as monotherapy for the treatment of active disease was less effective in the rapidly progressive MRL/lpr model.23 In conflict with the effectiveness of pharmacologic iNOS inhibition in murine lupus is the observation that iNOS-/- MRL/lpr mice, although having reduced signs of vasculitis and IgG rheumatoid factor production, had similar glomerular pathology in their MRL/lpr wild-type litter mates.24 The mechanisms behind the discrepancy of treatment response between pharmacologic and genetic manipulation of iNOS activity are still under investigation. One possibility is that inhibition of iNOS with arginine analogs may reduce pathology through non-iNOS-mediated mechanisms. For instance, L-NMMA may inhibit the y+ amino acid transporter system that translocates L-arginine substrate into the cytoplasm. Both L-arginine and L-NMMA were transported into activated RAW 264.7 macrophages via the same y+ transporter—an effect not seen in resting macrophages, suggesting that this transporter system is inducible. The transport of both molecules was inhibited by leucine.25 Whether L-NMMA inhibits transport of L-arginine in a similar manner is now known. Thus, intra- and extracellular concentrations of L-arginine may vary according to the presence of other co-transported amino acid derivatives and the level of activation of the transporter system. Supporting the importance of L-arginine availability as a rate-limiting step in NO synthesis in lupus is a study in which supplementation with L-arginine increased renal fibrosis in the MRL/lpr model. Another study dampened enthusiasm for targeting L-arginine by demonstrating no clinical or histopathologic improvement in renal disease with an L-arginine-free diet in MRL/lpr mice.23

NITRIC OXIDE BIOLOGY IN MURINE MODELS OF LUPUS

O2

303

NITRIC OXIDE IN SYSTEMIC LUPUS ERYTHEMATOSUS

Some interventions that do not directly inhibit iNOS activity may derive additional benefit by reducing expression of iNOS. For instance, chemical induction of heme oxygenase-1 and oral administration of mycophenolate mofetil are both effective therapies for treating glomerulonephritis in MRL/lpr mice (and both reduce iNOS expression in the kidney).26-28 In contrast, cyclophosphamide therapy increases 3NTyr formation in the setting of rodent models of bone marrow transplantation29 and cyclophosphamide-mediated bladder toxicity.30 Although the data regarding NOX formation in the setting of cyclophosphamide therapy are mixed, it is clear that oxidant stress is increased with its administration.31 This increased oxidative state can change the fate of NO from NOX to more toxic ONOO1. Thus, reductions in iNOS expression and ONOO1 production with MMF therapy may provide an additional therapeutic benefit over cyclophosphamide therapy for the treatment of lupus nephritis. This theory has not been tested in a rigorous fashion, however.

Potential Mechanisms for Pathogenicity of RNI in Lupus The mechanisms through which iNOS activity may be pathogenic in SLE and vascular disease have been studied in animal models and in vitro (Table 26.1). See Table 26.1 for examples of the effects of ONOO− on various cellular molecules. As mentioned previously, ONOO1 (a by-product of iNOS activity) can nitrate protein amino acids and reduce the activity of enzymes. One such enzyme is catalase, which serves to protect host tissues from free radical attack.17 In vascular tissue, prostacyclin synthase32 and eNOS33 are inactivated by ONOO1, leading to vasoconstriction. These observations

TABLE 26.1 PATHOGENIC EFFECTS OF ONOO- ON CELLULAR MOLECULES Proteins ●

Nitration of tyrosine residues with reduction in enzyme activity of: 32 ° Endothelial nitric oxide synthase 32 ° Prostacyclin synthase 17 ° Catalase

●

Damage to mitochondrial complex I/II with release of cytochrome c, resulting in apoptosis15

DNA ● ●

Formation of DNA strand breaks75 Nitration, leading to increased immunogenicity37-39

Lipids ●

304

●

Peroxidation of arachidonate to form isoprostanes76 Oxidation of LDL77

suggest that one mechanism through which iNOS activity is pathogenic is via deactivation of tissue-protective enzymes. Increasing attention is focusing on the manner in which immune tolerance is broken by presentation of autoantigens in a novel manner. Two such processes are noteworthy: presentation of nuclear antigens in the pro-inflammatory context of late apoptotic blebs and post-translational modification of self-antigens to form novel epitopes. Because nuclear antigens are presented in late apoptotic blebs,34 regulation of apoptosis and clearance of apoptotic cells is an important area of investigation. NO and ONOO1 are both integral in regulating non-receptor-mediated apoptosis in many cellular systems.15 To investigate the role of iNOS activity in apoptosis of splenocytes in MRL/lpr mice with active disease, iNOS activity was inhibited with L-NMMA. Compared to controls, those mice treated with L-NMMA exhibited reduced levels of splenocyte apoptosis. Treatment of splenocytes isolated from mice with active disease with an NO donor resulted in increased levels of apoptosis in vitro.35 NO or NO oxidative metabolites appeared to increase non-receptor-mediated apoptosis in this model of defective receptor-mediated apoptosis.36 Another mechanism for inducing autoimmunity is via modifications of self-antigens to form novel epitopes. ONOO1 can nitrate self-antigens in a manner that leads to a break in immune tolerance. For instance, normal mice immunized with nitrated IgG produced antinitrotyrosine antibodies that cross-reacted with singlestranded DNA (dsDNA).37 Human native DNA modified with ONOO1 had greater immunogenicity in experimental animals than native DNA without modifications. In two different studies, human anti-dsDNA antibodies produced a greater binding affinity for the ONOO1modified DNA than for unmodified dsDNA.38,39 The literature is rife with reports of the seemingly antithetic properties of NO. As discussed previously, many of the pathologic consequences of NO production arise from its synthesis in the setting of high reactive oxygen stress. NO can diffuse freely across membranes due to its uncharged nature, but it has a half-life of only approximately 30 seconds.2 Thus, iNOS activity can lead to ONOO1 production only if it occurs within or in close proximity to a cell with high reactive oxygen content. One mechanism for production of SO and NO in close proximity is through the parallel production of SO by the reductase domain of iNOS itself. This process has been observed in murine macrophages.4 Support of this mechanism in lupus comes from experiments of pharmacologic inhibition of iNOS in the MRL/lpr and NZB/W models. In these experiments, mice given L-NIL or L-NMMA demonstrated significant reductions in markers of systemic oxidant stress compared to mice treated with distilled water.40

NITRIC OXIDE BIOLOGY IN HUMAN SLE

Observational Studies Although there is compelling evidence for aberrant reactive nitrogen production in the pathogenesis of murine lupus nephritis, human studies provide only observational confirmation. Increased expression of the iNOS enzyme has been reported in multiple tissues among SLE subjects. Several laboratories have described increased expression in the glomeruli of subjects with proliferative lupus nephritis.41-43 In one study,42 glomerular iNOS staining co-localized with markers of apoptosis and staining for p53, a proapoptotic signaling molecule. These data suggest that one mechanism for iNOS-mediated glomerular damage is increased signaling for apoptosis. Immunostaining for iNOS protein and mRNA was elevated in SLE epidermal tissue in 33% of samples from cutaneous lupus subjects before exposure to ultraviolet B irradiation but in all samples after exposure.44 Among subjects with systemic disease, skin biopsy specimens from the buttocks revealed higher iNOS expression in endothelial cells and keratinocytes than in controls. Endothelial expression correlated with lupus disease activity. The presence of iNOS in unaffected skin endothelial cells suggests systemic expression.45 Studies of iNOS tissue expression are generally limited by practical concerns to organs that are frequently or easily biopsied. Therefore, serum and urine markers of systemic NO production have been employed to study RNI in larger SLE populations. In humans, the use of these surrogate markers is complicated by the genetic and dietary heterogeneity of the population and concurrent diseases. Diets high in NOX can dramatically influence the ability to accurately measure systemic

NO production through measures of serum or urine NOX.46 As discussed previously, the fate of NO in vivo (and thus its effects on surrounding or even distant tissues) is highly dependent on local concentrations of ROI. For example, individuals with the insulin resistance syndrome that is marked by increased systemic ROI production47 could be more likely to favor iNOSderived NO oxidation to ONOO1 rather than NOX. With these limitations in mind, several studies have reported increased serum levels of NOX in lupus patients in association with disease activity.43,48-51 In one study, this association was observed among subjects consuming a low NOX diet, reducing dietary sources of NOX as a confounding factor.43 Because ONOO1 has more pathogenic potential than NO itself, assays for 3-nitrotyrosine (3NTyr) were developed to measure the effect of ONOO1 production on serum proteins containing Tyr. In an Australian lupus cohort composed primarily of Caucasian and Asian subjects, serum 3NTyr levels were elevated in comparison to controls, and levels correlated with disease activity. Protein-bound carbonyls, markers of systemic oxidation, were also elevated during disease activity in this population.52 Serum 3NTyr levels correlated with disease activity, particularly renal disease activity, in African-American but not Caucasian SLE subjects in one cohort.43 One possible mechanism for the disparate outcomes in some African Americans with lupus is an increased predisposition toward RNI and ROI production in response to the inflammatory stimuli associated with lupus disease activity.43 Supporting this notion, a study of two NOS2 polymorphisms in African-American female SLE and control subjects revealed a significantly increased prevalence of these polymorphisms in those with SLE.53 The polymorphisms described have been associated with increased NO production and improved malaria survival in some populations in Western Africa.11,54 (See Table 26.2.)

NITRIC OXIDE BIOLOGY IN HUMAN SLE

This observation raises the possibility that some of the pathogenic effects of iNOS activity arise from its ability to produce ROI in proximity to NO.

TABLE 26.2 SELECTIVITY OF VARIOUS COMPOUNDS FOR iNOS AND MECHANISMS OF ACTION Selectivity in vitro (fold) Compound

IC50 for iNOS (μM)

iNOS vs. eNOS

iNOS vs. nNOS

Mechanism of Action

L-NMMAa

6.6

0.5

0.7

Competitive inhibition

1.6

49

23

Competitive inhibition

31

11

5.5

Competitive inhibitionc

1.4

333

104

Competitive inhibition

0.23

>4000

32

Competitive inhibition

0.028

1000

5

Prevents dimerization

L-NIL

a

Aminoguanidine GW274150a a

1400W BBS2

b

a

15

a. Results are from Boyd et al. b. Results are from Blasko et al.67 c. Aminoguanidine has NO-independent anti-inflammatory activities.78

305

NITRIC OXIDE IN SYSTEMIC LUPUS ERYTHEMATOSUS

Reactive Species and Atherosclerosis in SLE The presence of iNOS expression in endothelial cells of unaffected lupus skin45 raises the possibility that endothelial cells in other organs also have increased expression. Such overexpression of iNOS may be one mechanism behind the endothelial dysfunction seen in SLE subjects.55 In the setting of diabetes-related vascular disease, 3NTyr modifications reduced endothelial function by reducing the activity of prostacyclin synthase and eNOS.32 As with glomerular cells, ONOO1 can lead to apoptosis of vascular endothelial cells in vitro.56 In lupus patients, circulating apoptotic endothelial cells have been described in association with abnormal vascular tone as measured by flowmediated dilation.57 Circulating activated endothelial cells, another marker for endothelial dysfunction, were also elevated among lupus subjects with disease activity. Staining for 3NTyr was also elevated in these cells in association with disease activity.58 Recent studies in non-SLE subjects demonstrated increased serum levels of 3NTyr in those with atherosclerotic disease. In this population, 3NTyr levels reduced with statin therapy,59 suggesting a pathogenic role for iNOS and/or myeloperoxidase activity in atherosclerosis that is treatment responsive. These observations offer one possible mechanism for the observed accelerated atherosclerosis in SLE patients.60 One significant risk factor for atherosclerosis among SLE subjects is the presence of circulating antiphospholipid antibodies.61 To study mechanisms behind this observation, mice were injected with human monoclonal antibodies to β2-glycoprotein I or an isotype control antibody. Plasma 3NTyr levels were increased after β2-glycoprotein I antibody exposure. These data further implicate ONOO1 production in endothelial dysfunction.18 Studies evaluating serum 3NTyr as a biomarker of atherosclerosis and response to therapy in lupus patients have not been published to date.

Translation of Current Knowledge into Human Therapies

306

Expression of iNOS is an important arm of the innate immune response when it occurs in the setting of infectious stimuli. In the setting of lupus, its expression occurs outside this context (with additional expression in nonimmune cells such as endothelial cells and keratinocytes).45 It is generally accepted that ONOO1 is one of the more pathogenic and abundant of the RNI derived from iNOS activity. Both eNOS- and nNOSderived NO can combine with SO produced in close proximity to produce ONOO1. However, because iNOS produces log-fold higher amounts of NO iNOS is the most logical isoform target for prevention of ONOO1 production.1 Pharmacologic inhibition of iNOS has been

performed in murine models of lupus using a number of competitive inhibitors of the L-arginine substrate. For an inhibitor to be highly selective, it must have 50- to 100-fold more selectivity for iNOS than for eNOS or nNOS. See Table 26.2 for reports of in vitro selectivity of compounds for iNOS vs. other isoforms. This is important for development of drugs in humans, as inhibition of eNOS can lead to hypertension and reduced glomerular filtration rate62 and inhibition of nNOS can lead to reduced cognitive function.63 L-NMMA, L-NIL, and aminoguanidine (all effective in treating murine lupus16,22,64) do not have the necessary specificity for iNOS over eNOS or nNOS (0.5-, 30-, and 10-fold selectivity, respectively).1 However, newer compounds such as GW273629 and GW274150 have selectivities for iNOS that are 125 and 330 times greater than for eNOS. Their selectivity for iNOS is 1.5 and 100 times greater than nNOS. Given its superior overall selectivity, GW27415 offers the most hope for use in humans and is being developed by Glaxo-Smith-Kline for the treatment of rheumatoid arthritis, asthma,65 and migraine headaches.66 Another approach to inhibiting iNOS activity is to prevent dimerization of monomers to form the active homodimer. Using combinatorial chemistry, a pyrimidine imidazole compound known as BBS2 was discovered to be a potent selective inhibitor of iNOS that binds to the surface of the oxygenase domain and prevents dimer formation. Its IC50 is approximately 1 nM in cellbased assays, and its selectivity for inhibiting iNOS is >1000-fold greater than for eNOS. However, its selectivity versus nNOS is only fivefold.67 It is effective in preventing endotoxemic shock68 and smoke inhalation injury in animal models.69 The effect of its low selectivity for nNOS after chronic administration in humans is not known. Scavenging ONOO1 or SO directly offers an alternative approach to preventing iNOS-mediated pathology. This approach could reduce direct injury from ROI but also may prevent formation of ONOO1. A direct ONOO1 scavenger could also reduce the pathogenic effects of ONOO1 derived from both iNOS and MPO activity. Superoxide dismutases (endogenous enzymes that catalyze removal of SO in host tissues) have reaction rates ten times less than that of SO and NO, making this or any mimetic with a similar reaction rate less attractive as a therapy.70 Several glyoxylate- and glyoxamide-derived metalloporphyrin compounds have had favorable biologic activity as SOD mimetics. One compound, Mn(III) tetrakis(N-ethylpyridinium2-yl)porphyrin (AEOL-10113), had significant ONOO1 scavenging properties—offering hope that it may remove ONOO1 from tissues before it produces tissue injury or modification.71 This compound was effective in preventing the development of diabetes after adoptive transfer of

CONCLUSIONS Production of NO from NOS signals vasodilation and neurotransmission under physiologic circumstances. Increased expression of iNOS in response to infection

or malignancy is an important arm of the innate immune response. However, increased expression of iNOS in response to inflammatory stimuli present in SLE may lead to increased tissue damage, altered enzyme activity, and increased expression of altered self-antigens. There is compelling evidence that pharmacologic inhibition of iNOS leads to reduced disease activity and damage in murine models of lupus. Observational studies indicate that RNI are overproduced during lupus disease activity in humans and that expression of iNOS occurs in tissues damaged during such activity. Studies of therapies designed to inhibit iNOS or scavenge pathogenic ROI and RNI have not been performed in humans with lupus. Several compounds designed to inhibit iNOS activity, prevent dimerization of iNOS, or scavenge ROI and RNI are in development and offer hope that such studies will be forthcoming in the next few years.

REFERENCES

diabetogenic T-cell clones to NOD-SCID recipient mice.72 AEOL-10113 not only prevents damage due to an activated immune response but prevents the initiation of the innate immune response. In one study, the compound inhibited activation of macrophages by LPS by preventing binding of the redox-sensitive transcription factor NF-κB to DNA.73 A similar compound, AEOL-10150, prevented progression of disease in a murine model of amyotrophic lateral sclerosis.74 All of the compounds discussed previously are outlined in Table 26.2. Although these compounds offer promise, extensive testing in humans has not been performed.

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27

Systemic Lupus Erythematosus and Vasculitis Tsutomu Takeuchi, MD

INTRODUCTION Vasculitis is defined as the inflammation and necrosis of vessel walls as a primary process or as a complication of some other underlying pathologic condition, such as collagen-vascular, rheumatic, infectious, or malignant diseases.1-3 Vasculitis is now integrated into a family of “vasculitis syndromes,” which can be classified into two groups: primary vasculitis syndromes and secondary (associated with an underlying disease) vasculitis syndromes (Table 27.1). Accumulating evidence now supports the involvement of autoimmune processes in primary vasculitis syndromes [such as Wegener’s granulomatosis (WG), Churg-Strauss syndrome (CSS), and microscopic polyarteritis] in which anti-neutrophilic cytoplasmic antibodies can be detected. Primary vasculitis and vasculitis associated with collagen-vascular diseases have been shown to involve immunopathologic mechanisms.4 Immune complex formation and subsequent complement activation are the main steps in the pathogenesis of systemic lupus erythematosus (SLE). In addition, increasing evidence supports the notion that mechanisms other than those involving immune complexes may be involved in the pathogenesis of vasculitis associated with SLE.1 In this chapter, we summarize the clinico-pathologic features and discuss the pathogenesis of vasculitis in the prototype autoimmune disease SLE.

CALIBER OF AFFECTED VESSELS AND CLINICAL CONDITIONS

310

Vasculitis syndromes can be divided into several subgroups based on the size of the affected vessels (Table 27.1 and Fig. 27.1).5,6 Given the size of the affected vessels, one can speculate on the clinical manifestations7 (Fig. 27.1). Vasculitis in the capillaries, arterioles, or venules in the dermis may result in erythema, palpable purpura, or livedo reticularis. The pathologic features of these conditions are largely leukocytoclastic vasculitis, including hypersensitivity vasculitis (HV),

mixed cryoglobulinemia, and Schönlein-Henoch syndrome. The prognosis for these conditions is fairly good. In contrast, vasculitis in small and medium-size muscular arteries sometimes leads to the infarction of vital organs (the most life-threatening condition in vasculitis syndromes). Necrotizing vasculitis is a typical pathologic picture in this subgroup, particularly in classical polyarteritis nodosa (PAN).8 Vasculitis affecting large vessels, such as the aorta or main branches, and occurring in conditions such as Takayasu aortitis and temporal arteritis (TA) produces unique clinical manifestations (e.g., pulselessness, hypertension, jaw claudication, and headaches). Necrotizing vasculitis is the most important entity in clinical settings in terms of prognosis because it is intractable and often fatal.9

INCIDENCE OF VASCULITIS IN SLE Vasculitis in SLE is a common complication.10 Cutaneous vasculitic lesions (representing small-vessel involvement) are most common, whereas necrotizing visceral mediumand large-vessel involvement (mimicking primary vasculitic syndromes) may also occur (Fig. 27.1).11 On the other hand, granulomatous large-vessel disease does rarely occur in connective tissue diseases (anecdotal evidence of an SLE case with granulomatous vasculitis complications has been reported).12 In a cohort of 540 SLE patients, vasculitis was reportedly observed in 194 cases (36%), and the calculated number of new cases was 0.053 persons/year. Vasculitis was cutaneous in 82.5% of these patients (29.6% overall) and visceral in 12.4% (5% overall). In our experiences with 188 SLE patients, 32.7% of the patients experienced vasculitis. The vasculitis was cutaneous in 78% of these patients (25.5% overall) and visceral in 22% (7.2% overall), suggesting a clinical picture similar to that seen in the previous study (Table 27.2). Among the first episodes of visceral vasculitis in a Mexican study,

Primary Vasculitis Aorta and main branches: ● ● ●

Takayasu aortitis (aortitis syndrome) Giant cell arteritis (temporal arteritis) Kawasaki disease

Medium and small muscular arteries: ● ● ● ●

Classical polyarteritis nodosa Wegener’s granulomatosis Churg-Strauss syndrome (allergic granulomatosis agitis) Microscopic polyarteritis

Capillaries, arterioles, venules: ● ● ●

Essential mixed cryoglobulinemia Henoch-Schönlein purpura Hypersensitivity vasculitis

Others ● ● ●

Cutaneous vasculitis syndromes Isolated central nervous system vasculitis Thronboangiitis obliterans (Burger disease)

Secondary Vasculitis ●

● ● ● ● ●

Vasculitis associated with collagen-vascular or rheumatic diseases (RA, SLE, PM/DM, SSc, overlap syndrome, MCTD, Sjogren syndrome, Cogan syndrome, anti-phospholipid syndrome, Behcet disease, relapsing polychodritis, spondyloarthropathies, sarcoidosis) Vasculitis associated with malignant diseases Vasculitis associated with infectious disease Vasculitis related to substance abuse Radiation vasculitis Transplant vasculitis

66% were mononeuritis multiplex, 17% were digital necrosis, 10% were large-artery vasculitis of the limbs, and 6% were organ infarctions. In contrast, organ infarction was observed in 59%, digital infarcts in 14%, large-artery vasculitis of the limbs in 14%, retinal arteritis in 9%, and mononeuritis in 5% of patients in our institution—indicating different patterns for distinct ethnic groups and environments. The Mexican study also reported that patients with vasculitis had a longer disease duration and follow- up period, a younger age of SLE onset, and a higher incidence in men than SLE patients without vasculitis. Manifestations associated with vasculitis include myocarditis, psychosis, the Raynaud’s phenomenon, serositis, leukopenia, lymphopenia, pleuritis, and anti-phospholipid syndrome. It should be noted that the most frequent manifestation of SLE, glomerulonephritis, was not associated with vasculitis in this series.13

DIFFERENTIAL DIAGNOSIS OF VASCULITIS AND VASO-OCCLUSIVE DISEASE Vasculitis occurs in more than half of all patients with SLE, whereas the incidence of anti-phospholipid syndrome (a vaso-occlusive disease) in patients with SLE is about 15%. Although both conditions can be life threatening, aggressive anti-inflammatory therapy is indicated for SLE vasculitis and anti-coagulant therapy is indicated for anti-phospholipid syndrome (requiring a correct diagnosis). However, a differential diagnosis between these two entities is often difficult, especially when a tissue biopsy cannot be easily obtained.14 The situation is much more complicated if both conditions are simultaneously found in a given biopsy sample, which is often the case. This situation also complicates the analysis of the pathologic mechanism in vasculitis patients. Diagnostic surrogate markers for vasculitis, including soluble adhesion molecules or soluble forms of the thrombin receptor thrombomodulin in the patient’s sera, can assist in a differential diagnoses.15

PATHOGENESIS OF VASCULITIS IN SYSTEMIC LUPUS ERYTHEMATOSUS

PATHOGENESIS OF VASCULITIS IN SYSTEMIC LUPUS ERYTHEMATOSUS

TABLE 27.1 CLASSIFICATION OF VASCULITIS

Immune Complexes The etiology of vascular inflammation is not completely understood. However, the basic pathogenic mechanisms can be explored. The role of immune complexes (ICs) in the inflammatory manifestations of SLE is well documented, particularly in lupus nephritis. In this respect, Churg suggested that evidence of the involvement of ICs in human vasculitis was circumstantial and indirect for the following reasons:1 serum ICs are rarely detected and the complement level is high in patients with vasculitis, ICs cannot be detected in the vessel wall but are sometimes positive in the healthy skin of patients, and patients with vasculitis rarely have glomerulonephritis complications (the hallmark of immune complex disease), as shown in the Drenkard study.

Autoantibodies One promising factor in the pathogenesis of vasculitis may be an autoantibody-mediated process. Although the anti-basement membrane antibody is obviously indispensable to the pathogenesis of Goodpasture syndrome, an autoantibody that directly induces vasculitis in SLE patients has not been reported.1 In fact, anti-neutrophil cytoplasmic antibodies (ANCAs) that have a key role in primary vasculitis syndromes such as Wegener’s granulomatosis, Churg-Strauss, and microscopic polyangitis2 are infrequent in SLE.16 However, anti-endothelial cell antibody (AECA) and anti-phospholipid antibody are frequently detected in patients with SLE,17 and

311

Erythema Livedo reticularis Palpable purpura Skin ulcers Glomerulonephritis

Organ infarcts

Skin

TA

Subcutaneous tissue

SYSTEMIC LUPUS ERYTHEMATOSUS AND VASCULITIS

CALIBER OF AFFECTED VESSELS IN THE VASCULITIS SYNDROMES

Aortitis

PAN

WG CSS

HV Mixed cryo SchönleinHenoch

SLE

Capillaries, venules, arterioles

Small muscular artery

Medium muscular artery Organ infarcts

Large arteries Aorta Ischemic symptoms

Fig. 27.1 Caliber of affected vessels in the vasculitic syndromes. Left: caliber of affected vessels in the given clinical manifestations. Right: an individual disease among the vasculitis syndromes.

accumulating evidence suggests that these antibodies may be involved in vascular injuries. Such antibodies may bind to their targets and crosslink neutrophils or lymphocytes, triggering subsequent inflammatory processes.18 Numerous pitfalls in the use of the cyto-ELISA assay to detect AECA have been

TABLE 27.2 CLINICAL MANIFESTATIONS OF VASCULITIS IN SLE PATIENTS

Clinical Manifestations

Dreskins Study (%, n=540)

Saitama Experience (%, n=188)

Total Vasculitis

36

33

Cutaneous Visceral

83 17

78 12

66 17 10 6 n.d.

5 14 14 59 9

Visceral Vasculitis

312

Mononeuritis multiplex Digital necrosis Large artery vasculitis Organ infarcts Retinal arteritis

reported because reproducible results are difficult to obtain. Moreover, the target antigens recognized by AECA have not been identified. Given these limitations, the binding of these antibodies may induce the up-regulation of adhesion molecules, the production of cytokines and chemokines, or apoptosis in the endothelial cells.19

New Concept One may speculate that alternative mechanisms, other than immune complexes, may be involved in the pathogenesis of vasculitic lesions because many lymphocytes are observed around the blood vessels and may be involved in vasculitis. We hypothesized that the expression and/or function of surface structures on these lymphocytes may be up-regulated, compared to the situation in healthy individuals. Using peripheral blood lymphocytes from a vasculitis patient with SLE, we attempted to develop monoclonal antibodies recognizing the candidate surface structures on lymphocytes involved in the pathogenesis of vasculitis. Among the hundreds of clones that were successfully developed, we focused on one clone (SM-27) that recognized VLA-4. VLA-4 is a member of the integrin adhesive

Lymphocytes

VLA-4 (CD49d/CD29)

D1 D2 D3 VCAM-1 D4 D5 D6 D7

Neutrophils

LF A-1 (CD1 1a/CD18)

D1 D2 ICAM-1 D3 D4 D5

Mac-1 (CD1 1b/CD18)

ICAM-2

D1 D2

Endothelial cells Fig. 27.2 Adhesive interaction between leukocytes and endothelial cells. Adhesion molecules in the lymphocytes (left) and neutrophils (right) are shown in the upper part of the figure. Those in the endothelial cells are shown at the bottom. The gray box in the VCAM-1, ICAM-1, and ICAM-2 is the binding sites to the individual counterpart, β1 and β2 integrins.

receptor family and is comprised of α4 and β1 chains, which mediate adhesion between lymphocytes and endothelial cells through an interaction between VLA-4 and VCAM-120 (Fig. 27.2). These results may imply that an increase in VLA-4 expression may be involved in the pathogenesis of vasculitis by virtue of enhancing the adhesion between peripheral blood lymphocytes and the endothelium. In this respect, a new pathogenesis concept (the Schwartzman reaction) has been proposed.

SCHWARTZMAN REACTION AND THE PATHOGENESIS OF VASCULITIS Immune complexes have been detected in the affected vessel walls of patients with Schonlein-Henoch syndrome and IgA nephropathy without systemic manifestations. In contrast, circulating or deposited immune complexes are not detected in many vasculitis patients with SLE. Moreover, immune deposits may be observed in vessel walls without accompanying vasculitic lesions, indicating that the mere presence of immune deposits does not necessarily result in tissue injury with an inflammatory infiltrate and fibrinoid necrosis of the vessel wall. These observations argue against the Arthus reaction model, where immune complexes have a key role. An alternative mechanism, the Schwartzman reaction, has been postulated to participate in the pathogenesis of vasculitis.21 This model proposes that endothelial cells are primed to up-regulate adhesion molecules; leukocytes are activated,

resulting in the up-regulation of adhesion molecules; leukocytes and endothelial cells adhere to each other; and this adhesive interaction leads to vascular injuries. Argenbright and Barton demonstrated this hypothesis in a rabbit model.21 The first injection of endotoxin primed the rabbit’s endothelial cells, successfully inducing the expression of ICAM-1 on these cells. Subsequently, zymosan was administered to activate the leukocytes. As a result, the leukocytes adhered to the endothelial cells, causing the development of vasculitis accompanied by platelet aggregation, fibrin deposition, and hemorrhage. These results suggest that intravascular LFA-1/ICAM-1 (leukocyte-endothelium) adhesion was necessary for the development of this type of cytokine-primed neutrophildependent vasculitis. In this regard, our results demonstrating the up-regulated expression and function of VLA-4 (in addition to that of LFA-1) are consistent with the Schwartzman reaction hypothesis. This notion is now widely accepted, not only for vasculitis associated with collagen-vascular diseases but for primary vasculitis syndromes.22-24 The pathogenesis of vasculitis in SLE is now regarded as consisting of the following steps: priming, up-regulation of adhesion molecules, and vascular injury (Table 27.3). Pro-inflammatory cytokines, autoantibodies, and immune complexes are reported to induce the expression of adhesion molecules on endothelial cells (as described previously). In particular, pro-inflammatory cytokines and lipopolysaccharides (LPSs) are the most potent primers of endothelial cells.25,26 As shown in Fig. 27.3,

SCHWARTZMAN REACTION AND THE PATHOGENESIS OF VASCULITIS

ADHESIVE INTERACTION BETWEEN LEUKOCYTES AND ENDOTHELIAL CELLS

313

SYSTEMIC LUPUS ERYTHEMATOSUS AND VASCULITIS

TABLE 27.3 SCHWARTZMAN REACTION MODEL OF VASCULITIS IN SLE PATIENTS 1. Priming ●

Pro-inflammatory cytokines (IL-1, IL-6, TNFα)

●

Lipopolysaccharides

●

Immune complexes

●

Autoantibodies ■

Anti-endothelial cell antibody (AECA)

■

Anti-phospholipid antibody (aPL)

■

Anti-neutrophil cytoplasmic antibody (ANCA)

UP-REGULATION OF ADHESION MOLECULES IN VASCULITIS PATIENTS WITH SLE

2. Up-regulation of adhesion molecules ●

ICAM-1 and VCAM-1 on endothelial cells

●

Integrins on circulating neutrophils

VCAM-1) are produced by T cells, macrophages, and even endothelial cells.

3. Vascular injuries ●

Neutrophils, macrophages, cytotoxic T lymphocytes

●

Factors inducing apoptosis and necrosi

●

Gas mediator (reactive oxygen, nitrogen metabolites)

LPS stimulation can induce human umbilical endothelial cells to induce P-selectin after 5 to 30 minutes, followed by the induction of E-selectin after 6 hours. Thereafter, neutrophils can adhere to the endothelial cells. After 12 to 48 hours, ICAM-1 and VCAM-1 are expressed on the endothelial cells, allowing monocytes and lymphocytes to adhere. At this stage, cytokines such as IL-1, IL-6, and TNFα (which can strongly induce ICAM-1 and

We analyzed the expression of a series of integrin adhesion molecules on peripheral blood lymphocytes (PBLs) from normal subjects and from SLE patients with or without vasculitis27 and found that the expression of β2 integrins such as LFA-1, CD11b, and p150/95 (CD11c) is significantly elevated in active SLE with or without vasculitis—suggesting that these adhesion molecules may be related to the active phase of disease (Fig. 27.4 and Table 27.4). In contrast, the expression of VLA-4 was enhanced in SLE patients with vasculitis, but its expression in SLE patients without vasculitis was comparable to that in normal controls (Fig. 27.4). Furthermore, the adhesive function of VLA-4 against the CS-1 domain of fibronectin [as well as that of VCAM-1 on cytokine-activated human umbilical vein endothelial cells (HUVECs)] was also significantly increased in SLE patients with vasculitis.27 Accumulating evidence now supports the previous observation that the expression of VLA-4 is up-regulated in patients with vasculitis, not only in collagen-vascular diseases28 but in primary vasculitis syndromes such as Wegener’s granulomatosis.29,30 These results suggest that the increased expression and function of VLA-4 is intimately associated with the pathogenesis of vasculitis4,23,24 (Fig 27.4). These results are further supported by the

EXPRESSION OF ADHESION MOLECULES IN ENDOTHELIAL CELLS Stimulation

E-selectin

ICAM-1 VCAM-1

P-selectin

0 hr

314

1 hr

Function

Procoagulant Permiability

Mediators

PAF, L TB4

24 hr Tethering

48 hr Strong adhesion Migration

TNF-a, INF-γ, IL-1b, IL-6, LPS

Fig. 27.3 Expression of adhesion molecules in endothelial cells. The X axis indicates the time course after stimulation of endothelial cells with LPS. The Y axis indicates the level of expression of adhesion molecules.

VLA-4

Normal T cells Inactive SLE T cells

Fig. 27.4 Expression of adhesion molecules in SLE T cells in patients with distinct disease activity and clinical manifestations.

Active SLE T cells

LFA-1

+ arthritis activated CD44

LFA-1 (αLβ2) CD11a/CD18

αEβ7 CD103

VLA-4 (α4β1) CD49d/CD29

CD44

αEβ7

findings of a study using inflamed glomerular tissues that showed LFA-1/VLA-4 and ICAM-1/VCAM-1 adhesive interactions to play a role in glomerular vasculitis.30

EXPRESSION OF INTEGRIN LIGANDS IN SLE PATIENTS WITH VASCULITIS Given the evidence that integrin adhesion molecules are up-regulated on peripheral blood lymphocytes and neutrophils, one can speculate that the ligands for integrins are also up-regulated. Adhesion molecules on vascular endothelial cells are independently regulated, as shown in Fig. 27.3 (see also Table 27.4). The levels of VCAM-1, ICAM-1, and E-selectin on vascular endothelial

TABLE 27.4 ADHESION MOLECULES ON CELLS AND TISSUES FROM SLE PATIENTS WITH VASCULITIS Effector Cells ● ● ●

Lymphocytes: LFA-1↑, VLA-4↑ Neutrophils: LFA-1↑, CD11b↑, CD11c↑ Monocytes: LFA-1↑, CD11b↑, CD11c↑

Target Cells and Structures ● ●

Vascular endothelial cells: ICAM-1↑, VCAM-1↑, E-selectin↑ Vascular smooth muscle cells: β1 integrins ↑

Serum ● ● ●

s-ICAM-1↑ s-VCAM-1↑ s-E-selectin↑

+ epithelial involvement

cells were up-regulated in skin or muscle biopsy specimens from SLE patients or kidney biopsy samples from renal vasculitis patients,31-33 suggesting that pairs of integrins and integrin-ligands are simultaneously up-regulated and participate in the pathogenesis of vasculitis. Soluble ICAM-1 and VCAM-1 levels are elevated during the active stage of vasculitis,34-38 implying that pro-inflammatory cytokines or LPS may stimulate vascular endothelial cells to up-regulate these integrin ligands and selectins as priming factors.

MACHANISM OF VASCULAR INJURIES BY LEUKOCYTE-ENDOTHELIAL ADHESION

+ vasculitis

MECHANISM OF VASCULAR INJURIES BY LEUKOCYTE-ENDOTHELIAL ADHESION One may ask, “What is the mechanism of the ultimate injury to the vessel wall in vasculitis?” The firm adhesion of neutrophils to endothelial cells as a result of the enhanced expression and function of adhesion molecules allows neutrophils or lymphocytes to be retained at the site of inflammation. The exact mechanism of endothelial cell injury must be further examined. Matrix degradation, such as the degradation of elastic fibers or the internal elastic lamina, may occur as a consequence of the prolonged inflammation of vessel walls through the activity of a variety of proteases released from neutrophils, monocytes, and lymphocytes.39 In addition, cytotoxic granules or direct cell-to-cell contact may induce apoptosis in the endothelial cells.40,41 Alternatively, enhanced adhesion between leukocytes and endothelial cells may transmit signals within the endothelial cells to generate secondary messengers into the vessel walls. As shown in Fig. 27.5, α1 integrins are

315

SYSTEMIC LUPUS ERYTHEMATOSUS AND VASCULITIS

Lymphocytes

VLA-4 (CD49d/CD29)

D1 D2 D3 VCAM-1 D4 D5 D6 D7

Neutrophils

LFA-1 (CD11a/CD18)

D1 D2 ICAM-1 D3 D4 D5

Fig. 27.5 Expression of adhesion molecules in the affected tissues. Skin biopsy samples from SLE patients with skin vasculitis (upper panel). Kidney biopsy samples in SLE patients with glomerular vasculitis (lower panel).

Mac-1 (CD11b/CD18)

ICAM-2

D1 D2

Endothelial cells

up-regulated on the smooth muscle cells of affected blood vessels, and focal adhesion kinase (FAK) is tyrosine-phosphorylated, consistent with the media thickening observed in vasculitis (upper part of Fig. 27.5). A similar view can be obtained in tissues affected by glomerular vasculitis (lower part of Fig. 27.5).

FUTURE THERAPEUTIC STRATEGY Immune mechanisms and subsequent inflammation play a central role in the process of vasculitis in patients with SLE. Thus, immunosuppressive therapy forms the foundation of treatment for almost all forms of systemic vasculitis.42,43 Cytotoxic agents such as cyclophosphamides, azathiopurines, and methotrexates are widely used for this purpose.9 Newer agents (such as mycophenolate mofetil, rituximab, and tumour necrosis factor-alpha inhibitors) are finding new indications in the treatment of conditions such as SLE, skin vasculitis, cytoplasmic anti-neutrophil antibody–positive vasculitis, Wegenr’s granulomatosis, and Takayasu’s aortitis.44

Given our molecular understanding of the pathogenesis of vasculitis (particularly the role of the enhanced expression of adhesion molecules arising, in part, through the activity of pro-inflammatory cytokines), molecular targets of therapeutic interventions should include regulators of the adhesion molecules on either endothelial cells or neutrophils. As shown in Figs. 27.2 and 27.3, pro-inflammatory cytokines are key regulators in the expression of ICAM-1 and VCAM-1 on endothelial cells—suggesting that biological agents against TNFα may be indicated in the treatment of vasculitis.45 In this respect, anti-TNFα therapy reportedly induced vasculitis in rheumatoid arthritis,46,47 arguing against this possibility. Anti-VLA-4 antibodies might also be candidate targets, although the monoclonal anti-VLA-4 antibody natalizumab is no longer available because of safety issues. Biological agents targeting adhesion molecules and other cell surface structures would not only provide new therapeutic modalities but might provide new insight into our understanding of the pathogenesis of vasculitis and SLE.48

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MECHANISMS OF TISSUE DAMAGE

28

Mechanisms of Skin Damage Markus Böhm, MD and Thomas A. Luger, MD

INTRODUCTION Understanding the mechanisms of skin damage in cutaneous lupus erythematosus (CLE) requires knowledge of the current categorization of cutaneous LE types, the histopathology and the immunopathology of the specific cutaneous manifestations of LE that occur in patients with this autoimmune disease, insight into the molecular genetics that predisposes individuals to the disease, elucidation of the cellular and molecular biologic abnormalities that underlie the increased ultraviolet light (UV) photosensitivity, and insight into the immune pathomechanisms that orchestrate an abnormal immune response against the skin.

PATHOLOGY

318

Cutaneous histopathology can provide a very useful clue for the diagnosis of CLE. However, it should be noted that correct classification of LE into the four major categories—systemic LE (SLE), subacute CLE (SLCE), chronic CLE—largely relies on the clinical picture and key laboratory findings. Variants of the aforementioned specific CLE manifestations include discoid LE (DLE), LE profundus (lupus panniculitis), bullous LE, hypertrophic LE and chilblain LE, all of which, like SCLE and CCLE, may present as isolated skin disorders or which may represent cutaneous manifestations of SLE. It must be emphasized that the histopathology of all of these forms depends on the age of the lesion and previous therapy. Accordingly, skin biopsy specimens from early lesions may yield nonspecific signs of inflammation and topical or systemic corticosteroids can easily attenuate the true histopathologic picture. In this chapter we will outline the dermatopathologic key findings of the specific cutaneous LE lesions followed by a summary of the major pathogenetic concepts that have been developed thus far to explain skin damage in patients with LE.

Cutaneous Histopathology Epidermal Changes Epidermal changes such as hydropic (vacuolar) degeneration of the basal layer, scattered “Civatte bodies” (dead keratinocytes), epidermal atrophy, and compact orthohyperkeratosis are key features in SLE (i.e., in ACLE, also known as “malar rash”), SCLE, and DLE lesions1-3 (Fig. 28.1). These changes are identified by routine hematoxylin and eosin (H & E) staining in formalin-fixed biopsy specimens. Neonatal LE displays similar epidermal findings as compared to SCLE, and is sometimes more pronounced with cleft formation between the dermis and epidermis. Prominent vacuolar epidermal degeneration together with massive accumulation of inflammatory cells in the basal membrane zone (“interface dermatitis”) can progress to subepidermal blister formations known as bullous LE.4 The epidermal blister roof of patients with bullous LE is mostly intact in contrast to toxic epidermal necrolysis-like ACLE, an extremely rare and only recently described subtype of LE characterized by a pan-necrotic epidermis.5 Vacuolar degeneration and keratinocyte injury of the basal layer may be regarded as early pathogenetic steps in the development of CLE. Using in situ nick translation and TUNEL it was demonstrated that apoptotic nuclei accumulate in skin of patients with CLE after ultraviolet (UV) light exposure supporting the pathogenetic concept of increased apoptosis in skin of patients with CLE.6 As a consequence, epidermal atrophy and possibly reactive epidermal orthohyperkeratosis may develop. Epidermal hyperkeratosis is very prominent and typically includes the adnexal structures (“follicular plugging”) in DLE1,2 (Fig. 28.2). In hypertrophic LE, epidermal hyperkeratosis is even more increased with parakeratosis and acanthosis. Another pathologic finding of the dermoepidermal junction, especially in long-standing CLE lesions, is thickening of the basement membrane.1,2 It is most apparent in DLE, less in SCLE, and often absent in ACLE lesions, and can be visualized by the periodic acid-Schiff (PAS) stain (Fig. 28.3).

PATHOLOGY

Fig. 28.1 Histopathology of acute cutaneous lupus erythematosus (classical malar rash) in a patient with systemic lupus erythematosus. Note hydropic degeneration of the basal layer, apoptotic keratinocytes (Civatte bodies), epidermal atrophy, and compact orthohyperkeratosis. In addition, inflammatory cells, mainly lymphocytes and few neutrophils are present in a patchy distribution in the papillary dermis (hematoxylin and eosin stain).

Hydropic degeneration of the basal membrane zone, apoptotic keratinocytes, and atrophy are also found in chilblain LE.7 In rare cases of verrucous chilblain LE, epidermal hyperkeratosis, patchy parakeratosis, acanthosis, and hypergranulosis are the dominant features.8 Hydropic degeneration of the basement membrane zone, epidermal atrophy, follicular plugging, and basement membrane thickening occur in the majority of patients with LE panniculitis (LE profundus),9,10 while such changes are only occasionally detected in patients with LE tumidus.11,12

Inflammatory Cells Another consistent finding in all CLE forms is the presence of an inflammatory infiltrate consisting

Fig. 28.2 Classical discoid lupus erythematosus. Note the prominent follicular plugging. The infiltrate consisting mainly of lymphocytes is accentuated around the hair follicles and blood vessels. There is also dense mucin deposition in the upper dermis (hematoxylin and eosin stain). (See Color Plate 1.)

Fig. 28.3 Basement membrane thickening in discoid lupus erythematosus as demonstrated by periodic acid-Schiff stain. Note also hydropic degeneration of the basal layer of the epidermis.

mostly of lymphocytes. The cutaneous location and pattern of the inflammatory infiltrate detected by H & E staining differs in its location depending on the category of LE. The lymphocyte infiltrate in cutaneous lesions of patients with SLE (malar rash) can be sparse especially in early lesions and is typically located in the upper dermis around the blood vessels2 (Fig. 28-1). In more advanced lesions it becomes more prominent, involving the dermoepidermal junction (interface dermatitis), sometimes with extravasation of erythrocytes, and deposition of fibrinoid material around blood vessels and between collagen fibers. In some biopsy specimens of ACLE from patients with SLE, there are also signs of leucocytoclastic vasculitis, that is, nuclear dust, fibrinoid necrosis of the vessel wall, neutrophil infiltration, and extravasation of erythrocytes. In bullous LE of patients with SLE, there is in addition a prominent mixed mononuclear/neutrophilic infiltrate along with dermal microabscesses.4 The blister fluid contains fibrin and neutrophils. SCLE lesions and neonatal LE share similar patterns of the inflammatory cell infiltration. The lymphocytes are mostly confined to the upper dermis leading to a band-like infiltrate with interface dermatitis1-3 (Fig. 28.4). Erythrocyte extravasation and dermal fibrin deposition can occur. A striking feature of DLE distinguishing all other CLE forms is the prominent periadnexal inflammatory infiltrate1,2 (Fig. 28-2). The epidermal changes of DLE and LE hypertrophicus as outlined above (follicular plugging due to hyperkeratosis) may represent a follicular response to proinflammatory and proliferative signals released by infiltrating lymphocytes. Besides its striking periadnexal location, the inflammatory infiltrate in classical CLE lesions displays a patchy, sometimes a band-like (lichenoid), pattern. While the inflammatory

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MECHANISMS OF SKIN DAMAGE Fig. 28.4 Histopathological changes in subacute cutaneous lupus erythematosus. Note less dense and prominent inflammatory infiltrates as compared to Fig. 28.2. Inflammatory cells, mainly lymphocytes, are primarily found in the upper dermis close to the dermo-epidermal junction (interface dermatitis) as well as perivascularly (insert). There is hydropic degeneration of the basal layer (hematoxylin and eosin stain).

Fig. 28.5 Dermal mucin deposition in subacute cutaneous lupus erythematosus as visualized by the colloidal iron stain. Acid mucopolysaccharides are stained in blue and located throughout the dermis.

be detected in many inflammatory and noninflammatory conditions of the skin.

Cutaneous Immunopathology infiltrate of chilblain LE is likewise situated in the upper dermis, around the blood vesicles, and occasionally around the hair follicles (especially in the verrucous subtype),7 the infiltrates in LE tumidus and LE panniculitis are mainly present in deeper layers of the skin. In LE tumidus, perivascularly situated lymphocytes are found in the superficial and deep dermis and only infrequently around the skin adnexal structures.11,12 In LE panniculitis, lymphocytic infiltration is present, sometimes together with eosinophils, of the subcutaneous fat leading to panniculitis, fat necrosis, and hyalinization of adipose lobules.9,10 The pattern of the panniculitis is lobular, and sometimes paraseptal. Lymphoid follicles and germinal centers are often detected. Periadnexal infiltrates are less frequently seen.

Mucin Deposition

320

Another consistent feature of virtually all specific CLE lesions is dermal mucin deposition.2 It can be visualized by colloidal iron stain or Alcian blue stain (Fig. 28.5). Mucin deposits are most prominent in LE tumidus and may give rise, when excessively prominent, to so-called papular mucinosis. The biochemical nature of the deposited mucopolysaccharides in CLE (as well as in other inflammatory skin disorders) is undefined. Proinflammatory cytokines such as interleukin-1 (IL-1) released by inflammatory cells may be involved in inducing increased mucopolysaccharid synthesis by dermal fibroblasts, but the exact pathogenesis remains unknown. However, mucin deposition alone is not a specific dermatopathologic finding and can frequently

Early immunodermatologic work on cutaneous lesions of patients with SLE strongly suggested a pathogenetic role of precipitated immunoglobulins at the dermoepidermal junction in this autoimmune disorder. Due to its characteristic band-like staining pattern this phenomenon in the skin of patients with LE has been coined “lupus band.” The technique now routinely performed to detect immunoglobulins, fibrin, and complement components in lesional and nonlesional skin specimens of patients with LE is called direct immunofluorescence (DIF). It is most reliably performed on snap-frozen skin specimens. The intensity of fluorescence in skin biopsy specimens (lupus band test [LBT]) depends on the biopsy site, the acuity of a lesion, and previous treatment. Facial lesions may give false positive results whereas very early ones and those pretreated with topical corticosteroids and immunomodulators or systemic medication may yield false negative results. Moreover, immune complexes and complement along the dermoepidermal junction can be detected in a number of other inflammatory skin disorders. Therefore, a positive lupus band test must be interpreted in the context of the clinical picture and laboratory data of the patient. Although the overall diagnostic relevance of DIF analysis has declined during the last several years and proper clinical characterization of CLE lesions, serologic tests, and routine histopathology may be sufficient for establishing the correct diagnosis, the LBT in nonlesional skin has still a high predictive value for the diagnosis of SLE. Moreover, DIF studies may be helpful in discriminating inflammatory skin disorders with similar histopathologic pictures as LE. Finally, DIF studies can

PATHOLOGY

provide some important information on the pathogenesis of CLE. In general, most DIF studies have been undertaken in patients with SLE, DLE, and SCLE while comparatively less information is available regarding the LBT in lesional skin of the other CLE subsets. At least three patterns of DIF in skin of patients with LE can be distinguished.13

Immune Deposits at Dermoepidermal Junction: Lupus Band The most striking immunopathologic feature in CLE (and SLE) is the presence of deposited immunoglobulins (IgG, IgM, and IgA), complement (especially C3), and other serum proteins (e.g., fibrin, albumin, factor B, and properdin) at the dermoepidermal junction. Ultrastructural studies using immune electron microscopy have shown that the immune deposition takes place in the sub–lamina densa region. Several morphologic variants of the immune deposits at the dermoepidermal junction have been described including linear, granular, or shaggy. In addition globular deposits consisting of immunoglobulins, complement, and fibrin can frequently be detected in lesional skin (and nonlesional skin of patients with SLE). These ovoid (cytoid) bodies are scattered along the dermoepidermal junction but can also be found in the superficial dermis. Numerous studies on the immune deposition in patients with the three major LE forms with cutaneous involvement have resulted in a typical distribution of the LBT positivity in lesional and nonlesional skin (Table 28.1). In cutaneous lesions of patients with SLE, the lupus band test is positive in 90 to 100%. IgG, IgM, IgA, C3, and fibrin are most often detected. Most importantly, the LBT is positive in 50 to 90% in nonlesional, sun-protected skin of patients with SLE. In lesional skin of DLE, immune deposits are present in about 60 to 95%. IgG3 and C3 are most frequently found and typically display a linear band-like pattern, and sometimes also a granular fluorescence, along the dermoepidermal junction (Fig. 28.6). The LBT is

TABLE 28.1 DIRECT IMMUNOFLUORESCENCE FINDINGS (IMMUNE DEPOSITS OF IgG) IN SKIN OF PATIENTS WITH MAJOR LUPUS ERYTHEMATOSUS FORMS LE Subtype

Lesional Skin

Nonlesional Skin

DLE

60%–90%

0%

SCLE

90%–100%

0%

SLE

90%–100%

50%–90%

DLE, discoid lupus erythematosus; SCLE, systemic cutaneous lupus erythematosus; SLE, systemic lupus erythematosus.

Fig. 28.6 Positive lupus band test in lesional skin from a patient with discoid lupus erythematosus. IgG deposits are visualized by an anti-human IgG antibody coupled to the fluorochrome FITC. A fluorescent bright green band is seen at the dermoepidermal junction. Note “nonspecific” immunostaining on collagen fibers in the dermis.

usually negative in nonlesional skin of patients with DLE, although in some cases deposits of C3 and IgM have been described. Lesional skin from patients with SCLE displays a similar pattern to that of DLE, and the composition of immunoglobulins and complement at the basement membrane zone is similar to that of DLE. The LBT is positive in 60 to 100% of skin biopsy specimens taken from lesional skin of patients with SCLE, and is consistently negative in nonlesional skin. In chilblain LE and LE panniculitis, the majority of patients have immune deposits (mostly IgM and/or IgG and/or C3) at the dermoepidermal junction in lesional skin,7,9,10 while in LE tumidus LBT positivity is rather heterogeneous.11,12 The precise pathomechanism of immune deposition at the dermoepidermal junction in the skin of patients with LE remains only partially understood. A pathogenetic role for deposited immunoglobulins has recently been emphasized in a fraction of patients with bullous SLE. These patients have circulating anti–basement membrane zone antibodies (mostly IgG, less frequently IgA) directed against type VII collagen.14,15 Using the salt-split skin technique, moreover, autoantibodies directed against several undefined proteins of 230, 200, 180, 130, and 97 kD from epidermal extracts, and 75 kD from dermal extracts, were identified.16 It has long been known that the fluorescence intensity of the LBT in SLE correlates with disease activity,17 as well as with the serum titer of antinuclear antibodies, suggesting a causal relationship.18 Both native and single-stranded DNA antibodies have an affinity for collagen present in the basement membrane, possibly leading to in vivo fixation of anti-DNA antibodies.13 As will be outlined below, autoantigens such as SSA/Ro are exposed on the

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surface of epidermal keratinocytes upon ultraviolet (UV) radiation. Although this phenomenon would not fully explain the band-like pattern of deposited immunoglobulins at the dermoepidermal junction in LE, it may suggest a contribution to its pathogenesis via in vivo fixation of antinuclear antibodies (ANAs) to exposed epidermal epitopes.

Therefore, certain haplotypes may predispose to the abnormal immune response as seen in CLE. Second, UV-mediated apoptosis and exposure of autoantigens appears to be a central pathogenetic step. Finally, immune cells including T cells and dendritic cells are important executers of the abnormal immune response leading to skin damage in CLE.

Epidermal Immune Deposits

Immunogenetics

In addition to the lupus band, epidermal immunofluorescence occurring as a cytoplasmic and/or nuclear fluorescence has been described.13 A nuclear speckled immunofluorescence pattern can be detected in epidermal cells of nonlesional skin in patients with SLE. The presence of this DIF pattern in nonlesional skin correlates with the titer of anti-RNP antibody, suggesting a causal relationship and in vivo fixation. These deposits contain mostly IgG, less frequently IgM and IgA. However, similar DIF patterns have been seen in patients with other autoimmune disorders such as Sjögren’s syndrome or mixed connective tissue diseases. In lesional skin of SCLE there is another epidermal DIF pattern.13 Accordingly, a fine granular, dust-like deposition of IgG in the cytoplasm, nuclei, and intercellular space of the basal epidermis is detectable. This DIF pattern has been associated with the presence of circulating antiRo/SSA antibodies. It is also present in skin of patients with Sjögren’s syndrome and neonatal LE. In vivo fixation of anti-Ro/SSA antibodies in the pathogenesis of this phenomenon is supported by the fact that injection of anti-Ro/SSA antibodies into nude mice leads to analogous epidermal immunostaining in grafted human skin.19

A genetic base for CLE has long been postulated based on initial observations on pairs of sisters and twins and subsequent case–control studies.20 Moreover, carriers and patients with certain hereditary disorders such as X-linked and autosomal-recessive chronic granulomatosis disease and non–X-linked hyper-immunoglobulin M syndrome have an increased risk to develop CLE,21,22 indicating that the genes affected in the above disorders could be crucially involved in the development of this autoimmune disorder. It is also of interest that patients with SCLE and DLE exhibit a higher prevalence of polymorphic light eruption suggesting a common genetic background.23,24 In recent years, the methodologic approach to study the genetic base of CLE has relied on association studies, family linkage analysis, and transmission equilibrium testing. These studies collectively indicate that the development of CLE is controlled by multiple genes (Table 28.2).

Immune Deposits on the Vessels Vascular immunostaining can occur in both lesional and nonlesional skin in patients with SLE and DLE.13 The deposited IgG, IgM, C3, and fibrin can exhibit homogenous staining of the entire vessel wall (the most common finding), or a more granular pattern in and around the vessel. Especially in SLE and in presence of histopathologic signs of leukocytoclastic vasculitis vascular DIF may indicate immune complex vasculitis. However, many other vascular and nonvascular diseases can display similar patterns of vascular DIF.

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322

The precise pathogenesis of the specific skin lesions in patients with LE is still incompletely understood. First, there is evidence that a genetic base confers susceptibility to LE-specific skin disease. Accordingly, genes located in hitherto identified susceptibility loci for LE have been implicated as crucial players of inflammatory responses.

Major Histocompatibility Complex The most important locus that confers genetic susceptibility to SCLE appears to the major histocompatibility complex (MHC) locus at 6p21.3. It includes a number of genes that control inflammatory and immune responses (Fig. 28.7). Region I contains the class I human leukocyte antigens (HLAs), including HLA-A, -B, and Cw; region II contains the HLA II antigens (DP, DQ, and DR); and region III several complement genes, tumor necrosis factor (TNF), and heat shock protein (HSP) 70. Accordingly, the HLA A1, DR3, B8, DQ2, DRw52, C4null haplotype has been identified to be highly associated with susceptibility for SCLE in whites with circulating anti-Ro/SSA antibodies.24 This haplotype is also linked with susceptibility to other autoimmune disorders including myasthenia gravis, dermatitis herpetiformis Duhring and insulindependent diabetes.25 In addition, the haplotype HLA DQ1 and DQ2 is associated with the highest-serum anti-Ro/SSA antibodies in patients with SCLE.26 These data suggest that the MHC complex controls the tendency of an individual to mount an immune response towards the Ro/SSA antigen being exposed on the surface of UV-irradiated keratinocytes, as will be outlined below. In contrast to SCLE, the studies investigating HLA associations with DLE have conflicted with some

Locus

Gene

Putative Pathogenetic Role

6p12.3

MHC HLA A1,B8,DR3,DQ2,DRw52,C4null

Susceptibility to autoimmune haplotype disease; generation of an immune response to the Ro/SSA antigen

C2,C4A, C4B, factor B

Impaired clearance of apoptotic keratinocytes as well as of immune complexes facilitating an autoimmune response

TNF

Increased TNFγ production by keratinocytes after UV exposure leading to an apoptosis rate

2q13

IL-1 cluster (IL-1A,B, IL-1-RA)

Increased UV photosensitivity leading to increased apoptosis and exposure of nuclear antigens of keratinocytes

1q31

IL-10

Increased production of immunoglobulins, up-regulation of adhesion molecules (ICAM-1, ESELE)

1q23

FCGR2A

Increased generation of ADCC involving anti-Ro/SSA antibodies and resulting in keratinocyte cytotoxicity

7q35

TCR

Breaking of immune tolerance to the Ro antigen

1p13

GSTM1

Impaired oxidative stress defense leading to increased apoptosis

10q24

Fas (TNFRSF6)

Increased photosensitivity and apoptosis of keratinocytes

6q25

ORα

B-cell activation and immunoglobulin (autoantibody) synthesis

PATHOGENESIS

TABLE 28.2 LOCI WITH CORRESPONDING GENES THAT APPEAR TO CONFER SUSCEPTIBILITY TO CUTANEOUS LUPUS ERYTHEMATOSUS

ADCC, antibody-dependent cellular cytotoxicity; C, complement; ESELE, E-selectin; FCGR, Fcγ receptor; GST, glutathione-S-transferase; HLA, human leukocyte antigen; ICAM-1, intercellular adhesion molecule; IL-1, interleukin-1; IL-1-RA, IL-1 receptor antagonist; MHC, major histocompatibility complex; ORα, oestrogen receptor α; TCR, T-cell receptor; TNF, tumor necrosis factor; UV, ultraviolet light.

studies reporting no HLA association, while others confirm the A1, B8, DR3, and the B7, DR7 haplotypes.

Non-HLA Genes of Major Histocompatibility Complex Region III of the MHC locus also contains genes for complement C2, C4A, C4B, and factor B. Inherited C2 and C4 deficiency is strongly associated with circulating anti-Ro/SSA antibodies and development of SCLE.27-31 Moreover, lupus profundus has been found to be associated with partial deficiency of C4.32 Since complement factors are involved in macrophage activation and clearance of antibody–antigen complexes, these findings may indicate defective clearing of

apoptotic cells or immune complexes containing ANAs including anti-Ro/SSA antibodies.33 Alternatively, linkage disequilibrium with the real disease-predisposing locus may exist. In addition to C2 and C4, the pro-inflammatory cytokine TNF-α has been implicated in the pathogenesis of CLE. TNF-α is strongly induced in epidermal keratinocytes upon UV irradiation, and stimulates expression of the Ro/SSA antigen in these cells.34 The TNF polymorphism -308A has been associated with increased TNF production after UVB irradiation of epidermal keratinocytes and with the development of SCLE.35 It has been shown that the above polymorphism independently of the HLA-DR3 haplotype confers susceptibility to SLE. Finally, polymorphism

Fig. 28.7 Genetic organization of the human major histocompatibility complex. C, complement; HLA, human leukocyte antigen; HSP70, heat shock protein-70; MHC, major histocompatibility complex; TNF, tumor necrosis factor.

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of the locus encoding HSP70 (HSPA1A, B and L) has been investigated in patients with LE.36,37 In some populations, the PstI site containing allele B was found to be significantly increased in SLE patients compared to healthy controls. Although the genetic role of HSP70 for CLE remains undefined, HSP70 expression is well known to be induced in keratinocytes during cellular stress, including UV irradiation. HSP immunostaining in lesional skin of patients with SLE and DLE was diffusely distributed in the whole epidermis, hair follicles, and sweat gland cells. Others could detect HSP70 immunoreactivity along the dermoepidermal junction and around papillary vessels in lesional skin of patients with SLE. Using double fluorescence labeling, the latter authors found that immune deposits of IgM, IgG, and C3 were co-localized with Hsp70, suggesting that Hsp70 could shuttle autoantigens to the dermoepidermal junction.38

Candidate Loci Outside Major Histocompatibility Complex Locus

324

Several loci outside the MHC complex have been found to confer susceptibility to CLE. The respective loci contain genes that encode for various components of the immune system (cytokines, cytokine receptors), of the oxidant defense system, and the apoptosis machinery, which is involved in UV-mediated damage of epidermal keratinocytes. The locus 2q13 encoding the interleukin (IL)-1 cluster (IL-1α, -β and IL-1 receptor antagonist, IL-RA) has been linked to UV photosensitivity and DLE.39 Single-nucleotide polymorphisms (SNPs) have been also found for the IL-10 promoter.40 The identified SNPs conferred reduced in vitro production of the IL-10 by mononuclear cells.41 Since IL-10 has potent immunosuppressive functions, failure to produce this cytokine in sufficient amounts may facilitate the development of an abnormal immune response involving the generation of anti-Ro/SSA antibodies. Genes encoding the T-cell receptor C (TCR) are also associated with susceptibility to SLE. Restriction fragment length polymorphisms for the TCR genes Cβ1 and Cβ2 were found in 76% of patients with SLE and circulating anti-Ro/SSA antibodies, and in 41% of patients without these antibodies. Linkage analysis using genome-wide scans further revealed a susceptibility locus at 1q23 encoding the Fc receptor-II gene which mediates binding of immunoglobulins to lymphocytes to generate antibody-dependent cellular cytotoxicity.42 Like the loci encoding the IL-1 cluster and the cytokine IL-10, it is possible that changes in both of the latter genes predispose to a break of the immune tolerance against the Ro/SSA antigen. Finally, polymorphisms of two genes involved in UV light–induced genotoxicity and apoptosis, glutathione S-transferase (GST) and Fas (CD95), have been found

to be associated with SLE.43,44 GST is crucially involved in detoxifying intracellular reactive oxidative species, which are induced by both UVA and UVB. Fas is a member of the TNF receptor family and is activated by both binding to TNF-α and by ligand-independent receptor aggregation following UVB exposure. The GSTM1 null polymorphism was highly associated with SLE and the presence of anti-Ro/SSA antibodies, suggesting that deregulation of oxidative stress is important in the break of tolerance to Ro/SSA antigens. The identified SNP at the -670 nucleotide position of Fas was associated with photosensitive SLE when homozygous for Mval*2, suggesting that correct Fas signaling is involved in controlling the extent of cutaneous UV sensitivity and possibly the tendency of cells to expose antigens such as Ro/SSA to the immune system. Supportive for an important role of Fas is the lpr LE mouse that carries a point mutation of Fas, subsequently leading to defective signaling and increased susceptibility to autoimmunity, including LE-like skin changes.45 Recently, there is evidence that estrogen receptor gene polymorphisms could play another role in susceptibility to skin involvement in patients with SLE.46 Estrogens are considered to be important environmental (and physiologic) LE triggers in patients with SLE and SCLE because they can activate mature peripheral B cells to produce immunoglobulins including antidsDNA antibodies. Pregnancy, oral contraceptives containing oestrogens, and hormonal replacement therapy are also well-known triggers of disease activity in patients with SLE and SCLE. The Pvull C and the Xbal G alleles of the estrogen receptor have been found to be associated with a milder form skin involvement in patients with SLE.46

UV-Induced Apoptosis One of the most consistent features of patients with CLE, especially SLE and SCLE, is photosensitivity to UVB light. It is furthermore known that skin lesions can be experimentally induced by skin exposure of patients with CLE to artificial light sources and that there is also a common genetic background in both patients with polymorphic light eruption and SCLE as outlined above. A concept that would explain both the impact of UV irradiation as a trigger for CLE lesions and the relevance of circulating ANAs was postulated some years ago.47-49 In this concept, UVB-induced apoptosis of epidermal keratinocytes, a physiologic process of the skin that is intended to eliminate keratinocytes with accumulated harmful DNA photoproducts, plays a key role (Fig. 28.8). Indeed, apoptotic keratinocytes are detectable in lesional skin of CLE forms involving the epidermis and can also be reproduced upon UVB photoprovocation.6 In vitro, it was

PATHOGENESIS

Fig. 28.8 Simplified scheme of the key pathogenetic steps of photosensitive cutaneous lupus erythematosus. Ultraviolet light irradiation physiologically triggers apoptosis of epidermal keratinocytes via DNA damage as well as via induction of cell death receptor ligands (e.g. tumor necrosis factor-a). During apoptosis nuclear antigens such as Ro/SSA are redistributed to the cell surface mounting an autoimmune response which involves cytotoxic T cells that are directed to sites of skin damage via increased expression of cellular adhesion molecules. Deviations in the apoptosis program, generation of anti-Ro/SSA antibodies, defective clearance of apoptotic cells and immune complexes appears to depend on a multigenic predisposition. ADCC, antibody-dependent cellular cytotoxicity; ESELE, E-selectin; ICAM-1, intercellular adhesion molecule, IL-1, interleukin-1; TNF, tumor necrosis factor; UV, ultraviolet.

shown by immunostaining experiments with LE antibodies that several lupus autoantigens including Ro/SSA, La/SSB, SnRNP, and Sm are strikingly redistributed from the intracellular nuclear compartment to the cell surface within a few hours following UVB irradiation and initiation of the apoptotic machinery in human (non-LE) keratinocytes. These autoantigens were found in apoptotic bodies and small surface blebs, the latter containing also phosphatidyl serine, an anionic phospholipid commonly known as an apoptosis marker. A similar phenomenon of antigen recognition on the cell surface has been likewise proposed for anti-DNA antibodies. However, apoptosis is a physiologic process and apoptotic cells are regarded as dominant tolerogens for the immune system.50,51 These observations raise the question as to whether abnormalities exist in the UVBmediated apoptotic process itself in keratinocytes (or other cell types) of patients with CLE, or whether aberrations exist in the clearance process of apoptotic cells. While the precise answer to both questions is still open, there is increasing evidence that abnormal clearance of apoptotic cells or immune complexes via soluble complement components, especially C1q, may render patients susceptible to initiation of an

autoimmune response. For example, it is well established that immune complex processing is insufficient in patients with hypocomplementemia. Antibodies against C1q are strongly associated with severe SLE (especially with kidney involvement) and with hypocomplementemic urticarial vasculitis. Moreover, mice deficient for C1q or SAP exhibit high titers of ANAs and develop immune complex vasculitis in the kidney resulting in glomerulonephritis.52 In analogy to human keratinocytes irradiated with apoptosis-inducing doses of UVB in vitro, C1q-deficient animals display apoptotic bodies within the inflamed kidneys and exhibit delayed clearance of apoptotic cells. Abnormal processing of apoptotic cells in LE is supported by the fact that patients with SLE have defects in macrophage differentiation in vitro from CD34+ stem cells and exhibit impaired clearance of apoptotic cells in lymph nodes and skin biopsies.53

Role of Inflammatory Cells As outlined above, inflammatory cells, mainly lymphocytes, are consistently found in dermal infiltrates of CLE lesions. Based on the photosensitivity in combination with the presence of circulating ANAs (especially anti-Ro/SSA antibodies), and the described exposure

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MECHANISMS OF SKIN DAMAGE

326

of nuclear autoantigens on the cell surface of keratinocytes exposed to UVB light, a model of antibodydependent cellular cytoxicity may be proposed, especially for SCLE, neonatal LE, and possibly for SLE. In LE patients without photosensitivity, those with absence of detectable circulating ANAs (e.g., in some patients with DLE) or patients with CLE variants without prominent epidermal damage, however, other mechanisms leading to dermal inflammation must be considered. Accordingly, a mechanism of delayed-type hypersensitivity involving autoantigen-specific lymphocytes may be proposed. Immunophenotyping of infiltrating dermal inflammatory cells in lesional skin of patients with CLE revealed high numbers of CD4+ and CD8+ lymphocytes.54 A role for CD4+ cells in cutaneous inflammation is supported by successful treatment of patients suffering from severe CLE with a chimeric CD4 monoclonal antibody, cM-T412.55 Studies investigating the specificity of the dermis-infiltrating lymphocytes in skin lesions of patients with SLE indicated that there is a clonal accumulation consistent with an autoantigen-driven response.56 In lesional skin of patients with DLE and acute CLE suffering from SLE, CD3+ cells were also detected, as well as CXCR3 on CD4+ and CD8+ cells. The latter surface molecule is a member of the chemokine receptor being a ligand for CXL9, CXL10, and CXL11, all of which were found to be expressed at the dermoepidermal junction of patients with CLE.57 CXCR3 is otherwise expressed by CD45RO+, cells which are found in both epidermal- and subepidermal-infiltrating lymphocytes of spontaneous and experimentally induced CLE lesions earlier than CD45RA+ and CD31+ cells. In addition to the above T-cell subtypes, a role for epidermal Langerhans cells in the pathogenesis of CLE has been suggested. Langerhans cells decrease in number during UV irradiation and reduced numbers of these cells were also found in the lesional epidermis of CLE. On the other hand, CD36+ dendritic macrophages were found to be increased in lesional skin of patients with CLE (similar to UV-irradiated skin), suggesting through their capability to activate CD45RA+ cells a stimulatory role for the autoimmune response.58 Recently, plasmocytoid dendritic cells which naturally produce IFN-α/β have been found to accumulate in CLE lesions of patients with both SLE and DLE.59 The number of these cells in lesional skin correlated with those cells expressing the IFN-α/β–inducible protein MxA. In addition, the number of plasmocytoid dendritic cells in lesional skin coincided with the P-selectin ligand peripheral lymph node address on dermal endothelial cells. Through their ability to stimulate lymphocyte extravasation and activation, plasmocytoid dendritic cells may contribute to the pathogenesis of skin lesions in LE.

Key mediators regulating the influx of T cells into the skin of patients with LE are cellular adhesion molecules (CAM). These are members of the immunoglobulin superfamily, and include intercellular CAM-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin (ESELE). All are strongly induced by proinflammatory signals such as IL-1, TNF-α, or UV irradiation. Accordingly, increased in situ expression of these CAMs has been detected in various forms of LE, but also in other inflammatory skin conditions such as polymorphic light eruption, scleroderma, and lichen planus.60-63 Efforts have been made to identify distinct patterns of these CAMs in lesional skin of SLCE, SLE, and DLE in comparison with other inflammatory skin diseases. However, the overall picture is not clear. The most consistent finding appears to be overexpression of ICAM-1 detectable throughout the epidermis, sometimes linear, in SCLE and SLE, versus a more focally distributed overexpression in the basal layers of the epidermis in DLE. VCAM-1 has been found to be overexpressed in endothelial cells of lesional skin of CLE, as well as in nonlesional skin of SLE, underscoring the role of an activated endothelium in the latter LE form. The increased in situ expression of these CAMs may reflect the behavior of these molecules to be up-regulated by TNF-α (which is strongly induced by UVB exposure) and/or UVA/B per se. In addition to overexpression of ICAM-1 and VCAM-1 in lesions in the skin of patients with LE, increased serum levels of the corresponding soluble forms of these CAMs have been detected.64 Interestingly, increased serum VCAM-1 levels have been found to correlate with disease activity of SLE. Likewise, elevated levels of ESELE have been reported in patients with active widespread CLE lesions suggesting the usefulness of both sCAMs as LE activity markers. Whereas the precise functional role of the increased serum levels of sCAM-1, sVCAM-1, and sSELE for the pathogenesis of skin damage of LE remains to be defined, the pathogenetic role for ICAM-1 is underscored by blockade of ICAM-1 in SLE-prone MRL/lpr mice. Intraperitoneal injection of an anti–ICAM-1 antibody prevented both neurologic disease as well as the development of vasculitic skin lesions in the treated animals.65

CONCLUSIONS Multiple pathogenetic events have been found to be involved in the mechanism of skin damage in patients with SLE. They include accumulation of apoptotic epidermal keratinocytes induced by UV irradiation, exposure of nuclear antigens on the cellular surface, impaired clearing of apoptotic cells, and initiation and maintenance of an autoimmune response in individuals

the identified pathogenetic steps in the former LE forms, however, it can be expected that targeting apoptosis, cytokine function, cellular adhesion, and humoral and cellular immune effector pathways will become promising novel treatment avenues in fighting LE.

REFERENCES

with a multigenic susceptibility. While until now most advances in elucidating these events have been made in SLE, SCLE, and DLE, the mechanisms leading to the mostly nonsystemic CLE forms remain largely descriptive. Future studies will have to address these CLE forms at the pathogenetic level as well. Based on

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36. Pablos JL, Carreira PE, Martin-Villa JM, Montalvo G, Arnaiz-Villena A, Gomez-Reino JJ. Polymorphism of the heat-shock protein gene HSP70-2 in systemic lupus erythematosus. Br J Dermatol 1995; 34:721-723. 37. Ghoreishi M, Katayama I, Yokozeki H, Nishioka K. Analysis of 70 KD heat shock protein (HSP70) expression in the lesional skin of lupus erythematosus (LE) and LE related diseases. J Dermatol 1993;20:400-405. 38. Villalobos-Hurtado R, Sanchez-Rogriguez SH, Avalos-Diaz E, Herrera-Esparza R. Possible role of Hsp70 in autoantigen shuttling to the dermo-epidermal junction in systemic lupus erythematosus. Reumatismo 2003;55:155-158. 39. Suzuki H, Matsui Y, Kashiwagi H. Interleukin-1 receptor antagonist gene polymorphism in Japanese patients with systemic lupus erythematosus. Arthritis Rheum 1997;40:389-390. 40. Eskdale J, Kube D, Tesch H, Gallagher G. Mapping of the human IL10 gene and further characterization of the 5’ flanking sequence. Immunogenetics 1997;46:120-128. 41. Lazarus M, Hajeer AH, Turner D, Sinnott P, Worthington J, Ollier WE, et al. Genetic variation in the interleukin 10 gene promoter and systemic lupus erythematosus. J Rheumatol 1997;24: 2314-2317. 42. 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. 43. Ollier W, Davies E, Snowden N, Alldersea J, Fryer A, Jones P, et al. Association of homozygosity for glutathione-S-transferase GSTM1 null alleles with the Ro+/La− autoantibody profile in patients with systemic lupus erythematosus. Arthritis Rheum 1996;39:1763-1764. 44. Huang QR, Danis V, Lassere M, Edmonds J, Manolios N. Evaluation of a new Apo-1/Fas promoter polymorphism in rheumatoid arthritis and systemic lupus erythematosus patients. Rheumatology (Oxford) 1999;38:645-651. 45. Furukawa F, Kanauchi H, Wakita H, Tokura Y, Tachibana T, Horiguchi Y, et al. Spontaneous autoimmune skin lesions of MRL/n mice: autoimmune disease-prone genetic background in relation to Fas-defect MRL/1pr mice. J Invest Dermatol 1996;107:95-100. 46. Johansson M, Arlestig L, Moller B, Smedby T, Rantapaa-Dahlqvist S. Oestrogen receptor a gene polymorphisms in systemic lupus erythematosus. Ann Rheum Dis 2005;64:1611-1617. 47. Casciola-Rosen LA, Anhalt G, Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 1994;179:1317-1330. 48. Casciola-Rosen L, Rosen A, Petri M, Schlissel M. Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus. Proc Natl Acad Sci U S A 1996;93:1624-1629. 49. Casciola-Rosen L, Rosen A. Ultraviolet light-induced keratinocyte apoptosis: a potential mechanism for the induction of skin lesions and autoantibody production in LE. Lupus 1997;6:175-180. 50. Henson PM, Bratton DL, Fadok VA. Apoptotic cell removal. Curr Biol 2001;11:R795-805.

51. Savill J, Fadok V. Corpse clearance defines the meaning of cell death. Nature 2000;407:784-788. 52. Botto M, Dell’Agnola C, Bygrave AE, Thompson EM, Cook HT, Petry F, et al. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 1998;19:56-59. 53. Gaipl US, Kuhn A, Sheriff A, Munoz LE, Franz S, Voll RE, et al. Clearance of apoptotic cells in human SLE. Curr Dir Autoimmun 2006;9:173-187. 54. Hasan T, Stephansson E, Ranki A. Distribution of naive and memory T-cells in photoprovoked and spontaneous skin lesions of discoid lupus erythematosus and polymorphous light eruption. Acta Derm Venereol 1999;79:437-442. 55. Prinz JC, Meurer M, Reiter C, Rieber EP, Plewig G, Riethmuller G. Treatment of severe cutaneous lupus erythematosus with a chimeric CD4 monoclonal antibody, cM-T412. J Am Acad Dermatol 1996;34:244-252. 56. Kita Y, Kuroda K, Mimori T, Hashimoto T, Yamamoto K, Saito Y, et al. T cell receptor clonotypes in skin lesions from patients with systemic lupus erythematosus. Invest Dermatol 1998;110: 41-46. 57. Flier J, Boorsma DM, van Beek PJ, Nieboer C, Stoof TJ, Willemze R, et al. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J Pathol 2001;194:398-405. 58. Andrews BS, Schenk A, Barr R, Friou G, Mirick G, Ross P. Immunopathology of cutaneous human lupus erythematosus defined by murine monoclonal antibodies. J Am Acad Dermatol 1986;15:474-481. 59. Farkas L, Beiske K, Lund-Johansen F, Brandtzaeg P, Jahnsen FL. Plasmacytoid dendritic cells (natural interferon-alpha/betaproducing cells) accumulate in cutaneous lupus erythematosus lesions. Am J Pathol 2001;159:237-243. 60. Stephansson E, Ros AM. Expression of intercellular adhesion molecule-1 (ICAM-1) and OKM5 in UVA- and UVB-induced lesions in patients with lupus erythematosus and polymorphous light eruption. Arch Dermatol Res 1993;285:328-333. 61. Nyberg F, Hasan T, Skoglund C, Stephansson E. Early events in ultraviolet light-induced skin lesions in lupus erythematosus: expression patterns of adhesion molecules ICAM-1, VCAM-1 and E-selectin. Acta Derm Venereol 1999;79:431-436. 62. Jones SM, Mathew CM, Dixey J, Lovell CR, McHugh NJ. VCAM-1 expression on endothelium in lesions from cutaneous lupus erythematosus is increased compared with systemic and localized scleroderma. Br J Dermatol 1996;135:678-686. 63. Belmont HM, Buyon J, Giorno R, Abramson S. Up-regulation of endothelial cell adhesion molecules characterizes disease activity in systemic lupus erythematosus. The Shwartzman phenomenon revisited. Arthritis Rheum 1994;37:376-383. 64. Nyberg F, Acevedo F, Stephansson E. Different patterns of soluble adhesion molecules in systemic and cutaneous lupus erythematosus. Exp Dermatol 1997;6:230-235. 65. Brey RL, Amato AA, Kagan-Hallet K, Rhine CB, Stallworth CL, Brey-R. Anti-intercellular adhesion molecule-1 (ICAM-1) antibody treatment prevents central and peripheral nervous system disease in autoimmune-prone mice. Lupus 1997;6: 645-651.

CLINICAL ASPECTS OF THE DISEASE

29

Constitutional Features of Systemic Lupus Erythematosus Caroline Gordon, MA, MD, FRCP

INTRODUCTION Constitutional features are a common but rather nonspecific aspect of SLE. This chapter will review the assessment, differential diagnosis, and treatment of these features of SLE. Initially, fatigue will be reviewed. This is a somewhat controversial area as many people do not believe that fatigue can be attributed to SLE; however, it is the most common symptom described by patients. The term “fatigue” should reflect a subjective feeling of extraordinary tiredness, often associated with weariness, exhaustion, or lassitude, and frequently but not necessarily associated with irritability, inefficiency, and decreased capacity for work, as defined in the glossary for the European Consensus Lupus Assessment Method (ECLAM).1 The chapter then covers fever, weight loss, lymphadenopathy, and anorexia. Splenomegaly, although sometimes considered part of the constitutional features of lupus, as in the British Isles Lupus Assessment Group (BILAG) index of disease activity for lupus,2 is usually considered part of the hematologic manifestations of lupus because it is often associated with thrombocytopenia (see Chapter 36). Hepatomegaly is discussed in the chapter on gastrointestinal features of SLE (see Chapter 36). Nausea and vomiting, which are sometimes considered with anorexia under general features (as in the BILAG index2), are discussed in Chapter 36. Finally, this chapter briefly reviews sicca symptoms due to secondary Sjögren’s syndrome in SLE patients, with some overlap with Chapters 34 and 39.

FATIGUE

Association with Disease Activity If you ask SLE patients what disturbed their quality of life most, the answer is fatigue, and it is one of the most common complaints by lupus patients, occurring in about 50 to 86% of patients.3-5 There has been much debate about whether fatigue is due to lupus. There is no doubt that fatigue (or malaise or lethargy) is often present during flares of active lupus disease. Patients will

say, “I feel as tired in the morning as when I go to bed,” or “I sleep at least 10 hours at night and get up in the morning for 1 or 2 hours and am ready to go back to bed and do actually sleep for another 2 or 3 hours.” The fatigue associated with active lupus disease is an overwhelming feeling of fatigue that is associated with an ability to sleep, and is quite distinct from the fatigue that comes from patients with sleep deprivation due to various sleep disorders. It tends to be more variable than the fatigue that occurs in patients with fibromyalgia or depression. These patients tend to complain that fatigue is present most of the time, irrespective of the amount of sleep that the patient has had. Patients with active lupus disease may describe a reduced capacity to undertake physical activities, but this is often associated with other manifestations of active lupus, such as arthralgia or arthritis with early morning stiffness, or myalgia with or without weakness due to a myositic component. The fatigue in these patients will often show variation over time in parallel with improvement or deterioration in the other manifestations of lupus. In patients prone to fever (discussed below), for example, the fatigue will often be most evident during the periods of fever and be relatively less troublesome when the fever abates. In these patients, hydroxychloroquine may be helpful in relieving fatigue and the tendency to fever flares (see Chapter 44). There is some evidence that stopping hydroxychloroquine results in increased fatigue, but no controlled trials show that it reduces fatigue. Therapy with hydroxychloroquine was associated with more fatigue in one study,5 but this probably reflects confounding by indication for therapy with hydroxychloroquine. Patients with inflammatory disease affecting their joints and muscles may well describe difficulty carrying out activities as a result of their inflammatory process. Patients who have been on long-term corticosteroids may develop a myopathy with proximal weakness in the upper and lower limbs. Similarly, patients with cardiorespiratory activity or damage, and those with neurologic problems may also feel that many activities are “rather an effort.” These difficulties related to activity

329

CONSTITUTIONAL FEATURES OF SLE

BOX 29.1 CONSTITUTIONAL FEATURES IN SYSTEMIC LUPUS ERYTHEMATOSUS ●

Fatigue

●

Fever

●

Lymphadenopathy

●

Weight loss

●

Sicca symptoms

or damage associated with specific system involvement need to be distinguished from the symptom fatigue. Although some studies have shown an association between disease activity and lupus,4,5 others have not shown a close association between fatigue and disease activity or damage with in SLE patients.6,7 The problem is that patients without active disease also complain about fatigue. This is likely to reflect the multifactoral nature of fatigue in lupus, and it is therefore important to consider other possible underlying causes (Table 29.1).

Association with Anemia or Thyroid Disease Anemia is the most common condition associated with and contributing to fatigue in lupus patients, but is rarely severe enough to account for the degree of fatigue experienced by the patients.5 A mild anemia of chronic disease due to lupus is often present. In addition, many women with SLE, particularly those on warfarin, develop iron-deficiency anemia as a result of menstrual blood loss. This may be compounded by deficient intake of iron in the diet or gastrointestinal

TABLE 29.1 CAUSES AND ASSOCIATIONS OF FATIGUE IN SYSTEMIC LUPUS ERYTHEMATOSUS Causes of Fatigue

blood loss, usually due to drugs rather than disease. Hemolytic anemia (see Chapter 36) is less common but can be severe. Occasionally anemia related to folic acid deficiency is seen, usually in association with high redcell turnover in hemolytic anemia. Otherwise, folate deficiency is rare and patients with this problem should be investigated for celiac disease, particularly if they have coincidental iron and calcium deficiency. Celiac disease is one of the autoimmune conditions that can coexist with SLE, because they are both associated with the human leukocyte HLA-B8 and HLA-DR3 histocompatibility antigens.8 Another autoimmune condition that can contribute to anemia is vitamin B12 deficiency. Pernicious anemia with antibodies to intrinsic factor resulting to impaired absorption of B12 may be present in patients before the diagnosis of SLE or appear after the diagnosis of SLE.9 There is also an increased frequency of pernicious anemia in the relatives of patients with SLE. Similarly, autoimmune hypothyroidism can either precede or follow a diagnosis of SLE, be present in the relatives of patients with SLE and can undoubtedly contribute to fatigue in SLE patients.10 In patients with chronic renal disease, fatigue may be associated with anemia that can be helped by treatment with erythropoietin, although they may also have an unresponsive degree of fatigue associated with uremia. Otherwise, treatment is dependent on the cause of the anemia or appropriate to the degree of hypothyroidism present (Table 29.2).

TABLE 29.2 POSSIBLE TREATMENTS FOR FATIGUE IN SYSTEMIC LUPUS ERYTHEMATOSUS (DEPENDING ON UNDERLYING CAUSE) Treatment for Fatigue

Comments and Examples

Treatment of active disease

Hydroxychloroquine Immunosuppression as appropriate to systems involved and severity

Correction of anemia

Immunosuppression Iron Folic acid B12 injections Erythropoietin

Underlying Conditions

Active lupus disease Anemia

Anemia of chronic disease Hemolytic anemia Iron-deficiency anemia Folate deficiency Pernicious anemia Celiac disease Renal failure

Treament of hypothyroidism

Hypothyroidism

Antidepressants

Fibromyalgia

Self-management program

To improve self-efficacy and problem solving

Psychoeducational intervention

With or without partner

Exercise program

Graded aerobic activity Cardiovascular training

Depression Sleep disorders Impaired aerobic capacity

330

Low anaerobic threshold Physical deconditioning

Patients with lupus may develop fibromyalgia.7 It is important to establish whether patients have had chronic fatigue for at least 3 months, with chronic widespread pain above and below the diaphragm and on both sides of the body associated with the tender points characteristic of fibromyalgia,3 as this will influence the management of fatigue. Previous studies have shown an association between fatigue and the number of tender points in lupus patients.7,11 In a cross-sectional study of 260 patients attending two lupus clinics in Birmingham and London, only 10% of the lupus patients fulfilled criteria for fibromyalgia syndrome, whereas 50% complained of fatigue.3 In North America, studies have shown a frequency of fibromyalgia closer to 20 to 25% in SLE patients.12,13 This gap may reflect differences in the ethnic population in various lupus cohorts, and a difference in psychosocial influences on the development of fatigue, fibromyalgia, and impaired quality of life in general in lupus patients (see Chapter 4). We and others have shown that fatigue which is part of the assessment of vitality in the SF36 health survey is determined, like other domains of the SF36 health survey, predominantly by psychosocial factors and only to a small extent by any aspects of disease activity or chronic damage.7,12,14-17 Patients that are depressed often have fatigue.5,6 The possibility that fatigue in lupus patients may be associated with neuropsychiatric disease and disturbances in cerebral blood flow has been assessed using SPECT scanning.18 However, no such association was found. Fatigue may be associated with sleep disorders,5,19 with or without associated fibromyalgia. The study by Costa and colleagues19 suggested that depressed mood, prednisone use, and lack of exercise contribute to decreased overall sleep quality. The potential for psychoeducational interventions to improve fatigue has been demonstrated in a randomized controlled trial involving 122 patients (plus their partners) designed to improve patient self-efficacy, couple communication about lupus, social support, and problem solving.20 Patients receiving the educational intervention demonstrated significantly higher scores in couple communication, self-efficacy, and mental health status, and lower fatigue scores compared with the control group.20

the management of fatigue in SLE patients.21,25 Tench and colleagues26 reported a randomized study with 93 female patients which showed that an appropriately prescribed, but largely unsupervised, graded aerobic exercise program can be useful in the management of lupus patients with fatigue in the absence of active lupus disease. A Brazilian group27 has shown that supervised cardiovascular training can significantly improve exercise tolerance, aerobic capacity, quality of life, and depression in a controlled study involving 60 female SLE patients. The concept of exercise programs to improve fatigue, physical deconditioning, depression, poor sleep quality, fibromyalgia, and cardiovascular fitness is attractive, and further research in this area is urgently needed.

FEVER Whereas fatigue is one of the most common complaints of SLE patients, fever is one of the least common, but it causes the greatest diagnostic uncertainty, particularly when it is one of the first major manifestations of active lupus in a patient not yet diagnosed as suffering from lupus. Before attributing fever to lupus, it is clearly essential to exclude infection and cancer, particularly in patients with lymphadenopathy (Table 29.3).

Exclusion of Infection It is important to remember that lupus patients, both on and off immunosuppressive therapy, are at increased risk of infections, and will need screening for not only common bacterial infections, but also for bacterial endocarditis and less common pathogens including tuberculosis and atypical mycobacteria that may cause lymphadenopathy in addition to fever (see Chapter 35). Infections that are often associated with HIV infection, such as Pneumocystis carinii, fungal and

TABLE 29.3 DIFFERENTIAL DIAGNOSIS OF FEVER AND WEIGHT LOSS IN SLE PATIENTS Cause of Fever and Weight Loss

Comments

Acute Lupus

Usually Associated with Other Manifestations of SLE

Infection

Viral Bacterial Mycobacterial Fungal Protozoal Nematodes

Malignancy

Lymphoma (Hodgkin’s or non-Hodgkin’s) Primary carcinoma (e.g., renal) Metastatic carcinoma Myeloma

Association with Impaired Aerobic Capacity and Role of Exercise Therapy A number of studies have shown that women with SLE have impaired aerobic capacity and low anaerobic threshold, which is strongly associated with the perception of severe activity-limiting fatigue.21-24 These observations led to pilot studies looking at the role of exercise in

FEVER

Association with Fibromyalgia, Impaired Health Status, and Sleep Disturbance

331

CONSTITUTIONAL FEATURES OF SLE

viral infections, nematodes, and toxoplasmosis and other protozoal infections, should be considered in SLE patients with fever. Lupus patients are at increased risk of such infections because they often have antibodies to CD4+ T cells (see also Chapter 35). Consequently, they can have low CD4+ T-cell counts and reversed CD4:CD8 lymphocyte ratio, and they may even be misdiagnosed initially as suffering from HIV. Before antibiotics are started, multiple blood cultures, appropriate swabs of any potential sites of infection, and cultures and examination of urine, stool, and sputum should be done. Further investigation may include white cell scans, ultrasound, and CT or MRI scans to exclude abscesses or cancers.

Exclusion of Cancer The possibility of malignancy and lymphoma as causes of fever should be considered, especially in patients with lymphadenopathy without evidence of infection (Table 29.3). Lymphadenopathy can occur in SLE patients, but cancer should be suspected if lymph nodes steadily increase in size. In lupus lymphadenopathy, the nodes usually fluctuate in size. Recent studies have confirmed that patients with SLE are at increased risk of malignancy, particularly non-Hodgkin’s lymphoma.28 Non-Hodgkin’s lymphoma appears to be particularly common around the time of presentation and diagnosis of SLE, but is not necessarily related to treatment with cytotoxic therapy.29 Apart from lymphoma causing fever in lupus patients, renal cell carcinoma in particular may present with fever and cause diagnostic confusion in SLE patients if red cells in the urine are attributed to lupus-related renal disease (Table 29.2).

Recording of Fever in Lupus Disease Activity Indices

332

Having excluded nonlupus causes of fever, pyrexia can be attributed to SLE and can be recorded as part of the assessment of disease activity in SLE, using standardized disease activity indices.1 It should be noted that the commonly used validated disease activity indices require different levels of fever to be observed before they can be recorded (see Appendices). The BILAG index of disease activity records a documented fever greater than 37.5°C.2 However, the ECLAM will record a fever of 37.5°C or more, and specifies that this should be the documented base or morning temperature.1 For the systemic lupus erythematosus disease activity index (SLEDAI), a fever higher than 38°C is required.30 In all cases, it is stated that infection must be excluded and that the temperature must have been documented and not estimated by the patient or doctor. It is much easier to attribute fever to lupus when there are other objective signs of active lupus disease, clinically with supporting serology. Patients presenting

for the first time with pyrexia of unknown origin and other nonspecific features, such as erythema nodosum or arthralgia and myalgia without overt synovitis, may cause diagnostic problems if a full history and screening tests for SLE are not performed. Patients presenting with pleuritic chest pain and fever need careful evaluation for pneumonia and pulmonary infarction due to pulmonary embolus as well as lupus activity. When assessed in emergency rooms, patients are often diagnosed as having infection and are treated with antibiotics without adequate investigation in the early stages. Only the persistence of the fever with general deterioration in the patient’s condition raises suspicion when the patient does not improve after two or three courses of antibiotics. Once a diagnosis of SLE is established, treatment with moderate- to high-dose steroids will usually result in resolution of the fever. Antimalarials, particularly hydroxychloroquine, have been found to be helpful in the long-term management of fever, but obviously take too long to work to be useful in the management of the initial presentation with fever. The fevers can be difficult to treat, and the possibility of missed infection or malignancy should never be forgotten; thus, re-investigation may be necessary if the patient fails to improve with immunosuppression or new features develop.

LYMPHADENOPATHY Lymphadenopathy is a recognized feature of SLE, although not specific. In the BILAG index, palpable lymph nodes greater than 1 cm in diameter are recorded.2 However, SLEDAI, the Systemic Lupus Activity Measure-Revised (SLAM-R), and ECLAM do not record lymphadenopathy.1 As with fever, it is always critically important to exclude underlying infection and malignancy. In lupus, the nodes usually fluctuate in size, and suspicion should be aroused if a single lymph node enlarges steadily without resolving. A few patients who present with fever and considerable, often localized, lymphadenopathy, on biopsy are found to have granulomatous necrotizing lymphadenitis, known as Kikuchi-Fujimoto syndrome.31,32,33 About 30% of patients presenting with this form of necrotizing lymphadenitis have or go on to develop SLE or discoid lupus.34-37 Recent papers have suggested that it may be possible to identify differences in the histology between those that are really SLE from the onset of lymphadenopathy and those that are true Kikuchi-Fujimoto syndrome.33,38 There has been no confirmed underlying viral cause for this syndrome, but the possibility that viruses are involved in triggering the granulomatous necrotizing lymphadenitis and the onset of SLE remains. A number of studies have confirmed a high frequency of previous EBV and CMV infection in SLE

WEIGHT LOSS Unexplained weight loss of at least 5% body weight can be a nonspecific finding of many inflammatory diseases including SLE. The triad of fever, weight loss, and lymphadenopathy is well recognized in infection, malignancy, and the vasculitides, as well as in lupus. Loss of appetite or anorexia usually precedes the development of weight loss. This symptom may be due to drug side effects, but certainly can be a manifestation of active lupus disease. The BILAG index will record both anorexia and unintentional weight loss (>5%) under constitutional or general features.2 Anorexia can be associated with gastrointestinal disturbance, particularly nausea and vomiting, and can certainly appear as an early manifestation of new onset lupus or lupus flare in the absence of gastrointestinal features (see Chapter 34). In the SLAM-R index,39 weight loss can be recorded as “mild” if it is up to 10% of previous body weight, and it is recorded as “severe” if greater than 10% of body weight. SLEDAI and ECLAM do not record weight loss (see Appendices).1 Weight loss usually responds to treatment given for other manifestations of lupus, particularly when corticosteroids are involved. However, as with fever and lymphadenopathy, such treatment should not be given or increased until infection and malignancy have been excluded. Specific investigations of the gastrointestinal tract may be required to exclude comorbid disease and to look for evidence of localized lupus involvement that is interfering with absorption of nutrients (see Chapter 34).

SICCA SYMPTOMS Dry eyes (keratoconjunctivitis) and dry mouth (xerostomia) are characteristic features of secondary Sjögren’s syndrome, which affects up to a third of SLE patients.40 Some patients also complain of vaginal dryness or dry cough for which no clear cause is found due to involvement of other mucosal tissues as in primary Sjögren’s syndrome. Sometimes it is difficult to establish whether the patient really has primary Sjögren’s syndrome or SLE with secondary Sjögren’s syndrome. A history of salivary gland swelling prior to the onset of dry eyes and dry mouth, and an association with antiRo and/or anti-La antibodies usually points to a

diagnosis of primary Sjögren’s syndrome. This can be confirmed by a labial gland biopsy, other histology, or objective evidence of salivary gland involvement. Such tests are essential for the diagnosis of primary Sjögren’s syndrome in the absence of anti-Ro and anti-La antibodies. Patients with features of SLE who have anti-Ro and antiLa antibodies, but no history of salivary or lacrimal gland swelling, nor dry eyes, dry mouth, or other dryness of mucosal surfaces at presentation are usually considered to have SLE. In time, these patients may develop secondary Sjögren’s syndrome.40 The possibility that drugs— for example, tricyclic antidepressants—are causing sicca symptoms should be considered. In order to prevent serious complications resulting from the loss of the tear film on the eye in patients with dry eyes due to secondary Sjögren’s syndrome, patients should be advised to use artificial tears/lubricant eye drops liberally from the onset of such symptoms (see Chapter 39). Dryness of the mouth is much harder to treat (see also Chapter 34). There are various oral sprays, pastilles, and lozenges on the market that are said to improve salivary flow, but for which there is little evidence and little support from most patients. More recently, there has been interest in the treatment of primary Sjögren’s syndrome with pilocarpine, but there is no study addressing the use of this drug in SLE patients. In patients with primary Sjögren’s syndrome, various side effects have limited the use of this drug, although the side effects are often dose dependent and it may be possible to find an appropriate dose for a given patient.

CONCLUSIONS

patients (see Chapter 35), but the role of these viruses in the lymphadenopathy and the onset of lupus remains unclear. Lymphadenopathy appears to be most common in the first few years of lupus and is rare later in the disease cause. In fact, the development of lymphadenopathy for the first time in a patient who has had SLE for over 5 years should in my opinion arouse great suspicion of malignancy.

CONCLUSIONS Constitutional features of systemic lupus erythematosus, although nonspecific, are important features of the disease to assess and treat because they cause considerable distress to the patient. As discussed in this chapter, it is essential to consider the differential diagnosis of each symptom and sign in turn. Only after excluding other possibilities can the various features be attributed to lupus and treated as such. In our cohort in Birmingham, we have studied 591 patients over the last 16 years. We have found that 9.1% of patients have had at least one A score representing severe disease in the constitutional or general system. In order to score A on the BILAG index in this system, the patients must have suffered from fever and have two other symptoms among fatigue, weight loss, lymphadenopathy, and anorexia; or they may have had four of these features. Thus, if they do not have fever, they will have had objective signs including weight loss and lymphadenopathy in addition to fatigue and anorexia. These patients required high-dose immunosuppression with prednisolone over 20mg/day or equivalent therapy, such as intravenous methylprednisolone

333

CONSTITUTIONAL FEATURES OF SLE

to treat these symptoms that significantly interfered with the patient’s lifestyle and overall quality of life. Immunosuppression and antimalarial therapy are important in the treatment of fever, weight loss, and lymphadenopathy due to lupus. The treatment of fatigue may require other measures such as an aerobic exercise program, self-management plan, or psychoeducational intervention. In all cases, a careful search for and treatment of comorbid conditions including

depression, fibromyalgia, and sleep disorders should be undertaken. Patients need to be encouraged to pace their activities with an appropriate amount of rest and exercise depending on the severity of their disease activity and the amount of accumulated damage that they have sustained from their disease and its therapy. Further work is required to fully understand all the underlying causes for fatigue in lupus patients and how to determine what will be the most effective management plan for each patient.

REFERENCES

334

1. Griffiths B, Mosca M, Gordon C. Assessment of patients with systemic lupus erythematosus and the use of lupus disease activity indices. Best Pract Res Clin Rheumatol 2005;19: 685-708. 2. Hay EM, Bacon PA, Gordon C, Isenberg DA, Maddison P, Snaith ML, et al. The BILAG index: a reliable and valid instrument for measuring clinical disease activity in systemic lupus erythematosus. QJM 1993;86:447-58. 3. Taylor J, Skan J, Erb N, Carruthers D, Bowman S, Gordon C, et al. Lupus patients with fatigue-is there a link with fibromyalgia syndrome? Rheumatology (Oxford) 2000;39:620-3. 4. Zonana-Nacach A, Roseman JM, McGwin G Jr, Friedman AW, Baethge BA, Reveille JD, et al. Systemic lupus erythematosus in three ethnic groups. VI. Factors associated with fatigue within 5 years of criteria diagnosis. LUMINA Study Group. Lupus in minority populations: nature vs nurture. Lupus 2000;9:101-9. 5. Tench CM, McCurdie I, White PD, D’Cruz DP. The prevalence and associations of fatigue in systemic lupus erythematosus. Rheumatology (Oxford) 2000;39(11):1249-54. 6. Wang B, Gladman DD, Urowitz MB. Fatigue in lupus is not correlated with disease activity. J Rheumatol 1998;25:892-5. 7. Bruce IN, Mak VC, Hallett DC, Gladman DD, Urowitz MB. Factors associated with fatigue in patients with systemic lupus erythematosus. Ann Rheum Dis 1999;58:379-81. 8. Marai I, Shoenfeld Y, Bizzaro N, Villalta D, Doria A, Tonutti E, et al. IgA and IgG tissue transglutaminase antibodies in systemic lupus erythematosus. Lupus 2004;13:241-4. 9. McDonagh JE, Isenberg DA. Development of additional autoimmune diseases in a population of patients with systemic lupus erythematosus. Ann Rheum Dis 2000;59:230-2. 10. Pyne D, Isenberg DA. Autoimmune thyroid disease in systemic lupus erythematosus. Ann Rheum Dis 2002;61:70-2. 11. Akkasilpa S, Goldman D, Magder LS, Petri M. Number of fibromyalgia tender points is associated with health status in patients with systemic lupus erythematosus. J Rheumatol 2005;32:48-50. 12. Gladman DD, Urowitz MB, Gough J, MacKinnon A. Fibromyalgia is a major contributor to quality of life in lupus. J Rheumatol 1997;24:2145-8. 13. Wang B, Gladman DD, Urowitz MB. Fatigue in lupus is not correlated with disease activity. J Rheumatol 1998;25:892-5. 14. Stoll T, Gordon C, Seifert B, Richardson K, Malik J, Bacon PA, et al. Consistency and validity of patient administered assessment of quality of life by the MOS SF-36: its association with disease activity and damage in patients with systemic lupus erythematosus. J Rheumatol 1997;24:1608-14. 15. Sutcliffe N, Clarke AE, Levinton C, Frost C, Gordon C, Isenberg DA. Associates of health status in patients with systemic lupus erythematosus. J Rheumatol 1999;26:2352-6. 16. Omdal R, Waterloo K, Koldingsnes W, Husby G, Mellgren SI. Fatigue in patients with systemic lupus erythematosus: the psychosocial aspects. J Rheumatol 2003;30:283-7. 17. Alarcon GS, McGwin G Jr, Uribe A, Friedman AW, Roseman JM, Fessler BJ, et al. Systemic lupus erythematosus in a multiethnic lupus cohort (LUMINA). XVII. Predictors of self-reported

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health-related quality of life early in the disease course. Arthritis Rheum 2004;51:465-74. Omdal R, Sjoholm H, Koldingsnes W, Sundsfjord JA, Jacobsen EA, Husby G, et al. Fatigue in patients with lupus is not associated with disturbances in cerebral blood flow as detected by SPECT. J Neurol 2005;252:78-83. Costa DD, Bernatsky S, Dritsa M, Clarke AE, Dasgupta K, Keshani A, et al. Determinants of sleep quality in women with systemic lupus erythematosus. Arthritis Rheum 2005;53:272-8. Karlson EW, Liang MH, Eaton H, Huang J, Fitzgerald L, Rogers MP, et al. A randomized clinical trial of a psychoeducational intervention to improve outcomes in systemic lupus erythematosus. Arthritis Rheum 2004;50:1832-41. Robb-Nicholson LC, Daltroy L, Eaton H, Gall V, Wright E, Hartley LH, et al. Effects of aerobic conditioning in lupus fatigue: a pilot study. Br J Rheumatol 1989;28:500-5. Sakauchi M, Matsumura T, Yamaoka T, Koami T, Shibata M, Nakamura M, et al. Reduced muscle uptake of oxygen during exercise in patients with systemic lupus erythematosus. J Rheumatol 1995;22:1483-7. Forte S, Carlone S, Vaccaro F, Onorati P, Manfredi F, Serra P, et al. Pulmonary gas exchange and exercise capacity in patients with systemic lupus erythematosus. J Rheumatol 1999;26: 2591-4. Keyser RE, Rus V, Cade WT, Kalappa N, Flores RH, Handwerger BS. Evidence for aerobic insufficiency in women with systemic Lupus erythematosus. Arthritis Rheum 2003;49:16-22. Ramsey-Goldman R, Schilling EM, Dunlop D, Langman C, Greenland P, Thomas RJ, et al. A pilot study on the effects of exercise in patients with systemic lupus erythematosus. Arthritis Care Res 2000;13:262-9. Tench CM, McCarthy J, McCurdie I, White PD, D’Cruz DP. Fatigue in systemic lupus erythematosus: a randomized controlled trial of exercise. Rheumatology (Oxford) 2003;42:1050-4. Carvalho MR, Sato EI, Tebexreni AS, Heidecher RT, Schenkman S, Neto TL. Effects of supervised cardiovascular training program on exercise tolerance, aerobic capacity, and quality of life in patients with systemic lupus erythematosus. Arthritis Rheum 2005;53:838-44. Bernatsky S, Boivin JF, Joseph L, Rajan R, Zoma A, Manzi S, et al. An international cohort study of cancer in systemic lupus erythematosus. Arthritis Rheum 2005;52:1481-90. Bernatsky S, Ramsey-Goldman R, Rajan R, Boivin JF, Joseph L, Lachance S, et al. Non-Hodgkin’s lymphoma in systemic lupus erythematosus. Ann Rheum Dis 2005;64:1507-9. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 1992;35:630-40. Nieman RB. Diagnosis of Kikuchi’s disease. Lancet 1990; 335(8684):295. Dikov DI, Staikova ND, Solakov PT. Differential diagnosis of Kikuchi’s disease and systemic lupus erythematosus lymphadenopathy: clinicopathologic algorithm. Folia Med (Plovdiv) 2000;42:34-6.

38. Hu S, Kuo TT, Hong HS. Lupus lymphadenitis simulating Kikuchi’s lymphadenitis in patients with systemic lupus erythematosus: a clinicopathological analysis of six cases and review of the literature. Pathol Int 2003;53:221-6. 39. Liang MH, Socher SA, Larson MG, Schur PH. Reliability and validity of six systems for the clinical assessment of disease activity in systemic lupus erythematosus. Arthritis Rheum 1989;32: 1107-18. 40. Prabu A, Marshall T, Gordon C, Plant T, Bawendi A, Heaton S, et al. Use of patient age and anti-Ro/La antibody status to determine the probability of patients with systemic lupus erythematosus and sicca symptoms fulfilling criteria for secondary Sjogren’s syndrome. Rheumatology (Oxford) 2003;42: 189-91.

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33. Bosch X, Guilabert A, Miquel R, Campo E. Enigmatic KikuchiFujimoto disease: a comprehensive review. Am J Clin Pathol 2004;122:141-52. 34. Dorfman RF, Berry GJ. Kikuchi’s histiocytic necrotizing lymphadenitis: an analysis of 108 cases with emphasis on differential diagnosis. Semin Diagn Pathol 1988;5:329-45. 35. Kapadia V, Robinson BA, Angus HB. Kikuchi’s disease presenting as fever of unknown origin. Lancet 1989;2:1519-20. 36. Lecoules S, Michel M, Zarrouk V, Gaulard P, Schaeffer A, Godeau B. [Recurrent Kikuchi’s disease in a patient with discoid lupus]. Rev Med Interne 2003;24:613-6. 37. Lin HC, Su CY, Huang CC, Hwang CF, Chien CY. Kikuchi’s disease: a review and analysis of 61 cases. Otolaryngol Head Neck Surg 2003;128:650-3.

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CLINICAL ASPECTS OF THE DISEASE

30

Kidney Involvement in Systemic Lupus Erythematosus Gabor G. Illei, MD and James E. Balow, MD

The kidney is the most commonly involved major internal organ in patients with systemic lupus erythematosus (SLE). It is important to recognize that lupus kidney disease is not expressed as a simple phenotype. Indeed, there are extraordinarily broad spectra of both clinical manifestations and pathologic categories of lupus kidney disease. Not surprisingly, no single paradigm can be offered to define the pathogenesis of all forms of lupus nephritis. Most patients with renal involvement have immune complex–mediated glomerulonephritis, but extraglomerular tubulointerstitial inflammation and vasculopathy are also relatively common components of lupus nephropathy. Vasculopathy is mostly due to inadequately controlled hypertension, but SLErelated microvascular thrombosis and rarely vasculitis can contribute significantly to lupus renal disease.

DIAGNOSIS AND ASSESSMENT OF DISEASE ACTIVITY

Clinical Presentation of Nephritis

336

Depending on the populations studied, nephritis occurs in about 50 to 75% of patients with SLE. In those affected, nephritis characteristically appears within the first year after diagnosis of SLE. Careful screening tests are critical because most patients present with asymptomatic urine abnormalities, such as hematuria or proteinuria, or with new onset or worsening hypertension. Nocturia (due to loss of renal concentrating capacity) and/or foamy urine (due to proteinuria) are common initial manifestations of renal involvement but are rarely recognized unless the patient is specifically queried by the clinician. Proteinuria reflects the extent of involvement of peripheral glomerular capillary loops and tends to increase incrementally from cases of mesangial to endocapillary proliferative to membranous forms of lupus nephropathy. The latter involves virtually all glomerular capillary loops and is characteristically accompanied by heavy, nephrotic range (>3.5 g/day) proteinuria.

Glomerular hematuria, recognized as dysmorphic erythrocytes in urine sediment, is common in lupus nephritis; it is usually accompanied by proteinuria except in early mesangial nephropathy where it is often an isolated finding. Full-blown nephritic syndrome (hematuria with cellular casts and variable proteinuria) is seen in 30 to 40% of patients, while rapidly progressive glomerulonephritis (doubling or more of serum creatinine within a 3-month period) is rare and accounts for less than 10% of initial presentations. Hypocomplementemia (especially C3) and anti-DNA antibodies are commonly found in proliferative forms of nephritis. Classic cases of proliferative and membranous forms of lupus nephritis have distinct clinical presentations (Table 30.1). However, it is important to keep in mind that overlapping and mixed classes may coexist and that the classes are not static. Indeed, transitions among the various forms of lupus nephritis are common over time. The clinical presentation does not always predict the underlying histologic class of nephritis. This is especially true in treated patients where therapy may modify both the clinical and pathologic findings. In general, patients with mesangial nephritis have small amounts of proteinuria (<1 g/day) with hematuria but typically no cellular casts. Patients with proliferative nephritis have hypertension, nephritic urine sediment with various degrees of proteinuria (often at nephrotic range), low C3 and typically high titers of anti-DNA antibodies, whereas patients with membranous glomerulopathy have proteinuria often at nephrotic range but otherwise bland urine sediments; C3 tends to be normal, and anti-DNA antibodies when present are usually found in low titers.

Urinalysis Careful laboratory monitoring is essential for early detection of lupus nephritis. Urinalysis is the most important and effective method to detect and monitor disease activity in lupus nephritis but special efforts are usually necessary to obtain accurate urinary microscopy.

Mesangial

Proliferative

Membranous

Glomerulosclerosis

Early signs/symptoms

Mostly asymptomatic

Frequently asymptomatic New onset or worsening hypertension Nocturia, peripheral edema Macroscopic hematuria (rare) Fatigue

Nocturia, frothy urine, peripheral edema, anasarca Nephrotic syndrome Frequently asymptomatic

Depends on degree of renal insufficiency

Hypertension

Rare

Common

Less common

Slowly progressing

Proteinuria

Low grade

Variable

Nephrotic range

Variable

Urinary sediment

Hematuria No cellular casts

Nephritic (hematuria, cellular casts)

Nephrotic (proteinuria, oval fat bodies)

Chronic renal insufficiency (hyaline and granular casts) No hematuria or cellular casts

Loss of renal function

Only if it progresses into other forms

Uniform, if untreated Can be rapid

Variable Slow progression

Uniform, progressive

Anti-dsDNA antibodies

Variable

Common

Commonly absent

Commonly absent

Hypocomplementemia

Usually normal

Common

Variable

Commonly absent

DIAGNOSIS AND ASSESSMENT OF DISEASE ACTIVITY

TABLE 30.1 CLINICAL CHARACTERISTICS OF VARIOUS FORMS OF LUPUS NEPHRITISa

Serologic markers

a

There may be a significant overlap of these features between proliferative and membranous lupus nephritis and they may coexist. A kidney biopsy is needed to establish definite histologic type. A mixed membranous and proliferative pattern is associated with worse prognosis.

A fresh clean catch, unrefrigerated, second-morning urine sediment should be stained and examined in the clinic, if possible. Alternatively, specimens should be flagged for expeditious processing in the service laboratory to minimize spuriously negative results due to breakdown of cellular casts. Hematuria (usually microscopic, rarely macroscopic) indicates inflammatory glomerular or tubulointerstitial disease. Erythrocytes are fragmented or misshaped (dysmorphic). Granular and fatty casts reflect proteinuric states, whereas red blood cell, white blood cell, and mixed cellular casts reflect nephritic states (Fig. 30.1). Broad and waxy casts reflect chronic renal failure.

the simpler protein/creatinine ratio on spot urine samples.4,5

Renal Biopsy In the absence of objective urinary abnormalities, renal biopsy has little value and ordinarily should not be performed. It rarely establishes the diagnosis of lupus, but is necessary for classifying renal pathology; this information that has important prognostic implications.

Renal Function Tests Renal function evaluation should include estimation of glomerular filtration rate. This can be accomplished by formulas, such as the Cockroft-Gault formula1 or Modification of Diet in Renal Disease study (MDRD) formula,2,3 or from timed (usually 24-hour) creatinine clearance. Indeed, many clinical laboratories around the world are incorporating formula-based estimates of glomerular filtration rate (GFR) concurrently with measures of serum creatinine. Methods of estimating proteinuria vary among institutions with some strongly preferring 24-hour collections and others preferring

Fig. 30.1 Red blood cell cast in situ within the distal nephron. (See Color Plate 1.)

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KIDNEY INVOLVEMENT IN SLE

glomerular disease activity (potentially reversible) and sclerosis (irreversible damage) should be considered in each class of lupus nephritis. This is done by a semiquantitative analysis (on a 0 to 3+ scale) of specific histologic features of activity and sclerosis. This information is captured by either indicating the predominant feature as a subheading (a, active; s, sclerosing; or a/s, mixed active and sclerosing) to each major class or in the widely used checklists of activity and chronicity indices (Table 30.2).8

Monitoring of Lupus Nephritis Renal Function Fig. 30.2 Class I, minimal mesangial lupus nephritis; glomerulus shows essentially normal architecture (periodic acid–Schiff stain).

338

In fact, it is recognized that knowledge of pathologic findings is a powerful impetus for clinicians to prescribe aggressive intervention, particularly when results of common clinical tests may be less compelling.6 Early biopsy (before treatment) is indicated in patients with nephritic urine sediment, glomerular hematuria with proteinuria higher than 0.5 to 1.0g/day, low C3 and/or positive anti-ds DNA, or proteinuria higher than 1.0 to 2.0g/day (especially if C3 is low and/or positive anti-ds DNA). Patients with clinical and laboratory evidence of severe lupus nephritis, including nephritic or nephrotic syndrome, azotemia, and hypertension, may not require a renal biopsy prior to treatment with cytotoxic drugs. On the other hand, in patients with concomitant serologic abnormalities (i.e., low C3, positive anti-ds DNA) or patients who had previous immunosuppressive treatment may be candidates for renal biopsy even with more subtle findings. A repeat biopsy during or after treatment is indicated for unexplained worsening of proteinuria (e.g., >2g/day increase if non-nephrotic at baseline or >50% increase if nephrotic), unexplained worsening of renal function (e.g., reproducible >33% increase in serum creatinine, representing a 25% decrease in GFR), or persistent glomerular hematuria with proteinuria higher than 2g/day or proteinuria higher than 3g/day (especially if C3 is decreased). Following a succession of versions of the World Health Organization (WHO) classification of lupus nephritis, a novel approach has been recently been promulgated in an attempt to provide a more concise description of various lesions and classes of lupus nephritis7 (Table 30.2). From the major histologic classes, class IV nephritis is the most common (approximately 40%), while classes III and V follow with an approximate frequency of 25% and 15%, respectively (see Figs. 30.2-30.5). Transformation from one class to another can occur, both spontaneously and as a result of treatment. The additional features of

As described above, it is important to establish the level of GFR that corresponds to the level of serum creatinine for baseline reference in any given patient with lupus nephritis. Following this, serum creatinine can be used to measure change in renal function, since it is the main variable to account for change in GFR. In clinical practice, changes in renal function are more important than absolute values of renal function and significant reproducible changes in serum creatinine (e.g., 25 to 33% increase) are of concern—even if the change occurs within the normal population range of serum creatinine.

Proteinuria Measurement of 24-hour protein excretion is the gold standard, although this method is cumbersome for patients and fraught with collection errors. Collections of urine containing creatinine concentrations that deviate significantly from population averages for males (~20 mg/kg/day) or females (~15 mg/kg/day) should raise suspicions about the adequacy of the urine collection. Spot urine protein/creatinine is a simpler method to estimate the severity of proteinuria, and is increasingly popular form of monitoring proteinuria.9-11 In general, the numeric ratio approaches the number of grams per day of proteinuria. For example, if the protein-to-creatinine ratio is 2.0, the 24-hour protein excretion is approximately 2.0 g/day.

Urinalysis Resolution of active urine sediment is a feature of renal remission, but to be clinically meaningful has to be reproducible and sustained for several weeks. Reappearance of cellular casts with significant proteinuria is an early and reliable predictor of renal relapse and in most patients usually precedes rises in anti-DNA titers or decreases in C3 by several weeks12.

Serology Anti-DNA antibodies and C3 and C4 complement components are useful in monitoring activity of lupus nephritis and in guiding treatment. In general, changes

Histologic Classification

Activity and Chronicity Indices

Class I

Minimal mesangial proliferative LN

Activity index (lesions are scored 0 to 3+ with maximum score of 24 points)

Class II

Mesangial proliferative LN

Hypercellularity: endocapillary proliferation compromising glomerular capillary loops

Class III

Focal LN

Leukocyte exudation: polymorphonuclear leukocytes in glomeruli

Class III (A)

Active lesions: focal proliferative LN

Karyorrhexis/fibrinoid necrosis (weighted x2): necrotizing changes in glomeruli

Class III (A/C)

Active and chronic lesions: focal proliferative an sclerosing LN

Cellular crescents (weighted x2): layers of proliferating epithelial cells and monocytes lining Bowman’s capsule

Class III (C)

Chronic inactive lesions with glomerular scars: focal sclerosing LN

Hyaline deposits: eosinophilic and PAS-positive materials lining (wire loops) or filling (hyaline thrombi) capillary loops

Diffuse LN

Interstitial inflammation: infiltration of leukocytes (predominantly mononuclear cells) among tubules

Class IV-S (A)

Active lesions: diffuse segmental LN

Chronicity index (lesions are scored 0 to 3+ with maximum score of 12 points)

Class IV-G (A)

Active lesions: diffuse global LN

Glomerular sclerosis: collapse and fibrosis of capillary tufts

Class IV-S (A/C)

Active and chronic lesions: diffuse segmental proliferative and sclerosing LNActive and chronic lesions: diffuse global proliferative and sclerosing LN

Fibrous crescents: layers of fibrous tissue lining Bowman’s capsule

Class IV-S (C)

Chronic inactive lesions with scars: diffuse segmental sclerosing LN

Tubular atrophy: thickening of tubular basement membranes, tubular epithelial degeneration, with separation of residual tubules

Class IV-G (C)

Chronic inactive lesions with scars: diffuse global sclerosing LN

Interstitial fibrosis: deposition of collagenous connective tissue among tubules

Class IV

Class V

Membranous LN

Class VI

Advanced sclerosis LN

DIAGNOSIS AND ASSESSMENT OF DISEASE ACTIVITY

TABLE 30.2 HISTOLOGIC OF RENAL BIOPSIES

Sources: Histologic classification, adapted from International Society of Nephrology/renal Pathology Society (ISN/RPS) 2003 classification of LN, in Weening JJ, D’Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, et al. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 2004;65:521-30; indices adapted from Austin HA, Boumpas DT, Vaughan EM, Balow JE. Predicting renal outcomes in severe lupus nephritis: contributions of clinical and histologic data. Kidney Int 1994;45:544-50. LN, lupus nephritis.

in anti-DNA titers are more valuable than their absolute values. Patients with rising titers of anti-DNA antibodies warrant close monitoring for evidence of lupus activity. Complement has an important role in the pathogenesis of LN. Traditional measures of complement activity, such as CH50, C3, and C4, have low sensitivity and specificity because plasma levels reflect the result of the dynamic state of complement synthesis and consumption, both of which are increased during inflammation. C3 is more useful clinically than C4 because C4 deficiency is common in lupus patients, values of C3 and C4 are rarely discordant, and C3 levels correlate best with renal histology on repeat renal biopsies. Activation of the complement system is characterized by the generation of activated breakdown products of

precursor molecules. Complement breakdown products may be a better index of complement activation than factor levels, and there is a good rationale to use them as markers of disease activity. However, the available studies show conflicting results with markers of the classic, alternative, or final common pathways, showing correlation with activity in some but not in other studies. Some of this may result from methodologic differences, such as the use of plasma versus serum and differences in the definition of disease activity. Further work and large-scale trials are needed in this area to help further define appropriate complement split products for assessing lupus disease activity and to determine whether any of these can be used as a reliable biomarker. Plasma and urinary cytokines or chemokines or urinary podocytes may reflect lupus

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Treatment

A

B Fig. 30.3 A, Class II, mesangial proliferative lupus nephritis; glomerular capillary loops are mostly patent and of normal thickness, but the tuft shows increased mesangial cellularity and matrix (PAS stain). B, Ultrastructure of mesangial immune complex deposits (green arrows), which are typical of class-II lupus nephritis. CL, capillary lumen. (Micrograph courtesy of Sharda Sabnis, MD, Armed Forces Institute of Pathology, Washington, DC.)

activity, but these tests are not used in routine clinical practice at present (Fig. 30.5).

Assessment of Prognosis

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Numerous demographic and clinical variables can affect prognosis. Careful assessment of the unique combinations of such risk factors in individual patients is essential to optimize long-term outcomes. Patient characteristics associated with bad outcomes include black race,13,14 azotemia, anemia, antiphospholipid syndrome,15,16 failure to respond to initial immunosuppressive therapy, and flares with worsening in renal function.17 Combinations of severe active (crescents and fibrinoid necrosis) with marked chronic changes (moderate to severe tubulointerstitial fibrosis and tubular atrophy, such as chronicity index >3) are particularly ominous.8,18 In general, patients with a greater number of risk factors carry worse prognoses, are less likely to respond to therapy, tend to respond more slowly, and thus, need more aggressive treatment.

The immunosuppressive treatment of proliferative lupus nephritis consists of a period of intensive immunosuppression (induction) followed by a longer period of less intensive maintenance therapy. Despite a consensus on the general approach, there are wideranging opinions on the details of either of these treatments. The cornerstone of treatment of lupus nephritis is corticosteroid therapy. For induction it is used as high dose daily treatment (prednisone 0.5 to 1.0 mg/kg/d) or as bolus intravenous therapy (methylprednisolone 0.5 to 1.0 g for 1 to 3 days), most commonly in combination with other immunosuppressive drugs. There have been no controlled clinical trials proving the benefit of corticosteroids over supportive therapy; nonetheless, the long clinical experience amply demonstrates the value of steroids in the management of patients with lupus nephritis. Lupus nephritis is the only major organ manifestation for which effective immunosuppressive treatment has been established in controlled clinical trials.19-32 However, many of these studies have been limited by generic problems, including small number of patients, diverse racial mixes and socioeconomic backgrounds, and relatively short follow-up. Advances in adjunctive treatments over time, such as the use of angiotensin antagonists to reduce proteinuria (an independent risk factor for progressive renal dysfunction) may have improved the overall outlook for patients with lupus nephritis, which further complicates comparison of various studies. The National Institutes of Health (NIH) pulse cyclophosphamide regimen, established in a series of controlled trials over several decades,19,20,25,29 has been the standard against which other treatments were compared, either directly or indirectly. Early studies showed comparable efficacy of daily oral and monthly intravenous pulse cyclophosphamide therapy. However, the greater risks for cumulative toxicity with daily administration, particularly hemorrhagic cystitis and bladder cancer, has led to abandonment of essentially all but short courses (2 to 6 months) of daily cyclophosphamide therapy in lupus nephritis. The most recent NIH study showed that renal remission was achieved somewhat more rapidly with the combination of pulse methylprednisolone and pulse cyclophosphamide therapy.25 Extended follow-up (median 11 years) of this cohort demonstrated persistent benefit of cyclophosphamide-containing regimens compared to methylprednisolone alone,33 without added toxicity. Pulse cyclophosphamide is effective in most patients but seems to be less so in blacks.13,14 A 6-month course of monthly bolus cyclophosphamide is effective19 in inducing renal response in

Severity

Histology/Clinical Features

Induction

Maintenance Proliferative

Mild

● ●

Mesangial LN Focal proliferative LN with no adverse prognostic factor

●

●

● ●

●

Moderate

●

●

Focal proliferative LN with adverse prognostic factors Diffuse proliferative LN, not fulfilling criteria for severe disease

●

●

●

Severe

●

●

●

●

●

●

Any histology with abnormal renal function (reproducible increase of at least 30% in serum creatinine levels) Diffuse proliferative LN with multiple adverse prognostic factors Mixed membranous and proliferative (focal or diffuse) histology Fibrinoid necrosis/crescents in >25% glomeruli High activity and chronicity index Moderate disease that does not respond to therapy

●

●

High-dose corticosteroids (i.e., 0.5–1 mg/kg/day prednisone for 4–6 weeks with gradual tapering to 0.125 mg/kg every other day within 3 months). If no remission within 3 monthsor increased activity upon tapering of corticosteroids, start other immunosuppressive agent Low-dose cyclophosphamide (500 mg) every 2 weeks for 3 months MMF (2–3 g/day) for at least 6 months AZA (1–2 mg/kg/day) for at least 6 months If no remission after the first 6–12 months, advance to next therapy

●

Low-dose corticosteroids (i.e., prednisone ≤0.125 mg/kg on alternative days) alone or with AZA (1–2 mg/kg/day) Consider further gradual tapering at the

Pulse CY alone or in combination with pulse MP for the first 6 months (total 7 pulses). Background corticosteroids 0.5 mg/kg/day for 4 weeks, then taper Low-dose cyclophosphamide (500 mg) every 2 weeks for 3 months with corticosteroids as above MMF (3 g/day) (or AZA) with corticosteroids as above. If no remission after the first 6–12 months, advance to next therapy

●

Quarterly pulses of CY for 1 year beyondremission AZA (1–2 mg/kg/day)

●

If remission after the first 6–12 months, MMF may taper to 1.0 g/day bid for 6–12 months. Consider further taperingat the end of each year in remission or switching to AZA

Monthly pulses of CY in combination with pulse MP for 6–12 months If no response, consider MMF or rituximab

●

Quarterly pulses of CY for at least 1 year beyond remission, or azathioprine (1–2 mg/kg/day) MMF (2-3 g/day) Optimal length of MMF or AZA therapyis not known. We recommend using both for at least 1 year beyond complete remission. Once a decision is made to stop them, they should be tapered off gradually with close monitoring of patients

●

DIAGNOSIS AND ASSESSMENT OF DISEASE ACTIVITY

TABLE 30.3 RECOMMENDED TREATMENT ALTERNATIVES FOR LUPUS NEPHRITIS

Membranous

Mild

●

Non-nephrotic range proteinuria and normal renal function

●

High-dose corticosteroids alone or in combination with AZA

●

Low-dose corticosteroids alone or with AZA

Moderate/ severe

●

Nephrotic range proteinuria or abnormal renal function (reproducible increase of at least 30% in serum creatinine levels)

●

Bi-monthly pulse CY for 1 year (7 pulses) Cyclosporine A (3–5 mg/kg/day) for 1 year with gradual tapering thereafter MMF (2–3 g/day) for 6–12 months

●

Low-dose corticosteroids AZA MMF (1-2 g/day)

●

●

● ●

AZA, azathioprine; CY, cyclophosphamide; LN, lupus nephritis; MMF, mycophenolate mofetil.

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A

B

C

D

Fig. 30.4 A, Class III, focal proliferative lupus nephritis. A segmental area of solidification is observed (black arrow); this area shows fibrinoid necrosis and karyorrhexis with an early cellular crescent forming along Bowman’s capsule (hematoxylin and eosin stain). B, Class IV, diffuse proliferative lupus nephritis. The glomerulus is globally involved with endocapillary proliferation that compromises most of the capillary loops, and extensive fibrinoid necrosis and karyorrhexis are evident (hematoxylin and eosin stain). C, Class IV, diffuse proliferative lupus nephritis. The glomerulus shows irregular changes among different segments; wire loop lesions and hyaline thrombi (black arrow) represent massive subendothelial and intraluminal deposits of immune complexes; other tufts show variable degrees of proliferation and mesangial expansion. D, Ultrastructure of subendothelial immune complex deposits (green arrows) characteristic of both class III and IV lupus nephritis. CL, capillary lumen. (Micrograph courtesy of Sharda Sabnis, MD, Armed Forces Institute of Pathology, Washington, DC.)

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many patients, but maintenance therapy with prolonged courses of quarterly pulse cyclophosphamide is needed to maintain response.20 In an attempt to establish the optimal maintenance regimen, cyclophosphamide, azathioprine, and mycophenolate were compared as maintenance therapies after cyclophosphamide induction in a randomized controlled study in a mainly Hispanic and African-American population.22 Maintenance therapy with azathioprine or mycophenolate mofetil was as effective as quarterly cyclophosphamide in preserving renal function, but they appeared to be superior only when renal outcomes were combined with mortality (event-free survival).22 However, the median length of follow-up was less than 3 years. This is important because in previous studies most of the difference between various treatment regimens became apparent only after 5 years,19 emphasizing the importance of long-term follow-up to assess the real impact of any treatment in patients with lupus nephritis.

Protracted cyclophosphamide therapy decreases the flare rate and improves long-term outcomes, but is associated with significant treatment-related morbidities,17 most notably infertility, which is of major concern for the patients since the majority are women of child-bearing age. The risk of infertility increases with the cumulative dose and age of the patient.34 Therefore, several alternative regimens have been tested to replace or reduce the dose of cyclophosphamide, and there is a growing body of evidence that various immunosuppressive combinations are effective in the short and medium term in proliferative lupus nephritis. The Euro-Lupus nephritis study has recently demonstrated the utility of a short-course, low-cumulativedose cyclophosphamide regimen (cyclophosphamide 500 mg intravenously every 2 weeks for 3 months) followed by azathioprine maintenance in white patients.26 Long-term follow-up of the cohort revealed no difference between the low-dose and high-dose cyclophosphamide

A

B Fig. 30.5 A, Class V, membranous lupus nephritis; the capillary loops are nearly uniformly thickened with only a modest expansion of mesangial structures (periodic acid–Schiff stain). B, Ultrastructure of subepithelial immune complex deposits (white asterisks) characteristic of class V, membranous lupus nephritis. CL, capillary lumen. (Micrograph courtesy of Sharda Sabnis, MD, Armed Forces Institute of Pathology, Washington, DC.)

groups in the rate of renal impairment after 68 months of follow-up, with about 20% of the original cohort having some degree of renal impairment.35 Alternatives to pulse cyclophosphamide induction therapy commonly used at various centers around the world include azathioprine and mycophenolate mofetil. Mycophenolate mofetil (MMF) was claimed to be equivalent to daily oral cyclophosphamide in a small study of 42 patients with diffuse proliferative glomerulonephritis.21 In a more recent large randomized controlled study of 140 patients with lupus nephritis, MMF was at least as effective as pulse cyclophosphamide in inducing renal remission at 6 months and with fewer side effects in the MMF group. Shortterm follow-up of the two groups did not show any significant differences in renal flares or end-stage renal disease.32 Most studies indicate that azathioprine adds marginally to the efficacy of prednisone alone.36 Thus, at the present time, azathioprine is used as primary therapy mainly in milder forms of lupus nephritis, in patients

Preferred Approach Various treatment options and practical recommendations for management of lupus nephritis are summarized in Table 30.3. Corticosteroids are effective in the acute control of nephritis and are included in all treatment regimens. It is important not to withhold corticosteroids for fear of complications, but rather to test regularly the feasibility of reducing doses (preferably to alternate day), and to be willing to substitute alternative immunosuppressive strategies. The goal of treatment is to induce sustained remission that can be defined as normal renal function (less than 30% worsening of serum creatinine from baseline), no proteinuria (or at least <1 g/day) and inactive urine sediment. The time to reach remission varies from patient to patient but can be prolonged in those with severe disease. Stabilization at 3 months and significant improvement with the ability to reduce the dose of corticosteroids at 6 months are good indications of effective therapy. Practical guidelines for administration of pulse cyclophosphamide therapy, ovarian protection and monitoring for the most common side effects of immunosuppressives can be found elsewhere in this book (Chapter 46).

DIAGNOSIS AND ASSESSMENT OF DISEASE ACTIVITY

strongly opposed to use of cyclophosphamide, and as maintenance therapy after induction of remission with cyclophosphamide. Given the methodologic differences in various studies, it is impossible to give an unequivocal interpretation of the available data to determine a “one-fits-all” strategy to treat proliferative lupus nephritis. Several effective treatment options are available for the short and medium term and physicians can make their decisions based on the demographic and clinical characteristics of their patients, their personal experience with various treatments, and patient preferences.

Membranous Lupus Nephritis Treatment of membranous lupus nephritis (MLN) has emerged as a substantive clinical controversy. The relatively low risk of renal failure has long been the chief argument against the use of immunosuppressive drugs. The 10-year renal survival averages 80%, but renal survival estimates from individual studies range widely from 47 to 90%. More recently, recognizing that there are substantial morbidity and mortality risks associated with protracted nephrotic syndrome,37-39 the goal in MLN is to reduce proteinuria as the primary approach to reducing the thrombotic diathesis and dyslipidemia which confer substantial cardiovascular risks. Solid evidence-based recommendations for treatment of MLN are not available due to the lack of controlled studies. The treatment is based on the extrapolation of

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experiences with idiopathic membranous nephropathy and proliferative lupus nephritis. Despite negative results in a few small retrospective studies,40-42 many patients with MLN and high-grade proteinuria and/or progressive renal dysfunction receive an empiric trial of corticosteroids. In particular, high-dose alternate-day regimens of prednisone are widely used for 2 to 4 months. Other immunosuppressive agents, such as azathioprine,43 alkylating agents,44,45 mycophenolate mofetil,46,47 and cyclosporine48,49 are used based on retrospective or small open-label studies. The effects of adding alternate-month pulse cyclophosphamide or low-dose cyclosporine to alternate-day prednisone in 41 patients with LMN were evaluated in a prospective, randomized controlled trial.50 At 1 year, 46% of patients treated with prednisone and adjunctive immunosuppression achieved complete remission (proteinuria <0.3 g/day) compared to 13% in the prednisone-only group.50 Extended follow-up has shown that remissions tend to be more enduring with cyclophosphamide than with cyclosporine. Together these data suggest that combining immunosuppressive agents with prednisone may be more effective than prednisone alone in inducing renal remission. To date, the only randomized clinical trial in MLN supports the use of alternate-month pulse cyclophosphamide in combination with alternate-day prednisone. For patients who cannot receive cyclophosphamide, cyclosporine may be an alternative. The optimal combination and length of therapy and the role of other treatments, such as azathioprine, mycophenolate mofetil, or sirolimus has to be established in adequately powered, controlled randomized clinical trials. Better understanding of the pathogenesis of MLN is necessary to optimize the use of novel biologic agents.

Relapses of Lupus Nephritis

344

Approximately one-third to one-half of patients have a relapse of nephritis after achieving partial or complete remission of proliferative lupus nephritis and each major exacerbation is expected to leave residual and cumulative irreversible (often subclinical) damage. Nephritic exacerbations clearly have adverse effects on renal prognosis, while purely proteinuric exacerbations have much less prognostic importance.17 In general, we recommend aggressive treatment for patients with moderate or severe nephritic flares. These should be treated as new-onset nephritis if patients have not reached complete response. Mild nephritic flares and proteinuric flares could be treated less aggressively; however, close monitoring is essential with intensification of the therapy if patients do not respond promptly. A kidney biopsy is indicated in ambiguous situations.

Other Kidney Involvement in SLE Hypertensive nephropathy can be seen in patients with or without nephritis. Rapid worsening of renal function can occur with accelerated hypertension but most commonly uncontrolled hypertension is associated with slow progression of renal insufficiency and a bland urine sediment. Renal vein thrombosis classically presents as a sudden increase in proteinuria, rapid worsening of renal function, and flank pain. However, unusual presentations are not uncommon, and renal vein thrombosis should be excluded in patients with sudden increase in proteinuria and/or worsening of renal function with no clear explanation. It is more common in patients with nephrotic syndrome, especially membranous lupus nephritis, but has been described as well in proliferative lupus nephritis. Microangiopathic abnormalities can lead to renal failure in thrombotic microangiopathy, hypertensive crisis, and in antiphospholipid syndrome. These should be considered in the appropriate clinical settings. Antiphospholipid syndrome can be associated with cortical ischemia and infarction, thrombotic microangiopathy, and renal artery and renal vein thrombosis.51 Despite decades of research, the exact nature of antiphospholipid nephropathy in SLE is still controversial. Antiphospholipid antibody-positive patients undergoing kidney transplant are at increased risk of renal vascular thrombosis52 and graft loss.53

Adjunct Therapies Cardiovascular and cerebrovascular events account for approximately 50% of deaths among patients with lupus nephritis followed for an extended period of time.37-39 Therefore, cardiovascular risk factors should be managed aggressively. Hyperlipidemia is a common complication of the nephrotic syndrome, and there is evidence that it may contribute to the risk of progressive renal disease as well as the risk of atherosclerotic cardiovascular disease.54 Consequently, a comprehensive approach to the treatment of hyperlipidemia should be employed, including: treatment of the underlying immunemediated glomerular disease, diet, exercise, weight reduction (as indicated), and an effort to reduce proteinuria with angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers (ARBs), or the combination of these agents. Specific lipid-lowering agents are frequently required as well. Caution should be exercised when combining a statin with a fibric-acid derivative, cyclosporine, or tacrolimus. Blockade of the renin-angiotensin system with ACE inhibitors or receptor blockers (ARBs) is an important consideration for patients with lupus nephritis. In addition to lowering blood pressure, there is considerable

SLE and End-Stage Renal Failure None of the current regimens for treatment of lupus nephritis is fully effective in preventing renal failure. However, severe glomerulonephritis with uremia is not synonymous with irreversible, end-stage renal failure in lupus nephritis. The rate of evolution of renal failure has very important implications for treatment. Rapidly progressive renal failure, usually due to necrotizing and crescentic glomerulonephritis, is often reversible with effective treatment. Patients with evidence of active nephritis (specifically, nephritic urine sediment), even if oliguric and in advanced renal failure, warrant aggressive immunosuppressive treatment for approximately 3 months into maintenance dialysis therapy. On the other hand, in patients with “burnedout” lupus (e.g., renal failure in the context of contracted kidneys and urine sediment showing predominantly broad, waxy casts) who progress slowly and insidiously to irreversible end-stage renal failure, one should avoid the desperate and injudicious use of aggressive immunosuppressive drug therapy.

Approximately one-half of patients on maintenance hemodialysis continue to manifest or experience flares of lupus activity, which are clearly indications for continued treatment. A cautious, incremental prescription of prednisone and immunosuppressive drug therapy in such patients is warranted in order not to increase susceptibility to major infections in the uremic host. Kidney transplantation is a viable alternative for patients with end-stage renal disease caused by lupus nephritis. Clinically active lupus or evidence of recurrence of lupus nephritis in the allograft is rare if the transplant is performed after a sustained remission of SLE.

Emerging Therapies The use of immunosuppressive therapy has changed lupus nephritis from a progressive disease leading to end-stage renal disease to a chronic condition where renal function can be preserved in most patients. Recent advances in the development of more selective immunosuppressive agents and biologics targeting specific steps in the immunopathogenesis of lupus resulted in a surge of promising novel therapeutic candidates, which are at various stages of testing. Some intend to optimize existing immunosuppressive therapies by changing the dose or duration of commonly used drugs, to use these drugs in combination or sequentially to reduce adverse events, or to use more recently developed drugs that have been effective in other diseases. Others target specific steps in the pathogenesis of lupus nephritis such as interfering with T- and B-cell activation by blocking co-stimulatory molecules, preventing immune complex formation or deposition, and diverting the autoimmune response by inducing antigen-specific tolerance or by interfering with abnormal cytokine networks.

DIAGNOSIS AND ASSESSMENT OF DISEASE ACTIVITY

evidence that these agents slow the progression of chronic diabetic and nondiabetic kidney disease. A recent meta-analysis55 of 11 clinical trials showed that ACE inhibitors are renoprotective in nondiabetic patients with more than 0.5 g/day of proteinuria. While fewer data have been published about the effects of ARBs, they are reasonable alternatives for patients who develop an ACE inhibitor–associated nonproductive cough or angioedema. Many physicians are appropriately concerned about prescribing ACE inhibitors or ARBs for patients with chronic renal insufficiency. Acute increases in serum creatinine and/or potassium may occur, and serum chemistries should be checked within 1 week of starting these agents. A modest increase in serum creatinine likely reflects a salutary reduction in glomerular capillary pressure that may be associated with extended preservation of renal function. The risk of hyperkalemia may be reduced by adding a potassium-wasting diuretic and by avoiding potassium-rich foods. Combination antihypertensive regimens are frequently necessary in patients with chronic renal disease. ACE inhibitors (or ARBs) can be effectively combined with other antihypertensive agents, including diuretics, nondihydropyridine calcium-channel blockers and beta-blockers, depending on clinical circumstances. The combination of an ACE inhibitor and an ARB may be advantageous among patients with chronic renal insufficiency and persistent proteinuria.56 Preliminary evidence suggests the possibility that aldosterone antagonists may add further renoprotection, but this has not become a standard of clinical practice at the present time.57

Biologic Therapies Prevention of lymphocyte activation or selective depletion of well-defined lymphocyte subsets may lead to better understanding of the pathogenesis and potentially safer therapies. Treating patients with DNAse or interfering with the complement cascade by blocking C5, or neutralizing pathogenic antibodies by administering specific binding peptides or inducing specific antiidiotype antibodies may prevent immune complex formation and/or deposition. Breaking the established autoinflammatory circuit may be achieved by blocking important cytokines, such as interleukin-6 and -10, or the action of chemokines. Rituximab is a chimeric monoclonal antibody that binds specifically to the CD20 antigen. CD20 is expressed on B lymphocytes, from pre-B to activated B cells, but not on differentiated plasma cells. Although rituximab induces variable depletion of CD20+ cells in the peripheral blood, antibody production by plasma

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346

cells is maintained and peripheral B cells reappear 4 to 12 months after therapy.58 There is evidence that rituximab treatment of SLE patients has immunologic effects beyond autoantibody reduction, including deactivation of T cells and down-regulation of co-stimulatory molecule expression.59 To date, experience with rituximab treatment in SLE patients comes from uncontrolled trials and case series involving patients with various disease manifestations (renal, neurologic involvement, cytopenias, serositis) who are, in general, refractory to conventional immunosuppressive treatments.60-65 Different protocols were used, ranging from the usual full-dose regimen (375 mg/m2 rituximab weekly for 4 weeks) to various shorter and lower dosing schemes (500 to 1000 mg of rituximab twice 2 weeks apart). Most, but not all, patients received simultaneous immunosuppressive therapy with cyclophosphamide or MMF and corticosteroids. Generally, rituximab was well tolerated, and although infections have been reported, no major safety issues were reported. Infusion reactions (e.g., decreased blood pressure, fever, angioedema, chills, and bronchospasm) may occur; therefore, patients are premedicated with acetaminophen and diphenhydramine prior to the infusion of rituximab. For reasons that are not adequately understood at present, response to treatment did not correlate consistently with the degree of B-cell depletion. There were cases of disease relapse during the follow-up time. Large controlled studies comparing rituximab to standard treatment are underway to determine the most efficient treatment schedule, the need for adjuvant immunosuppressive therapy, and the safety of re-treating patients who relapse. CD 22, a modulator of B-cell signaling, is expressed on essentially the same B cells as CD20. Epratuzumab, a humanized anti-CD22 monoclonal antibody, was well tolerated and led to a decrease in BILAG scores in a small pilot study.66 The efficacy of epratuzumab is being evaluated in larger studies in nonrenal lupus. Specific inactivation or tolerization of autoreactive B cells would provide a more selective alternative to global B-cell depletion. LJP 394 (abetimus sodium) consists of four double-stranded oligonucleotides attached to a polyethylene glycol platform and binds avidly to anti-dsDNA antibodies. Several studies showed decreases in anti-dsDNA antibody levels.67 Although a trend toward a decreased rate of renal flares was seen in a subgroup of patients with high-affinity anti-dsDNA antibodies recently, objective clinical benefit has not been demonstrated to date.67,68 Antigen-specific T-cell activation requires a twostage signaling process. If T lymphocytes are activated through their antigen-specific T-cell receptor in the absence of a co-stimulatory “second” signal mediated

by co-stimulatory receptor–ligand pairs, the T-cell receptor-antigen/MHC interaction induces apoptosis, clonal deletion of antigen-specific T cells and ultimately anergy or even tolerance. Studies in animal models and in humans have demonstrated the essential role of the co-stimulatory molecule CD40 and its ligand CD40L (CD154) in the production of pathogenic autoantibodies and tissue injury in lupus nephritis. Anti-CD40L therapy ameliorates renal disease in animal models of SLE, and improves survival even when used in animals with established disease. In humans, a short course of treatment with an antiCD40L antibody in patients with proliferative lupus nephritis reduced anti-dsDNA antibodies, increased C3 levels, and decreased hematuria.69 The abnormalities in the peripheral B-cell compartment at baseline suggested intensive germinal center activity driven via CD154–CD40 interaction. These aberrancies normalized with treatment,70 and there was also substantial reduction in the frequency of B cells secreting IgG and IgM anti-DNA antibodies.71 Unfortunately, the study had to be stopped prematurely because of an increased number of thromboembolic events in this and other clinical studies using the same antibody. Another study using a different anti-CD40L antibody found no clinical benefit in patients with extrarenal lupus.72 Blocking other co-stimulatory pathways, such as CD28 or its ligands or interfering with several co-stimulatory pathways simultaneously may increase efficacy. CTLA4-Ig is a fusion protein derived from the extracellular domain of CTLA4 and the Fc portion of IgG1 that blocks B7/CD28 interaction and inhibits T-cell activation. CTLA4-Ig prevents the progression of renal disease and prolongs survival in NZB/W mice.73 In mice with advanced renal disease, a combination of CTLA4-Ig and cyclophosphamide improved survival significantly more than either agent alone.74 Abatacept (CTLA4-Ig) has been approved by the Food and Drug Administration (FDA) for use in moderate to severe rheumatoid arthritis, and is currently in clinical trials of nonrenal lupus. Cytokines are important mediators of autoimmunity and inflammation, and although their exact role in lupus is not yet defined, some are attractive therapeutic targets. BLyS (B-lymphocyte stimulator—also known as BAFF, zTNF4, TALL-1 and THANK) is a recently described stimulator of B cells. In murine models of SLE, serum levels of BLyS are increased and BLyS transgenic mice develop lupus-like disease. Treatment with soluble BLyS receptors significantly decreased proteinuria and increased survival of these mice.75 Elevated levels of BLyS were also found in patients with SLE, although it did not correlate with disease activity.76 LymphoStat-B is a fully human antiBlyS monoclonal antibody. A phase II study has been

outcomes become available, patients with potentially life-threatening or disabling major organ involvement who are in acceptable general medical condition should be considered for autologous HSCT after they failed a reasonable course of standard immunosuppressive therapy. High-dose cyclophosphamide without HSCT was suggested as an alternative approach. In a recent randomized controlled study, however, highdose cyclophosphamide was not more effective than traditional monthly intravenous pulse cyclophosphamide.83 Despite apparent clinical benefits, many questions remain unanswered. It is not known if the benefit is due solely to the high-dose immunosuppression and temporary arrest of disease activity or if it leads to the frequently postulated “resetting of the immune system” and fundamental changes in the underlying pathogenesis.

DIAGNOSIS AND ASSESSMENT OF DISEASE ACTIVITY

completed but not published yet. Alternative approaches to inhibit BLyS, such as soluble receptors, are in various phases of development. Tumor necrosis factor (TNF) has both inflammatory and anti-inflammatory properties, and its overall effect on SLE is still controversial. In a small open-label safety trial, infliximab (a humanized anti-TNF monoclonal antibody) was well tolerated, and patients had improvement in their inflammatory organ involvement. Interestingly, anti-dsDNA levels increased but this was not associated with any obvious clinical consequences. Proteinuria decreased by more than 50% in the four patients with nephritis and remained low for at least 1 year.77 Additional studies are necessary to define the role of TNF inhibition in the treatment of lupus nephritis. Interleukin-1 and -6 (IL-1, IL-6) are other important mediators of inflammation. A recombinant form of interleukin-1 receptor antagonist (a natural inhibitor of IL-1) is approved for rheumatoid arthritis. The experience with anakinra in SLE is limited to small case series.78,79 IL-6 has an important role in maintaining the autoinflammatory loop in SLE. IL-6 levels are elevated in both human and murine lupus, and blocking IL-6 or its receptor had beneficial effect in all models of lupus tested to date. Blocking the effect of IL-6 in humans may be beneficial in lupus through the blockade of terminal differentiation of B cells and by interacting with the inflammatory processes both systemically and locally. A phase I clinical study using an anti-IL6R monoclonal antibody (tocilizumab) is underway to address some of these questions.80 Type-I interferons serve as a link between the innate and adaptive immune system. The recognition of increased expression of interferon-regulated genes in SLE along with the data showing beneficial effects of blocking interferon in animal models of lupus makes them an attractive target. The first studies in lupus patients have recently started to assess the safety of interferon-a blockade. High-dose immunosuppression followed by autologous hematopoietic stem cell therapy (HSCT) is explored to induce long-term response with a short, intense period of immunosuppressive therapy. The available data suggest that this procedure is clinically beneficial, at least in the short and medium term. Among over 100 SLE patients who failed standard therapies, HSCT induced major clinical responses in about 65%.81,82 In some of these patients, responses were durable for at least several years but the overall length of follow-up is still too short to determine long-term benefit. Procedure-related mortality varies among studies between 5 and 12%, and seems to be lower in relatively larger single-center studies.81,82 Until more reliable estimates of the actual risks and long-term

A

B Fig. 30.6 A, Mixed membranous and proliferative lupus nephritis; the glomerular capillary loops show nearly uniform thickening;there are two segmental lesions (black arrows) showing hypercellularity with an adhesion to Bowman’s capsule (upper left), as well as an area of proliferation with fibrinoid necrosis and karyorrhexis (bottom center) (hematoxylin and eosin stain). B, Ultrastructure of mixed membranous and proliferative lupus nephritis showing both subendothelial (yellow plus signs) and subepithelial (white asterisks). (Micrograph courtesy of Sharda Sabnis, MD, Armed Forces Institute of Pathology, Washington, DC.)

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The long-term risk–benefit ratio compared to conventional immunosuppressive therapies has to be better defined as well. To accomplish the best therapeutic and scientific results, it is necessary to treat all patients in carefully planned protocols by specialized teams of

lupus specialists and transplanters. All immunoablative protocols should incorporate carefully planned studies of immune reconstitution to understand the mechanisms of cure or failure. Many of these questions are being addressed in ongoing studies.

REFERENCES 1. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976;16:31-41. 2. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999; 130:461-70. 3. Manjunath G, Sarnak MJ, Levey AS. Prediction equations to estimate glomerular filtration rate: an update. Curr Opin Nephrol Hypertens 2001;10:785-92. 4. Christopher-Stine L, Petri M, Astor BC, Fine D. Urine protein-tocreatinine ratio is a reliable measure of proteinuria in lupus nephritis. J Rheumatol 2004;31:1557-9. 5. Sessoms S, Mehta K, Kovarsky J. Quantitation of proteinuria in systemic lupus erythematosus by use of a random, spot urine collection. Arthritis Rheum 1983;26:918-20. 6. Esdaile JM, Joseph L, MacKenzie T, Kashgarian M, Hayslett JP. The benefit of early treatment with immunosuppressive agents in lupus nephritis [see comments]. J Rheumatol 1994;21: 2046-51. 7. Weening JJ, D’Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, et al. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 2004;65:521-30. 8. Austin HA, Boumpas DT, Vaughan EM, Balow JE. Predicting renal outcomes in severe lupus nephritis: contributions of clinical and histologic data. Kidney Int 1994;45:544-50. 9. Hogg RJ, Furth S, Lemley KV, Portman R, Schwartz GJ, Coresh J, et al. National Kidney Foundation’s Kidney Disease Outcomes Quality Initiative clinical practice guidelines for chronic kidney disease in children and adolescents: evaluation, classification, and stratification. Pediatrics 2003;111:1416-21. 10. Levey AS, Coresh J, Balk E, Kausz AT, Levin A, Steffes MW, et al. National Kidney Foundation practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Ann Intern Med 2003;139:137-47. 11. Morales JV, Weber R, Wagner MB, Barros EJ. Is morning urinary protein/creatinine ratio a reliable estimator of 24-hour proteinuria in patients with glomerulonephritis and different levels of renal function? J Nephrol 2004;17:666-72. 12. Hebert LA, Dillon JJ, Middendorf DF, Lewis EJ, Peter JB. Relationship between appearance of urinary red blood cell/white blood cell casts and the onset of renal relapse in systemic lupus erythematosus. Am J Kidney Dis 1995;26:432-8. 13. Bakir AA, Levy PS, Dunea G. The prognosis of lupus nephritis in African-Americans: a retrospective analysis. Am J Kidney Dis 1994;24:159-71. 14. Dooley MA, Hogan S, Jennette C, Falk R. Cyclophosphamide therapy for lupus nephritis: poor renal survival in black Americans. Glomerular Disease Collaborative Network. Kidney Int 1997;51:1188-95. 15. Moroni G, Ventura D, Riva P, Panzeri P, Quaglini S, Banfi G, et al. Antiphospholipid antibodies are associated with an increased risk for chronic renal insufficiency in patients with lupus nephritis. Am J Kidney Dis 2004;43:28-36. 16. Tektonidou MG, Sotsiou F, Nakopoulou L, Vlachoyiannopoulos PG, Moutsopoulos HM. Antiphospholipid syndrome nephropathy in patients with systemic lupus erythematosus and antiphospholipid antibodies: prevalence, clinical associations, and long-term outcome. Arthritis Rheum 2004;50:2569-79. 17. Illei GG, Takada K, Parkin D, Austin HA, Crane M, Yarboro CH, et al. Renal flares are common in patients with severe proliferative lupus nephritis treated with pulse immunosuppressive therapy: long-term followup of a cohort of 145 patients participating in

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56. Nakao N, Yoshimura A, Morita H, Takada M, Kayano T, Ideura T. Combination treatment of angiotensin-II receptor blocker and angiotensin-converting-enzyme inhibitor in non-diabetic renal disease (COOPERATE): a randomised controlled trial. Lancet 2003;361:117-24. 57. Bianchi S, Bigazzi R, Campese VM. Antagonists of aldosterone and proteinuria in patients with CKD. An uncontrolled pilot study. Am J Kidney Dis 2005;46:45-51. 58. Anolik JH, Barnard J, Cappione A, Pugh-Bernard AE, Felgar RE, Looney RJ, et al. Rituximab improves peripheral B cell abnormalities in human systemic lupus erythematosus. Arthritis Rheum 2004;50:3580-90. 59. Sfikakis PP, Boletis JN, Lionaki S, Vigklis V, Fragiadaki KG, Iniotaki A, et al. Remission of proliferative lupus nephritis following B cell depletion therapy is preceded by down-regulation of the T cell costimulatory molecule CD40 ligand: an open-label trial. Arthritis Rheum 2005;52:501-13. 60. Jacobson SH, Vollenhoven RV, Gunnarsson I. Rituximab-induced long-term remission of membranous lupus nephritis. Nephrol Dial Transplant 2006;21:1742-3. 61. Lambotte O, Durbach A, Kotb R, Ferlicot S, Delfraissy JF, Goujard C. Failure of rituximab to treat a lupus flare-up with nephritis. Clin Nephrol 2005;64:73-7. 62. Leandro MJ, Cambridge G, Edwards JC, Ehrenstein MR, Isenberg DA. B-cell depletion in the treatment of patients with systemic lupus erythematosus: a longitudinal analysis of 24 patients. Rheumatology (Oxford) 2005;44:1542-5. 63. Leandro MJ, Edwards JC, Cambridge G, Ehrenstein MR, Isenberg DA. An open study of B lymphocyte depletion in systemic lupus erythematosus. Arthritis Rheum 2002;46: 2673-7. 64. Looney RJ, Anolik JH, Campbell D, Felgar RE, Young F, Arend LJ, et al. B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/II dose-escalation trial of rituximab. Arthritis Rheum 2004;50:2580-9. 65. Marks SD, Patey S, Brogan PA, Hasson N, Pilkington C, Woo P, et al. B lymphocyte depletion therapy in children with refractory systemic lupus erythematosus. Arthritis Rheum 2005;52: 3168-74. 66. Kaufman J, Wegener WA, Horak ID, Muhammad QU, Ding C, Goldenberg DM, et al. Initial clinical study of immunotherapy in SLE using Epratuzumab (humanized anti-CD22 antibody). Arthritis Rheum 2004;50:S414. 67. Lorenz HM. Abetimus (La Jolla Pharmaceuticals). Curr Opin Investig Drugs 2002;3:234-9. 68. Alarcon-Segovia D, Tumlin JA, Furie RA, McKay JD, Cardiel MH, Strand V, et al. LJP 394 for the prevention of renal flare in patients with systemic lupus erythematosus: results from a randomized, double-blind, placebo-controlled study. Arthritis Rheum 2003;48:442-54. 69. Boumpas DT, Furie R, Manzi S, Illei GG, Wallace DJ, Balow JE, et al. A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum 2003;48:719-27. 70. Grammer AC, Slota R, Fischer R, Gur H, Girschick H, Yarboro C, et al. Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. J Clin Invest 2003;112:1506-20. 71. Huang W, Sinha J, Newman J, Reddy B, Budhai L, Furie R, et al. The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum 2002;46: 1554-62. 72. Kalunian KC, Davis JC Jr, Merrill JT, Totoritis MC, Wofsy D. Treatment of systemic lupus erythematosus by inhibition of T cell costimulation with anti-CD154: a randomized, double-blind, placebo-controlled trial. Arthritis Rheum 2002;46:3251-8. 73. Finck BK, Linsley PS, Wofsy D. Treatment of murine lupus with CTLA4Ig. Science 1994;265:1225-7. 74. Daikh DI, Wofsy D. Cutting edge: reversal of murine lupus nephritis with CTLA4Ig and cyclophosphamide. J Immunol 2001;166:2913-6. 75. Gross JA, Johnston J, Mudri S, Enselman R, Dillon SR, Madden K, et al. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 2000;404: 995-9.

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34. Boumpas DT, Austin HA, Vaughan EM, Yarboro CH, Klippel JH, Balow JE. Risk for sustained amenorrhea in patients with systemic lupus erythematosus receiving intermittent pulse cyclophosphamide therapy. Ann Intern Med 1993;119:366-9. 35. Houssiau FA, Vasconcelos C, D’Cruz D, Sebastiani GD, de Ramon Garrido E, Danieli MG, et al. Early response to immunosuppressive therapy predicts good renal outcome in lupus nephritis: lessons from long-term followup of patients in the Euro-Lupus Nephritis Trial. Arthritis Rheum 2004;50:3934-40. 36. Flanc RS, Roberts MA, Strippoli GF, Chadban SJ, Kerr PG, Atkins RC. Treatment of diffuse proliferative lupus nephritis: a metaanalysis of randomized controlled trials. Am J Kidney Dis 2004; 43:197-208. 37. Appel GB, Pirani CL, D’Agati V. Renal vascular complications of systemic lupus erythematosus. J Am Soc Nephrol 1994;4: 1499-515. 38. Ordonez JD, Hiatt RA, Killebrew EJ, Fireman BH. The increased risk of coronary heart disease associated with nephrotic syndrome. Kidney Int 1993;44:638-42. 39. Radhakrishnan J, Appel AS, Valeri A, Appel GB. The nephrotic syndrome, lipids, and risk factors for cardiovascular disease. Am J Kidney Dis 1993;22:135-42. 40. Donadio JV Jr. Treatment of membranous nephropathy in systemic lupus erythematosus. Nephrol Dial Transplant 1992; 7(Suppl 1):97-104. 41. Donadio JV Jr, Holley KE, Anderson CF, Taylor WF. Controlled trial of cyclophosphamide in idiopathic membranous nephropathy. Kidney Int 1974;6:431-9. 42. Gonzalez-Dettoni H, Tron F. Membranous glomerulopathy in systemic lupus erythematosus. Adv Nephrol Necker Hosp 1985;14:347-64. 43. Mok CC, Ying KY, Lau CS, Yim CW, Ng WL, Wong WS, et al. Treatment of pure membranous lupus nephropathy with prednisone and azathioprine: an open-label trial. Am J Kidney Dis 2004;43:269-76. 44. Chan TM, Li FK, Hao WK, Chan KW, Lui SL, Tang S, et al. Treatment of membranous lupus nephritis with nephrotic syndrome by sequential immunosuppression. Lupus 1999;8: 545-51. 45. Moroni G, Maccario M, Banfi G, Quaglini S, Ponticelli C. Treatment of membranous lupus nephritis. Am J Kidney Dis 1998;31:681-6. 46. Karim MY, Pisoni CN, Ferro L, Tungekar MF, Abbs IC, D’Cruz DP, et al. Reduction of proteinuria with mycophenolate mofetil in predominantly membranous lupus nephropathy. Rheumatology (Oxford) 2005;44:1317-21. 47. Spetie DN, Tang Y, Rovin BH, Nadasdy T, Nadasdy G, Pesavento TE, et al. Mycophenolate therapy of SLE membranous nephropathy. Kidney Int 2004;66:2411-5. 48. Hallegua D, Wallace DJ, Metzger AL, Rinaldi RZ, Klinenberg JR. Cyclosporine for lupus membranous nephritis: experience with ten patients and review of the literature. Lupus 2000;9: 241-51. 49. Radhakrishnan J, Kunis CL, D’Agati V, Appel GB. Cyclosporine treatment of lupus membranous nephropathy. Clin Nephrol 1994;42:147-54. 50. Austin HA, Vaughan EM, Balow JE. Lupus membranous nephropathy: randomized controlled trial of prednisone, cyclosporine and cyclophosphamide. J Am Soc Nephrol 2000; 11(Suppl 9):81A. 51. D’Cruz DP. Renal manifestations of the antiphospholipid syndrome. Lupus 2005;14:45-8. 52. Vaidya S, Wang CC, Gugliuzza C, Fish JC. Relative risk of posttransplant renal thrombosis in patients with antiphospholipid antibodies. Clin Transplant 1998;12:439-44. 53. Stone JH, Amend WJ, Criswell LA. Antiphospholipid antibody syndrome in renal transplantation: occurrence of clinical events in 96 consecutive patients with systemic lupus erythematosus. Am J Kidney Dis 1999;34:1040-7. 54. Kasiske BL, Velosa JA, Halstenson CE, La Belle P, Langendorfer A, Keane WF. The effects of lovastatin in hyperlipidemic patients with the nephrotic syndrome. Am J Kidney Dis 1990;15:8-15. 55. Jafar TH, Schmid CH, Landa M, Giatras I, Toto R, Remuzzi G, et al. Angiotensin-converting enzyme inhibitors and progression of nondiabetic renal disease. A meta-analysis of patient-level data. Ann Intern Med 2001;135:73-87.

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76. Zhang J, Roschke V, Baker KP, Wang Z, Alarcon GS, Fessler BJ, et al. Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus. J Immunol 2001;166:6-10. 77. Aringer M, Graninger WB, Steiner G, Smolen JS. Safety and efficacy of tumor necrosis factor alpha blockade in systemic lupus erythematosus: an open-label study. Arthritis Rheum 2004;50:3161-9. 78. Moosig F, Zeuner R, Renk C, Schroder JO. IL-1RA in refractory systemic lupus erythematosus. Lupus 2004;13:605-6. 79. Ostendorf B, Iking-Konert C, Kurz K, Jung G, Sander O, Schneider M. Preliminary results of safety and efficacy of the interleukin 1 receptor antagonist anakinra in patients with severe lupus arthritis. Ann Rheum Dis 2005;64:630-3.

80. Tackey E, Lipsky PE, Illei GG. Rationale for interleukin-6 blockade in systemic lupus erythematosus. Lupus 2004;13:339-43. 81. Burt RK, Traynor A, Statkute L, Barr WG, Rosa R, Schroeder J, et al. Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematosus. JAMA 2006;295: 527-35. 82. Jayne D, Passweg J, Marmont A, Farge D, Zhao X, Arnold R, et al. Autologous stem cell transplantation for systemic lupus erythematosus. Lupus 2004;13:168-76. 83. Petri M, Brodsky R, Jones R, Gladstone D, Brodsky I, Johnson L, et al. High-dose cyclophosphamide vs monthly (NIH) cyclophosphamide: one year results. Arthritis Rheum 2004;50:S406.

CLINICAL ASPECTS OF THE DISEASE

31

Cutaneous Lupus Erythematosus Thomas A. Luger, MD, Gisela Bonsmann, MD, Annegret Kuhn, MD, and Markus Böhm, MD

INTRODUCTION Cutaneous involvement in patients with lupus erythematosus (LE) is very common, and it is estimated that 85% of patients with SLE develop such changes during the course of their disease. In many cases, skin lesions are of special relevance because they frequently represent one of the initial symptoms of the disease and also may serve as an indicator for systemic disease activity. Moreover, skin manifestation in LE can be associated with a high degree of morbidity and severe impact on quality of life.1-3 Skin changes have been divided into two groups— those that are histologically specific for LE (LE-specific skin lesions) and those that do not express these characteristic features (LE-nonspecific skin lesions). The classification of the specific cutaneous LE (CLE) lesions is complex because similar types of skin involvement can occur in both SLE and patients with nonsystemic forms of LE. A widely accepted classification divides LE-specific skin disease into three major clinical subtypes according to their disease activity: acute CLE (ACLE), subacute CLE (SCLE), and chronic cutaneous LE (CCLE).4-6 However, clinical, histologic, and photobiologic evidence indicates several specific characteristics for LE tumidus. Therefore, it is now considered as a distinct entity, and is referred to as an intermittent subtype of LE (ICLE)7,8 (Box 31.1). The subtypes of CLE may be further specified according to the extent of their skin involvement (localized vs. generalized) and the localization of the inflammatory infiltrate in the skin (e.g., LE panniculitis, indicating LE-specific infiltration of the adipose tissue). It is important to note that in patients with SLE, different forms of LE-specific skin lesions can be present simultaneously. For example, a butterfly rash as a manifestation of localized ACLE and chilblain LE as a variant of CCLE may coincide in one and the same patient.9 On the other hand, nonspecific cutaneous manifestations such as Raynaud’s phenomenon, urticarial vasculitis, or calcinosis cutis may also occur in patients with different forms of CLE and SLE.10

This chapter is focused on the clinical features of specific cutaneous manifestations of LE. In addition, LE nonspecific skin involvement is briefly discussed.

SPECIFIC MANIFESTATIONS OF CUTANEOUS LUPUS ERYTHEMATOSUS

Acute Cutaneous Lupus Erythematosus The typical skin manifestations of ACLE are confluent symmetric erythema and edema, with a characteristic distribution in the central part of the face, commonly referred to as the malar dermatitis or butterfly rash (Fig. 31.1). It may present as a localized, occasionally transient form, or as a generalized more widespread variety (Box 31.2). Both forms of ACLE commonly occur in association with systemic disease and may develop several months before the onset of systemic organ involvement. ACLE lesions may be observed in 20 to 60 % of patients with SLE and represent one of the ACR diagnostic criteria for SLE.11,12 Localized ACLE typically commences as symmetric macules or papules on the cheeks and nose that later become confluent and last from several hours to weeks. This malar dermatitis frequently is misdiagnosed as sunburn. Occasionally, the lesions may be very discrete, demonstrating only erythema with little or no edema. Generalized ACLE—also referred to as SLE rash, maculopapular rash of SLE, or photosensitive lupus dermatitis—is less common (5 to 10%) and often overlaps with the occurrence of systemic manifestations. It is characterized by a symmetrically distributed, maculopapular erythematous to violaceous and sometimes pruritic rash that often involves the trunk with accentuation of the UV-exposed areas, but may also be localized elsewhere including the hands or feet where knuckles are typically spared.13,14 In many cases induction, or exacerbation of the rash after exposure to ultraviolet light (UV) has been observed indicating photosensitivity as an important diagnostic clue and pathogenetic component. In some patients, even

351

CUTANEOUS LUPUS ERYTHEMATOSUS

BOX 31-1 CLASSIFICATION OF LUPUS ERYTHEMATOSUS–SPECIFIC SKIN LESIONS (DÜSSELDORF CLASSIFICATION) A. Acute cutaneous lupus erythematosus (ACLE) Localized ACLE Generalized ACLE Toxic epidermal necrolysis-like ACLE B. Subacute cutaneous LE (SCLE) Annular Papulosquamous/psoriasiform C. Chronic cutaneous LE (CCLE) Discoid LE (DLE) Lupus panniculitis/LE profundus (LEP) Chilblain LE (CHLE) D. Intermittent cutaneous LE (ICLE) LE tumidus (LET)

aggravation of the systemic disease after UV exposure has been reported.15,16 Exacerbation or aggravation of ACLE also may be observed after (viral) infections and drug intake (hydralazine, isoniazide, procainamide).17,18 An unusual very acute form of generalized ACLE characterized by toxic epidermal necrolysis (TEN)–like lesions is seen on rare occasions. The cause of this

BOX 31-2 CLINICAL CHARACTERISTICS OF ACUTE CUTANEOUS LUPUS ERYTHEMATOSUS Localized form ● Butterfly rash (malar dermatitis) ● In 20 to 60% of SLE ● In 15% of SCLE Generalized form ● Erythematous to violaceous confluent rash on entire integument ● Rarely TEN-like ● Oral cavity Erythema, erosions, superficial ulcerations; in 7 to 45% during an acute flare Localization: hard palate, gingiva, buccal mucosa Histology: LE-specific (interface mucositis) or LE-nonspecific ● Erosive cheilitis Characteristics ● Photosensitivity ● Nonscarring ● Increased disease activity of SLE; in 40 to 90% antibodies against native dsDNA; in 10 to 30% antibodies against Sm LE, lupus erythematosus; SCLE, subacute cutaneous lupus erythematosus; SLE, systemic lupus erythematosus; TEN, toxic epidermal necrolysis.

bullous variant of ACLE is disruption of the damaged epidermis from the underlying dermis as a result of exaggerated keratinocyte apoptosis and severe interface dermatitis.19-22 ACLE lesions usually heal without scarring, but sometimes they become hyperkeratotic and poikiloderma can result from dyspigmentation. The concurrent incidence of ACLE with SCLE may be observed but is unusual. Mucocutaneous manifestations in patients with SLE involving the oral and nasal cavity are very common in patients with SLE. Typically, they consist of painful ulcerations, especially on the lips and buccal and palatal mucosa, but may be localized elsewhere in the oral cavity (Fig. 31.2).23,24

352

Fig. 31.1 Typical acute cutaneous lupus erythematosus (“malar rash”) in a young woman with systemic LE. (See Color Plate 1.)

Fig. 31.2 Mucosal aphthoid lesions in a patient with systemic lupus erythematosus. (See Color Plate 2.)

Subacute Cutaneous Lupus Erythematosus Subacute cutaneous lupus erythematosus is associated with specific clinical, serologic, and genetic characteristics (Box 31.3), and was recognized as a distinct entity by Gilliam in 1977.6 SCLE frequently is associated with systemic symptoms such as arthritis and a pronounced photosensitivity but normally has a favorable prognosis. Patients with SCLE usually have circulating antiRo/SSA and anti-La/SSB antibodies and an increased association with the HLA B8, DR3 haplotype as well as a 308A TNF-α promoter polymorphism.27-29 Data from several studies further indicate that approximately half

BOX 31-3 CHARACTERISTICS OF SUBACUTE CUTANEOUS LUPUS ERYTHEMATOSUS

Fig. 31.3 Systemic cutaneous lupus erythematosus with annular psoriasiform pattern. (See Color Plate 2.)

of patients with SCLE fulfill four or more ACR criteria for SLE.14 Initially, SCLE develops as erythematous papules or macules that expand and merge into hyperkeratotic papulosquamous or annular and sometimes polycyclic plaques. Thus, two types can be distinguished, including a papulosquamous variant consisting of psoriasislike or eczema-like lesions and an annular variant consisting of slightly raised erythemas with central clearing (Figs. 31.3 and 31.4).2,30 Mixed types do occur. SCLE lesions typically are located on UV-exposed skin, including the lateral aspects of the face, the V of the neck (often with sparing of the area under the chin), the upper ventral and dorsal part of the trunk, and the dorsolateral aspects of the forearms. SCLE lesions never lead to scarring, but hypo- or de-pigmentation is common, resulting in vitiligo-like depigmentation.31 In some cases, the disease may be very mild with the appearance of a few distinct scaly plaques after sun exposure. Several drugs may induce SCLE, such as hydrochlorothiazide, terbinafine, diltiazem hydrochloride, ACE

SPECIFIC MANIFESTATIONS OF CUTANEOUS LUPUS ERYTHEMATOSUS

The histopathologic changes in ACLE in early lesions are less characteristic than those seen in SCLE or DLE lesions. The lymphohistiocytic cellular infiltrate of the interface dermatitis is relatively modest and hydropic changes of basal keratinocytes only occasionally can be seen. Edema and fibrinoid deposits in the upper dermis as well as vasodilatation with extravasation of erythrocytes is a common feature. In severe forms, necrotic keratinocytes and epidermal necrosis resembling TEN may be observed. In the majority of patients with ACLE, band-like granular deposits of immunoglobulins and complement at the dermoepidermal junction of lesional as well as nonlesional skin known as the lupus band test (LBT) can be detected.25,26 The differential diagnosis of ACLE lesions includes erythema solare (sunburn), photoallergic and phototoxic drug eruptions, dermatomyositis, atopic eczema, seborrheic dermatitis, contact eczema, and rosacea.

Annular form ● Annular erythematous plaques with slight scaling and central clearing Papulosquamous form ● Psoriasis or eczema-like lesions Characteristics ● Localization: symmetric on UV-exposed areas ● Polycyclic confluent lesions ● High photosensitivity ● Nonscarring; hyperpigmentation, or vitiligo-like depigmentation ● Arthralgias ● In 70 to 90% anti-Ro/SSA antibodies; in 30 to 50% antiLa/SSB antibodies ● Rheumatoid factor positive in more than 30% ● Immunogenetic predisposition: HLA-A1, -B8, -DR3 ● ACR criteria in 50% fulfilled ● SLE only in 10 to 15% ACR, American College of Rheumatology; ANA, antinuclear antibodies.

Fig. 31.4 Classical annular variant of systemic cutaneous lupus erythematosus with central clearing. (See Color Plate 2.)

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inhibitors, and griseofulvin, as well as leflunomide30,32-38 and biologic agents such as etanercept.39 Conversely, after starting etanercept therapy in a patient with rheumatoid arthritis and SCLE, the skin eruption of SCLE went into remission.40 Discontinuation of the medication does not always result in the clearance of skin lesions. There is some evidence for SCLE being a paraneoplastic syndrome in association with solid tumors.41 A substantial proportion of patients with SCLE is sensitive to UV-light and may exhibit mild systemic symptoms, such as arthralgias and musculoskeletal complaints, especially if the disease occurs in association with Sjögren’s syndrome. Autoimmune thyroiditis also may be observed in connection with SCLE.25,30,42 In the same patient with SCLE, other forms of LEspecific skin lesions may occur, including ACLE as well as CCLE. In addition, these patients can also develop LE-nonspecific skin disease such as Raynaud’s phenomenon, livedo reticularis, or leukocytoclastic vasculitis. There is some evidence that SCLE patients who develop either ACLE or LE-nonspecific skin lesions more often acquire systemic disease.14 Some patients with SCLE have been reported to develop erythema multiforme–like lesions, occasionally with involvement of the mucous membranes in the presence of anti-Ro/SSA and/or La/SSB antibodies, a speckled ANA pattern, and a positive rheumatoid factor. This variant also may be regarded as Rowell’s syndrome, which was originally described in patients with DLE, but apparently also occurs in association with other forms of CLE and SLE. The overlap in Rowell’s syndrome and SCLE in many features suggests that LE with erythema multiforme-like rashes represents SCLE with targetoid lesions rather than a distinct entity.20,43 The prognosis of SCLE in general appears to be good, although approximately 50% of the patients meet the ACR criteria for SLE. However, only a low percentage (~15%) will develop manifestations of severe SLE.44 In newborns from mothers having anti-Ro/SSA autoantibodies, an SCLE-like rash has been observed, called neonatal lupus erythematosus (NLE). The annular erythematous lesions with central clearing predominantly are localized on the face especially the periorbital area. Although sensitivity to UV light has been reported, NLE lesions usually develop without sun exposure. Most infants with NLE have maternal anti-Ro/SSA autoantibodies. The skin lesions normally resolve within 6 months, correlating with the disappearance of the anti-Ro/SSA autoantibodies. In some children residual dyspigmentation and telangiectasias may persist.45,46 The histopathologic findings of NLE are identical to those seen in adult SCLE. In approximately 2%, heart involvement with congenital

atrioventricular block may develop. Therefore, careful monitoring of SCLE patients during pregnancy is required. Serial echocardiograms of fetuses should be performed between 18 and 24 weeks of pregnancy.45,47 Histopathology of SCLE lesions usually reveals LE-specific findings, such as keratinocyte damage, epidermal atrophy, and interface dermatitis. In the epidermis, hydropic or eosinophilic degeneration of keratinocytes is commonly seen. The upper dermis typically contains an infiltrate of lymphohistiocytic cells in an interface, perivascular, and periadnexal pattern. In comparison to CCLE, lesions in SCLE have little hyperkeratosis, basement membrane thickening, follicular plugging, and lymphohistiocytic cellular infiltrate. Using histopathologic criteria, it is not possible to differentiate the annular from the papulosquamous form of SCLE.25 The lupus band test of lesional skin is positive in the majority of cases. A distinct pattern of granular IgG and IgM deposits in the epidermis rather than at the dermoepidermal junction has been described in SCLE.26 There is evidence that these epidermal deposits reflect the presence of in vivo bound anti-Ro/SSA autoantibodies.48 The differential diagnosis of SCLE lesions consists of photoallergic and phototoxic drug eruptions, as well as other forms of annular erythemas such as erythema annulare centrifugum, erythema gyratum repens, granuloma annulare, eczema, psoriasis, and tinea.

Chronic Cutaneous Lupus Erythematosus Discoid Lupus Erythematosus The typical skin lesion of the classical form of CCLE, also known as discoid LE (DLE), is an erythematous to violaceous discoid plaque that becomes hyperkeratotic and eventually leads to atrophy and scarring (Fig. 31.5). The disease usually begins with erythematous

Fig. 31.5 Classical discoid lupus erythematosus. Note erythematosus and hyperkeratotic margins and central scarring. (See Color Plate 2.)

also involves the adnexal structures sometimes resulting in atrophy of hair follicles. Further findings include dermal edema, vasodilatation, and mucin deposition. Upon direct immunofluorescence, granular deposits of immunoglobulins at the dermoepidermal junction usually occur. Deposits are more likely to be detected if the lesions are not scarred and clinical signs of inflammation are present.25,26 Rare variants of CCLE that can occur in patients with SLE and nonsystemic LE also may be observed.52 Hypertrophic/verrucous DLE is an exaggerated form of DLE that is characterized by epidermal hyperkeratosis resulting in nodular lesions with a thick scaling frequently observed in the periphery of discoid lesions. These hypertrophic lesions are often localized on the extensor arms, but the face and upper trunk also may be involved and they often are resistant to conventional local therapy. Usually, classical discoid lesions can be detected in other sites.2,53 Lupus erythematosus teleangiectoides typically presents as reticular telangiectasias that frequently occur in discoid plaques and may result in a poikiloderma-like appearance. Long-term use of topical corticosteroids may be a causative factor.54

SPECIFIC MANIFESTATIONS OF CUTANEOUS LUPUS ERYTHEMATOSUS

macules that expand into discoid plaques with follicular hyperkeratosis and hyperesthesia as well as peripheral hyperpigmentation. Later, central atrophy, scarring, telangiectasia, and hypopigmentation lead to disfiguring plaques. Discoid lesions have a predilection for the face, ears, and neck, but may be widespread without a clear-cut relation to UV exposure.2,14 However, mechanical irritation or UV irradiation may lead to the initiation or the exacerbation of CLE lesions.15,49 Disfigurement due to scarring can be a serious problem, especially in patients with facial and scalp (scarring alopecia) involvement (Fig. 31.6). Mucosal membranes, including the lips, mucosal surfaces of the mouth, nasal membranes, conjunctivae, and genital mucosa may also be involved with characteristic discoid lesions resembling leukoplakia.2,14 Although DLE is primarily considered to be a cutaneous form of LE without systemic involvement, patients with SLE may have classical DLE lesions. The prognosis of patients with DLE usually is good, although long-term follow-up is needed because 5 to 10% will develop SLE. In general, the prognosis of the disseminated form is less favorable compared to the localized form.14,50,51 Histopathology of DLE lesions is characterized by keratinocyte damage and hyperkeratosis. In the epidermis and upper dermis, damaged keratinocytes present as eosinophilic colloid bodies. Macrophages in the upper dermis containing melanin from damaged epidermal melanocytes are a characteristic feature. A typical pathologic finding in DLE lesions is the thickening of the basement membrane zone that can be visualized on periodic acid-Schiff–stained sections. In the dermis, a band-like lymphohistiocytic cellular infiltrate with interface dermatitis in the upper dermis as well as in perivascular and periadnexal sites typically occurs. Hyperkeratosis may be very prominent and

Lupus Erythematosus Profundus Lupus erythematosus profundus (LEP) or lupus panniculitis is characterized by a dense inflammatory infiltration of the subcutaneous fat, resulting in single or multiple indurated sometimes painful plaques or nodules (Box 31.5). Subsequently, they often develop into depressed and disfiguring lesions (Fig. 31.7). Calcification and occasionally ulceration also may result.

BOX 31-4 CHARACTERISTICS OF DISCOID LUPUS ERYTHEMATOSUS Localized form (~80%) ● Face and scalp Disseminated form (~20%) ● Additionally upper trunk and arms Oral cavity ● Buccal mucosa and palate Characteristics ● Discoid erythematous plaques with follicular hyperkeratosis ● Peripheral growth ● Central scarring with hypopigmentation, active erythematous border with hyperpigmentation, hair loss, and scarring alopecia ● In <5% ANA of high titer; usually no anti-dsDNA antibodies; rarely anti-Ro/SSA or anti-U1-RNP antibodies ● Coincidence with SLE, SCLE, CHLE, and LEP possible

Fig. 31.6 Scarring alopecia in a patient with discoid lupus erythematosus. (See Color Plate 2.)

ANA, antinuclear antibodies; CHLE, chilblain lupus erythematosus; LEP, lupus erythematosus profundus; SLE, systemic lupus erythematosus.

355

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BOX 31-5 CHARACTERISTICS OF LUPUS ERYTHEMATOSUS PROFUNDUS AND CHILBLAIN LUPUS ERYTHEMATOSUS Lupus erythematosus profundus (lupus panniculitis) Localization: face, upper arms, shoulder, breast, buttocks, upper legs Subcutaneous indurated nodules or plaques adhering to overlying epidermis Skin over the nodules: red normal or features of discoid lupus erythematosus Calcification or ulceration may occur Scarring and deep lipatrophy ANA in up to 75%; usually no anti-dsDNA antibodies; if anti-dsDNA antibodies occur, development of SLE is possible ACR criteria in 35 to 50% fulfilled Chilblain lupus erythematosus Localization: cold-exposed acral areas (toes, heel, knees, finger, ears, nose) Violaceous painful swelling; large nodules Central erosion or ulceration Occasionally ANA and anti-Ro/SSA antibodies; usually no anti-dsDNA antibodies SLE in ~20% ACR, American College of Rheumatology; ANA, antinuclear antibodies; SLE, systematic lupus erythematosus.

Fig. 31.7 Lupus erythematosus profundus. Note prominent lipatrophy. (See Color Plate 3.)

cutaneous lupus erythematosus (ICLE) comprising lupus erythematosus tumidus (LET) has been included as a distinct variant into the classification of CLE7 (Box 31.6).

Lupus Erythematosus Tumidus Lesions of lupus panniculitis usually occur on the face, trunk, buttock, upper arms, breasts, and thighs. Rarely, lesions develop on the scalp and may resemble alopecia areata.2,14 The major histopathologic finding is a lobular lymphohistiocytic panniculitis in the subcutaneous tissue. Vessel wall thickening and a perivascular infiltrate also may be observed.55

Chilblain Lupus Erythematosus

LET is characterized by a remarkable photosensitivity, a mild course, and very rare development of SLE. Typically, photosensitive erythematosus, and sometimes urticarial plaques and nodules without epidermal hyperkeratosis or follicular plugging are observed (Fig. 31.9). Lesions are usually located on the face, upper trunk, and extremities, and heal without scarring or dyspigmentation. The majority of patients

Chilblain lupus erythematosus (CHLE), a rare variant of CCLE, may be a consequence of microvascular injury after cold exposure (Box 31.5). The pernio-like lesions usually occur on the toes and fingers, and occasionally on the nose, elbows, and knees. The lesions are characterized by circumscribed, purple, pruriginous, or painful plaques that sometimes may become hyperkeratotic or ulcerate (Fig. 31.8). Patients with chilblain lupus have a higher risk of developing SLE (up to 24%).56-58

Intermittent Cutaneous Lupus Erythematosus 356

Because of recently defined specific, clinical, histologic, and immunohistochemical criteria, intermittent

Fig. 31.8 Chilblain lupus erythematosus of the plantar surface of the toes. (See Color Plate 3.)

●

Lupus erythematosus tumidus Localization: UV-exposed areas, face, V of the neck, upper trunk, arms Erythematosus urticaria-like plaques without involvement of the epidermis High photosensitivity In ~10 to 30% ANA; in ~5% anti-Ro/SSA or anti-La/SSB antibodies; no anti-dsDNA antibodies Very good prognosis, spontaneous remission possible

ANA, antinuclear antibodies.

with LET do not have antinuclear antibodies, and diagnosis relies mainly on the clinical and histomorphologic picture. LET sometimes my be difficult to differentiate from other skin diseases such as lymphocytic infiltration Jessner Kanof, polymorphous light eruption, cutaneous mucinosis, and pseudolymphoma.59,60 Histologic findings include a dense perivascular and periadnexal lymphohistiocytic infiltration, usually without vacuolar degeneration of the dermoepidermal junction and without epidermal changes. Edema of the papillary dermis and dense deposits of mucin in the dermis are characteristic. In approximately 20% of patients with LET, deposits of immunoglobulin and complement at the basement membrane zone have been observed.61

NONSPECIFIC MANIFESTATIONS OF LUPUS ERYTHEMATOSUS In addition to the above-described LE-specific skin lesions, there is a plethora of nonspecific skin signs and associated skin diseases that can be present in patients with SLE and CCLE (Box 31.7). These skin signs include harmless vascular changes such as nailfold abnormalities (large and tortuous capillaries together with

Fig. 31.9 Lupus erythematosus tumidus of the face. Note wheal-like nodules without epidermal changes.

BOX 31-7 NONSPECIFIC MANIFESTATIONS OF CUTANEOUS LUPUS ERYTHEMATOSUS Leucocytoclastic vasculitis Urticarial vasculitis Palpable purpura Vasculopathy Atrophy blanche Degos’ disease-like lesions Livedo reticularis Raynaud’s phenomenon Periungual telangiectasia Calcinosis cutis Papulonodular mucinosis Rheumatoid nodules Scleroderma-like changes Sclerodactily Erythema multiforme-like lesions Bullous skin lesions Nonscarring alopecia

areas of avascularity), but also more serious complications such as vasculitis (leukocytoclastic vasculitis as palpable purpura, urticarial vasculitis, and nodular vasculitis) and other forms of vasculopathy (atrophy blanche, livedo reticularis, Degos’ disease-like lesions, ulcerations, thromboses); the latter may develop especially in patients with antiphospholipid antibody syndrome.10,64 Bullous skin lesions in lupus erythematosus were found to be associated with various subtypes of LE. According to the presence or absence of an LE-specific histopathology, distinct subsets have been separated. In active cases of ACLE and SCLE, and occasionally CCLE, bullous lesions may occur due to a severe vacuolar degeneration of basal cells (LE-specific bullous skin lesions). On the other side, a blistering disease is described in active SLE, characterized by a widespread, vesiculo-bullous nonscarring eruption with a dermatitis herpetiformis-like histology and immunopathologic features resembling epidermolysis bullosa acquisita (EBA). Bullous LE (BLE) responds to dapsone. Upon immunoblotting, the sera of many patients with BLE reacts with the same antigen—type VII collagen—as the sera of EBA patients. Furthermore, immunobullous diseases may be primarily associated with SLE.21,62,63 Papulonodular mucinosis may be observed in the presence or absence of typical LE skin lesions, and appears to be triggered by UV light. It presents as skincolored nodules that are mostly located on the trunk and the upper extremities.64 Nonscarring alopecia (“lupus hair”) may be seen in patients with SLE, while scarring alopecia typically occurs in patients with DLE involving the scalp. Raynaud’s phenomenon, calcinosis cutis, sclerodermalike changes, and rheumatoid nodules may indicate the presence of an overlap syndrome.2

NONSPECIFIC MANIFESTATIONS OF LUPUS ERYTHEMATOSUS

BOX 31-6 CHARACTERISTICS OF INTERMITTENT CUTANEOUS LUPUS ERYTHEMATOSUS

357

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TREATMENT OF CUTANEOUS LUPUS ERYTHEMATOSUS In patients with any form of cutaneous lupus erythematosus, systemic disease must be ruled out initially. Since UV light is a major factor known to initiate or exacerbate skin lesions it is very important that all patients receive adequate instructions for sun protection. This is further supported by an increased risk of immunocompromised individuals to develop skin cancer especially in chronic discoid lesions. UV protection includes sun avoidance, protective clothing, physical sun block, and broad spectrum sun screens with a high sun protecting factor. There is also some evidence that smoking may function as a trigger factor and interferes with the effectiveness of antimalarial therapy.65-67 The mainstay of local therapy is the topical use of corticosteroids. Superpotent topical class-IV corticosteroids are highly effective for the treatment of CLE lesions. In the case of thick, hyperkeratotic nodular lesions, the intralesional application may be helpful. However, complications such as atrophy and telangiectasias may occur. Therefore, long-term application of corticosteroids should be avoided, and in sensitive skin areas such as the face only low- to mid-potency

corticosteroids should be used.68,69 Alternatively, topical calcineurin inhibitors such as tacrolimus and pimecrolimus may be used.70 There is evidence for efficacy of these compounds in SLE and SCLE skin lesions, whereas DLE lesions do not respond significantly. Other reported local treatment modalities include kryotherapy and laser surgery.54,69 Systemic therapy is discussed in the treatment section of this book, and therefore is only mentioned briefly. Usually skin lesions refractory to topical treatment respond well to drugs used for systemic therapy of lupus erythematosus. LE-specific skin lesions generally respond well to treatment with antimalarials such as hydroxychloroquine or chloroquine, which may be combined with quinacrine.71 In the case of poor clinical response to antimalarials, dapsone, retinoids, thalidomide, or clofazimine may be helpful. In widespread refractory CLE, immunosuppressive drugs such as systemic corticosteroids (pulse therapy), methotrexate, azathioprine, cyclophosphamide, mycophenolate mofetil, or leflunomide were used with variable response.72-74 Experimental approaches include intravenous immunoglobulin (IVIG), photopheresis, anticytokine antibodies, targeting T or B cells, and co-stimulatory molecules.75,76

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69. Lehmann P. Topical treatment of cutaneous lupus erythematosus. In: Kuhn A, Lehmann P, Ruzicka T, eds. Cutaneous Lupus Erythematosus. Berlin: Springer, 2004:337-346. 70. Böhm M, Gaubitz M, Luger TA, Metze D, Bonsmann G. Topical tacrolimus as a therapeutic adjunct in patients with cutaneous lupus erythematosus. A report of three cases. Dermatology 2003;207:381-385. 71. Ochsendorf FR. Antimalarials. In: Kuhn A, Lehmann P, Ruzicka T, eds. Cutaneous Lupus Eryrthematosus. Berlin: Springer, 2004: 347-372. 72. Bacman D, Kuhn A, Ruzicka T. Dapsone and retinoids. In: Kuhn A, Lehmann P, Ruzicka T, eds. Cutaneous Lupus Erythematosus. Berlin: Springer, 2004:373-390.

73. Sticherling M. Immunosuppressive drugs in cutaneous lupus erythematosus. In: Kuhn A, Lehmann P, Ruzicka T, eds. Cutaneous Lupus Erythematosus. Berlin: Springer, 2004:403-418. 74. Karim Y, Cuadrado MJ. Thalidomide in cutaneous lupus erythematosus. In: Kuhn A, Lehmann P, Ruzicka T, eds. Cutaneous Lupus Erythematosus. Berlin: Springer, 2004:391-402. 75. Anolik JH, Aringer M. New treatments for SLE. cell-depleting and anti-cytokine therapies. Best Pract Res Clin Rheumatol 2005;19:859-878. 76. Luger TA, Böhm M. Skin targets for new biological agents in systemic autoimmune disease. In: Sarzi-Puttini P, Doria A, Girolomoni G, Kuhn A, eds. The Skin in Systemic Autoimmune Diseases. Amsterdam: Elsevier, 2006:313-324.

CLINICAL ASPECTS OF THE DISEASE

32

The Heart and Systemic Lupus Erythematosus Jennifer R. Elliott, MD, Amy H. Kao, MD, MPH, and Susan Manzi, MD, MPH

INTRODUCTION Systemic lupus erythematosus (SLE, lupus) is a multiorgan autoimmune disease that commonly involves the cardiovascular system. Inflammation associated with SLE and treatment-related complications may affect all structures of the heart, including the pericardium, conduction system, myocardium, endocardium, and coronary arteries.

PERICARDIUM

Epidemiology and Pathogenesis Pericardial disease is the most common and first recognized cardiovascular manifestation of SLE. In 1924, Keefer and Felty1 first described two lupus women with pericardial disease, one with a pericardial rub and another with fibrous adhesions at autopsy. Prevalence ranges from 16 to 100% have been reported (Table 32.1), depending on the type of diagnostic study and symptomatology of the patients. Echocardiographic evidence of pericardial abnormalities has been shown in 16 to 54%,2-7 whereas autopsy findings of pericardial disease have been reported in up to 100% of SLE patients.8-11 While the majority of lupus patients with pericardial effusions are asymptomatic, approximately 25% will develop symptomatic pericarditis during their disease course.12 Older age of disease onset13 and the presence of SSB/La autoantibodies in adults2,14 are risk factors that have been associated with pericardial disease. Pericardial disease may also be seen with druginduced SLE. Immunopathology of the pericardium15 and pericardial fluid16 have revealed immune complexes of IgG, IgM, C3, and C1q, confirming the inflammatory pathogenesis. Pathology of pericardial disease has changed with the advent of corticosteroids. Focal or diffuse active fibrinous changes were observed prior to corticosteroids,17 and healed fibrous lesions predominate since the introduction of steroid therapy.8

Clinical Manifestations and Diagnosis The symptomatology of acute pericarditis is not specific for SLE. Patients can experience substernal or precordial chest pain that is positional in nature. Symptoms are worse with inspiration, supine position, coughing, or swallowing, and are relieved with sitting up or bending forward. Fever, dyspnea, and tachycardia are other common symptoms. Pericarditis may be associated with generalized serositis, typically pleuritis,18 or inflammation in other organs.19 On auscultation, diminished heart sounds and a pericardial friction rub may be heard. This highpitched scratchy sound is variable, and is best heard with the patient leaning forward at end-expiration. Hypotension, elevated jugular venous distension (JVD), hepatic congestion, and pulsus paradoxus (a drop in systolic blood pressure of >10 mmHg during inspiration) can be seen with pericardial tamponade. Tamponade is an unusual complication of pericardial effusions in SLE patients, and may be secondary to an acute accumulation of small or moderate amounts of pericardial fluid or subacute accumulation of large amounts of fluid. In large SLE patient series, 20 reports of tamponade were seen, with an estimated rate of 2.5%.5 Many other case reports have been cited in the literature.20 Tamponade may be the initial presenting symptom of SLE or may occur throughout the disease course. Drug-induced pericardial tamponade has occurred with procainamide,21 isoniazid,22 hydralazine,23 and carbamazepine.24 Constrictive pericarditis is rarely associated with SLE. Chronic inflammation and subsequent thickening of the pericardium lead to impairment of diastolic filling and ventricular function. Patients may have a third heart sound (pericardial knock) and Kussmaul’s sign (persistent elevation of venous pressure during inspiration) along with hepatic congestion, ascites, and leg edema. Constrictive pericarditis has also been associated with procainamide use.15,25

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TABLE 32.1 CARDIOVASCULAR DISEASES IN SYSTEMIC LUPUS ERYTHEMATOSUS Area of Involvement

Diagnostic Modality

Prevalence

Pericardium

Autopsy8-11 Clinical/ECHO2-7

46–100% in pre-steroid era; ~53% in steroid era 16–54%

Conduction system

EKG8,30-32

10–75%

7

Myocardium

Autopsy Clinical/ECHO43-45

57% in pre-steroid era; 7% in steroid era 5–20%

Endocardium (valves)

Autopsy8,10,11,18,46 Transthoracic ECHO6-8,44,68-74 Transesophageal ECHO45

13–74% 12–60% 61%

Coronary artery

Autopsy8,89,93 Clinical CHD2,82-88 Subclinical CHD (using imaging techniques)94-100

22–54% 6–11% 17–40%

CHD, coronary heart disease; ECHO, echocardiography; EKG, electrocardiogram.

Septic or purulent pericarditis is a rare complication in SLE patients. All reported cases occurred in patients taking steroids.12 Staphylococcus aureus,26 Candida,27 and tuberculosis have been identified in septic pericarditis. Therefore, infection should be part of the differential diagnosis in immunosuppressed patients with SLE who present with acute pericarditis. Electrocardiographic (EKG) findings reveal a classic PR interval depression with diffuse concave ST segment elevation (Fig. 32.1). Tachyarrhythmias (atrial fibrillation and flutter) and rarely bradyarrhythmias can occur. Electrical alternans, with beat-to-beat irregularity, and reduced QRS voltage can occur with large pericardial effusions and tamponade. The chest radiograph (CXR) may be normal or reveal a classic “water-bottle” cardiac silhouette (Fig. 32.2), pericardial fat lines, or pleural effusions. Echocardiography is the diagnostic modality of choice and is the best test to evaluate for pericardial tamponade. Pericardial effusions are recorded as a

relatively echo-free space between the pericardium and ventricular epicardium. With large effusions, the heart may swing freely within the pericardial sac. Pericardial tamponade may reveal right ventricular diastolic collapse and paradoxical septal motion. Pericardial thickening and calcifications are seen in constrictive pericarditis. Computed tomography (CT) and magnetic resonance imaging (MRI) are better than echocardiography in identifying loculated effusions, but will also reveal the pericardial thickening seen on echocardiography in chronic disease and constrictive pericarditis. The pericardial fluid in patients with SLE-related pericarditis is generally straw-colored, but may be serosanguinous or hemorrhagic. The fluid is exudative with elevated protein, normal to low glucose levels, and elevated white blood cell count with a predominance of polymorphonuclear cells. Autoantibodies (antinuclear [ANA] and double-stranded DNA [dsDNA]) and LE cells have been detected in the pericardial fluid of some patients with SLE.28 While ANA-positive pericardial Fig. 32.1 Electrocardiogram of acute pericarditis showing sinus tachycardia, diffuse ST elevation, and PR depression in lead II, aVF, and lateral precordial leads. (Courtesy of John C. Bailey, MD, Indiana University School of Medicine, Indianapolis, IN.)

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Clinical Manifestations and Diagnosis

Fig. 32.2 Chest radiograph (AP view) of acute pericarditis showing enlarged cardiac silhouette with both pericardial and pleural effusions.

fluid may be suggestive of SLE, it is not specific, and has been seen in other autoimmune diseases (rheumatoid arthritis), infections (tuberculosis), and malignancies.28 The diagnosis of pericarditis in lupus patients is often a clinical one based on typical signs and symptoms since it may occur without a detectable effusion on echocardiography or an abnormal EKG. Careful consideration should also be given to other diagnostic possibilities, including pulmonary embolus, myocardial infarction (MI), aortic dissection, purulent pericarditis, and pneumonia.

Treatment

Epidemiology and Pathogenesis Cardiac rhythm and conduction abnormalities occur in 10 to 75% of SLE patients8,30-32 (Table 32.1). This variability in prevalence is a consequence of different

Patients may experience a spectrum of symptoms from fatigue, weakness, and palpitations to heart failure and syncope. Asymptomatic abnormalities may be found coincidentally on an EKG, revealing arrhythmias or conduction abnormalities. Etiologies other than SLE

CONDUCTION SYSTEM

Asymptomatic and hemodynamically stable patients with pericardial effusions are generally not treated. Therapy of symptomatic disease varies upon severity of symptoms. Mild pericarditis often responds to nonsteroidal anti-inflammatory agents (NSAIDs). Corticosteroid therapy (prednisone 20 to 80mg/day or 0.5 to 1.0mg/kg/day) is used for severe or refractory pericarditis and pericardial tamponade. Improvements in symptoms, ventricular function, and tamponade have been reported with the use of intravenous methylprednisolone, immunosuppressive agents, and immunomodulating agents, such as intravenous immunoglobulin (IVIg).29 Pericardiocentesis is a necessary intervention for pericardial tamponade, suspicion of septic pericarditis, or in symptomatic patients refractory to medical treatment. A pericardial window may also be required. In patients with chronic disease and constrictive pericarditis, pericardial stripping or pericardiectomy may be performed.

case definitions. Sinus tachycardia is the most common arrhythmia; however, bradycardia, atrial arrhythmias, and first- and second-degree atrioventricular (AV) heart block also occur. Third-degree or complete heart block is rare in adult lupus patients. Cardiac autonomic dysfunction is also a recognized complication of SLE.30,33 Complete heart block and other conduction abnormalities are rare as a presenting manifestation, but more commonly occur later in the disease course.34 The major difficulty is attributing conduction abnormalities to SLE alone, as other more common causes such as atherosclerotic disease, electrolyte disturbances, and thyroid dysfunction are commonly seen in SLE patients. When conduction abnormalities are related to lupus,35 it is commonly associated with other disease manifestations such as pericarditis, myocarditis, and antibodies to ribonucleoprotein (U1-RNP)36,37 and SSA/Ro.32 Adult complete heart block has been associated with antimalarial drug-induced cardiotoxicity38; only 13 case reports of complete heart block without concomitant hydroxychloroquine use have been reported.37 When conduction abnormalities are believed to be SLE related, immune-mediated injury to conduction tissues and small vessels of the myocardium are felt to be the underlying pathogenesis. Pathologic specimens have revealed focal degeneration and fibrosis of the sinus node, AV node, and AV bundles, as well as necrotizing arteritis and occlusion of the central sinus node artery.8,39 This chronic inflammation and fibrosis may lead to irreversible defects in the conduction system. Neonatal conduction abnormalities and congenital heart block (CHB) are well-recognized complications of neonatal lupus. CHB in infants (before or after birth) is associated with maternal autoantibodies to SSA/Ro and SSB/La in more than 85% of cases, independent of whether the mother has evidence of SLE.40 Approximately 2 to 3% of anti-SSA/Ro–positive mothers will have a child with CHB.41 Maternal IgG antibody–mediated CHB is most often detected at 18 to 24 weeks of gestation.42 Fetuses of mothers with SSA/Ro and SSB/La autoantibodies should be followed by maternal-fetal specialists and monitored by serial fetal echocardiograms. Dexamethasone (fluorinated corticosteroid), IVIg, plasmapheresis, and in utero cardiac pacing are used when evidence of arrhythmias or heart failure is detected. Mortality rates are close to 20%, with 60% of children requiring pacemakers.42

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(ischemia, thyroid dysfunction, electrolyte imbalance, and medications) must be investigated.

Treatment Asymptomatic tachyarrythmias and first- or seconddegree AV block do not require intervention and may be observed, assuming that other nonlupus causes have been investigated and treated. Third-degree and any conduction abnormality that results in hemodynamic instability requires intervention with medical therapy, such as antiarrhythmic medications or pacemaker placement. Corticosteroid therapy has successfully treated conduction blocks in a small number of case reports.35 One should keep in mind the association of drug-induced conduction abnormalities, such as antimalarials, and the offending medication should be discontinued.

MYOCARDIUM

Epidemiology and Pathogenesis

364

Myocardial abnormalities are observed in 5 to 57% of patients7,43-45 (Table 32.1), of which 10% are clinically apparent.18,43,46 Subclinical myocarditis has been reported in as many as 57% of autopsy series from the 1950s and 1960s.43 With the widespread use of corticosteroids, prevalence of myocarditis on autopsy has decreased to 7%. Myocarditis may progress to ventricular dysfunction, cardiomyopathy, and heart failure.46 The majority of myocardial abnormalities are due to lupus-related comorbidities such as ischemic heart disease, hypertension, renal failure, valvular disease, and medication toxicity. A 5-year prospective study demonstrated progressive abnormalities of systolic and diastolic left ventricular function in lupus patients with coexisting hypertension and coronary artery disease.47 The degree to which myocardial dysfunction is due to myocarditis or direct immune-mediated damage to myocardium is unknown. Abnormal cardiac hemodynamics on catheterization have been described in lupus patients without evidence of cardiac disease. Strauer and colleagues revealed increased biventricular end-diastolic pressures, decreased cardiac output, decreased cardiac contractility, decreased ejection fraction, increased wall stiffness, and reduction of coronary artery reserve.48 Del Rio and colleagues,49 described left ventricular dysfunction with higher heart rates, shortened left ventricular ejection time, and a prolonged pre-ejection period when compared to controls. These abnormalities were independent of age, disease duration, hypertension, corticosteroid use, and renal involvement, suggesting disease-related chronic inflammation as the cause. Ventricular dysfunction has also been associated with elevated anti-DNA antibody titers, suggesting a correlation with disease activity.

The pathogenesis of myocardial dysfunction is an immune-complex mediated process, with complement deposition and cytokine activation within the myocardial blood vessels, and to a lesser degree, myocardial tissue.50,51 Small foci of interstitial plasma cells and lymphocytes can be seen within the myocardial tissue, but diffuse inflammation is uncommon.8,12 Subsequent necrosis and fibrosis of myocytes leads to myocardial depression and conduction system abnormalities. In corticosteroid-treated patients, small foci of patchy myocardial fibrosis are observed, likely representing healed myocarditis.8 Circulating autoantibodies have also been hypothesized to play a role in the pathogenesis of myocardial dysfunction in SLE. Antimyocardial52 and anti-SSA/Ro32 autoantibodies have been associated with myocarditis. Borenstein and colleagues reported an association between the presence of anti–U1-RNP autoantibodies, myositis, and myocarditis in five SLE patients, suggesting a generalized inflammatory myopathic process.53

Clinical Manifestations and Diagnosis Clinical signs and symptoms are not specific for SLE, but are based on the severity of myocardial dysfunction. Lupus patients with myocarditis may be asymptomatic, or present with fever, chest pain, dyspnea, palpitations, and tachycardia out of proportion to fever. Presentation may be acute or chronically progressive with ultimate heart failure. Patients may experience fatigue, cough, orthopnea, paroxysmal nocturnal dyspnea, and signs of congestive heart failure. A cardiac murmur, third heart sound, or gallop rhythm may be auscultated, with irregular heart rate, displaced apex, elevated jugular venous distension (JVD), and lower extremity edema. Elevation of creatine kinase (CK) and troponin has not consistently correlated with myocardial involvement. Myocardial dysfunction may accompany other manifestations of SLE, specifically pericarditis. However, myocarditis is an unusual initial manifestation of SLE.54 Electrocardiograms reveal nonspecific ST segment changes, premature atrial or ventricular contractions, arrhythmias, and conduction abnormalities. Asymptomatic patients may first be discovered on an EKG evaluation performed for other reasons. Chest radiographs may be normal or have evidence of cardiomegaly, with or without pulmonary edema. Echocardiographic findings are nonspecific for lupus myocardial dysfunction, but are helpful in assessing cardiac function. Decreased ejection fraction, increased chamber size, prolonged relaxation time, decreased deceleration of early diastole, and decreased E/A ratio (filling of left ventricle in early and late diastole) have been observed in lupus patients.43 Cardiac catheterization has revealed abnormalities previously

reported, including myopathy, neuropathy, and retinotoxicity. Cardiotoxicity from both CQ and HCQ has been reported in 25 cases in the English-language literature to date38,62,63; however, less than 50% of these are biopsy proven. Systolic and diastolic dysfunction, as well as conduction abnormalities (first-degree AV block, right bundle branch block, left bundle branch block, and complete heart block) have been reported. Antimalarial drug-induced cardiotoxicity is a diagnosis of exclusion; however, endomyocardial biopsy may be diagnostic for it. Endomyocardial histology reveals large secondary lysosomes, and myeloid and curvilinear bodies (lipid-rich structures representing abnormal lysosomes), with variable myofiber atrophy and necrosis62-64 (Figs. 32.3 and 32.4). The majority of cases are older women with long duration of antimalarial

MYOCARDIUM

described by Strauer and colleagues,48 and should be considered to rule out coronary heart disease (CHD), particularly in patients with chest pain or cardiovascular risk factors. New imaging techniques have suggested the ability to detect myocardial injury; unfortunately the number of patients evaluated is small and the findings are not specific for lupus. Cardiac MRI has revealed higher T2 relaxation times, suggestive of myocardial edema, in active lupus patients when compared to asymptomatic lupus patients and controls.55 Diffuse accumulation of gallium-67 citrate scintigraphy has been reported in a lupus patient with clinical evidence of myocarditis.56 Indium-111 antimyosin Fab imaging in lupus patients has suggested more widespread disease than initially described by echocardiography.57 Endomyocardial biopsy reveals fibrous thickening of arterial walls with luminal narrowing and IgG deposits in perivascular areas; foci of plasma cells, lymphocytes, and neutrophils in the myocardium; and myocardial fibrinoid necrosis, fibrosis, and scarring.8,12,43,50,51,58,59 These findings are suggestive of lupus myocarditis in the appropriate clinical setting, but are not specific. The American College of Cardiology/American Heart Association (ACC/AHA)60 states that “endomyocardial biopsy can be useful in patients presenting with heart failure when a specific diagnosis is suspected that would influence therapy” and that “endomyocardial biopsy should not be performed in the routine evaluation of patients with heart failure.” The sensitivity and specificity of endomyocardial biopsy in lupus is unclear. Ardehali and colleagues recently evaluated the utility of diagnosis by endomyocardial biopsy in patients with unexplained cardiomyopathy.61 The sensitivity of endomyocardial biopsy compared to clinical diagnosis was 100% versus 66%, with equal specificities (86% vs. 87%). Seven patients were diagnosed with SLE, all of whom were diagnosed based on other clinical manifestations without endomyocardial biopsy. Prior to making a diagnosis of SLE-related inflammatory myocardial disease, other non-SLE etiologies must be investigated. Ischemic heart disease, which is the leading cause of cardiomyopathy and myocardial dysfunction, occurs at increased frequency in SLE patients. Renal disease and hypertension, two conditions common to SLE patients, also contribute to myocardial dysfunction. Infectious etiologies, both viral and bacterial, must be considered in a new diagnosis of myocarditis. Finally, treatment-related cardiotoxicity from cyclophosphamide and antimalarial medications must also be considered. Antimalarial medications, chloroquine phosphate (CQ) and especially hydroxychloroquine sulfate (HCQ), have become a part of standard therapy in SLE. Rare but potentially serious toxicities have been

A

B Fig. 32.3 Light microscopy of endomyocardial biopsy tissue for hydroxychloroquine-induced cardiotoxicity. A, Diffusely enlarged and vacuolated myocytes (paraffin-embedded, hematoxylin-eosin stain, magnification x180). B, Extensive sarcoplasmic accumulation of darkly staining material (plastic-embedded toluidine blue stain, magnification x540). (From Keating RJ, Bhatia S, Amin S, et al. Hydroxychloroquine-induced cardiotoxicity in a 39-year-old woman with systemic lupus erythematosus and systolic dysfunction. J Am Soc Echocardiogr 2005;18:981.)

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therapy; however, identification of risk factors is difficult given the small number of case reports.63 This diagnosis should be considered in the clinical scenario of myocardial dysfunction, past or concurrent use of CQ or HCQ, and endomyocardial biopsy.

Treatment

A

B

Therapy is based upon the underlying etiology of myocardial dysfunction, and treatment can be markedly different for coronary artery disease, medication-induced cardiotoxicity, and lupus myocarditis. Treatment of inflammatory myocardial disease with corticosteroids, in the setting of ischemic CHD, may worsen heart failure and hypertension. Corticosteroid therapy in large doses (1 mg/kg/day) or pulse intravenous therapy is the initial mainstay of therapy. Immunosuppressive and immunomodulating medications are also routinely used; however, there are no controlled clinical trials, only anecdotal case reports. Restoration of cardiac function has been reported using intravenous cyclophosphamide,65 azathioprine,51,66 IVIg,67 and plasmapheresis.59 Cases with endomyocardial fibrosis are less responsive to therapeutic interventions.51,53,59 Therapy with afterload reduction (ACE inhibitors or angiotensin-receptor blockers), diuretics, and betablockers, as well as water and salt restriction, is the standard of care for heart failure and cardiomyopathy.60 Aggressive management of hypertension and dyslipidemia for ischemic cardiomyopathy, and anticoagulation for depressed ejection fraction should also be considered. Antiarrhythmic medications, defibrillators, or ventricular assist devices are used when there are associated arrhythmias or hemodynamic instability. Scant information is available on long-term outcomes of SLE myocardial dysfunction. Law and colleagues followed 11 patients (median of 4 years) with acute lupus myocarditis.65 All patients were treated with high-dose corticosteroids and seven received intravenous cyclophosphamide. Nine patients had no recurrence of myocarditis but two deaths from sepsis occurred.

ENDOCARDIUM

Epidemiology and Pathogenesis C

366

Fig. 32.4 Electron microscopy of endomyocardial biopsy tissue for hydroxychloroquine-induced cardiotoxicity. A, Extensive accumulation of membrane-like material within myocytes (x38000). B, Myelinoid bodies (x10,000). C, Curvilinear bodies (x45,000). (From Keating RJ, Bhatia S, Amin S, et al. Hydroxychloroquine-induced cardiotoxicity in a 39-year-old woman with systemic lupus erythematosus and systolic dysfunction. J Am Soc Echocardiogr 2005;18:981.)

Valvular heart disease is common in SLE patients, with echocardiographic abnormalities reported in 12 to 61% of patients6-8,44,45,68-74 (Table 32.1). Autopsy studies reveal valvular abnormalities in 13 to 74%.6,10,11,18,46 Corticosteroid therapy has reduced the frequency of valvular lesions. Verrucous lesions were identified in 59% of autopsied patients in the pre-steroid era; while only 36% are reported with corticosteroid use.12 Transesophageal echocardiographic findings suggest

Fig. 32.5 Pathology of Libman-Sacks endocarditis (gross specimen). Two light-yellow verrucae can be seen on the endocardium of the left ventricle immediately beneath the insertions of chordae tendineae to the mitral valve. (Courtesy of American College of Rheumatology.)

with valvular abnormalities. Antiphospholipid antibodies are hypothesized to directly affect the endothelium, activate clotting factors, and mediate valvular damage.44 Recently, Leszczynski and colleagues observed aPL positivity in 77% of SLE patients with valvular abnormalities.71 This positive association has been noted by others.7,68,69 In contrast to these reports, other investigators have reported no difference in valvular abnormalities between SLE and primary APS patients.73,74 The pathogenesis of valvular disease in SLE has not been clearly elucidated; however, it is hypothesized that both aPL and immune complexes play a crucial role. Antiphospholipid antibodies directly bind to phospholipids on endothelial cells and platelets, leading to platelet aggregation and thrombus. Deposits of immunoglobulin and complement have been demonstrated histopathologically in Libman-Sacks lesions as well as in primary APS patients with valvular disease.75 Formation of these inflammatory prothrombotic lesions leads to damage, scarring, and deformities of the valvular structures.

ENDOCARDIUM

that valvular thickening is the most common endocardial lesion seen. Roldan and colleagues revealed valvular thickening on transesophageal echocardiography in 51% of SLE patients; 43% had Libman-Sacks vegetations, 25% with regurgitations, and 4% with stenosis.45 In 1924, Libman and Sacks first described the pathognomic valvular lesion in SLE, “atypical verrucous endocarditis.”9 Libman-Sacks endocarditis, also called marantic or noninfectious verrucous endocarditis, was initially described on the tricuspid valve,9,10 and later on the mitral valve8,11; however, any valve can be involved. The lesions are usually pea-sized (1 to 4 mm), pink or gray granular projections on valve leaflets, rings, and commissures, extending over both sides, and less commonly to the atrial or ventricular endocardium, chordae tendinae, and papillary muscles12 (Fig. 32.5). These noninfectious lesions may be visually similar to infective endocarditis, but they differ histopathologically. Lesions in Libman-Sacks endocarditis have an outer layer of fibrin, proliferating cells, and hematoxylin bodies, with an inner granular layer of immunoglobulin and complement lining the vascular wall.8,50 Bulkley and Roberts8 described different histologic stages of Libman-Sacks endocarditis. The active stage was described as having clumps of fibrin, lymphocytes, and plasma cells within or on the endocardium. These active lesions were described in younger patients with recent onset of disease and were not hemodynamically significant. The healed lesions consisted of fibrous tissue with calcifications, which were more commonly hemodynamically significant and observed in patients with long-standing disease and corticosteroid use. Active valvulitis and subsequent fibrotic lesions after therapy can lead to valvular insufficiency.50 Antiphospholipid syndrome (APS) and antiphospholipid antibodies (aPL) are commonly associated

Clinical Manifestations and Diagnosis Patients are usually asymptomatic and have abnormalities that lead to minor functional limitations. LibmanSacks endocarditis may present similarly to infective endocarditis with fever, tachycardia, murmur, splinter hemorrhages, embolic phenomenon, anemia, and elevation of inflammatory markers (erythrocyte sedimentation rate and C-reactive protein) may be seen. Echocardiography is key in the investigation. Valvular thickening, vegetations, regurgitation, and stenosis may be seen. Unfortunately, these findings are not specific for SLE, and only histopathology provides a definite diagnosis. Since many of the presenting symptoms can be similar, and the lesions may be indistinguishable by echocardiography, infectious endocarditis must be considered in the differential diagnosis. Outcomes of valvular disease in SLE suggest that these abnormalities have more long-term consequences than originally thought. Roldan and colleagues followed 69 SLE patients with echocardiograms every 2 years for 5 years.45 Valvular lesions changed over time; some resolved, some persisted, and new lesions occurred, suggesting an intermittent valvulitis. No correlation was found between valvular lesions and other clinical features of SLE. Complications such as cerebral vascular accident (CVA), peripheral embolism, heart failure, infective endocarditis, and necessity for valvular replacement occurred in 22% of lupus patients with valvular abnormalities as compared to only 8% without valvular lesions. Most cases of death were attributed to valvular disease. In a lupus cohort, 73% of 61 patients were reported to have valvular abnormalities, but only 7 patients (12%) developed severe valvular regurgitation

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over a 14-year period.76 Among the SLE patients with valvular lesions, 86% had complications such as CVA, peripheral embolism, need for valve surgery, and death. These complications were reported in only 25% of SLE patients without valvular lesions. Morelli and colleagues72 evaluated 71 SLE patients for the risk of ischemic CVA over 5.9 years. Nearly one-half (43%) had left-sided valve abnormalities, with ischemic CVA developing in 26% of these lupus patients (odds ratio 10.8, 95% confidence interval 2.33–50.52) from thromboembolism. A recent report suggested that valve lesions (vegetations and regurgitation) and aPL were independent predictors of cerebrovascular disease with odds ratios ranging from 5.3 to 10.6.77 Valvular abnormalities have also been associated with CHD.70 Twenty-six SLE patients with CHD were compared to 26 SLE patients without CHD and 26 healthy controls. Fifty percent (13 cases) of SLE patients with CHD had valve lesions, as compared to only 3.8% (1 case) of SLE patients without CHD and of healthy controls. Valvular abnormalities were associated with higher triglycerides and homocysteine levels.

Treatment

368

There is no specific therapy for Libman-Sack endocarditis. Treatment with corticosteroids and immunosuppressive agents is appropriate in patients with other manifestations of disease activity. The effect of immunosuppressive therapy is not known; however, complete healing with fibrous thickening has occurred with corticosteroid therapy.18 Valvular replacement may be indicated in the setting of hemodynamic compromise. Both mechanical and biologic valve replacements have been preformed with variable success rates.78 Recurrent valvulitis has been reported on biologic valves. Mechanic valve replacement is usually recommended; however, this requires lifelong anticoagulation. Anticoagulation is generally recommended for valvular lesions in patients with known antiphospholipid syndrome.79 There are no clear guidelines for therapy in aPL-positive SLE patients without thrombosis, but antiplatelet therapy has been used. Although no formal guidelines exist, SLE patients with prosthetic valves or valvular vegetations are considered at high risk for endocarditis with dental, genitourinary, and gastrointestinal procedures, and should receive prophylactic antibiotic therapy.80 Patients with mitral valve prolapse are considered to be at moderate risk and also warrant consideration for prophylactic antibiotics. Surveys of infectious disease specialists reveal that nearly 95% rarely or never recommend endocarditis prophylaxis to SLE patients undergoing invasive dental procedures, suggesting that this important preventive intervention may need to be initiated by the rheumatologist.81

CORONARY ARTERY

Epidemiology and Pathogenesis Coronary artery involvement is the most recent cardiovascular manifestation to be recognized in SLE patients. Pathology may be secondary to atherosclerosis, arteritis, thrombosis, embolization, or coronary artery spasm. While MI is the most common clinical manifestation, atherosclerotic heart disease is the most prevalent pathologic finding. The impact of atherosclerotic CHD in SLE patients was first emphasized by Urowitz and colleagues.82 A bimodal pattern of mortality was described in SLE patients, with early deaths occurring secondary to complications of disease activity and therapy, and late deaths occurring as a consequence of cardiovascular events. Autopsy studies confirmed the significance of coronary atherosclerosis in these cardiac deaths. Bulkley and Roberts reported that 22% of SLE patients had greater than 50% occlusion of one major coronary artery by atherosclerotic plaque.8 All of these patients had received corticosteroid therapy for at least 1 year. With improvements in diagnosis and treatment (antibiotics, antihypertensive medications, corticosteroids, and hemodialysis) of SLE, CHD has emerged as one of the leading causes of morbidity and mortality. The prevalence of CHD events (MI and angina) ranges from 6 to 11%2,82-88 (see Table 32.1), whereas cerebrovascular events (stroke and transient ischemic attacks) have been reported in 10 to 26%45,72,86-89 of SLE patients. The most striking feature of CHD in SLE is the predilection for young premenopausal women. Manzi and colleagues compared women with SLE to agematched women in the Framingham Offspring Cohort and found that lupus women aged 35 to 44 years were over 50 times more likely to have an MI as compared to controls.90 In addition, young women (aged 18 to 44 years) with SLE were 2.27 times more likely to be hospitalized for an MI, 3.8 times more likely to be hospitalized for congestive heart failure, and 2.05 times more likely to be hospitalized for stroke than age-matched controls.91 While women in the general population succumb to cardiovascular events around 60 years of age, the average age of first cardiovascular event in SLE women is 49 years.92 Similar to other cardiac manifestations of SLE, subclinical CHD is more prevalent than clinical events. Autopsy reports indicate that 22 to 54% of SLE patients have atherosclerotic CHD.8,89,93 Histopathologically, coronary atherosclerosis appears the same as that of the general population. Haider and Roberts89 reported an association between pericardial and valvular disease and coronary artery narrowing in SLE, suggesting an inflammatory or immunologic component. Newer noninvasive imaging techniques can function as

Fig. 32.6 Electron-beam computed tomography revealing coronary calcification in the left main artery and left anterior descending artery.

The pathogenesis of atherosclerosis in SLE is complex and multifactoral, and is an area of intense active research. Inflammation, while playing a prominent role in the pathogenesis of SLE, is also the underlying mechanism of atherosclerosis in both general and lupus populations. In addition, other factors, such as traditional CV risk factors, lupus-specific factors, and medications, also play a prominent role. When compared to age-matched controls, SLE patients have an increased burden of traditional cardiovascular risk factors,96,98,101,102 including hypertension, hypertriglyceridemia, hyperhomocysteinemia, diabetes mellitus, tobacco use, and sedentary lifestyle. In some reports, 27 to 64% of lupus patients are hypertensive and 52 to 56% have hypercholesterolemia (>200 mg/dL).83,94,99,101,103 Frequencies of other traditional risk factors vary from cohort to cohort: 16 to 48% are obese (body mass index [BMI] >27.5 kg/m2), 15 to 83% have a sedentary lifestyle, and 15 to 54% use tobacco. Traditional cardiovascular risk factors do not, however, completely account for the increased risk of CHD in SLE patients. Esdaile and colleagues104 reported that the increased risk of CHD and stroke cannot fully be explained by traditional Framingham risk factors alone. Nephritis, antiphospholipid antibodies, hyperhomocysteinemia, and other “lupus” factors and medications are thought to contribute to premature atherosclerosis in SLE patients. Renal disease and lupus nephritis have been associated with hypertension and increased atherosclerosis in SLE patients. Antiphospholipid antibodies have been associated with vascular events, including myocardial infarction and stroke; however, their role in atherogenesis is unclear.105 Homocysteine has been identified as a potentially modifiable risk factor for occlusive vascular disease in both general and lupus populations.106 Elevated homocysteine levels have been reported in 15% of lupus patients and have been associated with both cardiovascular events and subclinical atherosclerosis.101,103,106 A “lupus factor” has also been hypothesized, as patients with chronic, lowlevel “smoldering” disease activity seem to have increased frequencies of atherosclerosis.98 Corticosteroid therapy, while instrumental in the management of disease activity, has numerous metabolic side effects. Hypertension, hyperglycemia, obesity, and dyslipidemia are well-known sequelae of steroid therapy. Petri and colleagues reported that a change of 10 mg of prednisone leads to an increase of 7.5 mg/dL of total cholesterol, a 1.1-mmHg increase in mean arterial blood pressure, and 5.5-pound weight gain.107 Conversely, hydroxychloroquine therapy has been shown to have several beneficial cardiovascular effects in SLE patients. Hydroxychloroquine can reduce total cholesterol levels in SLE patients taking corticosteroids, suggesting that it may offset the dyslipidemia associated with

CORONARY ARTERY

surrogate markers of cardiovascular disease (CVD). B-mode carotid ultrasound, electron-beam computed tomography (EBT), and myocardial perfusion scans demonstrated subclinical CVD in 17 to 40% of the SLE cohorts.94-100 Manzi and colleagues detected carotid plaque by B-mode ultrasound in 40% of 175 lupus women (mean age 44.9 years).94 The independent risk factors for carotid plaque were higher systolic blood pressure, higher levels of LDL cholesterol, older age, prolonged treatment with prednisone, and a previous coronary event. Carotid plaque formation was greater in SLE patients when compared to age-, sex-, race-, and hypertensive-status matched controls, as shown by Roman and colleagues.98 They reported carotid plaque in 37.1% of SLE patients as compared to 15.2% of controls with similar baseline cardiovascular risk factors. Older patients with longer disease duration and disease-related damage were more likely to develop carotid plaques. In addition, SLE patients treated with prednisone, cyclophosphamide, and hydroxychloroquine were less likely to have plaque formation. Their findings suggested that controlling the underlying inflammation with immunosuppressive medications might protect against development of carotid atherosclerosis.98 Coronary artery calcification, as detected on EBT, is another surrogate marker for subclinical CHD (Fig. 32.6). Manger and colleagues97 reported coronary artery calcification in 28% of young SLE women (mean age of 38.8 years). In a recent study by Asanuma and colleagues,96 SLE patients had an increased prevalence of coronary artery calcification compared to age-similar controls (31% vs. 9%). Finally, Sella and colleagues99 reported defects in myocardial perfusion scintigraphy, a functional measure of subclinical CHD, in 28% of SLE patients. Prevalence figures for subclinical CVD in SLE are strikingly similar (30 to 40%) regardless of the imaging technique used to detect it.

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steroids.107,108 Hydroxycholoroquine has also been associated with improved glycemic control in poorly controlled diabetes.109 Roman and colleagues98 reported that SLE patients taking hydroxycholorquine were less likely to develop carotid plaques. Regardless of the underlying etiology, an important key to intervention and prevention of atherosclerosis in SLE is awareness of the increased risk in young lupus patients. Other coronary artery manifestations, such as coronary arteritis, aneurysms, vasospasm, and embolic phenomenon, have been described in lupus patients. Coronary arteritis is a rare cardiovascular manifestation of SLE and primarily a pathologic diagnosis at autopsy.12,46 Vasculitis in other organ systems can be suggestive of coronary arteritis, but coronary arteritis may occur without other signs of SLE activity. Angiography may reveal aneurysms or evolving areas of stenosis or occlusion on serial evaluations. Coronary artery spasm and microvascular disease are well-recognized phenomena in SLE patients with pathologic evidence of microthrombi and hyalinization of small coronary arteries.8,12,18 These small vessel changes are felt to be responsible for the cardiomyopathy seen in SLE. Finally, case reports of valvular embolization causing coronary artery disease have been reported in SLE patients.8

Clinical Manifestations and Diagnosis Signs and symptoms of angina or MI do not differ in SLE patients as compared to the general population. Chest discomfort may be described as heavy, tight, or crushing, and is typically located in the center of the chest or epigastrium. The pain can radiate to the arm, neck, jaw, back, or abdomen, and may be associated with dyspnea, dizziness, diaphoresis, heartburn, or a sense of impending doom. Hypotension or new-onset arrhythmia may be the presenting manifestation. Given the female predominance of SLE, it is important to be aware of other “atypical” symptoms of CVD, such as nausea, dyspepsia, fatigue, or anxiety. Diagnostic testing, including EKG, cardiac enzymes, and cardiac imaging, is the same in SLE patients as non-SLE populations. The most critical aspect in the diagnosis of atherosclerotic heart disease is to maintain a high index of suspicion, particularly in young women.

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The ACC/AHA has defined guidelines for the aggressive treatment of acute MI.110 After a complete, but efficient history and physical examination, prompt initiation of oxygen, nitroglycerine, aspirin, betablockers, and reperfusion therapy should be initiated. Lupus patients have been successfully treated with percutaneous angioplasty and coronary artery bypass surgery. Ward recently reported no difference in risk

of in-hospital mortality, length of stay, or congestive heart failure between SLE and non-SLE patients admitted for MIs.111 The most important aspect of premature CHD in SLE patients is prevention. Potentially modifiable traditional cardiovascular risk factors must be recognized and treated. Historically, physicians have done a suboptimal job of identifying these risk factors. Bruce and colleagues112 retrospectively reviewed the medical records of 24 SLE patients who developed a MI or acute coronary insufficiency, and identified several potentially modifiable CV risk factors that were not addressed. Al-Herz and colleagues reported that only one-third of patients in a lupus clinic cohort were evaluated for possible dyslipidemia.113 Perhaps some of these discrepancies are secondary to the lack of established screening and prevention guidelines specifically for SLE patients. Most lupus experts believe that SLE patients should be considered as CHD risk equivalents, similar to patients with diabetes mellitus. At initial presentation and then annually, SLE patients should be evaluated with a fasting lipid panel and serum glucose. Blood pressure and body mass index (BMI) (or waist circumference) should be calculated at each visit. Patients should also be screened for tobacco use and family history of MIs and strokes. Wajed and colleagues114 have recently proposed guidelines for cardiovascular risk factor management. If SLE is considered a CHD risk equivalent, they suggested the following target goals: systolic blood pressure less than 130 mmHg, diastolic blood pressure less than 80 mmHg, LDL cholesterol less than 100 mg/dL, fasting blood glucose less than 126mg/dL, and BMI less than 25 kg/m2.

CONCLUSIONS All structures of the heart can be involved in SLE, including the pericardium, conduction system, myocardium, endocardium, and coronary arteries. Prevalence figures from the literature vary depending on definitions and surveillance mechanisms (autopsy, imaging techniques, and symptomatic disease). In most cases, asymptomatic involvement does not require intervention. Pericardial disease seems to be the most common cardiovascular manifestation, while atherosclerotic coronary heart disease has the most significant impact on morbidity and mortality, especially in young premenopausal patients with SLE. With the advent of advanced imaging techniques, we may be in a position to identify those patients at greatest risk for cardiovascular disease. In the meantime, awareness of premature cardiovascular disease, screening, and management of modifiable risk factors is warranted.

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33

The Lung in Systemic Lupus Erythematosus Rapti Mediwake, MD and Roland M. du Bois, MD

INTRODUCTION Systemic lupus erythematosus (SLE) can affect all parts of the respiratory system, including airways (upper and lower), lung parenchyma, pleura, pulmonary vasculature, and respiratory muscles (Table 33.1). The lungs and the pleura are commonly affected in SLE with 50 to 70% of patients developing pleuropulmonary manifestations during the course of their disease.1-3 It may be asymptomatic and is frequently not associated with significant morbidity; however, life-threatening disease can occur. Patients can develop more than one pulmonary manifestation over the course of the disease. Pulmonary involvement diagnosed within the first year of disease is reported to be associated with increased mortality at 10 years.4

PLEURAL DISEASE

374

Pleuritis is present in 45 to 60% of patients, and is the most common thoracic manifestation of SLE.1,5 It tends to be painful and can be a presenting feature in 20%,6 but occurs more commonly (50%) during a disease exacerbation. Pleural abnormalities are found at autopsy in 50 to 100% of patients.7 Pleuritis is associated with a pleural effusion in 50% of patients.1,8 Effusions are commonly small or moderate in size, bilateral more often than unilateral, and equally distributed between right and left hemithoraces (Fig. 33.1).9 Pleural fluid is commonly exudative, serosanguinous, and neutrophilic during acute attacks, and lymphocytic in chronic effusions. It often contains antinuclear antibodies, although this is not tested routinely unless the diagnosis is unclear.10 Pleural effusions can occur in lupus secondary to cardiac or renal involvement but tend not to be painful. Pleural effusions can resolve spontaneously and small asymptomatic effusions may require no specific treatment. Symptomatic pleurisy may require nonsteroidal anti-inflammatory drugs (NSAIDS). Moderate effusions

may require introducing corticosteroids or stepping up their dosage.3 Long-term treatment may require immunosuppressive drugs or hydroxychloroquine. Pleurodesis or chest drain insertion is rarely required.

ACUTE PNEUMONITIS Acute lupus pneumonitis occurs in up to 4% of patients.1,11 Rarely it can be a presenting feature of lupus. It presents abruptly with fever, cough, dyspnea, pleuritic chest pain, hypoxia, and occasionally hemoptysis. Chest radiography typically shows patchy unilateral or bilateral pneumonic-like infiltrates, usually at the bases, commonly associated with pleural effusions. These features are seen more graphically via computed tomography (CT). Histopathologic findings include diffuse alveolar damage and necrosis, edema, inflammatory cell infiltrate, hemorrhage, and hyaline membrane formation.12 Alveolar damage is immune complex mediated. Immunofluorescence studies have shown granular deposits of IgG and C3 along the alveolar walls.13 Symptoms mimic bacterial infection or alveolar hemorrhage, and these should be excluded using bronchoalveolar lavage. Patients require corticosteroids,2 and in some cases require cyclophosphamide in addition.3 Plasmapheresis is also useful.11 This acute presentation carries a mortality of 50%.11,12 In survivors, more chronic disease is characterized by persistent radiographic shadowing and a restrictive ventilatory defect and reduced gas transfer for carbon monoxide (DLCO).

DIFFUSE ALVEOLAR HEMORRHAGE Alveolar hemorrhage is rare and occurs in 2% of lupus patients,1 but accounts for 20% of SLE hospital admissions.14 Alveolar hemorrhage can be the presenting feature in 20% of SLE patients and carries a

DIFFUSE ALVEOLAR HEMORRHAGE

TABLE 33.1 PULMONARY MANIFESTATIONS OF SYSTEMIC LUPUS ERYTHEMATOSUS Primary

Pleural

Pleuritis Pleural effusion Pleural thickening

Diffuse lung disease

Acute pneumonitis Diffuse alveolar hemorrhage Usual interstitial pneumonia Nonspecific interstitial pneumonia Lymphocytic interstitial pneumonia Organizing pneumonia

Small airways disease

Bronchiolitis obliterans

Vascular

Pulmonary hypertension Vasculitis Thromboembolism with or without antiphospholipid syndrome Acute reversible hypoxemic syndrome Secondary

Respiratory muscle weakness

Shrinking lung syndrome

Pneumonia Drug-induced lupus Pulmonary edema (renal/cardiac)

mortality of 50 to 60%.14,15 The typical presentation is of acute severe dyspnea with fever, and crepitations are heard on auscultation. Hemoptysis occurs in up to twothirds of patients.16 Chest radiographs can show widespread pulmonary infiltrates, ground-glass opacities,

Fig. 33.1 Pleural effusion. Chest radiograph shows blunting of both costophrenic angles, compatible with small pleural effusions.

Fig. 33.2 Diffuse alveolar haemorrhage. CT shows patchy ground glass attenuation in a bronchocentric distribution, consistent with intra-alveolar blood.

and areas of consolidation, usually bilateral with a lower zone predominance that clear within a few days either spontaneously or with treatment.17 CT confirms widespread patchy ground-glass attenuation with or without consolidation (Fig. 33.2). Arterial hypoxemia is common, and many patients need ventilatory support. Bronchoscopy and bronchoalveolar lavage are required to exclude a site of bleeding, concurrent infection, and other causes of acute respiratory failure. Blood in the large airways, serosanguinous lavage with incremental amounts of blood from serial aliquots, hemosiderin-laden macrophages, and lack of purulent sputum support the diagnosis, especially when hemoptysis is not present.18 A decrease in hemoglobin, seen in the first 1 to 2 days, is a characteristic feature, and is a useful clue in patients who do not have hemoptysis. Alveolar hemorrhage usually occurs in patients with active extra-pulmonary disease, commonly arthropathy and renal disease, and high levels of dsDNA antibodies.1,15 Alveolar hemorrhage is often associated with glomerulonephritis in lupus patients.19,20 Lupus nephritis is normally a pre-existing condition rather than an acute co-presentation with alveolar hemorrhage.15,16 Histopathologic features include polymorphonuclear and mononuclear cell interstitial inflammation, alveolar necrosis, hemosiderin-laden macrophages, and an acute necrotizing capillaritis.21 Immune complexes IgG and C3 are found in 50% of cases. Alveolar hemorrhage can have a fatal outcome with mortality rates reported of 40 to 90%.14,16 Patients who have a fatal course usually die rapidly within the first few days. There are no randomized controlled trials regarding treatment. The treatment of choice is high-dose corticosteroids with cyclophosphamide.15 Plasmapheresis should be used for patients with severe alveolar hemorrhage.16,22

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376

CHRONIC INTERSTITIAL LUNG DISEASE Chronic interstitial lung disease (ILD) is a rare manifestation of SLE. Most cases are asymptomatic. In some studies, up to two-thirds of patients have abnormalities of pulmonary function tests,1,23 and in one series, a third of patients was found to have chronic interstitial infiltrate at autopsy.24 Clinically significant pulmonary fibrosis is found in 3 to 13% of patients.25,26 In some cases, pulmonary fibrosis is preceded by acute pneumonitis, and is said to show radiologically the organizing phase of diffuse alveolar damage that has an interstitial pattern.11 Chronic ILD is characterized by progressive dyspnea, cough, bibasal crackles, diffuse interstitial infiltrates, and a restrictive lung defect. Bronchoalveolar lavage (BAL) reveals an increased lymphocyte count and slight increase in neutrophils and occasional eosinophils.27 Chest radiographs usually show bilateral pulmonary infiltrates affecting the lower lobes.28 Parenchymal opacities and interstitial abnormalities have been described. A prospective study by Fenlon and colleagues29 assessed high-resolution chest tomography (HRCT) in 34 patients with SLE, of whom 23% had respiratory symptoms. The most common features were thickened interlobular septa, parenchymal bands, subpleural bands, pleural tags, and thickening. Only 6% had ground-glass opacities, consolidation, and honeycombing. In 21% of patients, there was bronchiectasis and bronchial wall thickening. Pleural thickening was noted in 15% of patients. A lung biopsy will help to confirm diagnosis, and the site of biopsy should be chosen after review of HRCT scans to select the most optimal sites in discussion with the thoracic surgeon. Histopathologically, the pattern is of interstitial pneumonia, either nonspecific interstitial pneumonia (NSIP) or usual interstitial pneumonia (UIP). The NSIP pattern is more common,29 and encompasses a wide morphologic spectrum with varying degrees of alveolar septal inflammation and fibrosis. The more cellular pattern features mild to moderate alveolar mononuclear cell infiltrate, with involvement of the peribronchial region, interlobular septa, and visceral pleura. Lung architecture is preserved without associated fibrosis. Organizing pneumonia may be present. Fibrotic variants are characterized by alveolar septal fibrosis, with less cellularity and little or no honeycombing present (Fig. 33.3). Less commonly in lupus, a patchy temporal and spatial distribution denotes the presence of the usual interstitial pneumonia pattern of histopathology. Normally interstitial lung disease in lupus runs a slow course. Rarely, it can be severe with rapidly progressive disease. This tends to occur in patients who have overlap syndromes, especially with scleroderma.30 Treatment involves corticosteroids in low doses

Fig. 33.3 Nonspecific interstitial pneumonia (NSIP). CT scan at the lung bases shows widespread ground glass opacification with traction bronchiectasis compatible with a fibrotic NSIP histopathologic pattern. Note the absence of honeycombing.

together with immunosuppressive agents, generally azathioprine as the first choice. Lymphocytic interstitial pneumonia is rarely associated with SLE.31-33 It is a relatively more benign lymphoinfiltrative disorder characterized by interstitial widening due to increased numbers of small lymphocytes and plasma cells with additional infiltration of bronchovascular bundles, interlobular septa, and pleura. It is associated with hypergammaglobulinemia.34 Symptoms include cough, chest pain, fatigue, low-grade fever, and weight loss. Chest radiographs commonly show diffuse ground-glass, and reticular and nodular opacities with lower-zone predominance35 and occasional nodular collections. HRCT findings include diffuse ground-glass attenuation, septal thickening, ill-defined centrilobular nodules (1 to 2 cm), and scattered thin-walled cysts. Mediastinal lymphadenopathy is seen. In advanced cases, architectural distortion and honeycombing occur.36 Treatment includes corticosteroids and immunosuppression.

AIRWAYS DISEASE Upper airway involvement is uncommon in SLE. Occasionally, hypopharyngeal ulceration, laryngeal inflammation, epiglotitis, and subglottic stenosis occur.37,38

Small Airways Disease Abnormalities in pulmonary function tests are present in up to 70% of SLE patients with normal chest radiographs.30 Reductions in the FEV1/FVC ratio, characteristic of expiratory airflow obstruction, occurs

in less than 10% of patients.39,40 However, in many of these studies, smoking status was not taken into account. A study by Andonopoulos and colleagues41 excluded smokers and compared SLE patients to controls. They found no significant difference in the prevalence of airflow obstruction between the two groups. Several case reports of severe airways obstruction have been reported.42-44 In these cases, there was no improvement with bronchodilators, but in one case there was response to oral corticosteroids. In one case, histopathology showed focal bronchiolitis, with complement and immunoglobulin deposition.44 Intraluminal organizing pneumonia with inflammation in the terminal bronchioles extending into the alveoli has been described in several cases.11,45,46 Organizing pneumonia in SLE is probably underdiagnosed due to lack of lung biopsies in symptomatic patients. The clinical symptoms include fever, cough, and dyspnea, and can mimic infection. Chest radiography shows nonspecific diffuse interstitial infiltrates with a restrictive lung defect on lung function testing. A surgical lung biopsy, usually thoracoscopic, is normally required to make the diagnosis and to exclude other causes of consolidation, including alveolar cell carcinoma. Histopathology shows inflammation of the lung parenchyma and bronchioles, which is associated with plugging of small airways and alveolar ducts with granulation tissue. Treatment includes high-dose corticosteroids, and in some more refractory cases, cyclophosphamide.

PULMONARY VASCULAR DISEASE

Pulmonary Hypertension Pulmonary hypertension in SLE is relatively common with prevalence rates of 5 to 14%.47-49 Clinical manifestations are similar to primary pulmonary hypertension,

Main pulmonary artery

and include progressive dyspnea, diminished exercise tolerance, right heart strain on echocardiogram, and eventually cor pulmonale. Cardiac catheterization confirms pulmonary arterial hypertension, but CT scanning may raise the suspicion (Fig. 33.4). Pulmonary emboli as a cause should be excluded via CT pulmonary angiography (CTPA) or ventilation/perfusion (VQ) scan. Mild pulmonary hypertension is common. A screening study using Doppler echocardiography to assess pulmonary artery pressure by Simonson and colleagues,49 investigated 36 patients randomly selected from their local rheumatology clinic. Fourteen percent had a pulmonary artery systolic pressure greater than 30 mmHg, and one patient had a pulmonary artery systolic pressure of 70 mmHg. A 5-year follow-up of the same cohort showed that the prevalence of pulmonary hypertension increased from 14 to 43%. Of the 12 new patients with pulmonary hypertension at follow-up, 11 had normal pressure at the start of the study. The mean systolic pulmonary artery pressure increased from 23.4 mmHg to 27.5 mmHg. The mechanisms for nonembolic pulmonary hypertension in SLE patients remain unclear, but genetic predisposition,50 high incidence of associated livedo reticularis and digital ulcers,51 and Raynaud’s phenomenon in up to 75% have been reported, which implies vasoconstriction.52 Vasculitis has also been reported with reversal of pulmonary hypertension with immunosuppression.53 Histopathology shows intimal thickening, onion skin subintimal fibrosis, and medial hypertrophy together with in situ thrombosis.30,54 The prognosis in severe SLE-induced pulmonary hypertension is similar to primary pulmonary hypertension with high mortality and sudden death.47 Treatment includes vasodilator therapy (prostacyclins)55 and anticoagulation, as in primary pulmonary hypertension.

PULMONARY VASCULAR DISEASE

Aorta

Fig. 33.4 Pulmonary hypertension. CT scan shows a pulmonary artery diameter greater than than the adjacent aorta, indicating likely pulmonary hypertension. Note the presence of bilateral breast implants.

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There are reports of the use of corticosteroids improving outcome.56,57 Anticoagulation, corticosteroids, and cyclophosphamide in combination have also been shown to be useful in one case report.58

Thromboembolism Antiphospholipid antibodies are twice as common in lupus patients with pulmonary hypertension than in those without.59 Antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies) are associated with acute and recurrent pulmonary thromboembolism and pulmonary artery thrombosis and subsequent pulmonary hypertension.60,61 A review by Cervera and colleagues62 investigated 842 SLE patients for IgG and IgM anticardiolipin antibodies. Of these, 204 patients had IgG antibodies and 108 had IgM antibodies. There was an increased prevalence of thrombosis in patients with anticardiolipin antibodies, compared to anticardiolipin-negative patients (30% with IgG compared to 9% without, and 31% with IgM compared to 11% without).62 There also seems to be an association of antiphospholipid antibodies with pulmonary artery hypertension in the absence of thrombotic disease, possibly on the basis of local small vessel thrombosis that does not present with the classical acute features.60,63 Lifelong anticoagulation is required for patients with recurrent thromboembolism.64,65 When anticoagulation fails to control thromboembolism, corticosteroids or immunosuppression may be required.1

Acute Reversible Hypoxemic Syndrome This syndrome has been described in lupus patients who present with severe hypoxemia and show diffuse infiltrates on chest radiographs.66 The abnormalities are associated with a vasculopathy. There is no evidence of thromboembolism. Most cases respond to high doses of corticosteroids, or low doses of corticosteroids and aspirin.

RESPIRATORY MUSCLES

378

Respiratory muscle dysfunction or “the shrinking lung syndrome” occurs in lupus patients, and is a cause of dyspnea, which may be progressive without demonstrable evidence of parenchymal or pulmonary vascular disease.67,68 Chest radiographs are characterized by small lung volumes, elevated diaphragms, and basal atelectasis. Bilateral elevation of the diaphragm is common (Fig. 33.5), but in the early stages chest radiography may be normal.67 Secondary elevation of the diaphragm can be also be associated with interstitial lung disease.69 The loss in lung volume was initially thought to be occult parenchymal disease or adhesions from pleurisy.

A

B Fig. 33.5 A, Shrinking lung. Chest radiograph shows bilateral diaphragmatic elevation with some patchy basal atelectasis. B, Shrinking lung. Coronal reconstruction of CT scan shows bilateral basal linear atelectasis.

However, recent evidence suggests that it is likely due to true diaphragmatic weakness, although an alternative theory suggests that it is due to an ill-defined abnormality of chest wall expansion.70 The diaphragmatic weakness may be secondary to a myopathy,71 and this is supported by a postmortem report of diaphragm fibrosis.72 Corticosteroid myopathy as a cause of respiratory muscle weakness has been suggested, but a study by Evans and colleagues73 has shown a high incidence of respiratory muscle weakness in steroid-naive patients. Phrenic nerve dysfunction as a cause of diaphragmatic weakness has also been suggested, but a study by Wilcox and colleagues74 showed normal phrenic nerve latency in all their study patients with demonstrable diaphragmatic weakness. Dyspnea is the most common symptom, and orthopnea may be present. Shrinking lung syndrome should be suspected when symptoms do not seem to reflect chest radiograph findings. Patients do not normally have significant generalized muscle weakness.75

PULMONARY INFECTIONS Pneumonia is the most common form of pleuropulmonary involvement in SLE, occurring in approximately 50% of patients.65,81,82 Although SLE itself suppresses the immune system, it is thought that the high rate of infection is more likely related to immunosuppressive treatment.83,84 Fatal infections are commoner in patients who have had cytotoxic therapy in the preceding 3 months.85 Most infections are due to bacterial pathogens, including Mycobacterium tuberculosis. Opportunistic infections are also being seen more commonly. Of these, pneumocystis, cytomegalovirus, cryptococcus, nocardia, and aspergillus are the most prevalent. Any patient presenting with new pulmonary infiltrates must be presumed to have an infection until proven otherwise, and be started on broad-spectrum antibiotics following the collection of appropriate samples for culture. Routine blood and sputum cultures must be taken. Bronchoscopy and lavage are often required to exclude opportunistic and other infections.

DRUG-INDUCED LUPUS Lung involvement is common in drug-induced lupus.86 The lung manifestations do not differ from idiopathic SLE. Affected patients tend to be older and there is no female preponderance. Antihistone antibodies are

present in up to 95% of patients with drug-induced lupus,86 but dsDNA and hypocomplementemia are uncommon. The medications commonly implicated are procainamide and hydralazine.87 Isoniazid, methyldopa, chlorpromazine, penicillamine, and quinidine are also well-recognized triggers. Minocycline,88 interferons,89 and biologic therapy90 have recently also been implicated. Development of antibodies does not necessarily mean development of disease, which occurs in only a minority of patients. Up to 90% of patients taking procainamide and 50% of patients taking hydralazine develop antibodies, but only 33% and 10% develop drug-induced lupus, respectively. Genetic predisposition affects development of drug-induced lupus, with slow acetylators more likely to develop antinuclear antibodies and clinical manifestations.91 Pleural effusions are the most common manifestations. Pleuritis, pneumonitis, pulmonary hypertension,92 antiphospholipid antibodies,93 and pulmonary embolism94 also occur. Treatment includes stopping the offending drug. Most symptoms resolve over 1 to 2 weeks.95 Nonsteroidal anti-inflammatory drugs can be used. Corticosteroids may be required in severe disease.96

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Pulmonary function tests show decreased lung volumes, decreased diffusing capacity for carbon monoxide (DLCO) but with a normal or even elevated gas transfer index (Kco) as a result of extrapulmonary restriction, and a decrease in mouth pressures.73 Diaphragmatic weakness can be assessed by esophageal/ gastric pressure measurements or noninvasive phrenic nerve stimulation.76 Inspiratory and expiratory mouth pressure measurements provide a useful screening tool. Improvement with corticosteroids has been described in some case reports.77,78 A short trial of corticosteroids is normally suggested in patients with significant symptomatic respiratory muscle weakness with serial measurements of vital capacity and inspiratory mouth pressures. Improvement has also been seen with the use of theophylline79 and β agonists.80

PULMONARY EDEMA Pleural effusions and pulmonary edema secondary to renal lupus and nephrotic syndrome can occur.65 The effusions tend to be painless. Pulmonary edema can also rarely occur as a consequence of pericarditis or myositis associated with cardiac lupus.

CONCLUSIONS Pleuropulmonary involvement is more common in SLE than any other collagen vascular disease, and is an important cause of morbidity and mortality. Lung involvement can range from pleuritic chest pain to life-threatening alveolar hemorrhage or pneumonitis, and may be the first manifestation of the systemic disease. Thorough investigations are required to exclude infection because it can be life-threatening in immunosuppressed patients. Nonsteroidal drugs, corticosteroids, and cytotoxic therapy are the mainstays of treatment.

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outcome in patients with systemic lupus erythematosus. Br J Rheumatol 1996;35:248-254. Fishback N, Koss MN. Pulmonary involvement in systemic lupus erythematosus. Curr Opin Pulm Med 1995;1:368-375. Winslow WA, Ploss LN, Loitman B. Pleuritis in systemic lupus erythematosus: its importance as an early manifestation in diagnosis. Ann Intern Med 1958;49:70-88. Bell R, Lawrence DS. Chronic pleurisy in systemic lupus erythematosus treated with pleurectomy. Br J Dis Chest 1979; 73:314-316. Gould DM, Daves ML. A review of roentgen findings in systemic lupus erythematosus (SLE). Am J Med Sci 1958;235:596-610. Good JT, King TE, Anthony VB, et al. Lupus pleuritis. Clinical features and pleural fluid characteristics with special reference to pleural fluid antinuclear antibodies. Chest 1983;84:714-718. Matthay RA, Schwartz MI, Petty TL, et al. Pulmonary manifestations of systemic lupus erythematosus: review of twelve cases of acute lupus pneumonitis. Medicine 1975;54:397-409. Murin S, Wiedemann HP, Matthay RA. Pulmonary manifestations of systemic lupus erythematosus. Clin Chest Med 1998; 19:641-665. Inoue T, Kanayama Y, Ohe A, et al.Immunopathologic studies of pneumonitis in systemic lupus erythematosus. Ann Intern Med 1979;91:30-34. Abud-Mendoza C, Diaz-Jouanen E, Alarcon-Segovia D. Fatal pulmonary haemorrhage in systemic lupus erythematosus. Occurrence without haemoptysis. J Rheumatol 1985;12:558-561. Schwab EP, Schumaker HR, Freundlich B, et al. Pulmonary alveolar haemorrhage in systemic lupus erythematosus. Semin Arthritis Rheum 1993;23:8-15. Zamora MR, Warner ML, Tuder R, et al. Diffuse alveolar haemorrhage and systemic lupus erythematosus. Clinical presentation, histology, survival and outcome. Medicine (Baltimore) 1997; 76:192-202. Onomuro K, Nakata H, Tanaka Y, et al. Pulmonary haemorrhage in patients with systemic lupus erythematosus. J Thorac Imaging 1991;6:57-61. Leatherman JW, Davies SF, Hoidal JR. Alveolar haemorrhage syndromes: diffuse microvascular lung haemorrhage in immune and idiopathic disorders. Medicine (Baltimore) 1984; 63:343-361. Liu MF, Lee JH, Weng TH, et al. Clinical experience of 13 cases with severe pulmonary haemorrhage in systemic lupus erythematosus with active lupus nephritis. Scand J Rheumatol 1998; 27:291-295. de Andrade J, Kennedy JIJ. The lung in systemic lupus erythematosus. Semin Respir Crit Care Med 1999;20:169-77. Myers JL, Katzenstein AA. Microangiitis in lupus induced pulmonary haemorrhage. Am J Clin Pathol 1986;85:552-556. Mintz G, Galindo LF, Fernandez-Diez J, et al. Acute massive pulmonary haemorrhage in systemic lupus erythematosus. J Rheumatol 1978;5:39-50. Millman RP, Cohen TB, Levinson AI, et al. Systemic lupus erythematosus complicated by acute pulmonary haemorrhage: recovery following plasmapheresis and cytotoxic therapy [letter]. J Rheumatol 1981;8:1021-3. Miller LR, Greenberg SD, McLarty JW. Lupus lung. Chest 1985; 88:265-269. Holgate ST, Glass DN, Haslam P, et al. Respiratory involvement in in systemic lupus erythematosus. A clinical and immunopathological study. Clin Exp Immunol 1976;24:385-395. Eisenberg H, DuBois EL, Sherwin RP, et al. Diffuse interstitial lung disease in systemic lupus erythematosus. Ann Intern Med 1973; 79:37-45. Nagai S, Kitaichi M, Itoh H, Nishimura K, Izumi T, Colby TV. Idiopathic non-specific interstitial pneumonia/fibrosis: comparison with idiopathic pulmonary fibrosis and BOOP. Eur Respir J 1998;12:1010-1019. Kim TS, Lee SK, Chung MP, et al. Non-specific interstitial pneumonia with fibrosis: high resolution CT and pathological findings. AJR Am J Roentgenol 1998;171:1645-1650. Gross M, Esterly JR, Earle RH. Pulmonary alterations in systemic lupus erythematosus. Am Rev Respir Dis 1972;105:572-577. Fenlon HM, Doran M, Sant SM, et al. High resolution chest CT in systemic lupus erythematosus. Am J Roentgenol 1996; 166:301-307.

30. Haupt HM, Moore GW, Hutchins GM. The lung in systemic erythematosus. Analysis of the pathologic changes in 120 patients. Am J Med 1981;71:791-798. 31. Groen H, ter Borg EJ, Postma DS, et al. Pulmonary function in systemic lupus erythematosus related to distinct clinical, serological and nail fold capillary patterns. Am J Med 1992;93:619-627. 32. Filipek MS, Thompson ME, Wang PL, Gosselin MV, Primack S. Lymphocytic interstitial pneumonitis in patient with systemic lupus erythematosus: radiographic and high resolution CT findings. J Thorac Imaging 2004;19:200-203. 33. Benish B, Peison B. The association of lymphocytic interstitial pneumonia and systemic lupus erythematosus. Mt Sinai J Med 1979;46:398-401. 34. Strimlan CV, Rosenow EC, Weiland LH. Lymphocytic interstitial pneumonitis: review of 13 cases. Ann Int Med 1978;88:616-621. 35. Kim EA, Lee KS, Johkoh T, et al. Interstitial lung diseases associated with collegen vascular diseases: radiographic and histopathology findings. RadioGraphics 2002;22:S151-S165. 36. Johkoh T, Muller N, Pick ford H, et al. Lymphocytic interstitial pneumonia: thin section CT findings in 22 patients. Radiology 1999;212:567-572. 37. Martin L, Bedworth SM, Ryan JP, et al. Upper airway disease in systemic lupus erythematosus: a review of 4 cases and a review of the literature. J Rheumatol 1992;19:1186. 38. Toomey JM, Synder GGD, Maenza RM, et al. Acute epiglottis due to systemic lupus erythematosus. Laryngoscope 1974;84:522. 39. Gold WM, Jennings DB. Pulmonary function in patients with systemic lupus erythematosus. Am Rev Respir Dis 1966;93:556-567. 40. Grennan DM, Howie AD, Moran F, et al. Pulmonary involvement in systemic lupus erythematosus. Ann Rheum Dis 1978; 376:536-539. 41. Andonopoulous AP, Constantopoulos SH, Galanopoulou V, et al. Pulmonary function of non-smoking patients with systemic lupus erythematosus. Chest 1988;94:312-315. 42. Kallenbach J, Zwi S, Goldman HI. Airways obstruction in a case of disseminated lupus erythematosus. Thorax 1978;33:814-815. 43. Venizelos PC, Al-Bazzaz F. Pulmonary function abnormalities in systemic lupus erythematosus responsive to glucocorticoid therapy. Chest 1981;79:702-704. 44. Kinney WW, Angelillo VA. Broncholitis in systemic lupus erythematosus. Chest 1982;82:646-649. 45. Gammon RB, Bridges TA, al-Nazier H, et al. Bronchiolitis obliterans organizing pneumonia associated with systemic lupus erythematosus. Chest 1992;102:1171-1174. 46. Guerry-Force ML, Muller NL, Wright JL, et al. A comparison of bronchiolitis obliterans with organizing pneumonia, usual interstitial pneumonia, and small airways disease. Am Rev Respir Dis 1987;135:705-712. 47. Asherson RA, Higenbottam TW, Dinh Xuan AT, et al. Pulmonary hypertension in a lupus clinic: Experience with twenty four patients. J Rheumatol 1990;17:1292-1298. 48. Perez HD, Kramer N. Pulmonary hypertension in systemic lupus erythematosus: a report of four cases and review of the literature. Semin Arthritis Rheum 1981;11:177-181. 49. Simonson JS, Schiller NB, Petri M, et al. Pulmonary hypertension in systemic lupus erythematosus. J Rheumatol 1989;16:918-925. 50. Wilson L, Tomita T, Braniecki M. Fatal pulmonary hypertension in identical twins with systemic lupus erythematosus. Hum Pathol 1991;22:295. 51. Rubin LA, Geran A, Rose TH, et al. A fatal pulmonary complication in lupus in pregnancy. Arthritis Rheum 1995;38:710-714. 52. Asherson RA, Mackworth-Young CG, Boey ML, et al. Pulmonary hypertension in systemic lupus erythematosus. BMJ 1983; 287:1024-1025. 53. Ronconori AJ, Alvarez C, Molinas F. Plexogenic arteriopathy associated with pulmonary vasculitis in systemic lupus erythematosus. Respiration 1992;59:52. 54. Fayemi AO. The lung in systemic lupus erythematosus: a clincopathologic study of 20 cases. Mt Sinai J Med 1975;42:110-118. 55. Ignaszewski AP, Percy JS, Humen DP. Successful treatment of pulmonary hypertension associated with systemic lupus erythematosus with prostaglandin I2 and prostaglandin E1 [letter]. J Rheumatol 1993;20:595-596. 56. Kawaguchi Y, Hara M, Harigai M, et al. Corticosteroid pulse therapy in a patient with SLE and pulmonary hypertension. Clin Exp Rheumatol 1998;16:510.

76. Gibson CJ, Edmonds JP, Hughes GR. Diaphragm function and lung involvement in systemic lupus erythematosus. Am J Med 1977;63:926-932. 77. Walz- Leblanc BA, Urowitz MB, Gladmann DD, et al. The “shrinking lung syndrome” in systemic lupus erythematosus— improvement with corticosteroid therapy. J Rheumatol 1992; 19:1970-1972. 78. Stevens WM, Burdon JB, Clemens LE, et al. The “shrinking lung syndrome”: An infrequently recognised feature of systemic lupus erythematosus. Aust N Z J Med 1990;20:67-70. 79. Van Veen S, Peeters AJ, Sterk PJ, et al. The “shrinking lung syndrome” in SLE: treatment with theophylline. Clin Rheumatol 1993;12:462-465. 80. Munoz-Rodriguez FJ, Font J, Badia JR, et al. Shrinking lung syndrome in systemic lupus erythematosus: improvement with inhaled beta-agonist therapy. Lupus 1997;6:412-414. 81. Hunninghake GW, Fauci AS. Pulmonary involvement in the collagen vascular diseases. Am Rev Respir Dis 1979;119:471-530. 82. Purnell DC, Baggenstos AH, Olsen AM. Pulmonary lesions in disseminated lupus erythematosus. Ann Intern Med 1955; 42:619-628. 83. Toews G, Lynch JP. Pathogenesis and clinical features of pulmonary infections. In: Cannon G, Zimmerman G, eds. The Lung in Rheumatic Diseases. New York: Marcel Dekker, 1990:. 84. Ginzler E, Diamond H, Kaplan D, et al. Computer analysis of factors influencing frequency of infection in systemic lupus erythematosus. Arthritis Rheum 1978;21:37-44. 85. Hellmann DB, Petri M, Whiting-O’Keefe Q. Fatal infections in systemic lupus erythematosus: the role of opportunistic organisms. Medicine (Baltimore) 1987;66:341-348. 86. Yung RL, Richardson BC. Drug-induced lupus. Rheum Dis Clin North Am 1994;20:61-86. 87. Cush JJ, Goldings EA. Drug-induced lupus: clinical spectrum and pathogenesis. Am J Med Sci 1985;290:36-45. 88. Masson C, Chevailler A, Pascaretti C, et al. Minocycline related lupus. J Rheumatol 1996;23:2160-2161. 89. Schilling PJ, Kurzrock R, Kantarjian H, et al. Development of systemic lupus erythematosus after interferon therapy for chronic myelogenous leukaemia. Cancer 1991;68:1536-1537. 90. Sarzi-Puttini P, Atzeni F, Capsoni F, et al. Drug induced lupus erythematosus. Autoimmunity 2005;38:507-518. 91. Woosley RL, Drayer DE, Reidenberg MM, et al. Effect of acetylator phenotype on the rate of at which procainamide induces antinuclear antibodies and lupus syndrome. N Engl J Med 1978;298:1157-1159. 92. Asherson RA, Benbow AG, Speirs CJ, et al. Pulmonary hypertension in hydralazine induced systemic lupus erythematosus: association with C4 null allele. Ann Rheum Dis 1986;45:771-773. 93. Gastineau DA, Holcomb GR. Lupus anticoagulant in drug induced systemic lupus erythematosus (SLE) [letter]. Arch Intern Med 1985;145:1926-1927. 94. Asherson RA, Zulman J, Hughes GR. Pulmonary thromboembolism associated with procainamide induced lupus syndrome and anticardiolipin antibodies. Ann Rheum Dis 1989; 48:232-235. 95. Harmon CE, Portanova JP. Drug-induced lupus: clinical and serological studies. Clin Rheum Dis 1982;8:121-138. 96. Harmon KR, Leatherman JW. Respiratory manifestations of connective tissue disease. Semin Respir 1988;3:258-273.

REFERENCES

57. Goupille P, Fauchier L, Babuty D, et al. Precapillary pulmonary hypertension dramatically improved with high doses of corticosteroids during systemic lupus erythematosus [letter, comment]. J Rheumatol 1994;21:1976-1977. 58. Groen H, Bootma H, Postma DS, et al.. Primary pulmonary hypertension in a patient with systemic lupus erythematosus: partial improvement with cyclophosphamide. J Rheumatol 1993; 20:1055-1057. 59. Love PE, Santoro SA. Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-SLE disorders. Prevalence and clinical significance. Ann Intern Med 1990;112:682-698. 60. Asherson RA, Cervera R. Review: antiphospholipid antibodies and the lung. J Rheum 1995;22:62-66. 61. Goldhaber SZ. Pulmonary embolism. N Engl J Med 1998;339: 93-104. 62. Cervera R, Khamashta MA, Font J, et al. Systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 1000 patients. The European Working Party on Systemic Lupus Erythematosus. Medicine (Baltimore) 1993;72:113-124. 63. Haupt HM, Moore GW, Hutchins GM. The lung in systemic lupus erythematosus. Analysis of the pathologic changes in 120 patients. Am J Med 1981;71:791-798. 64. Khamashta MA, Cuadrado MJ, Mujic F, et al. The management of thrombosis in the antiphospholipid-antibody syndrome. N Engl J Med 1995;332:993-997. 65. Rosove MH, Brewer PM. Antiphospholipid thrombosis: clinical course after the first thrombotic event in 70 patients. Ann Intern Med 1992;117:303-308. 66. Abramson SB, Dobro J, Eberle MA, et al. Acute reversible hypoxaemia in systemic lupus erythematosus. Ann Intern Med 1991;114:941-947. 67. Gibson CJ, Edmonds JP, Hughes GR. Diaphragm function and lung involvement in systemic lupus erythematosus. Am J Med 1977;63:926-932. 68. Thompson PJ, Dhillon DP, Ledingham J, et al. Shrinking lungs, diaphragmatic dysfunction and systemic lupus erythematosus. Am Rev Respir Dis 1985;132:926-928. 69. Weinrib L, Sharma OP, Quismorio FP. A long- term study of interstitial lung disease in systemic lupus erythematosus. Semin Arthritis Rheum 1990;20:48-56. 70. Laroche CM, Mulvey DA, Hawkins PN, et al. diaphragm strength in the shrinking lung syndrome of systemic lupus erythematosus. QJMed 1989;71:429-439. 71. Gibson GJ. Diaphragmatic paresis: pathophysiology, clinical features, and investigation. Thorax 1989;44:960-970. 72. Rubin LA, Urowitz MB. Shrinking lung syndrome in SLE. A clinical pathologic study. J Rheumatol 1983;10:973-976. 73. Evans SA, Hopkinson ND, Kinnear WJ, et al. Respiratory disease in systemic lupus erythematosus: correlation with results of laboratory tests and histological appearance of muscle biopsy specimens. Thorax 1992;47:957-60. 74. Wilcox PG, Stein HB, Clarke SD, et al. Phrenic nerve function in patients with diaphragmatic weakness and systemic lupus erythematosus. Hum Pathol 1988;93(2):352-358. 75. Martens J, Demedts M, Vanmeenen MT, et al. Respiratory muscle dysfunction in systemic lupus erythematosus. Chest 1983;84: 170-175.

381

CLINICAL ASPECTS OF THE DISEASE

34

Gastrointestinal Disease in Systemic Lupus Erythematosus Chi Chiu Mok, MD, FRCP

INTRODUCTION Gastrointestinal (GI) manifestations of systemic lupus erythematosus (SLE) are protean. Any part of the GI tract, liver, biliary tract, and pancreas can be involved (Tables 34.1 and 34.2). Recognition is important because some of these manifestations carry significant mortality and morbidity. Presentations of GI lupus are nonspecific and have to be distinguished from infective, thrombotic, therapy-related, and non-SLE causes. Endoscopic procedures, biopsies, and imaging investigations, which can be invasive, may be needed. The prevalence of GI manifestations of SLE varies widely, depending on study design, clinical characteristics of patients studied, and whether screening examinations are routinely performed. Oral symptoms and mucosal lesions appear to be most frequent, whereas acute abdominal pain is the most serious. Because of the lack of controlled trials, treatment of GI lupus is largely based on anecdotal experience and uncontrolled observational studies. The mainstay of treatment is immunosuppression. Anticoagulation is indicated when thrombosis is the underlying mechanism.

CLINICAL MANIFESTATIONS

Oral Cavity

382

Oral ulceration is a common feature of SLE, occurring in 6 to 52% of patients.1 Oral ulcers is one of the 11 American College of Rheumatology (ACR) revised criteria for the classification of SLE, and is a marker for disease activity. Typically, these ulcers are superficial, painless, and mostly found on the hard palate, buccal cavity, and vermiform border. Less commonly, ulcers may also develop in the nasal cavity and the pharyngeal wall. Histology is usually nonspecific. Chronic discoid lupus erythematosus (DLE) may develop in the oral mucosa. Up to 24% of patients with chronic cutaneous LE had concomitant mucous membrane lesions.2 Mucosal DLE usually starts as a painless

erythematosus patch that slowly matures into a chronic plaque-like lesion. It is frequently found in the buccal mucosa, but the palate and tongue may also be involved. DLE lesions can be severely painful, and their morphology may be confused with lichen planus or leukoplakia. Tissue biopsy may show lupus-specific histopathology similar to that of the skin. Oral ulceration may also be caused by infection and therapy of SLE. Viral infection such as herpes simplex and fungal infections such as candidiasis may lead to painful oral ulcers and plaque-like lesions. Immunosuppressive agents such as cyclophosphamide and methotrexate may induce mucositis and mucosal ulceration. Sicca symptoms such as dry mouth and dry eyes are fairly common in SLE patients. Manoussakis and colleagues3 reported a 9.2% prevalence of secondary Sjögren’s syndrome in 283 unselected SLE patients using the American-European classification criteria. The clinical presentation of Sjögren’s syndrome in SLE was no different from that of primary Sjögren’s syndrome, but older age, Raynaud’s phenomenon, anti-Ro, anti-La, and rheumatoid factor were more frequent in SLE patients, whereas renal disease, lymphadenopathy, and thrombocytopenia were less common. SLE patients are prone to poor dental health. This is a result of multiple factors, including disease activity, reduced salivary flow, bleeding diathesis, and the use of medications such as corticosteroids (risk of gingival infection), aspirin, and nonsteroidal anti-inflammatory drugs (NSAIDs) (platelet dysfunction), cyclosporin A (gingivitis, gingival hypertrophy), methotrexate (stomatitis and mucositis), antiepileptic agents (gum hypertrophy), and tricyclic antidepressants (which worsen sicca). Meyer and colleagues4 studied the frequency of oral, dental, and periodontal findings in 46 patients with SLE. Compared with healthy matched controls, oral mucosal lesions such as aphthous ulcers, erythema, gingival overgrowth, and hemorrhage were more frequently found in SLE patients (48% vs. 25%). The extent of periodontal disease was related to the severity and duration

Oral cavity

Oral ulceration Mucosal discoid lupus Sicca symptoms Chronic periodontitis

Esophagus

Hypomotility Esophageal reflux with or without ulceration

Stomach

Gastritis, gastric ulceration Pernicious anemia Gastric antral vascular ectasia

Small bowel and peritoneum

Intestinal vasculitis (enteritis) Mesenteric insufficiency Intestinal pseudo-obstruction Malabsorption Protein-losing gastroenteropathy Peritonitis/ascites (serositis)

Large bowel

Colitis Inflammatory bowel diseases Collagenous colitis

of SLE. In addition to disease and treatment-related factors, chronic periodontitis in SLE has also been linked to genetic factors such as the FcgammaRIIa polymorphisms5 and the antineutrophil cytoplasmic antibodies.6 Periodontal disease may pose a potentially serious health risk in SLE patients because a recent systematic review suggested a modest association between periodontitis and cardiovascular diseases.7

Esophagus Dysphagia occurs in 1 to 13% and heartburn in 11 to 50% of patients with SLE.1 These may be attributed to dry mouth, esophageal hypomotility, esophagitis, or

TABLE 34.2 HEPATOBILIARY AND PANCREATIC DISEASE IN LUPUS Liver

Liver function derangement Autoimmune hepatitis (lupus hepatitis) Chronic viral hepatitis Hepatic vein thrombosis Veno-occlusive disease Nodular regenerative hyperplasia

Biliary tract

Acalculous cholecystitis Primary biliary cirrhosis Autoimmune cholangiopathy Sclerosing cholangitis

Pancreas

Pancreatitis

esophageal ulceration because of acid reflux and infection. Manometry studies reveal functional abnormalities of the esophagus in 10 to 32% of SLE patients.8,9 Aperistalsis or hypoperistalsis is most frequently found in the upper one-third of the esophagus,9 and is associated with Raynaud’s phenomenon in some studies.8 The reasons for esophageal hypomotility in SLE remain elusive. Skeletal muscle fiber atrophy, inflammatory reaction in the esophageal muscles, and ischemic or vasculitic damage of the Auerbach plexus have been postulated. Esophagitis with ulceration was reported in 3 to 5% of patients with SLE.1 This may be caused by gastroesophageal reflux or infections such as Candida, herpes simplex, and cytomegalovirus (CMV). Endoscopic examination with biopsy is necessary to establish the diagnosis. A true vasculitis leading to esophageal ulceration is probably rare.10 In addition, medications such as NSAIDs and the bisphosphonates are occasionally associated with esophagitis and bleeding esophageal ulcers.

CLINICAL MANIFESTATIONS

TABLE 34.1 GASTROINTESTINAL MANIFESTATIONS OF LUPUS

Stomach Gastritis, gastric erosion, and ulceration in SLE patients may result from treatment with high-dose corticosteroids and NSAIDs. In two studies of acute abdomen in SLE patients, perforated peptic ulcer was diagnosed in 6 to 8% of cases.11,12 While the exact incidence of peptic ulcer disease in SLE patients is unknown, adverse effects of medications are the most common causes. Vasculitis of the gastric mucosa related to active SLE causing ulceration and bleeding is exceedingly rare. Although pernicious anemia has been reported in patients with SLE, its prevalence is low. A study of 30 SLE patients reported that only one patient (3%) suffered from pernicious anemia characterized by low serum cobalamin level, macrocytic anemia, and the presence of antibody against intrinsic factor.13 Another study indicated that 19% of female SLE patients had low serum cobalamin levels, but none developed overt anemia.14 Gastric antral vascular ectasia (GAVE) is a rare vascular malformation in the GI tract that may cause acute or chronic bleeding. The characteristic endoscopic appearance is a collection of red spots of ectatic vessels arranged in stripes along the antral rugal folds. GAVE is mostly found in patients with systemic sclerosis, but has been reported in SLE.15 Although the stomach is relatively resistant to infection, CMV gastritis has been reported in heavily immunocompromised patients. Renal transplant recipients who receive mycophenolate mofetil (MMF)based immunosuppressive protocols are prone to disseminated CMV infections. As MMF is increasingly used in patients with SLE, CMV infection of the GI tract should not be overlooked.

383

GASTROINTESTINAL DISEASE IN SLE

Small Intestine Mesenteric/Intestinal Vasculitis The prevalence of intestinal vasculitis in patients with SLE ranged from 0.2 to 1.1%.15 In SLE patients presenting with acute abdominal pain, intestinal vasculitis was diagnosed in 5 to 60% of patients.11,16-18 Most patients with mesenteric vasculitis present with cramping or persistent abdominal pain, a variable degree of nausea and vomiting, fever, diarrhea, and bloody stools. Abdominal distension, tenderness, and rebound tenderness are usually present, and bowel sound may be diminished or absent. In severe cases, mucosal ulceration with bleeding, bowel edema with paralytic ileus, hemorrhagic ileitis, intussusception, and even bowel gangrene and perforation may develop.19,20 Active SLE in other organs is usually evident. Abdominal radiographs in patients with lupus mesenteric vasculitis may reveal changes such as pseudoobstruction of the gastric outlet, duodenal hypomotility, bowel loop distension, effacement of the mucosal folds, and thumb-printing appearance (submucosal edema as a result of bowel ischemia). Intra-abdominal free gas may appear after intestinal perforation, or because of pneumatosis cystoids intestinalis. Ultrasound and computed tomography (CT) scan of the abdomen are important in excluding intra-abdominal abscesses, pancreatitis, and other intra-abdominal pathologies. In addition, a contrast CT scan may reveal bowel wall changes, mesenteric vascular and fat changes, fluid collection, retroperitoneal lymphadenopathy, peritoneal enhancement, and hepatomegaly. Conspicuous prominence of mesenteric vessels with a palisade pattern or comb-like appearance supplying focal or diffuse dilated bowel loops, ascites with slightly increased peritoneal enhancement, and bowel wall thickening with double halo or target sign (enhancing outer and inner rim with hypoattenuation in the center) are characteristic early CT findings of lupus mesenteric vasculitis.21 The typical histopathologic findings of lupus mesenteric vasculitis usually occur in the arterioles and venules of the submucosa of the small bowel wall rather in the medium-sized mesenteric arteries.20,22,23 Vasculitic lesions tend to be segmental and focal.19 Immunohistochemical staining of the tunica adventitia and media may reveal immune complex, C3 complement, and fibrinogen deposition. Fibrinoid necrosis, intraluminal thrombosis of affected vessels, acute or chronic inflammatory infiltrates consisting of lymphocytes, plasma cells, histiocytes, and neutrophils may also be demonstrated.23

Mesenteric Insufficiency 384

Patients with SLE are prone to premature atherosclerosis. Chronic mesenteric insufficiency, or “intestinal

angina,” should be considered in patients who present with chronic intermittent abdominal pain. Symptoms usually start in the postprandial state and persist for several hours. Abdominal pain may be mild at onset and progress in severity over weeks or months. Fear of eating often leads to weight loss. Concomitant atherosclerotic disease in the coronary and carotid vessels is usually present. SLE patients at risk are those with long-standing disease, renal insufficiency, persistent proteinuria, antiphospholipid positivity, chronic corticosteroid therapy, and traditional risk factors for atherosclerosis. The diagnosis of chronic mesenteric insufficiency relies on a high index of suspicion. Conventional angiography is the gold-standard imaging procedure. Digital subtraction angiography, Doppler ultrasonography, and magnetic resonance imaging with angiography are adjunctive diagnostic modalities.24 Acute mesenteric ischemia can result from impaired blood flow within the mesenteric arterial or venous systems. Classically, abdominal pain is persistent and disproportionately severe relative to physical signs. Patients may also present with acute abdomen with distention, rigidity, fever, bloody diarrhea, melena, and hypotension. SLE patients with underlying chronic mesenteric insufficiency due to atherosclerosis or secondary antiphospholipid syndrome are particularly prone to acute intestinal ischemia, which may be precipitated by hypoperfusion states. Acute mesenteric thrombosis may result in bowel infarction, perforation, and peritonitis.

Intestinal Pseudo-Obstruction Intestinal pseudo-obstruction (IPO) is a clinical syndrome characterized by impaired intestinal motility as a result of dysfunction of the visceral smooth muscle or the enteric nervous system. IPO may be the initial presentation of SLE and usually occurs in the setting of active lupus.25 The small bowel is more commonly involved than the large bowel. Common presenting symptoms of IPO are a subacute onset of abdominal pain, nausea, vomiting, abdominal distention, and constipation. Physical examination often reveals a diffusely tender abdomen with sluggish or absent bowel sound. Rebound tenderness is usually absent. Radiologic examinations may demonstrate dilated, fluid-filled bowel loops, with thickened bowel wall and multiple fluid levels (Fig. 34.1). Organic causes for intestinal obstruction should be sought, preferably by nonsurgical assessment but laparotomy may be necessary in some patients. Manometry motility studies in patients with IPO may demonstrate esophageal aperistalsis and intestinal hypomotility.26 Interestingly, 63% of the reported cases of SLErelated IPO had concomitant ureterohydronephrosis

CLINICAL MANIFESTATIONS

Mader and colleagues28 screened 21 SLE patients for malabsorption by the D-xylose absorption test (DXT), microscopic examination of the stool for fat droplets, and biopsy from the second part of the duodenum. Two patients (10%) were found to have an abnormal DXT and excessive fecal fat excretion. In one of these patients, histologic examination revealed flattened and deformed villi with an inflammatory infiltrate. Immunoperoxidase staining did not reveal excessive deposition of immunoglobulins and light chains within the intestinal mucosa in these patients. Up to 23% of patients with SLE may be tested positive for either the IgA or IgM antigliadin antibodies,29 but biopsy-proven celiac disease (gluten-sensitive enteropathy) is exceedingly uncommon.

Protein-Losing Gastroenteropathy

Fig. 34.1 An SLE patient with intestinal pseudo-obstruction. Plain abdominal radiograph shows multiple dilated small bowel loops with fluid levels.

and contracted urinary bladder, and around one-third of these patients had documented histologic features of interstitial cystitis.25 Lupus interstitial cystitis may lead to bladder wall thickening and reduced bladder capacity. This may in turn induce ureterohydronephrosis because of detrusor muscle spasm and secondary vesiculo-ureteric reflux. The pathogenesis of IPO in SLE is unclear. The association with autoimmune cystitis and the demonstration of antibodies against proliferating cell–nuclear antigen in some patients27 suggests that vasculitis of the visceral smooth muscles is a mechanism which may lead to muscular damage and hypomotility. The simultaneous presence of ureterohydronephrosis in many patients with SLE-related IPO and the association of hypomotility of other parts of the GI tract indicate that the basic pathology may be dysmotility of the intestinal musculature. Whether this is caused by a primary myopathy, neuropathy, vasculitis, or antibodies directed against the smooth muscle of the gut wall requires further study.

Malabsorption Intestinal malabsorption in SLE may result in protein losing enteropathy, hypoalbuminemia, and ascites.

Protein-losing gastroenteropathy (PLGE) is characterized by hypoalbuminemia secondary to loss of protein from the GI tract. It is usually identified by an elevated clearance of stool α1-antitrypsin or the technetium99mlabeled human serum albumin scan (Fig. 34.2). Significant loss of protein from the kidneys should be ruled out. A variety of pathologies from the stomach down to the colon may be responsible for protein loss. Investigations into the causes of PLGE such as gastrointestinal lymphoma, malabsorption state, bacterial overgrowth, chronic infection, polyposis, and lymphatic obstruction are essential. Endoscopic examination with mucosal biopsies, barium studies, radiologic examinations, and absorption tests may be required. PLGE is a rare manifestation of SLE, and fewer than 50 cases have been described. We recently reported 16 cases of SLE-related PLGE and reviewed 32 other patients in the literature.30 PLGE was the presenting feature in three-quarters of our patients and most patients had active SLE in other organs. The most common presentation was generalized or dependent edema, and abdominal symptoms, such as pain, nonbloody diarrhea, nausea, vomiting, and anorexia. Protein leakage occurred more frequently from the small bowel (69%) than the large bowel (31%). Specific endoscopic, imaging, and histologic findings were often absent. The most common endoscopic appearance was mucosal edema.31 The biopsy was either normal or revealed nonspecific findings such as villous atrophy, submucosal edema, dilated lacteals, and inflammatory infiltrates. Definite lymphangiectasia, vasculitis, or C3 deposition in the capillary walls of the lamina propriae of villi was uncommon. The exact pathogenesis of PLGE remains elusive. Mucosal disruption and increase in mucosal capillary permeability as a result of complement- or cytokinemediated damage, mesenteric venulitis, and dilated/ ruptured mucosal lacteals have been postulated.30

385

GASTROINTESTINAL DISEASE IN SLE

Fig. 34.2 99mTc-labelled human serum albumin (HSA) scan in a patient with SLE showing leakage of protein from the small bowel. A, An area of diffuse activity in upper and central lower abdomen at 2 hours (left). B, Intense activity at the ileocecal valve region at 7 hours (middle). C, Intense activity in cecum, ascending colon, and transverse colon at 24 hours (right).

Infective Enteritis Infective enteritis should be considered in SLE patients presenting with abdominal symptoms. Bacterial enteritis is the most common, with nontyphoidal Salmonella infection being most frequently reported.32 Campylobacter jejuni infection and CMV enteritis may lead to ileal perforation.

Ascites and Peritonitis

386

Ascites in SLE may be inflammatory or noninflammatory in nature. Acute peritonitis with and without ascites can be caused by mesenteric vasculitis or serositis as a result of active SLE, infection, bowel infarction, perforated viscera, and pancreatitis. On the other hand, subacute or chronic peritoneal effusion can be the result of lupus peritonitis, hypoalbuminemia (nephrotic syndrome, protein-losing enteropathy, and liver cirrhosis), right heart failure, constrictive pericarditis, hepatic venous thrombosis, malignancy, and more indolent infections such as tuberculosis. Inflammatory peritonitis in SLE is generally painful, but clinical signs may be masked by corticosteroid and immunosuppressive treatment. Conversely, lupus peritonitis may present with severe abdominal pain mimicking acute surgical abdomen. In a recent cross-sectional study of 310 patients with SLE, 69 episodes of SLErelated serositis were reported in 37 patients (12%).33 Thirty percent of these episodes were peritonitis/ ascites. All patients presented with abdominal pain, but physical signs (abdominal distension, voluntary guarding, rebound tenderness and ascites) were present in less than 20% of patients. One patient presented with acute abdomen, but laparotomy did not reveal any significant pathologies. On follow-up, recurrence of peritonitis was more common than pericarditis and pleuritis. The exact pathogenesis of SLE-related peritonitis remains obscure. Inflammatory infiltrates, immunoglobulin, and complement deposits may be demonstrated in peritoneal tissues and the peritoneal vessels.34 Imaging studies such as contrast CT scan may reveal ascites and asymmetric thickening of the small bowel wall.

Large Intestine Lupus Colitis Although lupus enteritis mainly involves the small bowel, the large bowel may also be affected. In the series by Zizic and colleagues35 and Medina and colleagues,11 colonic involvement by SLE with perforation was described. Most patients had active SLE in other organs, and mortality was high. In addition, vasculitic ulcers of the rectal mucosa that may perforate and lead to septicemia have been described in patients with SLE.36

Inflammatory Bowel Disease Crohn’s disease and ulcerative colitis (UC) are rarely associated with SLE. Whether there is a true association between SLE and inflammatory bowel disease is unclear. Clinically and pathologically, lupus colitis may be indistinguishable from UC. Symptoms include lower abdominal discomfort, perirectal bleeding, and persistent diarrhea that may be bloody. The prevalence of UC in SLE patients is around 0.4%.15 AlarconSegovia and colleagues37 reported SLE in 3% of patients with UC. However, this prevalence figure might have been overestimated because cases of sulfasalazineinduced lupus were likely to be included in this early series.

Collagenous Colitis Collagenous colitis is a disorder characterized by colonic intraepithelial lymphocytosis, expansion of the lamina propria with acute and chronic inflammatory cells, and a thickened subepithelial collagen band. Patients usually present with chronic watery diarrhea despite normal radiologic and endoscopic findings. Collagenous colitis has been reported in association with DLE and SLE.15

Infective Colitis Colonic infections should be considered in SLE patients presenting with lower GI symptoms. CMV and amoebic colitis has been reported in patients with SLE.10 Lymphopenia, cytotoxic treatment, presence of renal disease, and a travel history to endemic areas are predisposing factors.

The incidence of diverticular disease does not appear to be higher in SLE patients than in the general population. Diverticular disease is expected to occur in older individuals with SLE, and is an important differential diagnosis in patients who present with fever, abdominal pain, and tenderness.

Liver Liver Function Abnormalities Liver function abnormalities are common but usually mild in patients with SLE. Multiple factors may contribute, such as the use of aspirin, NSAIDs, azathioprine, and methotrexate; fatty infiltration of liver as a result of corticosteroid treatment; diabetes mellitus; obesity; viral hepatitis; and alcoholism. In around onequarter of cases, no causes other than active SLE itself are responsible. Persistent and severe liver function abnormalities are uncommon, but require further investigations such as ultrasonography and liver biopsy to delineate the underlying causes. In a series of 206 SLE patients, Runyon and colleagues38 reported that 124 (60%) patients had abnormal liver function test results. However, significant liver disease was diagnosed in 43 (21%) patients only. Liver biopsy in 33 patients revealed steatotic hepatitis (36%), cirrhosis (12%), chronic active hepatitis (9%), chronic granulomatous hepatitis (9%), centrilobular necrosis (9%), chronic persistent hepatitis (6%), and microabscesses (6%). Eight patients improved with corticosteroid treatment, but three patients died of liver failure. Gibson and Myers39 studied 81 patients with SLE, and reported that 45 (55%) of them had abnormal liver function results at some point. The majority of these patients had mild liver function derangement. No causes other than SLE itself for the liver dysfunction were present in 19 (23%) patients. Of the patients with liver biopsy performed, seven showed normal histology, five had portal inflammatory infiltrates, one had fatty liver, and one had chronic active hepatitis. In a prospective study, Miller and colleagues40 reported liver function abnormalities in 23% of their SLE patients. One-third of them did not have identifiable causes. In 80% of these patients with persistent “unexplained” transaminase elevations during follow-up visits, changes in SGPT levels were concordant with SLE activity.

against hepatic antigens or liver–kidney microsomal proteins such as ANA, antismooth muscle antibodies (SMA), and anti-LKM antibodies. AIH may be classified into three types based on their immunoserologic markers. Type I AIH (the classical “lupoid hepatitis” described in the 1950s) is the most common form worldwide and is associated with ANA and/or SMA. Type II AIH is associated with the anti-LKM1 antibody, while type III AIH is associated with antiSLA/LP antibodies. Patients with AIH usually present with insidious onset of nonspecific symptoms such as fatigue, malaise, and anorexia. Liver enlargement, jaundice, and ascites may be present in severe cases. AIH is also associated with lupus-like features such as positive ANA, hypergammaglobulinemia, and joint symptoms. However, only around 10% of patients with AIH fulfill the ACR criteria for SLE.41 The term “lupus hepatitis” should be reserved for patients fulfilling the ACR criteria for SLE who have chronic active hepatitis, with documented lymphocytic infiltration of periportal areas on histology (Fig. 34.3). Other causes of liver function derangement such as viral infection, alcoholism, metabolic or genetic liver diseases, and effects of drugs have to be excluded. The incidence of AIH in SLE patients is unclear because not all patients will have the diagnosis confirmed by liver biopsy. In one study, evidence for chronic active hepatitis was present in 4.7% of patients who fulfilled the ACR criteria for SLE.42 Arnett and Reichlin43 reported that 4 (3%) of their 131 SLE patients had a clinical picture of chronic active hepatitis. Evidence for chronic viral infection was absent, and only 1 patient had low-titer SMA. Compared with non-SLE patients, SLE patients with AIH are more likely to have autoantibodies against dsDNA, Sm, and antiribosomal P.

CLINICAL MANIFESTATIONS

Diverticular Disease

Lupus Hepatitis Autoimmune hepatitis (AIH) is characterized histologically by interface hepatitis and portal plasma cell infiltration, hypergammaglobulinemia, and the presence of a variety of autoantibodies that direct

Fig. 34.3 Liver biopsy in an SLE patient showing active interface hepatitis with prominent plasmacytic infiltrates (hematoxylin and eosin stain, x200).

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GASTROINTESTINAL DISEASE IN SLE

Chronic Viral Hepatitis The prevalence of chronic hepatitis-B virus (HBV) infection does not seem to be higher in patients with SLE when compared to the general population, even in endemic areas. In a study from Taiwan, the prevalence of HBV infection was reported to be significantly lower than that in the general population (3.5% vs. 14.7%).44 Patients with coexistent SLE and chronic HBV infection had less lupus activity, including less proteinuria and a lower serum titer of anti-dsDNA than HBsAgnegative lupus patients. Another study from the Middle East did not find HBV infection in 96 SLE patients, compared to prevalence of 2% in the general population.45 Perlemuter and colleagues46 reported that 19 (3%) of their 700 SLE patients had chronic hepatitis C (HCV) infection. Compared with age- and gender-matched control patients, SLE patients with HCV infection had a higher prevalence of asymptomatic cryoglobulinemia. Ramos-Casals and colleagues47 reported an increased prevalence of HCV infection in their SLE patients compared to healthy blood donors (13% vs. 1%). SLE patients with HCV infection were less likely to have cutaneous disease and anti-dsDNA, but more likely to have hepatic dysfunction, low complement levels, and cryoglobulinemia than those without SLE.

Drug-Induced Hepatotoxicity Aspirin, NSAIDs, methotrexate, and leflunomide may cause elevation of parenchymal liver enzymes. Corticosteroids may induce fatty liver disease (steatotic hepatitis). Azathioprine and hydroxychloroquine occasionally cause hepatitis. Of interest is minocycline, a drug used in the treatment of rheumatoid arthritis and acne, which may induce a syndrome of druginduced lupus and autoimmune hepatitis. The statins are increasingly used in patients with SLE. Isolated case reports of statin-induced lupus-like syndrome and hepatitis should be noted.

Other Liver Diseases

388

Thromboembolic disorders of the liver may occur in patients with SLE, especially in the presence of the antiphospholipid antibodies. Budd-Chiari syndrome, a disease caused by occlusion of the hepatic veins, hepatic veno-occlusive disease, and hepatic infarction have been reported in patients with SLE and secondary antiphospholipid syndrome.48 Nodular regenerative hyperplasia (NRH) is characterized by diffuse nodularity of the liver with little or no fibrosis. It is a cause of noncirrhotic portal hypertension and may lead to ascites and variceal bleeding. NRH has been described in patients with SLE and primary antiphospholipid syndrome.49,50 The association

with the antiphospholipid antibodies suggests that NRH may result from liver regeneration to maintain its functional capacity after ischemia-induced injury.50 In an autopsy study of 160 livers, Matsumoto and colleagues51 described 7 cases of NRH, 5 of which were found in patients with SLE. NRH should be suspected in SLE patients with unexplained portal hypertension, and confirmed by liver biopsy. Hepatic nodules may be better visualized with magnetic resonance imaging (MRI) of the liver.49 Many patients with NRH of the liver are asymptomatic with normal liver function. Treatment should target at control of portal hypertension and its related complications.

Biliary Tract Disease Gallbladder disease appears to be no more frequent in SLE patients than in the general population. Cholecystitis in SLE may be confused with serositis. Acute acalculous cholecystitis has been described in patients with SLE.52 Patients usually present with acute abdomen, and cholecystectomy specimens may reveal vasculitis of the gall bladder. Although successful conservative treatment with corticosteroids has been reported,52 most patients were diagnosed after surgical treatment, especially if there was evidence of septicemia. On the other hand, primary biliary cirrhosis (PBC), autoimmune cholangiopathy (antimitochondrial antibody-negative PBC), and primary sclerosing cholangitis, a rare disorder associated with the inflammatory bowel diseases, have also been reported in patients with SLE.15

Pancreas Pancreatitis is an uncommon manifestation of SLE. The prevalence of pancreatitis in SLE patients ranges from 0 to 4%.15,53 Medications such as corticosteroids, azathioprine, and thiazide diuretics have been attributed to cause pancreatitis in some cases. Pascual-Ramos and colleagues53 analyzed 49 episodes of acute pancreatitis in 35 SLE patients. Seventeen episodes were considered idiopathic, and disease activity scores were significantly higher than those with identified causes of pancreatitis. Compared with non-SLE controls, “idiopathic” pancreatitis was more frequent in SLE patients. Medication use did not seem to be associated with the development of pancreatitis. Saab and colleagues54 reported eight SLE patients with pancreatitis. All responded to corticosteroid treatment. Derk and De Horatius55 studied 25 SLE patients diagnosed to have acute pancreatitis in a 20-year period. Threequarters of the patients had active SLE in other systems. Pancreatitis improved in most patients with systemic corticosteroids. These studies suggest that lupus pancreatitis is likely to be a distinct entity that

ABDOMINAL PAIN IN SLE PATIENTS Abdominal pain is a fairly common symptom in patients with SLE, but the actual incidence is unclear. Depending on the setting in which patients are assessed (e.g., in the emergency room, surgical ward, or outpatient clinic), abdominal pain is reported in 8 to 37% of patients with SLE,1,42 and can be due to SLE-related, treatment-related, or non-SLE-related causes (Table 34.3). Zizic and colleagues16 reported that 15 (11%) of 140 SLE patients developed signs and symptoms of acute surgical abdomen. Eleven (73%) patients underwent exploratory laparotomy, with nine showing intraabdominal arteritis and two showing polyserositis.

Mortality was high (53%), which was partially related to delay in the diagnosis of the underlying condition. Medina and colleagues11 studied 51 SLE patients who presented with acute abdomen and underwent surgical exploration. Intestinal vasculitis and intra-abdominal thrombosis was diagnosed in 9 (37%) and 3 (6%) patients, respectively. Patients with inactive SLE were more likely to have non–SLE-related causes for their acute abdominal pain. High SLE activity and a delay in surgical exploration were associated with higher mortality. Another study of 13 SLE patients who presented with acute abdominal pain in a teaching hospital reported that most patients had conventional surgical diagnoses rather than SLE-related causes.12 More recently, Lee and colleagues17 reported that 38 (22%) of 175 SLE patients admitted to their hospital were due to acute abdominal pain. Lupus enteritis (intestinal vasculitis) was the most common diagnosis (45%), but the mean SLEDAI (Systemic Lupus Erythematosus Disease Activity Index) scores in these patients were not significantly higher than those SLE patients without enteritis. In contrast, in another study of 56 SLE patients presenting with subacute abdominal pain (without peritoneal signs), intestinal vasculitis was diagnosed in 5% of patients only.18 These patients had SLEDAI scores higher than 8. Lian and colleagues59 reported that among 45 patients with SLE who presented with acute abdominal pain, serositis and bowel involvement were diagnosed by CT examination of the abdomen in 63% of patients who underwent this investigation. Abdominal pain and tenderness in SLE patients may precede an intra-abdominal disaster. Classical physical signs such as rebound tenderness may be masked by the use of corticosteroids and immunosuppressive agents.

ABDOMINAL PAIN IN SLE PATIENTS

occurs in patients with active disease and responds to immunosuppressive treatment. In fact, in the presteroid era, cases of pancreatic vasculitis were documented histopathologically.15 Autopsy studies have also demonstrated vascular damage consisting of severe intimal proliferation in the pancreatic vessels in patients with lupus pancreatitis.56 Hasselbacher and colleagues57 studied 25 patients with SLE, and demonstrated that 20% of patients had elevated amylase level and 25% had macroamylase activity. None of the patients had clinical pancreatitis. Eberhard and colleagues58 measured serum immunoreactive cationic trypsinogen (IRT) in 35 asymptomatic patients with SLE. Fifteen patients (43%) had elevated IRT levels on at least one occasion. There was no apparent association with the use of drugs such as prednisone and azathioprine. This suggested that subclinical pancreatic dysfunction might be present in some patients with SLE.

TABLE 34.3 DIFFERENTIAL DIAGNOSES OF ABDOMINAL PAIN IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS Disease Related

Therapy Related

Non-SLE Etiologies

Serositis Intestinal vasculitis/colitis Malabsorption

Gastritis, duodenitis Peptic ulcer with or without perforation

Infective gastroenteritis Inflammatory bowel disease Cholecystitis/cholangitis

Intestinal pseudo-obstruction

Pancreatitis

Pancreatitis

Protein losing gastroenteropathy

Intra-abdominal sepsis Infective enteritis

Viral hepatitis Surgical adhesions

Ischemic bowel disease

Infective colitis

Appendicitis

Mesenteric thrombosis

Bacterial peritonitis

Diverticulitis

Hepatic vein thrombosis

Intussusception

Hepatitis

Gynecologic conditions

Pancreatitis Acalculous cholecystitis (rare)

Rupture of vascular aneurysms (rare)

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Acute, persistent, or severe abdominal symptoms in patients with SLE should be promptly investigated. Blood counts, serum amylase level, renal and liver function tests, anti-dsDNA, complement levels, and abdominal radiography are basic investigations. Depending on the severity of clinical signs and symptoms, further investigations such as endoscopy, paracentesis, ultrasound scan, contrast CT scan, MRI, gallium scan, and angiography are indicated. A surgical opinion is required and exploratory laparotomy should be considered in patients with clinical and radiologic suspicion of visceral perforation or intra-abdominal collections.

TREATMENT OF GASTROINTESTINAL AND HEPATIC MANIFESTATIONS OF SLE Because of the lack of controlled trials, treatment advice on GI and hepatic manifestations of SLE is largely based on clinical experience and uncontrolled observational studies. Acute abdominal symptoms in SLE should be promptly evaluated. When infection and other important causes have been ruled out, immunosuppressive treatment should be considered, preferably under coverage with broad-spectrum antibiotics. Anticoagulation should be considered when thrombosis is the underlying mechanism. For patients who do not respond to corticosteroids alone, cyclophosphamide may be required, especially if there is a vasculitic component. Early surgical intervention should be instituted when conservative management fails.

Oral Cavity Lesions

390

Oral ulcers related to active SLE are often self-limiting or responsive to corticosteroid therapy for systemic disease. Symptomatic treatment includes chlorhexidine or soluble prednisone mouthwashes and corticosteroid-impregnated gels. Colchicine and pentoxifylline, which have been used in the treatment of Behcet’s disease, may be considered in patients with recurrent aphthous ulcers. For ulcers caused by herpes and fungal infection, appropriate antiviral and antifungal therapy should be instituted. Treatment of mucosal discoid lupus is similar to that of cutaneous lupus. Topical corticosteroids and antimalarials are the mainstays, but intralesional corticosteroids, azathioprine, thalidomide, dapsone, retinoids, and MMF may be required in difficult cases.60 Dry mouth in patients with SLE can be alleviated by air humidification, stimulation of salivary flow by sugarless mints or chewing gums, and artificial saliva preparations. Additional treatment includes the muscarinic receptor agonists such as pilocarpine and cevimeline. Regular dental checkups and treatment of early periodontitis are necessary to prevent local complications as well as to reduce the risk of cardiovascular disease.

The management of esophageal hypomotility and reflux symptoms in SLE patients is no different from those in patients with systemic sclerosis. High-dose H2-blockers, proton pump inhibitors, and prokinetic agents are the mainstay therapies. Immunosuppressive treatment is warranted for esophageal lesions that are proven histologically to be vasculitic in origin.

Mesenteric Vasculitis The mortality of lupus mesenteric vasculitis is high.16 Aggressive treatment has to be instituted early. Highdose intravenous methylprednisolone is the initial treatment of choice. Surgical intervention is indicated when response is not rapid or when there are clinical and radiologic signs of bowel perforation. Intravenous pulse cyclophosphamide has been used with success in an SLE patient with relapsing intestinal vasculitis that was refractory to corticosteroid treatment.22

Mesenteric Insufficiency Surgical revascularization has been shown to give longterm symptom relief in most patients with chronic mesenteric ischemia. Recently, percutaneous transluminal mesenteric angioplasty with or without a stent has become an alternative for selected patients.24 Acute mesenteric thrombosis causing bowel gangrene should be treated by surgical exploration and embolectomy.

Intestinal Pseudo-Obstruction This SLE-related condition usually responds to treatment with high-dose corticosteroids.25,61 Additional immunosuppression in the form of azathioprine, cyclosporin A, and cyclophosphamide was used with success in some reports.61,62 Despite maintenance therapy, some patients may have a relapsing course. Other adjunctive therapies in patients with IPO include broad-spectrum antibiotics and prokinetic agents such as erythromycin and octreotide (a long-acting somatostatin analog).63 Early recognition of IPO in SLE patients is important because the condition is potentially reversible with nonsurgical measures and early institution of immunosuppressive therapy.

Protein-Losing Gastroenteropathy Protein-losing gastroenteropathy in SLE is often corticosteroid-responsive. No controlled trials are available regarding the additional benefit of azathioprine. Based on the experience of the reported cases in the literature, it appears that the relapse rate of SLE-related PLGE is lower with maintenance therapy comprised of low-dose prednisolone and azathioprine than with prednisolone alone.30 An open-labeled study of 16 patients with SLEPLGE by our group reported that an initial regimen of high-dose prednisolone and azathioprine was well tolerated and effective in most patients.30 Relapse was very

Lupus Peritonitis Most patients with lupus peritonitis respond rapidly to moderate doses of corticosteroids. In patients with massive or refractory ascites, intravenous pulse methylprednisolone and additional immunosuppressive agents such as azathioprine, cyclosporin A, and cyclophosphamide may be needed.64

Lupus Hepatitis High-dose prednisone alone or a lower dose of prednisone in conjunction with azathioprine is the mainstay of treatment for AIH. Remission can be achieved in the majority of patients in the first 3 years of diagnosis. The use of azathioprine is corticosteroid sparing and reduces relapses. Maintenance therapy with low-dose prednisone and azathioprine is preferred for patients with multiple relapses. Newer treatment modalities of AIH include cyclosporine A, tacrolimus, MMF, and budesonide.

Lupus Pancreatitis Management of pancreatitis in SLE patients includes fluid resuscitation, bowel resting, discontinuation of nonessential but potentially offending drugs, and the use of antibiotics if necessary. Secondary causes of pancreatitis such as cholelithiasis, alcoholism, and hypertriglyceridemia have to be excluded. Close observation and serial-contrast CT scan of the abdomen are needed to monitor for the progress of pancreatitis and its complications. Corticosteroids should be considered in idiopathic cases of pancreatitis, particularly if SLE is active in other systems.

REFERENCES

uncommon with low-dose prednisolone and azathioprine maintenance therapy. For patients with refractory disease, intravenous pulse cyclophosphamide may be necessary. Prophylaxis for thromboembolic complications should be considered in patients with severe and persistent protein loss, especially if antiphospholipid antibodies are present.

CONCLUSIONS The gastrointestinal and hepatic manifestations of SLE are protean and not well understood. Despite the advances in imaging techniques, the diagnosis of GI lupus results from the exclusion process. As in active SLE in other systems, the mainstay of treatment is immunosuppression. Anticoagulation is indicated when thrombosis is the underlying etiology. More studies on quantitative assessment of the activity of GI lupus and controlled trials on its therapy are needed.

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with intravenous pulse cyclophosphamide: a clinical case report and review of the literature. Br J Rheumatol 1998;37:1023-1028. Weiser MM, Andres GA, Brentjens JR, et al. Systemic lupus erythematosus and intestinal venulitis. Gastroenterology 1981; 81:570-579. Sreenarasimhaiah J. Diagnosis and management of intestinal ischaemic disorders. BMJ 2003;326:1372-1376. Narvaez J, Perez-Vega C, Castro-Bohorquez FJ, et al. Intestinal pseudo-obstruction in systemic lupus erythematosus. Scand J Rheumatol 2003;32:191-195. Perlemuter G, Chaussade S, Wechsler B, et al. Chronic intestinal pseudo-obstruction in systemic lupus erythematosus. Gut 1998;43:117-122. Nojima Y, Mimura T, Hamasaki K, et al. Chronic intestinal pseudoobstruction associated with autoantibodies against proliferating cell nuclear antigen. Arthritis Rheum 1996;39: 877-879. Mader R, Adawi M, Schonfeld S. Malabsorption in systemic lupus erythematosus. Clin Exp Rheumatol 1997;15:659-661. Rensch MJ, Szyjkowski R, Shaffer RT, et al. The prevalence of celiac disease autoantibodies in patients with systemic lupus erythematosus. Am J Gastroenterol 2001;96:1113-1115. Mok CC, Ying KY, Mak A, et al. Outcome of protein losing gastroenteropathy in systemic lupus erythematosus treated with prednisolone and azathioprine. Rheumatology (Oxford) 2006;45:425-429. Kobayashi K, Asakura H, Shinozawa T, et al. Protein-losing enteropathy in systemic lupus erythematosus. Observations by magnifying endoscopy. Dig Dis Sci 1989;34:1924-1928. Lim E, Koh WH, Loh SF, et al. Non-thyphoidal salmonellosis in patients with systemic lupus erythematosus. A study of fifty patients and a review of the literature. Lupus 2001;10:87-92. Man BL, Mok CC. Lupus-related serositis: prevalence and outcome. Lupus 2005;14:822-826. Schocket AL, Lain D, Kohler PF, et al. Immune complex vasculitis as a cause of ascites and pleural effusions in systemic lupus erythematosus. J Rheumatol 1978;5:33-38. Zizic TM, Shulman LE, Stevens MB. Colonic perforations in systemic lupus erythematosus. Medicine (Baltimore) 1975;54:411-426. Yuasa S, Suwa A, Hirakata M, et al. A case of systemic lupus erythematosus presenting with rectal ulcers as the initial clinical manifestation of disease. Clin Exp Rheumatol 2002;20:407-410. Alarcon-Segovia D, Herskovic T, Dearing WH, et al. Lupus erythematosus cell phenomenon in patients with chronic ulcerative colitis. Gut 1965;28:39-47. Runyon BA, LaBrecque DR, Anuras S. The spectrum of liver disease in systemic lupus erythematosus. Report of 33 histologically-proved cases and review of the literature. Am J Med 1980;69:187-194. Gibson T, Myers AR. Subclinical liver disease in systemic lupus erythematosus. J Rheumatol 1981;8:752-759. Miller MH, Urowitz MB, Gladman DD, et al. The liver in systemic lupus erythematosus. QJM 1984;53:401-409. Hall S, Czaja AJ, Kaufman DK, et al. How lupoid is lupoid hepatitis? J Rheumatol 1986;13:95-98. Pistiner M, Wallace DJ, Nessim S, et al. Lupus erythematosus in the 1980s: a survey of 570 patients. Semin Arthritis Rheum 1991;21:55-64. Arnett FC, Reichlin M. Lupus hepatitis: an under-recognized disease feature associated with autoantibodies to ribosomal P. Am J Med 1995;99:465-472. Lu CL, Tsai ST, Chan CY. Hepatitis B infection and changes in interferon-alpha and -gamma production in patients with systemic lupus erythematosus in Taiwan. J Gastroenterol Hepatol 1997;12:272-276. Abu-Shakra M, El-Sana S, Margalith M. Hepatitis B and C viruses serology in patients with SLE. Lupus 1997;6:543-544.

46. Perlemuter G, Cacoub P, Sbai A, et al. Hepatitis C virus infection in systemic lupus erythematosus: a case-control study. J Rheumatol 2003;30:1473-1478. 47. Ramos-Casals M, Font J, Garcia-Carrasco M, et al. Hepatitis C virus infection mimicking systemic lupus erythematosus: study of hepatitis C virus infection in a series of 134 Spanish patients with systemic lupus erythematosus. Arthritis Rheum 2000; 43:2801-2806. 48. Asherson RA, Thompson RP, MacLachlan N, et al. Budd Chiari syndrome, visceral arterial occlusions, recurrent fetal loss and the “lupus anticoagulant” in systemic lupus erythematosus. J Rheumatol 1989;16:219-224. 49. Horita T, Tsutsumi A, Takeda T, et al. Significance of magnetic resonance imaging in the diagnosis of nodular regenerative hyperplasia of the liver complicated with systemic lupus erythematosus: a case report and review of the literature. Lupus 2002;11:193-196. 50. Klein R, Goller S, Bianchi L. Nodular regenerative hyperplasia (NRH) of the liver—a manifestation of “organ-specific antiphospholipid syndrome”? Immunobiology 2003;207:51-57. 51. Matsumoto T, Kobayashi S, Shimizu H, et al. The liver in collagen diseases: pathologic study of 160 cases with particular reference to hepatic arteritis, primary biliary cirrhosis, autoimmune hepatitis and nodular regenerative hyperplasia of the liver. Liver 2000;20:366-373. 52. Kamimura T, Mimori A, Takeda A, et al. Acute acalculous cholecystitis in systemic lupus erythematosus: a case report and review of the literature. Lupus 1998;7:361-363. 53. Pascual-Ramos V, Duarte-Rojo A, Villa AR, et al. Systemic lupus erythematosus as a cause and prognostic factor of acute pancreatitis. J Rheumatol 2004;31:707-712. 54. Saab S, Corr MP, Weisman MH. Corticosteroids and systemic lupus erythematosus pancreatitis: a case series. J Rheumatol 1998;25:801-806. 55. Derk CT, DeHoratius RJ. Systemic lupus erythematosus and acute pancreatitis: a case series. Clin Rheumatol 2004; 23:147-151. 56. Serrano Lopez MC, Yebra Bango M, Lopez Bonet E, et al. Acute pancreatitis and systemic lupus erythematosus: necropsy of a case and review of the pancreatic vascular lesions. Am J Gastroenterol 1991;86:764-767. 57. Hasselbacher P, Myers AR, Passero FC. Serum amylase and macroamylase in patients with systemic lupus erythematosus. Br J Rheumatol 1988;27:198-201. 58. Eberhard A, Couper R, Durie P, et al. Exocrine pancreatic function in children with systemic lupus erythematosus. J Rheumatol 1992;19:964-967. 59. Lian TY, Edwards CJ, Chan SP, et al. Reversible acute gastrointestinal syndrome associated with active systemic lupus erythematosus in patients admitted to hospital. Lupus 2003; 12:612-616. 60. Fabbri P, Cardinali C, Giomi B, et al. Cutaneous lupus erythematosus: diagnosis and management. Am J Clin Dermatol 2003;4:449-465. 61. Mok MY, Wong RW, Lau CS. Intestinal pseudo-obstruction in systemic lupus erythematosus: an uncommon but important clinical manifestation. Lupus 2000;9:11-18. 62. Hill PA, Dwyer KM, Power DA. Chronic intestinal pseudoobstruction in systemic lupus erythematosus due to intestinal smooth muscle myopathy. Lupus 2000;9:458-463. 63. Perlemuter G, Cacoub P, Chaussade S, et al. Octreotide treatment of chronic intestinal pseudoobstruction secondary to connective tissue diseases. Arthritis Rheum 1999; 42:1545-1549. 64. Provenzano G, Rinaldi F, Le Moli S, et al. Chronic lupus peritonitis responsive to treatment with cyclophosphamide. Br J Rheumatol 1993;32:1116.

CLINICAL ASPECTS OF THE DISEASE

35

Systemic Lupus Erythematosus and Infections William R. Gilliland, MD and George C. Tsokos, MD

Despite improved treatment and overall survival of patients with systemic lupus erythematosus (SLE), infection remains a major cause of morbidity and mortality.1-3 Although many of the infections are attributed to common pyogenic organisms such as Staphylococcus sp and Escherichia coli, opportunistic pathogens such as uncommon bacteria, fungi, viruses, and parasites are increasingly being recognized in critically ill SLE patients.4 Features of SLE itself (including global dysregulation of the immune system, a hallmark of SLE) appear to play a role in the increased susceptibility of these patients. In addition, immunosuppressive agents (most notably corticosteroids and cyclophosphamide) also significantly increase the risk for infections. Unfortunately, established predictors of impending infection or identifiers of a subgroup of patients prone to infections remain unclear. In lupus patients presenting with unexplained fever, confusion, or pulmonary infiltrates, differentiating between a disease flare and superimposed infections remains a clinical problem. This chapter reviews infections in SLE, including the impact of infections, possible pathogenic mechanisms, the spectrum of infectious agents, and diagnostic considerations.

IMPACT OF INFECTION IN SLE PATIENTS Differences in study design make it difficult to determine and compare the mortality rates in various studies. However, as summarized in Table 35.1 it is clear that infection remains a major cause of death.1-3,5-15 As recorded in the studies listed in Table 35.1, infection and active disease are clearly the top two primary causes of death in lupus patients. When available, autopsy data is important and virtually always shows undiagnosed infections.5,9,16 Although the importance of atherosclerosis on late mortality is being emphasized, death due to infection tends to occur early in the disease but continues throughout the duration of the patient’s illness.17

In the latest multicenter European study of over 1000 patients for a 10-year period, active SLE and infections each accounted for 28.9% of deaths during the first five years of disease, whereas thrombosis (26.1%) was the leading cause of death during the last five years.18 Depending on the study cited, the frequency of major infections in lupus patients ranges from 14 to 77%.19,20 Attempts to identify risk factors for infections in lupus cohorts have often yielded conflicting results. The clinical predictors most frequently cited are summarized in Table 35.2. Interestingly, lymphopenia was not identified as a risk factor in any of these studies. More recently, in a case-control study that investigated the risk factors associated with infection in lupus patients univariate analysis identified corticosteroid use at the time of or prior to infection, active renal disease, central nervous system involvement, and SLE disease activity index (SLEDAI) at the time of infection as risk factors. However, the use of corticosteroids was the only factor that remained statistically significant on multivariate analysis.18 In a monocentric cohort of 87 adults with SLE over a 37-year period (1960 to 1997), severe disease flares, renal disease, corticosteroid use, pulse cyclophosphamide, and/or plasmapharesis were identified as significant risk factor for infection. Multivariate analyses retained intravenous corticosteroids and/or immunosuppressants as independent risk factors for infection.3 Superimposed infections in lupus patients can also trigger disease exacerbation.6,22 Induction23,24 and aggravation25 of SLE have been reported with both parvovirus B19 and cytomegalovirus.26 Superantigens released from certain common pathogens, such as mycoplasma species,27 can independently activate B and T lymphocytes (causing SLE exacerbation).28,29 Polyclonal B-cell activation initiated by lipoploysaccharide from gram-negative bacteria can exacerbate autoimmune disease as well.30

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SYSTEMIC LUPUS ERYTHEMATOSUS AND INFECTIONS

TABLE 35.1 INFECTION AS A CAUSE OF DEATH IN SLE PATIENTSa Author

Years Covered

No. of Patients

Harvey et al., 1954 (5)b

1949-1954

138

Ginzler et al., 1978 (6)

1966-1976

Wallace et al., 1981 (7)

1950-1980

Rosner et al., 1982 (8)

No. of Deaths

Deaths Caused by Infections (%)

Country

38

United States

39

223

55

United States

36

609

128

United States

21

1965-1978

1103

222

United States

33

Rubin et al., 1985 (9)

1970-1983

417

51

Canada

28

Pistiner et al., 1991 (2)

1980-1989

464

26

United States

19

Janwityanuchit et al., 1993 (10)

1980-1989

537

77

Thailand

30

Massardo et al., 1994 (1)

1970-1991

218

48

Chile

12

Huicochea Grobet al., 1996 (11)

1970-1993

65

14

Mexico

29

Kim et al., 1999 (12)

1993-1997

544

43

Korea

33

Jacobsen et al., 1999 (13)

1975-1995

513

122

Denmark

21

Mok et al., 2000 (14)

1975-1999

186

9

China

67

Rodriquez et al., 2000 (15)

1960-1994

662

161

Puerto Rico

27

Noel et al., 2001 (3)

1960-1997

87

10

France

20

a. Only studies that included large numbers of patients were selected. b. Series in which autopsies were required.

PATHOGENESIS OF INFECTION: DEFECTS OF IMMUNE DEFENSE IN SLE Patients with SLE have numerous defects in both humoral and cellular immunity, which have been described in reviews.31-33 Several of these defects could partially explain the inadequacy of the immune defense in these patients (Table 35.3). Inherent defects

of the immune effector cells may be supplemented by the possibility that the preexisting activation of the effector cells of immune defense in SLE render them refractory to any stimulation,22,34 and the plethora of circulating autoantibodies may interfere with various functions of the cellular and humoral arms of the immune system (Table 35.4). The resulting immune defects are not universal, and there is heterogeneity in

TABLE 35.2 RISK FACTORS FOR INFECTION IDENTIFIED FROM CLINICAL STUDIES

394

Author

Type of Infection

Confirmed Risk Factors

Not Confirmed Risk Factors

Ginzler et al., 1978 (6)

All infections

Active lupus nephritis, prednisone dose

Leukopenia

Nived et al., 1985 (20)

All infection

Disease activity

Leukopenia, low complement

Rubin et al., 1985 (9)

Major, at times of death

Prednisone dose

Cytotoxic drugs, disease activity, disease duration

Hellmann et al., 1987 (16)

Fatal opportunistic

Prednisone dose

Cytotoxic drugs, CH50 levels

Duffy et al., 1991 (21)

During hospitalization

Disease activity

Prednisone dose

Petri and Genovese, 1992 (19)

Hospitalization for infection

Disease activity, prednisone, cytotoxics drugs

Cervera et al., 1999 (18)

All infections

Corticosteroid use

Noel et al., 2001 (3)

All infections

Intravenous corticosteroids and/or immunosuppressive drugs

Cellular and Humeral Defects in: ●

Monocyte/macrophage function

●

PMN number and function

●

CD4+ T cells, number and function

●

CD8+ T-cell cytolytic activity

●

NK cells, number and function

●

Cytokine production and receptors

●

Serum complement levels

●

Function of the Fcγ receptor

●

Function of the CR1, CR2, CR3

●

Balance among IgG subclasses

a. From reference 35. PMN = polymorphonuclear cells, NK = natural killer, IgG = immunoglobulin G.

their expression among lupus patients with variable susceptibility to different pathogens.

Macrophage Defects Multiple defects of the macrophage/monocyte system affect its antigen-presenting function. An important component of this defect is the diminished pahagocytic activity of lupus monocytes36 that does not increase upon stimulation in vitro with lipopolysaccharide.37 Decreased tumor necrosis factor production by mononuclear cells may contribute to the deficient phagocytic ability and predisposition to bacterial infections.38 Superoxide generation induced by phagocytosis by Fcγ receptor is also decreased in lupus patients.39

Circulating IgG and IgM autoantibodies against this receptor may interfere with its function.40 Furthermore, different patients may have autoantibodies directed against each of the three subclasses of Fcγ receptor, which may affect the phagocytic function of the macrophages and neutrophils.41 Last, monocytes from SLE patients have impaired capacity to adhere to plastic and ability to engulf apoptotic cells, which may indicate an intrinsic cellular defect.42

Neutrophil Defects Both quantitative and qualitative deficiencies may be seen in SLE (Table 35.5). Neutropenia, although not an American College of Rheumatology classification criterion, is a common finding in SLE.43 This is at least partially immune mediated and has been correlated with the presence of complement-activating antineutrophil antibodies.44 Antibodies to myeloid precursors have also been identified.45 The first component of neutrophil function, chemotaxis, is abnormal in SLE.46 Several mechanisms have been identified to include reduced complement-derived chemotactic factors47 and abnormal migration to a chemotactic stimulus.48 Proximal white subungual onychomycosis, a rare nail infection, has been described in immunocompromised individuals (including SLE). It is associated with a defect in neutrophil chemotaxis.49 Membrane recognition and attachment is also defective in SLE. Nived and colleagues50 found reduced opsonization of protein A containing Staphylococcus aureus by sera from lupus patients with active disease. Hartman and Wright51 demonstrated that some lupus patients have circulating autoantibodies directed against neutrophil adhesion glycoproteins (CD11b/CD18, Mac-1),

PATHOGENESIS OF INFECTION: DEFECTS OF IMMUNE DEFENSE IN SLE

TABLE 35.3 IMMUNE ABNORMALITIES IN SLE THAT MAY RESULT IN DEFECTIVE IMMUNE DEFENSEa

TABLE 35.5 NEUTROPHIL DISORDERS IN SLEa TABLE 35.4 ANTIBODIES DIRECTED AGAINST CELLS AND CELLULAR COMPONENTS OF THE IMMUNE SYSTEM THAT MAYCONTRIBUTE TO IMMUNE DYSFUNCTIONa

Neutrophil Disorder

Effect on Immune Defense

Decreased chemotaxis

Decrease

Cells

Surface Membrane Molecules

Decreased phagocytic activity

Decrease

Neutrophils

CD11b/CD18 (Mac-1, CR3) HLA-I heavy chains

High neutrophil clustering activity

Possible decrease

Monocytes/Macrophages

CR1, CR2 surface IgM and IgD

NK cells

FcγRI, FcγII, FcγIII, FcγIII HLA-DR framework epitope

T lymphocytes

β2-Microglobulin isoforms of CD45

B lymphocytes

IL-2 receptor

a. From reference 35. IgM = immunoglobulin M, IgD = immunoglobulin D, NK = natural killer, IL-2 = interleukin 2.

Existence of anti-lactoferrin, anti-elastase, and anti-lysozyme antibodies Increased spontaneous, and decreased after FMLP-stimulation, release of cytidine deaminase

?

Neutropenia

Decrease

Dysregulation of CD11b/CD 18 expression

Possible decrease

a. From reference 35.

395

SYSTEMIC LUPUS ERYTHEMATOSUS AND INFECTIONS

and some of these antibodies blocked adhesion or opsonin receptor function of the Mac-1 proteins. However, the existence of these antibodies did not correlate with the presence of neutropenia.52 The clinical importance of other autoantibodies against cytoplasmic neutrophil components (ANCA), such as lactoferrin, is unknown.53,54 Defective phagocytosis by neutrophils has been noted in SLE patients since the 1970s55 and is more prominent in untreated than in treated patients.38 Defective phagocytosis of apoptotic bodies leads to impaired disposal of autoantigens on dying cells that could enhance the autoimmune process.56 Excess of circulating immune complexes is probably the main reason for the persistent activation of neutrophils during active diseases. Because prior neutrophil activation results in subsequent defective response to secondary stimuli, lupus patients may exhibit defective neutrophil function against superimposed infection during active phases of disease.34

T-cell Defects T cells display multiple abnormalities that are crucial in the pathogenesis and in the natural course of SLE. CD4+ T-cell lymphopenia is the most commonly observed disorder in untreated patients.33 Lymphopenia correlates with disease flares57 and may also contribute to the development of infections. Defective production of cytokines may also contribute to the increased rate of infections (Table 35.6). A complete description of the T-cell defects in SLE is presented elsewhere in this volume (see Chapter 10).

Natural Killer Cell Defects Decreased numbers of NK cells have been reported in SLE patients that were more pronounced in patients with active disease.58 Circulating antilymphocytic and anti-NK autoantibodies may contribute to the decreased NK cell activity.57,59

TABLE 35.6 THE EFFECT OF CYTOKINE ABNORMALITIES ON IMMUNE DEFENSES IN PATIENTS WITH SLEa

396

Cytokine

Abnormality in SLE

Effect on Immune Defense

IFN-gamma

Decreased production

Decrease

IL-1

Decreased production

Decrease

IL-2

Decreased production in certain patients

Decrease

IL-10

Increased production

Possible decrease

TNF

Decreased production

Decrease

a. From reference 35.

B-cell and Immunoglobulin Defects Pronounced polyclonal B-cell activation and hyperglobulinemia are hallmarks of SLE. B cells seem to function adequately, as shown in several studies that reported normal antibody production and successful60,61 (or almost successful62) immunization. However, B-cell immunologic disorders have been described in SLE (see Chapter 11). Some SLE patients have hypogammaglobulinemia,63 IgG subclass deficiencies,64 or IgA deficiency. SLE patients with IgA deficiency are especially susceptible to infections.65

Complement Defects Normal function of the complement system is essential for host defense. Congenital deficiency of the earlier complement proteins (C1q, C1r, C1s, and C4) has a high prevalence in SLE (75%), which is often severe. C2 deficiency is also associated with SLE, but less commonly so (30%). C3 deficiency is rarely associated with SLE.66 The presence of normal-functioning early complement proteins may protect against SLE by allowing normal processing of immune complexes.67 Although the vast majority of SLE patients do not have inherited complement deficiencies, consumption of complement proteins by circulating and fixed immune complexes limit the amount of complement available for host defense.68-70 The number of complement receptors for C3b (CR1) on the surface membranes of erythrocytes is low in most patients with SLE, and this number is further decreased during disease flares.71 A decreased expression of CR1 has also been recognized on polymorphonuclear cells.72 Decreased expression of CR1 on polymorphonuclear cells resulted in an impaired recognition phase of phagocytosis.38

Spleen/Reticuloendothelial System Defects The spleen is the major component of the reticuloendothelial system (RES), and splenic dysfunction has been described in SLE patients. Several cases of functional asplenia with a high incidence of bacterial septicemia have been described.73 In many cases, the functional asplenia subsides without treatment. Defective clearance of IgG-sensitized erythrocytes for the circulation by the RES correlates with disease activity.74

Vascular Defects Anatomic lesions in SLE patients, resulting from the impact of the primary disease or accelerated atherosclerosis, represent another risk factor for infection. Disseminated damage in the microcirculation has been found.75 Small renal vessel injury and glomerular scarring may contribute to the increased susceptibility to urinary tract infections. Likewise, capillary vasculitis in

DRUG THERAPY Various immunosuppressive medications have been used over the past several decades to treat lupus patients. Along with corticosteroids, cyclophosphamide, azathioprine, and methotrexate remain the most commonly used medications. The impact of these agents on the immune system is fairly well established, particularly in those patients with lupus nephritis.78,79 Less is known about the potential infectious complications of newer immunosuppressive medications such as mycophenylate mofetil and TNFα blocking agents. Other drugs commonly used to treat SLE, such as nonsteroidal anti-inflammatory agents and antimalarial agents, are thought to have a lesser effect on immune defence.

It has been known for decades that SLE patients treated with immunosuppressive agents are more susceptible to infections than patients with other systemic rheumatic diseases treated comparably.80 Numerous studies indicate that administration of corticosteroids and other immunosuppressive medications are also at least partially responsible for the high infection rate, although the extent of that increased risk is unclear (as evidenced in Table 35.7). Immunosuppressive medications have a dual effect on the immune system in SLE patients. Suppression of abnormally functioning cells may normalize some aspects of the immune system. For example, neutrophil migration is significantly depressed in untreated SLE patients but normal in the treated patients,86 and treatment with a high dose of pulse methylprednisolone enhances Fcγ receptor-mediated mononuclear phagocyte function.87 The increased infection rate in some clinical studies of patients receiving immunosuppressive therapy may be attributed to active or advanced disease. High doses of corticosteroids and cytotoxics drugs are used almost exclusively in patients with organthreatening disease.2 When comparing the rate of infection between treated and untreated patients,

DRUG THERAPY

the gastrointestinal mucosa facilitates transudation of pathogens (such as salmonella) into the bloodstream.76 Synovitis decreases the resistance of synovium to penetration of macromolecules and consequently increases the risk for septic arthritis. The same pathogenic mechanism is probably responsible for the development of septic pericarditis with lupus pericarditis.77 Lupus skin lesions provide an uncontrolled site of entrance for microbes.

TABLE 35.7 CORTICOSTEROIDS, IMMUNOSUPPRESSIVE AGENTS, AND INFECTIONS IN SLE Author

Findings and Comments

Ginzler et al., 1978 (6)

Corticosteroids predispose to infection. Opportunistic Infections only with high steroid dose. Azathioprine predisposes to herpes zoster infection.

Nived et al., 1985 (20)

Steroid-independent increase of bacterial infections in SLE in comparison to RA.

Rubin et al., 1985 (9)

Mean prednisone doses slightly higher in infection group (63 mg versus 50 mg).

Austin et al., 1986 (79)

Cyclophosphamide associated with localized herpes zoster infection.

Hellman et al., 1987 (16)

Fatal infections correlated with prednisone and cytotoxic therapy.

Duffy et al., 1991 (21)

Infection rate did not correlate with prednisone dose.

Oh et al., 1993 (81)

Pulse methylprednisolone and cytotoxics did not increase the risk of infection.

Janwityanuchit et al., 1993 (10)

Steroid therapy predisposed to opportunistic infections. Fatal infections were more common in cyclophosphamide-treated group.

Paton et al., 1996 (82)

Risk of major infection 20 times higher and incidence of minor infection 10 times higher in the month following a course of pulse methylprednisolone. No additional risk with azathioprine, oral or intravenous cyclophosphamide.

Pryor et al., 1996 (83)

Higher maximum corticosteroid dose (195 vs. 73 mg) in infection group. Infection occurred with equal prevalence in those patients treated with intravenous vs. oral cyclophosphamide.

Zonana-Nacach et al., 2001 (84)

Higher accumulative dose of prednisone and treatment with intravenous cyclophosphamide associated with more infections.

Noel et al., 2001 (3)

Intravenous corticosteroids and immunosuppressants were independent risk factors for infection.

Badsha et al., 2002 (85)

Low dose methylprednisolone pulse (< or = 1500 gm over 3 days) had fewer serious infection compared to high dose (> 3 gm over 3 days).

SLE = systemic lupus erythematosus, RA = rheumatoid arthritis.

397

SYSTEMIC LUPUS ERYTHEMATOSUS AND INFECTIONS

the contribution of disease activity is often ignored. The studies from the National Institutes of Health (NIH)78,79 included only patients with lupus nephritis without end-stage renal disease, which may independently contribute to the increased infection rate.88 Although these studies failed to demonstrate an additive effect of cytotoxics drugs and prednisone in increasing the infection rate, they showed that cyclophosphamide increases the incidence of localized herpes zoster infection.79 Patients treated with only low doses of prednisone (<10 mg/day) also have an increased infection rate.6,89 Interestingly, the same dose did not increase the infection rate in patients with rheumatoid arthritis treated with similar doses.90 However, Nived and colleagues concluded that when matched for corticosteroids dose rheumatoid arthritis patients had only a slightly reduced risk of infection rate compared to SLE patients.20 Although the optimal treatment for lupus nephritis remains unclear, the use of mycophenylate mofetil (MMF, which selectively inhibits activated lymphocytes and renal mesangial cells) is being used more frequently as an agent for induction and maintenance in SLE nephritis. In a recent study of proliferative nephritis, Contreras and colleagues compared the infectious risks of pulse cyclophosphamide as an induction agent, followed by quarterly intravenous cyclophosphamide, daily azathioprine, or daily MMF. MMF and azathioprine maintenance therapy were associated with a significantly lower risk of infections (2% in each MMF and azathioprine, and 25% in cyclophosphamide).91 The safety and efficacy of using TNFα blockade is SLE is not yet known, although a recent small case series using infliximab suggests that it may be relatively safe.92

THE SPECTRUM OF INFECTIONS Although all major types of pathogens (including bacteria, mycobacteria, viruses, fungi, and parasites) are known to cause infections in lupus patients, it is

difficult to determine the precise contribution of each. Several reasons may explain part of this difficulty. Isolation of pathogens may be difficult because of prompt initiation of broad-spectrum antibiotics. The empiric early use of broad-spectrum antibiotics that are effective against gram-negative organisms has changed the pattern of infections in immunosuppressed patients over the last decade. Gram-positive infections are now more common than gram-negative ones, and fungal infections are the leading factor in the morbidity and mortality in immunocompromised patients with cancer.93 It is also possible that older studies underestimated the accurate number of infections or at least the number of causative organisms. Futrell and colleagues94 showed that autopsy in lupus patients with central nervous system disease frequently showed unsuspected systemic and brain infections. In addition, newly recognized pathogens were not included in earlier studies. For example, Chlamydia and Legionella species, relatively common causes of pneumonia now, were not known pathogens of the respiratory system 30 years ago. Hellmann and colleagues16 reported that only 10% of fatal opportunistic infections in lupus patients were diagnosed before autopsy. Therefore, the spectrum of infections in lupus patients has changed. Although the methodology of studies investigating the site of infection in SLE patients varies widely, Table 35.8 provides evidence that the most common sites of infection in decreasing frequency are the lung, bladder, blood, skin, central nervous system (CNS), and vagina. Pneumonia is the most common major infection of hospitalized SLE patients.21 Bates and colleagues95 showed that the determination of the pathogen is possible in only 50% of hospitalized and community-acquired infections, despite the use of modern laboratory techniques, invasive procedures, and autopsy. Futrell and colleagues showed that autopsy in SLE patients with CNS disease frequently shows

TABLE 35.8 SITES OF INFECTION Pulmonary (%)

Bladder (%)

de Luis et al., 1990 (97)

26

31

Andonopoulos, 1991(98)

35

Gomez et al., 1991 (99)

19

47

Massardo et al., 1991 (100)

30

23

Yuhara et al., 1996 (101)

29

Zonana-Nachach et al., 2001 (84) Gladman et al., 2002 (102)

Author

398

Bacteremia (%)

Skin (%)

CNS (%) 0

8

29

17

18

41

12

19

14 17

1

12

24

18

6

12

26

0

23

0

29

18

5

23

3

Vaginal (%)

9

Other (%)

39 22

Infections from Common Bacteria Common bacteria cause most infections in SLE patients.19 Staphylococcus aureus and Escherichia coli are the leading bacterial pathogens. Studies evaluating the most common bacterial pathogens vary widely in terms of diagnostic evaluation and patient population. Table 35.9 provides an overview of the most common bacteria. Compared to outpatients, hospitalized patients presented with a broader spectrum of pathogens (with a predominance of Pseudomonas aeruginosa in major and E coli in minor infections).21 Aside from these typical bacterial pathogens in patients with defective immunity and frequent hospitalization, several bacteria are worthy of special consideration. Streptococcus pneumoniae typically causes pneumonia in SLE patients,3,84,107 but meningitis,107 sepsis,107 and soft tissue infections have also been reported. This encapsulated organism requires intact splenic function and IgG2 production for opsonization and

phagocytosis,108 which may partially explain why SLE patients are particularly prone to this bacterium. Although the association is uncommon, both Neiserria meningitides and Neiserria gonorrhea have been reported in SLE patients. Susceptibility to these encapsulated organisms may be increased because of complement deficiency, RES dysfunction, and functional asplenia.109 Neisseria meningitides monoarthritis has also been described.110 More recently, N meningitides Group Y (considered an organism of relatively low virulence) was isolated from the blood or cerebrospinal fluid in three patients.111 Numerous cases of Salmonella infections are reported in SLE patients. Largely because of functional hyposplenism and RES dysfunction, this bacterium is overrepresented in several case series.100,106 The high susceptibility to Salmonella in sickle cell anemia patients presents a similar clinical condition to lupus patients with RES dysfunction. Salmonella frequently presents with bacteremia,112 but (less commonly) other locations have been reported (including the pericardium,113 an ovarian abscess,114 and septic arthritis,10,99) especially from areas with endemic salmonellosis. Abramson and colleagues showed that Salmonella was the single

THE SPECTRUM OF INFECTIONS

unsuspected systemic and brain infections in most patients.94 In addition, CNS infections frequently mimic signs and symptoms of SLE,96 delaying treatment that may lessen morbidity and mortality.

TABLE 35.9 PREVALENCE OF BACTERIAL ORGANISM IN SLE Author

Gram-negative

Gram-positive

Staples et al., 1974 (103)

E coli- 43% Klebsiella- 14% Enterococcus- 7%

S aureus- 21% S pneumococcus- 7% Strep pyogenes- 28%

Nived et al., 1985 (20)

E coli- 29% Proteus- 9% Enterobacter- 3%

S aureus- 40%

Harisdangkul et al., 1987 (104)

Klebsiella- 7% Enterobacter- 7%

S aureus- 14% S pneumococcus- 7%

Andonopoulos et al., 1988 (98)

E coli- 24% Klebsiella- 12% Serratia- 6% Proteus- 6% Enterococcus- 6% Bacteroides- 2%

S aureus- 18% S. pneumococcus- 18% Peptococcus- 6%

Massardo et al., 1991 (100)

E coli- 24% Klebsiella- 12% Salmonella- 5% Enterobacter- 4% Proteus- 3% Citrobacter- 2% Pseudomonas- 2%

S aureus- 16% S pneumococcus- 6%

Yuhara et al., 1996 (101)

Klebsiella- 6%

S aureus- 35% Strep pyogenes- 6%

Li et al., 1998 (105)

40%

32%

Bouza et al., 2000 (106)

Salmonella- 13% E coli- 10%

Others

10%

399

SYSTEMIC LUPUS ERYTHEMATOSUS AND INFECTIONS

most gram-negative isolate from the blood of SLE patients at Bellevue Hospital.112 Several case reports suggest that infection with Salmonella may lead to previously undiagnosed SLE.115,116 Listeria monocytogenes is a classic intracellular bacterium to which patients with acquired immune deficiency disorder (AIDS) and Hodgkin’s disease are highly susceptible.127 The major protective response against this bacterium is cell-mediated immunity, which as already discussed is impaired in SLE. Despite the increased theoretical susceptibility, only 13 cases have been described in the literature in the last 40 years.128-132 Although bacteremia was the most common syndrome, peritonitis, endocarditis, and arthritis have all been reported. The majority of these patients were on prednisone or other immunosuppressive drugs.36

organic brain syndrome, vasculitis, and nephritis, and are more likely to have received intravenous “pulse” methylprednisolone or a high accumulated dose of prednisolone.126 Furthermore, cumulative dose of prednisolone and presence of nephritis are independent risk factors for the development of TB.126 The role of prophylactic isoniazid in SLE patients from endemic areas taking corticosteroids remains controversial. A recent case-controlled study from Hong Kong evaluating the usefulness of isoniazid prophylaxis to prevent recurrences of TB in SLE patients failed to show any advantage in taking isoniazid versus placebo.129 Numerous cases of atypical mycobacteria (including M avium intracellulare,130 M kansasii,131 and M marinum132) have also been reported in SLE patients.

Viral Infections Infections from Mycobacteria Like L monocytogenes, Mycobacterium tuberculosis (TB) is an infection caused by intracellular bacteria. Impaired cell-mediated immunity and especially macrophage system defects predispose SLE patients to tuberculosis. Although the low numbers of cases in Western countries most likely reflect the low prevalence of mycobacteria in these populations, several reasons may be cited for clinicians to be increasingly vigilant. It is again considered a public health hazard, especially in areas with high prevalence of AIDS. Hypothetically, the off-label use of newer biologic medications such as the TNF alpha blockers may additionally predispose lupus patients to tuberculosis. Table 35.10 shows that in countries where tuberculosis is endemic the incidence of infection among SLE patients may exceed 5%. As demonstrated in Table 35.10, TB in SLE patients commonly presents in the miliary form with high mortality. Patients who have TB are more likely to have

Patients with SLE are at increased risk of viral infections, most of which are nonfatal but can be associated with significant morbidity. Previously cited defects in interferon production, CD4+ T lymphopenia, and NK cell number and function contribute to their susceptibility to viral agents. Altered cell-mediated immunity, coupled with the use of corticosteroids and other immunosuppressive agents, also contributes to susceptibility. Among the viruses, herpes zoster is the leading viral opportunistic infection. In a case-central retrospective study of 348 patients with SLE, 55 episodes of herpes zoster occurred in 47 patients (13.5%).133 More recently, in a retrospective analysis of Japanese patients with SLE 46.6% had a history of herpes zoster.134 A similar study of a Korean SLE cohort revealed an incidence of 13.9%.135 Risk factors for herpes zoster include serious disease manifestations such as nephritis, thrombocytopenia, or hemolytic anemia; treatments with

TABLE 35.10 TUBERCULOSIS IN SLE PATIENTS

400

Author

Years and Country

SLE Patients

Harvey et al., 1954 (5)

1949-1954 USA

38 Deaths

6 (16%)

Ginzler et al., 1978 (6)

1966-1976 USA

223

3 (1.3%)

Feng and Tan, 1982 (123)

1963-1979 Singapore

311

16 (5%)

Wong, 1992 (124)

1985-1989 Hong Kong

156

9 (5.7%)

Janwityanuchit et al., 1993 (10)

1980-1989 Thailand

537

22 (4%)

Cardenas et al., 1995 (125)

1987-1994 Mexico

80

5 (6.2%)

TB caused death in 3/5

Balakrishnan et al., 1998 (126)

India

146

17 (11.6%)

TB caused death in 1/17

Tam et al., 2002 (127)

Hong Kong

526

57 (11%)

67% had extrapulmonary or military TB

Sayarlioglu et al., 2004 (128)

1978-2001 Turkey

556

20 (3.6%)

45% had extrapulmonary TB

TB, tuberculosis.

SLE Patients with TB

Comments Autopsy data

TB was cause of death in 5/16

of both disorders has led to the speculation that SLE may be protective against HIV infection.145,146 However, it is very clear that SLE and HIV have similar laboratory and clinical features. HIV infections are frequently associated with a variety of serum autoantibodies, including antinuclear antibodies, anti-DNA, anti-ENA, ANCA, and anticardiolipin antibodies.145 Clinical symptoms may also be similar. A recent case series described 8 patients who presented with weight loss, anemia, and candidiasis who were mistakenly assumed to have HIV and a diagnosis of SLE was not considered (delaying appropriate treatment).147 Last, the presence of falsepositive tests for HIV infection in lupus patients may cause tragic errors in patient management. Wart virus infections are also common in SLE patients. In a case-controlled study, almost 50% of patients with SLE had warts compared to approximately 12% of controls.148 In a study comparing the prevalence of PAP smears in patients with SLE and controls, the overall prevalence of human papillomavirus infections was higher in patients with SLE compared to controls (11.8% versus 7.3%).149 Multiple cases of parvovirus B-19 infections have been described with similar clinical and serologic features that make them difficult to distinguish from SLE.150,151

THE SPECTRUM OF INFECTIONS

cyclophosphamide or azathioprine; and a concurrent or previous history of malignancy.133,136 A recent study also suggested that the majority of cases of herpes zoster cluster within two years prior to or two years after the diagnosis of SLE.137 Localized herpes zoster accounts for the majority of cases and typically occurred in patients with mild or inactive disease when they were receiving less than 20 mg of prednisone daily or no immunosuppressive therapy.133 Although the majority of patients recover without long-term sequelae, dissemination (11%) and bacterial overgrowth may occur (especially in those patients receiving immunosuppressive or prednisone at dose of 60 mg per day or higher).133 (See Table 35.11.) Unlike herpes zoster, cytomegalovirus (CMV) is not a common infectious agent in SLE patients. A recent report of 10 patients with SLE and CMV proposed three unique aspects of this viral infection: CMV infection may be difficult to distinguish from a SLE flare, CMV infection may trigger the development of SLE, and CMV may lead to an exacerbation of SLE.138 Seropositivity for CMV is higher in SLE patients than in patients with rheumatoid arthritis or normal controls, a fact that has led some investigators to speculate on its role in pathogenesis.139 Clinical manifestations include pneumonia140 and vasculitis,141 with other cases diagnosed postmortem.94 The incidence of Epstein-Barr virus (EBV), a common infection, in SLE populations is unknown. For many years, it has been suspected that EBV may be involved in the pathogenesis or in disease exacerbation. Various mechanisms have been proposed, including molecular mimicry of the EBV particle,142 differential role of CTLA-4 gene promoter genotype influencing immune responsiveness,143 and aberrant expression of viral lytic and latency leading to abnormal regulation of the EBV infection.144 Human immunodeficiency virus (HIV) infection is not common in SLE. In fact, the low rate of coexistence

Parasitic Infections Defects in cellular immunity result in increased susceptibility to parasitic infections. Furthermore, the use of corticosteroids increased the infections rate from P carinii and Toxoplasma gondii.93 Reactivated T gondii infections with encephalitis and generalized disease are well recognized in the immunocompromised host, especially in patients with AIDS or who have undergone organ transplantation. Fatal toxoplasmosis has been reported with SLE,16 but is uncommon. Toxoplasmosis may be difficult to differentiate from an SLE flare, especially neuropychiatric manifestations

TABLE 35.11 HERPES ZOSTER IN SLE PATIENTS Author

SLE Patients

Total Infections

Herpes Zoster

Nived et al., 1985 (20)

102

162

5

Wong, 1992 (124)

156

32

11

Janwityanuchit et al., 1993 (10)

537

220

41

Features of Patients with Herpes Zoster

The most common infection 39 patients were receiving immunosuppressive drugs a

Kahl, 1994 (133)

348

ND

47 (55)

Manzi et al., 1995 (136)

321

ND

48

Immunosuppressive drugs, nephritis, previous malignancy

Kang et al., 2005 (135)

303

ND

42

Nephritis, anti-Sm antibodies

a. Number of episodes. CPM = cyclophosphamide.

Nephritis, CPM, hemolytic anemia

401

SYSTEMIC LUPUS ERYTHEMATOSUS AND INFECTIONS

of SLE.152 Interestingly, interpretation of toxoplasma antibodies using dye and indirect hemagglutinin tests may also lead to confusion because toxoplasma may enhance the production of autoantibodies.153 Other systemic parasitic diseases have been described in SLE patients. These include strongyloides,154 paragonimiasis,155 and visceral leishmaniasis.156

Fungal Infections Historically, Candida albicans is the most common opportunistic pathogen at the time of death in SLE patients, although it may not be the cause of death.16 The most frequent sites of fatal cases of Candida were disseminated, abdominal, pulmonary, and esophageal. In many cases, candidiasis is found in patients with endorgan damage.16 Onychomycosis caused by Candida sp and other dermatophytes are also more common in SLE patients, with the duration of nail infection being twice as long as in non-autoimmune patients.157 Although Pneumocystis carinii, now classified as a fungus, gained great importance as an opportunistic pathogen during the AIDS epidemic it has been increasingly diagnosed in SLE patients in the last several decades (Table 35.12). This reflects the greater awareness of the disease and broader use of diagnostic procedures. The majority of SLE patients with P carinii pneumonia (PCP) have been treated with high-dose corticosteroids and other immunosuppressive drugs. However, cases of PCP have also been described in untreated SLE patients, suggesting that the immunologic dysregulation in SLE patients may in itself predispose SLE to developing PCP. Lymphopenia with low CD4+ T cells and reversed CD4+/CD8+ ratio may also be a risk factor for the development of PCP.158 In addition, PCP has been described in three children with SLE (all of whom were on immunosuppressive agents).159

Cryptococcus neoformans causes opportunistic infections in immunocompromised SLE patients. According to a review of these cases, the neurologic findings are often mistakenly diagnosed as neuropsychiatric SLE.163 In a recent retrospective study of patients with SLE and CNS infections, cryptococcal infection accounted for 10 of 17 cases, followed by bacterial infections in the remaining 7 cases. Mortality was extremely high (41.2%) in this group of patients.164 Although Aspergillus fumingatus is ubiquitous in nature, aspergillosis is seldom described in SLE patients. In a review of 23 cases, the most common clinical presentation was cough and fever (and fatality was high, at 95%).165 However, a recent case of an SLE patient on peritoneal dialysis was successfully treated with amphotericin B and removal of the catheter.166 Nocardia asteroides, a gram-positive aerobic bacterium with fungal characteristics, has also been reported infrequently in SLE patients. A review of 32 SLE patients with nocardiasis indicated that the lung was the most common site of infection (81%), followed by the CNS (13%). Mortality was high (35%), especially when the CNS was involved (75%).167

DIAGNOSTIC AND CLINICAL CONSIDERATIONS Fever and other symptoms suggestive of infection in SLE patients always pose the problem of differentiating between SLE flare and superimposed infection. Caregivers must frequently decide, without the aid of laboratory testing, to initiate empiric antibiotics and/or adjust the dose of immunosuppressive agents. Frequently, the timing of these therapeutic decisions is crucial. Frequently, the diagnosis of infection is based on a combination of clinical and laboratory findings and

TABLE 35.12 PNEUMOCYSTIS CARINII IN SLE PATIENTS Author

402

Years

SLE Patients

Patients with PCP

Treatment Before Infection

Mortality

Ginzler et al., 1987 (6)

1966-1976

223

1

ND

ND

Hellman et al., 1987 (16)

1969-1987

44 deaths

3

ND

3/ND

Porges et al., 1992 (158)

1989-1990

20 at comparable risk

6

3 CS + CPM, 3 CS + AZA

4/6

Liam and Wang, 1992 (160)

1987-1988

351

9

4 CS + CPM, 4 CS, 1 NT

5/9

Godeau et al., 1994 (161)

10

750

6

3 CS + CPM, 1 CS, 2 NT

2/6

Page, 1995 (162)

1989-1994

60

3

3 CS, 1 AZA + cy

0/3

SLE = systemic lupus erythematosus, ND = not determined, CS = corticosteroids, CPM = cyclophosphamide, AZA = azathioprine, NT = no treatment, cy = cyclosporine.

Prophylaxis should be considered in a several settings. Patients at high risk of developing P carinii may benefit using a regimen of low-dose trimethoprine/sulfamethoxazole three times weekly, or monthly-inhaled pentamidine in selected individuals.179 However, the role for PCP prophylaxis in these patients remains controversial. Because of concern that SLE patients may have an increased risk of adverse effects with sulfa-containing antibiotics, Petri of the Hopkins Lupus Cohort does not recommend PCP prophylaxis unless the patient has signs of severe immunosuppression (such as thrush).67 Prophylaxis with isoniazid in patients with a positive tuberculin test being placed on high-dose corticosteroids therapy is a wise precaution. Strategies similar to those recommended with AIDS patients may also be considered in SLE patients whose active disease is being treated with immunosuppressive drugs.180 SLE patients with heart murmurs and valvular vegetations should be given antibiotic prophylaxis before invasive dental or genitourinary procedures to decrease the risk of endocarditis.181 Others have argued that because endothelial damage to heart valves may be seen in up to 50% of SLE patients (and that there is no method of identifying that subpopulation at greatest risk) all SLE patient should be given antibiotic prophylaxis as outlined by the American Heart Association.181 Immunizations should also be considered in SLE patients, but throughout the years routine immunizations have also been controversial for two reasons: efficacy and safety. In regard to efficacy, the ability to mount a protective antibody response varies depending on the type of vaccination and the study. In terms of pneumococcal vaccines, older studies have demonstrated an impaired response182,183 or no immunologic response.60 On the other had, a recent study demonstrated a fourfold antibody response in 47% of SLE patients given a pneumococcal vaccine.184 Another study examined the mean concentration of pneumococcal polysaccharide-specific IgG to the seven serotypes tested and found that the vast majority of SLE patients had significant increases in all serotypes tests (215 of the SLE patients responded to either none or only one of the seven polysaccharides).185 Similarly, responses to influenza and tetanus vaccines in the older literature are highly variable. However, a more recent study demonstrated that protective antibodies were seen in 90% of patients with SLE immunized with tetanus toxoid (TT) and 88% of SLE patients immunized with Haemophilus influenzae type B (HIB).184 It seems reasonable that SLE patients receive immunizations according to the recommendations of the Centers for Disease Control and Prevention and the Immunization Practices Advisory committee. It may also be prudent to measure the antibody response in SLE patients, in order to determine if they need to be repeated.

DIAGNOSTIC AND CLINICAL CONSIDERATIONS

the clinical gestalt of the physician. In a study from Japan, disease flare accounted for approximately 66% of febrile episodes compared to 33% attributed to infection.168 Similarly, a prospective study of fevers in SLE patients found that increased disease activity accounted for 55% of episodes, with the remaining episodes being attributed to infection (45%).169 Clinical signs and symptoms may also be difficult to interpret in SLE patients because both infections and disease flare can have similar clinical presentations. For example, it may be difficult to determine if the source of pleurisy is infection or SLE activity in a febrile patient. Another difficulty is the effect of corticosteroids and other immunosuppressive therapies to impair the inflammatory response. Some authors suggest that clinical and laboratory similarities between the current febrile episode and previous lupus flares offer some assurance that lupus is the cause of the fever.170 Interestingly, few studies have addressed laboratory methods to distinguish between the infection and disease flare. Routine laboratory testing such as peripheral white blood counts with differentials (and gram stains and cultures of appropriate fluids and tissue) may be helpful. The presence of high titers of anti-dsDNA and decreased complement levels may favor disease flare. The use of C-reactive protein (CRP) levels to distinguish between the two has been controversial.171-173 A study of 28 episodes of febrile episodes in Asian patients with SLE found that CRP levels in those patients with SLE flares were only modestly elevated compared to infections that provoked more substantial elevations in CRP.174 A prospective study of CRP levels in SLE patients found that in the absence of serositis CRP levels exceeding 60 mg/dl were always associated with infection.175 Recently, in a retrospective study of lupus patients diagnosed with non-typhoidal salmonellosis, elevated CRP levels were valuable in distinguishing between fever from a pure lupus flare and one complicated by infection.176 On the other hand, normal CRP levels may be found in SLE patients with infections.177 To further complicate the issue, SLE may also be associated with false-positive serologic tests (including syphilis, HIV, Lyme disease, toxoplasmosis, and other infections). Polyclonal hyperglobulimemia may partially explain this phenomenon. In addition, corticosteroids and other immunosuppressive agents may result in false-negative skin tests for TB.178 Can the infection rate be reduced? Obviously, maintaining appropriate personal hygiene and minimizing contact with ill individuals may be helpful in decreasing viral and other infections. Theoretically, controlling the underlying immunologic defects may also be helpful, but unfortunately one of the main side effects of these medications is an increased risk of infection.

403

SYSTEMIC LUPUS ERYTHEMATOSUS AND INFECTIONS

404

In regard to safety, several case reports and small case series have suggested that SLE may develop de novo or a disease exacerbation may be seen in SLE patients after being immunized.186-188 On the other hand, double-blind trials of Pneumovax189 and influenza vaccine190 found no increase in flares. More recent studies evaluating the safety of immunizations of pneumococcal,184,185 TT, and HIB184 conclude that they may be safely administered to SLE patients. Live viral vaccines should probably not be administered to severely immunocompromised SLE patients. Infections, along with increased activity, remain the major sources of morbidity and mortality in SLE patients. In part because of the underlying immunologic

defects and the judicious use of immunosuppressive agents, patients with SLE have a higher infection rate than the general population. Although the spectrum of infectious agents and the anatomic location of infection may have changed over the past several decades, the percentage of deaths due to infections has not changed significantly. Exclusion of infections is mandatory to adequately care for SLE patients who present with fever or other infectious manifestations. Unfortunately, the current diagnostic tools used to differentiate between infections and disease flares are limited. Hopefully, better diagnostic tools and treatment of superimposed infections in SLE patients will be available in the future.

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113. Sanchez-Guerero J, Alacon-Segovia D. Salmonella pericarditis with tamponade in systemic lupus erythematosus. Br J Rheumatol 1990;29:69-71. 114. Li KE, Cohen MG. Nontyphoidal salmonella ovarian abscess in systemic lupus erythematosus. Arthritis Rheum 1985;28:75-79. 115. Li EK, Cohen MG, Ho AK, et al. Salmonella bacteraemia occurring concurrently with the first presentation of systemic lupus erythematosus. Br J Rheumatol 1993;32:66-67. 116. Martinez JT, Vidaller A, Pac V, et al. Systemic lupus erythematosus presenting as Salmonella enteritidis bacteremia. J Rheumatol 1991;18:785. 117. Chanock S. Evolving risk factors for infectious complications of cancer therapy. Hematol Oncol Clin North Am 1993;7:771-793. 118. Perez DH, Andron RI, Goldstein IM. Infection in patients with systemic lupus erythematosus. Arthritis Rheum 1979; 22:1326-1333. 119. Kraus A, Cabral AR, Sifuentes-Osornio J, et al. Listeriosis in patients with connective tissue diseases. J Rheumatol 1994; 21:635-638. 120. Jansen TL, van Heereveld HA, Laan RF, et al. Septic arthritis with Listeria monocytogenes during low-dose methotrexate. J Intern Med 1998;244:87-90. 121. Tse KC, Li FK, Chan TM, et al. Listeria monocytogenes peritonitis complicated by septic shock in a patients on continuous ambulatory peritoneal dialysis. Clin Nephrol 2003;60:61-62. 122. Harisdangkul V, Songchareon S, Lin A. Listerial infections in patients with systemic lupus erythematosus. South Med J 1992; 85:957-960. 123. Feng PH, Yan TH. Tuberculosis in patients with systemic lupus erythematosus. Ann Rheum Dis 1982;41:11-14. 124. Wong KL. Patterns of SLE in Hong Kong Chinese: A cohort study. Scand J Rheumatol 1992;21:289-296. 125. Cardenas D, Salazar-Parano M, Orozco-Barocio G, et al. Prevalence of tuberculosis in a Mexican population of patients with systemic lupus erythematosus. Lupus 1995;4:85. 126. Balakrishnan C, Mangat G, Mittal G, et al. Tuberculosis in patients with systemic lupus erythematosus. J Assoc Physicians India 1998;46:682-683. 127. Tam LS, Le EK, Wong SM, et al. Risk factors for clinical features for tuberculosis among patients with systemic lupus erythematosus in Hong Kong. Scand J Rheumatol 2002;31:296-300. 128. Sayarlioglu M, Inanc M, Kamali S, e al. Tuberculosis in Turkish patients with systemic lupus erythematosus: Increased frequency of extrapulmonary localization. Lupus 2004;13:274-278. 129. Mok MY, Lo Y, Cha TM, et al. Tuberculosis in systemic lupus erythematosus in an endemic area and the role of isoniazid prophylaxis during corticosteroid therapy. J Rheumatol 2005; 32:609-615. 130. Yang WK, Fu LS, Lan JL, et al. Mycobacterium avium complexassociated hemophagocytic syndrome in systemic lupus erythematosus patients: Report of a case. Lupus 2003;12:312-316. 131. Hsu PY, Yang YH, Hsiao CH, et al. Mycobacterium kansasii infection presenting as cellulites in a patient with systemic lupus erythematosus. J Formos Med Assoc 2002;101:581-584. 132. Enzenauer RJ, McKoy J, Vincent D, et al. Disseminated cutaneous and synovial Mycobacterium marinum infection in a patient with systemic lupus erythematosus. South Med J 1990; 83:471-474. 133. Kahl LE. Herpes zoster infections in systemic lupus erythematosus: Risk factors and outcome. J Rheumatol 1994;21:84-86. 134. Ishikawa O, Abe M, Miyachi Y. Herpes zoster in Japanese patients with systemic lupus erythematosus. Clin Exp Dermatol 1999;24:327-328. 135. Kang TY, Lee HS, Kim TH, et al. Clinical and genetic risk factors of herpes zoster in patients with systemic lupus erythematosus. Rheumatol Int 2005;25:97-102. 136. Manzi S, Kuller LH, Kutzer J, et al. Herpes zoster in systemic lupus erythematosus. J Rheumatol 1995;22:1254-1258. 137. Pope JE, Krizova A, Ouimet JM, et al. Close association of herpes zoster reactivation and systemic lupus erythematosus (SLE) diagnosis: Case-control study of patients with SLE or noninflammatory musculoskeletal disorders. J Rheumatol 2004; 32:274-279. 138. Sekigawa I, Nawata M, Seta N, et al. Cytomegalovirus infection in patients with systemic lupus erythematosus. Clin Exp Rheumatol 2002;20:559-564.

165. Gonzalez-Crespo MR, Gomez-Reino JJ. Invasive aspergillosis in systemic lupus erythematosus. Semin Arthritis Rheum 1995; 24:304-314. 166. Schattner A, Kagan A, Zimhony O. Aspergillus peritonitis in a lupus patients on chronic peritoneal dialysis. Rheumatol Int 2005. 167. Mok CC, Yuen KY, Lau CS. Nocardiosis in systemic lupus erythematosus. Semin Arthritis Rheum 1997;26:675-683. 168. Inoue T, Takeda T, Koda S, et al. Differential diagnosis of fever in systemic lupus erythematosus using discriminant analysis. Rheumatol Int 1986;6:69-77. 169. Grana J, Freire M, DeToro J, et al. Prospective study of 20 episodes of fever in 13 patients with systemic lupus erythematosus. Arthritis Rheum 1994;37:S322. 170. Stahl NI, Klippel JH, Decker JL. Fever in systemic lupus erythematosus. Am J Med 1979;67:935-940. 171. Becker GJ, Waldburger M, Hughes GR, et al. Value of serum C-reactive protein measurements in the investigation of fever in systemic lupus erythematosus. Ann Rheum Dis 1980;39:50-52. 172. Middleton GD, McFarlin JE, Sipe JD, et al. C reactive protein in systemic lupus erythematosus: Elevation does not predict infection. Arthritis Rheum 1994;37:S321. 173. Hind CR, Ng SC, Feng PH, et al. Serum C-reactive protein measurement in the detection of intercurrent infection in Oriental patients with systemic lupus erythematosus. Ann Rheum Dis 1985;44:260-261. 174. Borg EJ, Horst G, Limburg PC, et al. C-reactive protein levels during disease exacerbations and infections in systemic lupus erythematosus: A prospective longitudinal study. J Rheumatol 1990;17:1642-1648. 175. Lim E, Koh WH, Loh SE, et al. Non-typhoidal salmonellosis in patients with systemic lupus erythematosus: A study of fifty patients and the review of the literature. Lupus 2001;10:87-92. 176. Roy S, Tan KT. Pyrexia and normal C-reactive protein (CRP) in patients with systemic lupus erythematosus: Always consider the possibility of infection in febrile patients with systemic lupus erythematosus regardless of CRP levels. Rheumatology (Oxford) 2001;40:349-350. 177. Hanson CA, Reichman LB. Tuberculosis skin testing and preventive therapy. Semin Respir Infect 1989;4:182-188. 178. Sneller MC, Hoffman GS, Talar-Williams C, et al. An analysis of forty-two Wegener’s granulomatosus patients treated with methotrexate and prednisone. Arthritis Rheum 1995; 38:608-613. 179. Clumeck N. Primary prophylaxis against opportunistic infection in patients with AIDS. N Engl J Med 1995;332:739-740. 180. Zysset MK, Montgomery MT, Redding SW, et al. Systemic lupus erythematosus: A consideration for antimicrobial prophylaxis. Oral Surg Oral Med Oral Pathol 1987;64:30-44. 181. Jarrett MP, Schiffman G, Barland P, et al. Impaired response to pneumococcal vaccine in systemic lupus erythematosus. Arthritis Rheum 1980;23:1287-1293. 182. McDonald E, Jarrett MP, Schiffman G, et al. Persistence of pneumococcal antibodies after immunization in patients with systemic lupus erythematosus. J Rheumatol 1984;11:306-308. 183. Battafarano DF, Battafarano NJ, Larsen L, et al. Antigen-specific antibody responses in lupus patients following immunizations. Arthritis Rheum 1998;41:1828-1834. 184. Elkayam O, Paran D, Caspi D, et al. Immunogenicity and safety of pneumococcal vaccination in patients with rheumatoid arthritis or systemic lupus erythematosus. Clin Infect Dis 2002;34:147-153. 185. Ayvazian LF, Badger TL. Disseminated lupus erythematosus occurring among student nurses. N Engl J Med 1948;239:565-570. 186. Ristow SC, Douglas RG, Condemi JJ. Influenza vaccination in patients with systemic lupus erythematosus. Ann Intern Med 1978;88:786-789. 187. Older SA, Battafarano DF, Enzenauer RJ, et al. Can immunization precipitate connective tissue disease? Report of five cases of systemic lupus erythematosus and review of the literature. Semin Arthritis Rheum 1999;29:131-139. 188. Klippel JH, Karsh J, Stahl NI, et al. A controlled study of pneumococcal polysaccharide vaccine in systemic lupus erythematosus. Arthritis Rheum 1979;22:132-135. 189. Williams GW, Steinberg AD, Reinertsen JL, et al. Influenza immunization in systemic lupus erythematosus: A double-blind trial. Ann Intern Med 1978;88:729-734.

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139. Rider JR, Olliver WER, Lock RJ, et al. Human cytomegalovirus infection and systemic lupus erythematosus. Clin Exp Rheumatol 1997;15:405-409. 140. Ross CN, Beynon HLC, Savill JS, et al. Gancyclovir for cytomegalovirus infections in immunocompromised patients with renal disease. Q J Med 1991;81:929-936. 141. Bulpitt KJ, Brahn E. Systemic lupus erythematosus and concurrent cytomegalovirus: Diagnosis by antemortem skin biopsy. J Rheumatol 1989;16:677-680. 142. James JA, Neas BR, Moser KL, et al. Systemic lupus erythematosus in adults is associated with previous Epstein-Barr virus exposure. 2001;44:1122-1126. 143. Parks CG, Cooper GS, Hudson LL, et al. Association of EpsteinBarr virus with systemic lupus erythematosus: Effect modification by race, age, and cytotoxic T lymphocyte-associated antigen 4 genotype. Arthritis Rheum 2005;52:1148-1159. 144. Gross AJ, Hochberg D, Rand WM, et al. EBV and systemic lupus erythematosus: A new perspective. J Immunol 2005; 174(11):6599-6607. 145. Kaye BR. Rheumatic manifestations of infection with human immunodeficiency virus. Ann Intern Med 1989;111:158-167. 146. Barthel HR, Wallace DJ. False-positive human immunodeficiency virus testing in patients with lupus erythematosus. Semin Arthritis Rheum 1993;23:1-7. 147. Chowdhry IA, Tan IJ, Mian N, et al. Systemic lupus erythematosus presenting with features suggestive of human immunodeficiency virus infection. J Rheumatol 2005;32:1365-1368. 148. Johansson E, Pyrhonen S, Rostila T. Warts and wart virus antibodies in patients with systemic lupus erythematosus. Br Med J 1977;1:74-76. 149. Tam LS, Chan AY, Chan PK, et al. Increased prevalence of squamous intraepithelial lesions in systemic lupus erythematosus: Association with human papillomavirus infection. Arthritis Rheum 2004;50:3619-3625. 150. Trapani S, Ermini M, Falcinin F. Human parvovirus B19 infection: Its relationship with systemic lupus erythematosus. Semin Arthritis Rheum 1999;28:319-325. 151. Seve P, Ferry T, Koenig M, et al. Lupus-like presentation of parvovirus B19 infection. Semin Arthntis Rheum 2005;34:642-648. 152. Zamir D, Amar M, Groisman G, et al. Toxoplasma infection in systemic lupus erythematosus mimicking lupus cerebritis. Mayo Clin Proc 1999;74:575-578. 153. Noel I, Balfour AH, Wilcox MH. Toxoplasma infection and systemic lupus erythematosus: Analysis of the serological response by immunoblotting. J Clin Pathol 1993;46:628-632. 154. Potter A, Stephens D, De Keulenaer B. Strongyloides hyperinfection: A case for awareness. Ann Trop Med Parasitol 2003;97:855-860. 155. Kraus A, Guerra-Bautista G, Chavarria P. Paragonimiasis: An infrequent but treatable cause of hemoptysis in systemic lupus erythematosus. J Rheumatol 1990;17:244-246. 156. Ravelli A, Viola S, De Benedetti F, et al. Visceral leishmaniasis as a cause of unexplained fever and cytopenia in systemic lupus erythematosus. Acta Paediatr 2002;91:246-247. 157. Boonchai W, Kulthanan K, Maungprasat C, et al. Clinical characteristics and mycology of onychomycosis in autoimmune patients. J Med Assoc Thai 2003;86:995-1000. 158. Porges AJ, Beattie SL, Ritchlin C, et al. Patients with systemic lupus erythematosus at risk for pneumocystis carinii pneumonia. J Rheumatol 1992;19:1191-1194. 159. Foster HE, Malleson PN, Petty RE, et al. Pneumocystis carinii pneumonia in childhood systemic lupus erythematosus. J Rheumatol 1996;23:753-756. 160. Liam C-K, Wang F. Pneumocystis carinii pneumonia in patients with systemic lupus erythematosus. Lupus 1992;1:379-385. 161. Godeau B, Coutant-Perronne V, Houng DLT, et al. Pneumocystis carinii pneumonia in the course of connective tissue disease: Report of 34 cases. J Rheumatol 1994;21(2):246-251. 162. Page B. Pneumocystis carinii pneumonia and systemic lupus erythematosus. Lupus 1995;4:87. 163. Zimmerman B, Spiegel M, Lally EV. Cryptococcal meningitis in systemic lupus erythematosus. Semin Arthritis Rheum 1992; 22:18-24. 164. Hung JJ, Ou LS, Lee WI, et al. Central nervous system infections in patients with systemic lupus erythematosus. J Rheumatol 2005;32:40-43.

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Hematologic and Coagulation Abnormalities of Systemic Lupus Erythematosus and the Antiphospholipid Syndrome E. Nigel Harris, MD, Wendell A. Wilson, MD, and Silvia S. Pierangeli, PhD

INTRODUCTION Hematologic abnormalities such as anemia, leucopenia, thrombocytopenia, and clotting abnormalities are well recognized features of systemic lupus erythematosus (SLE). However, it is only within the past two decades that a group of autoantibodies, designated antiphospholipid (aPL) antibodies, has been shown to be intimately associated with these features of SLE.1 APL antibodies are detected by the anticardiolipin (aCL) antibody2 or lupus anticoagulant (LA) test.3 Although important, they are certainly not the sole autoantibodies associated with blood cell destruction in SLE, nor are they the sole reason for arterial or venous thrombosis (as discussed in material following). It should also be borne in mind that aPL antibodies and the hematologic and clotting abnormalities with which they are associated can occur independently of SLE [often manifested in the primary antiphospholipid syndrome (APS)]. This chapter discusses abnormalities of blood cells in SLE and examines the APS.

ANEMIA

408

There are a variety of causes of anemia in patients with SLE. These include anemia of chronic disease, iron deficiency anemia, autoimmune hemolytic anemia, pure red cell aplasia, and other causes. Anemia (less than 12 g/dl) of chronic disease is the most common cause of low hemoglobin in these patients.4 Iron deficiency anemia is not infrequent and doubtless relates to the frequency of women with menstrual abnormalities and consequent menorrhagia, which is not uncommon in women with chronic diseases such as SLE. Diagnosis depends on demonstrations of low serum iron and serum ferritin (<20 μg/dl) and high total iron binding capacity (TIBC).

Although anemia of chronic disease and iron deficiency anemia are rarely life threatening, the third cause of anemia in SLE (namely, autoimmune hemolytic) can cause severe morbidity and even mortality.5 It is recognized by a precipitous fall in hemoglobin (>3 g/dl), elevated reticulocyte count (>5%), a rise in bilirubin and invariably, a positive Coombs test. In the mid 1980s,6 an association of hemolytic anemia with elevated aCL antibodies was first recognized and this association was confirmed in many subsequent studies. Although IgG aCL antibodies (particularly at high levels6) are most frequently associated with the condition, IgM aCL antibodies (also at high levels) have been similarly associated.7-9 There is speculation that aPL antibodies might bind phospholipid-protein complexes on red cell membranes, resulting in increased uptake and destruction by the reticuloendothelial system.8 The presence of aPL antibodies in patients with autoimmune hemolytic anemia should prompt treating physicians to seek a history of thrombocytopenia, venous or arterial thrombosis, or recurrent pregnancy loss. In addition, they should be alert to the possibility that any of these events (manifestations of the APS1) may occur in the future. A sizable percentage of patients with autoimmune haemolytic anemia are aCL antibody negative, suggesting that autoantibodies with specificities for a variety of red cell membrane antigens may also account for red cell destruction. Treatment of autoimmune hemolytic anemia relies acutely on high-dose corticosteroid. The dose is tapered as the hemoglobin level rises.4,5 For patients unresponsive to corticosteroids, immunosuppressive drugs, danazol,10 and more recently introduced agents such as mycophenolate mofetil11 may be effective.

THROMBOCYTOPENIA The finding of a platelet count between 100,000/dl and 150,000/dl is not uncommon in SLE, occurring in 20 to 30% of patients.13 However, severe thrombocytopenia (platelet counts <50,000/dl) is less common, occurring in about 5% patients at some time in the course of their disease.5 A small number of patients may present first with immune thrombocytopenic purpura (ITP) and later develop other features of SLE. A number of studies have demonstrated an association of thrombocytopenia with IgG and/or IgM aCL antibodies or the LA.6,7,14,15 As noted previously, the presence of thrombocytopenia with aPL antibodies should trigger an examination of the patient’s previous history (or an anticipation in the future) for complications of APS.1 The association of thrombocytopenia with aPL antibodies might be explained by autoantibodies interacting with phospholipid-protein antigens in platelet membranes, resulting in enhanced uptake and destruction. Absorption of aPL antibodies by platelet membranes has been demonstrated in one study.16 Of relevance, too, are studies showing that these antibodies in the presence of low doses of ADP, collagen, and thrombin can result in platelet activation.17 Studies from our group have shown that affinity purified aCL antibodies from patients with APS-enhanced activation of platelets treated with suboptimal doses of ADP, thrombin, or collagen.17 In another study, rabbit aCL antibodies were shown to enhance collagen-induced platelet activation.18 The platelet GPIIb/IIIa receptor mediates platelet aggregation induced by all physiologic agonists and is the receptor for fibrinogen and a marker of platelet activation. We have shown that aPL antibodies enhance the expression of platelet membrane glycoproteins, particularly GPIIb/IIIa and GPIIIa, when platelets are pretreated with suboptimal doses of TRAP (a thrombin receptor agonist peptide).19 In a recent study, the intracellular events resulting from activation of platelets by antiphospholipid antibodies and subthreshold doses of thrombin have been examined and involve the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK).20 The nature of the receptor(s) for aPL antibodies in platelets has not been completely elucidated. Studies have shown that the exposure of phosphatidylserine is necessary for aPL antibodies to enhance activation/aggregation of platelets.16 Another

publication indicated that a 70-kDa protein in the platelet membrane is a receptor for rabbit aCL antibodies.18 Recently, investigators suggested that dimers of β2GPI (a putative target antigen for aPL antibodies) bind to the receptor for apolipoprotein E (aPOER2′) on the membrane of platelets and enhance the phosphorylation of p38MAPK and release of thromboxane B2 initiated by collagen.21 APL antibodies cannot be the only explanation for thrombocytopenia in SLE, in that antiplatelet antibodies have been demonstrated in patients who are aCL antibody negative. Treatment of patients with immune thrombocytopenia often relies on high-dose corticosteroids initially, with introduction of immunosuppressive agents such as cyclophosphamide if patients are unresponsive to corticosteriods. Newer agents such as mycophenolate mofetil (less toxic than cyclophosphamide) may be beneficial.22 In those unresponsive to all of these drugs, splenectomy is recommended. Highdose immunoglobulin therapy is often effective in the acute management of life-threatening thrombocytopenia, but its effect is short-lived and treatment is expensive. Hence, if such therapy is used the physician should have a longer-term management strategy.

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THE ANTIPHOSPHOLIPID SYNDROME AND VASCULAR COMPLICATIONS OF SLE

Pure red cell aplasia is an uncommon cause of anemia in SLE, presumably secondary to auto antibodies targeting red cell precursors in the bone marrow.12 Corticosteroid or immunosuppressive agents have proven useful in these circumstances.

Background In 1952, Conley and Hartmann first recognized an unusual coagulation abnormality in two patients with SLE who presented with hemorrhage and prolonged clotting tests.23 In the subsequent decade, other SLE patients with this disorder were described, but it became increasingly evident that bleeding abnormalities noted initially were uncommon (even when these patients underwent surgery). The clotting abnormality came to be called the “lupus anticoagulant (LA).” In 1963, Bowie and colleagues reported that patients with the LA were subject, paradoxically, to recurrent thrombosis.24 By the early 1980s, the association with both venous and arterial thrombosis was confirmed in several studies. Affected women were also found subject to recurrent pregnancy losses, usually occurring in the second or third trimester of pregnancy.25 As interest in the LA grew in the 1960s and 1970s, investigators demonstrated that this abnormality was due to an autoantibody that interrupts the conversion of prothrombin to thrombin, resulting in a prolonged clotting test. The prothrombin-thrombin conversion reaction is catalyzed by phospholipids and there was speculation that the LA autoantibody interacted with one or more of these phospholipids. This was based on the finding that addition of a phospholipid mixture (containing phosphatidylserine) corrected the

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clotting abnormality. Demonstration that a monoclonal antibody with LA activity reacted with phospholipids utilizing an Ouchterlony technique also suggested specificity for phospholipids.26 In addition, patients with the LA often had a biologic false-positive test for syphilis (BFP-STS), attributed to the presence of autoantibodies reacting with the phospholipid antigen cardiolipin. It was the association of the LA with the BFP-STS that led Harris and colleagues to develop a solid-phase immunoassay with cardiolipin as antigen27 to detect antibodies with “LA activity.” Once developed, the aCL test correlated with LA positivity and proved to be far more sensitive than the LA test. This test also correlated with recurrent venous or arterial thrombosis, pregnancy loss, and/or thrombocytopenia in a population of SLE patients.27 Although this laboratory symptom complex was first largely detected in those with SLE, later studies showed that it could occur independently of SLE. In addition, it became evident that “aCL antibodies” cross-reacted with other negatively charged phospholipids and hence these antibodies were designated “aPL antibodies.” The disorder with which these antibodies were associated was called the APS.28,29 In the early 1990s there came another surprise, in that aPL antibodies were found to bind the serum protein β2-glycoprotein I (β2GPI).30,31 β2GPI binds negatively charged molecules, including phospholipids. About 60 to 80% of patients positive for the aCL test will bind β2GPI presented on oxidized polystyrene plates.32 In some patients with aPL antibodies, binding to other serum proteins (including prothrombin) has been demonstrated.33 Hence, antiphospholipid antibodies are best regarded as consisting of diverse subpopulations specific for β2GPI, other serum proteins, negatively charged phospholipids, and proteinphospholipid complexes.

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Because patients with APS may be subject to thrombosis at any venous or arterial sites, involving vessels of any size, this disorder may present in many ways. In the venous system, deep-vein thrombosis is most frequent, but thrombosis of the inferior vena cava, hepatic, portal, renal, pulmonary, and sagital veins have all been described. In the arterial circulation, presentation with stroke is most frequent, but thrombosis of mesenteric, coronary, brachial, digital, and ocular arteries have all been reported. Hence, in addition to deep-vein thrombosis or stroke other presentations may include gangrene of the fingers, bowel infarction, loss of vision in an eye, myocardial infarction, and so on. Some patients have pulmonary hypertension, either secondary to pulmonary vascular thrombosis or multiple pulmonary emboli.

In most instances, thrombosis is episodic, but antibody levels are persistently present—suggesting that thrombosis is triggered by a site-specific vascular event in the presence of aPL antibodies. Some patients present with an aggressive thrombotic diathesis, where multiple sites are affected either simultaneously or over a period of weeks. This presentation has come to be known as the catastrophic antiphospholipid syndrome (CAPS).34 Occlusion of small vessels appear to be most frequent, leading to ischemia of multiple organs such as the kidneys, bowels, lungs, heart, adrenal glands, and skin. This presentation is very similar to TTP or to disseminated intravascular coagulation (DIC).1 The presence of a moderate to high positive aCL test and/or an unequivocally positive LA test would help clinch the diagnosis of CAPS. Pregnancy loss is a frequent occurrence in women with the APS.35 Although the earlier literature emphasized losses in the second and third trimester, this complication can occur at any time during pregnancy. In many but not all instances, placental infarction secondary to thrombosis of placental vessels is present. Some studies suggest that placental vascular thrombosis occurs because aPL antibodies displace annexin A5, a phospholipid binding protein in the placental trophoblast.36 This results in “exposure” of the negatively charged phospholipid template of the placental vasculature, which serves as a catalytic surface for coagulation protein interactions, resulting in thrombosis. There are a number of other clinical features associated with APS not attributable to thrombosis. The occurrence of thrombocytopenia (and less frequently hemolytic anemia) has already been discussed. Cardiac valvular abnormalities are also frequently observed. These include valvular vegetations (Libman-Sachs endocarditis), valvular thickening, valvular stenosis, and regurgitation. Whereas abnormalities of any of the four valves have been described, mitral involvement is most frequent, followed by the aortic valve.37 These are a variety of non-thromobotic neurologic abnormalities described in APS. These include transverse myelopathy, chorea, Guillan-Barre syndrome, psychosis, and migraine headaches. Skin manifestations have also been described, including livedo reticularis and leg ulcers, often located in the pre-tibial or ankle areas. Most of the non-thrombotic manifestations of APS described prevously (such as thrombocytopenia, hemolytic anemia, valvular abnormalities, neurologic, and skin manifestations) can occur in SLE in the absence of aCL or LA positivity. If these non-thrombotic complications are explained by autoimmunity, it suggests that APS and SLE share common autoantibodies that are not limited to aPL antibodies.

The diagnosis of APS is confirmed by finding a moderate to high positive aCL test or a positive LA test.28,29 ACL test results are reported according to isotype, IgG, IgM, or IgA.2 This test can be positive in a host of other autoimmune-, infectious-, or drug-induced disorders without clinical manifestations of APS being present. Both isotype and level of positivity are important in distinguishing APS from other disorders. In general, an IgG aCL test at medium to high levels is most specific for the diagnosis,6 but it should be borne in mind that patients with APS may be only IgM isotype positive and rarely only IgA isotype positive. APL antibodies often bind β2GPI, and when β2GPI is “presented” on oxidized polystyrene plates the antiβ2GPI test is positive in about 70 to 80% of patients with positive aCL tests.32 A few patients with APS may be positive for anti-β2GPI and negative for aCL antibodies.38 The anti-β2GPI test is more specific for APS than is the aCL test, but it is less sensitive. Another modification of the aCL test in which a defined mixture of negatively charged phospholipids have been used in place of cardiolipin has also yielded a test (the APhL ELISA) that is more specific and nearly as sensitive as the aCL test for diagnosis of APS.2 Hence, in patients where the aCL test is equivocal, the anti-β2GPI and/or the APhL ELISA may be used to confirm the diagnosis. The LA is the second test used for diagnosis of APS.3 It identifies a subset of autoantibodies in APS that functionally disrupt prothrombin-thrombin conversion and thus delay clot formation. It is recognized by first finding a prolonged partial thromboplastin time (PTT) or Russell viper venom time (RVVT). However, such prolongation can also result from a clotting factor deficiency or presence of other anticoagulant proteins. Confirmation of the LA is made by mixing patient plasma with normal plasma. The test remains prolonged if the LA is present, but becomes normal if prolongation is due to a clotting factor deficiency. A second confirmatory test is required to distinguish the LA from other naturally occurring anticoagulants. A phospholipid mixture is added to the patient plasma, and if the test normalizes the LA is present (it will remain prolonged with other anticoagulants). The LA is not reliable in patients receiving anticoagulant therapy or in those with other coagulation abnormalities.

Pathogenic Effects of Antiphospholipid Antibodies There is strong evidence that aPL antibodies are pathogenic in vivo from studies that utilized animal models of thrombosis, endothelial cell activation, and pregnancy loss.39-42 However, the mechanisms by which aPL antibodies mediate disease are only partially

understood and the understanding is limited by the apparent polyspecificity of the antibodies, the multiple potential end-organ targets, and the variability of clinical context that disease may present. APL antibodies are heterogeneous and it is known that more than one mechanism may be involved in causing thrombosis.43 In fact, in vitro studies have reported that aPL may cause thrombosis by interfering with activation of protein C (or inactivation of factor V by activated protein C) by inhibiting endothelial prostacyclin production, by impairment of fibrinolysis, by activating endothelial cells, and by exerting stimulatory effects on platelet function.16,17,44-47 There is now also convincing evidence that activation of complement mediates aPL-induced fetal loss, thrombosis, and EC activation (discussed later in a separate section).48-52

Treatment Treatment of patients with venous or arterial thrombosis is directed at preventing recurrent events, to which APS patients are prone. Most authorities favor prolonged use of warfarin, but there is controversy about the target INR (international normalized ratio). Earlier studies suggested that a target INR of greater than 3.0 was important in preventing recurrence of thrombosis.53 Recently, investigators have shown that lower target INRs (2.5 to 3.0) may be just as effective and the risk of hemorrhage reduced.54-56 One study of patients with stroke and positive aCL antibody tests suggested that warfarin was no more effective than a daily aspirin in preventing recurrent stroke. This study should be viewed with great caution, in that many of the patients did not fulfill the criteria for APS.57 If patients have recurrent arterial or venous thrombosis despite an INR of 2.5 to 3.0, increasing the warfarin to achieve a target INR of 3.0 to 3.5 is recommended. Anecdotal information suggests that lowmolecular-weight heparin prophylaxis may be effective in patients with recurrence despite warfarin therapy. Treatment of CAPS is based largely on anecdotal data. Use of plasmapheresis, IVIG, and immunosuppressive therapy (high-dose corticosteroids and/or cyclophosphamide)—singly or in combination—have proven successful in some but not all affected patients.34 There is uncertainty about management of patients with persistent high positive aCL or positive LA tests who have never had thrombosis. A study comparing low-dose warfarin versus aspirin is ongoing, but until that study is complete authorities favor the use of one aspirin daily (see Chapter 47). In patients with a history of pregnancy loss attributable to APS, use of heparin (5,000 to 10,000 units bid if no history of thrombosis and 15,000 units bid if a history of thrombosis) plus a low-dose aspirin daily is favored by most authorities.35 If pregnancy loss occurs

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Laboratory Tests

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HEMATOLOGIC AND COAGULATION ABNORMALITIES OF SLE AND THE ANTIPHOSPHOLIPID SYNDROME

412

despite heparin and aspirin therapy, use of intensive intravenous immunoglobulin (IVIG) therapy has been reported to be successful in some studies58 (disputed in others59). See Chapter 47. As indicated, the treatment of the APS can be directed toward preventing thromboembolic events by using antithrombotic medications or by modulating the immune response with immunotherapy. In the case of thrombotic manifestations, both approaches have been used with considerable side effects. In patients with a history of thrombosis, there is a high risk of recurrence and oral anticoagulation at a relative INR is frequently used for a long period of time. Furthermore, there is a need for frequent monitoring and patient compliance with diet and lifestyle to minimize the risk of thrombosis recurrence. Hence, there is a need for new, more efficient, and less harmful modalities of treatment for APS. In the last few years, significant knowledge has been gained on the effects of aPL antibodies on platelets and endothelial cells and the molecular events these antibodies trigger. The data strongly suggest activation of p38 MAPK in platelets and endothelial cells by antiphospholipid antibodies, which may justify the use of specific inhibitors as a new approach to the treatment and prevention of thrombosis.20,60 Most importantly,

data indicate that statins (currently used to treat hypercholesterolemia) and the antimalarial drug hydroxcychloroquine are also good candidates for the treatment and prevention of thrombosis in APS.19,61-63 Furthermore, antagonists of GPIIb/IIIa may be useful, particularly in treatment of acute thrombotic events. Recent findings provide convincing evidence that activation of complement contributes to aPL-mediated thrombosis and pregnancy loss, and suggest that specific inhibitors of complement activation may be used to ameliorate clinical manifestations of APS.48-52 Management of thrombocytopenia or hemolytic anemia in APS has been discussed earlier in this chapter. Vascular complications in SLE are not solely attributable to coexistence of the APS. Venous thrombosis is more frequent in patients with nephrotic syndrome. Stroke and myocardial infraction also occur with increased frequency in SLE, perhaps related to prolonged corticosteroid usage resulting in atherosclerosis. Alternatively, immune complex-mediated vasculitis may cause neurologic, myocardial, and other organ damage not unlike that seen in APS. Distinction between these conditions is vitally important because short- and long-term treatment and prognosis will be different depending on the cause.

REFERENCES 1. Harris, EN, Khamashta MA. Antiphospholipid sydrome. In Hochberg, Silman, Smolen, Weinblatt, Weisman (eds.), Rheumatology, Forth Edition. London: Elsevier Limited (in press). 2. Pierangeli SS, Harris EN. Clinical laboratory testing for the antiphospholipid syndrome. Clin Chim Acta 2005;357:17-33. 3. Brandt JT, Triplett DA, Alving B, et al. Criteria for diagnosis of lupus anticoagulant: An update on behalf of the subcommittee on Lupus Anticoagulant/Antiphospholipid antibody of the scientific and standardization committee of the ISTH. Thromb Haemost 1995;74:1185-1195. 4. Vaulgarelis M, Kokori SI, Ioanndis JP, et al. Anaemia in systemic lupus erythematosus: Aetological profile and the role of erythropoietin. Ann Rheum Dis 2000;59:217-222. 5. Sultan SM, Begum S, Isenberg DA. Prevalence, patterns of disease and outcome in patients with systemic lupus erythematosus who develop severe hematologic problems. Rheumatology 2003;42:230-234. 6. Harris EN, Gharavi AE, Chan JKH, et al. Thrombosis recurrent fetal loss and thrombocytopenia. Arch Intern Med 1986;146:21532156. 7. Deleze M, Alarcon-Segovia D, Oria CV, et al. Hemocytopenia in systemic lupus erythematosus: Relationship to antiphospholipid antibodies. J Rheumatol 1989;16:926-930. 8. Sthoeger Z, Sthoeger D, Green L, Geltner D. The role of anticardiolipin auto-antibodies in the pathogenesis of autoimmune haemolytic anemia in systemic lupus erythematosus. J Rheumatol 1993;20:2058-2061. 9. Lang B, Straub RH, Weber S et al. Elevated anticardiolipin antibodies in autoimmune haemolytic anemia irrespective of underlying systemic lupus erythematosus. Lupus 1997;6: 652-655. 10. Avina-Zubieta JA, Galindo-Rodriquez G, Robledo I, et al. Long term effectiveness of danazol, carticosteriods and cytotoxic drugs in the treatment of hematologic manifestations of systemic lupus erythematosus. Lupus 2003;12:52-57.

11. Alba P, Karim MY, Hunt BJ. Mycophenolate mofetil as a treatment for autoimmune haemolytic anemia inpatients with systemic lupus erythematosus and antiphospholipid syndrome. Lupus 2003;12:633-635. 12. Habib GS, Saliba WR, Froom P. Pure red cell aplasia and lupus. Semin Arthritis Rheum 2002;31:279-283. 13. Front J, Cervera R, Ramos-Casals M, et al. Clusters of clinical and immunologic features in systemic lupus erythematosus: Analysis of 600 patients from a single centre. Semin Arthritis Rheum 2004;33:217-230. 14. Hasselaar P, Derksen RhWM, Blokzijl L, DeGroot PG. Crossreactivity of antibodies directed against cardiolipin, DNA, endothelial cells and blood platelets. Thromb Haemost 1990;63:169-173. 15. Hakim AJ, Machin SJ, Isenberg DA. Autoimmune thrombocytopenia in primary antipospholipid syndrome and systemic lupus erythematosus: The response to splenectomy. Semin Arthritis Rheum 1993;28:20-35. 16. Khamashta MA, Harris EN, Gharavi AE, et al. Immune mediated mechanism for thrombosis: Antiphospholipid antibody binding to platelet membranes. Ann Rheum Dis 1988;47:849-854. 17. Campbell AL, Pierangeli SS, Wellhausen S, Harris EN. Comparison of the effect of anticardiolipin antibodies from patients with the antiphospholipid syndrome and with syphilis on platelet activation and aggregation. Thromb Haemost 1995;73:529-534. 18. Lin YL, Wang CT. Activation of human platelets by the rabbit anticardiolipin antibodies. Blood 1992;80:3135-3143. 19. Espinola RG, Pierangeli SS, Gharavi AE, Harris EN. Hydroxychloroquine reverses platelet activation induced by human IgG antiphospholipid antibodies. Thromb Haemost 2002;87:518-522. 20. Vega-Ostertag ME, Harris EN, Pierangeli SS. Intracellular events in platelet activation induced by antiphospholipid antibodies in the presence of low doses of thrombin. Arthritis Rheum 2004;50:2911-2919.

43. Roubey RAS. Mechanisms of autoantibody-mediated thrombosis. Lupus1998;7:S114-S119. 44. Esmon NL, Safa O, Smirnov M, Esmon CT. Antiphospholipid antibodies and the protein C pathway. J Autoimmun 2000;15: 221-225. 45. Arvieux J, Rousell B, Pouzol P, Colomb MG. Platelet activating properties of murine monoclonal antibodies to β2glycoprotein I. Thromb Haemost 1993;70:336-341. 46. Forastiero R, Martinuzzo M, Carreras LO, Maclouf J. Anti-β2 glycoprotein I antibodies and platelet activation in patients with antiphospholipid antibodies: Association with increased excretion of platelet-derived thromboxane urinary metabolites. Thromb Haemost 1998;79:42-45. 47. Martinuzzo ME, Maclouf J, Carreras LO, Levy-Toledano S. Antiphospholipid antibodies enhance thrombin-induced platelet activation and thromboxane formation. Thromb Haemost 1993;70:667-671. 48. Holers VM, Girardi G, Mo L, Guthridge JM, et al. C3 activation is required for anti-phospholipid antibody-induced fetal loss. J Exp Med 2002;195(2):211-220. 49. Salmon JE, Girardi G, Holers VM. Complement activation as a mediator of antiphospholipid antibody induced pregnancy loss and thrombosis. Ann Rheum Dis 2002;61:46-50. 50. Girardi G, Berman J, Redecha P, et al. Complement C5a receptors and neutrophils mediate fetal injury in the antiphophospholipid syndrome. J Clin Invest 2003;112(11):1644-1654. 51. Girardi G, Redecha P, Salmon J. Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nature Medicine 2004;10:1222. 52. Pierangeli SS, Girardi G, Vega-Ostertag ME, et al. Requirement of activation of complement C3 and C5 for antiphospholipid antibody-mediated thrombophilia. Arthritis Rheum 52: 2120-2124. 53. Khamashta MA, Cuadrado MJ, Mujic F, et al. The management of thrombosis in the antiphospholipid Syndrome. N Engl J Med 1995;332:993-997. 54. Krnic-Barrie S, O’Connor CR, Looney SW, et al. A retrospective review of 61 patients with antiphospholipid syndrome. Arch Intern Med 1997;157:2101-2108. 55. Crowther MA, Ginsberg JS, Julian J, et al. A comparison of two intensities of Warfarin for the prevention of recurrent thrombosis in patients with the Antiphospholipid Antibody Syndrome. N Eng J Med 1003;249:1133-1138. 56. Finazzi G, Marchioli R, Brancaccio V, et al. A randomized clinical trial of high intensity warfarin vs conventional anti-thrombotic therapy for the prevention of recurrent thrombosis in patients with the antiphospholipid syndrome (WAPS). J Thromb Haemost 2005;3:848-853. 57. Levine SR, Brey RL, Tilley BC, et al. Antiphospholipid antibodies and subsequent thrombo-occlusive events in patients with ischemic stroke. JAMA 2004;291:576-584. 58. Clark AL, Branch DW, Silver RM, et al. Pregnancy complicated by the antiphospholipid syndrome: Outcomes with intravenous immunoglobulin therapy. Obstet Gynecol 1999;93:437-441. 59. Branch DW, Peaceman AM, Druzin M, et al. A multicenter, placebo-controlled pilot study of intravenous immune globulin treatment of antiphospholipid syndrome during pregnancy: The Pregnancy Loss Study group. Am J Obstet Gynecol 2000;182:122-127. 60. Vega-Ostertag ME, Casper K, Swerlick R, et al. Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antbodies. Arthritis Rheum 2005;52:1545-1554. 61. Ferrara DE, Swerlick R, Casper K, et al. Fluvastatin inhibbitrs up-regulation of tissue factor expression by antiphospholipid antibodies on endothelial cells. J Thromb Haemost 2004;2: 1558-1563. 62. Ferrara DE, Kiu X, Espinola RG, et al. Inhibition of the thrombogenic and inflammatory properties of antiphospholipid antibodies by fluvastatin in an in vivo animal model. Arthritis Rheum 2003;48:3272-3279. 63. Pierangeli SS, Vega-Ostertag ME, Harris EN. Intracellular signaling triggered by antiphospholipid antibodies in platelets and endothelial cells: a pathway to targeted therapies. Thromb Res 2004;114:467-476.

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21. Lutters BCH, Derksen RHWM. Tekelenburg WL, Lenting PJ, Arnout J. de Groot PH. Dimers of β2glycoprotein I increase platelet deposition to collagen via interaction with phospholipids and the apolipoprotein E receptor 2’. J Biol Chem 2003; 278:33831-33838. 22. Vasoo S, Thumboo J, Fong KY. Refractory immune thrombocytopenia in systemic lupus erythematosus response to mycophenolate mofetil. Lupus 2003;12:630-632. 23. Conley CL, Hartmann RC. A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest 1952;12:621-622. 24. Bowie WEF, Thompson JH, Pascuzzi CA, Owen GA. Thrombosis in systemic lupus erythematosus despite circulating anticoagulants. J Clin Invest 1963;62:416-430. 25. Carreras LO, Defreyn G, Machin SJ, et al. Arterial thrombosis, intrauterine death and lupus anticoagulant: Detection of immunoglobulin interfering with prostacyclin formation. Lancet 1981;I:244-246. 26. Pengo V, Thiagarajan P, Shapiro SS, Heine MJ. Immunological specificity and mechanism of action of IgG lupus anticoagulants. Blood 1987;70:69-76. 27. Harris EN, Gharavi AE, Boey ML, et al. Anticardiolipin antibody: Detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;II:1211-1214. 28. Harris EN. Syndrome of the Black Swan. Br J Rheumatol 1987;26:324-327. 29. Wilson WA, Gharavi AE, Koike T, et al. International consensus statement on preliminary classification for definite antiphospholipid syndrome. Arthritis Rheum 1999;42:1309-1311. 30. Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;335:1544-1547. 31. McNeil HP, Simpson RJ, Chesterman CN, Kriks SA. Anti-phospholipid antibodies are directed against a complex antigen which includes a lipid-binding inhibitor of coagulation: β2 glycoprotein 1 (apolipoproteinH). Proc Natl Acad Sci USA 1990;87: 4120-4124. 32. Matsuura E, Igarashi Y, Yasuda T et al. Anticardiolipin antibodies recognize β2-glycoprotein 1 structure altered by interacting with an oxygen and modified solid phase surface. J Exp Med 1994;179;457-462. 33. 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. 34. Asherson RA, Cervera R, Piette JC, et al. Catastropic antiphospholipid syndrome: Clinical and laboratory features of 50 patients. Medicine 1998;77:195-207. 35. Branch DW, Khamashta MA. Antiphospholipid syndrome: Obstetric diagnosis, management and controversies. Obstet Gynecol 2003;101:1333-1344. 36. Rand JH, Wu S, Andree HAM, et al. Pregnancy loss in the antiphospholipid syndrome: A possible thrombogenic mechanism. N Engl J Med 1997;337:154-160. 37. Nesher G, Ilani J, Rosenman D, Abraham AS. Valvular dysfunction in antiphospholipid syndrome: Prevalence, clinical features and treatment. Semin Arthritis Rheum 1997;27:27-35. 38. Nash MJ, Camilleri RS, Kunka S, Mackie IJ, et al. The anticardiolipin assay is required for sensitive screening for antiphospholipid antibodies. J Thromb Haemost 2004;2:1077-1081. 39. Branch DW, Dudley DJ, Mitchell MD, et al. 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. 40. Pierangeli SS, Liu XW, Anderson GH, et al. Induction of thrombosis in a mouse model by IgG, IgM and IgA immunoglobulins from patients with the antiphospholipid syndrome. Thromb Haemost 1995;74:1361-1367. 41. Pierangeli SS, Colden-Stanfield M, Liu X, et al. Antiphospholipid antibodies from antiphospholipid syndrome patients activate endothelial cells in vitro and in vivo. Circulation 1999;99: 1997-2000. 42. Jankowski M, Vreys I. Wittevrongel C, et al. Thrombogenicity of β2-glycoprotein I-dependent antiphospholipid antibodies in a photochemically induced thrombosis model in the hamster. Blood 2003;101:157-162.

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37

The Nervous System in Systemic Lupus Erythematosus John G. Hanly, MD

INTRODUCTION Although neurological (N) and psychiatric (P) events are frequently encountered in patients with systemic lupus erythematosus (SLE), they remain one of the more perplexing aspects of the disease and pose diagnostic and therapeutic challenges for clinicians. The difficulties are due in part to the lack of specificity of the majority of NP manifestations, uncertainty regarding the pathogenic mechanisms, and a paucity of controlled data to support therapeutic strategies.

CLASSIFICATION OF NP-SLE

414

The ACR classification criteria for SLE include NP manifestations, seizures, and psychosis. However, it is widely acknowledged that a much broader range of NP disease manifestations occur in SLE patients. With a preference for affecting the central nervous system, individual NP events may reflect either a diffuse disease process (e.g., psychosis and depression) or focal process (e.g., stroke and transverse myelitis), depending on the anatomic location of pathology. Several classifications have been developed for NP-SLE,1-3 but until recently most have lacked definitions for individual manifestations and the approach to investigation and diagnosis has been inconsistent. In 1999, the ACR research committee produced a standard nomenclature and diagnostic criteria for 19 NP syndromes known to occur in SLE patients4 (Table 37.1). For each of the 19 NP syndromes, potential etiologies other than SLE are identified either for exclusion or recognition as an “association” (acknowledging that in some clinical presentations definitive attribution is not possible). The identification of other non-lupus causes for NP events in SLE patients is of critical importance and has not been adequately addressed in previous classification systems. Guidelines for reporting NP events were also developed by the ACR research committee, and specific diagnostic tests were recommended for each syndrome. Although these criteria were developed primarily to facilitate research

studies of NP-SLE, they also provide a practical guide to the assessment of individual SLE patients with NP disease.

EPIDEMIOLOGY OF NP-SLE In a representative selection of studies utilizing the ACR nomenclature and definitions,5-9 the overall prevalence of NP disease has varied between 37 and 95%. The most common of the 19 NP syndromes in each of these five SLE cohorts were cognitive dysfunction (55 to 80%), headache (24 to 72%), mood disorder (14 to 57%), cerebrovascular disease (5 to 18%), seizures (6 to 51%), polyneuropathy (3 to 28%), anxiety (7 to 24%), and psychosis (0 to 8%). Most of the

TABLE 37.1 NEUROPSYCHIATRIC SYNDROMES IN SLE AS DEFINED BY THE ACR NOMENCLATURE Central Nervous System

Peripheral Nervous System

●

Aseptic meningitis

Guillain Barré syndrome

●

Cerebrovascular disease

Autonomic neuropathy

●

Demyelinating syndrome

Mononeuropathy

●

Headache

Myasthenia gravis

●

Movement disorder

Cranial neuropathy

●

Myelopathy

Plexopathy

●

Seizure disorders

Polyneuropathy

●

Acute confusional state

●

Anxiety disorder

●

Cognitive dysfunction

●

Mood disorder

●

Psychosis

From The American College of Rheumatology nomenclature and case definitions for neuropsychiatric lupus syndromes. Arthritis Rheum 1999;42(4):599–608, with permission.

PATHOGENESIS OF NP-SLE Although the diversity of NP manifestations reported in SLE patients makes it unlikely that there is a single pathogenic mechanism, there are at least three primary immunopathogenic mechanisms implicated in NPSLE: vasculopathy of predominantly small intracranial blood vessels, autoantibody production, and the generation of inflammatory mediators (Fig. 37.1).

Primary NP-SLE +++

Vasculopathy

+

Autoantibodies Antineuronal

–

+

Antiribosomal

–

++

Antiphospholipid

+++

+

Inflammatory mediators

+

++

Focal NP disease

Diffuse NP disease

Secondary NP-SLE Complications of SLE (e.g. uremia, hypertension) Complications of SLE therapy (e.g. steroids, infection)

Vasculopathy In a limited number of neuropathological studies,10-13 the predominant finding was a bland noninflammatory vasculopathy. In contrast, inflammatory disease of small or large vessels was rare. Brain microinfarcts occurred in close anatomical proximity to the microangiopathy.10 Although instructive, the majority of neuropathologic studies in SLE have significant limitations due to a bias in patient selection, a temporal disconnect between the NP event and tissue sampling, and the potential impact of confounding factors such as infection, hypertension, and corticosteroids on neuropathology.

PATHOGENESIS OF NP-SLE

other NP syndromes were infrequent, with a prevalence of less than 1% in most studies, emphasizing the rarity of many of these entities. The attribution of individual NP events to SLE per se or to an alternative etiology remains a challenge. In the absence of a diagnostic gold standard for most of the NP-SLE syndromes, attribution is determined case by case on the basis of exclusion using the best available clinical, laboratory, and imaging data. The ACR NPSLE classification4 provides a basis for addressing this issue in a systematic manner because for each NP syndrome there is a comprehensive list of exclusions and associations, the presence of which may indicate an etiology other than SLE. Utilizing this approach and taking into consideration the temporal relationship between the NP event and the diagnosis of SLE, a recent study has reported that up to 41% of all NP events in SLE patients may be attributed to factors other than lupus.7 This finding is in keeping with data from a Finnish study5 that concluded that five NP syndromes (headache, anxiety, mild depression, mild cognitive impairment, and polyneuropathy, without electrophysiologic confirmation), should not be considered primary manifestations of the disease.

Autoantibodies A humoral immune response directed against neuronal antigens, ribosomes, and phospholipid-associated proteins has been implicated in the pathogenesis of NP-SLE. The data from human studies on antineuronal antibodies is largely circumstantial. This includes the temporal relationship between clinical events and serologic findings,14 the presence of autoantibodies in the cerebrospinal fluid,15 and to a very limited extent their identification in neuronal tissues from patients succumbing to the disease.16 Autoantibodies gain access to the CSF of SLE patients by means of passive transfer from the circulation through a permeabilized blood/brain barrier17,18 and independently by direct intrathecal production.14,17 The fine specificity of antineuronal antibodies has been studied extensively but in general has not resulted in greater diagnostic specificity. Most recently, attention has been focused on anti-NR2 glutamate receptor antibodies as a potentially novel system that could Fig. 37.1 Factors contributing to the pathogenesis of neuropsychiatric (NP) disease in SLE. (Modified from Hanly JG. Neuropsychiatric lupus. Current Rheumatology Reports 2001;3:205-212.)

Concurrent non-SLE NP disease

415

THE NERVOUS SYSTEM IN SYSTEMIC LUPUS ERYTHEMATOSUS

explain some of the complexities of NP-SLE. Although of considerable interest, the findings to date are largely derived from animal studies and require confirmation in human subjects with NP-SLE. The limited studies in human lupus examining the association between this subset of antineuronal antibodies and cognitive impairment have yielded conflicting results.19,20 Anti-ribosomal P (anti-P) antibodies were first described in SLE patients in 1985 and are quite specific for SLE, with a prevalence of 13 to 20% depending on ethnic group.21 In 1987, these autoantibodies were first linked to NP-SLE, in particular psychosis.22 Subsequent work either supported, refuted, or extended this initial observation to include depression.21,23-25 Potential explanations for the differences in study outcomes include variability in diagnostic criteria for psychiatric disease, variance in the temporal relationship between clinical events and serologic testing, and differences in assay technique (particularly antigen preparation and purity). Autoimmune antiphospholipid antibodies, which are directed against phospholipid-binding proteins such as β2-glycoprotein I and prothrombin,26 are associated with predominately focal manifestations of NP-SLE. The most common neurologic disorders are those of vascular origin (such as transient cerebral ischemia or stroke), but other associations include seizures, chorea, transverse myelitis, and cognitive dysfunction.8 In a review of more than 1000 SLE patients, NP manifestations occurred in 38% of patients with lupus anticoagulant compared to 21% of patients without these antiphospholipid antibodies.27 In a study of 118 SLE patients, 33% of whom were positive for the LA,28 there was a significantly greater proportion of cognitive impairment in LA-positive (50%) compared to LAnegative (25%) patients. The favored pathogenic mechanism for this subset of autoantibodies in NP-SLE is thrombosis within vessels of different caliber and subsequent downstream cerebral ischemia. A procoagulant state may be induced through acquired resistance to protein C and protein S, platelet aggregation, and direct activation of endothelial cells.26 However, the intrathecal production of antiphospholipid antibodies in patients with NP-SLE,17 their association with diffuse cognitive impairment,29,30 and in vitro evidence indicating modulation of neuronal cell function31 raise the possibility of an alternative pathogenic mechanism.

Inflammatory Mediators

416

Studies from Japan were the first to report an association between enhanced intracranial production of interleukin (IL)-6 with seizures32 and interferon-alpha with lupus psychosis.33 Subsequent studies have provided further evidence for the intrathecal production

of IL-634-37 and have identified other potential candidate cytokines such as IL-10,37,38 IL-2,39 and IL-8.36 The sources of intrathecal production of these cytokines include neuronal33,35 and glial cells.33 The stimulus for and regulation of this enhanced cytokine response remain to be determined. Although potentially an epiphenomenon, it could be a consequence of cell activation mediated by autoantibodies within the intrathecal space. However, measuring CSF cytokine levels unselectively in patients with any manifestation of NP-SLE is unlikely to be of diagnostic value in individual cases.40 Other potentially important inflammatory mediators are matrix metalloproteinases (MMPs), a family of endoperoxidases that can degrade extracellular matrix components.41 MMP-9 is a gelatinase and is secreted by a variety of cells (including macrophages, T lymphocytes, and endothelial and smooth muscle cells) in the blood vessel wall.42 Implicated in the pathogenesis of plaque rupture,43 elevated levels have also been associated with other conditions [including multiple sclerosis (MS),44 Guillain-Barré syndrome,45 rheumatoid arthritis,46 and SLE].47 A recent study48 has examined the association between circulating levels of MMP-9 and NP-SLE. Although there was no difference in the levels of MMP-9 between SLE patients and healthy population controls, elevated levels of MMP-9 were associated with NP-SLE and in particular with cognitive impairment. It is of interest that increased expression of MMP-9 is found in the disrupted blood/brain barrier following cerebral ischemia and may facilitate lymphocyte migration into and possibly through the arterial wall.49 Elevated MMP-9 levels have also been detected in CSF samples of patients with NP-SLE compared to SLE patients without NP manifestations and normal controls.50 Furthermore, the positive correlation among CSF MMP-9 levels, proinflammatory cytokines, and biomarkers of neuronal and glial degradation50 supports the suggestion that the enhanced production of MMP-9 is under cytokine control and is responsible for central nervous system damage.

CLINICAL MANIFESTATIONS OF NP-SLE

Headache The association between SLE and headaches, including migraine, is controversial. The reported prevalence of headache has varied widely (between 24 and 72%5-9), but the prevalence of headache in the general population is also high (with up to 40% of individuals reporting a severe headache at least once per year).51 Two of the most recent studies52,53 found no increase in the prevalence of headache in SLE, which was also the conclusion of a recent metanalysis.54 Furthermore, there is

Psychosis, Mood Disorders, and Anxiety Psychosis is reported in up to 8% of SLE patients5-9,61 and is characterized by either the presence of delusions (false belief despite evidence to the contrary) and/or hallucinations (perceptual experiences occurring in the absence of external stimuli). The latter are most frequently auditory. Psychosis is a rare but dramatic manifestation of NP-SLE and when present it must be distinguished from other causes, including high doses of corticosteroids, non-prescribed drug abuse, schizophrenia, and depression. Depression and anxiety are common symptoms in lupus and occur in 24 to 57% of patients.5-9,25 However, as there are no features of these syndromes that are unique to SLE patients there is often uncertainty about the etiology and attribution in individual cases. The association between psychosis depression and anti-P antibodies in SLE is supported by some but not all studies.21,22,24,25

Cerebrovascular Disease The many forms of cerebrovascular disease are reported in 5 to 18% of SLE patients5-9 and are likely multifactorial in etiology. Accelerated atherosclerosis is well recognized in SLE, particularly in relation to coronary heart disease (which is 5 to 10 times more frequent in SLE patients compared to control populations).62 This also contributes to the increased rate of cerebrovascular events in SLE. An additional etiologic factor is the prothrombotic state as a consequence of antiphospholipid antibodies,26 which provides a rationale for therapeutic intervention with anticoagulants in selected cases.

Seizures Generalized and focal seizures are reported in 6 to 51%5-9 of SLE patients and may occur either in the setting of active generalized multisystem lupus or as isolated neurologic events. Their occurrence is frequently associated with the presence of antiphospholipid antibodies,8 which co-occur with microangiopathy, arterial thrombosis, and subsequent cerebral infarction.

Demyelination, Transverse Myelopathy, and Chorea These are rare manifestations of central nervous system disease in SLE and occur no more frequently than in 1 to 3% of patients.5-9,63 Clinical and neuroimaging evidence of demyelination has been described and is frequently indistinguishable from multiple sclerosis.64 Thus, this particular syndrome may represent a concordance or overlap of two autoimmune conditions. Transverse myelopathy65 and chorea66 present acutely and are frequently associated with antiphospholipid antibodies.65,66 Although an arterial thrombotic event is a likely contributory mechanism for transverse myelopathy, the cause of chorea is less clear and there has been speculation that it may be a consequence of a direct interaction of antiphospholipid antibodies with neuronal structures in the basal ganglia.67

CLINICAL MANIFESTATIONS OF NP-SLE

only one study55 reporting an association between headache and other clinical features of active lupus. Aseptic meningitis is a relatively uncommon but well documented cause of headache in SLE56,57 and requires confirmation by analysis of CSF. Other potential causes must be considered, including infection and idiosyncratic reactions to medications such as antibiotics and nonsteroidal anti-inflammatory drugs.58-60 Thus, although headache may be a component of active SLE in rare individual patients it is more likely that the majority of headaches in SLE patients are due to non-SLE causes.

Neuropathy and Myasthenia Gravis A sensorimotor neuropathy is the most common neuropathy and has been reported in up to 28%5-9 of SLE patients. It frequently occurs independently of other disease characteristics.68 The abnormalities are persistent, but in one study 67% of patients had no change in their neuropathy over a 7-year period.68 Other less frequent forms of neuropathy include cranial neuropathy,7 autonomic neuropathy,69,70 plexopathy,71 mononeuritis multplex,72 and Guillain-Barré syndrome.73,74 Myasthenia gravis has been reported in SLE but is rare.75,76

Acute Confusional State This term has replaced what was previously called “organic brain syndrome” and is synonymous with “encephalopathy” and “delirium.” It encompasses a state of impaired consciousness or level of arousal that can progress to coma. Characteristics include reduced ability to focus, disturbed mood, and impaired cognition. It has been reported in 4 to 7% of SLE patients5,7,8 and must be distinguished from other causes, including metabolic abnormalities and hypertensive encephalopathy.

Cognition and Cognitive Dysfunction Cognition is the sum of intellectual functions that result in thought. It includes reception of external stimuli, information processing, learning, storage, and expression. Disturbance of even one of these functions can result in disruption of normal thought production and present as cognitive dysfunction.

Assessment of Cognitive Function in SLE Patients No simple screening test for cognitive dysfunction in SLE patients is currently available for use in the

417

THE NERVOUS SYSTEM IN SYSTEMIC LUPUS ERYTHEMATOSUS

418

clinical setting. Subjective reports of cognitive dysfunction lack reliability, and neuropsychologic test batteries are long and cumbersome and require expert administration and interpretation. Although easily administered, the Modified Mini Mental Status exam is not very sensitive for detecting mild (albeit clinically significant) cognitive dysfunction (especially problems with executive function, which are common in SLE patients). Self-report of cognitive difficulties is currently the only means by which clinicians can screen patients who may have significant cognitive dysfunction. The Cognitive Symptoms Inventory is a 21-item selfreport questionnaire designed for patients with rheumatic disease to assess self-perception of one’s ability to perform everyday activities.77 Although it has not been validated against neuropsychological testing, a recent report suggests that this instrument may be useful as a bedside screening tool to identify SLE patients at risk for cognitive impairment.78 However, formal testing of cognitive function is the only definitive way of diagnosing cognitive impairment and applicable patients should be referred to a neuropsychologist. Cognitive dysfunction, assessed using neuropsychological assessment techniques, has been reported in up to 80% of SLE patients,5 although most studies have found a prevalence between 17 and 66%.79,80 Many individual patients have subclinical deficits. For example, a review of 14 cross-sectional studies of cognitive function in SLE revealed subclinical cognitive impairment in 11 to 54% of patients.79 In non-SLE populations there are several potential causes of cognitive dysfunction, many of which may also be present in and exacerbated by SLE (Table 37.2). Whether they contribute to or even cause cognitive dysfunction in SLE patients requires individual consideration. However, the prevalence, severity, course, and impact of SLEassociated cognitive dysfunction are greater than that of both healthy controls81-83 and several non-CNS disease populations,83-85 strongly suggesting that SLEspecific events may play a significant role in the development of cognitive dysfunction. A single pattern of SLE-associated cognitive dysfunction has not been found, but commonly identified cognitive abnormalities include overall cognitive slowing, decreased attention, impaired working memory, and executive dysfunction (e.g., difficulty with multitasking, organization, and planning). As the majority of SLE patients with cognitive impairment have relatively mild deficits, the careful selection and assessment of cognitive performance in control groups is of critical importance in defining expected levels of function in healthy individuals and those with other chronic diseases. Although cognitive impairment may be viewed as a distinct subset of NP-SLE, it can also serve as a surrogate of overall

TABLE 37.2 NON-SLE CAUSES OF COGNITIVE DYSFUNCTION Causes

Examples

Direct CNS disease or injury

● ● ● ●

Systemic illness

● ● ● ●

Medication

● ● ● ●

Psychological or psychiatric disturbance

● ● ● ●

Metabolic disturbance

Ischemia Traumatic brain injury Cerebral hemorrhage Neurodegenerative disorders Hypertension Hyperthyroidism Hypothyroidism Fever Beta blockers Antihistamines Antidepressants Non-steroidal anti-inflammatory drugs Mania Depression Anxiety Psychosis

●

Hyper- or hypocalcemia Hyper- or hyponatremia Uremia Hypoxemia

Pain

●

Acute or chronic

Fatigue

●

Acute or chronic

Sleep disturbance

●

Fatigue/daytime somnolence Sleep apnea

● ● ●

●

Modified from Hanly JG, Harrison MJ. Management of neuropsychiatric lupus. Bailliere’s Best Practice & Research Clinical Rheumatology 2005;19(5):799-821.

brain health in SLE patients who may be affected by a variety of factors including other NP syndromes.

Clinical Associations with Cognitive Impairment The association between cognitive impairment in SLE and other clinical variables has been examined in a number of studies. It is intuitive that certain clinically overt NP-SLE events such as stroke and antiphospholipid antibody-associated multifocal infarction would likely be accompanied by cognitive dysfunction. Thus, it is not surprising that the prevalence of cognitive dysfunction in patients with past or current clinically overt NP-SLE is usually greater than in those with no such history.81,82,86,87 Mood and psychological distress, even in the absence of a frank psychiatric diagnosis, are known to influence cognitive functioning as well as alter performance on neuropsychologic tests. The high prevalence of

The Identification of Risk Factors for Cognitive Impairment Predictors of cognitive decline over time have also been examined. In one study,87 when patients who were cognitively impaired at the initial assessment were compared to those who were not impaired the differences between groups in tests of recent memory and delayed free recall decreased over five years. A similar result was reported by Waterloo and colleagues.94 However, patients who had clinically overt NP-SLE at any time in their disease course had a statistically significant decline in memory performance over five years when compared to patients without a history of clinically overt NP-SLE.87 These results suggest that the occurrence of clinically overt NP events, rather than the identification of isolated subclinical cognitive impairment, is a more reliable predictor of deterioration in selective aspects of memory function over time. The association between cognitive function and anticardiolipin (aCL) antibodies has been examined in a number of cross-sectional and prospective studies. For example, 51 SLE patients studied prospectively were divided into those who were persistently aCL antibody positive or negative on the basis of up to seven antibody determinations over a five-year period.30 The relative change in performance on individual neuropsychological tests was then compared between patients who were antibody positive and negative. Those who were persistently IgG aCL antibody positive demonstrated a greater reduction in psychomotor speed compared to those who were antibody negative. In contrast, patients who were persistently IgA aCL antibody positive had significantly poorer performance

The Evolution of Cognitive Impairment The outcome of cognitive impairment in SLE patients has been examined in a number of studies. For example, in a five-year prospective study of 70 SLE patients using a standardized panel of neuropsychological tests87 the prevalence of overall cognitive impairment in SLE patients fell from 21 to 13% over the period of study. Five patterns of cognitive performance were observed over the five-year period (Fig. 37.2). Eighty-three percent of patients were either never impaired or had resolution of cognitive impairment without specific therapeutic interventions. An additional 13% of patients demonstrated either an emerging or fluctuating pattern of impairment, and only 4% (2 patients) showed persisting deficits which were stable over time. Similar benign changes in cognitive performance over time have been reported by Waterloo and colleagues94 in 28 patients over five years,

Fig. 37.2 Change in cognitive function in 47 SLE patients assessed prospectively on three occasions over five years. [Derived from Hanly JG, Cassell K, Fisk JD. Cognitive function in systemic lupus erythematosus: Results of a 5-year prospective study. Arthritis Rheum 1997;40(8):1542–1543 (reference 87).]

CHANGE IN COGNITIVE FUNCTION IN SLE PATIENTS OVER 5 YEARS (n = 47) 80 70

CLINICAL MANIFESTATIONS OF NP-SLE

by Hay and colleagues95 in a two-year prospective study, and by Carlomagno and colleagues.96

psychological disturbance in SLE has led some to hypothesize that SLE-associated cognitive dysfunction is primarily due to the psychological impact of the underlying disease. Studies to date have both supported88 and refuted89 this hypothesis. Longitudinal data suggest that SLE patients with psychiatric involvement experience an improvement in cognition with improved psychiatric status at one-year follow-up that is not observed in patients with persistent psychiatric disorders.90 Most studies, using several methods to measure global SLE disease activity, have detected no association between the presence of cognitive impairment and disease activity.88,90,91 Furthermore, no association has been found between cognitive dysfunction and the use88,90,92,93 or dose of corticosteroid88,90,93 in patients with SLE.

64

% patients

60 50 40 30 19

20

9

10

4

4

Fluctuated

Persisted

0 Absent

Resolved

Emerged

419

THE NERVOUS SYSTEM IN SYSTEMIC LUPUS ERYTHEMATOSUS

Collectively, these data indicate that NP events in SLE patients, regardless of their etiology and attribution, have a negative impact on a patient’s quality of life. Although the overall clinical impact of NP-SLE may be detrimental, it is likely that individual NP manifestations differ in their prognostic implications. For example, the subtle cognitive deficits detected by formal neuropsychological testing have not been associated with a negative impact on health-related quality of life, at least as determined by self-report questionnaires.86,87 In another study,100 70% of patients with cognitive difficulties were able to maintain their work capacity and 86% had no change in their social functioning. Relatively few studies have examined the course of NP-SLE over time. Karassa and colleagues101 examined the prognosis of NP disease in 32 patients who had been hospitalized for NP-SLE and followed for two years. The outcome was generally favorable, with either substantial improvement (69%) or stabilization (19%) accounting for the majority of cases. A high number of prior NP events and the occurrence of the antiphospholipid syndrome were predictors of an unfavorable clinical outcome at two years. There is no consensus in the literature on the association between NP-SLE and mortality. Some studies report increased mortality102-106 in SLE patients with NP disease and others report no

in conceptual reasoning and executive ability. Similar results have been reported by Menon and colleagues in a two-year prospective study of 45 SLE patients.29 These data suggest that IgG and IgA aCL may be responsible for long-term subtle deterioration in cognitive function in SLE patients. No such association has not been found between cognitive function and other lupus autoantibodies such as antineuronal, lymphocytotoxic, anti-P, and anti-DNA antibodies.30,97,98

CLINICAL IMPACT AND PROGNOSIS OF NP-SLE The clinical impact of NP events in SLE has been determined by examining the association with a variety of clinical indicators, including quality of life. In a recent study,7 NP events were associated with significantly lower scores on most subscales of the SF-36 (a generic self-report measure of health-related quality of life) and with higher fatigue scores. Of interest, these associations were present regardless of the attribution of the NP event to SLE or an alternative etiology (Fig. 37.3), but did not occur in patients with a history of renal disease. Jonsen and colleagues99 also reported a higher frequency of disability in SLE patients with NP disease compared to patients without NP events and the general population.

ATTRIBUTION OF NP EVENTS AND QUALITY OF LIFE 100

*p<0.05, **p<0.01 *

SF-36 subscale score

80

**

*

* *

** *

60

40

20

0 Physical function

Social function

Role– physical

NP events (SLE)

420

Role– emotional

Mental health

NP events (non-SLE)

Bodily pain

Vitality

General health

No NP events

Fig. 37.3 Association between cumulative neuropsychiatric events in SLE patients and self-report health-related quality of life as reflected by the SF-36. [Derived from Hanly JG, McCurdy G, Fougere L, Douglas JA, Thompson K. Neuropsychiatric events in systemic lupus erythematosus: Attribution and clinical significance. J Rheumatol 2004;31(11):2156-2162 (reference 7).]

DIAGNOSTIC IMAGING AND NP-SLE When considering neuroimaging in NP-SLE, it is helpful to incorporate an assessment of both brain structure and brain function. Whereas computerized tomography (CT) scanning is the preferred technique for the diagnosis of acute intracranial hemorrhage, it has largely been replaced by magnetic resonance imaging (MRI) for the detection of other abnormalities due to MRI’s greater sensitivity.112 Abnormalities on MRI scanning may be found in 19113 to 70%114 of SLE patients. T2-weighted MRI imaging identifies pathologic processes that cause edema and is more sensitive than T1-weighted imaging for the detection of abnormalities in patients with NP-SLE. Applying the technique of fluid-attenuating inversion recovery (FLAIR) to dampen the CSF signal and highlight areas of edema further enhances the utility of T2-weighted images.64,115 Focal neurological disease is associated with predominately fixed lesions in the periventricular and subcortical white matter, usually in the territory of a major cerebral blood vessel.116 However, these multiple white matter lesions are quite nonspecific and are more commonly attributed to hypertension, disease duration, and age-related small-vessel disease rather than to the presence of NP-SLE.117-119 If the lesions are larger, occur in the corpus collosum, and are seen on T1-weighted images, the diagnosis of multiple sclerosis has to be considered.64 Diffuse NP clinical presentations are associated with transient subcortical white matter lesions and patchy hyperintesities in the gray matter, which are not usually confined to the territories of major cerebral blood flow.116 Other abnormalities detected on MRI scanning in SLE patients include cerebral infarction, venous sinus thrombosis, and increased signal in the spinal cord accompanying the clinical presentation of myelopathy.64 MRI also provides quantitative volumetric analysis of brain atrophy. The most objective neuroimaging study of brain function is positron emission tomography (PET) scanning, but practical considerations limit its applicability.2 Single-photon-emission computed tomography (SPECT) scanning2 provides semiquantitative analysis of regional cerebral blood flow and metabolism. It is exquisitely sensitive, and in studies of SLE patients120-124 SPECT imaging has identified both diffuse and focal deficits that may be fixed or reversible. However, the findings are not specific for SLE120 and do not always correlate with clinical NP manifestations.125 In fact, it is important to remember

that up to 50% of SLE patients without clinical manifestations of NP disease may have an abnormal SPECT scan.112 The significance of these imaging abnormalities is not always clear cut. The most common explanation is that they reflect a primary or secondary reduction in blood flow. However, in the brain there is sometimes disassociation between metabolism and blood flow. Changes in blood flow and metabolism can also occur in sites distant from those of the pathologic lesion, a phenomenon known as diaschisis.126 The application of several technologies to MRI scanning has provided additional opportunities to assess brain metabolism and function. Magnetic resonance angiography (MRA) permits a noninvasive visualization of cerebral blood flow, although it is probably not optimal for visualization of flow in small-caliber vessels which are those primarily involved in NP-SLE. Magnetic resonance spectroscopy (MRS) allows the identification and quantification of brain metabolites, thereby providing indirect evidence of cellular changes.112 Thus, the amount of N-acetyl (NA) compounds, which reflect the quantity and integrity of neuronal cells, is reduced in lupus brains. Studies of SLE patients have found an association between reduced NA brain levels with neurocognitive dysfunction127 and independently with elevated IgG antiphospholipid antibodies.128 Brain lactate levels are also elevated, indicating ischemia and inflammation, whereas choline compounds are increased reflecting damaged cell membranes and myelin destruction. Magnetization transfer imaging (MTI) is a structural imaging modality that allows for detection of tissue damage especially in terms of integrity of white matter tracks that cannot be easily visualized using conventional MRI.129 It is particularly suited to the detection and quantification of diffuse brain damage. This technique quantifies the exchange of protons between water within a macromolecule such as myelin and protons in free water. Either the loss of myelin or the accumulation of edema will alter the transfer, which is expressed as the magnetization transfer ratio (MTR).130 Studies to date have revealed a lower MTR in patients with NP-SLE and multiple sclerosis, whereas there was no difference between healthy controls and SLE patients without NP disease.114,131,132 The findings in SLE patients correlated with the results of cognitive assessment and psychiatric functioning.133 As both MRS and MTI identified abnormalities in SLE patients with normal MRI scans, these techniques provide a means of detecting and quantifying brain injury in patients with NP-SLE that is not apparent with other imaging modalities. Diffusion-weighted imaging (DWI) is very effective in the detection of hyperacute brain injury, in particular acute ischemia following stroke when the diffusion

DIAGNOSTIC IMAGING AND NP-SLE

such association.107-110 One cause of mortality in SLE is suicide, which has been reported in association with NP manifestations in a recent study involving a small number of patients.111

421

THE NERVOUS SYSTEM IN SYSTEMIC LUPUS ERYTHEMATOSUS

of water is highly restricted due to the acute shift of fluid into the intracellular compartment and cytotoxic edema.64,130 DWI also provides additional information on white matter homogeneity and connectivity. The technique is based on the principle of isotropy (Brownian motion), which refers to unrestricted chaotic movement of proton-containing molecules in free water. However, in the highly structured tissue of the brain (such as white matter and white matter tracks) it is easier for molecules to move in some directions than in others thus creating preferential diffusion, or anisotropy. Pathologic conditions can disturb the highly structured integrity of the white matter fibers, causing loss of anisotropy and changing the diffusion behavior of the molecules.134 If radiofrequency pulses are applied at certain intervals, it is possible to obtain information about water molecule movement between pulses. This information is provided in the form of diffusion coefficients and contains vector information regarding the directions of diffusion. The level of fractional anisotropy (FA) can be calculated for individual pixels within a region of interest or for the entire brain, and the results are presented as a histogram (with lower FA peaks indicating more pixels with higher diffusion values).134 Lower FA peaks indicate damage or degeneration in white matter tracks.129 DWI has been used to examine brain tissue changes in NP-SLE in terms of degeneration of parenchymal structure. Although patients with NP-SLE have been found to have more pixels with low FA values than healthy controls,135,136 it is unclear whether there is a difference in diffusion pattern between patients with active disease and patients who have chronic brain damage. An additional MRI-based technique is functional MRI (fMRI), which assesses cerebral blood flow and neuronal activity through the measurement of oxygenation status of hemoglobin. Studies of SLE patients examined utilizing this technique are awaited with interest.

was increased in the same patient population, with a sensitivity of 48% and specificity of 87%. Moreover, the levels of both NFL and GFAP were associated with abnormalities on MRI scanning and were reduced following the successful treatment of a variety of NP manifestations with cyclophosphamide. Although elevated levels of NFL and GFAP are not restricted to SLE, these data indicate a potential objective biological indicator of nervous system disease in lupus patients.

DIAGNOSIS OF NP-SLE The first step in the management of a patient with SLE who presents with a NP event is to determine whether the event can be convincingly attributed to SLE (i.e., a complication of the disease or its therapy) or whether it reflects a coincidental disease. This is achieved largely by a process of exclusion, given the absence of a diagnostic gold standard for most of the NP manifestations that occur in SLE. Thus, the correct diagnosis is derived from a careful analysis of the clinical, laboratory, and imaging data on a case-by-case basis and these may be utilized to a varying extent depending on the clinical circumstances (Table 37.3). Examination of the CSF should be considered primarily to exclude infection. Measurement of CSF autoantibodies, cytokines, and biomarkers of neurologic damage is still a subject of research. In considering circulating autoantibodies, those most likely to

TABLE 37.3 MANAGEMENT OF NP EVENTS IN PATIENTS WITH SLE Treatment Strategy

Examples

Establish diagnosis of NP-SLE

●

●

●

BIOLOGICAL MARKERS OF NERVOUS SYSTEM DAMAGE

422

Nonspecific abnormalities may be found in the CSF of 33% of patients with NP disease137 and include pleocytosis and elevated protein levels. A recent study138 has indicated more specific markers of cellular damage. Thus, elevated levels of CSF neurofilament triplet protein (NFL), which reflect neuronal and in particular axonal damage, were increased in SLE patients with NP-SLE compared to SLE patients without NP disease and healthy controls. The sensitivity was 74% and specificity 65%. Likewise, the level of CSF glial fibrillary acidic protein (GFAP), which indicates astrogliosis or scarring,

●

CSF examination primarily to exclude infection Autoantibody profile (antiphospholipid, antiribosomal P) Neuroimaging to assess brain structure and function Neuropsychological assessment

Identify aggravating factors

●

Hypertension, infection, metabolic abnormalities

Symptomatic therapy

●

Anticonvulsants, psychotropics, anxiolytics

Immunosuppression

●

●

Corticosteroids, azathioprine, cyclophosphamide, mycophenolate mofetil B lymphocyte depletion

●

Heparin, warfarin

Anticoagulation

Modified from Hanly JG. Neuropsychiatric lupus. Current Rheumatology Reports 2001;3:205-212.

TREATMENT OF NP-SLE Management will need to be tailored to the needs of individual patients (Table 37.3), and there remains a paucity of controlled studies to inform treatment decisions. Once a diagnosis of NP-SLE is established, the first step is to identify and treat potential aggravating factors such as hypertension, infection, and metabolic abnormalities. Symptomatic therapy with, for example, anticonvulsants, antidepressants, and antipsychotic medications should be considered if appropriate. Immunosuppressive therapy with high-dose corticosteroids, azathioprine, and cyclophosphamide is used to varying degrees. With the exception of one study,139 there are no placebo-controlled studies examining the benefit of either oral or intravenous corticosteroids140,141 in NP-SLE. Similarly, pulse intravenous cyclophosphamide therapy,142-150 akin to that which has been used in the treatment of lupus nephritis, has also been reported to be beneficial in NP-SLE although only one controlled study has been performed.151 A recent open-label study of 13 patients with lupus psychosis reported a favorable outcome in all patients treated with oral cyclophosphamide for six months followed by maintenance therapy with azathioprine.152 Another study by Barile-Fabris and colleagues151 compared intermittent intravenous cyclophosphamide to intravenous methylprednisone given for up to two years in SLE patients with predominantly neurologic disease and reported a significantly better response rate with cyclophosphamide (95%) compared to methylprednisone (54%) (p < 0.03). In virtually all of these studies, immunosuppressive therapy was used in conjunction with corticosteroids in addition to symptomatic therapies, such as antipsychotic medications. More targeted immunosuppressive therapies (for example, B lymphocyte depletion with anti-CD20 used alone or in combination with cyclophosphamide153,154) are promising but require further study. Anticoagulation is strongly indicated for focal disease when antiphospholipid antibodies are implicated, and such therapy will usually be lifelong.155-157

The identification of a potentially reversible cause is the first step in initiating treatment for SLE patients with cognitive impairment. Simple causes of new cognitive difficulties are often identified by review of the patient’s history. Recent changes in medication are among the most common. Antidepressants, anticonvulsants, and antihypertensive treatments frequently used in SLE may cause reversible cognitive problems. Adjustments in drug selection and dose may result in cognitive improvement. Treatment of even mild anxiety and depression may also improve cognitive symptoms. At present, any additional attempt to address the issue of treatment of cognitive dysfunction in SLE is at best speculative. Two approaches, pharmacologic treatment and cognitive rehabilitation, can be considered (although neither has yet been systematically attempted in SLE, let alone established evidence of efficacy). Only one placebo-controlled study of pharmacologic therapy for SLE-associated cognitive dysfunction has been performed.139 Ten SLE patients who were not receiving corticosteroids were enrolled in an N of one doubleblind controlled trial using 0.5 mg/kg prednisone daily. Except for complaints related to cognition, these patients presumably had inactive SLE at enrollment. The authors reported improved cognition in five of the eight subjects who completed the trial. The use of antiplatelet or anticoagulant therapy in SLE patients with antiphospholipid antibodies for the treatment of cognitive dysfunction without evidence of thromboembolic phenomena has a theoretical basis but lacks evidence for efficacy and remains controversial. Pharmacologic treatment aimed at “cognitive enhancement” has not yet been studied in SLE and has only recently been attempted in conditions such as multiple sclerosis (MS).158 Such treatments may ultimately prove efficacious in disorders such as MS159 and may have potential applications in SLE. Other pharmacologic agents have been developed for the treatment of cognitive dysfunction associated with conditions such as Alzheimer’s disease and attention deficit disorder. However, the variability in the presence and persistence of cognitive deficits in SLE patients, as well as the lack of biologic plausibility for efficacy, remain major hurdles for the design of clinical trials. Although the actions of such medications are not disease-specific, there is currently no data to support or refute their use in the treatment of SLE-associated cognitive dysfunction. Cognitive rehabilitation, which typically involves intensive retraining of cognitive skills, suffers from the same problems of variability in the nature, persistence, and biologic basis when considering the design and implementation of a trial of efficacy. Although individualized cognitive rehabilitation programs may indeed prove useful for some SLE patients, demonstrating the

TREATMENT OF NP-SLE

provide the greatest diagnostic yield are antiphospholipid antibodies. The value of measuring anti-P antibodies remains uncertain given the conflicting results to date, whereas the role of anti-NR2 antibodies in NPSLE is currently unknown. Neuroimaging should include a modality for assessing brain structure and another for assessing brain function. Neuropsychological testing should only be done to address specific concerns about cognitive ability, as the detection of isolated subclinical deficits appears to have little clinical significance.

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generalized effectiveness of this approach is challenging. Cognitive rehabilitation programs have been employed in other conditions (e.g., stroke, dementia, traumatic brain injury, and MS) to teach patients with cognitive dysfunction means of functionally adapting to their impairments so that they can maintain, if not regain, some level of independence. Until recently, no cognitive rehabilitation programs specifically intended for SLE patients have been developed. A novel psycho-educational group intervention targeted specifically at SLE patients with self-perceived cognitive dysfunction was designed to improve the performance of common cognitive activities they found problematic.160 Results of a pilot study of this program demonstrated that participation may result in improvement in memory self-efficacy, memory function, and ability to perform daily activities that require cognitive function.161,162 Although rehabilitation programs such as this are not generally available, lupus patients with verified cognitive dysfunction can be referred for cognitive rehabilitation to a neuropsychologist or occupational therapist with expertise in cognitive retraining.

CONCLUSIONS Nervous system disease in SLE patients poses significant diagnostic and therapeutic challenges. The use of a standard nomenclature with clear definitions and diagnostic criteria for individual NP syndromes provides a uniform platform for clinical studies and a more rational approach to the assessment of individual patients. The correct diagnosis of NP-SLE is largely based on clinical assessment supported by a variety of investigative tools, including neuroimaging. Determining the attribution of NP events is difficult, but it is clear that a significant proportion do not have a direct immunopathogenic link to SLE—an observation that has implications for both treatment and prognosis. Regardless of attribution, the occurrence of most NP events in SLE patients is associated with a significant negative impact on a patient’s health-related quality of life. Thus, treatment must be tailored to the needs of individual patients and consists of a combination of pharmacologic and nonpharmacologic interventions.

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patients with neuropsychiatric systemic lupus erythematosus, demonstrated by magnetization transfer imaging. Arthritis Rheum 2000;43(1):48–54. Bosma GP, Rood MJ, Huizinga TW, de Jong BA, Bollen EL, van Buchem MA. Detection of cerebral involvement in patients with active neuropsychiatric systemic lupus erythematosus by the use of volumetric magnetization transfer imaging. Arthritis Rheum 2000;43(11):2428–2436. Bosma GP, Middelkoop HA, Rood MJ, Bollen EL, Huizinga TW, van Buchem MA. Association of global brain damage and clinical functioning in neuropsychiatric systemic lupus erythematosus. Arthritis Rheum 2002;46(10):2665–2672. Huizinga TW, Steens SC, van Buchem MA. Imaging modalities in central nervous system systemic lupus erythematosus. Curr Opin Rheumatol 2001;13(5):383–388. Bosma GP, Huizinga TW, Mooijaart SP, Van Buchem MA. Abnormal brain diffusivity in patients with neuropsychiatric systemic lupus erythematosus. AJNR Am J Neuroradiol 2003; 24(5):850–854. Moritani T, Shrier DA, Numaguchi Y, Takahashi C, Yano T, Nakai K, et al. Diffusion-weighted echo-planar MR imaging of CNS involvement in systemic lupus erythematosus. Acad Radiol 2001;8(8):741–753. Small P, Mass MF, Kohler PF, Harbeck RJ. Central nervous system involvement in SLE. Diagnostic profile and clinical features. Arthritis Rheum 1977;20(3):869–878. Trysberg E, Nylen K, Rosengren LE, Tarkowski A. Neuronal and astrocytic damage in systemic lupus erythematosus patients with central nervous system involvement. Arthritis Rheum 2003;48(10):2881–2887. Denburg SD, Carbotte RM, Denburg JA. Corticosteroids and neuropsychological functioning in patients with systemic lupus erythematosus. Arthritis Rheum 1994;37(9):1311–1320. Barile L, Lavalle C. Transverse myelitis in systemic lupus erythematosus: The effect of IV pulse methylprednisolone and cyclophosphamide. J Rheumatol 1992;19(3):370–372. Eyanson S, Passo MH, Aldo-Benson MA, Benson MD. Methylprednisolone pulse therapy for nonrenal lupus erythematosus. Ann Rheum Dis 1980;39(4):377–380. Trevisani VF, Castro AA, Neves Neto JF, Atallah AN. Cyclophosphamide versus methylprednisolone for the treatment of neuropsychiatric involvement in systemic lupus erythematosus. Cochrane Database Syst Rev 2000;3. Baca V, Lavalle C, Garcia R, Catalan T, Sauceda JM, Sanchez G, et al. Favorable response to intravenous methylprednisolone and cyclophosphamide in children with severe neuropsychiatric lupus. J Rheumatol 1999;26(2):432–439. Leung FK, Fortin PR. Intravenous cyclophosphamide and high dose corticosteroids improve MRI lesions in demyelinating syndrome in systemic lupus erythematosus. J Rheumatol 2003;30(8):1871–1873. Boumpas DT, Yamada H, Patronas NJ, Scott D, Klippel JH, Balow JE. Pulse cyclophosphamide for severe neuropsychiatric lupus. Q J Med 1991;81(296):975–984. Neuwelt CM, Lacks S, Kaye BR, Ellman JB, Borenstein DG. Role of intravenous cyclophosphamide in the treatment of severe neuropsychiatric systemic lupus erythematosus. Am J Med 1995; 98(1):32–41. Ramos PC, Mendez MJ, Ames PR, Khamashta MA, Hughes GR. Pulse cyclophosphamide in the treatment of neuropsychiatric systemic lupus erythematosus. Clin Exp Rheumatol 1996; 14(3):295–299. Mok CC, Lau CS, Chan EY, Wong RW. Acute transverse myelopathy in systemic lupus erythematosus: Clinical presentation, treatment, and outcome. J Rheumatol 1998;25(3):467–473. Galindo-Rodriguez G, Avina-Zubieta JA, Pizarro S, Diaz de Leon V, Saucedo N, Fuentes M, et al. Cyclophosphamide pulse therapy in optic neuritis due to systemic lupus erythematosus: An open trial. Am J Med 1999;106(1):65–69. McCune WJ, Golbus J, Zeldes W, Bohlke P, Dunne R, Fox DA. Clinical and immunologic effects of monthly administration of intravenous cyclophosphamide in severe systemic lupus erythematosus. N Engl J Med 1988;318(22):1423–1431. Barile-Fabris L, Ariza-Andraca R, Olguin-Ortega L, Jara LJ, FragaMouret A, Miranda-Limon JM, et al. Controlled clinical trial of IV cyclophosphamide versus IV methylprednisolone in severe

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111. Karassa FB, Magliano M, Isenberg DA. Suicide attempts in patients with systemic lupus erythematosus. Ann Rheum Dis 2003;62(1):58–60. 112. Sibbitt WL, Jr, Sibbitt RR, Brooks WM. Neuroimaging in neuropsychiatric systemic lupus erythematosus. Arthritis Rheum 1999;42(10):2026–2038. 113. McCune WJ, MacGuire A, Aisen A, Gebarski S. Identification of brain lesions in neuropsychiatric systemic lupus erythematosus by magnetic resonance scanning. Arthritis Rheum 1988; 31(2):159–166. 114. Rovaris M, Viti B, Ciboddo G, Gerevini S, Capra R, Iannucci G, et al. Brain involvement in systemic immune mediated diseases: magnetic resonance and magnetisation transfer imaging study. J Neurol Neurosurg Psychiatry 2000;68(2):170–177. 115. Sibbitt WL Jr, Schmidt PJ, Hart BL, Brooks WM. Fluid attenuated inversion recovery (FLAIR) imaging in neuropsychiatric systemic lupus erythematosus. J Rheumatol 2003;30(9):1983–1989. 116. Bell CL, Partington C, Robbins M, Graziano F, Turski P, Kornguth S. Magnetic resonance imaging of central nervous system lesions in patients with lupus erythematosus: Correlation with clinical remission and antineurofilament and anticardiolipin antibody titers. Arthritis Rheum 1991;34(4):432–441. 117. Gonzalez-Crespo MR, Blanco FJ, Ramos A, Ciruelo E, Mateo I, Lopez Pino MA, et al. Magnetic resonance imaging of the brain in systemic lupus erythematosus. Br J Rheumatol 1995;34(11): 1055–1060. 118. Stimmler MM, Coletti PM, Quismorio FP Jr. Magnetic resonance imaging of the brain in neuropsychiatric systemic lupus erythematosus. Semin Arthritis Rheum 1993;22(5):335–349. 119. Cauli A, Montaldo C, Peltz MT, Nurchis P, Sanna G, Garau P, et al. Abnormalities of magnetic resonance imaging of the central nervous system in patients with systemic lupus erythematosus correlate with disease severity. Clin Rheumatol 1994;13(4): 615–618. 120. Nossent JC, Hovestadt A, Schonfeld DH, Swaak AJ. Singlephoton-emission computed tomography of the brain in the evaluation of cerebral lupus. Arthritis Rheum 1991;34(11): 1397–1403. 121. Oku K, Atsumi T, Furukawa S, Horita T, Sakai Y, Jodo S, et al. Cerebral imaging by magnetic resonance imaging and single photon emission computed tomography in systemic lupus erythematosus with central nervous system involvement. Rheumatology (Oxford) 2003;42(6):773–777. 122. Rubbert A, Marienhagen J, Pirner K, Manger B, Grebmeier J, Engelhardt A, et al. Single-photon-emission computed tomography analysis of cerebral blood flow in the evaluation of central nervous system involvement in patients with systemic lupus erythematosus. Arthritis Rheum 1993;36(9):1253–1262. 123. Rogers MP, Waterhouse E, Nagel JS, Roberts NW, Stern SH, Fraser P, et al. I-123 iofetamine SPECT scan in systemic lupus erythematosus patients with cognitive and other minor neuropsychiatric symptoms: A pilot study. Lupus 1992;1(4):215–219. 124. Kovacs JA, Urowitz MB, Gladman DD, Zeman R. The use of single photon emission computerized tomography in neuropsychiatric SLE: A pilot study. J Rheumatol 1995;22(7):1247–1253. 125. Waterloo K, Omdal R, Sjoholm H, Koldingsnes W, Jacobsen EA, Sundsfjord JA, et al. Neuropsychological dysfunction in systemic lupus erythematosus is not associated with changes in cerebral blood flow. J Neurol 2001;248(7):595–602. 126. Feeney DM, Baron JC. Diaschisis. Stroke 1986;17(5):817–830. 127. Brooks WM, Jung RE, Ford CC, Greinel EJ, Sibbitt WL Jr. Relationship between neurometabolite derangement and neurocognitive dysfunction in systemic lupus erythematosus. J Rheumatol 1999;26(1):81–85. 128. Sabet A, Sibbitt WL Jr, Stidley CA, Danska J, Brooks WM. Neurometabolite markers of cerebral injury in the antiphospholipid antibody syndrome of systemic lupus erythematosus. Stroke 1998;29(11):2254–2260. 129. Diwadkar VK. MS: New techniques in magnetic resonance imaging add their potential for neuropsychiatric research. J Psychosom Res 2002;53:677–685. 130. Peterson PL, Howe FA, Clark CA, Axford JS. Quantitative magnetic resonance imaging in neuropsychiatric systemic lupus erythematosus. Lupus 2003;12(12):897–902. 131. Bosma GP, Rood MJ, Zwinderman AH, Huizinga TW, van Buchem MA. Evidence of central nervous system damage in

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prevention of recurrent thrombosis in patients with the antiphospholipid antibody syndrome. N Engl J Med 2003; 349(12):1133–1138. Krupp LB, Christodoulou C, Melville P, Scherl WF, MacAllister WS, Elkins LE. Donepezil improved memory in multiple sclerosis in a randomized clinical trial. Neurology 2004;63(9):1579–1585. Doraiswamy PM, Rao SM. Treating cognitive deficits in multiple sclerosis: Are we there yet? Neurology 2004;63(9):1552–1553. Harrison MJ, Morris K, Barsky J, Horton R, Toglia J, Robbins L, et al. Development of a unique and novel psycho-educational program for active systemic lupus erythematosus (SLE) patients with self-perceived cognitive difficulties. Arthritis Rheum 2003;49(9):S1685. Harrison MJ, Morris K, Barsky J, Horton R, Toglia J, Robbins L, et al. Preliminary results of a novel psycho-educational program designed for systemic lupus erythematosus (SLE) patients with self-perceived cognitive difficulties. Arthritis Rheum 2003;49(9):S1058. Harrison MJ, Toglia J, Chait S, Morris K, Barsky J, Horton R, et al. MINDFULL: Mastering the Intellectual Navigation of Daily Functioning and Undoing the Limitations of Lupus, A pilot study to improve quality of life in SLE. Arthritis Rheum 2004;50(9):S242.

CLINICAL ASPECTS OF THE DISEASE

38

Overlap Syndromes, Mixed Connective Tissue Disease, and Undifferentiated Connective Tissue Disease Robert W. Hoffman, DO

INTRODUCTION Classification of the rheumatic diseases continues to be challenging for a number of reasons, including the fact that clinical features of these diseases can be protean and overlapping and that despite advances in our understanding of disease pathogenesis the etiology of most autoimmune rheumatic diseases remains elusive. The presence of features of more than one well-defined connective tissue disease “overlapping” in the same patient is widely recognized to occur. Such patients are frequently identified as suffering overlap syndrome. The term overlap syndrome has been used in the literature to describe patients with features of more than one distinctive rheumatic disease, either simultaneously or sequentially.

In addition, there are a number of so-called overlap syndromes that have distinctive autoantibodies associated with them. A summary of some of these overlap syndromes and autoantibodies is presented in Table 38.1. Mixed connective tissue disease (MCTD) is a distinctly identifiable rheumatic disease characterized clinically by the presence of overlapping features of SLE, scleroderma, and polymyositis and characterized serologically by the presence of high levels of antibodies against U1-ribonucleoprotein (U1-RNP).1 Although there is no uniformly accepted diagnostic criteria for MCTD, validated classification criteria have been published for MCTD.2-7 Two of the most widely recognized classification criteria for MCTD are outlined in Table 38.2.

TABLE 38.1 AUTOANTIBODIES AND OVERLAP SYNDROMESa Antibody

Antigen Specificity

Clinical Features

PM/Scl or PM-1 20-110kD of unknown function

Complex nuclear antigen with multiple proteins

Polymyositis-scleroderma overlap

Ku

70kD/80kD peptides involved in repair of DNA double helix breaks

Overlap syndrome, SLE, MCTD & scleroderma

RNA polymerase

RNA polymerase I, II, III

SLE & scleroderma

Heterogeneous nuclear

hnRNP A2 and other species

MCTD, SLE & RA ribonucleoprotein (hnRNP)

SSA/Ro

Ro antigen

Primary or secondary sub-acute cutaneous lupus & apparently normal individuals

U1-RNP (nRNP, RNP)

U1-70kD, A & C polypeptides

MCTD & less commonly scleroderma or other rheumatic disease

a. Systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), heterogeneous nuclear ribonucleoprotein (hnRNP), rheumatoid arthritis (RA).

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TABLE 38.2 CLASSIFICATION CRITERIA FOR MIXED CONNECTIVE TISSUE DISEASEa Criteria of Alarcon-Segovia and Villarreal Serologic Criteria ●

Anti-RNPb antibody must be present at a moderate-high level in serum

Clinical Criteria

There must be at least 3 out of the 5 of the following clinical findings (and this must include either synovitis or myositis): ● Edema of the hand ● Synovitis ● Myositis ● Raynaud’s ● Acrosclerosis Criteria of Sharpc Major Criteria ● ●

● ● ●

Severe myositis Pulmonary involvement with diffusion capacity for carbon monoxide below 70% of predicted for age or pulmonary hypertension or proliferative vascular lesion Raynaud’s phenomenon or esophageal hypomotility Swollen hands observed or sclerodactyly Highest observed anti-U1 RNP anti-extractable nuclear antigen by passive hemagglutination testing of at least 1:10,000 with positive anti-U1 RNP by immunodiffusion and negative for anti-Sm antibodies by immunodiffusion testing

Minor Criteria ● ● ● ● ● ● ● ● ● ● ●

Alopecia Leukopenia Anemia Pleuritis Pericarditis Arthritis Trigeminal neuropathy Malar rash Thrombocytopenia Mild myositis History of swollen hands

a. Alarcon-Segovia and Cardiel (6). b. Anti-RNP was defined in this study using hemagglutination. A titer equal to or greater than 1:1,600 was required in their study to be considered “moderate or high.” c. Requires four major criteria filled with anti-U1 RNP of at least 1:4,000 by passive hemagglutination assay for definite MCTD or three major criteria for probable or two major criteria from 1, 2, and 3 and two minor criteria with anti-U1 RNP at least 1:1,000 by passive hemagglutination assay.

The term undifferentiated connective tissue disease (UCTD), as introduced in the literature by LeRoy and colleagues, was used to describe the early phase of connective tissue disease when the features of the disease were nonspecific or “undifferentiated.”8 Currently, there is no uniformly accepted definition of the term UCTD and various investigators have applied it quite differently.8-19 It would, however, appear to remain a potentially useful designation for the large number of patients who initially fail to meet established classification or diagnostic criteria for another rheumatic disease. Furthermore, longitudinal epidemiologic studies have demonstrated that many patients initially classified as UCTD remain undifferentiated without progression to another currently identifiable rheumatic disease despite careful observation over time.10-12 See Chapter 34.

OVERLAP SYNDROMES As previously stated, the presence of features of more than one well-defined connective tissue disease (including SLE, scleroderma, polymyositis/dermatomyositis, Sjogren’s syndrome, and rheumatoid arthritis) overlapping in the same patient is widely recognized to occur and such patients have frequently been described in the literature using the term overlap syndrome.20-24 An alternative approach to classification of such patients has been the identification of patients by the presence of a distinctive autoantibody.25-36 It has been proposed that the combination of autoantibodies and genetics may be more precise methods of classification of patients with rheumatic disease, including those with overlapping clinical features.25

●

Raynaud’s phenomenon

●

Arthralgia/arthritis

●

Swollen hands

●

Sclerodactyly

●

Esophageal dysfunction

PM/Scl

●

Mild myositis

●

Pulmonary dysfunction with a risk of developing pulmonary hypertension

As shown in Table 38.3, a number of distinct autoantibodies have been recognized that define groups of patients that can have clinically overlapping features of more than one systemic rheumatic disease (Fig 38.1).

Anti-tRNA Synthetase Syndrome Antibodies directed against the Jo-1 antigen (later shown to be anti-histidyl-tRNA synthetase antibodies) were the first anti-synthetase antibodies identified.26,27 These anti-synthetase antibodies were reported to occur in patients with overlapping clinical features of myositis, arthritis, and intestinal lung disease.26-28 Some patients have had an erosive arthritis that mimics rheumatoid arthritis, and some patients have had features of scleroderma (including Raynaud’s phenomenon, sclerodactyly, dysphagia, and telangiectasias). Although antibodies to the Jo antigen (or histidyl-tRNA synthetase) were the first reported for an anti-synthetase antibody (the Jo antigen appearing to be the most common), patients have been described with similar clinical features and antibodies directed

The U1 RNP: A

U1-70kD

•Spliceosome component (processes pre-mRNA) •Common autoantigen in SLE •Found in some patients with scleroderma • Defining autoantigen in MCTD

U1-RNA C Sm

against other tRNA synthetases [including threonyl (PL-7), alanyl (PL12), isoleucyl (OJ), and glycyl (EJ)] have been reported.28

B/B', D

Fig. 38.1 Schematic of the U1-RNP. The U1-70kD protein binds specifically to the first stem loop of the U1 RNA. The U1-A protein binds similarly to the second stem loop. The Sm proteins bind to an area on a fourth stem loop. The signal recognition (SR) and U1-C proteins participate in proteinprotein interactions with other members of the U1-RNA. Sera from patients with MCTD, SLE, and scleroderma have been found that react with various components of the U1-RNP.

Antibodies against the PM/Scl (also known as PM1) antigen are found in patients that exhibit features of both polymyositis and scleroderma.29,30 These patients typically have Raynaud’s phenomenon, arthralgia, and myositis. They frequently have tendon involvement, mechanic’s hands, and skin lesions of dermatomyositis.29,30 They may have interstitial lung disease and have a risk of severe renal disease with renal crisis.29,30 Diffuse scleroderma skin involvement is uncommon in patients with this antibody reactivity, however (Fig. 38.2).

OVERLAP SYNDROMES

TABLE 38.3 COMMON CLINICAL FEATURES OF MCTD

Ku Anti-Ku antibodies were first described by Mimori and colleagues, who reported that patients with this antibody specificity had features of both scleroderma and polymyositis.31 Subsequently, anti-Ku reactivity has also been detected in patients classified as having SLE, Sjogren’s syndrome, polymyositis, autoimmune thyroid disease, and MCTD.31,32 Anti-Ku antibodies appear to be uncommon.

RNA Polymerase There is a series of RNA polymerases that have been identified, and autoantibodies variously reactive with RNA polymerases I, II, and III have been described.33,34 Patients with antibodies to RNA polymerases I and III have been described as having features of scleroderma, whereas patients with isolated antibodies to RNA polymerase II have been reported to have an SLE-overlap syndrome.33,34 Testing for these antibodies has revealed that it appears to be a common reactivity in patients with features of diffuse scleroderma.34

Heterogeneous Nuclear RNP Antibodies and T cells reactive with heterogeneous nuclear ribonucleoprotein (hnRNP) antigens have been reported among patients with SLE, rheumatoid arthritis, and MCTD.35-36 Recently, Skriner, and colleagues have identified autoantibodies reactive with different epitopes on hnRNP A2 antigen that appear to distinguish patients with MCTD from those with RA or SLE.36 Greidinger and colleagues have reported that T cells reactive with hnRNP could be cloned from MCTD patients and that these T cells could provide help for autoantibody production in vitro.37

SS-A (Ro) Anti-SS-A/Ro antibodies may be detectible in patients with primary or secondary Sjogren’s syndrome, in subacute cutaneous lupus, and in some cases in apparently

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OVERLAP SYNDROMES, MIXED CONNECTIVE TISSUE DISEASE, AND UNDIFFERENTIATED CONNECTIVE TISSUE DISEASE

Surface Ig, FcR or other receptors capturing apoptic debris including cleaved 70kD Ag antigen Antigen presented T cell in context receptor of HLA-DR Abnormal Ag processing? Ag

Abnormal activation threshold for cell signaling? Signal

Cytokines Process Accessory Ag molecules Debris HLA synthesis Excess co-stimulation? including DNA Antigen and RNA presenting cell stimulating T cell help? CD4’, CD8’ Dendritic cell, ACP through T cells, B cells, B cell or toll-like neutrophils, macrophage receptor (TLR) other cells signaling

Tissue damage

Fig. 38.2 A model of MCTD pathogenesis. Constituents of dead cells (apoptotic debris) that fail to be cleared through noninflammatory pathways provide pro-inflammatory innate immune signals (including through toll-like receptors, TLRs) and are recognized by autoantibodies, thus activating B cells and opsonizing the dead cell material for more efficient uptake and presentation by antigen-presenting cells (potentially including dendritic cells and B cells themselves). Through antigen-specific signals with MHC-bound autoantigen and nonspecific pro-inflammatory second signals, T cells become activated, resist apoptosis, and mature into memory and effector cells. These in turn produce pro-inflammatory mediators, induce differentiation and maturation of B cells, and directly mediate cell injury. B cells under these influences avoid apoptosis and produce high quantities of avid complement-fixing antibodies that induce tissue injury at sites of autoantigen recognition. (From Greidinger EL, Hoffman RW. Autoantibodies in the pathogenesis of mixed connective tissue disease. Rheum Dis Clin North Am 2005;31(3): 437-450.)

otherwise healthy individuals.38-41 Sjogren’s syndrome has been found to frequently coexist in overlap patients or in patients classified as having another connective tissue disease such as SLE, MCTD, RA, scleroderma, autoimmune liver disease, and others.

U1-RNP As reviewed in material following, the presence of high levels of anti-U1-RNP antibodies is a characteristic feature of MCTD and is required for classification of patients with MCTD.1-7 The presence of high levels of antibodies reactive with the U1-70kD polypeptide of U1-RNP has been reported to occur early in the course of disease and to have particularly strong association with MCTD.42-44 Low levels of anti-U1-RNP antibodies and antibodies against other RNP polypeptides [particularly Smith (Sm)-B/B′ and Sm-D] have been observed in a number of other rheumatic diseases, including SLE and scleroderma.43-45

MIXED CONNECTIVE TISSUE DISEASE

Clinical Features 432

CD4+ T cell

The primary clinical features of MCTD include Raynaud’s phenomenon, swollen fingers or hands,

arthralgia or arthritis, sclerodactyly, myositis (typically mild), and pulmonary dysfunction that can take a variety of forms.1,46-56 These are outlined in Table 38.4. Clinical features of MCTD that may also frequently be present include trigeminal neuralgia, lymphadenopathy, mild anemia, leukopenia, malar rash, and mild anemia. The initial presentation of MCTD often includes swollen fingers or swelling of the hands. Raynaud’s phenomenon is an early and characteristic feature of MCTD, being present in over 90% of patients. Sclerodactyly may often appear as a later manifestation of disease, and telangiectasias over the hands and face may also be seen. A variety of rashes may occur, including the malar rash of SLE and dermatomyositis-like rashes with Gottren’s papules or a heliotrope rash. Sicca complex has been found to occur in up to a quarter of patients.55 Arthralgia is present in most patients, and some develop a nonerosive polyarthritis. Less commonly, an erosive arthritis may occur.52 Myalgias are common, occurring in a quarter to half of all patients. In most patients, myositis is mild (although it can be severe in some patients).52,54 Pulmonary involvement can be a serious complication of MCTD, with pulmonary hypertension the most ominous problem. This is the main

●

High levels of antinuclear antibodies in a speckled patter of staining by immunofluorescence

●

High levels of antibodies reactive with extractable nuclear antigen (ENA) (this complex antigenic mixture contains U1-RNP and Smith [Sm] antigens)

●

Anti-U1-RNP antibodies present at high levels and generally remain stable over time

●

Reactivity with U1-70kD polypeptide of U1-RNP complex has a strong association with MCTD in many studies

●

Anti-U1-RNA antibodies present and changes in levels of these in the serum may correlate with disease activity (not routinely available in clinical laboratories at this time, however)

●

Absence of antibodies to Smith (Sm) antigen (including Sm-D polypeptides) or double-stranded DNA is typical of MCTD

●

Leukopenia and mild anemia often present

●

Rheumatoid factor present in approximately 25% of patients and may be present at high levels

disease-related cause of death in MCTD.52 Pulmonary symptoms can be absent until pulmonary hypertension is advanced. Pulmonary symptoms can include cough, the sensation of shortness of breath (at rest or with exertion), and pleuritic chest pain. The single breath diffusion of carbon monoxide has been shown to be a sensitive indictor of pulmonary disease in MCTD, including pulmonary hypertension in some patients.56 Patients can also experience pleuritis, or less commonly interstitial lung disease. There is reasonable evidence in MCTD that the treatment of pulmonary hypertension can prolong survival, and based on this it has been felt that careful monitoring to identify and treat pulmonary hypertension is warranted. The performance of annual pulmonary function testing and two-dimensional echocardiography have been advocated as screening tests for early identification of pulmonary hypertension in MCTD.52,54,56 Esophageal motility abnormalities, including symptomatic esophageal reflux, are very common in MCTD.57-58 In contrast to SLE, serious renal involvement in MCTD is distinctly uncommon, although mild renal abnormalities have been commonly recognized to be present. In a small percentage of patients classified as MCTD, serious membranoproliferative glomerulonephritis can occur.59-65

Serology and Immunology Antibodies against U1-RNP (also known as nuclear ribonucleoprotein [nRNP]) are a hallmark of the

disease and are required for diagnosis by both the original definition of the syndrome and in subsequent classification criteria.1-7 Patients will typically have a high-titer speckled antinuclear antibody pattern on immunofluorescence using the HEp-2 human cell line or murine tissue as the substrate for the test. Patients will have high levels of antibodies reactive with extractable nuclear antigen (ENA), which contains both the RNP and Sm antigens. Anti–U1-RNP antibodies develop in close temporal relationship to the onset of the disease and demonstrate an ordered progression of reactivity with individual U-small nuclear ribonucleoprotein (snRNP) polypeptides. The close linkage between the development of anti-RNP immunity and the onset of clinical disease has been used to support the hypothesis that T- and B-cell immunity against RNP plays a central role in disease pathogenesis.62,66

MIXED CONNECTIVE TISSUE DISEASE

TABLE 38.4 SEROLOGIC AND IMMUNOLOGIC FEATURES OF MCTD

Pathogenesis B cells and T cells have been implicated in the pathogenesis of MCTD.68-76 High circulating levels of autoantibodies directed against RNP are required for the classification of patients with the disease.2-7 Anti-RNP antibodies have been identified as an early feature of disease and their emergence is chronologically linked to the onset of clinical symptoms.65 Immunohistologic studies have demonstrated the presence of antibodies and B cells at sites of tissue injury,73 although studies have not proven that anti-RNP antibodies directly mediate tissue injury in MCTD. The recent development of an experimental animal model of MCTD, however, may facilitate such research.76,77 Anti-RNP antibodies may be reactive with a series of U1-associated polypeptides, including 70kD, A, and C. MCTD patients may also have antibodies reactive with U1 RNA, which is noncovalently associated with the U1 polypeptides in the spliceosome complex present in nucleated cells.42,43,51 A link between innate immune signaling and MCTD has been suggested by the association of the disease with anti-RNA antibodies and the fact that human U1 RNA has recently been shown to be able to activate antigen-presenting cells and that T-cell epitopes on U1-70kD all reside within the RNA binding domain of the protein.69,77,78 MCTD patients frequently have antibodies and T cells that react with the spliceosomal antigen heterogeneous nuclear ribonucleoprotein A2.37 MCTD patients have been reported to have a pattern of autoantibody reactivity with heterogeneous nuclear ribonucleoprotein A2 antigen that distinguishes them from patients with SLE or rheumatoid arthritis.36 MCTD patients frequently exhibit rheumatoid factor activity in their sera and often have antibodies reactive with phospholipid antigens, including cardiolipin.78

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OVERLAP SYNDROMES, MIXED CONNECTIVE TISSUE DISEASE, AND UNDIFFERENTIATED CONNECTIVE TISSUE DISEASE

The precise role (if any) these antibodies play in pathogenesis is not known. T cells are thought to have a central role in pathogenesis of MCTD (for a review of this topic see Chapter 10).76 These antiboides have been indirectly and directly implicated through a series of observations, including the fact that anti-RNP autoantibodies are present in high levels and have features of cytokine driven affinity maturation, that RNP-reactive T cells can be isolated from the peripheral blood of MCTD patients, that T cells are commonly present at sites of tissue injury, that RNP-reactive T cells can provide help to autoantibody-producing cells in vitro and ex vivo, and that T cells are found at the site of tissue injury in an inducible animal model of MCTD. The topic of autoantibody-reactive T cells in MCTD and SLE is reviewed in detail in Chapter 10.

Course of the Disease At the onset of disease, patients with MCTD typically have Raynaud’s phenomenon, arthralgia, swollen hands, and mild myositis.52 Initially, patients may be classified as UCTD, overlap syndrome (see also material following) or another rheumatic disease such as rheumatoid arthritis.52-54 When carefully sought, myositis, pulmonary abnormalities, or esophageal dysfunction have frequently been found to be present prior to the onset of symptoms and may not be initially recognized.52,56 Prospective, long-term outcome studies have identified the fact that substantial pulmonary dysfunction is a common feature of MCTD and that pulmonary hypertension is the most common diseaserelated cause of death in MCTD.52 Serious renal and central nervous system involvement are uncommon in MCTD, although they can be present in some patients.52 Overall, the prognosis for most patients with MCTD has been shown to be favorable. Monitoring for the development of pulmonary hypertension in MCTD using echocardiography and pulmonary function testing has been advocated by some authors based on the facts that pulmonary hypertension is a major cause of mortality in patients, can be difficult to identify early based on either history or physical examination, and may be a reversible complication of MCTD.52,56

UNDIFFERENTIATED CONNECTIVE TISSUE DISEASE

434

The term UCTD has been used to describe patients lacking adequate features to meet classification or diagnostic criteria for a well-recognized rheumatic disease. Often the term has been applied to patients early in the course of their disease before it has become fully manifest. The term was first used in the literature by LeRoy and colleagues in 1980 to describe the early

phases of a connective tissue disease when the clinical and diagnostic findings were not yet specific.8 Subsequently, the term has been used similarly by a number of authors, but the exact criteria for inclusion in these studies have varied widely.9-19 The term as first applied was not intended to describe “overlap syndrome” patients who met established criteria for one or more connective tissue disease. It has been recognized in prospective longitudinal studies of UCTD that a substantial number of patients will not progress to a well-defined connective tissue disease. In fact, in one large prospective study where patients were followed for up to 10 years less than onethird of patients eventually met classification criteria for a definite rheumatic disease. Although comprehensive epidemiologic data are not yet available, it appears that substantial numbers of patients may fall into the category of UCTD and that many such patients may remain without further differentiation. In one large study of 410 patients with connective tissue disease of less than one year in duration, over half had UCTD.9

TREATMENT OF MCTD, UCTD, AND OVERLAP SYNDROMES Current treatment of MCTD, UCTD, and overlap syndromes is based primarily on information derived from clinical experience and controlled trials that have been done on other rheumatic diseases. Unfortunately, there are no large controlled clinical trials that can be used to direct therapy in these disorders. Treatment is typically focused on the primary organ systems affected. The course of any individual patient can be quite variable, although long-term studies on MCTD and UCTD provide some guidance for clinicians and patients. An outline of suggested treatment is presented in Table 38.5. Mild UCTD, MCTD, or an overlap syndrome with the predominant feature being arthritis without major organ system involvement can often be treated conservatively with non-steroidal antiinflammatory agents or hydroxychloroquine. If symptoms are more refractory, corticosteroids may be needed in low doses, with response to therapy guiding the titration of the dose. If significant synovitis is present and/or is refractory to conservative treatment, or if bony erosions are detected, methotrexate may be indicated. Agents that block tumor necrosis factor alpha (TNF) have not been well studied in MCTD and should be used cautiously because they have been reported to induce anti-DNA antibodies and induce SLE.79,80 Although Aringer and colleagues have recently reported the safe and effective treatment of a group of SLE patients with anti-TNF agents, Christopher-Stine and Wigely reported that anti-TNF agents used in

Arthralgia/Arthritis ● ● ●

Non-steroidal anti-inflammatory agents, hydroxychloroquine, and low-dose corticosteroids Methotrexate for severe or erosive disease Role of agents that block tumor necrosis factor alpha not clearly defined

Raynaud’s Phenomenon ● ●

Protect from cold and trauma to hands/feet; warm clothing to prevent central cooling Vasodilators including long-acting calcium channel blockers

Skin ● ●

Avoidance and protection from sun Topical or oral corticosteroids and hydroxychloroquine

Pulmonary Manifestations ●

● ●

Pulmonary hypertension and pulmonary interstitial disease are associated with increased mortality; monitor for evidence of these, as symptoms may be minimal early in disease, using pulmonary function testing, echocardiography and high-resolution computerized tomography Corticosteroids and cytotoxic drugs appear to benefit interstitial pulmonary fibrosis and may benefit pulmonary hypertension Consider early consultation with a pulmonary hypertension specialist as treatment is complex and evolving

TREATMENT OF MCTD, UCTD, AND OVERLAP SYNDROMES

TABLE 38.5 TREATMENT OF MCTD, UCTD, AND OVERLAP SYNDROMES

Gastrointestinal ● ● ● ● ●

●

Proton-pump inhibitors are very effective in many patients with reflux symptoms Dilatation benefits some patients and monitoring for Barrett’s esophagus may be appropriate Muscle Myalgias with normal or modestly elevate serum creatinine kinase may require no treatment Inflammatory myositis treatment is similar to polymyositis with high-dose corticosteroids, immunosuppressive agents such as methotrexate, and intravenous immune globulin (which is usually reserved for refractory myositis) Fibromyalgia may be present and contribute to pain; a multimodal chronic-pain management approach should be used for fibromyalgia symptoms

Sicca ● ● ● ● ●

Provide careful ophthalmologic and dental care Ocular lubricants beneficial for ocular dryness Pilocarpine may benefit patients with oral and/or ocular dryness who fail conservative measures Water-soluble lubricants for vaginal dryness Topical skin lubricant for symptoms of itching and dryness

Reproductive Health ● ● ●

High-risk obstetrical monitor during pregnancy Avoidance of drugs that me be harmful to the fetus when anticipating pregnancy and when pregnant Sildenafil may be beneficial for male or female sexual dysfunction; prevalence sexual dysfunction may be quite high

Hematologic ● ●

Mild anemia and leukopenia are common and usually require no specific treatment Mild to moderate thrombocytopenia usually responds to low-dose corticosteroids

Cardiovascular ● ●

Pericarditis and other forms of serositis usually respond to moderate doses of corticosteroids Monitor for development of hypertension and treat aggressively, as patients are at risk for scleroderma renal crisis (especially in scleroderma-spectrum overlap syndromes; see also Table 38.1)

Neuropsychiatric ● ● ● ●

Depression is common in chronic illness, including rheumatic diseases, and can significantly impact quality of life Fatigue may be a symptom of depression in some patients Trigeminal neuropathy is usually a self-limiting problem Peripheral neuropathy treated symptomatically with supportive measures, such as amitryptiline and gabapentin Continued

435

OVERLAP SYNDROMES, MIXED CONNECTIVE TISSUE DISEASE, AND UNDIFFERENTIATED CONNECTIVE TISSUE DISEASE

TABLE 38.5 TREATMENT OF MCTD, UCTD, AND OVERLAP SYNDROMES—cont’d Renal ● ● ● ●

Careful and frequent blood pressure monitoring with aggressive treatment of hypertension when detected ACE inhibitors in appropriately high doses used for renal crisis Serial monitoring of renal function and urinalysis indicated Standard therapy for glomerulonephritis when present in overlap syndrome patients is similar or the same as that used in lupus nephritis patients

patients with scleroderma overlap/MCTD did in fact induce a lupus-like syndrome among their patients.81,82 In many patients, hydroxychloroquine and/or low to intermediate doses of corticosteroids will usually control mild to moderate SLE-like features. Higher doses of corticosteroids (e.g., prednisone 1 mg/kg) may be required for treatment of more serious systemic involvement (e.g., myositis, myocarditis, pericarditis, glomerulonephritis, or thrombocytopenia). There are also reports of the use of anti-CD20 monoclonal antibodies in refractory SLE, suggesting that this might be of use in treating highly select patients with severe and refractory disease.83 It has been shown that high titers of anti-U1 RNP are associated with a high risk of esophageal dysfunction, myositis, pulmonary disease, and pulmonary hypertension, which can be detected by functional tests and biopsies early in the evolution of MCTD when patients are still asymptomatic.52,56-58 Thus, presence of high levels of anti-RNP antibodies might guide the clinician to careful evaluation of such patients, as clinically indicated, with studies of esophageal motility, muscle enzymes, electromyography, pulmonary function (including diffusion capacity), and pulmonary imaging. Echocardiography may be useful to detect early pulmonary hypertension (even in asymptomatic patients). It may be used to indirectly measure pulmonary artery pressure. In instances where there is evidence of pulmonary hypertension, a right heart catheterization can provide useful information by precisely defining pulmonary artery pressure and by determining if any right heart valvular dysfunction is present. There is evidence that early pulmonary abnormalities, including pulmonary hypertension and interstitial lung disease, in MCTD may respond to corticosteroids alone or in combination with cyclophosphamide.52,54,56 Early pulmonary hypertension in a mildly to moderately symptomatic phase in MCTD may be successfully

treated with nifedipine, captopril, or hydralazine in addition to immunosuppressive drugs. Antidotal experience and uncontrolled trials reported in the literature appears to favor the use of cyclophosphamide,52,54,56 which may potentially avoid more severe disease. When pulmonary hypertension is more advanced and very severe, corticosteroids, cytotoxic drugs, calcium channel blockers, anticoagulants, and angiotensinconverting enzyme (ACE) inhibitors have all been employed as therapeutic agents, but the responses have not always been favorable.52,54,56 Most recently, prostaglandins, prostacyclin analogs, and the dual endothelin-receptor antagonist bosentan have been used to treat patients with severe pulmonary hypertension.84-88 Esophageal dysfunction is common in MCTD and appears to respond well to proton pump blockers. Early treatment of myositis in MCTD may require lower doses of corticosteroids for a shorter duration that in cases of polymyositis.52,54 More severe disease may require the use of methotrexate or intravenous immune globulin.54 In a prospective longitudinal study from a tertiary referral center, a significant number of the 34 patients studied eventually developed significant major organ system involvement and were treated with moderate to high doses of corticosteroids and/or cyclophosphamide.52 Two-thirds of those treated responded favorably, whereas one-third with very severe disease were less responsive to corticosteroids (four of which died). As previously described, in MCTD inflammatory changes such as rash, pleuritis, pericarditis, and myositis responded to corticosteroids, whereas pulmonary and scleroderma-like features were less responsive to therapy. Among some patients, the long-term prognosis was good [with disease becoming inactive in 38% of the patients, including 10 with a sustained remission and 9 off all therapy for a mean of 16 years (range 7 to 20 years)].52

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The Eye in Systemic Lupus Erythematosus Alastair K. O. Denniston, MRCP, MRCOphth and Philip I. Murray, PhD, FRCOphth

INTRODUCTION About a third of patients with SLE may have ocular involvement. The majority have the complication of dry eye syndrome, which may cause discomfort but is usually responsive to treatment and rarely affects vision. A significant minority may have sight-threatening complications, such as retinal vaso-occlusive disease, scleritis, or severe ocular surface disease. In addition, these serious ocular complications may indicate significant disease activity elsewhere. Thus, the ophthalmologist may play a valuable part both in recognizing and managing the SLE patient’s ocular disease and in warning of increasing systemic disease activity. Vigilance and appropriately aggressive therapy may be life saving as well as vision-preserving.

CLASSIFICATION AND ASSESSMENT OF DISEASE ACTIVITY Although ocular involvement is not one of the 11 features in the American College of Rheumatologists classification criteria for SLE,1 recognizing ocular manifestations may be a useful marker of disease activity. In particular, severe retinopathy strongly correlates with active CNS disease.2 The updated British Isles Lupus Assessment Group disease activity index (BILAG 2004) includes the “ophthalmic system” as one of the nine categories considered. In their validation study, good reliability and physician agreement were seen for the ocular activity index in the one series that was sufficiently powered for ocular patients.3

CLINICAL PRESENTATION

External and Anterior Segment Keratoconjunctivitis Sicca

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Keratoconjunctivitis sicca (KCS, or “dry eye syndrome”) due to secondary Sjögren syndrome is the most common ocular manifestation of SLE, affecting about 25% of patients. Secondary Sjögren syndrome is strongly

associated with HLA-DRw52 and the autoantibodies anti-Ro (SSA) and anti-La (SSB). Symptoms may vary from slight irritation and burning in milder cases to severe pain and blurred vision in significant corneal involvement. Clinical examination using slit-lamp biomicroscopy reveals a small or absent concave tear meniscus with a tear film break-up time of under 10 seconds. Corneal abnormalities, which may be highlighted with fluorescein drops and a cobalt blue light, include punctate epitheliopathy, mucus filaments, strands, and plaques. Additional staining with Rose Bengal drops reveals a characteristic interpalpebral pattern, with greatest staining nasal and temporal to the corneal limbus. Tear production, as measured by the Schirmer test, is reduced. Wetting of the test strip by less than 5 mm after 5 minutes in the un-anaesthetized eye indicates severe tear deficiency. In one series of patients with SLE, the median value was 7.5 mm.4 It should be noted that the correlation of dry eye symptoms with observed disease is poor. Many more patients report “dry eyes” than have visible disease,4 and many asymptomatic patients do have some degree of keratoconjunctivitis sicca.

Other Keratopathies and Anterior Segment Complications Superficial punctate keratitis may be observed in patients with SLE in the absence of visible KCS. One series noted corneal staining in as many as 21 of 24 (88%) SLE patients.5 Similarly, superficial punctate keratitis (and recurrent erosions) has been noted in DLE. Interestingly, this responded well to the immunomodulator quinacrine hydrochloride, suggesting that this may be a direct autoimmune phenomenon. Deeper corneal involvement may occasionally occur, with both interstitial keratitis and keratoendotheliitis reported. In the latter, polyrefringent crystals in the deep stroma with associated edema are seen. Other rare anterior segment complications include iris neovascularization (rubeosis iridis, usually indicating severe retinal ischemia) and uveitis.

Episcleritis is observed in 1 to 2% of SLE patients. Patients present with a red eye with mild, if any, discomfort. Upon examination, either sectoral or diffuse injection of the superficial vessels is seen. The superficial nature of the inflammation may be confirmed by the instillation of topical 10% phenylephrine. When only the more superficial vessels are injected (as occurs in episcleritis), constriction of these vessels will result in visible blanching. This can helpfully distinguish the condition from scleritis, where deeper vessels would be unaffected and the redness would be maintained. Although not serious in itself, episcleritis may indicate increased systemic activity. (See Box 39.1.) Scleritis is observed in 1% of SLE patients, and may be the first manifestation of their systemic disease.6 The more common anterior scleritis usually presents with a severely painful red eye, which may be so severe that it wakes the patient up at night. Upon examination, the deeper vascular plexus is injected. This may be widespread (diffuse non-necrotizing scleritis) or focal and associated with a nodule (nodular nonnecrotizing scleritis). Necrotizing scleritis is rare. It is important to also examine the eye in day or room light, as the bright light of the slit lamp often underestimates the degree of scleral inflammation (and thinning). Posterior scleritis may present with mild to severe ocular pain (which may be referred to brow or jaw), reduction of vision, hypermetropic shift (i.e., becoming more long-sighted), photopsia, and diplopia. Ocular signs include choroidal folds or detachment (may be annular), exudative retinal detachments, and edema of the macula or optic disc. In addition, there may be associated periocular and orbital signs, such as lid edema, proptosis, lid retraction, and restricted ocular motility. B scan ultrasound is the investigation of choice (see “Investigations”). Scleritis may be sight threatening and is an indicator of significant systemic activity requiring commencement or increase of systemic therapy (as “Treatment”). Conversely, the scleritis improves as the systemic disease is brought under control. As the scleritis resolves, it becomes thinner and allows the blue-black color of the uvea to show

BOX 39-1 CAUSES OF RED EYE IN SLE Common ● Keratoconjunctivitis sicca Less common ● Episcleritis ● Scleritis Rare ● Keratitis (other than keratoconjunctivitis sicca) ● Anterior uveitis

through. Scleral thinning may induce high degrees of astigmatism, and in necrotizing disease possibly globe perforation. Scleritis requires urgent referral to an ophthalmologist.

Lid Disease Both SLE and DLE may involve the eyelids, resulting in raised scaly lesions resembling chronic blepharitis.7 Lid biopsy may be necessary to confirm the diagnosis. This shows hyperkeratosis, focal intracellular edema and degeneration of the basal layer, and lymphocytic infiltrates (especially around the vessels). Interestingly, a mouse model for SLE inoculated with human monoclonal anti-DNA antibodies demonstrated acute and chronic inflammation of the lids with immune complex deposition. The lids may also be involved in the classic malar rash of SLE.

CLINICAL PRESENTATION

Episcleritis and Scleritis

Orbital Disease Uncommonly, SLE may be associated with orbital inflammation or periorbital edema. Orbital inflammation may present with acute proptosis, lid edema, conjunctival injection and chemosis, reduced motility, and raised intraocular pressure. It may be misdiagnosed as orbital cellulitis, thyroid eye disease, or another form of orbital inflammation. Orbital inflammation may include myositis or rarely panniculitis (lupus erythematosus profundus). Myositis may be demonstrated by imaging using CT and B scan ultrasound showing enlargement of extraocular muscles, and upon biopsy. Episodes of orbital myositis may occur concomitantly with a generalized myositis.8 Orbital inflammation may also be associated with acute ocular ischemia and posterior scleritis.9 Rare complications include episodic periorbital edema and septic cavernous sinus thrombosis in the presence of severe immunosuppression. (See Box 39.2.)

Posterior Segment Classic Lupus Retinopathy Bergmeister first described retinal involvement in SLE in 1929. He noted cotton-wool spots and other lesions arising several weeks after a flare-up of the patient’s cutaneous manifestations. The retinopathy is usually bilateral, but may be asymmetric. The presence of retinopathy is commonly associated with systemic activity, active systemic disease being present in 88% of one series of retinopathy patients.10 Although older series reported retinopathy in up to 28% of SLE patients, more recent studies suggest that its prevalence is nearer 10% in adults11 and in children. This reduction is likely to reflect the overall improved control of systemic SLE. Mild retinopathy is usually asymptomatic, but severe disease (and its complications) may cause reduced visual acuity, field defects, distortion,

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BOX 39-2 CAUSES OF LOSS OF VISION IN SLE Anterior Segment ● Severe keratoconjunctivitis sicca Lens ● Cataract (secondary to inflammation/corticosteroids) ● Vitreous ● Vitreous hemorrhage (secondary to proliferative retinopathy) Retina ● Severe vaso-occlusive retinopathy ● Central or branch retinal vein occlusion (CRVO/BRVO) ● Central or branch retinal arteriole occlusion (CRAO/BRAO) ● Exudative retinal detachment Choroid ● Choroidopathy ● Choroidal effusion ● Choroidal infarction

A

Neuro-ophthalmic ● Optic neuritis ● Anterior ischemic optic neuropathy ● Posterior ischemic optic neuropathy ● Optic chiasmopathy ● Cortical infarcts

or floaters. The classic clinical picture is of cotton-wool spots and retinal hemorrhages with abnormalities of the vasculature, such as arterial narrowing, capillary dilation, venous dilation, and tortuosity (see Figs. 39.1, 39.2, and 39.3). Additional features include retinal edema, hard exudates, and microaneurysms.

B

Severe Vaso-occlusive Retinopathy (“Retinal Vasculitis”) The more severe vaso-occlusive retinopathy of SLE is much less common. Significant visual loss is reported in up to 80% of such patients, with final acuity of <20/200 in 50%.2 In addition, severe vaso-occlusive retinopathy is strongly associated with the lifethreatening complication of CNS lupus.2 The risk of

C Fig. 39.2 Fundus fluorescein angiography of the same patient: (A) early, (B) arteriovenous, and (C) late phases demonstrating capillary “drop-out,” vessel wall staining, and leakage.

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Fig. 39.1 Acute lupus retinopathy with cotton-wool spots, arterial narrowing, venous dilation, and tortuosity.

vaso-occlusive disease (ocular and CNS) is increased in the presence of antiphospholipid antibodies, with some observers noting a fourfold increase for ocular disease.12 Interestingly, most of the features seen in SLE-associated retinopathy may occur in the presence of antiphospholipid antibodies without SLE (primary antiphospholipid syndrome or Hughes syndrome). Cotton-wool spots are, however, less common.

CLINICAL PRESENTATION

A Fig. 39.3 Detail of the same patient showing venous beading.

This form of retinopathy is often termed retinal vasculitis, although true vasculitic changes are often not present upon histologic examination. It is characterized by widespread arteriolar occlusions and capillary nonperfusion with subsequent neovascularization in 40 to 72% of cases.2 Such proliferative retinopathy may in turn be complicated by vitreous hemorrhage (up to 63%), retinal traction, and retinal detachment (up to 27%).2 Rarely, frank vasculitis does occur. Reports include a “frosted branch”-type periphlebitis with exudative maculopathy. Fluorescein angiography may reveal areas of arterial and capillary nonperfusion, staining of vessel walls, and leakage notably from neovascular fronds (see Figs. 39.4 and 39.5). Angiography commonly shows more extensive disease than clinically suspected. At the histologic level there is hyaline thrombus formation with thickening of the vessel walls and perivascular infiltrate, but inflammation of the vessel wall itself is not seen, in contrast to a “true” vasculitis.13 Immune complex

B

C Fig. 39.5 Fundus fluorescein angiography of the same patient: (A) early, (B) arteriovenous, and (C) late phases demonstrating the presence of active new vessels. These did not resolve with immunosuppression and required sectoral argon photocoagulation.

Fig. 39.4 The same patient one year later, showing resolution of cotton-wool spots. However, careful examination reveals two areas of abnormal neovascularization.

deposition containing IgG, C1q, and C3 may be seen upon immunofluroescence staining. This unexpected observation of immune complex deposition without apparent vasculitis has also been seen in mouse models. Proposed explanations included the relative impermeability of the retinal vasculature

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BOX 39-3 DIFFERENTIAL DIAGNOSIS OF LUPUS RETINOPATHY AND SEVERE VASO-OCCLUSIVE DISEASE ● ● ● ● ● ● ● ● ● ● ● ●

Behçet’s disease Wegener’s granulomatosis Polyarteritis nodosa (PAN) Scleroderma Polymyositis/dermatomyositis Sarcoidosis Syphilis Lyme disease Human immunodeficiency virus (HIV) retinopathy Cytomegalovirus (CMV) retinitis Accelerated hypertension Central retinal vein occlusion (CRVO)

(as compared to the choroid) and the antigen:antibody ratio. Increasing awareness of the prothrombotic role of antiphospholipid antibodies and the observed similarities to the retinopathy of antiphospholipid syndrome would suggest that these changes may reflect multifactorial vaso-occlusion due to antiphospholipid antibodies and hypertension, with a minor role for vascular or perivascular inflammation. (See Box 39.3.) Ophthalmic disease may be more extensive upon histologic examination than is clinically apparent. A recent histologic study of a lupus patient without known ocular disease noted degenerative changes of the pericytes and smooth muscle cells of the retinal blood vessels, with partial obliteration of capillary lumen and IgG deposition.14

Arteriole and Venule Occlusions Occlusion of the larger retinal vessels is a well-recognized complication of SLE and is strongly associated with the presence of antiphospholipid antibodies, such as anticardiolipin IgG. As noted previously, these thrombogenic autoantibodies are also associated with lupus retinopathy, ocular vaso-occlusive disease, and CNS vaso-occlusive disease. Diseases of larger vessels include central and branch retinal artery occlusions (see Fig. 39.6), central and branch retinal vein occlusions, and combined retinal artery and vein occlusions.2,13

Fig. 39.6 Branch retinal arteriole occlusion in a patient with SLE and antiphospholipid syndrome. (See Color Plate 3.)

immunosuppressive treatment. Exudative retinal detachment may arise due to underlying lupus choroidopathy.16 As discussed previously, tractional retinal detachment may occur secondarily to neovascularization from severe retinal vaso-occlusive disease.

Lupus Choroidopathy Although less common than lupus retinopathy, choroidal involvement is an increasingly recognized cause of visual morbidity in SLE. Choroidopathy appears to be due to ischemia and may result in single or multifocal serous detachments of the retina and retinal pigment epithelium (RPE). These may mimic typical central serous chorioretinopathy (CSR)—with macular involvement causing visual loss17,18—or may

Other Retinal Manifestations

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An unusual manifestation of retinal disease in lupus is of a bilateral peripheral pigmentary retinopathy that may mimic retinitis pigmentosa. It is speculated that this may arise from peripheral vaso-occlusive disease.15 Hypertension is common and may result in features of hypertensive retinopathy, most dramatically in the accelerated phase. Cases of retinal necrosis in SLE patients have been reported, such as varicella zoster virus acute retinal necrosis (see Fig. 39.7) and CMV retinitis. These infections may result from the patient’s abnormal immune system or a complication of their

Fig. 39.7 Acute retinal necrosis secondary to varicella zoster virus in a patient with SLE. (See Color Plate 3.)

● ● ● ● ●

Vogt-Koyanagi-Harada syndrome Central serous chorioretinopathy Sympathetic ophthalmia Sarcoidosis Choroidal metastases

be eccentric with minimal symptoms. Either type may progress to extensive exudative retinal detachment.18 The serous detachments may reverse with control of the systemic disease.18 Fluorescein angiography shows site(s) of leakage from the choroid into the subretinal and sub-RPE spaces (akin to typical CSR), but also areas of choroidal ischemia (suggestive of the underlying cause).18 Other choroidal manifestations include choroidal effusions, visual loss from choroidal infarction, and choroidal neovascular membranes. (See Box. 39.4.)

Neuro-ophthalmic Optic Nerve Disease Optic nerve disease occurs in about 1% of patients with SLE. Clinical presentations include acute optic neuritis and anterior or posterior ischemic optic neuropathy. Disease of the optic nerve or chiasm may be the presenting feature of SLE. Upon histologic examination either demyelination or axonal necrosis may be seen, with both cases thought to arise from ischemia.19 Chronic retinal or optic nerve disease may result in slowly progressive visual loss and optic atrophy.19 In addition, bilateral swelling of the optic disc may signify papilledema associated with idiopathic intracranial hypertension or accelerated hypertension, both of which are more common in SLE.

Ocular Motility Abnormalities Disturbance of eye movements is not uncommon in SLE. This is most commonly due to brain stem or other central disease, although cranial neuropathies, tenosynovitis, and myositis are also seen. In one series of 33 patients with ocular motility abnormalities, 16 had brain stem infarcts, 4 had isolated sixth nerve palsies, 3 had meningitis, 2 had idiopathic intracranial hypertension, and 2 had myositis.20 Isolated ptosis and isolated third-nerve palsy may also be seen. Acquired Brown’s syndrome has been reported and may respond to systemic corticosteroids.21 Severe ophthalmoplegia due to Miller-Fisher syndrome has also been reported in a patient with SLE. This responded to plasmapheresis but not to conventional immunosuppression. Unilateral internuclear ophthalmoplegia (INO),22 and less commonly bilateral INO, skew deviations and one-and-a-half syndrome23 are reported. Transient diplopia either in isolation or

with vertigo may occur. This is usually associated with antiphospholipid antibodies, possibly due to disturbance of the posterior cerebral circulation. Horizontal and vertical nystagmus may also be seen.

TREATMENT

BOX 39-4 DIFFERENTIAL DIAGNOSIS OF SLE CHOROIDOPATHY

Retrochiasmal CNS disease in SLE can cause a range of visual disorders. Permanent visual field defects may present acutely or subacutely, and occasionally as the first presentation of the disease. Occipital involvement may result in cortical blindness.24 Migraine is common, and both amaurosis fugax and typical fortification spectra are reported. Other retrochiasmal complications include idiopathic intracranial hypertension.25

INVESTIGATIONS

General General investigations for suspected SLE are discussed elsewhere but include tests toward a diagnosis (e.g., ANA, anti-dsDNA antibodies, anti-Sm antibodies, and so on), tests for thrombotic risk (antiphospholipid antibodies such as anticardiolipin and lupus anticoagulant), and tests for disease activity (e.g., complement levels, anti-dsDNA, and antibody titer).

Ophthalmic Specific ophthalmic tests are used when clinically indicated. For ocular surface disease, Schirmer’s test may be useful to indicate the level of tear production and to help direct appropriate tear replacement therapy. The diagnosis and monitoring of posterior scleritis may be greatly assisted by B-scan ultrasonography. Signs include posterior scleral thickening (usually > 1.5 mm), with fluid in Tenon’s space (the “T-sign”). Posterior scleritis will also be apparent upon CT and MRI. In retinal disease, fundus fluorescein angiography (FFA) is often helpful—particularly in assessing vasoocclusion and ongoing leakage and detecting new vessels (see Fig. 39.4). When choroidal disease is suspected, indocyanine green (ICG) angiography may give further information regarding the choroidal circulation. Centrally located fluid (e.g., macular edema, central serous chorioretinopathy) may now be identified noninvasively with optical coherence tomography (OCT). Orbital and neuro-ophthalmic manifestations of SLE often require cranial imaging by CT and/or MRI.

TREATMENT

General Control of the systemic disease activity has a beneficial effect on the ophthalmic status. The more severe ophthalmic complications, such as lupus retinopathy (including severe vaso-occlusive disease), are less

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common in well-controlled patients. In addition, remission of scleritis, retinopathy, choroidopathy, and CNS-lupus usually coincides with the systemic disease coming under control. As stressed earlier, severe ophthalmic disease may indicate that systemic disease has worsened (or is about to) and requires urgent rheumatologic assessment. Systemic treatment may need to be increased for worsening systemic disease or for severe ophthalmic disease in isolation. This is usually coordinated by the patient’s rheumatologist in conjunction with the ophthalmologist. Immunosuppression should only be managed by those with appropriate experience and facilities for monitoring. Systemic therapies are discussed elsewhere but include non-steroidal anti-inflammatory drugs, anti-malarials, corticosteroids, other immunosuppressive agents, intravenous immunoglobulin, plasmapheresis, and the newer biologics. Some patients may also need antihypertensives, renal support, and/or anticoagulation to optimize both their systemic and ophthalmic prognosis.

Ophthalmic External and Anterior Segment

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Most patients with keratoconjunctivitis sicca secondary to SLE may be controlled with tear replacement therapy adjusted according to the needs of the patient. In very mild cases, low-viscosity drops (e.g., hypromellose, hydroxy-ethylcellulose, and polyvinyl alcohol) may be sufficient. More severe cases usually require combination therapy; for example, daytime use of a carbomer (medium viscosity, temporary blurring of vision, moderate duration of effect) and a paraffin at night (high viscosity, long-lasting effect but significant blurring of vision). Preservative free preparations are preferred when frequent administration is necessary or intolerance develops. Emerging products include those based on hyaluronic acid. These appear to be effective in reducing dry eye symptoms and improving the health of the ocular surface. In extreme cases, autologous serum may be used. These more severe cases should be under the care of an ophthalmologist. Tear replacement therapy may be augmented by punctal occlusion, initially in the form of punctual plugs. Any coexistent lid margin disease (such as blepharitis) should be treated. Where there is active ocular surface inflammation, judicious use of topical corticosteroid or ciclosporin may be necessary. Environmental modifications include lowering room temperatures, using humidifiers, and using moist chamber goggles— although these measures are often impractical. Mild anterior segment inflammation as in most cases of keratitis (non-KCS), anterior uveitis, and episcleritis may respond to topical corticosteroids (or alternatively topical NSAIDs for episcleritis). However, it is

usually necessary to improve disease control with systemic therapy in order to maintain remission even in these milder cases. In more severe anterior segment inflammation (such as scleritis) or orbital inflammation, systemic therapy is always necessary. In mild scleritis, oral non-steroidal anti-inflammatory drugs (e.g., flurbiprofen 100 mg 3x/d) are usually used first line. More severe disease usually requires oral corticosteroids (e.g., prednisolone 1 mg/kg/d), but may be preceded by intravenous methylprednisolone (e.g., 500 mg to 1g daily for 3 days). This is later supplemented with, or replaced by, other immunosuppresive agents (e.g., cyclophosphamide, azathioprine, methotrexate, cyclosporin, or mycophenolate) as part of a steroid-sparing strategy or where disease is unresponsive to steroids.

Posterior Segment Retinopathy or choroidopathy requires systemic treatment, as described previously. In unilateral or asymmetric retinal or choroidal disease, this is sometimes supplemented by regional corticosteroid injections. Significant vascular occlusions in the presence of antiphospholipid antibodies may be treated with corticosteroids, warfarin, and low-dose acetylsalicylic acid. Neovascularization is usually treated with pan-retinal photocoagulation. Subsequent anterior segment neovascularization has been reported. Non-clearing vitreous hemorrhage or tractional retinal detachment may require vitreo-retinal surgery.

Neuro-ophthalmic Neuro-ophthalmic complications reflect active CNS disease and require systemic therapy, as outlined previously. In one small series of SLE-associated optic neuropathy, four out of seven patients responded to corticosteroid therapy.19 Two pilot studies have suggested that cyclophosphamide is effective either in conjunction with corticosteroids or in steroid-resistant cases.26 Assuming that the final lethal insult in these cases is ischemia, it is likely that there is a relatively short therapeutic window during which the underlying immune process can be subverted and axonal injury prevented. If antiphospholipid antibodies are present, anticoagulation may be necessary as well to treat and prevent recurrence of optic neuropathy.

Ophthalmic Complications of Systemic Therapy Although much attention is often directed toward the possible complications of chloroquine or hydroxychloroquine therapy, it should be remembered that visual morbidity is more frequently associated with corticosteroid treatment. The association between posterior subcapsular cataracts and exogenous corticosteroids is

patients need be seen no more often than the general population. High-risk patients include those on a dose of >6.5 mg/kg, with a duration of >5 years, obese, with renal or hepatic disease, with preexisting retinal disease, or over the age of 60.29 In contrast, the UK’s Royal College of Ophthalmologists suggests that low-risk patients do not need a baseline examination by an ophthalmologist. They advise that the prescribing rheumatologist should ask about visual symptoms and record near visual acuity for each eye. Patients with visual impairment should then be referred to an optometrist, who can refer any significant abnormality to the local ophthalmologist in the usual way. They also advise calculation of lean body weight and testing of renal and liver function to ensure safe dosing.28 When patients are referred to the ophthalmologist due to an abnormality at baseline or a deterioration in visual function, they should have a full ophthalmic examination, including distance and near visual acuity, color vision, visual fields (red target), macular function (Amsler grid), anterior segment, and dilated fundoscopy. As with the AAO recommendations, additional tests are used as indicated.28 With regard to the type of Amsler grid used, a recent study recommended the use of the threshold Amsler grid (which utilizes cross-polarizing filters to reduce the perceived luminance of the grid). This had an increased sensitivity for subtle hydroxychloroquine-associated scotomas (detected scotomas in 45% of asymptomatic patients) compared to the conventional white on black (scotomas found in 3.6%) or red on black (scotomas in 8.9%) Amsler grids.30

REFERENCES

well established. Cataract surgery in patients with SLE is generally successful, although prognosis will be worse if there is visually significant posterior segment disease. Increased intraocular pressure due to exogenous corticosteroids may occur in up to 30% of the normal population, with 5% experiencing an increase of more than 15 mmHg. This steroid-induced ocular hypertension must be monitored (and often treated) to reduce progression to secondary glaucoma, with classical pathologically cupped optic disc and corresponding visual field loss. The aminoquinolones chloroquine and hydroxychloroquine have been widely used in the treatment of lupus. Both drugs can cause a reversible visually insignificant keratopathy (cornea verticillata), and more importantly an irreversible sight-threatening maculopathy. Initially, there is loss of the foveal reflex that may proceed to a fine granular appearance of the macula and finally a “bull’s-eye” maculopathy accompanied by a fall in visual acuity and a central scotoma. End-stage disease includes generalized atrophy, peripheral pigmentation akin to retinitis pigmentosa, arteriolar attenuation, and optic atrophy.27 Although both drugs may cause an identical retinopathy, this is extremely rare with hydroxychloroquine when used at currently recommended doses (<6.5 mg/kg/d). At these levels, only 20 cases of retinopathy have been reported in more than a million patients who have taken the drug. All of these cases had been taking the drug for more than five years. Combined data from case series found two cases of visually significant toxicity in more than 2500 patients.28 Although an equivalent threshold dose is sometimes quoted for chloroquine (<3.5 mg/kg/d), this is much less well established and thus should not be assumed to be a “safe” dosing range. Risk increases with increasing dose, increasing duration, and reduced renal function. Screening is recommended for all patients taking chloroquine. For patients taking hydroxychloroquine, the American Academy of Ophthalmology (AAO) recommends a baseline examination followed by screening according to risk. The initial examination incorporates a complete ophthalmic review (including dilated fundoscopy) and baseline visual field testing (Amsler or Humphrey 10-2). Special tests (including color vision testing, fundus photography, fluorescein angiography, and multifocal electroretinography) are optional. High-risk patients are seen annually, whereas low-risk

CONCLUSIONS Ocular manifestations of SLE, although relatively infrequent, are important both in terms of their direct effects (pain, loss of vision, and so on) and in the information they provide as to overall disease activity. Severe retinal vaso-occlusive disease and optic neuropathy are strongly associated with active CNS involvement. As control of systemic disease activity has improved, severe ocular complications have become uncommon. However, SLE is still a potentially blinding condition. Any visual symptoms require urgent ophthalmic assessment to identify sight-threatening disease requiring systemic therapy.

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3. Isenberg DA, et al. BILAG 2004: Development and initial validation of an updated version of the British Isles Lupus Assessment Group’s disease activity index for patients with systemic lupus erythematosus. Rheumatology (Oxford) 2005; 44(7):902-906.

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4. Jensen JL, et al. Oral and ocular sicca symptoms and findings are prevalent in systemic lupus erythematosus. J Oral Pathol Med 1999;28(7):317-322. 5. Spaeth GL. Corneal staining in systemic lupus erythematosus. N Engl J Med 1967;276(21):1168-1171. 6. Foster CS. Immunosuppressive therapy for external ocular inflammatory disease. Ophthalmology 1980;87(2):140-150. 7. Huey C, et al. Discoid lupus erythematosus of the eyelids. Ophthalmology 1983;90(12):1389-1398. 8. Grimson BS, Simons KB. Orbital inflammation, myositis, and systemic lupus erythematosus. Arch Ophthalmol 1983;101(5): 736-738. 9. Stavrou P, et al. Acute ocular ischaemia and orbital inflammation associated with systemic lupus erythematosus. Br J Ophthalmol 2002;86(4):474-475. 10. Stafford-Brady FJ, et al. Lupus retinopathy: Patterns, associations, and prognosis. Arthritis Rheum 1988;31(9):1105-1110. 11. Ushiyama O, et al. Retinal disease in patients with systemic lupus erythematosus. Ann Rheum Dis 2000;59(9):705-708. 12. Asherson RA, et al. Antiphospholipid antibodies: A risk factor for occlusive ocular vascular disease in systemic lupus erythematosus and the “primary” antiphospholipid syndrome. Ann Rheum Dis 1989;48(5):358-361. 13. Graham EM, et al. Cerebral and retinal vascular changes in systemic lupus erythematosus. Ophthalmology 1985;92(3):444-448. 14. Nag TC, Wadhwa S. Histopathological changes in the eyes in systemic lupus erythematosus: An electron microscope and immunohistochemical study. Histol Histopathol 2005;20(2): 373-382. 15. Sekimoto M, et al. Pseudoretinitis pigmentosa in patients with systemic lupus erythematosus. Ann Ophthalmol 1993;25(7): 264-266. 16. Chan WM, et al. Bilateral retinal detachment in a young woman. Lancet 2003;361(9374):2044. 17. Cunningham ET Jr., et al. Central serous chorioretinopathy in patients with systemic lupus erythematosus. Ophthalmology 1996;103(12):2081-2090.

18. Jabs DA, et al. Choroidopathy in systemic lupus erythematosus. Arch Ophthalmol 1988;106(2):230-234. 19. Jabs DA, et al. Optic neuropathy in systemic lupus erythematosus. Arch Ophthalmol 1986;104(4):564-568. 20. Keane JR. Eye movement abnormalities in systemic lupus erythematosus. Arch Neurol 1995;52(12):1145-1149. 21. McGalliard J, Bell AL. Acquired Brown’s syndrome in systemic lupus erythematosus: Another ocular manifestation. Clin Rheumatol 1990;9(3):399-400. 22. Galindo M, et al. Internuclear ophthalmoplegia in systemic lupus erythematosus. Semin Arthritis Rheum 1998;28(3): 179-186. 23. Yigit A, et al. The one-and-a-half syndrome in systemic lupus erythematosus. J Neuroophthalmol 1996;16(4):274-276. 24. Brandt KD, et al. Cerebral disorders of vision in systemic lupus erythematosus. Ann Intern Med 1975;83(2):163-169. 25. Carlow TJ, Glaser JS. Pseudotumor cerebri syndrome in systemic lupus erythematosus. JAMA 1974;228(2):197-200. 26. Rosenbaum JT, et al. Successful treatment of optic neuropathy in association with systemic lupus erythematosus using intravenous cyclophosphamide. Br J Ophthalmol 1997;81(2): 130-132. 27. Hobbs HE, Freedman A. Retinopathy following chloroquine therapy. Lancet 1959;2:478-480. 28. Buckley R, et al. Ocular toxicity and hydroxycychloroquine: guidelines for screening. 2004. Available at www.ncophth.ac.uk/ cloos/publications/oculartoxicity2004.pdf 29. Marmor MF, et al. Recommendation on screening for chloroquine and hydroxychloroquine retinopathy: A report by the American Academy of Ophthalmology. Ophthalmology 2002;109:1377-1382. 30. Almony A, et al. Threshold Amsler grid as a screening tool for asymptomatic patients on hydroxychloroquine therapy. Br J Opthalmol 2005;89:569-574.

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40

Pregnancy Megan E. B. Clowse, MD, MPH and Michelle Petri, MD, MPH

INTRODUCTION As systemic lupus erythematosus (SLE) treatment has improved, more women with lupus are able to become pregnant. Whereas pregnancy used to be discouraged in these women, it is now a frequent issue raised by patients to their physicians. The majority of women with lupus can successfully carry a pregnancy and deliver a healthy infant. The chances for this are improved if the lupus is well controlled and the woman is followed carefully by both a rheumatologist and an obstetrician trained in high-risk pregnancies. There are some women, however, who should carefully consider the risks of pregnancy prior to conception. These include women taking immunosuppressant medications (especially cyclophosphamide, mycophenolate mofetil, and methotrexate) and women with a prior history of arterial thrombosis from antiphospholipid syndrome (APS). Cyclophosphamide, mycophenolate mofetil, and methotrexate are contraindicated during pregnancy (particularly in the first trimester), as they frequently lead to fetal anomalies. Women with prior arterial thrombosis are at high risk for repeated (sometimes catastrophic) arterial thrombosis during pregnancy despite adequate anticoagulation. This chapter discusses the risks of pregnancy to lupus, the risks of lupus to pregnancy, and recommended methods of monitoring and treating these women in order to decrease these risks.

EPIDEMIOLOGY

The Effect of Lupus on Fertility The majority of women with SLE maintain their fertility and can even conceive during a time of severe lupus activity. The number of pregnancies that occur in a woman with lupus is similar to that seen in healthy women of the same age.1,2 However, the majority of the pregnancies occurred prior to the onset of lupus in the studies that compare fertility rates. It is not clear if the reason for fewer pregnancies after diagnosis is because of the increased age of the mother, the personal

choice of a woman with a serious illness, the warnings of physicians, or ovarian failure due to cyclophosphamide treatment.

The Effect of Lupus on Pregnancy Women with lupus have an increased rate of pregnancy loss over women without lupus (Table 40.1). In a comparison study of women with lupus compared with their friends and relatives, 60% of pregnancies that occurred after the onset of lupus ended in a live birth versus 80% of those in healthy friends and 87% in relatives (p<0.01).1 A population study in the United Kingdom confirmed this increase in pregnancy loss, with 23% of pregnancies after the onset of lupus ending in loss compared to 8% of those in controls (p<0.01).2 Similarly, a Greek case-control study demonstrated a fourfold increase in pregnancy loss in women with lupus versus healthy women.3 The rate of pregnancy loss varies between the reported cohorts of lupus pregnancies, ranging from 13 to 59% and averaging about 20%.4-6 The few studies with loss rates over 30% had an unusually high rate of elective abortions, perhaps inflating these rates beyond that physiologically associated with lupus.5,7 The timing for each woman’s entry into the cohort, whether it is prior to conception or in the midst of pregnancy, may also impact this rate. A woman who presents to a tertiary center for care in the second or third trimester is not at the same risk for pregnancy loss as a woman who is seen in her first trimester. The standard of care of lupus, high-risk pregnancies, and premature infants has improved over the decades in which the cohorts of lupus pregnancies have been collected. A comparison of lupus pregnancy loss rates from the 1960s (43%) to the twenty-first century (17%) demonstrates the benefits of these improvements.8,9 Miscarriage, defined as a pregnancy loss prior to 20 weeks of gestation, is the most common form of pregnancy loss in all pregnancies, including those affected by lupus. However, the rate of stillbirths (defined as a pregnancy loss after 20 weeks gestation) is particularly

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450

TABLE 40.1 THE EFFECT OF LUPUS ON PREGNANCY Fertility rate

Similar to women without lupus

Pregnancy loss rate

Increased over women without lupus Between 15 and 30% of pregnancies end with a loss

Increased risk for loss: High activity lupus Antiphospholipid syndrome Hypertension Lupus nephritis Prior pregnancy losses

Preterm birth rate

Increased over women without lupus At least a third of lupus pregnancies deliver before 37 weeks gestation

Increased risk for preterm delivery: High activity lupus Antiphospholipid syndrome Hypertension Prednisone use Immunosuppressive medications

elevated in lupus pregnancies. In developed countries, the stillbirth rate is generally under 1.5%. However, in pregnancies affected by lupus this rate can be as high as 11%.10-12 The strongest risk factors for pregnancy loss in lupus are increased lupus activity at conception or during pregnancy, and secondary antiphospholipid syndrome (APS).10,13-15 Other risk factors include hypertension, hypocomplementemia, and renal disease.10,16 Women who have the onset of lupus during pregnancy (“gestational lupus”) are also at high risk for pregnancy loss.14 A preterm birth is usually defined as a live birth before 37 weeks of gestation, which occurs in about 12% of all pregnancies in the United States.17 A third of lupus pregnancies, however, end with a preterm birth.6 Some of the increase in prematurity is iatrogenic: the physician decides to deliver a pregnancy early because of the presence of lupus. There is, however, also an increased risk of preterm premature rupture of membranes in women with lupus.18 Risk factors for preterm delivery are similar to those that cause pregnancy loss. These include increased lupus activity, secondary antiphospholipid syndrome (APS) and the presence of antiphospholipid antibodies, and prednisone use during pregnancy.6,10,13 The long-term effects of maternal lupus on offspring have not been systematically studied. There does not appear to be an increase in the rate of congenital abnormalities in these offspring.19 Several reports, however, demonstrate an increase in learning disabilities in the sons of lupus patients, particularly women with Ro or La antibodies.20-23 APS is associated with repeated pregnancy failure, preterm birth, and preeclampsia. This syndrome is further discussed in Chapter 36. The presence of Ro and/or La antibodies puts an infant at risk for congenital lupus, which is discussed in Chapter 42.

The Effect of Pregnancy on Lupus There is considerable debate as to whether pregnancy increases lupus activity or not. Some studies show that the rate of lupus flare in women who become pregnant increases several-fold over that of lupus patients who are not pregnant.12,24 Other studies have not found an increased risk for lupus flare during pregnancy.25-28 Fortunately, most lupus flares during pregnancy are mild to moderate and involve the skin and joints.3,4,24 There are a few women, however, who will develop a severe lupus flare during pregnancy. The risk for lupus flare exists in each trimester, and also in the weeks following delivery. The risk of flare during pregnancy is increased by the presence of lupus activity at the time of conception.14,15 In the Hopkins Lupus Cohort, women with increased activity in the six months prior to conception had a fourfold risk for increased lupus activity during pregnancy.13 Other risks for increased lupus activity in pregnancy include cessation of anti-malarial medication, prior history of lupus nephritis, and a history of three or more lupus flares.10,29,30 Proteinuria is common during lupus pregnancy and can be related to chronic renal disease, preeclampsia, or increased lupus activity. In the Hopkins Lupus Cohort, 25% of women had over 500 mg/24 hour of protein in their urine at some point during pregnancy. This rate is similar to that reported in other cohorts, with rates ranging between 5 and 20%.3,10,26 The rate of renal failure after pregnancy is low. There are isolated reports of dialysis dependence after a lupus nephritis flare during pregnancy. However, the vast majority of women regain renal function with aggressive medical therapy after delivery.3,10,24,31,32 The risk of maternal death after pregnancy, although low, may be higher than in the general population. A population-based study of all lupus-related births in

PATHOGENESIS Placental damage from inflammation and ischemia are likely the main pathogenic mechanisms for complications in lupus pregnancy. Placentas from pregnancies affected by lupus are smaller than those resulting from healthy pregnancy, with over 25% of them weighing less than 2 standard deviations below normal size.35 Placentas in pregnancies affected by active lupus may show signs of chronic inflammation. With immunofluorescence, deposits of immunoglobulins and complement may be found in uterine blood vessels and within the placenta.36,37 Inflammation in the uterus may damage the placenta, leading to poor delivery of blood, nutrients, and oxygen to the fetus—resulting in intrauterine growth retardation. Inflammation may also mimic the cytokine cascade found in infectious chorioamniitis, which can prompt premature rupture of membranes and preterm labor.38 The placental damage caused by secondary APS may be more related to complement activation by antiphospholipid antibodies than by thrombosis. A murine model has demonstrated that an intact complement cascade is required for pregnancy loss from exposure to antiphospholipid antibodies.39 This model also demonstrates that the main function of heparin may be to decrease complement activation, rather than to serve as an anticoagulatant.40 Ischemia can also lead to poor placental function. This may occur because of poor spiral artery recruitment early in pregnancy, leading to decreased blood flow as the demands of the fetus increase later in pregnancy.36 Ischemia may also occur from artery occlusion by thrombosis, particularly in patients with secondary APS. Up to a third of placentas from women with lupus and antiphospholipid antibodies have greater than 20% of their volume consumed by infarction, and almost half have diffuse ischemia.35 As the placenta ages, the degree of atherosis increases, which can lead to placental insufficiency. In lupus pregnancies, however, there is a much greater increase in atherosis, contributing to diffuse ischemia within the placenta.36

Monitoring Pregnancy The combined skills of a high-risk obstetrician and rheumatologist are best suited to following a woman

with lupus during pregnancy. The patient must be monitored for increased lupus activity, preeclampsia, and preterm labor. The fetus must be closely watched to ensure adequate growth and development, particularly in the third trimester.

PATHOGENESIS

California in a two-year period revealed 555 births and no maternal deaths.33 In the Hopkins Lupus Cohort, 3 out of 265 pregnancies ended with a maternal death within six weeks of delivery. Few other cohorts report any maternal deaths.3,11 The causes of death include HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, thrombosis associated with APS, pulmonary hypertention, and infection following severe lupus flare.34

Clinical Many symptoms of normal pregnancy can mimic symptoms of active lupus, and vice versa. In normal pregnancy, many women suffer significant fatigue. Pregnancy can also lead to skin changes such as palmar erythema, facial blushing, and melasma. Melasma (or the “mask of pregnancy”) results in a macular, photosensitive, hyperpigmented area over the cheeks and forehead. Hair loss is very common in the months after delivery, as is hair regrowth that can be most obvious in the temples. A pregnant woman may develop mild shortness of breath early in pregnancy from increased progesterone and late in pregnancy because of the enlarging uterus, although they are generally not hypoxic. Many pregnant women will develop back pain as their gravid uterus increases their lumbar lordosis. They may also develop noninflammatory knee effusions. Finally, pregnancy complications such as hypertension and preeclampsia may cause seizures or headaches. Signs and symptoms of active lupus in pregnancy are similar to those of non-pregnant lupus patients. In the Hopkins Lupus Pregnancy Cohort of 265 pregnancies, 26% of lupus patients had cutaneous findings during pregnancy—including malar rash, discoid lesions, and photosensitivity. Arthritic complaints were also common, occurring in 20% of pregnancies, with findings of joint swelling and morning stiffness. Serositis, manifesting as pleuritic chest pain (pain that increases with inspiration) and shortness of breath, is also an indication of increased lupus activity and occurred in 9% of pregnancies in the Hopkins cohort. Less common lupus findings included neurologic changes in 3% and pulmonary disease in 1%.

Laboratory Laboratory data can be helpful in predicting pregnancy complications, assessing lupus activity, and distinguishing between lupus activity and preeclampsia.

Predicting Complications There are several autoantibodies and laboratory changes that can either precede pregnancy or develop in the course of pregnancy. These should be assessed as early in pregnancy (or prior to pregnancy if possible) and repeated as needed. Ro and La Antibodies SSA/Ro antibodies are found in 35% and SSB/La antibodies are found in 15% of SLE patients.41 They can also

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be clinically associated with Sjogren’s syndrome. The presence of Ro and La antibodies places a fetus at risk for neonatal lupus; namely, congenital heart block or neonatal lupus rash (see Chapter 42). These autoantibodies should be assessed early in pregnancy. If they are positive in pregnancy or at any point prior to pregnancy, the women will need to undergo weekly fetal four-chamber echocardiography between weeks 16 and 28 of gestation. Antiphospholipid Antibodies APS is associated with a significant increase in pregnancy loss and preterm birth. (See Chapter 36.) At the onset of pregnancy, anticardiolipin antibodies and the lupus anticoagulant should be tested. If either of these is positive, they should be confirmed six weeks later. Double-stranded DNA Antibodies These antibodies are positive in 40% of patients with lupus.41 Antibodies to double-stranded DNA (dsDNA) vary over time, and their ability to predict lupus flare is debated.42,43 In the Hopkins Lupus Pregnancy Cohort, the presence of the dsDNA antibody increased the risk for preterm birth whether or not there was increased clinical lupus activity (44% without dsDNA versus 60% with dsDNA). In women with both highly active lupus clinically and dsDNA antibodies, the risk for perinatal mortality increased more than fivefold.44 Complement Complement activation is an important component of lupus pathophysiology, and a decrease in complement levels may signal an increase in lupus activity. Complement is best measured with C3 and C4 levels, both of which may fall during a lupus flare.45,46 During pregnancy in healthy patients, complement can increase by 10 to 50%, and there may be an increase in the degree of complement activation.47 Half of the pregnancies in the Hopkins Lupus Pregnancy Cohort were hypocomplementemic. Hypocomplementemia during pregnancy led to a small increase in the risk for pregnancy loss. However, women with both increased lupus activity clinically and low complement had a fivefold increase in risk for stillbirth. Only 16% of pregnancies with increased lupus activity clinically and low complement resulted in a full-term birth.44 C3 and C4 levels should be monitored monthly during pregnancy.

Monitoring Lupus Activity

452

Complete Blood Counts Thrombocytopenia, anemia, and lymphopenia can all be signs of increased lupus activity. Changes related to pregnancy, however, may also trigger changes in blood counts. During normal pregnancy, the hematocrit can

fall several points because of hemodilution from the woman’s increased vascular volume. This can lead to a diagnosis of anemia in up to half of pregnant women.48 The neutrophil count may increase during pregnancy, but the lymphocyte count remains fairly unchanged.49 Up to 8% of normal pregnancies will have a platelet count between 100,000 to 130,000. A platelet count below 100,000 may be associated with active lupus, APS, intrauterine demise, HELLP syndrome, or preeclampsia. Renal Function In normal pregnancy, the glomerular filtration rate is increased due to increased maternal blood volume. This frequently leads to a decrease in serum creatinine during pregnancy. The increased renal flow may also prompt a mild increase in proteinuria, which can be more dramatic in women with prior renal disease. An increasing serum creatinine is worrisome for increased lupus nephritis during pregnancy. A urinalysis should also be performed monthly to monitor for proteinuria. Any indication of proteinuria should be followed with either a 24-hour urine for protein or a spot urine protein:creatinine ratio.50 Proteinuria in the first half of pregnancy is likely from lupus activity. New-onset proteinuria in the latter half of pregnancy, however, may be from lupus activity, preeclampsia, or both. Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) The ESR is markedly elevated in most pregnant women, and therefore does not play a role in monitoring lupus activity during pregnancy. The CRP has not been widely studied during pregnancy, particularly in lupus patients, and thus its utility is unknown.

Differentiating Between SLE and Preeclampsia One of the biggest challenges in caring for women with lupus during pregnancy can be determining whether the presence of hypertension and proteinuria in the latter half of pregnancy is from preeclampsia or lupus. The treatment for these two problems is different: preeclampsia requires delivery for recovery and lupus activity requires immunosuppression. Preeclampsia is defined as the presence of proteinuria ≥300 mg/24 hours and hypertension over 140/90 that both develop after at least 20 weeks of pregnancy. Women with preeclampsia may also develop symptoms of headache, peripheral edema, and rapid weight gain. Preeclampsia occurs in 2 to 3% of all pregnancies. Risk factors for preeclampsia include prior hypertension, diabetes, obesity, multiple gestations, a prior pregnancy with preeclampsia, thrombophilia, and lupus.51 It is one of the leading causes of preterm birth

Radiology Ultrasonography is the mainstay for fetal monitoring and assessment. An ultrasound is indicated early in pregnancy to estimate the date of conception and date of delivery, and to provide a baseline for fetal growth. All pregnancies should have a thorough anatomy screening ultrasound between weeks 18 and 20 to assess for any fetal anomalies. Every three to four weeks thereafter an ultrasound to assess fetal growth and amniotic fluid levels should be obtained in lupus pregnancies, as these can be early markers for placental insufficiency.57 Fetal surveillance is started about week 26 and continued weekly or twice weekly until delivery in all

lupus pregnancies. The purpose is to assess for fetal distress and hypoxia, as well as to ascertain the stability of the cervix. The “modified” biophysical profile is the most common measure used to perform this surveillance in the United States. This is comprised of a non-stress test, amniotic fluid volume, and an assessment of fetal breathing and movement. Other techniques, such as umbilical artery Doppler velocimetry waveform analysis, can also be used. In this analysis, an increase in vascular resistance may indicate placental insufficiency, which can lead to fetal hypoxia. Consideration of delivery should be made if these studies reveal significant abnormalities. Pregnancies at risk for congenital heart block from SSA/Ro and/or SSB/La antibodies should be screened with four-chamber fetal echocardiography. (See Chapter 42.) These echocardiograms should be obtained weekly between weeks 16 and 28 of gestation in order to identify the earliest stages of fetal heart block. If identified, treatment with dexamethasone or betamethasone should be initiated. Betamethasone is preferred over other corticosteroids because it easily passes through the placenta to treat the fetus and it may have a smaller impact on the neuropsychiatric development of the offspring than dexamethasone. Although third-degree congenital heart block is usually irreversible, an attempt at treatment with betamethasone is warranted. First- and second-degree heart block may be reversible with steroid therapy.58,59 For a more detailed discussion of neonatal lupus syndrome and its treatment, see Chapter 42.

MEDICATIONS IN LUPUS PREGNANCY

and rarely can lead to the death of the infant and/or mother. When the hypertension and proteinuria are accompanied by grand mal seizures, it is considered eclampsia. Severe preeclampsia is characterized by more dramatic elevations in proteinuria (≥5000 mg/24 hr) and hypertension (≥160/110).53 Women with severe preeclampsia may also have confusion, strokes, thrombocytopenia, rising creatinine, and worsening liver function. HELLP syndrome is a variant of preeclampsia with hemolysis, elevated liver tests, and low platelets. Women with HELLP have microangiopathic hemolytic anemia, but do not always have hypertension or proteinuria.52 Based on the presentations of preeclampsia, severe preeclampsia, and HELLP syndrome, it is easy to see how preeclampsia or HELLP could be confused with active lupus. Many women with lupus can develop thrombocytopenia, proteinuria, and hypertension. The timing of the symptoms can be helpful, as preeclampsia does not usually start until after 20 weeks of gestation. Several markers may help to distinguish lupus from these pregnancy-related complications. ● The presence of other symptoms of active lupus: Arthritis, lupus rashes, and mouth ulcers all may be indications that increased lupus activity is to blame. A thorough review of symptoms and physical examination may reveal these clues. ● Hypocomplementemia or rising dsDNA antibodies: These laboratory indications of increased lupus activity may be helpful in diagnosing lupus activity during pregnancy. ● Serum uric acid: Some studies show that the uric acid level will rise over 5.0 mg/dl in women with preeclampsia or HELLP syndrome.54 Similar elevations have not been identified in lupus flares in the absence of renal failure. ● Urine calcium level: Women with preeclampsia have low levels of calcium in their urine. A 24-hour urine for calcium or a spot calcium to creatinine ratio can identify preeclampsia.55,56

MEDICATIONS IN LUPUS PREGNANCY The management of lupus during pregnancy requires a balance of the benefits of treatment to the mother and the risks to the fetus. Frequently, medications should be maintained during pregnancy in order to prevent the reactivation of lupus activity that will put the pregnancy at risk. There are, however, some medications that are completely contraindicated during pregnancy. (See Table 40.2.).

Prenatal Multivitamin All women, prior to and during pregnancy, should take a prenatal multivitamin daily with at least 400 mg of folic acid to decrease the risk of neural tube defects.

Non-steroidal Anti-inflammatories The safety of NSAIDs during pregnancy has been well researched. There appears to be a small increase in the risk of miscarriage for women who take NSAIDs around the time of conception.60 There is also concern about the use of NSAIDs in the final weeks of pregnancy, as they may prompt premature closure of the ductus arteriosis

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TABLE 40.2 MEDICATIONS FOR LUPUS DURING PREGNANCY Medication

Uses

Risks to the Fetus

Prenatal multivitamin

All pregnant women to prevent neural tube defects and iron-deficiency

None

Non-steroidal antiinflammatories (NSAIDs)

Pain control from arthritis, serositis, myalgias Tocolytic agent for preterm labor

May increase rate of miscarriage if taken around the time of conception (60) Likely not associated with congenital abnormalities Avoid in third trimester as may promote closure of the ductus arteriosis leading to fetal pulmonary hypertension (61) May cause oligohydramnios (63) Decreases uterine activity, prolongs labor

Acetaminophen (Paracetamol)

Pain control

Considered safe during pregnancy

Hydroxychloroquine

Prevention and treatment of lupus activity

No increase in congenital abnormalities (29, 72, 91) Decrease in lupus activity during pregnancy (29) No increase in pregnancy loss or preterm birth (72)

Azathioprine

Prevention and treatment of lupus activity, especially lupus nephritis

No clear increase in a specific congenital abnormality (73) Increase in preterm birth and growth retardation may be caused by underlying disease process (92)

Low-dose aspirin

Antiphospholipid Syndrome

No increase in congenital abnormalities (85) No increase in ductus arteriosus closure, increased risk of neonatal bleeding, or adverse pregnancy outcomes (93)

Heparin and low-molecularweight heparin

Antiphospholipid Syndrome

No increase in adverse pregnancy outcomes (94) Caution with Epidural anesthesia

Mycophenolate Mofetil

Avoid in pregnancy

Very few reported cases of pregnancy exposure; several reports of congenital anomalies (82)

Cyclophosphamide

Avoid in pregnancy

Reported cases of fetal demise and congenital abnormalities (79)

Methotrexate

Avoid in pregnancy

Used as abortifacient; causes congenital anomalies (95)

and may decrease uterine contractility.61,62 In some circumstances, NSAIDs have been associated with oligohydramnios, likely from suppression of blood flow to fetal kidneys.63 The judicious use of NSAIDs in the second trimester is considered safe. Acetaminophin is a safe alternative to NSAIDs for pain control during pregnancy.

Corticosteroids

454

Corticosteroids are the mainstay of lupus therapy during pregnancy. In the placenta, the enzyme 11[beta]-hydroxysteroid dehydrogenase (11[beta]HSD) deactivates prednisone and prednisolone, only allowing 10% of the drug to pass to the fetus. Therefore, these are good medications for treating active lupus in the mother. On the other hand, betamethasone is not significantly metabolized by 11(beta)-HSD, and passes easily through the placenta.

Betamethasone is a better drug for treating the fetus, as in cases of neonatal lupus and fetal lung maturation in anticipation of a preterm birth.64 When rodents are exposed to high doses of corticosteroids during pregnancy, their offspring have an increased incidence of cleft palate. Some reports show a small increased risk for cleft palate in human fetuses exposed to corticosteroids, but other studies do not bear this out.65,66 Prednisone exposure may increase the risk for intrauterine growth retardation, although this effect may be explained by the underlying illness requiring the medication.67 Offspring exposed to corticosteroids in utero do not have complications in studies up to age 20, other than slightly higher blood pressure levels.68 Prednisone use during pregnancy can increase the incidence of maternal hypertension, diabetes mellitus, and preeclampsia. In studies of the use of prednisone

Hydroxychloroquine Many women with lupus conceive while taking hydroxychloroquine. The cessation of hydroxychloroquine either prior to or during pregnancy places a woman at a several-fold risk for lupus flare.29,71 Although most of these flares will be cutaneous or arthritis in nature, they will likely require the use of further immunosuppression as treatment. Both the flare and the other immunosuppressants may put the mother and fetus at risk of complications. The half-life of hydroxychloroquine is up to three months. Therefore, pregnancies in which this medication was stopped just prior to or after conception will still have exposure to it. Hydroxychloroquine has not been demonstrated to increase birth defects, nor has it been associated with increased pregnancy loss or preterm birth.72 For these reasons, we recommend continuation of hydroxychloroquine in women trying to conceive and those who become pregnant. Chloroquine, however, is not recommended due to the risks of congenital abnormalities.73

Azathioprine The use of azathioprine during pregnancy has been well documented in women with prior solid-organ transplants. Azathioprine is transformed by the placenta to thiouric acid, an inactive metabolite. Because of this, there is little damage to the fetus despite use throughout pregnancy. There has been no specific birth defect associated with azathioprine identified in over 400 pregnancies.73 The rate of miscarriage is not increased with azathioprine. Despite a few reports of an increase in growth retardation, neonatal immunosuppression, leukopenia, and pancytopenia,74 this appears to be very rare in practice. There is an increased rate of preterm births in women on azathioprine, but the explanation may be other medications or the underlying disease that requires the use of azathioprine.75 In the Hopkins Lupus Pregnancy Cohort, 31 pregnancies in women with lupus on azathioprine were seen. A higher proportion of these pregnancies resulted in pregnancy loss (19 versus 7% without azathioprine), but this was confounded by the increase in lupus activity in the women on azathioprine.76

Cyclophosphamide Cyclophosphamide is a known teratogen with significant limb abnormalities and cognitive impairment resulting from first-trimester exposure.77 In reports of pregnant women treated with cyclophosphamide for breast cancer in the second and third trimesters, few pregnancy complications or congenital abnormalities are noted.78 Among lupus patients, however, the experience of cyclophosphamide use during pregnancy is more worrisome. Two cases of inadvertent cyclophosphamide administration in the first weeks after conception led to two miscarriages.79 Three cases of second- and third-trimester use of cyclophosphamide for severe lupus flare are published in the literature and two of these pregnancies resulted in pregnancy loss shortly after administration of the drug.79-81 When lupus activity is severe enough during pregnancy to require cyclophosphamide, the viability of the pregnancy is in doubt irrespective of the therapy taken. Prior to the use of this medication, a candid discussion of the risks must be undertaken with the mother.

MEDICATIONS IN LUPUS PREGNANCY

for APS, pregnancies exposed to prednisone had higher rates of preterm birth, hypertension, and diabetes mellitus.69,70 Because of these side effects, prophylactic use of prednisone during pregnancy is not recommended. With lupus flares, however, pulse dose and higher doses of daily prednisone may be necessary to regain control of the disease. The risk of unchecked lupus activity is greater than the use of moderate to high-dose corticosteroids during pregnancy.

Cellcept Mycophenolate mofetil causes fetal resorption in animals given therapeutic levels of the medication. Only a handful of pregnancies with exposure to mycophenolate mofetil have been reported in humans, none of which have been born at full term.82 There is one case report of severe facial and midline congenital abnormalities after first-trimester exposure to the medication.83 Another case report describes mild limb abnormalities.82 Given the small amount of data available, we do not recommend the use of mycophenolate mofetil during pregnancy. Current advice is that it should be stopped at least three months before conception if possible.

Methotrexate A pregnancy with first-trimester exposure to methotrexate is at increased risk for fetal abnormalities. Although there are case reports of healthy offspring after first-trimester exposure, there are also reports of severe cranial and CNS abnormalities, limb defects, developmental delay, and intellectual impairment.67 The risk to the pregnancy of methotrexate exposure is best demonstrated by its use as an abortifacient for tubal pregnancies. Women taking methotrexate should protect against pregnancy and stop the medication three to six months prior to conception.

Thalidomide Thalidomide is associated with severe limb abnormalities in offspring exposed to the drug in utero. For this reason, the medication should not be used in women

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with reproductive potential without the concomitant use of reliable contraception.

ANTIPHOSPHOLIPID SYNDROME The treatment of APS during pregnancy requires the use of heparin and aspirin to prevent pregnancy loss. Women with APS who are trying to conceive should take daily low-dose aspirin prior to and throughout pregnancy. Despite the complications associated with higher doses of NSAIDs, there are minimal side effects to the pregnancy and fetus from low-dose aspirin. Studies have documented that women at risk for preeclampsia may lower their blood pressure and decrease the incidence of preeclampsia through the use of daily low-dose aspirin.84 A follow-up of children born in a large study of low-dose aspirin use showed no increase in congenital defects or developmental delay up to 18 months.85 Low–molecular-weight heparin, either at prophylactic doses if the woman has no history of arterial or venous thrombosis, or at full anticoagulant doses if the woman does, should be started when the pregnancy is diagnosed (see Chapter 42). Although there are reports of increased pregnancy loss, prematurity, and infant intraventricular hemorrhage in pregnancies exposed to heparin, it is difficult to assess the effect of the medication versus the indication for it in causing these problems.86-88 Low–molecular-weight heparin should be switched to unfractionated heparin before delivery, because low–molecular-weight heparin has a longer duration of action. Caution must be used when spinal epidural anesthesia is planned for women on anticoagulants because of the risk for hemorrhage leading to a compressive spinal

lesion and paralysis.89 It is recommended that heparin be discontinued 12 hours prior to the insertion of an epidural catheter. Protamine sulfate may be used to reverse the anticoagulant effects of heparin if necessary.34 Anticoagulation must be continued for six weeks after delivery, as women with APS are at increased risk for thrombosis during this period.90

RECOMMENDED MEDICATIONS FOR LUPUS IN PREGNANCY In women without active lupus immediately prior to or during pregnancy, no specific medications are required. (See Table 40.3.) If a woman is on hydrochloroquine pre-conception, she should continue it during pregnancy to avoid reactivation of lupus activity. Women who are on azathioprine to control lupus activity prior to pregnancy should continue this medication during pregnancy. Mild lupus activity during pregnancy, particularly arthritis and skin disease, may be best treated with hydroxychloroquine or low-dose prednisone (i.e., up to 20 mg per day). Modest increases in proteinuria, serositis, thrombocytopenia, or other signs of moderate lupus activity may require somewhat higher doses of corticosteroids. Persistently rising proteinuria or falling platelets could prompt initiation of azathioprine. A pregnancy complicated by severe lupus activity with dangerously low platelet counts, rising creatinine, CNS disease, and/or pulmonary disease is at great risk for fetal demise. Pulse-dose intravenous corticosteroids and higher doses of azathioprine should be employed. Another option is intravenous immunoglobulin (IVIg), which has few fetal side effects and may regain control of severely active lupus in some organs.96

TABLE 40.3 TREATMENT OF LUPUS DURING PREGNANCY No Lupus Activity

Mild Lupus Activity

Moderate Lupus Activity

Severe Lupus Activity

Pre-natal Multivitamin

X

X

X

X

Hydroxychloroquine

X (a)

X

X

X

NSAID (during second trimester)

X

X

X

Corticosteroids

X Low dose

Azathioprine

456

X High dose

X High dose and IV pulse

X

X

Cyclophosphamide

X (b)

Intravenous Immunoglobuline (IVIg)

X

(a)Continue Hydroxychloroquine, but do not start it unless there is lupus activity. (b)Cyclophosphamide puts the pregnancy at great risk and should only be used when the life of the mother is at risk.

CONCLUSIONS The prognosis for both mother and baby is good in lupus pregnancy in the twenty-first century. There is a place for optimism among physicians and patients, as

many of the complications of lupus and preterm delivery are well managed today. However, women with risk factors such as active lupus, hypertension, renal disease, and APS still may have difficult pregnancies and are at risk for pregnancy loss. When a woman with lupus becomes pregnant, she must be closely monitored by a rheumatologist and high-risk obstetrician to improve her chances of a successful pregnancy.

REFERENCES

The health of the mother and fetus must be weighed if the decision is made to use cyclophosphamide in these situations.

REFERENCES 1. Petri M, Allbritton J. Fetal outcome of lupus pregnancy: A retrospective case-control study of the hopkins lupus cohort. J Rheumatol 1993;20:650. 2. Hardy CJ, Palmer BP, Morton SJ, et al. Pregnancy outcome and family size in systemic lupus erythematosus: A case-control study. Rheumatology (Oxford) 1999;38:559. 3. Georgiou PE, Politi EN, Katsimbri P, et al. Outcome of lupus pregnancy: A controlled study. Rheumatology (Oxford) 2000;39:1014. 4. Carmona F, Font J, Cervera R, et al. Obstetrical outcome of pregnancy in patients with systemic lupus erythematosus: A study of 60 cases. Eur J Obstet Gynecol Reprod Biol 1999;83:137. 5. Kiss E, Bhattoa HP, Bettembuk P, et al. Pregnancy in women with systemic lupus erythematosus. Eur J Obstet Gynecol Reprod Biol 2002;101:129. 6. Clark CA, Spitzer KA, Nadler JN, et al. Preterm deliveries in women with systemic lupus erythematosus. J Rheumatol 2003;30:2127. 7. Wong KL, Chan FY, Lee CP. Outcome of pregnancy in patients with systemic lupus erythematosus: A prospective study. Arch Intern Med 1991;151:269. 8. Clark CA, Spitzer KA, Laskin CA. Decrease in pregnancy loss rates in patients with systemic lupus erythematosus over a 40-year period. J Rheumatol 2005;32:1709. 9. Buyon JP. Dispelling the preconceived notion that lupus pregnancies result in poor outcomes. J Rheumatol 2005;32:1641. 10. Cortes-Hernandez J, Ordi-Ros J, Paredes F, et al. Clinical predictors of fetal and maternal outcome in systemic lupus erythematosus: A prospective study of 103 pregnancies. Rheumatology (Oxford) 2002;41:643. 11. Varner MW, Meehan RT, Syrop CH, et al. Pregnancy in patients with systemic lupus erythematosus. Am J Obstet Gynecol 1983;145:1025. 12. Lima F, Buchanan NM, Khamashta MA, et al. Obstetric outcome in systemic lupus erythematosus. Semin Arthritis Rheum 1995;25:184. 13. Clowse M, Witter FR, Magder LC, et al. The impact of increased lupus activity on obstetrical outcomes. Arthritis Rheum 2005;52:514. 14. Bobrie G, Liote F, Houillier P, et al. Pregnancy in lupus nephritis and related disorders. Am J Kidney Dis 1987;9:339. 15. Moroni G, Ponticelli C. The risk of pregnancy in patients with lupus nephritis. J Nephrol 2003;16:161. 16. Martinez-Rueda JO, Arce-Salinas CA, Kraus A, et al. Factors associated with fetal losses in severe systemic lupus erythematosus. Lupus 1996;5:113. 17. Dattel BJ, Chescheir N, Lockwood C, et al. (eds.). Your Pregnancy and Birth, Fourth Edition. Washington DC: Meredith Books 2005. 18. Johnson MJ, Petri M, Witter FR, et al. Evaluation of preterm delivery in a systemic lupus erythematosus pregnancy clinic. Obstet Gynecol 1995;86:396. 19. Bonaminio P, de Regnier RA, Chang E, et al. Minor physical anomalies are not increased in the offspring of mothers with SLE. Ann Rheum Dis 2006;65(2)246.

20. Ross G, Sammaritano L, Nass R, et al. Effects of mothers’ autoimmune disease during pregnancy on learning disabilities and hand preference in their children. Arch Pediatr Adolesc Med 2003;157:397. 21. McAllister DL, Kaplan BJ, Edworthy SM, et al. The influence of systemic lupus erythematosus on fetal development: Cognitive, behavioral, and health trends. J Int Neuropsychol Soc 1997;3:370. 22. Lahita RG. Systemic lupus erythematosus: Learning disability in the male offspring of female patients and relationship to laterality. Psychoneuroendocrinology 1988;13:385. 23. Neri F, Chimini L, Bonomi F, et al. Neuropsychological development of children born to patients with systemic lupus erythematosus. Lupus 2004;13:805. 24. Petri M. Hopkins lupus pregnancy center: 1987 to 1996. Rheum Dis Clin North Am 1997;23:1. 25. Urowitz MB, Gladman DD, Farewell VT, et al. Lupus and pregnancy studies. Arthritis Rheum 1993;36:1392. 26. Tincani A, Faden D, Tarantini M, et al. Systemic lupus erythematosus and pregnancy: A prospective study. Clin Exp Rheumatol 1992;10:439. 27. Lockshin MD, Reinitz E, Druzin ML, et al. Lupus pregnancy: Casecontrol prospective study demonstrating absence of lupus exacerbation during or after pregnancy. Am J Med 1984;77:893. 28. Lockshin MD. Pregnancy does not cause systemic lupus erythematosus to worsen. Arthritis Rheum 1989;32:665. 29. Clowse M, Magder LC, Petri M. The effect of hydroxychloroquine on pregnancy outcomes and disease activity in lupus patients. Arthritis Rheum 2004;50:1846. 30. Levy RA, Vilela VS, Cataldo MJ, et al. Hydroxychloroquine (hcq) in lupus pregnancy: Double-blind and placebo-controlled study. Lupus 2001;10:401. 31. Rubbert A, Pirner K, Wildt L, et al. Pregnancy course and complications in patients with systemic lupus erythematosus. Am J Reprod Immunol 1992;28:205. 32. Julkunen H. Pregnancy and lupus nephritis. Scand J Urol Nephrol 2001;35:319. 33. Yasmeen S, Wilkins EE, Field NT, et al. Pregnancy outcomes in women with systemic lupus erythematosus. J Matern Fetal Med 2001;10:91. 34. McMillan E, Martin WL, Waugh J, et al. Management of pregnancy in women with pulmonary hypertension secondary to SLE and anti-phospholipid syndrome. Lupus 2002;11:392. 35. Magid MS, Kaplan C, Sammaritano LR, et al. Placental pathology in systemic lupus erythematosus: A prospective study. Am J Obstet Gynecol 1998;179:226. 36. Abramowsky CR. Lupus erythematosus, the placenta, and pregnancy: A natural experiment in immunologically mediated reproductive failure. Prog Clin Biol Res 1981;70:309. 37. Grennan DM, McCormick JN, Wojtacha D, et al. Immunological studies of the placenta in systemic lupus erythematosus. Ann Rheum Dis 1978;37:129. 38. Salafia CM, Parke AL. Placental pathology in systemic lupus erythematosus and phospholipid antibody syndrome. Rheum Dis Clin North Am 1997;23:85.

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39. Girardi G, Salmon JB. The role of complement in pregnancy and fetal loss. Autoimmunity 2003;36:19. 40. Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nat Med 2004;10:1222. 41. Buyon J. Systemic Lupus Erythematosus B: Clinical and Laboratory Findings. In Klippel JH, Crawford LJ, Stone JH, et al. (eds.), Primer on the Rheumatic Disease. Atlanta: Arthritis Foundation 2001. 42. Ho A, Magder LS, Barr SG, et al. Decreases in anti-double-stranded DNA levels are associated with concurrent flares in patients with systemic lupus erythematosus. Arthritis Rheum 2001;44:2342. 43. ter Borg EJ, Horst G, Hummel EJ, et al.. 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. 44. Clowse M, Magder LC, Petri M. Complement and anti-dsdna titers are predictive of pregnancy outcomes. Arthritis Rheum 2004;50:1011. 45. Ho A, Barr SG, Magder LS, et al. A decrease in complement is associated with increased renal and hematologic activity in patients with systemic lupus erythematosus. Arthritis Rheum 2001;44:2350. 46. Zonana-Nacach A, Salas M, Sanchez ML, et al. Measurement of clinical activity of systemic lupus erythematosus and laboratory abnormalities: A 12-month prospective study. J Rheumatol 1995;22:45. 47. Richani K, Soto E, Romero R, et al. Normal pregnancy is characterized by systemic activation of the complement system. J Matern Fetal Neonatal Med 2005;17:239. 48. Samuels P. Hematologic complications of pregnancy. In Gabbe SG (ed.), Obstetrics: Normal and Problem Pregnancies, Fourth Edition. New York: Churchill Livingstone 2002. 49. Buyon JP, Kalunian KC, Ramsey-Goldman R, et al. Assessing disease activity in SLE patients during pregnancy. Lupus 1999;8:677. 50. Christopher-Stine L, Petri M, Astor BC, et al. Urine protein-tocreatinine ratio is a reliable measure of proteinuria in lupus nephritis. J Rheumatol 2004;31:1557. 51. Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science 2005;308:1592. 52. Baxter JK, Weinstein L. HELLP syndrome: The state of the art. Obstet Gynecol Surv 2004;59:838. 53. Schroeder, BM. ACOG practice bulletin on diagnosing and managing preeclampsia and eclampsia: American College of Obstetricians and Gynecologists. Am Fam Physician 2002;66:330. 54. Williams KP, Galerneau F. The role of serum uric acid as a prognostic indicator of the severity of maternal and fetal complications in hypertensive pregnancies. J Obstet Gynaecol Can 2002;24:628. 55. Taufield PA, Ales KL, Resnick LM, et al. Hypocalciuria in preeclampsia. N Engl J Med 1987;316:715. 56. Ramos JG, Martins-Costa SH, Kessler JB, et al. Calciuria and preeclampsia. Braz J Med Biol Res 1998;31:519. 57. Branch DW. Pregnancy in patients with rheumatic diseases: Obstetric management and monitoring. Lupus 2004;13:696. 58. Saleeb S, Copel J, Friedman D, et al. Comparison of treatment with fluorinated glucocorticoids to the natural history of autoantibody-associated congenital heart block: Retrospective review of the research registry for neonatal lupus. Arthritis Rheum 1999;42:2335. 59. Ishimaru S, Izaki S, Kitamura K, et al. Neonatal lupus erythematosus: Dissolution of atrioventricular block after administration of corticosteroid to the pregnant mother. Dermatology 1994;189(1):92. 60. Li DK, Liu L, Odouli R. Exposure to non-steroidal anti-inflammatory drugs during pregnancy and risk of miscarriage: Population based cohort study. BMJ 2003;327:368. 61. Paladini D, Marasini M, Volpe P. Severe ductal constriction in the third-trimester fetus following maternal self-medication with nimesulide. Ultrasound Obstet Gynecol 2005;25:357. 62. King J, Flenady V, Cole S, et al. Cyclo-oxygenase (cox) inhibitors for treating preterm labour. Cochrane Database Syst Rev 2005;CD001992. 63. Hill LM, Lazebnik N, Many A. Effect of indomethacin on individual amniotic fluid indices in multiple gestations. J Ultrasound Med 1996;15:395.

64. van Runnard Heimel PJ, Franx A, Schobben AF. Corticosteroids, pregnancy, and HELLP syndrome: A review. Obstet Gynecol Surv 2005;60(1):57. 65. Park-Wyllie L, Mazzotta P, Pastuszak A, et al. Birth defects after maternal exposure to corticosteroids: Prospective cohort study and meta-analysis of epidemiological studies. Teratology 2000;62:385. 66. Fraser FC, Sajoo A. Teratogenic potential of corticosteroids in humans. Teratology 1995;51:45. 67. Rayburn WF. Connective tissue disorders and pregnancy: Recommendations for prescribing. J Reprod Med 1998;43:341. 68. Doyle LW, Ford GW, Davis NM, et al. Antenatal corticosteroid therapy and blood pressure at 14 years of age in preterm children. Clin Sci (Lond) 2000;98:137. 69. Laskin CA, Bombardier C, Hannah ME, et al. Prednisone and aspirin in women with autoantibodies and unexplained recurrent fetal loss. N Engl J Med 1997;337:148. 70. Vaquero E, Lazzarin N, Valensise H, et al. Pregnancy outcome in recurrent spontaneous abortion associated with antiphospholipid antibodies: A comparative study of intravenous immunoglobulin versus prednisone plus low-dose aspirin. Am J Reprod Immunol 2001;45:174. 71. Canadian Consensus Conference on Hydroxychloroquine. J Rheumatol 2000;27:2919. 72. Costedoat-Chalumeau N, Amoura Z, Huong du LT, et al. Safety of hydroxychloroquine in pregnant patients with connective tissue diseases: Review of the literature. Autoimmun Rev 2005;4:111. 73. Petri M. Immunosuppressive drug use in pregnancy. Autoimmunity 2003;36:51. 74. DeWitte DB, Buick MK, Cyran SE, et al. Neonatal pancytopenia and severe combined immunodeficiency associated with antenatal administration of azathioprine and prednisone. J Pediatr 1984;105:625. 75. Sgro MD, Barozzino T, Mirghani HM, et al. Pregnancy outcome post renal transplantation. Teratology 2002;65:5. 76. Clowse M, Magder LC, Witter FR, et al. Azathioprine use in lupus pregnancy. Arthritis Rheum 2005;52:S386. 77. Paladini D, Vassallo M, D’Armiento MR, et al. Prenatal detection of multiple fetal anomalies following inadvertent exposure to cyclophosphamide in the first trimester of pregnancy. Birth Defects Res Part A Clin Mol Teratol 2004;70:99. 78. Berry DL, Theriault RL, Holmes FA, et al. Management of breast cancer during pregnancy using a standardized protocol. J Clin Oncol 1999;17:855. 79. Clowse ME, Magder L, Petri M. Cyclophosphamide for lupus during pregnancy. Lupus 2005;14:593. 80. Clowse M, Petri MP. Cyclophosphamide for lupus in pregnancy: Seventh International Congress on SLE and Related Conditions. Lupus 2004:115. 81. Kart Koseoglu H, Yucel AE, Kunefeci G, et al. Cyclophosphamide therapy in a serious case of lupus nephritis during pregnancy. Lupus 2001;10:818. 82. Pergola PE, Kancharla A, Riley DJ. Kidney transplantation during the first trimester of pregnancy: Immunosuppression with mycophenolate mofetil, tacrolimus, and prednisone. Transplantation 2001;71:994. 83. Le Ray C, Coulomb A, Elefant E, et al. Mycophenolate mofetil in pregnancy after renal transplantation: A case of major fetal malformations. Obstet Gynecol 2004;103:1091. 84. Duley L, Henderson-Smart DJ, Knight M, et al. Antiplatelet agents for preventing pre-eclampsia and its complications. Cochrane Database Syst Rev 2004;CD004659. 85. CLASP Collaborative Group. Low dose aspirin in pregnancy and early childhood development: Follow up of the collaborative low dose aspirin study in pregnancy. Br J Obstet Gynaecol 1995;102:861. 86. Hall JG, Pauli RM, Wilson KM. Maternal and fetal sequelae of anticoagulation during pregnancy. Am J Med 1980;68:122-140. 87. Nageotte MP, Freeman RK, Garite TJ, Block RA. Anticoagulation in pregnancy. Am J Obstet Gynecol 1981;141:472-473. 88. Malloy MH, Cutter GR. The association of heparin exposure with intraventricular hemorrhage among very low birth weight infants. J Perinatol 1995;15:185-191. 89. Sternlo JE, Hybbinette CH. Spinal subdural bleeding after attempted epidural and subsequent spinal anaesthesia in a

93. CLASP Collaborative Group. CLASP: A randomised trial of low-dose aspirin for the prevention and treatment of pre-eclampsia among 9364 pregnant women. Lancet 1994;343:619. 94. Ginsberg JS, Hirsh J, Turner DC, et al. Risks to the fetus of anticoagulant therapy during pregnancy. Thromb Haemost 1989;61:197. 95. Yedlinsky NT, Morgan FC, Whitecar PW. Anomalies associated with failed methotrexate and misoprostol termination. Obstet Gynecol 2005;105:1203. 96. Gordon C, Kilby MD. Use of intravenous immunoglobulin therapy in pregnancy in systemic lupus erythematosus and antiphospholipid antibody syndrome. Lupus 1998;7:429.

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patient on thromboprophylaxis with low molecular weight heparin. Acta Anaesthesiol Scand 1995;39:557. 90. Erkan D. The relation between antiphospholipid syndrome-related pregnancy morbidity and non-gravid vascular thrombosis: A review of the literature and management strategies. Curr Rheumatol Rep 2002;4:379. 91. Parke A, West B. Hydroxychloroquine in pregnant patients with systemic lupus erythematosus. J Rheumatol 1996;23:1715. 92. Armenti VT, Radomski JS, Moritz MJ, et al. Report from the national transplantation pregnancy registry (ntpr): Outcomes of pregnancy after transplantation. Clin Transpl 2002;121.

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41

Fertility in Systemic Lupus Erythematosus Michael D. Lockshin, MD

INTRODUCTION Infertility, of either sex, is the inability to create an embryo. In patients with systemic lupus erythematosus (SLE), causes of infertility include severe illness, organ failure, and cytotoxic treatment. Most SLE patients are fertile. Many of those who are not avail themselves of assisted reproductive technologies (ART). Twenty-seven percent of SLE, antiphospholipid syndrome, and lupus-like disease pregnancies seen by the author in the past 2 years used ART to achieve conception. Among SLE patients, fetal loss (inability to carry a documented fetus to viability) is more prevalent than infertility. Early fetal losses (before 10 menstrual weeks) are often unexplained or attributable to genetic fetal abnormalities. Later fetal losses are usually due to active disease or to disease-induced damage. Infertility and fetal loss together constitute pregnancy failure, a classification of which is shown in Table 41.1.

TABLE 41.1 CLASSIFICATION OF PREGNANCY FAILURE Infertility (failure to conceive) Disease-related

Specific autoimmunity? Antiphospholipid antibody?

Treatment-related

Cyclophosphamide Other drugs

Male

Sperm count, quality

Age-related

Menopause

Fetal loss (recurrent pregnancy loss) Disease-unrelated

Disease-related

460

Unknown Maternal anatomic Fetal genetic/structural Maternal hormonal Infection Antiphospholipid antibody Toxemia Renal insufficiency Treatment-related Congenital heart block

This chapter reviews causes of infertility and of early fetal loss, and considers aspects of ART pertinent to SLE patients and their families. Late pregnancy loss has been reviewed in Chapter 12.1,2

INFERTILITY (FAILURE TO CONCEIVE) The definition of infertility is 1 year of unprotected regular coitus without conception. (Details of frequency and timing of coitus are not considered in the definition.) Infertility affects 10 to 15% of reproductiveage couples and remains unexplained in 10 to 20% of infertile couples.3 Common causes of infertility are outlined in Table 41.2. Only drug-induced ovarian failure is an SLE-specific cause.

Age-Related Infertility Advancing maternal age is a major contributor to infertility. Thirty-three percent of normal women are infertile at age 40, and 87% at age 45,2 even when menstrual cycles are still normal. In my rheumatology practice, which focuses on rheumatic disease pregnancy, 88% of patients seeking care for pregnancy-related issues are older than 30 years, 38% older than 35, and 15% older than 40. In this practice, the most common cause of infertility is advanced maternal age, followed by male factor infertility, and, uncommonly, cyclophosphamideinduced ovarian failure. No published studies have systematically analyzed causes of infertility in patients with SLE.

Disease-Related Infertility In vitro studies demonstrate that antiphospholipid antibodies (aPLs) are toxic to trophoblast growth and function; aPLs can inhibit implantation and trophoblast invasion, events that occur within the first few weeks of fetal life.4-6 These effects might be perceived as infertility or very early embryonic death, but because women with aPLs are generally fertile, the importance of the laboratory observations remains unclear.

Cause

Example

Menopause

Age

Primary ovarian failure

Antiovarian antibody, cyclophosphamide

Endocrine abnormality

Hypothyroidism (Hashimoto)

TABLE 41.3 EFFECTS OF SELECTED ANTIRHEUMATIC DRUGS ON FERTILITY Drug

Impairment of Fertility

NSAID

Cases of inhibition of follicle rupture

Prednisone

—

Dexamethasone

—

Uterine infection

Chlamydia

Betamethasone

—

Uterine anatomic abnormality

Septate uterus, myomata

Chloroquine/hydroxychloroquine

—

Sulfasalazine

In males

Fallopian anatomic abnormality

Endometriosis, salpingitis

Leflunomide

—

Parenteral chromosomal abnormality

Maternal XO

Azathioprine/6 MP

No

Methotrexate

Fetal chromosomal abnormality

Lethal mutation

Oligospermia at high doses

Male factor

Inadequate sperm number or function, infrequent intercourse

Autoimmune Disease as Cause of Infertility Diagnosable autoimmune disease is an infrequent cause of infertility in an apparently well population. One infertility clinic found only 1.5% of examined patients had SLE.7 Another found aPLs equally often in infertile and fertile women.8 A third clinic randomized antinuclear antibody-, anticardiolipin antibody-, and anti-β2 glycoprotein I antibody—positive infertile patients to a heparin-aspirin regimen, a treatment directed against antiphospholipid syndrome, during ART attempts.9 The treatment was ineffective.

Treatment-Related Infertility SLE patients treated with cyclophosphamide may develop ovarian failure. In general, the risk of ovarian failure correlates with age at first dose of (intravenous) cyclophosphamide and number of doses, and ranges from 12% for women aged under 25 years to 62% for women older than 30.10,11 Suppression of gonadal function during cyclophosphamide treatment for the purpose of protecting ovaries or testes is often advocated. Recommendations include use of gonadotropin-releasing hormone antagonists (leuprolide) or progestin/estrogen hormone replacement (oral contraceptive pills).12 Preliminary data support this recommendation.13 Methotrexate and other cytotoxic agents may also cause ovarian failure. Nonsteroidal anti-inflammatory drugs inhibit cilia motion in the fallopian tube, delaying transport of ova, and reducing chances for

Cyclophosphamide

In males and females

Cyclosporine

No

Tacrolimus

—

Mycophenolate mofetil

—

Intravenous immunoglobulin

—

Etanercept

—

Infliximab

No

Adalimumab

—

Anakinra

—

Rituximab

—

Alefacept

—

RECURRENT EMBRYONIC AND FETAL LOSS

TABLE 41.2 CAUSES AND EXAMPLES OF INFERTILITY

Source: Adapted from consensus documents developed at the Fourth International Conference on Sex Hormones, Pregnancy and the Rheumatic Diseases, Stresa, Italy, September 20—22, 2004.

fertilization. The effect on fertility of most drugs used in SLE patients is unknown (Table 41.3).

RECURRENT EMBRYONIC AND FETAL LOSS Recurrent losses of an established fetus occurring before 10 gestational weeks, or before identification of a fetal heartbeat, are embryonic losses; those occurring after 10 weeks or fetal heartbeat are fetal deaths. Recurrent pregnancy losses may be disease related or disease independent.

Disease-Independent Recurrent Pregnancy Losses Studies on recurrent pregnancy losses variously define “recurrent” as two or three, consecutive or not, embryonic and fetal losses, leading to some inconsistency of conclusions. In one highly rigorous study, that defined

461

FERTILITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

recurrence as three or more documented losses, and that used chromosomal/genetic, bacteriologic, radiographic, and hormonal assessments to find causes, approximately half of the women had no defined cause, 20% had maternal anatomic causes, 20% had antiphospholipid and related autoantibodies, 10% fetal structural or genetic abnormalities, and 1% infection.14 Although nonimmunologic coagulopathies (especially factor VLeiden, prothrombin 20210, and MTHFR mutations) may also cause fetal death, the risk imparted by these abnormalities is still debated.15,16

Lupus-Related Recurrent Pregnancy Loss Differing referral patterns and clinical mixes of patients make it difficult to apportion disease-related causes of recurrent pregnancy loss among patients with SLE. Antiphospholipid antibody—related losses account for a large percentage in all series; severe preeclampsia (often associated with maternal hypertension, nephritis, and/or renal insufficiency) account for many additional losses; and active SLE, premature rupture of membranes, and maternal diabetes, the latter two related to corticosteroid therapy, are responsible for fetal death in a minority of patients. Congenital complete heart block, associated with anti-Ro/SSA and anti-La/SSB antibodies, is a rare cause of fetal death. Causes of pregnancy loss have been reviewed extensively elsewhere,1 and in Chapter 40.

EVALUATION OF INFERTILITY IN SLE PATIENTS

Definitions of Assisted Reproductive Technology Procedures Evaluation for infertility, whether or not SLE is a suspected diagnosis, consists of excluding those items listed in Tables 41.1 and 41.2. A glossary of contemporary assisted reproductive technology procedures appears in Table 41.4; a brief description of the procedures undergone by mother, father, and child in ART appears in Table 41.5. The intent of presenting these data is to indicate the numerous processes to which patients and their progeny are subject. Procedural errors and mishaps may occur at any step. That these processes are successful and safe most of the time is a form of miracle. Causes of infertility that are more prevalent in SLE patients than in otherwise normal women include hypothyroidism due to coexistent Hashimoto thyroiditis,17 and early menopause due to cyclophosphamide treatment or, rarely, to antiovarian antibodies.

Risks of Evaluation for Infertility 462

The investigation of a lupus patient for infertility carries some risk. Medical risk occurs because of invasive procedures (transvaginal ultrasounds,

TABLE 41.4 GLOSSARY OF TERMS USED IN PROCEDURES USED TO ACHIEVE PREGNANCY Acronym

Definition

ART

Assisted reproductive technologies All infertility treatment procedures: Laboratory culture sperm/ova, transcervical embryo transfer Sperm/ova placement into fallopian tube Ovulation induction with monitoring Washed sperm placement

COH

Controlled ovarian hyperstimulation

GIFT

Gamete intrafallopian transfer

ICSI SZI

Intracytoplasmic spermatozoa injection Partial zonal dissection Subzonal insemination

IUI/ICI

Intrauterine/cervical insemination

IVF

In vitro fertilization

OD

Oocyte donation

OI

Ovulation induction

ZIFT

Zygote intrafallopian transfer

PZD

hysterosalpingography, and laparoscopy may result in infection or hemorrhage). Other types of risk include emotional and financial (procedures are very expensive and often not covered by insurance). The procedures are embarrassing and uncomfortable. Other than immunosuppression or thrombocytopenia, lupus patients harbor no unusual risks or contraindications for these procedures.

RISKS OF TREATMENT FOR INFERTILITY The greatest potential risk to the SLE patient occurs during ovulation induction (OI). No standard process for OI exists; most regimens consist of, first, cycling ovulation by administering a gonadotropin-release inhibitor such as leuprolide (this causes an initial surge of estrogen), and then a gonadotropin to induce simultaneous maturation of several ova.18 Estrogen levels must be monitored during this period (controlled ovarian stimulation [COI]). Uncontrolled ovarian hyperstimulation may result in a large number of ova, massive increase in ovarian size, marked fluid retention, cytokine release syndrome, and renal failure (ovarian hyperstimulation syndrome). At the point of maximum ova production, follicle maturation is induced and the ova donor, either the patient or surrogate, undergoes surgical harvesting, usually by transvaginal puncture.

At risk

Procedure

Female (pre-embryo)

Administer GnRH inhibitory analogue Administer gonadotropin Harvest oocytes (laparoscopy or transvaginal)

Female (post-embryo)

Implant embryo transcervical Administer progesterone supplement

Male

Harvest semen With/without seminal fluid From live/dead donor Wash semen Freeze semen Thaw semen Activate semen Inject into oocyte Partial zona dissection Subzonal insemination

Oocyte

Embryo

Laboratory culture, placement into fallopian tube Injection of single sperm into oocyte, used for male factor infertility Laboratory culture of aspirated oocytes with sperm Transfer Fertilize ovum Grow gamete/zygote/ embryo in vitro Assess “quality” Remove cell for genetic analysis Implant transcervical

Two groups have reported small retrospective studies detailing effects of these procedures in lupus and antiphospholipid syndrome (APS) patients. Our group in New York examined 19 women who underwent 68 OI cycles, resulting in 22 pregnancies and 14 live-born children.19 Four of 16 cycles in SLE patients (25%) were complicated by SLE flare, and 2 in ovarian hyperstimulation syndrome (1 with renal failure). With more rigorous control of ovarian stimulation, no maternal complications have occurred since 2002. A Paris group reported 114 cycles in 21 SLE or APS patients, with 18 pregnancies and 9 live births.20 Three of the SLE and 5 of the APS patients were newly diagnosed at the time of the OI procedure; 5 of them had concealed known diagnoses from their physicians, an unfortunate occurrence also seen in New York. Thirteen of 62 SLE cycles (23%) resulted in SLE flare and two women developed phlebitis. Both studies concluded that ART procedures were not innocuous, but SLE patients were at no special risk compared to others with similar organ involvement,

such as renal insufficiency. In particular, the hyperestrogenemia associated with OI is trivial compared to that of late pregnancy. A normally menstruating woman has a circulating estradiol level of about 150 pg/mL, a woman at the end of OI 1500 pg/mL, but a woman at the end of pregnancy has 20,000 pg/mL.21 None of these levels is demonstrably associated with change in disease activity, nor with thrombosis in women with antiphospholipid antibody. Procoagulant states do not predispose women to special thrombosis during OI. Of 747 cycles in 305 women with aPL, factor VLeiden, MTHFR and prothrombin mutations, and deficient antithrombin III, protein C, or protein S, only 4 (0.5%) cycles were complicated by ovarian vein thrombosis. Risk for thrombosis was greatest in women 40 years of age or older and with high plasma homocysteine.22

RISKS OF TREATMENT FOR INFERTILITY

TABLE 41.5 ART PROCEDURES FOR MALE, FEMALE, AND EMBRYO

Risks to Pregnancies Resulting from Treatment for Infertility Risks to Mother Pregnancies resulting from ART present more hazards to the mothers than do normally conceived pregnancies, in part because of the frequent occurrence of multiple gestation pregnancies. Mothers in our experience suffered osteopenia (from heparin), Mallory-Weiss tear (during hyperemesis gravidarum), diabetes, and severe pre-eclampsia. One attempted suicide after the deaths of her triplets.

Risks to Embryo, Fetus, and Child Known Risks From the point of view of the embryo, in vitro fertilization imparts modest additional risk. Half of surviving children in our clinic were delivered prematurely, all of one triplet set had neonatal lupus, and one child had pulmonary stenosis. A meta-analysis of more than 12,000 in vitro fertilization singleton pregnancies compared to 1.9 million spontaneous singleton pregnancies found odds ratios of 1.6 to 2.7 for perinatal mortality, pre-term delivery, low birth weight, very low birth weight, and small-for-gestational age babies.23 Another study found 30 to 40% increased risk for fetal malformation,24 although controversy exists on this point.25 Unknown Risks Quite a number of aspects of the effects of ART on the embryo are unknown. Between fertilization and implantation, the following processes take place: sex differentiation, spatial organization of the embryo, brain organization, imprinting, and X inactivation. It is assumed, but not known, that these processes proceed as normally in vitro as in vivo.26 When oocyte nuclei are transplanted into (usually younger, assumed

463

FERTILITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

healthier) cytoplasm, the mitochondria of the reconstituted ovum are genetically different from the nucleus. In one of the few studies on this topic, 6 of 13 embryos carried donor mitochondrial DNA.27 Paternal mitochondria may appear in the embryo.28 Whether any of these phenomena impart a long-term biological effect of any importance is unknown. At the intracellular and intranuclear levels, spindle organization and first-cycle DNA replication may differ between products of in vitro and in vivo fertilization. The signals for, or imparted by, initial cell fusion are not fully understood. Whether altered biology imparts an effect is unknown. Nonetheless, long-term follow-ups of children born of ART have not revealed any important consequences.

RISKS FROM EVALUATION AND TREATMENT FOR RECURRENT PREGNANCY LOSS

Risks to Mother The risks to the mother for evaluation of recurrent pregnancy loss are similar to those for evaluation for infertility, since hysteroscopy, hysterosalpingography, and other invasive procedures may be advised. The risks for treatment are those of the drugs administered, complicated by increased fluid volume, relative thrombocytopenia, and other physiologic changes of pregnancy. Thus, osteoporosis is a high risk for women treated with heparin, and hypertension and congestive heart failure for those treated with intravenous immunoglobulin. An additional risk is continuation beyond a safe time, rather than early termination, of a toxic pregnancy despite advanced pre-eclampsia or HELLP syndrome because the fetus is considered

“valuable.” Rapid deterioration of maternal renal, hepatic, or cerebral function may ensue.

Risks to Embryo, Fetus, and Child A high proportion of children of women with recurrent fetal loss, although brought to live birth, are nonetheless premature, with the same risks as comparably premature infants of women without rheumatic disease. Small-for-gestation babies are common. Except for those with complete congenital heart block, children of lupus mothers followed long term have no special risks for malformation or developmental abnormalities. However, in studies from Italy and from the United States, compared to socioeconomic-, race-, gestational, age- and weight-matched control infants, boys but not girls born of mothers with SLE have more verbal learning disabilities.29,30 The meaning of this observation is unknown. These findings have not been analyzed for effects of treatments for infertility.

CONCLUSIONS Infertility is not a specific problem of SLE or other autoimmune disease. While complicated by risks imparted by specific aspects of an individual woman’s illness, ART conceptions are possible and successful in patients with SLE. Risks of lupus and antiphospholipid antibody pregnancies continue after ART; multiple gestation pregnancies add to maternal and fetal complications. Live-born children of ART appear to suffer no abnormalities beyond those normally associated with prematurity or multiple gestation, but long-term follow-up has not been done, and many theoretical biological differences between these and normally conceived children have not been explored.

REFERENCES

464

1. Lockshin MD. Pregnancy and rheumatic diseases. In: Koopman WJ, Moreland LW, eds. Arthritis and Allied Conditions. Philadelphia: Lippincott Williams & Wilkens, 2005;1719-1728. 2. Lockshin MD, Sammaritano LR, Wartzman S. Lupus pregnancy. In: Lahita R, ed. Systemic Lupus Erythematosus. New York: Academic Press, 2004. 3. Gurtcheff S, Hatasaka H. Fertility and autoimmune disease. In: Lockshin MD, Branch DW,eds. Reproductive and Hormonal Aspects of Systemic Autoimmune Diseases. Vol. 4, Handbook of Systemic Autoimmune Diseases. Amsterdam and Boston: Elsevier, 2005;29-42. 4. Di Simone N, Raschi E, Testoni C, et al. Pathogenic role of anti-beta 2-glycoprotein I antibodies in antiphospholipid associated fetal loss: characterisation of beta 2-glycoprotein I binding to trophoblast cells and functional effects of anti-beta 2-glycoprotein I antibodies in vitro. Ann Rheum Dis 2005;64:462-467. 5. Sebire NJ, Fox H, Backos M, et al. Defective endovascular trophoblast invasion in primary antiphospholipid antibody syndrome-associated early pregnancy failure. Hum Reprod 2005;17:1067-1071.

6. Quenby S, Mountfield S, Cartwright JE, et al. Antiphospholipid antibodies prevent extravillous trophoblast differentiation. Fertil Steril 2005;83:691-698. 7. Geva E, Lerner-Geva L, Burke M, et al. Undiagnosed systemic lupus erythematosus in a cohort of infertile women. Am J Reprod Immunol 2004;51:336-340. 8. Balasch J, Reverter JC, Creus M, et al. Human reproductive failure is not a clinical feature associated with beta(2) glycoprotein-I antibodies in anticardiolipin and lupus anticoagulant seronegative patients (the antiphospholipid/cofactor syndrome) Hum Reprod 1999;14:1956-1959. 9. Stern C, Chamley L, Norris H, et al. A randomized, double-blind, placebo-controlled trial of heparin and aspirin for women with in vitro fertilization implantation failure and antiphospholipid or antinuclear antibodies. Fertil Steril 2003;80:376-383. 10. Huong du L, Amoura Z, Duhaut P, et al. Risk of ovarian failure and fertility after intravenous cyclophosphamide. A study in 84 patients. J Rheumatol 2002;29:2571-2576. 11. Boumpas DT, Austin HA 3rd, Vaughan EM, et al. Risk for sustained amenorrhea in patients with systemic lupus erythematosus

13.

14. 15. 16. 17.

18.

19.

20.

21. Speroff L, Glass RH, and Kase NG. The Endocrinology of Pregnancy. In: Mitchell C, editor. Clinical Gynecologic Endocrinology and Infertility, 5th ed. Baltimore: Williams and Wilkins. 1994;251-289. 22. Grandone E, Colaizzo D, Vergura P, et al. Age and homocysteine plasma levels are risk factors for thrombotic complications after ovarian stimulation. Hum Reprod 2004;19:1796-1799. 23. Jackson RA, Gibson KA, Wu YW, et al. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103:551-563. 24. Hansen M, Bower C, Milne E, et al Assisted reproductive technologies and the risk of birth defects—a systematic review. Hum Reprod 2005;20:328-338. 25. Kurinczuk JJ, Hansen M, Bower C. The risk of birth defects in children born after assisted reproductive technologies. Curr Opin Obstet Gynecol 2004;16:201-209. 26. Lucifero D, Chaillet JR, Trasler JM. Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology. Hum Reprod Update 2004;10:3-18. 27. Brenner CA, Barritt JA, Willadsen S, et al. Mitochondrial DNA heteroplasmy after human ooplasmic transplantation. Fertil Steril 2000;74:573-578. 28. Sutovsky P, Van Leyen K, McCauley T, et al. Degradation of paternal mitochondria after fertilization: implications for heteroplasmy, assisted reproductive technologies and mtDNA inheritance. Reprod Biomed Online 2004;8:24-33. 29. Ross G, Sammaritano L, Nass R, et al. Effects of mothers’ autoimmune disease during pregnancy on learning disabilities and hand preference in their children. Arch Pediatr Adolesc Med 2003;157:397-402. 30. Neri F, Chimini L, Bonomi F, et al. Neuropsychological development of children born to patients with systemic lupus erythematosus. Lupus 2004;13:805-811.

REFERENCES

12.

receiving intermittent pulse cyclophosphamide therapy. Ann Intern Med 1993;119:366-369. Blumenfeld Z. Preservation of fertility and ovarian function and minimalization of chemotherapy associated gonadotoxicity and premature ovarian failure: the role of inhibin-A and -B as markers. Mol Cell Endocrinol 2002;187:93-105. Somers E, Marder W, Christman G, Ognenovski V, McCune WJ. Use of a gonadotropin-releasing hormone analog for protection against premature ovarian failure during cyclophosphamide therapy in women with severe lupus. Arthritis Rheum 2005;52:2761-2767. Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996;66:24-29. Robertson L, Wu O, Greer I. Thrombophilia and adverse pregnancy outcome. Curr Opin Obstet Gynecol 2004;16:453-458. Kupferminc MJ. Thrombophilia and pregnancy. Curr Pharm Design 2005;11:735-748. Criswell LA, Pfeiffer KA, Lum RF, et al. Analysis of families in the multiple autoimmune disease genetics consortium (MADGC) collection: the PTPN22 620W allele associates with multiple autoimmune phenotypes. Am J Hum Genet 2005;76:561-571. Ragni G, Vegetti W, Riccaboni A, et al. Comparison of GnRH agonists and antagonists in assisted reproduction cycles of patients at high risk of ovarian hyperstimulation syndrome. Hum Reprod 2005;2421-2425. Guballa N, Sammaritano L, Schwartzman S, et al. Ovulation induction and in vitro fertilization in systemic lupus erythematosus and antiphospholipid syndrome. Arthritis Rheum 2000;43:550-556. Huong du LT, Wechsler B, Vauthier-Brouzes D, et al. Importance of planning ovulation induction therapy in systemic lupus erythematosus and antiphospholipid syndrome: a single center retrospective study of 21 cases and 114 cycles. Semin Arthritis Rheum 2002;32:174-188.

465

CLINICAL ASPECTS OF THE DISEASE

42

Neonatal Lupus: Clinical Perspectives Anca D. Askanase, MD, MPH and Jill P. Buyon, MD

INTRODUCTION

466

Neonatal lupus (NL) is a model of passive autoimmunity; anti-SSA/Ro-SSB/La antibodies (Ab) cross the placenta and presumably injure the fetus. The most serious manifestation is congenital heart block (CHB). Other manifestations of NL are transient, disappearing with the clearance of maternal Ab from the neonatal circulation. NL is uncommon (~1:15,000 births) and little information has been available on health outcomes. Congenital heart block, absent structural abnormalities and detected in the second trimester, is almost universally associated with maternal autoantibodies reactive with the intracellular soluble ribonucleoproteins 48 kD SSB/La, 52 kD SSA/Ro, or 60 kD SSA/Ro.1,2 An erythematosus skin rash with a predilection for the scalp and periorbital region, most often apparent in the first 8 weeks after birth, is also strongly linked to these maternal antibodies as well as to antibodies against U1 RNP.2,3 Permanent cardiac and transient cutaneous disease are the most common manifestations of neonatal lupus, initially named because of the resemblance of the skin rash to subacute cutaneous lupus erythematosus (SCLE). Less often, abnormalities of the liver or blood affect newborns exposed in utero to maternal anti-SSA/Ro-SSB/La antibodies.4-7 Fetal and neonatal injury are presumed to result from the transplacental passage of IgG anti-Ro/La antibodies from the mother into the fetal circulation.8 Fetal/ neonatal disease is independent of maternal disease; mothers may have systemic lupus erythematosus (SLE), Sjögren’s syndrome (SS) or other autoimmune symptoms, or may be entirely asymptomatic.9 The fetal heart appears to be uniquely vulnerable, since complete block has only been reported in a single mother,10 despite mothers’ exposure to identical circulating levels of the autoantibodies. CHB carries a significant mortality (15% to 30%, primarily fetal and neonatal) and morbidity (67% of surviving affected children require permanent pacing before adulthood).9 The recurrence rate of CHB in subsequent pregnancies is ~19%11

(Table 42.1), or at least nine-fold greater than the risk for CHB in a primigravida with the candidate antibodies.10 Extensive work from several laboratories has resulted in the molecular characterization of the maternal autoantibody responses and the cloning of genes expressing the cognate antigens whose structural features suggest a role in transcriptional regulation. Anecdotal cases support the use of dexamethasone for the treatment of in utero effusions, hydrops, and incomplete block, but prospective evaluation of dexamethasone or other prophylactic therapy for the at-risk pregnancy remains to be reported. Bridging the gap from identification of the target antigens recognized by the maternal autoantibodies to the mechanism by which these antibodies result in tissue damage and overt clinical disease represents a major challenge. The necessity of anti-SSA/Ro-SSB/La antibodies is supported by their presence in more than 85% of mothers whose fetuses are identified with conduction abnormalities in a structurally normal heart.1,2

TABLE 42.1 OUTCOME OF 100 PREGNANCIES IMMEDIATELY SUBSEQUENT TO BIRTH OF CHILD WITH CHBa Outcome

n

%

Healthy

73

73

Manifestations of Neonatal Lupus

CHB only

15

15

CHB + rash

3

3

Rash only

7

7

Fetal demise

1

1

Neonatal death (heart valve dysfunction and heart failure)

1

1

a

Data obtained from the Research Registry for Neonatal Lupus, September 15, 2005. CHB, congenital heart block.

CLINICAL OBSERVATIONS AND CONSIDERATIONS

Classification and Progression of Heart Block The issue of “complete” versus “incomplete” heart block warrants clarification. It is recognized that heart block might progress through various stages. Therefore, cases have been loosely assigned as CCHB (congenital complete heart block) when in fact the rhythm was second-degree block. It is anticipated that, as echocardiograms are done more frequently during pregnancies of mothers with anti-SSA/Ro-SSB/La antibodies, this point will be clarified. Presently, CHB is best used to describe congenital heart block, which can be first, second, or third degree. To ascertain the spectrum of arrhythmias associated with maternal anti-SSA/Ro-SSB/La antibodies, Askanase and colleagues13 reviewed records of all children enrolled in the Research Registry for Neonatal Lupus (RRNL). Of 187 children with CHB whose mothers had anti-Ro/La antibodies, 9 had a prolonged PR interval on EKG at birth, 4 of whom progressed to more advanced AV block. A child whose younger sibling had third-degree block was diagnosed with first-degree block at age 10 years at the time of surgery for a broken wrist. Two children diagnosed in utero with seconddegree block were treated with dexamethasone and reverted to normal sinus rhythm by birth, but ultimately progressed to third-degree block. Four children had second-degree block at birth: of these, two progressed to third-degree block. These data have important research and clinical implications. Perhaps many fetuses sustain mild inflammation, but resolution is

variable, as suggested by the presence of incomplete AV block. Since subsequent progression of less-advanced degrees of block can occur, an EKG should be performed on all infants born to mothers with anti-Ro/La antibodies. The Toronto registry group retrospectively analyzed the single-center outcome of children with isolated CHB presenting from 1965 to 1998, up to age 20 years.14 Cases were diagnosed from fetal life through childhood. There were a total of 102 cases, divided in three groups. Fetal presentation in 29 cases had 43% mortality rate; neonatal presentation in 33 cases had 6% mortality. There were no deaths in the 40 cases presenting in childhood, but it should be noted that 19 out of 20 tested had negative maternal antibody levels. Risk factors for death were a fetal diagnosis, the presence of hydrops, gestational age under 33 weeks, or the development of endocardial fibroelastosis (EFE) with ejection fraction of 40% of less. Three neonates who had been diagnosed before birth were treated with corticosteroids. Most of the cases (88 to 89%) were paced by age 20. Late cardiomyopathy occurred in 5% overall, but in the antibody-positive cases was 11%. There was progression noted also in the degree of heart block over time. Pacemaker insertions were associated with complications in 25% of cases, and there was a frequent need for pacemaker revisions. The Toronto group also retrospectively reviewed the clinical history, echocardiography, and pathology of fetuses and children from five medical centers who had EFE associated with CHB and were born to mothers positive for anti-SSA/Ro or anti-SSB/La antibodies.15 Thirteen patients were identified, six with a prenatal and seven with a postnatal diagnosis. Severe ventricular dysfunction was seen in all fetal and postnatal cases. Four fetal and three postnatal cases had EFE at initial presentation. However, two fetal and four postnatal cases developed EFE 6 to 12 weeks and 7 months to 5 years from CHB diagnosis, respectively, even despite ventricular pacing in six postnatal cases. Eleven (85%) either died (n=9) or underwent cardiac transplantation (n=2) secondary to the EFE. Pathologic assessment of the explanted heart, available in 10 cases, revealed moderate to severe EFE in seven cases and mild EFE in three cases, predominantly involving the left ventricle. Immunohistochemistry in four cases (including three fetuses) demonstrated deposition of IgG in four, IgM in three, and T-cell infiltrates in three cases, suggesting an immune response by the affected fetus or child. The important conclusion from this study was that EFE occurs despite adequate ventricular pacing and is associated with significant mortality, whether developing in fetal or postnatal life. Of interest, this same group of investigators also described a unique clinical series of three cases of isolated

CLINICAL OBSERVATIONS AND CONSIDERATIONS

However, when Brucato and colleagues10 prospectively evaluated 118 pregnancies in 100 patients with antiSSA/Ro antibodies, the frequency of CHB in a fetus was only 1.7%. Gladman and colleagues12 reported no cases of CHB in 100 live births in 96 women with anti-SSA/Ro and/or anti-SSB/La antibodies and no history of a previous child with NL. This low frequency suggests that a fetal factor and/or the in utero environment are likely to amplify the effects of the antibody, which may be necessary but insufficient to cause fibrotic replacement of the atrioventricular (AV) node and in some cases cardiomyopathy. Notably, one mother in the series reported by Brucato and colleagues,10 who gave birth to two healthy children, developed complete heart block herself, raising the possibility that her heart had acquired “fetal factors.” Clearly, this is a unique situation and one that needs to be further studied, since it is likely to contribute important clues on pathogenesis.

467

NEONATAL LUPUS: CLINICAL PERSPECTIVES

468

EFE (one fetus, two infants, all female) with antibodypositive mothers in the absence of CHB.16 Two died and one received a heart transplant. Histologic evaluation revealed EFE with diffuse IgG deposition and T-cell infiltration but, surprisingly, no detectable apoptosis. This latter observation is curious and remains unexplained.

Management of At-Risk Pregnancy and Congenital Heart Block Detected In Utero The clinical approach to cardiac manifestations of NL includes obstetric and rheumatologic management of (1) the fetus with a normal heart rate but at risk of developing CHB, and (2) the fetus identified with heart block. All pregnant women with anti-SSA/Ro-SSB/La antibodies should have serial fetal echocardiography done by an experienced pediatric cardiologist weekly from 16 to 26 weeks, and every other week until about 34 weeks. Until recently, the in utero detection of firstdegree block was not technically feasible. However, the EKG equivalent of the PR interval can now be measured by echocardiography.17 Using the gated-pulsed Doppler technique, time intervals from the onset of the mitral A wave (atrial systole) to the onset of the aortic pulsed Doppler tracing (ventricular systole) within the same left ventricular cardiac cycle may be measured. This time interval represents the “mechanical” PR interval. Its validity was confirmed by neonatal electrocardiographic correlation to the pulsed Doppler mechanical PR interval.18 The normal mechanical PR interval in the fetus is 0.12 ±0.02 seconds (95% confidence interval, 0.10–0.14). A prospective National Institutes of Health–supported multicenter study, PR Interval and Dexamethasone Evaluation (PRIDE), in CHB is underway to examine the mechanical PR interval weekly in pregnant woman with anti-SSA/Ro and/or anti-SSB/La antibodies. One of the goals of this trial is to identify the prevalence of first-degree block and to determine whether it is a marker for more advanced destruction of the conducting system. Such information will provide the optimal opportunity for reversibility. It is also strongly recommended that all neonates born to mothers with anti-SSA/Ro-SSB/La antibodies have an EKG at birth to detect first-degree block. The initiation of dexamethasone or plasmapheresis as a preventive measure has been considered. With regard to prophylactic therapy of the high-risk mother (documentation of high-titer anti-SSA/Ro and SSB/La antibodies, anti-48kD SSB/La and 52kD SSA/Ro on immunoblot, and a previous child with NL), administration of prednisone, dexamethasone, or plasmapheresis is not justified at the present time. Maternal prednisone (at least in low and moderate doses) early

in pregnancy does not prevent the development of CHB.19 This might be anticipated since prednisone given to the mother is not active in the fetus,20 and levels of anti-SSA/Ro and anti-SSB/La antibodies remain relatively constant during steroid therapy. Kaaja and Julkunen21 recently reported their experience using intravenous immunoglobulin (IVIG) and corticosteroids in highest-risk mothers (those with anti-SSA/Ro antibodies and a previous child with CHB). Conclusions are limited by the small number of treated patients (n=8) and the absence of a control group. The one mother who did have a second baby with CHB received only IVIG and no corticosteroids. Effectiveness of treatment is difficult to assess in this study. To formally evaluate the efficacy of treatment, a randomized, placebo-controlled trial would need to be conducted, and probably only include mothers who had a previous child with CHB. An example of a power analysis follows: if one accepts a clinically meaningful outcome as a reduction of the recurrence rate of CHB from 19 to 10%, 261 mothers will be needed in each group; and from 19 to 5%, 97 mothers would be needed (α=80%). The mechanism of potential efficacy is unknown, but effective decrease of circulating antibody in the fetus might involve idiotype/anti-idiotype regulation, a decrease in placental transport, or perhaps induction of surface expression of the inhibitory Fc receptor, FcγRIIB, on macrophages.22 Precedent for decrease of anti-Ro/La transport has been provided by a murine model.23,24 Modulation of inhibitory signaling could be a potent therapeutic strategy for attenuating autoantibody-triggered inflammatory diseases. The rationale for treatment of identified heart block and prevention of potential heart block is to diminish a generalized inflammatory insult and reduce or eliminate maternal autoantibodies. Accordingly, several intrauterine therapeutic regimens have been tried including dexamethasone, which is not metabolized by the placenta and is available to the fetus in an active form. In the largest retrospective study published to date, it was observed that fluorinated glucocorticoids ameliorated incomplete AV block and hydropic changes in autoimmune-associated congenital heart block but did not reverse established third-degree block.25 Maternal risks of dexamethasone are similar to any glucocorticoid and include infection, osteoporosis, osteonecrosis, diabetes, hypertension, and pre-eclampsia. Fetal risks include oligohydramnios, intrauterine growth retardation, and adrenal suppression. Intervention with glucocorticoids might decrease acute inflammation but not necessarily prevent subsequent fibrosis. A second goal of the PRIDE trial, noted above, is to assess the efficacy of maternal oral dexamethasone (4 mg/d) in reversing or preventing the progression of AV block

Transient Manifestations of Neonatal Lupus: Skin, Liver, and Blood Neonatal lupus skin lesions generally become manifest several weeks into postnatal life; less commonly the rash is present at birth. Ultraviolet exposure may be an initiating factor and can exacerbate an existing rash.2,27 Cutaneous activity, inclusive of erythema and the continued appearance of new lesions, is generally present for several weeks with resolution by 6 to 8 months of age coincident with the clearance of maternal autoantibodies from the baby’s circulation. The rash frequently involves the face and scalp with a characteristic predilection for the upper eyelids, but may be present in other locations and in some instances covers virtually the entire body. Lesions are superficial inflammatory plaques resembling subacute cutaneous lupus erythematosus of the adult, typically annular or elliptical with erythema and scaling. Hypopigmentation is frequent and may be a prominent feature, and may persist into the second year of life. The more characteristic lesions of adult discoid lupus such as follicular plugging, dermal atrophy, and scarring are generally not observed in the neonatal skin rash. A recent assessment of NL rash (absent heart disease) in 57 infants in the RRNL found no significant difference with regard to residual sequelae (hypopigmentation, telengectasias, pitting, scarring, atrophy) between untreated infants and those treated with topical corticosteroids. It is most important for the clinician to consider the outcomes of pregnancies subsequent to the birth of a child with NL rash: among RRNL mothers, 25% of such pregnancies resulted in a child with CHB (Table 42.3). To extend the information base on the hepatobiliary manifestations of NL, Lee and colleagues6 evaluated records from 219 children enrolled in the RRNL. Nineteen (9%) had probable or possible hepatobiliary disease, 16 of whom also had cardiac or cutaneous

CLINICAL OBSERVATIONS AND CONSIDERATIONS

newly detected in utero. Under the PRIDE protocol, if third-degree block has remained present with no improvement through 6 weeks of dexamethasone therapy, the drug is tapered and discontinued. (Continued use of dexamethasone is warranted in such a case only if hydropic changes are present.) Available data support serial cardiac monitoring of all fetuses with any bradyarrhythmias detected in utero and of neonates with incomplete blocks at birth whose mothers are previously known or currently identified to have anti-SSA/Ro-SSB/La antibodies. Fetal echocardiogram is essential to diagnose and follow the course of disease, and may suggest the presence of an associated myocarditis by the finding of decreased contractility in addition to the secondary changes associated with myocarditis such as an increase of cardiac size, pericardial effusions, and tricuspid regurgitation. The obstetric management should be guided by the degree of cardiac failure noted on the ultrasound images. The in utero environment is preferred as long as possible because of the low resistance circulatory pathways, thereby affording minimal work to maintain cardiac output. A case report in which ritodrine was given intravenously to the mother to increase the heart rate of a 28-week fetus with CHB and a ventricular rate of 54 beats per minute reminds us that nonimmunosuppressive approaches may have a role in therapy (sympathomimetics can transiently increase fetal heart rate, although they do not restore coordination of AV conduction on which the heart is dependent for adequate filling).26 Current treatment recommendations for CHB diagnosed in utero are summarized in Table 42.2. Figure 42.1 provides an overview of the translational approach to prevention and therapy, including both current and investigational strategies.

TABLE 42.2 THERAPEUTIC APPROACHES TO CONGENITAL HEART BLOCK DIAGNOSED IN UTERO Situation (Degree of Block at Presentation)

Treatment

3rd degree (>2 weeks from detection)

Evaluation by serial echos; no therapy

3rd degree (<2 weeks from detection)

4 mg p.o. dexamethasone daily for 6 weeks. If improvement to 2nd degree or better, continue until delivery

Alternating 2nd degree/3rd degree 2nd degree Prolonged mechanical PR interval (1st degree)

4 mg dexamethasone to delivery, unless block progresses to 3rd degree and remains so for 6 weeks (then taper and withdraw)

Block associated with signs of myocarditis, congestive heart failure and/or hydropic changes

4 mg dexamethasone until improvement

Severely hydropic fetus

4 mg dexamethasone plus apheresis to rapidly remove maternal antibodies

469

NEONATAL LUPUS: CLINICAL PERSPECTIVES

Weekly fetal echocardiograms Mother with anti-Ro/La Abs

Prophylactic therapy: IVIG to decrease placental transport of anti-Ro/La

16 weeks

23 weeks

32 weeks

Treatment: 1st and 2nd degree heart block: 4 mg dex to decrease inflammation 3rd degree heart block: TGFβ inhibition to forestall fibrosis

Birth

EKG: If 1st degree: Follow by cardiologist If normal sinus rhythm: To date, no reports of later conduction abnormalities

Fig. 42.1 Translational approach to congenital heart block. This schematic outlines monitoring and treatment options for pregnancy and birth in women with anti-Ro/La antibodies, including both recommended practice as well as approaches under investigation. Current recommendations include weekly echocardiograms between 16 and 32 weeks of gestation, treatment with 4 mg dexamethasone to the mother (see Table 42.2), and an EKG at birth (any conduction abnormality at birth should be followed by a cardiologist, but no later conduction abnormalities have been reported to date in an infant with normal sinus rhythm at birth). Treatment of the mother with intravenous immune globulin (IVIG) to reduce transplacental transport of anti-Ro/La antibodies is currently being evaluated. Recent laboratory findings point to inhibition of transforming growth factor (TGF)-β production as a promising therapeutic target to forestall fibrosis in the fetus with third-degree block in utero.

manifestations of NL. Three clinical variants of hepatobiliary disease were observed: (1) severe liver failure present during gestation or in the neonatal period, often with the phenotype of neonatal iron storage disease; (2) conjugated hyperbilirubinemia with mild or no elevations of aminotransferases, occurring in the first few weeks of life; and (3) mild elevations of aminotransferases occurring at approximately 2 to 3 months of life. The prognosis for the children in the last two categories was excellent.

TABLE 42.3 OUTCOMES OF 47 PREGNANCIES IMMEDIATELY SUBSEQUENT TO BIRTH OF CHILD WITH CUTANEOUS MANIFESTATIONS OF NLa Outcome

n

%

Healthy

18

38

Manifestations of NL

CHB only

7

15

CHB + rash

6

13

Rash only

15

32

1

2

Fetal demise a

470

Data obtained from the Research Registry for Neonatal Lupus, September 15, 2005. CHB, congenital heart block; NL, neonatal lupus.

Thrombocytopenia has been observed together with other manifestations of NL,5 but may in some cases result from antiplatelet rather than anti-SSA/Ro-SSB/La antibodies targeting the surface of fetal cells. Kanagasegar and colleagues7 recently reported an infant with neutropenia and mildly abnormal liver functions, but no cardiac or cutaneous manifestations of NL, born to a mother with anti-SSA/Ro-SSB/La antibodies. The child’s neutropenia improved as maternal antibody was metabolized. Sera from this child and mother, as well as sera from two RRNL mothers who had given birth to infants with CHB and neutropenia, were shown to bind the cell surface of intact neutrophils.7 Binding to neutrophils was then inhibited (>80%) by incubating the sera with 60-kD Ro antigen, suggesting that anti-60kD SSA/Ro is directly involved in the pathogenesis of neutropenia.

Breast-feeding Finally, some comment on breast-feeding is warranted since this is so often a concern among physicians and their patients. In order to first determine whether human breast milk contains anti-Ro/La antibodies, breast milk was obtained from nine antibody-positive mothers and evaluated by ELISA and immunoblot.28 All mothers had anti-Ro/La antibodies of both IgG and IgA isotypes in their breast milk. These results paralleled those obtained from the respective sera, with lower titers measured in the milk.

CONCLUSIONS Currently, the presence of anti-SSA/Ro antibodies in a primigravid woman predicts a risk of about 2% for CHB in an offspring. This suggests the contribution of fetal factors. In considering the pathogenesis of CHB, unresolved challenges remain; the antigen system is considered by definition to be associated with RNA

and not normally expressed on cell surface membranes, and the maternal heart is not affected. Apoptosis may explain, in part, accessibility of antigen to maternal antibody and developmental vulnerability. Fetal factors are being sought, with focus on those that promote fibrosis. Postnatal development of cardiomyopathy continues to be reported and carries a poor prognosis. Incorporation of the mechanical PR interval in serial fetal echocardiographic assessments of mothers with anti-SSA/Ro-SSB/La antibodies may provide a window of opportunity for treatment. Because postnatal progression of less-advanced degrees of block can occur, an EKG should be performed on all infants born to mothers with the candidate antibodies. With the establishment of two large research registries, one in the United States and the other in Canada, significant advances in the management of high-risk pregnancies and assessment of long-term outcomes of both mothers and children should be made.

REFERENCES

A questionnaire was sent to all mothers in the RRNL (by definition, all mothers have anti-Ro/La antibodies and a child with NL), to ascertain precise details of their breast-feeding history for both their affected and unaffected children. Based on a questionnaire returned by 129 of 237 mothers, of 77 children with NL rash, 45 were breast-fed, compared to 60 of 117 unaffected siblings (p value was not significant). There were insufficient numbers of patients with cardiomyopathies to ascertain the influence of breast-feeding on this serious complication.

REFERENCES 1. Buyon JP. Neonatal lupus syndrome. In: Lahita RG, ed. Systemic Lupus Erythematosus. 4th ed. Boston: Elsevier Academic Press, 2004:449-484. 2. Lee LA. Neonatal lupus erythematosus. J Invest Dermatol 1993;100:9s-13s. 3. Solomon BA, Laude TA, Shalita AR. Neonatal lupus erythematosus: discordant disease expression of U1RNP-positive antibodies in fraternal twins. Is this a subset of neonatal lupus erythematosus or a new distinct syndrome? J Am Acad Dermatol 1995;32:858-862. 4. Laxer RM, Roberts EA, Gross KR, et al. Liver disease in neonatal lupus erythematosus. J Pediatr 1990;116:238-242. 5. Watson R, Kang JE, May M, Hudak M, Kickler T, Provost TT. Thrombocytopenia in the neonatal lupus syndrome. Arch Dermatol 1988;124:560-563. 6. Lee LA, Sokol RJ, Buyon JP. Hepatobiliary disease in neonatal lupus erythematosus: Prevalence and clinical characteristics in cases enrolled in a national registry. Pediatrics 2002;109:e11. 7. Kanagasegar S, Cimaz R, Kurien BT, Brucato A, Scofield RH. Neonatal lupus manifests as isolated neutropenia and mildly abnormal liver functions. J Rheumatol 2002;29:187-191. 8. Story CM, Mikulska JE, Simister NE. A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to the fetus. J Exp Med 1994, 180:2377-2381. 9. Buyon JP, Hiebert R, Copel J, et al. Autoimmune-associated congenital heart block: mortality, morbidity, and recurrence rates obtained from a national neonatal lupus registry. J Am Coll Cardiol 1998;31:1658-1666. 10. Brucato A, Frassi M, Franceschini F, et al. Risk of congenital heart block in newborns of mothers with anti-Ro/SSA antibodies detected by counterimmunoelectrophoresis. Arthritis Rheum 2001;44:1832-1835. 11. Solomon DG, Rupel A, Buyon JP. Birth order and recurrence rate in autoantibody-associated congenital heart block: implications for pathogenesis and family counseling. Lupus 2003;12:646-647. 12. Gladman G, Silverman ED, Yuk-Law, et al. Fetal echocardiographic screening of pregnancies of mothers with anti-Ro and/or anti-La antibodies. Am J Perinatol 2002;19:73-80.

13. Askanase AD, Friedman DM, Copel J, et al. Spectrum and progression of conduction abnormalities in infants born to mothers with anti-Ro/La antibodies. Lupus 2002;11:145-151. 14. Jaeggi ET, Hamilton RM, Silverman ED, et al. Outcome of children with fetal, neonatal or childhood diagnosis of isolated congenital atrioventricular block. J Am Coll Cardiol 2002; 39:130-137. 15. Nield LE, Silverman ED, Taylor GP, et al. Maternal anti-Ro and anti-La antibody-associated endocardial fibroelastosis. Circulation 2002;105:843-848. 16. Nield LE, Silverman ED, Smallhorn JF, et al. Endocardial fibroelastosis associated with maternal anti-Ro and anti-La antibodies in the absence of atrioventricular block. J Am Coll Cardiol 2002;40:796-802. 17. Glickstein JS, Buyon JP, Friedman D. The fetal PR interval: pulsed Doppler echocardiographic assessment. Am J Cardiol 2000;86:236-239. 18. Glickstein J, Buyon J, Kim M, Friedman D, PRIDE Investigators. The fetal Doppler mechanical PR interval: a validation study. Fetal Diagn Ther 2004;19:31-34. 19. Waltuck J, Buyon JP. Autoantibody-associated congenital heart block: Outcome in mothers and children. Ann Intern Med 1994;120:544-551. 20. Blanford AT, Pearson Murphy BE. In vitro metabolism of prednisolone, dexamethasone, betamethasone, and cortisol by the human placenta. Am J Obstet Gynecol 1997;127:264-267. 21. Kaaja R, Julkunen H. Prevention of recurrence of congenital heart block with intravenous immunoglobulin and corticosteroid therapy: comment on the editorial by Buyon et al. Arthritis Rheum 2003;48:280-281. 22. Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatory activity of IVIG mediated through the inhibitory Fc receptor. Science 2001;291:445-446. 23. Tran HB, Ohlsson M, Beroukas D, et al. Subcellular redistribution of La(SS-B) autoantigen during physiologic apoptosis in the fetal mouse heart and conduction system: a clue to the pathogenesis of congenital heart block. Arthritis Rheum 2002;46:202-208. 24. Tran HB, Macardle PJ, Hiscock J, et al. Anti-La (SS-B) antibodies transported across the placenta bind apoptotic cells in fetal organs targeted in neonatal lupus. Arthritis Rheum 2002;46:1572-1579.

471

NEONATAL LUPUS: CLINICAL PERSPECTIVES

472

25. Saleeb S, Copel J, Friedman D, Buyon JP. Comparison of treatment with fluorinated glucocorticoids to the natural history of autoantibody-associated congenital heart block: retrospective review of the Research Registry for Neonatal Lupus. Arthritis Rheum 1999;42:2335-2345. 26. Matsushita H, Higashino M, Sekizuka N, et al. Successful prenatal treatment of congenital heart block with ritodrine administered transplacentally. Arch Gynecol Obstet 2002;267:51-53.

27. Neiman AR, Lee LA, Weston WL, Buyon JP. Cutaneous manifestations of neonatal lupus without heart block: characteristics of mothers and children enrolled in a national registry. J Pediatr 2002;37:674-680. 28. Askanase AD, Miranda-Carus ME, Tang X, Katholi M, Buyon JP. The presence of IgG antibodies reactive with components of the SSA/Ro-SSB/La complex in human breast milk: Implications in neonatal lupus. Arthritis Rheum 2002;46:269-271.

CLINICAL ASPECTS OF THE DISEASE

43

Incomplete Lupus Erythematosus Tom J.G. Swaak, MD and Johannes C. Nossent, MD

INTRODUCTION The abundant use of classification criteria in rheumatology is a mixed blessing for practicing physicians.1 In the case of systemic lupus erythematosus, the introduction and subsequent widespread application of the initial, revised and updated American College of Rheumatology (ACR) criteria2-4 has been instrumental in delineating well-defined patient cohorts for epidemiologic, outcome, and intervention studies. However, as clearly stated in relevant publications, these criteria are the result of extensive statistical modeling to reach the lowest amount of heterogeneity in study cohorts. SLE on the other hand remains a clinical syndrome with a diverse phenotype in each individual patient that is in addition often variable over time. In daily practice, this has (silently) evolved into a tradition, where the ACR criteria are needed to confirm the clinical diagnosis of SLE. This practice has never been validated and has several drawbacks. First, patients not fulfilling four of the required ACR criteria could possibly be not given the diagnosis (and not receive the required treatment). It is of the utmost importance to realize that a large amount of data reduction was applied to achieve the models supporting the ACR criteria. In the process, a lot of additional clinical information has been excluded.2 These clinical data may nonetheless prove useful in the management of an individual patient, regardless of the amount of classification criteria fulfilled. Table 43.1 clearly demonstrates the problem: not only may patients present with a multisystemic disease and still not fulfill the ACR criteria, but a patient may also fulfill four criteria while the diagnosis of SLE is quite questionable. For example, the current ACR criteria contain four dermatologic elements, and thus a patient with only skin involvement can be diagnosed with SLE. However, it is well known that only a few (5%) of chronic cutaneous lupus patients will develop a more severe systemic disease. As a partial solution to this, methods have been proposed of weighting the various ACR criteria,5,6 but even this system failed to take into account that various disease presentations

do not occur independently of one another. The criteria photosensitivity and malar rash are highly interrelated, while there also is a strong correlation between anti-dsDNA Ab and positive antinuclear antibody (ANA) results.7 In the past, patients with typical symptoms but not fulfilling the ACR criteria for SLE or another connective tissue disease (CTD), have been labeled with diagnoses such as subclinical lupus, variant lupus, latent lupus, or overlap syndromes, with the overall idea that these patients would develop a specific disease entity in the future.8-13 In our opinion, incomplete lupus (ILE) is the most fitting description for these patients’ condition and this chapter summarizes current knowledge on this group of patients. It is our aim to contribute to a uniform understanding of this genuine clinical entity, which is well known to experienced rheumatologists, but has largely been identified in earlier textbooks.14 Generating further interest and study can facilitate the management of these patients with better and earlier identification of patients at risk of progressing to SLE. Finally, knowledge about the manner in which immune disturbances in ILE patients translate into a less severe clinical phenotype may provide important clues for the whole field.

DELINEATING INCOMPLETE LUPUS ERYTHEMATOSUS SLE is usually a disease that evolves over time and only a minority of patients present with multisystem disease that can be promptly classified according to the ACR criteria. In the LUMINA study cohort, this occurred in only 15 % of patients.15 In the remainder of patients, the mean time from the fulfillment of the first ACR criterion to the fulfillment of at least four criteria varied from 39 months for patients with one presenting ACR criterion to 32 and 14 months, respectively, for patients presenting with two or three ACR-validated criteria. The majority of these SLE patients were thus initially not classified as having SLE, but rather as having ILE

473

INCOMPLETE LUPUS ERYTHEMATOSUS

474

TABLE 43.1 FREQUENT DISEASE FEATURES IN SLE AND ACR CLASSIFICATION CRITERIA Organ System

ACR Classification Criteria

Other Frequent Features

Constitutional

—

Fever, malaise, anorexia, weight loss

Cutaneous

1 Malar rash 2 Discoid rash 3 Photosensitivity 4 Oral/nasopharyngeal ulcers

Alopecia, Raynaud’s phenomenon, general rash, subacute cutaneous LE, urticaria, bullous lesions, vasculitis, panniculitis,

Musculoskeletal

5 Nonerosive arthritis

Arthralgia, myalgia, myositis, tendonitis, ligamentous laxity, osteonecrosis

Cardiopulmonary

6 Pleuritis/pericarditis

Pleural effusions, myocarditis, pneumonitis, verrucous endocarditis, interstitial fibrosis, pulmonary hypertension, dyspnea

Renal

7 Proteinuria (>500 mg/day) Urinary cellular casts

Nephrotic syndrome, renal insufficiency, renal vein thrombosis, hypertension

Neurologic

8 Psychosis Seizure

Organic brain syndrome, cranial neuropathies, peripheral neuropathies, cerebellar signs

Gastrointestinal

—

Pancreatitis, ascites, elevated liver enzymes, vasculitis

Hematologic

9 Hemolytic anemia Leucopenia (<4000) Lymphopenia (<1500) Thrombocytopenia (<100,000)

Anaemia of chronic disease, lupus anticoagulant, thrombosis, splenomegaly, lymphadenopathy

Other systems

—

Sicca syndrome, conjunctivitis, episcleritis

Laboratory

10 Anti-dsDNA Ab, anti-Sm Ab, False-positive VDRL/ApL Ab 11 Positive ANA

Lupus band test on skin biopsy, anti-C1q Ab, hypocomplementemia

for various periods of time, and it would be interesting to know if presenting with ILE had therapeutic consequences. Similar timeframes for ILE have been described in European studies as well.13,16,17 Also of note was the longest observed lag time in the LUMINA cohort of 328 months, illustrating the potential for late disease progression in ILE patients. While these and other data illustrate the gradual progression from ILE to SLE in selected patients, they do not provide much needed information on patients not progressing beyond three ACR criteria. There are limited data in the literature that address this question. A few studies have analyzed the frequency and course of ILE separately, while other studies have included ILE in the group of undifferentiated connective tissue diseases (UCTDs). The term “undifferentiated connective tissue diseases” was introduced in the 1980s as the widespread use of the ANA test identified increasing numbers of patients with a systemic disease and a positive ANA test, and the question was raised whether these patients all really had SLE or that milder disease forms were more often recognized with the widespread diagnostic ANA testing. The further refinement of serologic test systems to demonstrate more subgroups of ANA indicate that patient groups potentially could be characterized by

their specific antinuclear antibody as well as titer. However, criteria for the diagnosis of UCTD are not yet established and the name should be reserved for those patients with features strongly suggestive of an autoimmune rheumatic disease but not readily classified or related to a specific CTD.18 Published UCTD series contain variable numbers of ILE patients, but in addition, groups of patients with not yet fully recognized rheumatoid arthritis or scleroderma.19,20 The question whether ILE is a separate entity or only a subset of UCTD is difficult to answer due to a lack of data on the subject, and reflects our basic uncertainty how to regard this group of patients in the larger scheme of things (Figure 43.1). Only one study has reported epidemiologic data on ILE.13 In a population based in Denmark, the prevalence of ILE was estimated to be 5.2 per 100,000, which was about one-quarter the prevalence of definite SLE I in that region. This rate must be considered a minimum estimate given the selection bias towards definite SLE in the study design. In Sweden, 28 ILE patients were identified during a 10-year observation period; from the numbers given, an annual ILE incidence of almost 2 per 100.000 can be inferred.12,21 Other cross-sectional studies on ILE do not report epidemiologic figures, but conclude that

Fulfilment of formal classification criteria

UCTD (symptoms not in classification criteria) ? ?

SLE

SS

Scl

RA

Fig. 43.1 Theoretical framework of the interrelationship among systemic lupus erythematosus, incomplete lupus erythematosus, and undifferentiated connective tissue diseases.

ILE is not infrequent.9,10,22 Despite a larger number of studies on UCTD in the literature, these are all crosssectional in design and give no indication of the relative frequency of this condition.23-26

PRESENTATION OF INCOMPLETE LUPUS ERYTHEMATOSUS The demographic findings from the available studies on ILE (summarized in Table 43.2) show that despite methodologic differences in study design, ILE patients seem to have few specific characteristics to set them apart from SLE patients in general. In addition, it seems clear that there has been little difficulty in realizing adequate study cohorts, which illustrates the relative frequency of ILE. Clinical findings reported in these studies are summarized in Table 43.3, and arranged by the presence of ACR classification criteria and other clinical features. Malar rash, photosensitivity, and the presence of ANA, with few patients with anti-dsDNA Ab and absence of anti-Sm Ab, are the main presenting findings. With the exception of the Swedish study, there seems to be a very low frequency of renal involvement, which in most studies has not been further scrutinized by histologic investigations given that a wait-and-see approach sufficed in all cases. All studies report a virtual absence of neurologic ACR criteria, which is not surprising given their low sensitivity in the SLE classification.

ACCRUAL OF AMERICAN COLLEGE OF RHEUMATOLOGY CRITERIA IN INCOMPLETE LUPUS ERYTHEMATOSUS The rate of progression to an ACR criteria–based definition of SLE has been the focus in most reports and their findings are summarized in Table 43.4. The studies with the longest follow-up reported the highest progression rates, indicating that elapsed time is an important contributor to this process. There are, however,

few consistent clinical predictors for progression to SLE aside from a short symptomatic period and malar rash, while serologic findings seem more reliable as such indicators.27,28 This implies that ILE patients must be followed most closely for SLE progression in the first few years, but at the same time prolonged observation will be necessary before one can rest assured that the disease will not progress at all.

DISEASE OUTCOME IN INCOMPLETE LUPUS ERYTHEMATOSUS Two studies have reported measures for disease activity and damage development in ILE cohorts, although the validity of using these scoring systems in assessing ILE patients has not been documented, and underestimates of disease activity are likely. In the European multicenter study, which is the largest ILE study reported thus far, 122 ILE patients were followed for 3 years.11 The development of disease activity over time (as measured by the Systemic Lupus Erythematosus Activity Index [SLEDAI] and European Consensus Lupus Activity Measurement [ECLAM]) for these patients is represented in Table 43.5. It is clear that these ILE patients continued to have low disease activity over the 3-year period, while patients progressing to SLE had significant increases in disease activity. More detailed information on the type and number of ILE patients experiencing disease activity is presented in Table 43.6. Here the annual number of ILE patients experiencing specific clinical features is given as a function of similar disease activity in the previous year, with detailed information on the number of patients with increasing, stable, or decreasing symptoms. Such data provide better insight in the dynamic process of changing disease activity in individual patients than overall scores. Arthritis and fatigue were the most frequent symptomatic finding; while the prevalence of arthritis fluctuated (15%, 19%, and 13%), there was considerable variation in the number of patients remitting and experiencing new arthritic flares, demonstrating the dynamics of this type of disease activity over time. In contrast, the prevalence of pericarditis decreased over the years from 4 to 0% of the patients with a different pattern of dynamics. Only a minority of the patients had renal involvement, with a relatively stable overall prevalence as expected with this type of complication. Nonetheless, both proteinuria and hematuria were varied over time in severity in most patients. The most frequently observed laboratory finding was leucocytopenia: overall prevalence varied during follow-up in this cohort between 36% and 20% with considerable intrapatient variation. Nonhematolytic anemia (overall prevalence variation from 10 to 11% and 16%) showed a similar large intrapatient variation.

DISEASE OUTCOME IN INCOMPLETE LUPUS ERYTHEMATOSUS

ILE (at least one clinical ACR criterion)

475

122

1:121

40 ± 13

Patients (n)

Gender (male/female)

Age at onset years ± standard deviation

5:82 34 ± 13

45 (14-88)

2:26

28

1, 2, or 3 ACR criteria

South Sweden Population based

Stahl-Hellgren12

38

37

6:32

38

2 or 3 ACR criteria

United States Single tertiary center

Greer9

ACR, American College of Rheumatology; ESCISIT, European Standing Committee on International Clinical Studies Including Therapeutic Trials.

Symptomatic period (months)

1, 2, or 3 ACR criteria

ANA pos + one organ system affected

Inclusion criteria

87

Puerto Rico Single tertiary center

Villa10

Europe Multicenter

ESCISIT11

Setting

Demographic Features

37.5

2:20

22

1 or 2 ACR criteria + any of 14 own criteria; stable disease ≥5 yrs

Canada Single tertiary center

Ganczarczyk8

TABLE 43.2 DEMOGRAPHIC FEATURES OF ILE PATIENTS IN VARIOUS STUDIES

37.3

1:19

63

1, 2, or 3 ACR criteria

Denmark Population based

Voss13

INCOMPLETE LUPUS ERYTHEMATOSUS

476

ESCISIT11

Clinical Finding Constitutional symptoms

Villa10

Stahl-Hellgren12 Greer9

Ganczarczyk8

Voss13

35

NA

NA

NA

18

NA

Malar rash

4

15

21

13

5

15

Discoid lupus

4

6

7

34

0

0

Photosensitivity

NA

32

42

24

23

35

Oropharyngeal ulcers

NA

0

0

3

23

0

Arthritis

13

13

32

47

41

10

Serositis

4

1

14

16

18

35

Glomerulonephritis

4

3

18

0

0

0

Central nervous system disorder

3

0

0

3

0

0

Hematologic disorder

NA

NA

14

18

NA

25

Anemia (any type)

10

12.5

NA

NA

NA

NA

Hemolytic anemia

2

NA

NA

0

0

NA

36

3

NA

16

36

NA

Leucopenia Thrombocytopenia

8

4

NA

3

23

NA

Immunologic disorder

NA

8

18

0

41

60

Anti-dsDNA Ab

NA

8

NA

0

9

NA

Anti-Sm Ab

NA

0

NA

0

0

NA

APL

NA

NA

21

0

ANA

100

98

100

82

82

Alopecia

NA

NA

7

NA

NA

5

Raynaud

25

4

21

NA

NA

30

Anti-SSA/SSB Ab

NA

8

7

20

NA

NA

Hypocomplementemia

NA

3

7

14

18

NA

14 (BFP-STS)

DISEASE OUTCOME IN INCOMPLETE LUPUS ERYTHEMATOSUS

TABLE 43.3 BASELINE ACR CRITERIA AND OTHER CLINICAL FEATURES OF ILE PATIENTS IN PUBLISHED SERIES (%)

39 100

ACR, American College of Rheumatology; ESCISIT, European Standing Committee on International Clinical Studies Including Therapeutic Trials; ILE, incomplete lupus erythematosus; NA, not available.

TABLE 43.4 FOUR OR MORE ACR CRITERIA IN PUBLISHED SERIES ON ILE PATIENTS Study

ESCISIT11

Villa10

Stahl-Hellgren12

Greer9

Ganczarczyk8

Follow-up time (years)

3

2.2 (±2.4)

13 (10–20)

1.6 (1–3)

8 (5–15)

Patients developing ≥4 ACR criteria (%)

25 (20)

8 (9)

16 (57)

2 (5)

7 (32)

Time to reaching 4 ACR criteria (years)

1.2 (0.2–3)

4.4

5.3

1.5

≥5 (definition)

22 (88) 25 (100) NA

NA

8 (50) 8 (50)

1 (50) 2 (100) NA

0 (0) 0 (0) 7 (100)

None

Malar rash ApL Ab

Malar rash Oral ulcers Anti-dsDNA Hypocomplementemia

None

HLA-DR1

Patients reaching 4 ACR criteria (%) <1 year <5 years ≥5 years Risk factors for SLE progression

ACR, American College of Rheumatology; ESCISIT, European Standing Committee on International Clinical Studies Including Therapeutic Trials; ILE, incomplete lupus erythematosus; NA, not available; SLE, systemic lupus erythematosus.

477

INCOMPLETE LUPUS ERYTHEMATOSUS

TABLE 43.5 MEDIAN DISEASE ACTIVITY SCORES AT YEAR 1, 2, AND 3 IN 122 PATIENTS INITIALLY DIAGNOSED WITH ILE Remaining ILE (±SD) Observation Year

ECLAM

SLEDAI

Year 1

2.1 ±2.3

2.6 ±4.5

Year 2

2.1 ±2

Year 3

1.7 ±2

Progressed to SLE (±SD) Total patients (n)

ECLAM

SLEDAI

Total patients (n)

100

4.4 ±2.3

4.3 ±4

22

2.0 ±4.3

98

6.1 ±4.5

8.5 ±9

24

2.4 ±4.5

97

5.6 ±2.2

9.9 ±8.9

25

ECLAM, European Consensus Lupus Activity Measurement; SLEDAI, Systemic Lupus Erythematosus Activity Index; ILE, incomplete lupus erythematosus; NA, not available; SD, standard deviation; SLE, systemic lupus erythematosus.

Damage according to Systemic Lupus International Collaborating Clinics (SLICC) criteria was assessed in the Swedish cohort score and hardly increased during prolonged follow-up,12 indicating the previously mentioned persistent but low level of disease. Several other outcome measures are potentially relevant in the study of outcomes in ILE patients. Studies of ILE impact on mortality, cardiovascular morbidity (given the relation

between inflammatory conditions and atherosclerosis), and quality of life, however, have not been reported. Given the presumed frequency of the condition, such data should not prove very difficult to acquire. Table 43.7 gives an overview of the results extracted from the reported ILE studies. None of the studies reported fatalities during follow-up of ILE patients. Drug treatment of ILE clearly is not a rare

TABLE 43.6 CHANGES IN PERCENTAGE OF INCOMPLETE LUPUS ERYTHEMATOSUS PATIENTS WITH SPECIFIED CLINICAL Present in Year 1 Fever

No Change

Less Severe

More Severe

New

Remission

Present in Year 2

Present No in Year 3 Change

5

8

1

1

0

6

3

8

3

0

Fatigue

30

20

12

1

2

5

17

20

32

13

Arthritis

15

19

7

1

0

11

7

19

13

4

Malar rash

4

6

3

0

0

3

1

6

10

1

Generalized rash

1

2

0

0

0

2

1

2

1

0

Discoid rash

4

1

0

0

0

1

4

1

4

1

Pericarditis

4

2

0

1

0

1

3

2

0

0

Seizures

1

2

0

0

0

2

1

2

1

0

Stroke

2

3

0

1

0

2

1

3

1

0

Psychoses

0

1

0

0

0

1

0

1

2

0

Proteinuria

4

4

1

0

0

3

3

4

3

2

Raised serum creatinine

1

0

0

0

0

0

1

1

0

0

Urinary casts

3

1

0

0

0

1

3

5

8

1

Hematuria

5

5

2

0

0

3

3

0

0

0

10

11

4

0

0

7

6

11

16

4

2

0

0

0

0

0

2

0

0

0

36

29

18

1

2

8

15

29

20

11

8

4

3

0

0

1

5

4

5

2

Nonhemolytic anemia Hemolytic anemia Leucopenia

478

Present in Year 2

Thrombocytopenia

CONCLUSIONS In daily practice, the diagnosis of a definite specific CTD is made when the patient fulfills ACR classification criteria. The results of the reported studies here demonstrate that patients who do not fulfill sufficient ACR criteria for SLE should be considered to have ILE.

In most studies, ILE cohorts differ from SLE cohorts in the low number of individuals with renal and CNS involvement. Overall, they seem mostly characterized by the presence of antinuclear antibodies, Raynaud’s phenomenon, arthritis, leucocytopenia, and thrombopenia. ILE is clearly not a rare entity,13,29 and while many patients are likely to have a benign disease course, 10 to 50% of patients within an ILE cohort will progress to SLE in the near or distant future. Aside from the number of ACR criteria fulfilled, there are few distinguishing features for ILE patients. It should be stressed, however, that disease duration is a quite important determinant of the process of classification and subsequent management. As illustrated by the long lag time between ILE and SLE progression in the Swedish and Canadian cohorts, prolonged periods of observation are indicated in ILE patients. This is supported by the dynamic pattern of intra-patient changes seen over 3 years in individual patients in the ESCISIT study, even though the overall prevalence of specific symptoms remained low. While ILE can be considered

CONCLUSIONS

occurrence, given that considerable numbers of patients received NSAID, antimalarials, and oral corticosteroid therapy. In the European Standing Committee on International Clinical Studies Including Therapeutic Trials (ESCISIT) study, 38% of patients were at inclusion already on treatment with prednisolone (none with doses >10 mg/d). This figure changed to 43% and 27% in year 2 and year 3, respectively. For antimalarials, these figures were 17% and 32%, respectively, while azathioprine was prescribed in only 2%, 3%, 5%, and 4.5% of cases, respectively. The influence (if any) of these treatment regimens on disease expression and the progression to SLE has not been studied.

FINDINGS THROUGHOUT FIRST, SECOND, AND THIRD YEAR OF OBSERVATION Less Severe

More Severe

New

Remission

New

Remission

Present Present in in Year 2 Year 1

Without any Change

Decrease Increase Newly in in devel- Complete Severity Severity oped Cessation

2

0

1

8

1

6

5

8

1

1

0

6

3

1

1

17

5

17

5

30

20

12

1

2

5

17

0

0

9

15

9

15

15

19

7

1

0

11

7

0

0

9

5

9

5

4

6

3

0

0

3

1

0

0

1

2

1

2

1

2

0

0

0

2

1

0

0

3

0

3

0

4

1

0

0

0

1

4

0

0

0

2

0

2

4

2

0

1

0

1

3

0

0

1

2

1

2

1

2

0

0

0

2

1

0

1

0

2

0

2

2

3

0

1

0

2

1

0

0

2

1

2

1

0

1

0

0

0

1

0

0

0

1

2

1

2

4

4

1

0

0

3

3

0

0

0

1

0

1

1

0

0

0

0

0

1

0

0

7

4

7

4

3

1

0

0

0

1

3

0

0

0

0

0

0

5

5

2

0

0

3

3

0

1

11

0

11

0

10

11

4

0

0

7

6

0

0

0

0

0

0

2

0

0

0

0

0

2

4

0

5

14

5

14

36

29

18

1

2

8

15

1

1

1

4

1

4

8

4

3

0

0

1

5

479

INCOMPLETE LUPUS ERYTHEMATOSUS

TABLE 43.7 CLINICAL DISEASE OUTCOME IN PUBLISHED SERIES ON ILE PATIENTSa Study

ESCISIT11

Villa10

Stahl-Hellgren12

Greer9

Ganczarczyk8

NSAID usage

NA

NA

NA

47

41

Antimalarials usage

17

NA

NA

29

NA

Corticosteroid usage

43

NA

NA

32

9

Cytotoxic drugs usage

5

NA

NA

3

0

SLEDAI baseline

2.6

NA

NA

NA

NA

SLEDAI latest

2.4

NA

NA

NA

NA

SLICC score baseline

NA

NA

0.1

NA

NA

SLICC score latest

NA

NA

0.16

NA

NA

Death

0

0

0

0

0

a

Figures are recalculated to represent ILE patients who did not progress to systemic lupus erythematosus.

ESCISIT, European Standing Committee on International Clinical Studies Including Therapeutic Trials; ILE, incomplete lupus erythematosus; NA, not available; NSAID, nonsteroid anti-inflammatory drug; SLEDAI, Systemic Lupus Erythematosus Activity Index; SLICC, Systemic Lupus International Collaborating Clinics.

a subset of SLE characterized by often mild disease, its unpredictable course indicates that it should be considered a distinctive disease entity. ILE patients are often included in patient cohorts described by the term “undifferentiated” CTD (UCTD), which represents early disease that has not yet evolved into a disease such as SLE, scleroderma, Sjögren’s syndrome, or polymyositis-dermatomyositis (PM-DM). Earlier studies have shown that a minority of UCTD patients (mainly defined by rheumatic complaints in the

presence of ANA) could be classified as SLE a few years later.19,20,23,30,31 As these ANA-positive patients with undefined musculoskeletal symptoms often do not evolve into SLE, the term ILE seems preferable in cases where patients fulfill at least one clinical ACR criterion for SLE, while reserving the term UCTD for patients with other clinical manifestations. This will allow better delineation of patient groups for much needed future prospective studies on the natural course, immunology, and management of ILE patients.

REFERENCES

480

1. Bywaters EG. Systemic lupus erythematosus. Classification criteria for systemic lupus erythematosus, with particular reference to lupus-like syndromes. Proc R Soc Med 1967;60:463-464. 2. Cohen AS, Reynolds WE, et al. Preliminary criteria for the classification of systemic lupus erythematosus. Bull Rheum Dis 1971;21:643-648. 3. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271-1277. 4. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725. 5. Costenbader KH, Karlson EW, Mandl LA. Defining lupus cases for clinical studies: the Boston weighted criteria for the classification of systemic lupus erythematosus. J Rheumatol 2002;29: 2545-2550. 6. Clough JD, Elrazak M, Calabrese LH, Valenzuela R, Braun WB, Williams GW. Weighted criteria for the diagnosis of systemic lupus erythematosus. Arch Intern Med 1984;144:281-285. 7. Nossent HC, Rekvig OP. Is closer linkage between systemic lupus erythematosus and anti-double-stranded DNA antibodies a desirable and attainable goal? Arthritis Res Ther 2005;7:85-87. 8. Ganczarczyk L, Urowitz MB, Gladman DD. “Latent lupus.” J Rheumatol 1989;16:475-478. 9. Greer JM, Panush RS. Incomplete lupus erythematosus. Arch Intern Med 1989;149:2473-2476.

10. Vila LM, Valentin AH, Garcia-Soberal M, Vila S. Clinical outcome and predictors of disease evolution in patients with incomplete lupus erythematosus. Lupus 2000;9: 110-115. 11. Swaak AJ, Smeenk RJ, Manger K, Kalden JR, Tosi S, et al. Incomplete lupus erythematosus: results of a multicentre study under the supervision of the EULAR Standing Committee on International Clinical Studies Including Therapeutic Trials (ESCISIT). Rheumatology (Oxford 2001;40:89-94. 12. Stahl HC, Nived O, Sturfelt G. Outcome of incomplete systemic lupus erythematosus after 10 years. Lupus 2004;13:85-88. 13. Voss A, Green A. Systemic lupus erythematosus in Denmark: clinical and epidemiological characterization of a county-based cohort. Scand J Rheumatol 1998;27:98-105. 14. Lockshin MD. What is SLE? J Rheumatol 1989;16:419-420. 15. Alarcon GS, McGwin G Jr, Roseman JM, Uribe A, Fessler BJ, Bastian HM, et al. Systemic lupus erythematosus in three ethnic groups. XIX. Natural history of the accrual of the American College of Rheumatology criteria prior to the occurrence of criteria diagnosis. Arthritis Rheum 2004;15;51:609-615. 16. Stahl-Hallengren C, Jonsen A, Nived O, Sturfelt G. Incidence studies of systemic lupus erythematosus in Southern Sweden: increasing age, decreasing frequency of renal manifestations and good prognosis. J Rheumatol 2000;27:685-691. 17. Swaak AJ, Nieuwenhuis EJ, Smeenk RJ. Changes in clinical features of patients with systemic lupus erythematosus

19.

20.

21.

22. 23.

24.

25. Danieli MG, Fraticelli P, Franceschini F, Cattaneo R, Farsi A, Passaleva A, et al. Five-year follow-up of 165 Italian patients with undifferentiated connective tissue diseases. Clin Exp Rheumatol 1999;17:585-591. 26. Gilboe IM, Husby G. Application of the 1982 revised criteria for the classification of systemic lupus erythematosus on a cohort of 346 Norwegian patients with connective tissue disease. Scand J Rheumatol 1999;28:81-87. 27. Swaak AJ, Huysen V, Smeenk RJ. Antinuclear antibodies in routine analysis: the relevance of putative clinical associations. Ann Rheum Dis 1993;52:110-114. 28. Garred P, Voss A, Madsen HO. Association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients. Genes Immun 2001;2:442-450. 29. Calvo-Alen J, Bastian HM, Straaton KV, Burgard SL, Mikhail IS, Alarcon GS. Identification of patient subsets among those presumptively diagnosed with, referred, and/or followed up for systemic lupus erythematosus at a large tertiary care center. Arthritis Rheum 1995;38:1475-1484. 30. Dijkstra S, Nieuwenhuys EJ, Swaak AJ. The prognosis and outcome of patients referred to an outpatient clinic for rheumatic diseases characterized by the presence of antinuclear antibodies (ANA). Scand J Rheumatol 1999;28:33-37. 31. Vlachoyiannopoulos PG, Tzavara V, Dafni U, Spanos E, Moutsopoulos HM. Clinical features and evolution of antinuclear antibody positive individuals in a rheumatology outpatient clinic. J Rheumatol 1998;25:886-891.

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18.

followed prospectively over 2 decades. Rheumatol Int 1992;12: 71-75. Mosca M, Baldini C, Bombardieri S. Undifferentiated connective tissue diseases in 2004. Clin Exp Rheumatol 2004;22(suppl 33):S14-S18. Danieli MG, Fraticelli P, Salvi A, Gabrielli A, Danieli G. Undifferentiated connective tissue disease: natural history and evolution into definite CTD assessed in 84 patients initially diagnosed as early UCTD. Clin Rheumatol 1998;17:195-201. Williams HJ, Alarcon GS, Joks R, Steen VD, Bulpitt K, Clegg DO, et al. Early undifferentiated connective tissue disease (CTD). VI. An inception cohort after 10 years: disease remissions and changes in diagnoses in well established and undifferentiated CTD. J Rheumatol 1999;26:816-825. Stahl-Hallengren C, Jonsen A, Nived O, Sturfelt G. Incidence studies of systemic lupus erythematosus in Southern Sweden: increasing age, decreasing frequency of renal manifestations and good prognosis. J Rheumatol 2000;27:685-691. Ganczarczyk L, Urowitz MB, Gladman DD. “Latent lupus.” J Rheumatol 1989;16:475-478. Mosca M, Tavoni A, Neri R, Bencivelli W, Bombardieri S. Undifferentiated connective tissue diseases: the clinical and serological profiles of 91 patients followed for at least 1 year. Lupus 1998;7:95-100. Mosca M, Neri R, Bencivelli W, Tavoni A, Bombardieri S. Undifferentiated connective tissue disease: analysis of 83 patients with a minimum followup of 5 years. J Rheumatol 2002;29:2345-2349.

481

TREATMENT OF THE DISEASE

44

Nonsteroid Treatment of Systemic Lupus Erythematosus Robert G. Lahita, MD, PhD

INTRODUCTION There has not been a new treatment approved for systemic lupus erythematosus (SLE) in over 25 years. There are many forms of therapy available, however, and most are not approved for an SLE indication by the Food and Drug Administration. This chapter is focused on nonsteroidal anti-inflammatory drugs and a number of other noncytotoxic agents that are used in the treatment of SLE. Among these other agents are antimalarial agents, retinoids, thalidomide, dapsone, topical steroids, and androgenic compounds.

NONSTEROIDAL ANTI-INFLAMMATORY AGENTS Many patients with SLE can be treated initially with bed rest. Up to 80% of patients with SLE are treated at some time with nonsteroidal anti-inflammatory drugs (NSAIDs) for musculoskeletal symptoms, headaches, or inflammation of mucous membranes and serositis1 (Table 45.1). Standard anti-inflammatory agents range from aspirin, aspirin-like substances, to nonsteroidal anti-inflammatory agents. Therapeutic doses of these agents suppress inflammation by inhibiting the prostaglandin pathways and the cyclooxygenase enzymes in a selective or nonselective manner.2,3 The cyclooxygenase enzymes are labeled type 1 or type 2. Newer agents are cyclooxygenase-2 suppressants. The safety of the Cox-2 selective drugs and all other members of the class of Cox inhibitors is now questionable, subsequent to the description of increased cardiovascular complications such as acute myocardial infarction and hypertension.4-6 Although such agents are known to inhibit prostacyclin, an essential component of the normal clotting process,7 enhanced clotting has not been shown with these agents in SLE patients with or without coagulopathies. Nevertheless, agents are in development and currently on the market that retain the property of prostacyclin inhibition. Despite the finding that patients with SLE have a higher incidence of atherosclerotic heart disease,

there are no data on the use of these drugs in SLE patients and enhanced cardiovascular risk.8,9 Despite the lack of data, it would be prudent to avoid such agents in the treatment of patients with SLE who are at cardiovascular risk. These include all patients with SLE and rheumatoid arthritis who are at risk of developing accelerated vascular disease. Perhaps the greatest risk for SLE patients ingesting NSAIDs of both the selective and nonselective variety can be summarized as well-known renal toxic effects. Such agents lower glomerular filtration (GFR), and have significant effects on the ascending limb of the loop of Henle and the renal medulla, causing hypertension.10-13 There is also an effect from sodium retention. Azotemia quickly appears in patients with GFR less than 50 mL/min. Hypertension is also a risk in patients on any NSAID, and is dose dependent for both the Cox-1 and Cox-2 selective and nonselective agents.14 Cutaneous and allergic reactions have been seen in SLE patients ingesting these agents. These reactions include hepatotoxicity. Ovulation and pregnancy have also been affected by cyclooxygenase inhibitors.15,16 In animal model knockouts for the cyclooxygenase gene, there is total infertility. There are also modest data showing that animals exposed to high levels of the Cox-2 inhibitors fail to reproduce in comparison to their unexposed littermates. Finally, patients with SLE have a higher sensitivity to sulfur-containing drugs.17 One drug of the selective Cox-2 variety, Celecoxib, contains a sulfonamide moiety that produces an allergic reaction in many SLE patients.18 Severe allergic reactions to other NSAIDs have not occurred in SLE patients.

ANTIMALARIAL DRUGS Some of the most effective low-toxicity agents available to SLE patients are agents of the antimalarial class, including hydroxychloroquine, chloroquine, and quinacrine. The mechanism of action of these agents remains unknown.

483

NONSTEROID TREATMENT OF SYSTEMIC LUPUS ERYTHEMATOSUS

484

These agents are effective primarily as weak immunosuppressives. However, they have the added properties of lowering serum cholesterol and acting as an anticlotting agent.19 The antimalarials have immunomodulatory and anti-inflammatory properties. In vitro effects on macrophages and B cells have been described. Membrane phospholipids, phagocytosis, and the synthesis of certain cytokines have been inhibited with these agents. The secretion of interleukin-1, interleukin-6, and tumor necrosis factor-alpha is thought to be inhibited by these agents due to the effect of the drug on posttranslational events. The class activity within both the primary and secondary phospholipid syndrome revolves around the inhibition of fibrinogen binding, thrombin-induced platelet responses, and platelet aggregation.20 Despite all of these effects, these drugs are generally not useful for patients with severe SLE. The antimalarial drugs are very useful in the treatment of cutaneous SLE.21 These drugs are particularly effective in the early mild to moderate systemic disease, and they are often used in conjunction with steroids or chemotherapeutic agents.22 Hydroxychloroquine is also useful as a single agent in the treatment of antiphospholipid syndrome. Hydroxychloroquine is perfectly safe to use in pregnant SLE patients.23 Chloroquine and hydroxychloroquine are useful drugs in the treatment of discoid lupus. Clofazimine has also been used in place of hydroxychloroquine.24 The major toxicity of these agents is ophthalmologic. Corneal anesthesia and deposition of pigment in the retina are primary manifestations of toxicity.25 In the United States, patients with SLE who begin on these agents should have an ophthalmologic examination every 6 months26 (see Chapter 39) Additionally, these agents should not be used in anyone with glucose 6-phosphate dehydrogenase (G6PD) deficiency, underlying liver disease, or porphyria. Hydroxychloroquine is prescribed at 200 mg twice daily, although lower or higher doses might be used, and depend on both the size of the patient and tolerability.27 The hydroxychloroquine dose should not exceed 6.5 mg/kg/d. Chloroquine is used at concentrations of 250 mg/d and the same safety precautions apply. However, chloroquine is not routinely used at present because of the overall safety of the drugs hydroxychloroquine and quinacrine with respect to ocular toxicity. Both of these agents are often used in conjunction with quinacrine at 100 mg/d. However, Atabrine and Mepacrine, both brand names for quinacrine, are not widely available at this writing (Table 45.2).

lupus such as bullous SLE.28 It is also particularly useful in the treatment of lupus profundus. Some patients will be allergic to the sulfone component of dapsone, and care must be used when using this drug at doses above 100 mg/d. Use of this drug can result in long-lasting remission. Dapsone has been useful in one study in the therapy of thrombocytopenia.29 Caution must be exercised in prescribing this drug to patients with G6PD deficiency. In addition, hemolytic anemia resulting from dapsone can be a problem.

THALIDOMIDE There is renewed interest in the use of this drug for patients with SLE.30 Thalidomide is mainly used to treat discoid lupus, and its use in the SLE population is recent.31-34 The dosage is 1.5 to 5 mg/kg/d. Avoidance of doses higher than 100 mg/d is recommended in order to avoid profound neuropathy. However, SLE is a disease primarily of women of childbearing age, and a major concern for patients who take this drug is teratogenicity in pregnancy. In the United States, there are many restrictions to the use of this drug in young women. Teratogenicity is monitored by the STEPS program (System for Thalidomide Education and Prescribing Safety), which provides a follow-up review of most prescriptions dispensed in the United States. The primary reason for caution is the well-known incidence of phocomelia in patients who take this agent. The principal concern in the use of thalidomide is the onset of irreversible paresthesias. Thalidomide has also been associated with the onset of primary antiphospholipid syndrome,35 hypothyroidism, sedation, and constipation.

RETINOIDS Retinoids are vitamin-A derivatives, and are commonly used by some physicians to treat acute lupus rashes such as the malar rash,36 in addition to the discoid rashes often observed in conjunction with SLE or by themselves. These agents are also not used casually in women of childbearing age due to teratogenic effects. There is also potential for a variety of side effects, including dry skin, alopecia, eczema, skin fragility, and photosensitivity. Myalgia, hypertriglyceridemia, hepatitis, pancreatitis, and pseudotumor cerebri have also been observed. Isotretinoin is used at a dosage of 0.5 to 1 mg/kg/d in two doses with food. This regimen should not be continued longer than 15 to 20 weeks because of toxicity.

DAPSONE

ANDROGEN TREATMENT OF LUPUS ERYTHEMATOSUS

Dapsone is a sulfone drug used primarily to treat leprosy. However, it is useful in certain forms of refractory skin

There has been emphasis on the use of androgens for the treatment of SLE. The use of androgen therapy in

Other androgens such as danazol and cyproterone acetate have not been effective in the treatment of SLE. Thrombocytopenia does respond to levels of danazol at 400 mg per day, but mild to moderate lupus does not.43-45 Methyl testosterone in any form does not show efficacy in the treatment of SLE because of lack of response. In addition, testosterone has many unsightly side effects such as hirsutism, lowered voice, acne rash, and increased muscularity.

REFERENCES

the treatment of lupus comes from the observation that females predominate with this disease.37 Despite these findings, androgens have not shown as much promise in the treatment of disease as originally hoped. Dehydroepiandrosterone (DHEA) has shown steroid-sparing effects in one study and an overall reduction of flares in another study.38,39 Moreover, there was initial excitement about DHEA being able to produce bone growth in the presence of corticosteroids.40 Unfortunately, the drug has failed to meet the endpoints for the resolution of steroid-induced osteoporosis. DHEA is not suitable for the treatment of anything but mild to moderate lupus,41 although data from Taiwan would indicate that the drug can be used for moderate to severe SLE.42 Side effects from most androgens including DHEA include acne and mild hirsutism in some patients. There is also evidence that lipid profiles change and estrogen levels rise to levels found with usual hormone replacement. Health-food store–grade DHEA is not suitable for clinical use because of inconsistent levels of active drug and bioavailability. The effective oral dose of the drug is 200 mg.

CONCLUSIONS There are many nonsteroid drugs that can be used to treat the disease lupus. Most of these are quite safe and without serious side effects. These include the anti-inflammatory drugs that inhibit cyclooxygenases; the antimalarial agents that are effective immunosuppressants; and the retinoids, thalidomide, and dapsone used for skin manifestations. The androgens have yet to be shown as effective in the treatment of lupus. These drugs are preferred over the cytotoxic agents and may be a first-line therapy.

REFERENCES 1. Ostensen M, Villiger PM. Nonsteroidal anti-inflammatory drugs in systemic lupus erythematosus. Lupus 2001;10:135-139. 2. Infante R, Lahita RG. Rheumatoid arthritis. New disease-modifying and anti-inflammatory drugs. Geriatrics 2000;55:30-40. 3. Cryer B, Dubois A. The advent of highly selective inhibitors of cyclooxygenase-a review. Prostaglandins Other Lipid Mediators 1998;56:341-361. 4. Mukherjee D, Topol EJ. Cox-2: where are we in 2003? Cardiovascular risk and Cox-2 inhibitors. Arthritis Res Ther 2003;5:8-11. 5. Mukherjee D, Nissen SE, Topol EJ. Cox-2 inhibitors and cardiovascular risk: we defend our data and suggest caution. Cleve Clin J Med 2001;68:963-964. 6. Mukherjee D, Nissen SE, Topol EJ. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 2001;286: 954-959. 7. Catella-Lawson F, Crofford LJ. Cyclooxygenase inhibition and thrombogenicity. Am J Med 2001;110(3A):28S-32S. 8. Krotz F, Schiele TM, Klauss V, Sohn HY. Selective COX-2 inhibitors and risk of myocardial infarction. J Vasc Res 2005;42:312-324. 9. FitzGerald GA. Coxibs and cardiovascular disease. N Engl J Med 2004;351(17):1709-1711. 10. Szeto CC, Chow KM. Nephrotoxicity related to new therapeutic compounds. Ren Fail 2005;27:329-333. 11. Aw TJ, Haas SJ, Liew D, Krum H. Meta-analysis of cyclooxygenase-2 inhibitors and their effects on blood pressure. Arch Intern Med 2005;165:490-496. 12. Sowers JR, White WB, Pitt B, Whelton A, Simon LS, Winer N, et al. The effects of cyclooxygenase-2 inhibitors and nonsteroidal anti-inflammatory therapy on 24-hour blood pressure in patients with hypertension, osteoarthritis, and type 2 diabetes mellitus. Arch Intern Med 2005;165:161-168. 13. Hermann M, Shaw S, Kiss E, Camici G, Buhler N, Chenevard R, et al. Selective COX-2 inhibitors and renal injury in salt-sensitive hypertension. Hypertension 2005;45:193-197. 14. Wang K, Tarakji K, Zhou Z, Zhang M, Forudi F, Zhou X, et al. Celecoxib, a selective cyclooxygenase-2 inhibitor, decreases

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monocyte chemoattractant protein-1 expression and neointimal hyperplasia in the rabbit atherosclerotic balloon injury model. J Cardiovasc Pharmacol 2005;45:61-67. Sirois J, Sayasith K, Brown KA, Stock AE, Bouchard N, Dore M. Cyclooxygenase-2 and its role in ovulation: a 2004 account. Hum Reprod Update 2004;10:373-385. Chan VS. A mechanistic perspective on the specificity and extent of COX-2 inhibition in pregnancy. Drug Saf 2004;27: 421-426. Knowles S, Shapiro L, Shear NH. Should celecoxib be contraindicated in patients who are allergic to sulfonamides? Revisiting the meaning of ‘sulfa’ allergy. Drug Saf 2001;24:239-247. Lander SA, Wallace DJ, Weisman MH. Celecoxib for systemic lupus erythematosus: case series and literature review of the use of NSAIDs in SLE. Lupus 2002;11:340-347. Wallace DJ. The use of chloroquine and hydroxychloroquine for non-infectous conditions other than rheumatoid arthritis or lupus: a critical review. Lupus 1996;5(suppl):S59-S64. Edwards MH, Pierangeli S, Liu XW. Hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice. Circulation 1997;96:4380-4384. Wozniacka A, McCauliffe DP. Optimal use of antimalarials in treating cutaneous lupus erythematosus. Am J Clin Dermatol 2005;6:1-11. Fessler BJ, Alarcon GS, McGwin G, Roseman J, Bastian HM, Friedmajn AW, et al. Systemic lupus erythematosus in three ethnic groups. XVI. Association of hydroxychloroquine use with reduced risk of damage accrual. Arthritis Rheum 2005; 52:1473-1480. Costedoat-Chalumeau N, Amoura Z, Duhaut P, Huong du LT, Sebbough D, Wechsler B, et al. Safety of hydroxychloroquine in pregnant patients with connective tissue diseases: a study of one hundred thirty-three cases compared with the control group. Arthritis Rheum 2004;50:3056-3057. Bezerra EL, Vialr MJ, da Trindade Neto PB, Sato EI. Double blind, randomized, controlled clincal trial of clofazimine compared

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with chloroquine in patients with systemic lupus erythematosus. Arthritis Rheum 2005;52:3073-3078. Klippel JH. Immunosuppressive therapy: antimalarials, cytotoxic agents and azathioprine. In: Lahita RG, ed. Systemic Lupus Erythematosus. New York: Churchill Livingstone, 1992:933-948. Sfikakis PP, Mavrikakis M. opthlamologic monitoring for antimalarial toxicity. J Rheum 2004;31:1011-1012. Davis JC, Klippel JH. Antimalarials and Immunosuppressive Drugs. In: Lahita RG, ed. Systemic Lupus Erythematosus. San Diego: Academic Press, 1999:967-984. Burrows NP, Bhogal BS, Black MM, Rustin MH, Ishida-Yamamoto A, Kirtschig G, et al. Bullous eruption of systemic lupus erythematosus: a clinicopathological study of four cases. Br J Dermatol 1993;128:332-338. Park YH, Sunamoto M, Miyoshi T, Konaka Y. Effectiveness of dapsone on refractory immune thrombocytopenia in a patient with systemic lupus erythematosus associated with sarcoidosis. Rinsho Ketsueki 1993;34:870-875. Coelho A, Souto MI, Cardoso CR, Salgado DR, Schmal TR, Waddington CM, et al. Long-term thalidomide use in refractory cutaneous lesions of lupus erythematosus: a 65 series of Brazilian patients. Lupus 2005;14:434-439. Alfadley A, Al-Rayes H, Hussein W, Al-Dalaan A, Al-Aboud K. Thalidomide for treatment of severe generalized discoid lupus lesions in two patients with systemic lupus erythematosus. J Am Acad Dermatol 2003;48(suppl 5):S89-S91. Georgala S, Katoulis AC, Hasapi V, Koumantaki-Mathioudaki E. Thalidomide treatment for hypertrophic lupus erythematosus. Clin Exp Dermatol 1998;23:141. Hawkins DF. Thalidomide for systemic lupus erythematosus. Lancet 1992;339:1057. Bessis D, Guillot B, Monpoint S, Dandurand M, Guilhou JJ. Thalidomide for systemic lupus erythematosus. Lancet 1992;339:549-550. Tektonidou MG, Vlachoyiannopoulos PG. Antiphospholipid syndrome triggered by thalidomide in a patient with discoid lupus erythematosus. Lupus 2003;12:723-724.

36. Gergely P, Csaky L, Gonzalez-Cabello P. Immunological effects of retinoids. Tokai J Exp Clin Med 1990;15:235-239. 37. Lahita RG. Sex and age in systemic lupus erythematosus. In: Lahita RG, ed. Systemic Lupus Erythematosus. New York: John Wiley and Sons, 1986:523-539. 38. Petri MA, Lahita RG, Van Vollenhoven RF, Merrill JT, Schiff M, Ginzler EM, et al. Effects of prasterone on corticosteroid requirements of women with systemic lupus erythematosus: a double-blind, randomized, placebo-controlled trial. Arthritis Rheum 2002;46:1820-1829. 39. Petri MA, Mease PJ, Merrill JT, Lahita RG, Iannini MJ, Yocum DE, et al. Effects of prasterone on disease activity and symptoms in women with active systemic lupus erythematosus. Arthritis Rheum 2004;50:2858-2868. 40. Hartkamp A, Geenen R, Godaert GL, Bijl M, Bijlsma JW, Derksen RH. The effect of dehydroepiandrosterone on lumbar spine bone mineral density in patients with quiescent systemic lupus erythematosus. Arthritis Rheum 2004;50:3591-3595. 41. Lahita RG. Dehydroepiandrosterone (DHEA. and lupus erythematosus: an update [editorial]. Lupus 1997;6:491-493. 42. Chang DM, Lan JL, Lin HY, Luo SF. Dehydroepiandrosterone treatment of women with mild-to-moderate systemic lupus erythematosus: a multicenter randomized, double-blind, placebo-controlled trial. Arthritis Rheum 2002;46:2924-2927. 43. Ahn YS, Harrington WJ, Simon SR, Mylvaganam R, Pall LM, So AG. Danazol for the treatment of idiopathic thrombocytopenic purpura. N Eng J Med 1983;308:1396. 44. Ahn YS, Mylvaganam R, Garcia RO, Kim CI, Palow D, Harrington WJ. Low-dose danazol therapy in idiopathic thrombocytopenic purpura. Ann Intern Med 1987;107:177-181. 45. Agnello V, Pariser K, Gell J. Preliminary observation on Danazol therapy of systemic lupus: effects on DNA antibodies, thrombocytopenia and complement. J Rheumatol 1983;10:682-687.

TREATMENT OF THE DISEASE

45

Systemic Steroids Mustafa Al-Maini, MD and Murray Urowitz, MD

INTRODUCTION Corticosteroid medications are found in the therapeutic armamentarium of most medical specialties. Rheumatologists have had a long relationship with these agents1,2 since their first demonstrated therapeutic efficacy in rheumatoid arthritis.3 Systemic corticosteroids therapy is the cornerstone of the treatment of acute, active systemic lupus erythematosus (SLE) either alone or in combination with “steroid-sparing” (immunosuppressive) agents.2 Despite this, many issues regarding the methods of use of this class of agents remain controversial. Such issues include the appropriate initial dose, route of administration, duration of treatment, acceptable withdrawal algorithm, and impact, prevention, and treatment of the inevitable side effects of prolonged use. In this review, we incorporate the best evidence available as well as refer to our experience in the University of Toronto Lupus Clinic, a prospective long-term observational cohort study of 35 years duration.4 This chapter will review multiple aspects of using systemic corticosteroids in treating SLE. Historical Aspects of Corticosteroids Use in Treating Rheumatic Diseases and SLE In the early 1920s, Edward Calvin Kendall proposed that rheumatoid arthritis represented a deficiency of adrenal hormone. In the 1930s, Henry L. Mason and others isolated a number of steroid hormones from the adrenal cortex and special interest focused on Compound E, later named cortisol (hydrocortisone). Kendall had suggested that such hormones could be therapeutically useful in rheumatoid arthritis (RA), and research in this area was accelerated during World War II.1 In 1949, Philip Showalter Hench and associates at the Mayo Clinic obtained sufficient quantities of cortisol to administer it to a patient with rheumatoid arthritis.3 The results of this new treatment were dramatic. RA patients who until then were incapacitated experienced dramatic improvement. So great was the excitement over the new treatment, that the Nobel Prize committee altered its time-honored rule for delaying the award

for a number of years after any major discovery, and immediately in 1950 awarded the prize in medicine to Edward Calvin Kendall, Tadeus Reichstein, and Philip Showalter Hench for their “discoveries relating to the hormones of the adrenal cortex, their structure and biological effects.”1 Soon thereafter, oral corticosteroids were also used to treat SLE. In the early 1960s, Pollak and colleagues5,6 reported that high doses of oral prednisone helped to prevent or postpone the onset of renal failure. It was not until much later that the long-term side effects became obvious and muted the initial excitement over the therapeutic actions of corticosteroids. Oral corticosteroids became the mainstay of treatment for most clinical manifestations of SLE. Another important development took place in 1976, as Edward Cathcart and his colleagues treated seven SLE patients with diffuse proliferative glomerulonephritis with high-dose intravenous methylprednisolone “pulses.”7 This treatment led to a rapid and dramatic improvement in five of seven patients. This treatment regimen had initially been used for acute rejection in renal transplantation with impressive results.8,9 Unfortunately, 30 years later many uncertainties around this treatment approach still exist.

RATIONALE FOR SYSTEMIC CORTICOSTEROIDS USE IN SLE SLE is characterized by generalized inflammation and excessive autoantibody production. These findings constitute the rationale for the use of systemic corticosteroids in the treatment of SLE for their antiinflammatory and immunosuppressive actions. The effect of the inflammation on organ dysfunction is indisputable. It has been established that the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoids in particular play an essential natural role in aborting and limiting inflammation.10 This natural mechanism of suppression of inflammation is multiplied significantly when treating SLE with pharmacologic doses.

487

SYSTEMIC STEROIDS

The beneficial and therapeutic effects in SLE of administered systemic corticosteroids are likely due first to their anti-inflammatory effects in the short term and later to their immunosuppressive effects through inhibition of antibody synthesis.11 Once steroids have been initiated, one must use objective criteria to measure response to therapy to guide systemic corticosteroid tapering and withdrawal. Currently, four disease activity measures are in use in lupus clinics and in therapeutic trials: SLE Disease Activity Index (SLEDAI-2K),12 the British Isles Lupus Assessment Group (BILAG),13 SLE Activity Measure (SLAM),14 and European Consensus Lupus Activity Measurement (ECLAM)15 (see Chapter 2). We recommend using any of the above indices to monitor the disease activity in SLE patients to assess adjusting the systemic corticosteroids dose used. In therapeutic trials, predetermined systemic corticosteroid withdrawal algorithms are sometimes used as outcome measures and are applied irrespective of disease activity. The latter approach with its lack of flexibility is not appropriate for use in daily practice.

Mechanisms of Action The human glucocorticoid receptor (GR) gene is one locus on chromosome 5q31-32, and the human GR messenger RNA has alternative splice variants that produce the target proteins.16 The GR is a member of the steroid-hormone-receptor family of proteins. It binds with high affinity to cortisone, which initiates the dissociation of molecular chaperones, such as heat-shock proteins, from the receptor. The GR are intracellular structures and therefore most of the initial action is contained within the affected cells. In brief, the cortisol–GR complex penetrates the nucleus to bind to the DNA sequence called glucocorticoidresponsive elements. The latter complex initiates the transcription process mediated by RNA polymerase II.

The proteins produced are anti-inflammatory proteins,17 which are discussed below. Cortisol (hydrocortisone) is the major corticosteroid hormone found in the human adrenal cortex. It is derived from the hydroxylation of cortisone. Systemic corticosteroid agents mimic the endogenous steroid hormones produced in the adrenal cortex, mineralocorticoid (aldosterone), and glucocorticoid (cortisol). Mineralocorticoids are primarily regulated by the renin-angiotensin system and possess salt-retaining properties. Glucocorticoids are primarily regulated by corticotropin (ACTH), and have anti-inflammatory effects as well as metabolic and immunogenic effects on the body. While several corticosteroid agents possess properties of both hormones, fludrocortisone is most commonly used for its mineralocorticoid activity and hydrocortisone, cortisone, prednisone, and prednisolone are used for their glucocorticoid effects (Table 45.1).18,19 Although the broad mechanisms are known, the cellular pathways affected by the drugs are numerous and complex.20-22 As mentioned above, the initial therapeutic effects in SLE of administered systemic corticosteroids are likely due first to their anti-inflammatory effects on the cellular mechanism of the immune response and later to their immunosuppressive effects through inhibition of antibody synthesis as effects on humoral immune response.20 Corticosteroids exert their anti-inflammatory effect primarily by inhibiting the production of cytokines and cell-adhesion molecules. Anti-inflammatory effects of systemic corticosteroids include decrease of neutrophil margination, decrease macrophage migration, decrease accumulation of neutrophils at inflammatory sites, inhibition of neutrophil and macrophage phagocytosis, inhibition of production of proinflammatory cytokines IL-1 and TNF, inhibition of cycloxygenase-2 (COX-2), induction of expression of

TABLE 45.1 FORMS AND HALF-LIFE OF STEROIDS Hormone

488

Equivalent Commercial Tablet (mg)

Half-Life

Cortisone acetate

25

12 hours

Hydrocortisone

20

Relative Potency

Activity Short acting

12 hours

1

Short acting

Prednisone

5

12–36 hours

4

Intermediate acting

Prednisolone

5

12–36 hours

4

Intermediate acting

Methylprednisolone

4

12–36 hours

5

Triamcinolone

4

12–36 hours

Dexamethasone

0.75

48 hours

Betamethasone

0.65

48 hours

Intermediate acting Intermediate acting

25

Long acting Long acting

FORMS AND DELIVERY ROUTES OF SYSTEMIC CORTICOSTEROIDS The various types of corticosteroids that have been used in rheumatology and their potencies are summarized in Table 45.1. There is no reason to believe that there is a major difference among various oral corticosteroid preparations11,25,26; rather it is frequency of dosing and the dose used that determine the clinical effect. Hydrocortisone is the saturated physiologic form of the steroids, whereas prednisone is an unsaturated synthetic. Prednisone is the most frequently used preparation due to its shorter half-life and the availability of tablets in a variety of doses, which makes dose adjustments easy.27 Multiple websites provide instant conversions of corticosteroid dosages. (We recommend www.globalrph.com/corticocalc.htm.)

Topical Corticosteroids Topical corticosteroid preparations may be used for inflammatory skin rashes without evidence of vasculitis. Formulations include short acting hydrocortisone (0.125% to 1.0%), intermediate-acting products that contain triamcinolone (0.025% to 0.5%), and long-acting formulations with betamethasone (0.01% to 0.1%).

Intrasynovial Therapy with Corticosteroids In selected patients where the disease activity is limited to the soft tissue and joints, intrasynovial corticosteroid injections can be given. These preparations vary in potency, dosage, and formulations. Examples are methylprednisolone acetate, prednisolone tebutate, dexamethasone acetate, and betamethasone acetate. A local anesthetic is used with intrasynovial corticosteroids to produce immediate pain relief.

Intralesional Corticosteroids Intralesional delivery is mainly used to treat discoid lupus, and triamcinalone acetonide is the corticosteroid of choice for this indication.

Systemic Corticosteroids Oral Therapy For oral delivery, most clinicians prescribe prednisone (North America) or prednisolone (Europe) for the reasons mentioned above. Two dosing schedules are used: daily morning dose or divided dose regimen (two to four times per day). In patients with less active disease, the single daily dose may minimize side effects. Patients with more active disease will require divided dose regimens. In patients who experience significant mineralocorticoid side effects, particularly fluid retention, methylprednisolone (Medrol) may be used instead at a 20% lower dose. However, methylprednisolone is more expensive than prednisone. Patients with liver disease are sometimes unable to metabolize prednisone (which is technically a prohormone) and may be given prednisolone instead (in the same dose as prednisone).1,28,29 The typical approach to dosage regimens quoted in the rheumatology literature is based on body weight, which has no scientific basis. Corticosteroid responsiveness is more related to host factors, disease factors, and organ-specific factors. Clinical experience has shown that host responsiveness both in the disease and side effects is variable. Some patients may develop osteonecrosis on as little as 20 mg of prednisone for 6 months, and some never develop this lesion on much higher doses. It has also long been known that RA is much more sensitive as a disease to steroid effects than is SLE. Furthermore, within the spectrum of SLE, some organ systems are more sensitive to steroids (skin, joints, and serous membranes) and some much less sensitive (renal, central nervous system). Each of these factors should be considered (host, disease, and organ) in choosing an appropriate induction dose of prednisone. According to the literature, typical dosages of prednisone follow: low or maintenance dosage, approximately 0.1 to 0.25 mg/kg/d; moderate dosage, approximately 0.5 mg/kg/d; high dosage, 1 to 3 mg/kg/d; and massive (very high) dosage, 15 to 30 mg/kg/d.30 We would recommend that in adults induction doses could be defined as low (1 to 20 mg/d), moderate (20 to 40 mg/d), high (40 to 80 mg/d) and very high (>80 mg/d, which includes oral and intravenous “pulse therapy”/intermittent infusion). These doses are not based on body weight. Based on the past responsiveness of the host and organ involvement, the physician could choose a low, moderate, high, or very high dose as induction therapy. Therapeutic dilemmas occur when a patient does not respond to a high dose of prednisone. The current tendency is to then move to “pulse” therapy in massive doses rather than a lesser increase in dose such as 100 to 200 mg/d of prednisone or methylprednisolone. This dilemma is discussed below.

FORMS AND DELIVERY ROUTES OF SYSTEMIC CORTICOSTEROIDS

lipocortin-1, decrease of T-cell proliferation, and decrease of IL-2 synthesis.23 On the other hand, corticosteroids block antibody production by a number of mechanisms. Lymphopenia (T cells affected more than B cells and CD4 T cells more than CD8 T cells) may be induced through lymphocyte redistribution (mainly to bone marrow and spleen) and perhaps through enhanced apoptosis.23 In addition, corticosteroids may inhibit IL-2 synthesis and signaling, inhibit signal transduction events critical for T-cell activation, down-regulate cell surface molecules important for full T-cell activation and function, and inhibit antigen-presenting cell function.24

489

SYSTEMIC STEROIDS

490

Intravenous Therapy

Intramuscular Therapy

Methylprednisolone (trade names Depo-Medrol, Medrol, Solu-Medrol) is prednisolone with a methyl group attached at position 6, to produce a compound that is water soluble. Due to its solubility, this preparation can be given intravenously, and very large doses can be given in a very short time. This drug was used initially in treating diffuse lupus nephritis using the same protocol as in renal transplantation.9 This protocol is this still used in treating SLE and other rheumatic diseases in most clinics and hospitals despite the lack of evidence that it is superior to lower doses. In fact, evidence from several small studies suggests that moderate doses (defined by some authors as 500 mg) have equal efficacy and fewer side effects than high doses.31-34 The other advantage of the intravenous treatment is that it can be given intermittently when a flare occurs, rather than keeping patients on very high doses of oral corticosteroids for longer periods. Studies of RA patients showed that low and moderate doses are comparable to the efficacy of high doses, with no difference, for example, in terms of the reduction rate of lymphocytes counts.35-39 Based on this evidence and our experience from the lupus clinic, if the intravenous “pulse” therapy is chosen as the induction with a very-high-dose approach we would advocate the use of a stepwise increase of the pulse doses, depending on response, rather than beginning with megadoses. The evidence to support this approach includes the following: 1. There have been no randomized clinical trials to support the often-used 1000-mg dose or to determine the appropriate frequency of methylprednisolone infusion in SLE. 2. The reports that have appeared in the literature that compared moderate doses versus high doses did not show any superiority of the high doses and observed a lower infection incidence with moderate doses.31-34 3. The pharmacodynamics and pharmacokinetics of methylprednisolone suggest that a dose of 320 mg may be adequate for full immunosuppressive effects.40 The duration of “pulse therapy” typically varies from 3 to 5 consecutive days.43 Again, this is not evidence based, and because patients are usually continued on high doses of prednisone after the pulse there would not seem to be much rationale for the longer course of “pulse therapy.” Situations in which intravenous pulse therapy might be considered are generally those that have been refractory to treatment with high oral doses of prednisone such as severe lupus nephritis and neuropsychiatric disease, severe refractory thrombocytopenia or hemolytic anemia, and severe vasculitis.

Intramuscular (IM) administration is usually reserved for situations where oral and intravenous routes are not possible, such as when there is a limited vascular access or where there is some question of adequate absorption of the oral preparation. The FLOAT study, in which patients were randomized to receive either a rapid taper dose of oral methylprednisolone or an IM injection of triamcinalone for mild to moderate flares, found equal efficacy at 1 month. The IM injection resulted in a more rapid response at day 1.41

TREATMENT OF SPECIFIC MANIFESTATIONS OF SLE The goals of therapy are to control the inflammatory response in affected organs and to suppress the abnormal systemic enhanced autoimmune response.44 Table 45.2 provides a summary of specific organ treatment in SLE.

Cutaneous Involvement Various skin manifestations occur in SLE. Some forms can be treated with antimalarial medications (like hydroxychloroquine) with or without topical steroids. Occasionally, systemic corticosteroids are used in low doses as a bridge therapy until antimalarials have their effects. Skin vasculitis is an important entity in this group of manifestations. When it occurs as an isolated skin manifestation without major organ involvement, it is usually treated with oral prednisone in low to moderate doses.

Musculoskeletal Manifestations Nonsteroidal anti-inflammatory drugs (NSAIDs) and hydroxychloroquine are the mainstays of treatment for polyarthralgia-polyarthritis in SLE. Systemic corticosteroids, usually prednisone in doses of 5 to 20 mg/d, may be required for more severe polyarthritis. It should be noted that if the patient has severe arthritis, or prednisone cannot be tapered to less than 7.5 mg/d, the physician might consider adding methotrexate with folic acid to the treatment regimen.

Myositis Isolated muscle involvement is rare in SLE. It is usually accompanied by other musculoskeletal manifestations. Minimal weakness and increased creatine kinase (CK) levels may require only low-dose corticosteroids. Significant muscle weakness may require moderate to high doses of corticosteroids, along with a second-line immunosuppressive agent.

Cardiopulmonary Symptomatic serositis (pleuritis and pericarditis) with or without small effusions may require low to moderate

For moderate thrombocytopenia (50–120,000)

No

Thrombocytopenia

Vasculitic skin lesions with major organ involvement

Neuropsychiatric lupus

9

12

8

7

No

May respond No

Lupus nephritis (WHO I, II) Lupus nephritis (WHO III, IV, V)

Acute lupus pneumonitis

No

No

Constitutional

If unresponsive to NSAIDs

Usually dramatic response

Arthralgia and/or Arthritis Myositis

Serositis

Yes

Moderate to severe polyarthritis Mainly increased creatine kinase with little weakness

Vasculitic skin lesions ONLY

Initial or bridging until hydroxychloroquine in full effect

Skin and mucous membrane

Low (e.g., Prednisone 1–20 mg/day)

Myocarditis

6

5

4

3

2

1

Manifestations

Lupus headache

No

No

If unresponsive to low No

If unresponsive to low

May respond

May respond

No

No With significant muscle weakness

If unresponsive to low or bridging until hydroxychloroquine in full effect Yes, if unresponsive to lower doses

Moderate (e.g., Prednisone 20–40 mg/day)

Yes

Yes

For refractory thrombocytopenia or very low initial levels (<50,000)

No Induction dose

If unresponsive to moderate

If unresponsive to moderate

If unresponsive to moderate

No

No No

No

Severe discoid

High (e.g., Prednisone 40–80 mg/day)

Unresponsive to high dose

Unresponsive to high dose

Severe, life-threatening, refractory

No Unresponsive to high dose

No

If unresponsive to moderate

No

No

No No

No

No

Intravenous (e.g., Pulses x. 1–3)

TABLE 45.2 REGIMEN OF CORTICOSTEROID INDICATED IN TREATMENT OF SYSTEMIC LUPUS ERYTHEMATOSUS

TREATMENT OF SPECIFIC MANIFESTATIONS OF SLE

491

SYSTEMIC STEROIDS

doses of oral prednisone. While systemic corticosteroids are used to treat refractory cases of SLE serositis, most patients can be treated with NSAIDs (Table 45.2). Myocarditis requiring systemic corticosteroid therapy, is rare but when it occurs may require moderate or high doses of prednisone. Acute lupus pneumonitis can be a serious manifestation of SLE requiring the use of high or very high doses of steroids until improvement is achieved.42

Renal Manifestation Lupus nephritis is a major cause of morbidity and mortality in SLE. The specific contribution of corticosteroid treatment in patient prognosis is unclear since therapeutic trials focus on patients with proliferative nephritis where concurrent immunosuppressive therapy is the “standard of care.” Patients with mesangial or membranous lupus nephritis may have a good prognosis with steroids alone, but these studies have not been done.43 Patients with mild mesangial disease alone may respond to low-dose corticosteroids. Patients with more significant mesangial or membranous disease may respond to moderate (20 to 40 mg) doses of steroids as initial therapy, and after 6 to 8 weeks a decision can be made to either add immunosuppressive agents, increase the steroid dose, or both if the disease is not improving, or to begin to wean corticosteroids if improvement has occurred. Proliferative lupus nephritis will usually require high doses of corticosteroids as part of the induction therapy in combination with one of the following immunosuppressive agents—cyclophosphamide, azathioprine, or mycophenalate mofetil. Failure to control the disease with 40 to 80 mg of prednisone within 6 to 8 weeks may lead to the use of very high pulse therapy as discussed above.

Hematologic Manifestations Cytopenia Immune-mediated cytopenias are common in SLE patients, and if mild may not require specific therapy.

Hemolytic Anemia Mild hemolytic anemia may occur during a generalized flare of SLE, and is treated with the appropriate therapy for the flare. More severe hemolytic anemia may require moderate to high doses of prednisone with or without concomitant immunosuppressive agents.

Neutropenia

492

Typically, neutropenia as an isolated finding does not require corticosteroid therapy. If the patient requires corticosteroids for other manifestations, the neutropenia is concomitantly corrected.

Lymphopenia Lymphopenia alone does not require corticosteroid therapy, given that the count will normalize when disease activity is brought under control.

Thrombocytopenia Thrombocytopenia is usually not associated with bleeding unless platelet counts are below 35,000/mm3. Moderate thrombocytopenia may require low to moderate doses of prednisone with or without concomitant immunosuppressive agents. Severe, life-threatening, or refractory thrombocytopenia may require hospitalization and intravenous pulse therapy with methylprednisolone. The pulse therapy of methylprednisolone should be followed by high-dose oral prednisone.

Neuropsychiatric Manifestation Neuropsychiatric lupus44,45 often presents a therapeutic challenge. Meanwhile, there is a lack of randomized control trials that provide evidence on treating this manifestation. Systemic corticosteroids are the initial treatment for neuropsychiatric lupus, and usually in moderate to high doses. Some neuropsychiatric manifestations such as generalized seizures, transverse myelitis, and psychosis may require very high pulse therapy with corticosteroids. Severe headaches attributed to SLE after all other causes have been excluded may occasionally be treated with moderate dose of systemic corticosteroids.46 Unfortunately, the central nervous system side effects of high doses of systemic corticosteroids may make it difficult to differentiate between an exacerbation of the disease and side effects of the medication.

TAPERING AND WITHDRAWAL OF SYSTEMIC CORTICOSTEROIDS IN SLE Exogenous systemic corticosteroids will alter normal HPA axis regulation and suppress the ability of the HPA to respond to stress through increase in ACTH secretion.47 Given early in the morning, prednisone (the most common steroid used to treat SLE) in doses between 7.5 and 15 mg/d will suppress the HPA axis within a month48,49; suppression time will be shortened to within a week if the dose is more than 15 mg/d.50,51 The pattern of ACTH suppression will differ in relation to dosage time of day, quantity, and duration. It has been estimated that the adrenal gland may take 1 year to fully recover fully normal ACTH levels after corticosteroid treatment, regardless of dose. Reduction and withdrawal of corticosteroids therapy can lead to steroid reduction syndrome. An array of symptoms may be exhibited ranging from frank adrenal insufficiency, to pseudorheumatic complaints, to flares of the underlying disease.52 For this reason, it is

Side Effects of Steroids It is widely accepted that the toxicity of steroids varies directly with the dosage and duration of therapy,

although not all studies have supported this conclusion.57-59 As stated above, individual host differences and disease-specific differences are also important in susceptibility to corticosteroid side effects. The wide range of corticosteroids side effects may be explained by the presence of glucocorticosteroid receptors on many different cell types, although this is not likely the sole explanation.60 Van Vollenhoven1 recently classified the side effects into the following three categories: Immediate: These are almost universal among patients taking corticosteroids, even at low doses, and they occur relatively quickly. The list includes fluid retention, blurry vision, mild euphoria or other mood changes, insomnia, weight gain and redistribution of body fat, and immunosuppression. Gradual: These side effects are unlikely to present in the first days to weeks of treatment, but become almost universal with more prolonged therapy. These include metabolic effects (e.g., hyperglycemia, hypertension, and osteoporosis), thinning of the skin, muscle weakness (myopathy), acne, dyspepsia, Cushingoid habitus, adrenal suppression, hypertriglyceridemia, and dyslipidemia. Also included is the poorly understood proatherogenic effect of corticosteroids, which is partly explained by changes in levels of low-density or high-density lipoprotein cholesterol or triglycerides. Idiosyncratic: Some unpredictable side effects occur in patients treated with systemic corticosteroids of any dose or for any period of time, although clearly the risk increases with longer duration and higher dose. These effects include cataract formation, avascular necrosis of joints, psychosis, and adrenal failure on withdrawal of therapy. For details of the side effects and precautions, see Table 45.3.19 One of most important side effects that may require special attention is avascular necrosis (AVN) or osteonecrosis. There is some evidence that AVN may also be dose dependent.61-64 We have recently shown that early, more intensive corticosteroid therapy in inception SLE patients is the more important determining factor in the future development of AVN.65,66 Intermittent intravenous infusion of methylprednisolone has its own side effects profile, which differs from the prolonged use of oral corticosteroids, in part due to the high dose infused over a short period. Side effects reported include acute arthralgia, infection, gastrointestinal bleeding, metallic taste, hyperglycemia, palpitations, facial flushing, and elevated blood pressure.67 Rarer side effects have also included anaphylactic reaction,68,69 seizure,70 arrhythmias,71 and sudden death.72

TAPERING AND WITHDRAWAL OF SYSTEMIC CORTICOSTEROIDS IN SLE

important to determine the nature and etiology of the symptoms so that an appropriate adjustment in medications can be made.53 Patients usually respond to reinstitution of the lowest previous dose of corticosteroids. Once SLE disease activity is deemed to be under control (as determined clinically and serologically), either with corticosteroids alone or in conjunction with other agents, reduction of the corticosteroid dose should be attempted at the lowest dose possible. For patients on multiple daily dose regimens, it is reasonable to start by reducing the dose to a single daily dose given in the morning without changing the daily total dose.54 The number of doses should be reduced gradually until a single daily dose is reached. The pace of reduction will depend on initial disease manifestations, responsiveness of the patient’s disease, and the patient’s tolerance of the corticosteroids. Typically, the dose should not be lowered by more than 25% decrements. The final aim will be to totally discontinue corticosteroids, but that may not always be possible. Many patients may require prednisone at 10 to 15 mg/d to prevent recurrence.55 Some authors advocate tapering the steroids as a curve that appears “logarithmic” (e.g., 60, 40, 20, 15, 10, 7.5, 5, 2.5, 0 mg) rather than linear (e.g., 60, 50, 40, 30, 20, 10, 0 mg). When low-dose steroid levels have been achieved, and the decision has been made to attempt steroid withdrawal, we have used a very slow “7-week tapering schedule.” For example, at the University of Toronto Lupus Clinic, if the patient is on 10 mg of prednisone and the aim is to decrease to 7.5 mg, the patient will be instructed to lower the dose to 7.5 mg for only 1 day in the first week, 2 days in the second week, 3 days in the third week, and so forth until in the seventh week the patient is taking 7.5 mg/d on all 7 days. The main advantages of this slow tapering, we believe, are that it is associated with a lower rate of relapse and affords a better opportunity for adrenal recovery. An alternate-day regimen has been used in SLE with mixed results. The theory behind this approach is that the anti-inflammatory effect lasts longer than the catabolic and ACTH-suppressive effects,56 suggesting that it may be reasonable to withdraw the drug while there are still anti-inflammatory effects, but sparing the system the catabolic side effects. It is recommended that the disease be under good control before such a regimen is attempted. Patients with SLE generally do not tolerate alternate-day corticosteroid therapy well, because symptoms tend to become more significant by the end of the second day.1 We do not find this approach helpful for SLE patients because they tend to flare more quickly.

493

SYSTEMIC STEROIDS

TABLE 45.3 ADVERSE EFFECTS OF STEROID THERAPY AND CAUTIONS Systems

Adverse Effects

Cautions/Comments

Fluid/electolyte disturbances

Sodium retention Edema Increased potassium excretion Increased calcium excretion

Use with caution in congestive heart failure or hypertensive patients. Decreased salt intake. Potassium supplements may be necessary. Use with caution in patients at increased risk of developing osteoporosis; calcium supplements may be necessary, especially in postmenopausal women.

Gastrointestinal

Gastric irritation Nausea/vomiting, weight loss/weight gain, abdominal distention, peptic ulcer, ulcerative esophagitis, pancreatitis

Take with meals to prevent gastric upset. The risk of these effects increases with increased dosages and prolonged use; use of antiulcer agents is suggested only in patients requiring long-term steroid therapy at high dosages; use with caution or avoid in patients with gastrointestinal diseases in which perforation or hemorrhage are potential risks.

Endocrine

Hypercortisolism (Cushingoid state), secondary adrenal insufficiency Menstrual difficulties, including amenorrhea and postmenopausal bleeding Precipitation of diabetes mellitus Glucose intolerance, hyperglycemia

Associated with long-term use even at lower dosages.

Cardiovascular

Hypertension

Thromboembolism

In patients with diabetes, increased dosages of insulin or oral hypoglycemic agent and changes in diet should be expected. Use with extreme caution in patients with recent myocardial infarction because of an apparent association with left ventricular free-wall rupture. Use with care in patients with thromboembolic disorders because of reports of (rare) increased blood coagulability and risk of interactions with oral anticoagulants.

Thrombophlebitis, congestive heart failure exacerbation Ocular

494

Posterior subcapsular cataracts

Prolonged use may result in increased intraocular pressure or damaged ocular nerve.

Glaucoma May enhance secondary fungal or viral infections of the eye

Use in patients with ocular herpes simplex may cause corneal perforation unless antiviral agents are prescribed.

Musculoskeletal

Muscle pain or weakness, muscle wasting, pathologic long-bone or vertebral compression fractures, atrophy of protein matrix of bone, aseptic necrosis of femoral or humeral heads

Use with caution in patients prone to development of osteoporosis; risk versus benefit should be reassessed if osteoporosis develops; elderly, debilitated or poorly nourished patients may be more prone to these effects. Supplementation with calcium, 1500 mg per day, and vitamin D, 800 IU per day, is recommended. Alendronate (Fosamax) therapy should also be considered.

Neuropsychiatric

Headache, vertigo, seizures, increased motor activity, insomnia, mood changes, psychosis

Use with caution at high doses in patients with convulsive or psychiatric disorders. Use may aggravate pre-existing psychiatric conditions. Steroid-induced psychosis is dose related, occurs within 15 to 30 days of therapy, and is treatable if steroid therapy must be continued. Pseudotumor cerebri reported during withdrawal.

Dermatologic

Acne, impaired wound healing, hirsutism, skin atrophy/increased fragility, ecchymoses

Ecchymoses due to easy bruisability should be restricted to exposed, potentially traumatized extremities when associated with steroid use.

Other

Increased susceptibility to infections, masked symptoms of infections

Contraindicated in patients with systemic fungal infections (except to control drug reactions associated with amphotericin B [Fungizone] therapy). Do not use live virus vaccinations during therapy. Reactions to skin tests may be suppressed.

While controlled studies of steroids are rare,73,74 there is no doubt among practicing rheumatologists that systemic corticosteroids are highly effective and that small changes in dose can produce dramatic effects. It should be noted, however, that systemic corticosteroids are often prescribed in daily practice without much of the evidence-based medical scrutiny that most cytotoxic drugs are currently given. Thus, there are no widely accepted algorithms for steroid initiation or withdrawal. Because of their dramatic effect and widespread uses in rheumatology, it is very difficult to design clinical trials to study the effects of newer therapies in SLE. One can never use a placebo arm for systemic corticosteroids when steroids are known to be effective and often life-saving. In addition, corticosteroid withdrawal schemes must always have an escape mechanism should the disease flare because of corticosteroid withdrawal.75 This issue will have to be addressed in all future therapeutic trials in SLE in one of a variety of ways, including tabulating cumulative dose of steroids during the trial, measuring percent reduction of steroids during the trial as a secondary outcome, assessing the ability to wean steroids according to a predefined algorithm during the trial as a primary outcome, or keeping the dose of steroids constant and assessing

the effect of the new agent on global or organ specific activity measures.76 A recent published consensus by the American College of Rheumatology outlines in detail the future approach to the clinical trials that address steroid-sparing medications.77

REFERENCES

SYSTEMIC CORTICOSTERIODS, SLE, AND FUTURE CLINICAL TRIALS

CONCLUSIONS In this chapter, we summarized the uses of systemic corticosteroids in the treatment of SLE. We briefly described the history of its use and impact of that the treatment has made on rheumatic diseases. The mechanism of action is clearly twofold: anti-inflammatory and immunosuppressive. Oral prednisone and intravenous methylprednisolone are the most commonly used forms of systemic corticosteroids. We proposed a guideline for a dosing regimen for prednisone, and suggested a dose range regimen. For intravenous pulse methylprednisolone, we propose a lower dose than is commonly prescribed. The withdrawal of corticosteroids from a patient is also an important component of treatment and approaches to weaning were discussed. Finally, we reviewed the side effects of corticosteroids to emphasize the importance of measures that should be followed to prevent and treat these side effects. We highlighted the difficulties in managing corticosteroid therapy during therapeutic trials of newer agents in SLE.

REFERENCES 1. van Vollenhoven RF. Corticosteroids in rheumatic disease. Understanding their effects is key to their use. Postgrad Med 1998;103:137-142. 2. Kimberly RP. Treatment. Corticosteroids and anti-inflammatory drugs. Rheum Dis Clin North Am 1988;14:203-221. 3. Hench PS, Kendall EC, Slocumb CH, et al. The effect of a hormone of the adrenal cortex (17-hydroxy-11-dehydrocorticosterone: compound E) and of pituitary adrenocorticotropic hormone on rheumatoid arthritis. Proc Staff Meet Mayo Clin 1949;24:181-197. 4. Urowitz MB, Gladman DD. Contributions of observational cohort studies in systemic lupus erythematosus: the University of Toronto lupus clinic experience. Rheum Dis Clin North Am 2005;31:211-221. 5. Pollak VE, Pirani CL, Kark RM. The effect of large doses of prednisone on the renal lesions and life span of patients with lupus glomerulonephritis. J Lab Clin Med 1961;57:495. 6. Pollak VE, Pirani CL, Schwartz FD. The natural history of the renal manifestations of systemic lupus erythematosus. J Lab Clin Med 1964;63:537. 7. Cathcart ES, Idelson BA, Scheinberg MA, Couser WG. Beneficial effects of methylprednisolone “pulse” therapy in diffuse proliferative lupus nephritis. Lancet 1976;1(7952):163-166. 8. Kountz SL, Cohen R. Initial treatment of renal allografts with large intrarenal doses of immunosuppressive drugs. Lancet 1969;15:338-340. 9. Bell PR, Briggs JD, Calman KC, Paton AM, Wood RF, Macpherson SG, et al. Reversal of acute clinical and experimental organ rejection using large doses of intravenous prednisolone. Lancet 1971;:876-880.

10. Webster JI, Tonelli L, Sterberg EM. Neuroendocrine regulation of immunity. Annu Rev Immunol 2002;20:125-163. 11. Thiessen JJ. Prednisolone. J Am Pharm Assoc 1976;16:143-146. 12. Gladman DD, Ibañez D, Urowitz MB. SLEDAI 2000. J Rheumatol 2002;29:288-291. 13. Isenberg DA, Rahman A, Allen E, Farewell V, Akil M, Bruce IN, D’ et al. BILAG 2004. Development and initial validation of an updated version of the British Isles Lupus Assessment Group’s disease activity index for patients with systemic lupus erythematosus. Rheumatology (Oxford) 2005;44:902-906. 14. Bae SC, Koh HK, Chang DK, Kim MH, Park JK, Kim SY. Reliability and validity of systemic lupus activity measure-revised (SLAM-R) for measuring clinical disease activity in systemic lupus erythematosus. Lupus 2001;10:405-409. 15. Bencivelli W, Vitali C, Isenberg DA, Smolen JS, Snaith ML, Sciuto M, et al. Disease activity in systemic lupus erythematosus: report of the consensus study group of the European workshop for rheumatology research. III. Development of a computerized clinical chart and its application to the comparison of different indices of disease activity. Clin Exp Rheumatol 1992;10:549-554. 16. Lu Nz, Cidlowski JA. The origin and the function of multiple human glucocorticoid receptor isoforms. Ann N Y Acad Sci 2004;1024;102-123. 17. Rhen T, Cidlowski JA. Mechanisms of disease: antiinflammatory action of glucocorticoids-new mechanisms for old drugs. N Engl J Med 2005;353:1711-1723. 18. Drug Facts and Comparisons. St. Louis: Facts and Comparisons, 1997:122-123.

495

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19. Zoorob RJ, Cender D. A different look at corticosteroids. Am Fam Physician 1998;58:443-450. 20. Boumpas DT, Chrousos GP, Wilder RL, Cupps TR, Balow JE. Glucocorticoid therapy for immune-mediated diseases: basic and clinical correlates. Ann Intern Med 1993;119:1198-1208. 21. Chan L, O’Malley BW. Steroid hormone action: recent advances. Ann Intern Med 1978;89:664-701. 22. Parrillo JE, Fauci AS. Mechanisms of glucocorticoid action on immune processes. Annu Rev Pharmacol Toxicol 1979;19, 179-201. 23. Al-Maini MH, Mountz JD, Al-Mohri HA, et al. Serum levels of soluble Fas correlate with indices of organ damage in systemic lupus erythematosus. Lupus 2000;9:132-139. 24. Baxter JD. The effects of glucocorticoid therapy. Hosp Pract 1992;27:111-134. 25. Twabo AV, Hallmark MR, Sakmar E, et al. Bioavailability of prednisolone tablets. J Pharmacokin Biopharm 1977;5,257-270. 26. Disanto AR, Desante KA. Bioavailability and pharmacokinetics of prednisone in humans. J Pharma Sci 1975;64:109-112. 27. Burlingme MB, Delafuente JC. Treatment of systemic lupus erythematosus. Drug Intel Clin Pharm 1998;22:283-289. 28. Powell LW, Axelsen E. Corticosteroids in liver disease: studies on the biological conversion of prednisone to prednisolone and plasma binding protein. Gut 1972;13:690-696. 29. Jenkins JS, Sampson PA. Conversion of cortisone to cortisol and prednisone to prednisolone. BMJ 1967;12:205-207. 30. McEvoy GK, Litvak K, Welsh OH, eds. AHFS Drug Information. Bethesda, MD: American Society of Health-Systems Pharmacists, 1996. 31. Badsha H, Kong KO, Lian TY, Chan SP, Edwards CJ, Chang HH. Low-dose pulse methylprednisolone for systemic lupus erythematosus flares is efficacious and has a decreased risk of infectious complications. Lupus 2002;11:508-513. 32. Badsha H, Edwards CJ. Intravenous pulses of methylprednisolone for systemic lupus erythematosus. Semin Arthritis Rheum 2003;32:370-377. 33. Howe HS, Boey ML, Feng PH. Methylprednisolone in systemic lupus erythematosus. Singapore Med J 1990;31:18-21. 34. Edwards JC, Snaith ML, Isenberg DA. A double blind controlled trial of methylprednisolone infusions in systemic lupus erythematosus using individualised outcome assessment. Ann Rheum Dis 1987;46:773-776. 35. Vischer TL, Sinniger M, Ott H, Gerster JC. A randomized, doubleblind trial comparing a pulse of 1000 with 250 mg methylprednisolone in rheumatoid arthritis. Clin Rheumatol 1986;5:325-326. 36. Fan PT, Yu DT, Clementd PJ, et al. Effects of corticosteroids on the human immune response: comparison of one and three daily 1 gram intravenous pulses of methylprednisolone. J Lab Clin Med 1978;91:625-634. 37. Radia M, Furst DE. comparison of three pulse methylprednisolone regimens in the treatment of rheumatoid arthritis. J Rheumatol 1988;15:242-246. 38. Iglehart IW 3rd, Sutton JD, Bender JC, Shaw RA, Ziminski CM, Holt PA, et al. Intravenous pulsed steroids in rheumatoid arthritis: a comparative dose study. J Rheumatol 1990;17:159-162. 39. Smith MD, Ahern MJ, Roberts-Thomson PJ. Pulse steroid therapy in rheumatoid arthritis: can equivalent doses of oral prednisolone give similar clinical results to intravenous methylprednisolone? Ann Rheum Dis 1988;47:28-33. 40. Fan PT, Yu DT, Clements PJ, et al. Effect of corticosteroids on human immune response: comparison of one and three daily 1 gram intravenous pulses of methylprednisolone. J Lab Clin Med 1978;91(4):625-634. 41. Danowski A, Magder L, Petri M. Flares in lupus: Outcome Assessment Trial (FLOAT), a comparison between oral methylprednisolone and intramuscular triamcinolone. J Rheumatol 2006;33:57-60. 42. Abramson SB, Dobro J, Eberle MA, et al. Acute reversible hypoxemia in systemic lupus erythematosus. Ann Intern Med 1991;114:941-947. 43. Austin HA, Muenz LR, Joyce KM, et al. Prognostic factors in lupus nephritis. Contribution of renal histologic data. Am J Med 1983;75:382-389. 44. Wong KL, Woo EK, Yu YL, Wong RW. Neurologic manifestations of systemic lupus erythematosus: a prospective study. Q J Med 1991;81:857-870.

45. Sibley JT, Olszynski WP, Decoteau WE, Sundaram MB. The incidence and prognosis of central nervous system disease in systemic lupus erythematosus. J Rheumatol 1992;19:47-52. 46. Brandt KD, Lessell S. Migrainous phenomenon in systemic lupus erythematosus. Arthritis Rheum 1978;21:7-16. 47. Byyny RL. Withdrawal from glucocorticoid therapy. N Engl J Med 1976;295:30-32. 48. Danowski TS, Bonessi JV, Sabeh G, et al. Probabilities of pituitary adrenal responsiveness after steroid therapy. Ann Intern Med 1964;61:11-15. 49. Nichols T, Nugent CA, Tyler FH. Diurnal variation in suppression of adrenal function by glucocorticoids. J Clin Endocrinol Metab 1965;25:343-349. 50. Paris J. Pituitary adrenal suppression after protracted administration of adrenal cortical hormones. Mayo Clin Proc 1961;36:305. 51. Axelrod L. Glucocorticoid therapy. Medicine 1976;55:39-65. 52. Dickson RB, Christy NP. On the various forms of corticosteroid withdrawal syndrome. Am J Med 1980;68:224-230. 53. Morgan HG, Boulnois J, Burns-Cox C. Addiction to prednisone. BMJ 1973;14;2:93-94. 54. Kountz DS, Clark CL. Safely withdrawing patients from chronic glucocorticoid therapy. Am Fam Physician 1997;55:521-525. 55. Aranow C, Emy J, Barland P. Reactivation on inactive systemic lupus erythemaosus. Scand J Rheumatol 1996;25:282-286. 56. Fauci AS. Alternate-day corticosteroid therapy. Am J Med 1978;64:729-731. 57. Zonana-Nacach A, Barr SG, Magder LS, Petri M. Damage in systemic lupus erythematosus and its association with corticosteroids. Arthritis Rheum 2000;43:1801-1808. 58. Mont MA, Glueck CJ, Pacheco IH, et al. Risk factors for osteonecrosis in systemic lupus erythematosus. J Rheumatol 1997;4:654-662. 59. Gladman DD, Urowitz MB, Chaudhry-Ahluwalia V, et al. Predictive factors for symptomatic osteonecrosis in patients with systemic lupus erythematosus. J Rheumatol 2001;28: 761-765. 60. Saag KG, Furst DE. Major side effects of glucorticosteroids. In: Rose BD, editor. UpToDate. Wellesley, MA: UpToDate;2004. 61. Abeles M, Urman JD, Rothfield NF. Aseptic necrosis of bone in systemic lupus erythematosus. Relationship to corticosteroid therapy. Arch Intern Med 1978;138:750-755. 62. Massardo L, Jacobelli S, Leissner M, et al. High-dose intravenous methylprednisolone therapy associated with osteonecrosis in patients with systemic lupus erythematosus. Lupus 1992;1:401-405. 63. Zizic TM, Marcoux C, Hungerford DS, et al. Corticosteroid therapy associated with ischemic necrosis of bone in systemic lupus erythematosus. Am J Med 1985;79:596-604. 64. Oinuma K, Harada Y, Nawata Y, et al. Osteonecrosis in patients with systemic lupus erythematosus develops very early after starting high dose corticosteroid treatment. Ann Rheum Dis 2001;60:1145-1148. 65. Prasad R, Ibanez D, Gladman DD, Urowitz MB. Corticosteroid dose in early SLE is a determining factor for avascular necrosis: a nested case control study of inception patients. Arthritis Rheum 2004:50:40-90. 66. Gladman DD, Urowitz MB, Chaudhry-Ahluwalia V, Ibanez D, Bogoch ER. Outcomes of symptomatic osteonecrosis in 95 patients with systemic lupus erythematosus. J Rheum 2001;28:22262229. 67. Burlingme MB, Delafuente JC. Treatment of systemic lupus erythematosus. Drug Intel Clin Pharm 1998;22:283-289. 68. Mendelson LM, Meltzer EO, Hamburger RN. Anaphylaxis-like reactions to corticosteroid therapy. J Allergy Clin Immunol 1974;54:125-131. 69. Freedman MD, Schocket AL, Chapel N. Anaphylaxis after intravenous methylprednisolone administration. JAMA 1981; 245:607-608. 70. Suchman AL, Condemi JJ, Leddy JP. Seizure after pulse therapy with methyl prednisolone. Arthritis Rheum 1983;26:117. 71. Schmidt GB, Meier MA, Sadove MS. Sudden appearance of cardiac arrhythmias after dexamethasone. JAMA 1972;18:221: 1402-1404. 72. Moses RE, McCormick A, Nickey W. Fatal arrhythmia after pulse methylprednisolone therapy. Ann Intern Med 1981;95:781.

76. Schiffenbauer J, Simon LS. Randomized controlled trials in systemic lupus erythematosus: what has been done and what do we have to do? Lupus 2004;13:398-405. 77. Ad Hoc Working Group on Steroid-Sparing Criteria in Lupus. Criteria for steroid-sparing ability of interventions in systemic lupus erythematosus: report of a consensus meeting. Arthritis Rheum 2004 50:3427-3431.

REFERENCES

73. Strand V. Clinical trial design in systemic lupus erythematosus: lessons learned and future directions. Lupus 2004;13:406-411. 74. Liang MH, Corzillius M, Bae SC, Fortin P, Esdaile JM, Abrahamowicz M. A conceptual framework for clinical trials in SLE and other multisystem diseases. Lupus 1999;8:570-580. 75. Corzillius M, Bae SC. Methodological issues of corticosteroid use in SLE clinical trials. Lupus 1999;8:692-697.

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46

Cytotoxic Drug Treatment Eva D. Papadimitraki, MD and Dimitrios T. Boumpas, MD, FACP

INTRODUCTION Cytotoxic agents were initially introduced for their ability to interrupt nucleic acid and protein synthesis in malignant cells. The realization that these agents may also suppress the immune system led to their use in immune-mediated diseases including systemic lupus erythematosus (SLE). Ever since their introduction, management of the disease has dramatically changed and its prognosis has greatly improved. Because of their significant side effects, cytotoxic therapy in lupus is reserved only for patients with moderate to severe disease (Box 46.1). To date, most experts agree that the treatment of moderate to severe lupus consists of a period of intensive immunosuppressive therapy (induction therapy) followed by a longer period of less-intensive therapy (maintenance therapy). The primary objective of the induction therapy is to halt injury, recover function, and induce remission by controlling immunologic activity. On the other hand, maintenance therapy is used to consolidate remission and prevent flares with agents (or schedules) that are associated with a lower risk for complications and are more convenient to the patient. In this chapter, we discuss the use of cytotoxic/ immunosuppressive drug therapy in SLE. For each drug under discussion, we provide background information on the mode of action, pharmacokinetics, clinical use in lupus, and side effects. We also discuss monitoring for side effects and strategies to minimize them. Because azathioprine, cyclophosphamide, and mycophenolate mofetil are the most widely used cytotoxic drugs, we review these drugs in more detail.

AZATHIOPRINE

498

Azathioprine is a cycle-specific antimetabolite that is commonly included in maintenance regimens for lupus nephritis and in regimens against SLE. Azathioprine and its metabolite, 6-mercaptopurine, are believed to affect cell-mediated and humoral immune responses. These effects include decreasing

circulating lymphocytes, inhibiting lymphocyte proliferation, reducing antibody production, and suppressing natural killer (NK) cell activity.1

Pharmacokinetics Azathioprine is rapidly converted to 6-mercaptopurine through enzymatic and nonenzymatic mechanisms. 6-mercaptopurine is then converted (1) to active analogues such as thiopurine via hypoxanthine-guaninephosphoribosyl-transferase (HGPRT), and (2) inactive analogues such as 6-methylmercaptopurine (via thiopurine methyltransferase) and 6-methylthiouric acid (via xanthine oxidase) metabolites.2

BOX 46-1 GENERAL INDICATIONS FOR CYTOTOXIC DRUG USE IN SYSTEMIC LUPUS ERYTHEMATOSUS General ● Involvement of major organs and/or extensive involvement of non-major organs (i.e., skin) refractory to other agents ● Failure to respond to or inability to taper corticosteroids to acceptable doses for long-term use Specific organ involvement Renal ● Proliferative and/or membranous nephritis (nephritic or nephritic syndrome) ● Hematologic ● Severe thrombocytopenia (platelets less than 20,000 K) ● Thrombotic thrombocytopenic purpura (TTP)–like syndrome ● Severe hemolytic or aplastic anemia, or immune neutropenia not responding to corticosteroids Pulmonary ● Lupus pneumonitis and/or alveolar hemorrhage Cardiac ● Myocarditis with depressed left ventricular function, pericarditis with impeding tamponade Gastrointestinal ● Abdominal vasculitis Nervous system ● Transverse myelitis, cerebritis, psychosis refractory to corticosteroids, mononeuritis multiplex, severe peripheral neuropathy

Adverse Effects

CYCLOPHOSPHAMIDE

Drug Interactions

Gastrointestinal complaints such as nausea, vomiting, and diarrhea are the most common side effects of azathioprine, leading approximately 15 to 30% of patients to discontinuation of the drug within 6 months.5 Mild increases in liver-associated enzymes are not uncommon, but severe liver injury is uncommon. Reversible dose-related myelosuppression is not uncommon; leucopenia occurs in approximately 4.5% and thrombocytopenia in 2% of patients receiving low-dose azathioprine.6 Notably, azathioprine toxicity is highly idiosyncratic, and has been associated with genetic polymorphisms leading to decreased thiopurine methyltransferase (TPMT) activity, and thus, to impaired ability to detoxify 6-MP. Subjects with low or absent TPMT activity seem to be at increased risk of developing severe azathioprine-induced myelotoxicity, which when occurring, has a delayed (between 4 and 10 weeks after initiation of the treatment) but acute onset.6 Genetic testing for TPMT polymorphisms represents an option for identifying patients with impaired TPMT activity. Alternatively, TPMT activity can now be measured directly in red cell membranes using commercially available kits. In the case that neither of the abovementioned options are available, a low initial dose of azathioprine with frequent monitoring of the white blood cell count (WBC) every 1 to 2 weeks for the first 3 months of treatment and every 1 to 3 months thereafter. Less common side effects include acute hypersensitivity syndromes (usually within the first 2 weeks of the treatment), infection (albeit less common than with cyclophosphamide), and perhaps an increased risk for lymphoproliferative malignancies.5,7,8

Clinical Use The starting dose is 1 mg/kg/d with the usual dose at 2 to 3 mg/kg/d in one to three doses taken with food.

CYCLOPHOSPHAMIDE

Concurrent administration of allopurinol should be avoided since the combination of the two drugs may dramatically increase the toxicity of azathioprine.3 Resistance to warfarin has been associated with the administration of azathioprine.4

Monitoring includes complete blood cell count (CBC) with platelets, creatinine, and AST or ALT. Liver enzyme activity should be measured every 3 to 4 months (Table 46.1). The drug should be used cautiously in patients with renal or liver disease or in patients who use allopurinol. Controlled randomized studies in lupus nephritis have not shown superiority for azathioprine when compared to high-dose corticosteroids, and the drug is used as a corticosteroid-sparing agent in various manifestations of lupus. In moderate to severe lupus, azathioprine has been used as a maintenance therapy at doses ranging from 1 to 3 mg/kg/d, especially in women of reproductive age, because of its acceptable safety profile during pregnancy. Although discontinuation of the drug due to side effects is not uncommon, more often the drug is discontinued for lack of efficacy.

One daily dose is sufficient for therapeutic purposes. Measurement of neither azathioprine nor 6-mercaptopurine (6-MP) plasma levels is helpful in predicting the drug’s therapeutic or toxic effects. Approximately 1% of 6-MP is excreted in the urine; dose modification helps in reducing toxicity in the case of renal impairment (25% reduction of the dose if creatinine clearance [CrCl] occurs; 50% reduction if CrCl is below 10 mL/min). The drug is slightly dialyzable (5 to 20%) and is administered post-hemodialysis.

Cyclophosphamide (CY) is an alkylating agent whose administration results in cell death, which can occur at any stage during the cell cycle. CY depletes both T and B cells, and reduces the production of pathogenic autoantibodies.9

Pharmacokinetics Cyclophosphamide, an inactive merchlorethamine derivative, is rapidly metabolized to a variety of active metabolites, such as 4-hydroxycyclophosphamide, phosphoramide mustard, and acrolein, by cytochrome P-450 in the liver or other tissues such as transitional epithelial cells of the bladder or lymphocytes. Oral and intravenous administrations of CY result in similar plasma concentrations.10 Plasma concentrations of CY are not used as clinical predictors of either efficacy or toxicity. Approximately 20% of the drug is exerted by the kidney, whereas 80% is processed by the liver. A dose modification is necessary for patients with renal impairment (25% reduction in patients with creatinine clearance 25 to 50 mL/min; 30 to 50% reduction if creatinine clearance is below 25 mL/min). CY may be administered in patients with end-stage renal disease on dialysis with dialysis performed 8 to 12 hours later. No dose modification is usually required for patients with liver disease. Concurrent administration of other drugs such as allopurinol, carbamazepine, phenytoin, and succinylcholine may increase the toxicity of CY.

Modes of Administration and Monitoring Pulse Cyclophosphamide Therapy Because of a better efficacy-to-toxicity ratio, intermittent intravenous pulse therapy (IV-CY) has replaced daily oral use in lupus in most places (see Box 46.2

499

CYTOTOXIC DRUG TREATMENT

TABLE 46.1 RECOMMENDED MONITORING OF CYTOTOXIC THERAPY IN SYSTEMIC LUPUS ERYTHEMATOSUS Toxicities Requiring Monitoring

Baseline Evaluation

Laboratory Monitoring

50–100 mg/d in 1–3 doses with food

Myelosuppression, hepatotoxicity, lymphoproliferative diseases

CBC, platelets, creatinine, AST or ALT

CBC and platelets every 2 weeks, with changes in dosage; baseline tests every 1–3 months

Mycophenolate mofetil (Category C)

1–3 g/d in 2 divided doses with food

Myelosuppression, hematotoxicity, infection

CBC, platelets, creatinine, AST or ALT

CBCs and platelets every 1–2 weeks with changes in dosage; baseline tests every 1-3 months

Cyclophosphamide (Category D)

50–150 mg/d in a single dose with breakfast. Lots of fluids, empty bladder before bedtime

Myelosuppression, hemorrhagic cystitis, myeloproliferative disease, malignancies

CBC, platelets, creatinine, AST or ALT, urinalysis

CBC with differential every 1–2 weeks, with changes in dosage and then every 1–3 months. Keep WBC above 4000/mm3 with dose adjustment. Urinalysis, AST or ALT every 3 months. Urinalysis every 6–12 months following cessation

Methotrexate (Category X)

7.5–15 mg/wk in 1-3 doses with food or milk/water

Myelosuppression, hepatic fibrosis, pneumonitis

CXR, hepatitis B and C serology in high-risk patients, AST or ALT, albumin, alkaline phosphatase, and creatinine

CBC with platelets, AST, albumin, creatinine every 1–3 months

Cyclosporin A

100–400 mg/d in 2 doses at the same time every day with meal or between meals

Renal insufficiency, CBC, creatinine, anemia, hypertension uric acid, LFTs, blood pressure

Drug

Dosage

Azathioprine (Food and Drug Administration pregnancy category Da)

Creatinine every 2 weeks until dose is stable, then monthly; CBC, potassium, and LFTs every 1–3 months. Cyclosporin levels only with high doses

a

Azathioprine may be used during pregnancy if needed. CBC, complete blood count; AST, aspartate transaminase; ALT, alanine transaminase; LFTs, liver function tests; WBC, white blood cell count. Adapted from ACR Ad Hoc Committee on Clinical Guidelines for Monitoring Drug Therapy in Rheumatoid Arthritis. Arthritis Rheum 1996;39: 723-731.

for protocols). Reversible myelotoxicity is a common dose-related, adverse effect of CY. After pulse therapy, the nadir of lymphocyte count occurs on approximately day 7 to 10 and that of granulocyte count on approximately day 10 to 14. The WBC nadir is about 3000 cells/mm3 after a dose of 1 g/m2 and 1500 cells/mm3 after a dose of 1.5 g/m2.11 Because the risk of infection increases substantially with WBCs below 3000 cells/mm3, the dose is adjusted to keep it above this level. A prompt recovery from granulocytopenia usually occurs after 21 to 28 days. On the other hand, thrombocytopenia is extremely rare in monotherapy with CY.

500

with urinalysis, creatinine, and AST or ALT. Total WBC counts of less than 3500/mm3 should lead to dose reductions, because lower counts are associated with a markedly increased risk of opportunistic infection. Urinalysis is performed every 6 to 12 months after cessation (Table 46.1).

Adverse Effects Reversible alopecia and nausea are the most commonly observed side effects of CY, whereas myelotoxicity, gonadal toxicity, and malignancy represent less frequent, although much more serious, adverse effects.

Oral Cyclophosphamide

Infections

Monitoring for daily oral administration includes CBC with differential and platelet count every 1 to 2 weeks initially, and then every 1 to 3 months thereafter, along

A variety of different infections can occur, including bacterial infections, opportunistic infections (pneumocystitis carinii, fungal infections, and nocardia) and

Gonadal Toxicity Premature ovarian failure represents a well-documented side effect of CY. Several mechanisms have been implicated in its pathogenesis including marked acceleration in follicular maturation, depletion, and eventually exhaustion, and direct toxicity of the drug and its metabolites to gonadal cells. The risk of developing premature ovarian failure depends on the age of patient at the initiation of treatment and the cumulative dose of the drug as we first reported in 1993.15 In our study, the rates of sustained amenorrhea after a short course (seven or fewer pulses) of CY were 0% for patients aged under 25 years, 12% for those aged 26 to 30, and 25% for patients aged 30 and older. On the other hand, a long course (15 or more pulses) of CY induced sustained amenorrhea in 17% of patients under age 25, 43% of patients aged 26 to 30, and 100% of patients 30 and older. In males, gonadal toxicity may be observed with as little as a 7-gm cumulative dose corresponding to an approximately 2-month daily oral therapy.16 A number of strategies to preserve fertility in patients with SLE taking CY have been tried with encouraging initial results. Some authors have suggested that the co-administration of GnRH antagonists confers protection against premature ovarian failure, and therefore recommend a GnRH antagonist–based protocol in CY-treated female patients17-19 (Box 46.2). Other strategies for preserving fertility, such as cryopreservation of unfertilized ova and ovarian tissue germ cell transplantation, are currently under investigation, and should be considered experimental at best at the present time. In male patients receiving CY for malignancies, the frequency of azoospermia ranges from 50 to 90%.20,21 The administration of testosterone and sperm banking represent valid strategies for preservation of testicular function and fertility (Box 46.2).21,22

BOX 46-2 NATIONAL INSTITUTES OF HEALTH PROTOCOL FOR ADMINISTRATION AND MONITORING OF PULSE CYCLOPHOSPHAMIDE THERAPY Estimate creatinine clearance by standard methods. Calculate body surface area (m2): BSA= √height (cm) x weight (kg)/3600. CY dosing and administration Initial dose CY is 0.75 g/m2 (0.5 g/m2 of CY if creatinine clearance rate is less than one-third of expected normal). Administer CY in 150-ml normal saline IV over 30–60 min (alternative: equivalent dose of pulse CY may be taken orally in highly motivated and compliant patients). WBC at days 10 and 14 after each CY treatment (patient should delay prednisone until after blood tests drawn to avoid transient corticosteroid-induced leukocytosis). Adjust subsequent doses of CY to maximum dose of 1.0 g/m2 to keep nadir WBC above 1500/μL. If WBC nadir falls below 1500/μL, decrease next dose by 25%. Repeat CY doses monthly (every 3 weeks in patients with extremely aggressive disease) for 6 months (7 pulses), then quarterly for 1 year after remission is achieved (inactive urine sediment, proteinuria <1gram/d, normalization of complement [and ideally anti-DNA], and minimal or no extrarenal lupus activity). Alternative maintenance therapy: azathioprine or MMF for 1–2 years. Protection of bladder against CY-induced hemorrhagic cystitis Diuresis with 5% dextrose and 0.45% saline (e.g., 2 L at 250 mL/hr). Frequent voiding; continue high-dose oral fluids for 24 hours. Patients return to clinic if they cannot sustain adequate fluid intake. Consider Mesna (each dose 20% of total CY dose) intravenously or orally at 0, 2, 4, and 6 hours after CY dosing. Sustained diuresis may be difficult to achieve, or if pulse CY is administered in outpatient setting. If difficulty is anticipated with sustaining diuresis (e.g., severe nephrotic syndrome) or with voiding (e.g. neurogenic bladder), insert a three-way urinary catheter with continuous bladder flushing with standard antibiotic irrigating solution (e.g., 3 L) or normal saline for 24 hours to minimize risk of hemorrhagic cystitis. Antiemetics (usually administered orally) Dexamethasone 10 mg in single dose plus the following: Serotonin receptor antagonists: Granisetron (Kytril) 1mg with CY dose (usually repeat dose in 12 hours); Ondasetrone (Zofran) 8 mg tid for 1–2 days. Monitor fluid balance during hydration. Use diuresis if patient develops progressive fluid accumulation. Complications of pulse CY Expected: nausea and vomiting (central effect of CY) mostly controlled by serotonin receptor antagonists; transient hair thinning (rarely severe at CY doses ≤1 g/m2). Common: significant infection diathesis only if leukopenia not carefully controlled; modest increase in herpes zoster (very low risk of dissemination); infertility (male and female); amenorrhea proportional to age of the patient during treatment and to the cumulative dose of CY. In females at high risk for persistent amenorrhea, may consider using leuprolide 3.75 mg subcutaneously 2 weeks prior to each dose of cyclophosphamide. In males, may use testosterone 100 mg intramuscularly every 2 weeks.

CYCLOPHOSPHAMIDE

reactivation of latent Herpes zoster, Mycobacterium tuberculosis, and human papilloma virus. An increased rate of Herpes zoster and bacterial infections has been documented in patients receiving CY, and has been associated with higher doses of corticosteroids and a nadir of WBC less than 3000 cells/mm3 at some point during treatment.12 Oral CY regimens also may pose a greater risk of infection compared with intravenous pulse regimens.13 Opportunistic infections such as candidiasis or Pneumonocystis carinii pneumonia may be seen in patients on concomitant high-dose corticosteroid therapy. In this regard, recent studies have shown by multivariate analysis that the dosage of corticosteroids is the overriding independent determinant of the risk of infection among patients with SLE patients receiving CY with concomitant high doses of corticosteroids.14

501

CYTOTOXIC DRUG TREATMENT

Malignancy Patients with systemic lupus erythematosus are at increased risk of developing lymphoma independent of treatment. However, the administration of alkylating agents enhances this risk and also probably that of leukemia, skin cancer, and other malignancies as well. Mechanisms of alkylating agent–induced malignancy include direct chromosomal damage and decreased immune surveillance. The duration of therapy is an important risk factor; the incidence is greatest in patients treated for more than 2 to 3 years or patients with a cumulative dose over 100 g.23 Patients with previous exposure to cyclophosphamide are at increased risk for hematologic malignancies including myelodysplastic syndrome, and myeloproliferative disease including acute leukemia and multiple myeloma.

Bladder Toxicity Including Bladder Carcinoma The appearance of hemorrhagic cystitis and bladder carcinoma has been well documented in patients receiving long-term oral CY.24 The role of BK virus, present in the majority of adults in latent form in the urogenital tract, and its reactivation following CY therapy are currently been explored. In these patients, nonglomerular microscopic or gross hematuria represents the most common manifestation of CY-induced hemorrhagic cystitis. The value of urine cytology has been questioned. In our opinion, this does not represent a useful test to monitor for bladder cancer.24,25 The risk for bladder malignancy is life-long after CY therapy, and patients with nonglomerular hematuria should undergo cystoscopy no matter how late hematuria occurs. Although the absolute risk of bladder cancer is largely unknown, an up to 30-fold increase in the risk of developing bladder cancer has been documented in large trials. Among patients receiving CY, a cumulative dose of above 100 g of CY and smoking are well-documented risk factors for bladder cancer.25 The use of intermittent pulse CY, together with adequate hydration, has practically eliminated the cases of bladder carcinoma, although hemorrhagic cystitis may be seen in cases of inability to empty the bladder (i.e., neurogenic bladder in patients with transverse myelitis) or in cases where the practice of adequate hydration and frequent emptying of the bladder are not followed meticulously (Box 46.2). We use routinely sodium 2-mercaptoethane sulfonate (mesna)— an agent that has been advocated to reduce the concentration of acrolein and probably other toxic metabolites in the bladder—although controlled studies demonstrating its efficacy in lupus are not available.

Clinical Use 502

Although some centers still employ daily oral CY regimens for short periods of time (2 mg/kg/d every morning

in a single dose for 3 to 12 months until remission), the National Institutes of Health (NIH) protocol has become the protocol of choice for most physicians (Box 46.2). Randomized controlled studies (RCTs) are available only for lupus nephritis; recommendations for other indications are based on extrapolation from those data. RCTs with long-term follow-up have shown that IV-CY is effective for moderate to severe proliferative lupus nephritis, with a better toxicity profile than daily oral CY.27 Following induction therapy, a maintenance regimen is essential to decrease the risk of flares.28 Subsequent studies from the NIH have demonstrated that combination pulse therapy with CY and methylprednisolone (IV-MP) improves renal outcomes without increasing toxicity.29,30 Based on these studies, the NIH group has proposed seven monthly pulses of IV-CY (0.5 to 1 g/m2) followed by quarterly pulses for at least 1 year beyond remission. For patients with moderate to severe disease, monthly pulses of IV-MP are added during the induction period. Ovarian toxicity (found to be both age and dose related), infections (especially with herpes zoster), flares (observed in approximately one-third of patients), incomplete response, and in rare cases, refractoriness to treatment, have emerged from these studies as significant limitations of current cytotoxic therapy. Because of concerns about toxicity, together with the appreciation that the disease may be less severe in whites, European investigators sought alternative protocols to administer the drug (Euro-Lupus Nephritis Trial [ELNT]).31,32 In studies involving for the most part patients with milder forms of disease, less intensive regimens of CY (six fortnightly pulses at a fixed dose of 500 mg each in combination with three daily doses of 750 mg of IV-MP) followed by azathioprine as maintenance, had comparable efficacy but less toxicity than a short course of high-dose IV-CY (eight pulses).32 By multivariate analysis, early response to therapy at 6 months (defined as a decrease in serum creatinine level and proteinuria <1 g/d) was the best predictor of good long-term renal outcome. In addition to demonstrating that in patients with milder forms of lupus nephritis less intensive regimens of IV-CY may be involved, this study demonstrated that sequential therapy with a short course of IV-CY followed by azathioprine is a valid approach in lupus. In addition to proliferative and membranous lupus nephritis, case reports, case series, and uncontrolled clinical studies support the efficacy of IV-CY in severe thrombocytopenia, neurologic disease (myelitis, encephalitis, psychosis, mononeuritis multiplex, and polyneuropathy), abdominal vasculitis, acute pneumonitis/alveolar hemorrhage, dermatologic disease, and other severe manifestations of lupus.34-36

MYCOPHENOLATE MOFETIL Mycophenolate mofetil (MMF) is an antimetabolite immunosuppressive agent that reversibly inhibits purine synthesis. More specifically, MMF is a reversible, noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMP-DH) that more potently affects the type II enzyme isoform (i.e., the one expressed in activated T and B cells) as compared with the type I isoform (expressed in most cell types).37 In vitro actions of MMF include inhibition of T- and B-cell proliferation upon mitogenic stimulation, suppression of antibody production, and reduction of adhesion molecules that are necessary for the migration of lymphocytes into sites of inflammation.38,39 MMF decreases cytokine-induced nitric oxide production in mouse and rat endothelial cells; if extrapolated to human disease, this finding may explain its activity against tissue damage of inflamed tissue.40 Moreover, MMF may inhibit vascular smooth-muscle proliferation, and retard the development of atherosclerosis associated with organ transplantation, a feature highly desirable in lupus where premature atherosclerosis represents a major problem.41

Pharmacokinetics The drug is converted to an inactive metabolite in the liver, and is then excreted into the gastrointestinal tract, where after undergoing deglucuronidation, enters enterohepatic circulation and is excreted by the kidney. Peak levels of MMF occur within 1 to 2 hours after administration. Serum levels of MMF are increased in patients with impaired renal function, and the dose should be adjusted in such patients (1 g/d maximum if CrCl is less than 25 mL/min). Antacids and cholestyramine decrease the bioavailability of MMF, and the co-administration with azathioprine should be avoided.

Adverse Effects Gastrointestinal toxicity, including nausea, vomiting, and diarrhea—all of which can be alleviated by reducing or splitting the daily dose—appear to be the most common side effect of the drug in SLE patients. Although results of studies regarding infectious complications have been inconclusive, respiratory tract infections, Herpes zoster, and cellulitis—although generally rare—are the most frequent infections.42,43 MMF may impose an increased risk for skin and nonskin malignancies, but further data are needed to confirm this finding.44

Clinical Use Following its introduction in transplantation, MMF was used to treat lupus patients refractory to corticosteroids or cytotoxic agents in small case series. It was subsequently studied in RCTs in lupus nephritis where it was compared (in doses of 1 to 3 g/d for 6 to 24 months) to either oral or pulse CY both for the induction and maintenance of remission (Table 46.2). In these studies the drug demonstrated comparable efficacy and fewer side effects than CY. In the absence of long-term data and specific outcomes (such as doubling of serum creatinine or end-stage renal disease), claims of superiority in terms of efficacy to CY cannot be adequately substantiated at present.49-51 This is especially true for the most severe cases where CY has a track record of efficacy,27 something that hopefully will be also be demonstrated for MMF. In these patients, the combination of IV-CY with IV-MP is the treatment of choice.51 In addition to proliferative nephritis, MMF has been used for the treatment of a variety of other manifestations of lupus including membranous nephropathy, skin disease, refractory thrombocytopenia, and pulmonary hemorrhage.52-56 However, additional controlled trials are needed to establish the use of this agent for other disease manifestations.

CYTOTOXIC THERAPY IN SLE: WHICH AGENT AND FOR WHOM?

A randomized controlled trail in neuropsychiatric lupus has confirmed its efficacy in severe neuropsychiatric lupus.36

CYTOTOXIC THERAPY IN SLE: WHICH AGENT AND FOR WHOM? Although considered by many as a superior treatment to corticosteroids, in RCTs the superiority of azathioprine is marginal at best, and is currently being used as induction therapy in mild cases of lupus or as a maintenance therapy in patients with various degrees of severity. Although azathioprine and MMF have not been formally tested “head to head” as induction therapy, published and anecdotal experience suggests that some patients with disease refractory to IV-CY (which usually does not respond to azathioprine) may respond to MMF,55,56 an observation that underscores the potential superiority of MMF to azathioprine as an induction treatment. However, the initial data thus far have failed to demonstrate superiority of MMF as a maintenance treatment in the study by Contreras and colleagues.48 The significant difference in cost between the two drugs begs the question of comparison studies. There is no doubt that MMF is less toxic than CY, does not cause ovarian failure, and is more acceptable to patients than CY.51 Taken all together, at present we view MMF as an agent for moderate cases of lupus, where in the past, IV-CY may have been used, especially in patients for whom ovarian toxicity is an important consideration (Table 46.3). Although there

503

42

64

140

59

Chan (2000)45 (Induction)

Chan (2005)46 (Maintenance)

Ginzler (2003)47 (Induction)

Contreras (2004)48 (Maintenance)

5% white, 95% Hispanic or African American

56% African American

100% Chinese

100% Chinese

Class III–IV nephritis Nephrotic: 42% of MMF gp, 60% of CYC/MMF gps Mean serum Cr: AZA gp, 1.7 mg/dL; CYC gp, 1.5 mg/dL; MMF gp, 1.6 mg/dL Chronicity index: AZA gp, 3.2; CYC gp, 1.9 (p<0.01 vs. MMF); MMF gp, 3.8

Class IV nephritis Nephrotic: 42% of MMF gp; 46% of CYC gp Serum Cr >1.3 mg/dL: 18% in MMF gp, 27% in CYC gp

Class IV nephritis Elevated serum Cr: 27% Proteinuria: >3g/d: 63% Newly diagnosed disease: 50/64

Class IV nephritis Mean serum Cr: 1.2 mg/dL (29% with elevated Cr) Mean serum albumin: 2.8 mg/dL (both gps)

Baseline Characteristics

Induction therapy: monthly boluses of IVCY (0.5–1 g/m2 and prednizolone (mean oral dose 0.6 mg/kg/d) ± IVMP pulses (3–7) until remission (proteinuria <3 g/d, or reduction >50% if subnephrotic, + improved/stable Cr) Maintenance therapy: quarterly IVCY (0.5–1 mg/kg/d) AZA (1–3 mg/kg) MMF (0.5-3 G/D) All: oral prednizolone (≤0.5 mg/kg/d) for 1–3 years

MMF as induction treatment (1–3 g/d) IVCY (NIH protocol) Patients with no improvement >30% in more than one renal characteristic that had been abnormal on entry were crossed to the other regimen at 12 weeks

MMF 2 g/d for 6 mo, 1.5 g/d for 6 mo, 1 g/d for 1 year. Duration of treatment 12 mo in 20 pts, ≥24 mo in 12 pts CYC 2.5 mg/kg/d, replaced by AZA 1.5 mg/kg/d at 6 mo

MMF 1 g/d for 6 months, then halved CYC 2.5 mg/kg/d, replaced by AZA 1.5 mg/k/d at 6 mo For both gps: prednizolone 0.8 mg/kg/d tapered to 10 mg/d at 6 mo, then AZA 1 mg/kg/d at 12 mo

Randomized Treatments

34 mo

Evaluation: at 24 weeks of treatment

63 mo

12 mo

Follow-up

72-month, event-free survival rate for composite endpoint of death or chronic renal failure: higher in MMF gp (p=0.05) and AZA (p<0.01) than CYC gps Rate of relapse-free survival: higher in MMF than CYC gp (p<0.05) Lower rates of hospitalization, infections, and gastrointestinal side effects in MMF and AZA gps

More complete remissions in MMF gp than CYC gp (16 vs. 4, p<0.05) More partial remissions in MMF than in CYC (21 vs. 17) Mean Cr proteinuria or urinary sediment at 24 weeks: NS (intention to treat analysis) 3 patients (all randomized to CYC) died with severe SLE

Complete/partial remission: 90% in both gps MMF gp, fewer infections Composite endpoint of end-stage renal failure or death: 4 pts in the CYC gp vs 0 in MMF gp (p=0.06)

Complete remission rates: 81% in MMF gp, 76% in CYC gp Improvements in degree of proteinuria, serum albumin, and Cr also similar in first year Relapse rates in first year: 15% in MMF gp, 11% in CYC gp

Results

AZA, azathioprine; Cr, creatinine; CT, combination therapy; CYC, cyclophosphamide; gp, group; IV, intravenous; IVCY, intravenous cyclophosphamide; IVMP, intravenous methylprednizolone; MMF, mycophenolate mofetil; MP, methylprednizolone; NS, nonsignificant; pts, patients; SLE, systemic lupus erythematosus.

n

Trial

TABLE 46.2 RANDOMIZED CONTROLLED TRIALS FOR USE OF MMF IN LUPUS NEPHRITIS

CYTOTOXIC DRUG TREATMENT

504

OTHER AGENTS

Methotrexate Methotrexate, an antimetabolite, is a structural analogue of folic acid that inhibits the de novo synthesis of purine metabolites through the inhibition of dihydrofolate reductase (DHFR) and other folate-dependent enzymes. It has both anti-inflammatory and immunosuppressive effects.

OTHER AGENTS

are several uncertainties regarding optimal administration and its long-term safety and efficacy, the recent follow-up study by Chan and colleagues46 has resolved several of these issues. For severe cases or the cases in which the disease does not remit after the first 6 months of therapy with MMF (or does not improve substantially after the first 3 months of therapy), the combination of pulses of IV-CY and IV-MP until remission remains the treatment of choice (Table 46.3). On the other hand, initial data and anecdotal experience suggest that individual patients may respond unpredictably to one treatment or the other, and that it may be necessary to switch therapy if response is inadequate within the first few months of disease. For patients that enter remission within 6 months, maintenance therapy with MMF for the initial first 1 to 2 years of remission may be preferable to azathioprine, although unlike the quarterly pulses of IV-CY, the efficacy of this approach has not been tested in patients with severe disease.51,57 Regardless of the agent used, early effective cytotoxic therapy is of paramount importance, as first demonstrated in lupus nephritis by Esdaile and colleagues.58 Whether physicians, in view of the better toxicity profile of MMF, will be more likely to use cytotoxic therapy earlier in the course of the disease remains to be seen.

Pharmacokinetics and Adverse Effects Similar to RA treatment, methotrexate can be administered weekly either orally or parenterally in SLE.59,60 Hepatic, hematologic, and pulmonary toxicities are the most important side effects of methotrexate (Table 46.1). Concomitant administration of folic acid (2.5 to 5 mg/wk, 24 hours after intake of methotrexate) is also recommended, and has been proven to limit methotrexate-related toxicity.

Clinical Use in Lupus Although there has been substantial evidence supporting the effectiveness of methotrexate in RA, there are scarce data on its use in lupus. Current recommendations suggest its use as a steroid-sparing agent for articular and cutaneous manifestations of the disease.

TABLE 46.3 RECOMMENDED CYTOTOXIC THERAPY FOR MAJOR ORGAN INVOLVEMENT IN SYSTEMIC LUPUS ERYTHEMATOSUS Disease Severity

Induction Therapy

Maintenance Therapy

Mild

High-dose corticosteroids (i.e., 0.5–1 mg/kg/d prednisone for 4–6 weeks with gradual tapering to 0.125 mg/kg every other day within 3 months) alone or in combination with azathioprine (1–2 mg/kg/d). If no remission within 3 months, treat as moderately severe

Low-dose corticosteroids (i.e., prednisone ≤ 0.125 mg/kg on alternate days) alone or with azathioprine (1–2 mg/kg/d) Consider further gradual tapering at the end of each year of remission

Moderate

MMF (2 g/d) (or azathioprine) with corticosteroids as above. If no remission after the first 6–12 months, advance to next therapy OR Pulse cyclophosphamide alone or in combination with pulse corticosteroids for the first 6 months (background corticosteroids 0.5 mg/kg/d for 4 weeks, then taper) for 7 pulses

If remission after the first 6-12 months MMF may be tapered to 1.5 g/d bid for 6-12 months and then to 1 g/d. Consider further tapering at the end of each year in remission OR Quarterly pulses of cyclophosphamide OR Azathioprine (1–2 mg/kg/d)

Severe

Monthly pulses of cyclophosphamide in combination with pulse corticosteroids for 6–12 months If no response, consider adding rituximab or switch to MMF

Quarterly pulses of cyclophosphamide for at least 1 year beyond remission Azathioprine (1–2 mg/kg/d) MMF (1–2 g/d)

505

CYTOTOXIC DRUG TREATMENT

A randomized double-blind placebo controlled trial of methotrexate in lupus patients showed that a weekly dose of 15 to 20 mg for 6 months effectively controlled disease activity and allowed for corticosteroid reduction.61

guidelines to decrease toxicity are shown in Table 46.1. The drug has been associated with increased risk of skin cancer and lymphoma in transplant recipients; this, however, has not been confirmed for patients receiving cyclosporine for autoimmune disorders.

Cyclosporine

Clinical Use

Cyclosporine A is a fungus-derived lipophilic endecapeptide that inhibits calcineurin and T-cell activation. The therapeutic effects of cyclosporine in lupus are mainly linked to its ability to reduce antigen presentation and T-cell–mediated autoantibody production by B lymphocytes.

Cyclosporine is most commonly used for membranous lupus nephropathy at doses of 3 to 5 mg/kg/d. Small case series also suggest a clinical benefit in other manifestations of SLE such as skin rashes, thrombocytopenia, and aplastic anemia.65

Pharmacokinetics Peak concentration occurs within 1 to 8 hours after administration. If required, drug concentration should be measured in whole blood, which is rarely necessary in autoimmune diseases unless used in doses greater than 3 mg/kg/d. Clinical response to cyclosporine is relatively slow and occurs after 1 to 2 months of treatment. Cyclosporine is metabolized to more than 20 metabolites and excreted mainly in the bile. Although its elimination is not altered in the case of renal insufficiency, cyclosporine administration should be avoided in patients with impaired renal function due to its nephrotoxic effects.

Drug Interactions A variety of drugs interact with cyclosporine leading to reduced cyclosporine concentrations (rifampin, phenyntoin, phenobarbital, nafcillin, etc.), increased cyclosporine concentrations (erythromycin, clarithromycin, azoles, calcium channel blockers, amiodarone, allopurinol, colchicine, etc.), or augmenting its nephrotoxic effects (nonsteroidal anti-inflammatory drugs, aminoglycosides, quinolones, ACE inhibitors, amphotericin B).62

Adverse Effects

506

Transient gastrointestinal complaints, hypertrichosis, gingival hyperplasia, and clinically insignificant increases in serum levels of alkaline phosphatase are the most commonly observed side effects. Tremor, paresthesias, electrolyte disturbances (hyperkalemia and hypomagnesemia), and hyperuricemia may also occur. Hypertension occurs in approximately 20% of patients receiving cyclosporine, and is controlled either by reduction of the dose or by antihypertensive treatment.63 A major side effect of cyclosporine is nephrotoxicity, which is reversible after adjustment of the dose or discontinuation of the drug in the majority of cases. Risk factors are high-dose cyclosporine (>5 mg/kg/d) and an increase in serum creatinine of more than 50% of the baseline value.64 Monitoring

Intravenous Gammaglobulin Intravenous immunoglobulin (IVIG) is thought to represent an immunoregulatory agent, and has been used for the treatment of variety of severe manifestations of lupus. Mechanisms of action include Fc-gamma receptor blockade as well as immunomodulation of complement and T cells, all of which have been suggested as contributors to its therapeutic effect in SLE.66 Intravenous immunoglobulin is administered at doses of 400 mg/kg/d for 5 consecutive days, and is most commonly used for the treatment of severe, refractory thrombocytopenia, achieving a rapid rise in the number of platelets within hours of administration. Nephritis, arthritis, fever, rashes, and immunologic parameters improve with intravenous immunoglobulin.66,67 Side effects of intravenous gammaglobulin include fever, myalgias, headache, arthralgias, and rarely, aseptic meningitis. The drug is contraindicated in cases of known IgA deficiency.

General Issues Cytotoxic Drugs in Severe, Life-Threatening Disease Controlled trials and clinical experience suggest that IV-CY in combination with IV-MP is the treatment of choice for most patients.51,57 For refractory patients, based on initial experience, availability, and potential side effects, combinations of IV-CY with rituximab (see Chapter 48) may be an acceptable strategy at present.57 MMF may rescue a few refractory patients, but its efficacy in critically ill patients requires further documentation. For selected patients (patients with neurologic disease, antiphospholipid syndrome, thrombocytopenia), IVIG may be considered as an adjunct therapy. In critically ill patients, plasmapheresis may offer some benefit to selected patients. CY may be administered either orally or as pulse therapy in the intensive care unit. The major concern is bladder protection and infections from the respirator and intravenous lines. In such cases, bladder irrigation through a three-way catheter in case of urine output lower than

Prevention and Management of Infection and Immunizations Infections attributed predominantly to corticosteroids and cytotoxic drugs are an important cause for hospital admissions and death, accounting for approximately one-quarter of all deaths.68 Judicious use of corticosteroids and immunizations may decrease the frequency of infections. Strategies to decrease the impact of infections have been discussed elsewhere69 and include (1) simple hygienic measures and education aimed at both patients and doctors; (2) antimicrobial prophylaxis in cohorts of patients with increased prevalence of certain infections, patients who receive heavy doses of immunosuppressive agents, or undergo procedures associated with temporary bacteremia; and (3) immunizations similar to those available to the general population. Bacterial Endocarditis Clinical or subclinical valvular abnormalities are common in patients with moderate to severe lupus and may be predisposing to bacterial endocarditis. Patients with known valvular abnormalities should receive endocarditis prophylaxis prior to invasive dental, intestinal, or genitourinary procedures, according to the standard regimens of the American Heart Association.70 For patients without known valvular abnormalities, some investigators have also advocated the use of prophylactic antibiotic prophylaxis.71,72 In our opinion, this is a reasonable suggestion, especially when patients receive high-intensity immunosuppressive therapy, but firm data to support this are lacking at present. Tuberculosis and Pneumocystis carinii Widely applicable guidelines for tuberculosis prophylaxis in lupus do not exist. Purified protein derivative (PPD) testing may be considered in areas with a high prevalence of tuberculosis for patients who receive long-term prednisone equal to or greater than the equivalent of 15 mg/d with 5 mm representing the most realistic cut-off.69,73 For patients living in lowprevalence countries, current data do not support the routine use of PPD testing prior to cytotoxic therapy.69

In contrast to patients with systemic vasculitis,74 we do not routinely employ prophylaxis for Pneumocystis carinii in lupus. However, some authors recommend it for patients on high-dose corticosteroids alone or in combination with cytotoxic drugs if the CD4 count is below 300 cells/μL (one doublestrength tablet of trimethoprine-sulfomethoxazole three times a week or dapsone 100 mg/d if allergic to sulfamethoxazole).74,75

OTHER AGENTS

100 mL/hr is essential, along with diligent care of the lines and tapering of corticosteroids. An aggressive search for infection before and after therapy is essential. Until infection is ruled out, we typically use highdose corticosteroids. An aggressive tapering of corticosteroids, once the patient improves, is essential to minimize the risk of infections and other complications. The role of the multidisciplinary approach involving several medical subspecialties cannot be overemphasized.

Immunizations Although vaccination may theoretically induce polyclonal activation in lupus and induce a flare, it is felt to be safe. More specifically, influenza vaccine has been shown to be safe and effective,76 while pneumococcal vaccine is also safe but the resultant antibody titers may be decreased in patients with SLE as compared to controls.76,77 Use of corticosteroids such as prednisone may contribute to blunted antibody response. Protective immune response can be achieved safely in patients with SLE with both tetanus toxoid and Hemophilus influenza type B in addition to pneumococcus.77 Immunization of patients on corticosteroids alone (doses equivalent to 20 mg/d of prednisone for more than 2 weeks) or in combination with cytotoxic drugs with live vaccines (e.g., measles, mumps, rubella, polio, varicella, VZV [zoster vaccine] and vaccinia [smallpox]) is contraindicated. The efficacy and safety of hepatitis B vaccination in lupus patients requires further documentation. Rheumatic syndromes temporally related to vaccination, especially hepatitis B, have been described, but a causal relationship has not been established.78,79 Pulmonary Infiltrates Pulmonary complications remain a major cause of both morbidity and mortality in lupus patients receiving cytotoxic drugs. The differential diagnosis in this setting is broad, and includes both infectious and noninfectious processes. Rarely are the radiographic findings classic for one disease, and most potential etiologies have overlapping clinical and radiographic appearances. An aggressive approach to identifying a specific etiology is of paramount importance; this includes bronchoscopy and early use of CT scanning.80

Screening for Malignancy Certain cancers occur more frequently in SLE patients when compared with the general population, as recent data substantiate, and may be in part due to cytotoxic/immunosuppressive drugs. The risk appears to be greatest for lymphoma.81 Cooperative efforts to evaluate this risk have been undertaken by several groups. Increased frequency of abnormal cervical Papanicolaou (Pap) smears in women with SLE has

507

CYTOTOXIC DRUG TREATMENT

been reported by several groups. In one study, risk factors for development of an abnormal Pap smear include a history of sexually transmitted diseases, use of oral contraceptives, and use of immunosuppressive drugs, especially CY.82 Vigilance in cancer-preventive strategies similar to that of the general population is essential. This is particularly important in view of recent data suggesting that appropriate cancer screening may be overlooked in patients with SLE.83

Use of Cytotoxic Drugs in Pregnancy Pregnant patients with active lupus and mild disease are generally managed with corticosteroids. In moderate to severe disease, corticosteroids azathioprine,

cyclosporine, and IVIG may be acceptable for the fetus.84 In life-threatening disease, CY may be used only if there are no alternative therapies. CY and methotrexate are contraindicated (Food and Drug Administration category D, positive evidence for risk), while adequate information regarding MMF is lacking (category C, risk cannot be ruled out). Because azathioprine may be excreted in the breast milk, breast-feeding is not recommenced. The American Academy of Pediatrics recommends that nursing is permissible for women receiving corticosteroids, but the interval between dose and nursing should be at least 4 hours if the prednisone dose is more than 20 mg/d.

REFERENCES

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1. Pedersen BK, Beyer JM, Rasmussen A, et al. Azathioprine as a single drug in the treatment of rheumatoid arthritis induces complete suppression of natural killer cell activity. APMIS 1984; 92:221-225. 2. Van SK, Johnson CA, Porter WR. The pharmacology and metabolism of the thiopurine drugs 6-mercaptopurine and azathioprine. Drug Metabol Rev 1985;16:157-174. 3. Cummins D, Sekar M, Halil O, Banner N. Myelosuppression associated with azathioprine-allopurinol interaction after heart and lung transplantation. Transplantation 1996;61:1661-1662. 4. Walker J, Mendelson H, McClure A, Smith MD. Warfarin and azathioprine: clinically significant drug interaction. J Rheumatol 2002;29:398-399. 5. Singh G, Fries JF, Spitz P, Williams CA. Toxic effects of azathioprine in rheumatoid arthritis: a national port-marketing perspective. Arthritis Rheum 1989;32:837-843. 6. Leipold G, Schutz E, Haas JP, Oellerich M: Azathioprine-induced severe pancytopenia due to a homozygous two-point mutation of the thiopurine methyltransferase gene in a patient with juvenile HLA-B-27-associated spondylarthritis. Arthritis Rheum 1997; 40:1896-1898. 7. Fields CL, Robinson JW, Roy TM, et al. Hypersensitivity reaction to azathioprine. South Med J 1998;91:471-474. 8. Gaffney K, Scott DG. Azathioprine and cyclophosphamide in the treatment of rheumatoid arthritis. Br J Rheumatol 1998;37: 824-836. 9. Cupps TR, Edgar LC, Fauci AS. Suppression of human B lymphocyte function by cyclophosphamide. J Clin Invest 1982;128:2453-2457. 10. Struck RF, Alberts DS, Horne K, et al. Plasma pharmacokinetics of cyclophosphamide and its cytotoxic metabolites after intravenous versus oral administration in randomized, crossover trial. Cancer Res 1987;47:2723-2726. 11. Grochow LB, Colvin M. Clinical pharmacokinetics of cyclophosphamide. Clin Pharmacokinet 1979;4:380-394. 12. Pryor BD, Bologna SG, Kahl LE. Risk factors for serious infection during treatment with cyclophosphamide and high dose corticosteroids for systemic lupus erythematosus. Arthritis Rheum 1996;39:1475-1482. 13. Guillevin L, Cordier JF, Lhote F, et al. A prospective, multicenter, randomized trial comparing steroids and oral cyclophosphamide in the treatment of generalized Wegener’s granulomatosis. Arthritis Rheum 1997;40:2099-2104. 14. Gladman DD, Hussain F, Ibanez D, Urowitz MB. The nature and outcome of infection in systemic lupus erythematosus. Lupus 2002;11:234-239. 15. Boumpas DT, Austin HA 3d, Vaughan EM, et al. Risk for sustained amenorrhea in patients with systemic lupus erythematosus receiving intermittent pulse cyclophosphamide therapy. Ann Intern Med 1993;119:366-369.

16. Rivkees SA, Crawford JD. The relationship of gonadal activity and chemotherapy-induced gonadal damage. JAMA 1988; 259:2123. 17. Slater CA, Liang MH, McCune JW, Christman GM, Laufer MR. Preserving ovarian function in patients receiving cyclophosphamide. Lupus 1999;8:3-10. 18. Dooley MA, Patterson CC, Susan L, et al. Preservation of ovarian function using depot leuprolide acetate during cyclophosphamide therapy for severe lupus nephritis. Arthritis Rheum 2000;43:2858. 19. Somers EC, Marder W, Christman GM, Ognenovski V, McCune WJ. Use of a gonadotropin-releasing hormone analog for protection against premature ovarian failure during cyclophosphamide therapy in women with severe lupus. Arthritis Rheum 2005; 52:2761-2767. 20. Nicholson HS, Byrne J. Fertility and pregnancy after treatment for cancer during childhood or adolescence. Cancer 1993;71:3392-3399. 21. Masala A, Faedda R, Aigna S, et al. Use of testosterone to prevent cyclophosphamide-induced azoospermia. Ann Intern Med 1997;126:292-295. 22. Raptopoulou A, Sidiropoulos P, Boumpas D. Ovarian failure and strategies for fertility preservation in patients with systemic lupus erythematosus. Lupus 2004;13:887-890. 23. Stillwell TJ, Benson RCJ, DeRemee RA, et al. Cyclophosphamide induced bladder toxicity in Wegener’s granulomatosis. Arthritis Rheum 1988;31:465-470. 24. Radis CD, Kahl LE, Baker GL, et al. Effects of cyclophosphamide on the development of malignancy and on long-term survival on patients with rheumatoid arthritis. A 20-year follow-up study. Arthritis Rheum 1995;38:1120. 25. Talar-Williams C, Hijazi YM, Walther MM, et al. Cyclophosphamide induced cystitis and bladder cancer in patients with Wegener’s granulomatosis. Ann Int Med 1996;124:477-484. 26. Austin HA III, Klippel JH, Balow JE, et al. Therapy of lupus nephritis. Controlled trial of prednizone and cytotoxic drugs. N Engl J Med 1986;314:614-619. 27. Boumpas DT, Austin HA III, Vaughn EM, et al. Controlled trial of pulse methylprednisolone versus two regimens of pulse cyclophosphamide in severe lupus nephritis. Lancet 1992;340 (8822):741-745. 28. Gourley MF Austin HA III, Scott D, et al. Methylprednisolone and cyclophosphamide alone or in combination in patients with lupus nephritis. A randomised, controlled trial. Ann Intern Med 1996;125:549-557. 29. Illei GG Austin HA, Crane M, et al. Combination therapy with pulse cyclophosphamide plus pulse methylprednisolone improves long-term renal outcome without adding toxicity in patients with lupus nephritis. Ann Intern Med 2001;135: 248-257.

52. Gaubitz M, Schorat A, Schotte H, Kern P, Domschke W. Mycophenolate mofetil for treatment of systemic lupus erythmatosus: an open pilot trial. Lupus 1999;8:731-737. 53. Vasoo S, Thumboo J, Fong KY. Refractory immune thrombocytopenia in systemic lupus erythematosus: response to mycophenolate mofetil. Lupus 2003;12:630-632. 54. Samad AS, Lindsley CB. Treatment if pulmonary hemorrhage in childhood systemic lupus erythematosus with mycophenolate mofetil. South Med J 2003;96:705-707. 55. Dooley MA, Cosio FG, Nachman PH, et al. Mycophenolate mofetil therapy in lupus nephritis: clinical observations. J Am Soc Nephrol 1999;10:833-839. 56. Kapitsinou OO, Boletis JN, Skopouli FN, et al. Lupus nephritis treatment with mycophenolate mofetil. Rheumatology (Oxford) 2004;43:377-380. 57. Boumpas DT, Sidiropoulos P, Bertsias G. Optimum therapeutic approaches for lupus nephritis: what therapy and for whom? Nature Clinical Pract Rheumatol 2005;1:22-30. 58. Esdaile JM, Joseph L, MacKenzie T, Kashgarian M, Hayslett JP. The benefit of early treatment with immunosuppressive drugs in lupus nephritis. J Rheumatol 1994;21(11):1985-1986. 59. Herman RA, Vend-Pedersen P, Hoffman J, et al. Pharmacokinetics of low-dose methotrexate in rheumatoid arthritis patients. J Pharm Sci 1989;78:165. 60. Kremer JM, Alarcón GS, Lightfoot RW Jr, et al. Methotrexate for rheumatoid arthritis: suggested guidelines for monitoring liver toxicity. Arthritis Rheum 1994;37:316. 61. Carneiro JR, Sato EI. Double blind, randomized, placebo control trial of methotrexate in systemic lupus erythematosus. J Rheumatol 1999;26:1275-1279. 62. Campana C, Regazzi MB, Buggia I, Molinaro M. Clinically significant drug interactions with cyclosporin: an update. Clin Pharmacokinet 1996;30:141-179. 63. Landewe RB, Goei The HS, van Ritjhoven AW, et al. Cyclosporine in common clinical practice: an estimation of the benefit/risk ratio in patients with rheumatoid arthritis. J Rheumatol 1994;21:1631-1636. 64. Rodriguez F, Krayenbuhl JC, Harrison WB, et al. Renal biopsy findings and follow up of renal function in rheumatoid arthritis patients treated with cyclosporin A: an update from the International Kidney Biopsy Registry. Arthritis Rheum 1996;39:1491-1498. 65. Griffiths B, Emery P. The treatment of lupus with cyclosporin A [review]. Lupus 2001;10:165-170. 66. Levy Y, Sherer Y, Ahmed A, et al. A study of 20 SLE patients with intravenous immunoglobulin—clinical and serologic response. Lupus 1999;8:705-712. 67. Boletis JN, Ioannidis JP, Boki KA, Moutsopoulos HM. Intravenous immunoglobulin compared with cyclphosphamide for proliferative lupus nephritis. Lancet 1999;354:569-570. 68. Cervera R, Khamashta MA, Font J, et al. Morbidity and mortality in systemic lupus erythematosus during a 10-year period: a comparison of early and late manifestations in a cohort of 1,000 patients. Medicine (Baltimore) 2003;82:299-308. 69. Gilliland WR, Tsokos GC. Prophylactic use of antibiotics and immunisations in patients with SLE. Ann Rheum Dis 2002; 61: 191-192. 70. Dajani AS, Taubert KA, Wilson W, et al. Prevention of bacterial endocarditis. Recommendations by the American Heart Association. JAMA 1997;277:1794-1801. 71. Mitcell SR, Cupps TR, Nashel SJ, et al. Valvulitis in systemic lupus erythematosus. Am J Med 1989;86:510. 72. Zysset MK, Montgomery MT, Redding SW. Systemic lupus erythematosus. A consideration for antimicrobial prophylaxis. Oral Surg Oral Med Oral Pathol 1987;64:30-34. 73. Hamilton CD. Tuberculosis in the cytokine era: what rheumatologists need to know. Arthritis Rheum 2003;48 2085-2091. 74. Sneller MC, Hoffman GS, Talar-Williams C, et al. An analysis of forty-two Wegener’s granulomatosis patients treated with methotrexate and prednisone. Arthritis Rheum 1995;38:608-613. 75. Porges AJ, Beatie SL, Ritchlin C, et al. Patients with systemic lupus erythematosus at risk for Pneumonocystis carinii pneumonia. J Rheumatol 1992;19:1191-1194. 76. Brodman R, Gilfillan R, Glass D, Schur PH. Influenza vaccine response in systemic lupus erythematosus. Ann Intern Med 1978;88:735.

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30. Dooley MA, Hogan S, Jennette C, et al. Cyclophosphamide therapy for lupus nephritis: poor renal survival for black Americans. Glomerular Disease Collaborative Network. Kidney Int 1997;51:1188-1195. 31. Houssiau FA, Vasconcelos C, D’Cruz D, et al. Immunosuppressive therapy in lupus nephritis: the Euro-Lupus Nephritis Trial, a randomised trial of low dose versus high-dose intravenous cyclophosphamide. Arthritis Rheum 2002;46:2121-2131. 32. Houssiau FA, Vasconcelos C, D’Cruz D, et al. Early response to immunosuppressive therapy predicts good renal outcome in lupus nephritis: lessons from long-term follow-up of patients in Euro-Lupus Nephritis Trial. Arthritis Rheum 2004;50: 3934-3940. 33. Boumpas DT, Barez S, Klippel JH, Balow JE. Intermittent cyclophosphamide for the treatment of autoimmune thrombocytopenia in systemic lupus erythematosus. Ann Intern Med 1990;112:674-677. 34. Boumpas DT, Yamada H, Patronas NJ, et al. Pulse cyclophosphamide for severe neuropsychiatric lupus. Q J Med 1991;81:975-984. 35. Takada K, Illei GG, Boumpas DT. Cyclophosphamide for the treatment of systemic lupus erythematosus. Lupus 2001;10: 154-161. 36. Barile-Fabris L, Ariza-Andraca R, Olguin-Ortega L, et al. Controlled clinical trial of IV cyclophosphamide versus IV methylprednisolone in severe neurological manifestations in systemic lupus erythematosus. Ann Rheum Dis 2005;64:620-625. 37. Carr SF, Papp E, Wu JC, Natsumeda Y. Characterization of human type I and type II IMP dehydrogonases. J Biol Chem 1993:268:27286-27292. 38. Eugui EM, Almquist SJ, Muller CD, Allison AC. Lymphocyte-selective cytostatic and immunosuppressive effects of mycophenolic acid in vitro: role of deoxyguanosine nucleotide depletion. Scand J Immunol 1991;33:161-173. 39. Allison AC, Eugui EM. Immunosuppressive and other effects of mycophenolic acid and an ester prodrug, mycophenolate mofetil. Immunol Rev 1993;136:5-28. 40. Senda M, DeLustro B, Eugui E, Natsumeda Y. Mycophenolic acid, an inhibitor of IMP dehydrogonase that is also an immunosuppressive agent, suppresses the cytokine induced nitric oxide production in mouse and rat endothelial cells. Transplantation 1995;60:1143-1148. 41. Hu W, Liu Z, Chen H, et al. Mycophenolate mofetil versus cyclophosphamide in patients with diffuse proliferative lupus nephritis. Chin Med J (Engl) 2002;115:705-709. 42. Pisoni CN, Sanchez FJ, Karim Y, et al. Mycophenolate mofetil treatment in systemic lupus erythemstosus. Arthritis Rheum 2004;50:S415 (abstract). 43. Riskalla MM, Somer EC, Fatica RA, McCune WJ. Tolerability of mycophenolate mofetil in patients with systemic lupus erythematosus. J Rheumatol 2003;30:1508-1512. 44. Schiff MH, Leishman B. Long term safety of CellCept (mycophenolate mofetil), a new therapy for rheumatoid arthritis patients. Br J Clin Pharmacol 1996;41:513-516. 45. Chan TM, Li FK, Tang CS, et al. Efficacy of mycophenolate mofetil in patients with diffuse proliferative lupus nephritis. Hong KongGuangzhou Nephrology Study Group. N Engl J Med 2000; 343:1156-1162. 46. Chan TM. Wong WS, Lau CS, et al. Long-term study of mycophenolate mofetil as continuous induction and maintenance treatment for proliferative lupus nephritis. J Am Soc Nephrol 2005;16: 1076-1084. 47. Ginzler EM, Aranow C, Buyon J, et al. A multicenter study of mycophenolate mofetil (MMF) vs. intravenous cyclophosphamide (IVC) as induction therapy for severe lupus nephritis (LN): preliminary results. Arthritis Rheum 2003;48:1690. 48. Contreras G, Pardo V, Leclercq B, et al. Sequential therapies for proliferative lupus nephritis. N Engl J Med 2004;350: 971-980. 49. Balow JE, Austin HA III. Maintenance therapy for lupus nephritis— something old, something new. N Engl J Med 2004;350:1044-1046. 50. Boumpas DT. Sequential therapies with intravenous cyclophosphamide and oral mycophenolate mofetil or azathioprine are efficacious and safe in proliferative lupus nephritis. [commentary]. Clin Exp Rheumatol 2004;22:276-277. 51. McCune WJ. Mycophenolate mofetil for lupus nephritis. N Engl J Med 2005;353:2282-2284.

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77. Battafarano DF, Battafarano NJ, Larsen L, et al. Antigen-specific antibody responses in lupus patients following immunization. Arthritis Rheum 1998;41:1828. 78. Older SA, Batafarano DF, Enzenauer RJ, et al. Can immunization precipitate connective tissue disease? Report of five cases of systemic lupus erythematosus and review of the literature. Semin Arthritis Rheum 1999;29:131-139. 79. Ioannou, Y, Isenberg, DA. Immunization of patients with sytemic lupus erythematosus: the current state of play. Lupus 1999;8:497. 80. Shorr AF, Susla GM, O’Grady NP. Pulmonary infiltrates in the non-HIV-infected immunocompromised patient: etiologies, diagnostic strategies, and outcomes. Chest 2004;125: 260-271.

81. Bernatsky S, Ramsey-Goldman R, Rajan R, et al. Non-Hodgkin’s lymphoma in systemic lupus erythematosus. Ann Rheum Dis 2005;64:1507-1509. 82. Bernatsky S, Ramsey-Goldman R, Gordon C, et al. Factors associated with abnormal Pap results in systemic lupus erythematosus. Rheumatology (Oxford) 2004;43:1386. 83. Bernatsky SR, Cooper GS, Mill C, et al. Cancer screening in patients with systemic lupus erythematosus. J Rheumatol 2006;33:45-49. 84. Bermas BL, Hill JA. Effects of immunosuppressive drugs during pregnancy. Arthritis Rheum 1995;38:1722.

TREATMENT OF THE DISEASE

47

Treatment of Antiphospholipid Syndrome Munther A. Khamashta, MD, FRCP, PhD

INTRODUCTION Antiphospholipid syndrome (APS) is defined by the development of thrombosis and/or adverse obstetric events in the presence of antiphospholipid antibodies (aPLs).1 The clinical consequence of thrombosis, which is the main complication of APS, depends on the site and extent of the process. In most cases, larger vessels are involved, particularly thrombosis of the intracranial arteries (leading to cerebral ischemia) or the deep veins of the lower extremities (sometimes leading to pulmonary embolism). These events usually occur in isolation or, if recurrent, over months or years. Much less commonly, there is diffuse thrombotic occlusion of predominantly small vessels, which leads to damage and dysfunction of multiple organs concurrently or sequentially over days or weeks. The term “catastrophic” APS (CAPS) has been coined for this serious and often fatal complication.2 Data regarding management and prognosis are limited. The long-term prognosis for aPL-positive patients is most influenced by the risk of thrombosis. Thrombotic complications are unpredictable, and the mechanisms triggering these events in individual patients are ill-defined. Despite advances in studies of the mechanisms of thrombosis in APS, and the development of newer assays such as anti-β2 glycoprotein I and antiprothrombin antibodies, the serologic “fingerprint” of the patient most at risk for arterial (especially stroke) or venous thrombosis remains elusive.

PRIMARY THROMBOPROPHYLAXIS OF ANTIPHOSPHOLIPID ANTIBODY–POSITIVE SUBJECTS The controversy concerning whether prophylactic treatment is indicated for patients with aPL who have no history of thrombosis remains unresolved. Although epidemiologic studies suggest a risk of thrombosis in subjects positive for aPL,3 some of these individuals never develop these events. It is clear that

there are factors other than aPL that are necessary for the thrombosis to develop.4 Although low-dose aspirin (75 to 100 mg/d) has been considered a logical first option, the Physician Health Study showed that low-dose aspirin (325 mg/d) in men with aCL did not protect against deep venous thrombosis or pulmonary embolus.5 In contrast, hydroxychloroquine may be protective against the development of thrombosis in aPL-positive patients with SLE.6,7 A prospective, randomized clinical trial comparing aspirin alone with aspirin plus low-intensity warfarin (international normalized ratio [INR]~1.5) in patients with aPL who have never had thrombosis is currently underway in the United Kingdom. Until these results are available, we suggest that individuals with a persistently positive aCL (moderate to high titers) and/or unequivocally positive lupus anticoagulant tests take low-dose aspirin (75 to 100 mg/d) indefinitely. Cessation of estrogen-containing oral contraceptive use; treatment of hypertension, hyperlipidemia, or diabetes if present; and the avoidance of smoking are all additional recommended therapeutic measures. Prophylaxis with heparin administered subcutaneously should certainly be given to cover higher-risk situations, such as surgery and long-haul flights.

PREVENTION OF RECURRENT THROMBOSIS Despite the enormous amount of work that has focused on the pathogenesis and clinical manifestations of APS, there has been surprisingly little published on the management of thrombosis associated with aPL, and there are only limited data from prospective clinical trials on which to base treatment decisions. Acute management of venous or arterial thrombosis in patients with APS is no different from that of other patients with similar complications. Patients with venous thromboembolism are given heparin (low molecular weight in most countries) followed by warfarin.

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TREATMENT OF ANTIPHOSPHOLIPID SYNDROME

512

Fibrinolytic therapy has been used successfully in patients with APS.8 There is now good evidence from both retrospective9 and prospective10 studies that APS patients with thrombosis will be subject to recurrences, and that these can be prevented by long-term anticoagulation. Many patients with APS in whom anticoagulation has been stopped have had major recurrent thrombosis. It is not clear, however, whether prolonged anticoagulation is necessary in APS patients whose first thrombotic episode developed in association with surgery, oral contraceptive use, pregnancy, or other circumstantial thrombotic risk factors. The type of thrombosis is predictive. Retrospective analysis of patients with APS and recurrent thrombosis showed that a venous thrombosis is followed by another venous thrombosis in more than 70% of cases, and an arterial thrombosis is followed by another arterial thrombosis in more than 90% of cases.9,11 Most patients requiring long-term anticoagulant therapy respond well to warfarin targeted to an INR of 2.0 to 3.0. However, the optimal intensity of anticoagulation therapy is uncertain for patients with aPL-associated thrombosis. Retrospective studies in the 1990s suggested that thrombosis in APS patients should be managed by high-intensity (target INR 3.0 to 4.0) oral anticoagulation.9,11 The more recent prospective studies12,13 and a systematic review14 suggest only an INR of 2.0 to 3.0 is required. The major concern about these recent prospective and randomized studies is the fact that the majority of patients included had venous rather than arterial thrombosis. Furthermore, none of these studies achieved the expected sample size; a large number of patients were excluded because they had already had recurrent events on oral anticoagulation; and in the Crowther’s study,12 patients with recent stroke were excluded so that in the final study 76% of the patients had previous venous thrombosis only, and review of the high-intensity arm showed that the patients were below the therapeutic range 43% of the time. Oral anticoagulation therapy carries an inevitable risk of serious hemorrhage. In APS, serious bleeding complications may occur, but their risk is not higher than that observed in other thrombotic conditions warranting oral anticoagulation.15 In APS patients with previous arterial events, the dangers of thrombosis and stroke far outweigh the risk of anticoagulant-induced bleeding. The traditional fear of cerebral hemorrhage has almost certainly resulted in the undertreatment of many patients with cerebral APS.16 We recommend that APS patients with previous venous thrombotic events should have moderate-intensity (INR 2.0 to 3.0) anticoagulation. However, those with previous arterial events merit high-intensity (INR 3.0 to 4.0) anticoagulation until there is evidence to the contrary.16,17

Concerns exist over the validity of the INR in the control of oral anticoagulant dosing if lupus anticoagulant is present. The inhibitor occasionally increases the prothrombin time, and the INR may not reflect the true degree of anticoagulation.18 This phenomenon seems to be more likely when certain recombinant thromboplastin reagents are used, and can usually be circumvented by careful selection of the thromboplastin to be used for the prothrombin time test.19 The increasing use of oral anticoagulation therapy has increased the need for anticoagulant monitoring and encouraged a move towards “point of care” or “near-patient” testing and self-monitoring. Several trials have suggested that such monitoring might be equal to or even better than standard monitoring. A recent systematic review and meta-analysis of all randomized controlled trials showed that self-management improves the quality of oral anticoagulation. Patients capable of monitoring and self-adjusting therapy have fewer thromboembolic events and lower mortality.20 However, self-monitoring is not feasible for all patients, and requires identification and education of suitable candidates. Patients’ self-monitoring of oral anticoagulation have a greater degree of autonomy and particularly suits young patients with busy lifestyles who find attending the anticoagulation clinics irksome. Certainly, patient self-monitoring is associated with a higher level of patient satisfaction and quality of life than supervised management when assessed by questionnaires.21 We have used self-monitoring in motivated APS patients with success. However, all the data on self-monitoring relate to running an INR of 2.0 to 3.0, not 3.0 to 4.0. We propose to formally study this group to ensure that they are attaining satisfactory anticoagulation. Some patients with APS continue to have recurrent thrombotic events despite an INR of 3.0 to 4.0. Whether additional therapy with low-dose aspirin is efficacious in this situation is not known, but the risk of hemorrhage is increased when aspirin is used alongside oral anticoagulant therapy.22 Hydroxychloroquine use may aid preventing thrombosis in lupus patients. Due to its excellent safety profile, this drug might empirically be given to APS patients with insufficient control despite optimal oral anticoagulation. One of the features of APS is that some patients appear relatively resistant to warfarin, with some requiring up to 25 mg/d to maintain adequate anticoagulation. In our experience, most of these patients were receiving other drugs and, notably, azathioprine, at the same time as warfarin therapy. Azathioprine interacts with warfarin, reducing its efficacy by possible hepatic enzyme induction.23 Conversely, patients on warfarin who stop azathioprine may be at risk of bleeding and should be monitored carefully.

management of CAPS is not known. Most cases have been treated by corticosteroids, anticoagulation, plasmapheresis, high-dose intravenous immunoglobulin (IVIG), and cyclophosphamide, usually in some form of combination of these. The best outcome (70% survival) was seen in patients treated with a combination of corticosteroids, anticoagulation, plasmapheresis, and/or IVIG. The addition of cyclophosphamide was associated with a lower rate of survival, although these patients may have had more severe disease.2 Appropriate management plans when aPLs are found in association with various clinical features and in asymptomatic individuals are outlined in Tables 47.1 and 47.2.

PREVENTION OF RECURRENT PREGNANCY LOSS

PREVENTION OF RECURRENT PREGNANCY LOSS

Autoimmune thrombocytopenia is an accompanying problem in 25% of individuals with APS.24 Generally, it is mild (platelet counts of 50,000 to 150,000/mm3), but occasionally severe thrombocytopenia occurs. The treatment of thrombosis and thrombocytopenia in the same patient is a difficult clinical problem, and requires careful management. It is worth noting that thrombocytopenia does not necessarily protect patients against thrombosis,25 and platelet counts of 50,000 to 100,000/mm3 in APS should not modify the treatment policy of thrombosis with warfarin. The role of steroids and immunosuppressive drugs in the treatment of patients with aPL and thrombosis is uncertain. Such drugs do not always suppress aPL, and they have severe side effects when given for prolonged periods. Furthermore, in a large series of patients with APS, corticosteroids and immunosuppressive therapy, prescribed for some patients to control lupus activity, did not prevent further thrombotic events.9 The use of these drugs is probably justified only in patients with life-threatening conditions with repeated episodes of thrombosis despite adequate anticoagulation therapy, namely CAPS.2 Patients with CAPS frequently present to the intensive care unit with the challenging differential diagnosis of multisystem failure. The diagnosis requires a high degree of clinical awareness but, once the condition is suspected clinically, the diagnosis is generally confirmed by the demonstration of aPL (anticardiolipin antibody and/or lupus anticoagulant). CAPS is a serious complication with mortality in reported cases of approximately 50%.2,26 The optimal

The management of pregnancy in women known to have APS is the subject of much debate, and as yet there have been very few randomized controlled trials. Anticoagulation in one form or another is the preferred treatment, rather than steroids (once widely recommended27,28). Current choices include aspirin, heparin, or both (Table 47.2). Two prospective trials showed that heparin plus low-dose aspirin is more effective than aspirin alone for achieving live births among women with aPL and first-trimester recurrent pregnancy loss.29,30 However, such increased efficacy of the combination treatment was not seen in a later randomized controlled trial of similar design.31 Another prospective trial of aPL-positive women with repeated pregnancy loss but no history of thrombosis or SLE,

TABLE 47.1 PRIMARY THROMBOPROPHYLAXIS AND PREVENTION OF RECURRENT THROMBOSIS IN ANTIPHOSPHOLIPID SYNDROME Clinical Situation

Recommended Treatment

aPL-positive individuals with no history of thrombosis or pregnancy loss

Removal of additional thrombotic risk factors is essential. Long-term low-dose aspirin (although no evidence of benefit). Consider hydroxychloroquine, especially for those with systemic lupus erythematosus and other connective tissue diseases. Thromboprophylaxis with heparin in high-risk situations. Low-intensity warfarin (INR 1.5) is being tested in a clinical trial.

First thromboembolic event (venous)

Long-term warfarin (INR 2.0–3.0). Continue treatment even if aPL become negative.

First thrombotic event (arterial)

Long-term warfarin (INR 3.0–4.0). Continue treatment even if aPL become negative.

Recurrent thrombosis

Long-term warfarin (INR 3.0–4.0). Continue treatment even if aPL become negative.

Difficult (resistant) cases

Long-term warfarin (INR 3.0–4.0). Consider adding low-dose aspirin, hydroxychloroquine or immunosuppressive drugs. In catastrophic antiphospholipid syndrome patients, consider intravenous immunoglobulins and plasmapheresis.

aPL, antiphospholipid antibodies; INR, international normalized ratio.

513

TREATMENT OF ANTIPHOSPHOLIPID SYNDROME

TABLE 47.2 MANAGEMENT OF PREGNANT WOMEN WITH ANTIPHOSPHOLIPID SYNDROME Clinical Situation

Recommended Treatment

aPL-positive women with no history of thrombosis or pregnancy loss

Low-dose aspirin from pre-conception (although no evidence of benefit)

Recurrent first-trimester miscarriages

Low-dose aspirin from pre-conception. If LMW heparin is used, consider cessation at 20 weeks gestation if uterine artery Doppler scans are normal

Late pregnancy complications or first-trimester miscarriages despite aspirin

Low-dose aspirin from pre-conception plus LMW heparin (Dalteparin 5000 IU or Enoxaparin 40 mg once daily) from positive pregnancy test

Past venous thromboembolic events

Low-dose aspirin plus LMW heparin (Dalteparin 5,000 IU or Enoxaparin 40mg once daily) from positive pregnancy test, twice daily from 16–20 weeks gestation

Previous arterial or microvascular events

Low-dose aspirin plus LMW heparin (Dalteparin 5000 IU or Enoxaparin 40 mg twice a day). If symptoms are not controlled, consider warfarin therapy (INR 2.5) from the second trimester

aPL, antiphospholipid antibodies; INR, international normalized ratio; LMW, low molecular weight.

found similar live birth rates (~80%) using either lowdose aspirin or a placebo,32 suggesting that treatment may be unnecessary in some women. Although optimal treatment for women with one or more late pregnancy losses (second/third trimester) but no history of thromboembolism is controversial, most experts support the use of heparin therapy in addition to low-dose aspirin.27,28 IVIG has also been used during pregnancy, usually in conjunction with heparin and low-dose aspirin, especially in women with particularly poor past obstetric histories or recurrent pregnancy loss during heparin treatment.33 However, a randomized controlled pilot study of IVIG treatment during pregnancy in unselected APS cases found no benefit to this expensive therapy compared to heparin and low-dose aspirin.34 It is currently unclear whether IVIG may play a role in “refractory” cases. Therefore, it would seem prudent to limit its use to women with APS who have had pregnancy losses despite treatment with aspirin and heparin.35 For women with APS on warfarin because of previous thrombosis who want to become pregnant, it is important to note that there is a warfarin embryopathy, particularly on exposure of the fetus during weeks 6 to 12 of gestation. Hence, patients should be switched from

warfarin to subcutaneous heparin at an early enough time to ensure that there is no fetal exposure during that period. Some physicians prefer to switch before conception is attempted, whereas others do so as soon as pregnancy is determined.36,37 Heparin should be initiated at the time of warfarin cessation, and should be continued both intrapartum and postpartum until warfarin is reintroduced. Both warfarin and subcutaneous heparin are compatible with breast-feeding. Pregnancy complicated by APS requires expert care and a team approach by obstetricians and physicians. Close monitoring of both mother and fetus is essential. Ultrasound monitoring of fetal growth and uteroplacental blood flow is crucial. This allows for timely delivery. Some authorities use uterine artery waveforms at 20 and 24 weeks gestation, and those pregnancies with evidence of an early diastolic notch are monitored very closely with biweekly growth scans because of the high risk of intrauterine growth restriction and pre-eclampsia.38 When there are no notches, we recommend assessment of growth and amniotic fluid volume every 4 weeks. Doppler flow studies of the umbilical artery may be used, as in other pregnancies at high risk of fetal compromise through uteroplacental insufficiency.

REFERENCES

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1. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295-306. 2. Asherson RA, Cervera R, de Groot PG, et al. Catastrophic antiphospholipid syndrome: international consensus statement on classification criteria and treatment guidelines. Lupus 2003;12:530-534.

3. Mok CC, Tang SS, To CH, et al. Incidence and risk factors of thromboembolism in systemic lupus erythematosus: a comparison of three ethnic groups. Arthritis Rheum 2005;52:2774-2782. 4. Khamashta MA. Primary prevention of thrombosis in subjects with positive antiphospholipid antibodies. J Autoimmun 2000;15:249-253.

22. Meade TW, Miller GJ. Combined use of aspirin and warfarin in primary prevention of ischemic heart disease in men at high risk. Am J Cardiol 1995;75:23B-26B. 23. Rivier G, Khamashta MA, Hughes GRV. Warfarin and azathioprine: a drug interaction does exist. Am J Med 1993;95:342. 24. Cuadrado MJ, Mujic F, Munoz E, et al. Thrombocytopenia in the antiphospholipid syndrome. Ann Rheum Dis 1997;56: 194-196. 25. Krnic-Barrie S, CR OC, Looney SW, et al. A retrospective review of 61 patients with antiphospholipid syndrome. Analysis of factors influencing recurrent thrombosis. Arch Intern Med 1997;157:2101-2108. 26. Erkan D, Asherson RA, Espinosa G, et al. Long term outcome of catastrophic antiphospholipid syndrome survivors. Ann Rheum Dis 2003;62:530-533. 27. Branch DW, Khamashta MA. Antiphospholipid syndrome: obstetric diagnosis, management and controversies. Obstet Gynecol 2003;101:1333-1344. 28. Derksen RH, Khamashta MA, Branch DW. Management of the obstetric antiphospholipid syndrome. Arthritis Rheum 2004;50:1028-1039. 29. Kutteh WH. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low-dose aspirin is superior to low-dose aspirin alone. Am J Obstet Gynecol 1996;174:1584-1589. 30. Rai R, Cohen H, Dave M, et al. Randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies). BMJ 1997;314:253-257. 31. Farquharson RG, Quenby S, Greaves M. Antiphospholipid syndrome in pregnancy: a randomized, controlled trial of treatment. Obstet Gynecol 2002;100:408-413. 32. Pattison NS, Chamley LW, Birdsall M, et al. Does aspirin have a role in improving pregnancy outcome for women with the antiphospholipid syndrome? A randomized controlled trial. Am J Obstet Gynecol 2000;183:1008-1012. 33. Clark AL, Branch DW, Silver RM, et al. Pregnancy complicated by the antiphospholipid syndrome: outcomes with intravenous immunoglobulin therapy. Obstet Gynecol 1999;93:437-441. 34. Branch DW, Peaceman AM, Druzin M, et al. A multicenter, placebo-controlled pilot study of intravenous immune globulin treatment of antiphospholipid syndrome during pregnancy. The Pregnancy Loss Study Group. Am J Obstet Gynecol 2000;182:122-127. 35. Gordon C, Kilby MD. Use of intravenous immunoglobulin therapy in pregnancy in systemic lupus erythematosus and antiphospholipid antibody syndrome. Lupus 1998;7:429-433. 36. Hunt BJ, Gattens M, Khamashta M, et al. Thromboprophylaxis with unmonitored intermediate-dose low molecular weight heparin in pregnancies with a previous arterial or venous thrombotic event. Blood Coagul Fibrinolysis 2003;14:735-739. 37. Ruiz-Irastorza G, Khamashta MA. Management of thrombosis in antiphospholipid syndrome and systemic lupus erythematosus in pregnancy. Ann N Y Acad Sci 2005;1051:606-612. 38. Stone S, Hunt BJ, Khamashta MA, et al. Primary antiphospholipid syndrome in pregnancy: an analysis of outcome in a cohort of 33 women treated with a rigorous protocol. J Thromb Haemost 2005;3:243-245.

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5. Ginsburg KS, Liang MH, Newcomer L, et al. Anticardiolipin antibodies and the risk for ischemic stroke and venous thrombosis. Ann Intern Med 1992;117:997-1002. 6. Petri M. Hydroxychloroquine use in the Baltimore Lupus Cohort: effects on lipids, glucose and thrombosis. Lupus 1996;5(Suppl 1): S16-S22. 7. Erkan D, Yazici Y, Peterson MG, et al. A cross-sectional study of clinical thrombotic risk factors and preventive treatments in antiphospholipid syndrome. Rheumatology (Oxford) 2002;41:924-929. 8. Julkunen H, Hedman C, Kauppi M. Thrombolysis for acute ischemic stroke in the primary antiphospholipid syndrome. J Rheumatol 1997;24:181-183. 9. Khamashta MA, Cuadrado MJ, Mujic F, et al. The management of thrombosis in the antiphospholipid-antibody syndrome. N Engl J Med 1995;332:993-997. 10. Schulman S, Svenungsson E, Granqvist S. Anticardiolipin antibodies predict early recurrence of thromboembolism and death among patients with venous thromboembolism following anticoagulant therapy. Duration of Anticoagulation Study Group. Am J Med 1998;104:332-338. 11. Rosove MH, Brewer PM. Antiphospholipid thrombosis: clinical course after the first thrombotic event in 70 patients. Ann Intern Med 1992;117:303-308. 12. Crowther MA, Ginsberg JS, Julian J, et al. A comparison of two intensities of warfarin for the prevention of recurrent thrombosis in patients with the antiphospholipid antibody syndrome. N Engl J Med 2003;349:1133-1138. 13. Finazzi G, Marchioli R, Brancaccio V, et al. A randomized clinical trial of high-intensity warfarin vs. conventional antithrombotic therapy for the prevention of recurrent thrombosis in patients with the antiphospholipid syndrome (WAPS). J Thromb Haemost 2005;3:848-853. 14. Lim W, Crowther MA, Eikelboom JW. Management of antiphospholipid antibody syndrome: a systematic review. JAMA 2006;295:1050-1057. 15. Ruiz-Irastorza G, Khamashta MA, Hunt BJ, et al. Bleeding and recurrent thrombosis in definite antiphospholipid syndrome: analysis of a series of 66 patients treated with oral anticoagulation to a target international normalized ratio of 3.5. Arch Intern Med 2002;162:1164-1169. 16. Ruiz-Irastorza G, Khamashta MA. Stroke and antiphospholipid syndrome: the treatment debate. Rheumatology (Oxford) 2005;44:971-974. 17. Khamashta MA, Hunt BJ. Moderate dose oral anticoagulant therapy in patients with the antiphospholipid syndrome? No. J Thromb Haemost 2005;3:844-845. 18. Moll S, Ortel TL. Monitoring warfarin therapy in patients with lupus anticoagulants. Ann Intern Med 1997;127:177-185. 19. Lawrie AS, Purdy G, Mackie IJ, et al. Monitoring of oral anticoagulant therapy in lupus anticoagulant positive patients with the anti-phospholipid syndrome. Br J Haematol 1997;98:887-892. 20. Heneghan C, Alonso-Coello P, Garcia-Alamino JM, et al. Selfmonitoring of oral anticoagulation: a systematic review and meta-analysis. Lancet 2006;367:404-411. 21. Cromheecke ME, Levi M, Colly LP, et al. Oral anticoagulation selfmanagement and management by a specialist anticoagulation clinic: a randomised cross-over comparison. Lancet 2000;356: 97-102.

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48

New Treatments in Systemic Lupus Erythematosus Vasileios C. Kyttaris, MD and George C. Tsokos, MD

INTRODUCTION Immunosuppressive therapy, the main treatment modality for severe lupus, has been effective in reducing some of the sequelae of systemic lupus erythematosus (SLE), such as end-stage renal disease, but has not proven uniformly effective in reducing SLE-associated morbidity and mortality. Furthermore, the medications used currently are universally immunosuppressive, thus increasing the risk of serious infections, and at the same time have a variety of toxicities in tissues and organs outside the immune system.1-3 When designing a therapeutic regimen that will be both effective and safe, key pathophysiologic steps in the development of autoimmunity, perpetuation of the abnormal immune response and immune-mediated tissue damage need to be recognized and targeted in a highly specific manner. In the case of SLE, the precise sequence of events that lead to the break in self-tolerance and the development of autoimmunity remains elusive, but recent studies have shed light on pathophysiologic processes that are important in the continuation of the abnormal immune response and ensuing tissue damage. Researchers have capitalized on these findings and designed novel therapeutic approaches using a variety of antibodies, fusion molecules, small chemical compounds, and gene therapy techniques. These novel therapies discussed herein (Table 48.1), have been tried in ex vivo experiments, and in murine models of lupus as well as in patients with SLE with encouraging albeit preliminary results.

TARGETING SOLUBLE MEDIATORS

516

The successful use of tumor necrosis factor a (TNF-a) inhibitors in rheumatoid arthritis4 and Crohn’s disease speaks to the fact that the disruption of signaling among immune cells can lead to significant clinical improvement of autoimmune diseases without necessarily re-establishing tolerance. The recognition of such

pivotal cytokines in SLE may lead to the design of medications that can induce remission and prevent flares.

B-Lymphocyte Stimulator B-lymphocyte stimulator (BLyS) is a soluble molecule that belongs to the TNF ligand superfamily,5 and is secreted primarily by myeloid lineage cells such as monocytes and neutrophils. It binds exclusively to B cells via one of three surface receptors (BAFFR, BCDMA, or TACI),6 leading to activation of the nuclear factor (NF)-κB pathway, and by up-regulating survival molecules leads to increased B-cell survival.7,8 Several studies have linked BLyS to autoimmunity, and specifically to overactivation of humoral immunity with the production of autoantibodies. Initially, it was shown that mice genetically engineered to overproduce BLyS9 developed B-cell hyperplasia, hypergammaglobulinemia, and an array of autoantibodies including anti–double-stranded DNA antibodies, and had immunoglobulin deposition in their kidneys. Thus, these BLyS-transgenic mice developed a condition reminiscent of human SLE. It was thereafter shown that the levels of BLyS in lupus-prone mice (both NZB/NZW F1 and MRL-lpr/lpr) are elevated at the onset of disease,10 establishing BLyS as a potential therapeutic target. Indeed, a fusion molecule of TACI (the BLyS receptor) with the Fc portion of the immunoglobulin blocked the effects of BLyS in NZB/NZW F1 mice, resulting in a decrease in proteinuria and increased survival.10 Similar clinical improvement was also shown in a rheumatoid arthritis murine model with the use of TACI-Ig,11 pointing to the fact that BLyS plays a significant but not disease-specific role in autoimmunity. Carrying these observations to humans, the levels of BLyS were shown to be persistently increased in 25% of patients with SLE while another 25% of the patients had intermittent elevations12,13 of BLyS in their peripheral blood. In addition, BLyS levels showed a strong correlation with anti-dsDNA levels,14,15 further underscoring

Treatment

Molecule Used

Mode of Action

Status

Soluble Mediators

Anti–B-lymphocyte stimulator (BLyS)

Monoclonal antibody

Blocks BLyS-mediated increased B-cell survival

Successful animal trials. Safety established in phase I trial. Ongoing phase II trial

Anti-Interleukin6 receptor (IL-6R)

Monoclonal antibody

Blocks proinflammatory effects of IL-6 on B and T cells

Effective in murine lupus. Human phase I trial is ongoing

Anti-Interferon (IFN)

Receptor coupled to Ig

Blocks IFN effect

Successful anti–IFN-γ treatment in murine lupus. IFN-α may be a better target in humans

Anti-C5

Monoclonal antibody

Blocks activation of terminal complement components

Successful in mice. Effective and safe in patients with PNH

TARGETING SOLUBLE MEDIATORS

TABLE 48.1 NEW TREATMENTS FOR SYSTEMIC LUPUS ERYTHEMATOSUS

Cell Surface Molecules

Anti-CD20

Chimeric Antibody

B cell depletion

Phase II/III trial in patients with SLE is ongoing

Anti-CD22

Humanized antibody

Modulation of B cell signaling

Safe in phase I in SLE patients. Phase II is ongoing

Anti-CD40L

Monoclonal antibody

Blocks T:B cell cross-talk

Trials on halt due to thromboembolic side effects

LJP 394

Chemical compound

Blocks the production of anti-dsDNA antibodies

Some effect on quality of life; better effect on patients with high levels of anti-dsDNA antibodies

CTLA-4 Ig

Receptor coupled to Ig

Blocks co-stimulation of T cells

Effective in mice Ongoing phase I/IIA trial in SLE

Intracellular Molecules

T-cell receptor ζ chain

Sense plasmid

Normalizes calcium responses in T cells; increases IL-2 production

Ex vivo evidence

CaMKIV

Dominant negative plasmid

Increases IL-2 production by T cells

Ex vivo evidence

CREM

Anti sense plasmid

Increases IL-2 production by T cells

Ex vivo evidence

the potential importance of BLyS in the pathophysiology of human SLE. Both murine and human trials led to the proposal that blocking BLyS can produce clinical improvement of patients with SLE and may decrease the need for corticosteroids and other immunosuppressive drugs. A phase I trial using a monoclonal antibody against BLyS (BLySmAb) was successfully completed in patients with SLE. BLySmAb was no different than the placebo in both effectiveness and side effects, but given the limited time (one or two infusions of BLySmAb vs. placebo) of treatment, the results of the larger and longer phase II trial that is underway are expected to be more informative.12 This trial recruited patients with mild to

moderately active SLE and at least history of antidsDNA antibody positivity; if positive, this trial may prove BLySmAb to be an important medication for maintenance therapy and prevention of flares in SLE. In addition to BLySmAb, fusion molecules of the receptors TACI and BAFFR with immunoglobulin (TACI-Ig and BAFFR-Ig) are being assessed in normal individuals and primates, respectively. Furthermore, in a study using gene therapy techniques, MRL lpr/lpr mice were transfected with an adenovirus encoding TACI-Fc (a fusion molecule between TACI and the Fc portion of Ig). The transfected mice had milder nephritis and increased survival when compared to controls.16 All of these approaches to BLyS antagonism

517

NEW TREATMENTS IN SYSTEMIC LUPUS ERYTHEMATOSUS

can help us to elucidate the role of BLyS in the pathophysiology of SLE and ultimately to provide a useful medication in the management of SLE.

Interleukin-6 (IL-6) IL-6 is a pleiotropic proinflammatory cytokine that is mainly secreted by monocytes.17 IL-6 binds to the IL-6 receptor (IL-6R) on the surface of cells. In addition, IL-6 can bind to soluble IL-6R, and then the IL-6:IL-6R complex may directly activate cells. IL-6 promotes B-cell maturation and T-cell differentiation, while at the same time synergizes with TNF-a and IL-1 to promote systemic inflammatory response.18 In murine models of SLE, IL-6 has been shown to be increased and to contribute to autoantibody production. IL-6 was effectively blocked in NZB/NZW F1 mice using either an anti–IL-6 antibody19 or an anti–IL-6R antibody.20 Blocking IL-6 led to decreased anti-dsDNA antibodies, decreased proteinuria, and improved survival. This evidence from murine lupus led to the hypothesis that IL-6 neutralization may prove to be an important therapeutic option for patients with SLE. IL-6 levels are elevated in patients with SLE,21,22 and may contribute to immunoglobulin production. Neutralization of IL-6 in vitro decreases the spontaneous secretion of immunoglobulin by lupus B cells while exogenous IL-6 increases it.23 IL-6 may also play a significant role locally in the kidneys in lupus nephritis, as exhibited by increased IL-6 levels in urine of patients with active lupus nephritis24 and in situ expression of IL-6 in the glomeruli of patients with lupus nephritis.25 The anti–IL-6R monoclonal antibody, MRA, is currently being tested in phase I clinical trials in patients with SLE. MRA has been proven to be efficacious and in the lymphoproliferative Castleman’s disease and rheumatoid arthritis,26,27 making it an attractive therapeutic option for SLE as well.

Interferon

518

One of the earliest molecules to be targeted in the MRL lpr/lpr mouse was interferon (IFN)-α, a cytokine produced in large amounts by activated T cells. IFN- levels are high in the areas of inflammation in the MRL lpr/lpr mice, and play a significant role in the pathophysiology of murine lupus.28 For these reasons, IFN-α was specifically targeted using, among other techniques, gene therapy. A plasmid that encodes the receptor of IFN- (IFN-R) fused to the Fc portion of IgG1 was injected in mice leading to the production of a molecule that effectively blocks IFN-.29,30 MRL lpr/lpr mice transfected with this construct before disease onset were protected against early death, and had lower levels of autoantibodies and milder renal disease. Interestingly, initiation of treatment as late as 4 months after the onset of disease led to significant disease improvement.

In addition to the important findings in murine lupus models, IFNs received more attention recently after the finding that IFN-α-inducible genes were “turned on” in peripheral blood mononuclear cells (PBMCs) from patients with SLE.24 This “interferon signature” in gene expression in SLE was primarily seen in active lupus patients who also tended to have more severe disease manifestations such as brain and kidney involvement. In contrast to the mice models though, IFN-α and not IFN-α25 was found to be the instigating factor for the “interferon signature” in SLE. More specifically, the levels of mRNA transcribed from genes that are inducible by IFN-α but not IFN were significantly higher in SLE compared to control PBMC. Furthermore plasma from patients with SLE up-regulates IFN-α inducible genes in normal PBMCs. Despite these very intriguing findings, direct measurement of IFN-α in the serum of patients with SLE did not show any difference between patients with SLE and controls, pointing to the fact that other factors maybe mimicking the IFN effect in SLE. If indeed IFN-α is the culprit of these aberrations, it may prove important in the perpetuation of the autoimmune response in SLE because IFN-α is produced by antigen-presenting cells upon activation by immune complexes containing autoantibodies and apoptotic material.31 In turn, IFN-α further enhances the antigen-presenting function of monocytes,32 and activates T and B cells, and thus helps the vicious cycle of immune dysregulation in SLE.33 Further studies are clearly needed to establish the precise role of IFN-α in lupus, and then whether it can be specifically targeted directly or indirectly via its intracellular signaling pathway (e.g., STAT-1).

Tumor Necrosis Factor-α The use of TNF-α inhibitors has revolutionized the treatment of rheumatoid arthritis,4 but their use in SLE remains controversial. Patients without history of SLE have developed anti-dsDNA antibodies and even clinical lupus upon treatment with TNF-α inhibitors, and so it would seem counterintuitive to use such drugs in SLE. Furthermore, low overall production of TNF-α may be contributing to the decrease in the activationinduced cell death34 that is characteristic of SLE, while local production of TNF-α due to environmental factors (infection, UV light) may be contributing to triggering disease exacerbations. In particular, UVB—a known cause of disease exacerbation—is shown to trigger TNF-α production in the skin.35 In turn, TNF-α has been shown to up-regulate the expression of the 52kd Ro/SSA protein and mRNA in keratinocytes.36 Since 52kd Ro/SSA protein is expressed on apoptotic bodies, and anti-Ro antibodies have been associated with cutaneous lupus, these observations provide a link between UV-induced apoptosis and TNF-α production

Complement System The immune dysregulation in SLE leads to the activation of the complement system, cleavage of C5, and production of the potent proinflammatory mediators C5a and C5b-9.38,39 The terminal complement components are found deposited in various tissues such as the kidney and the skin, where complement can cause direct damage and also attract neutrophils through the anaphylatoxic action of C5a. A monoclonal antibody that prevents the cleavage of C5 and used in NZBxNZW F1 mice resulted in delayed onset of proteinuria and improved survival.40 Furthermore, a similar anti-C5 monoclonal antibody (eculizumab) was proven safe and effective for the treatment of paroxysmal nocturnal hemoglobinemia.41 A clinical trial of eculizumab in patients with SLE, especially patients with active nephritis, is thus needed to assess its usefulness in the management of SLE.

TARGETING CELL–SURFACE MOLECULES AND CELL–CELL INTERACTION Both B and T cells are targeted in SLE in an attempt to disrupt the cycle of immune-cell activation via upregulated or missing cell-surface signaling molecules.

Anti-CD20 It is almost intuitive to target B cells in SLE given the several lines of evidence that B cells play a pivotal role in the pathophysiology of SLE. Not only do hyperactive B cells produce a variety of autoantibodies, but they also produce proinflammatory cytokines and serve as antigen-presenting cells.42 A medication that can efficiently deplete B cells can therefore prove effective in the treatment of SLE. Rituximab, an anti-CD20 chimeric antibody that targets and depletes B cells but not plasma cells, and has a proven efficacy against lymphoma43 and rheumatoid arthritis,44 was used in patients with difficult-to-manage SLE. Several small phase I/II studies and case series have shown promising results with as many as 80% of patients with lupus nephritis achieving at least partial remission.45-50 The use of concomitant cyclophosphamide, various corticosteroid-tapering schedules, and various rituximab doses in these studies makes it difficult to ascertain the exact role that rituximab may play in the treatment of SLE. Furthermore, the high percentage of human

antichimeric antibodies (HACA) reported in SLE,50 as compared to those reported in patients with lymphoma or rheumatoid arthritis, may lower the efficacy of the drug over time or increase infusion reactions. There are also concerns as to what the effect of unpredictable and prolonged B-cell depletion will have on the ability of the already immunocompromised lupus patients to fight infectious agents. Currently underway is a large, double-blind, placebo control phase II/III trial, focused specifically on the effectiveness of rituximab in moderate to severe SLE (patients with active glomerulonephritis are excluded).

Anti-CD22 Given some of the concerns with anti-CD20 treatment such as prolonged B-cell depletion and development of HACA, other approaches to B-cell depletion are also being explored. CD22 is a molecule expressed on the surface of mature B cells and not plasma cells or memory B cells, and is involved in the regulation of signal transduction via the B-cell receptor and the adhesion of B cells to other immune cells via its ligand CD22L. CD22L is increased in circulating B and T cells in lupus-prone mice, and its expression paralleled the development of clinical disease.51 A humanized anti-CD22 antibody (epratuzumab) is currently undergoing phase II trial in patients with moderately active SLE after an initial phase I trial showed a favorable safety profile with minimal immunogenicity of the compound and only moderate decrease in the number of circulating B cells. Furthermore, the fact that epratuzumab is fully humanized and causes moderate B-cell depletion may make this a medication of choice for patients who are intolerant of rituximab.

TARGETING CELL–SURFACE MOLECULES AND CELL–CELL INTERACTION

with anti-Ro–associated cutaneous lupus. Further strengthening this argument, TNF-α has been found up-regulated in the skin of patients with refractory subacute cutaneous lupus.37 Taking these studies together, TNF-α neutralization may prove of benefit in the treatment of patients with certain manifestations of SLE such as severe cutaneous lupus.

LJP-394 Anti-dsDNA antibodies are implicated in the pathogenesis of SLE, and their levels correlate with disease activity in select patients.52 LJP-394 (abetimus sodium) is an artificial compound made of four deoxynucleotide-like molecules bound together and thus resembles ds-DNA, and at least in theory can bind to proteins that also bind ds-DNA. LJP-394 injection in murine models of lupus resulted in the decrease in dsDNA antibody levels. Phase II/III clinical trials in humans have not resulted in similar success.53-55 Although anti-dsDNA antibody titers were decreased in patients treated with LJP-394, the clinical effectiveness of the drug was not as impressive. Patients on LJP-394 showed improved quality-of-life measures and a trend towards improved disease control with longer duration to renal flare. If approved, LJP-394 would be a helpful add-on to the current treatment of patients with SLE and especially those with high-titer, high-affinity anti-dsDNA antibodies.

519

NEW TREATMENTS IN SYSTEMIC LUPUS ERYTHEMATOSUS

Anti-CD40L Although B cells can be activated in a T-cell independent manner, a significant line of evidence exists that T cells are important for the hyperactive B cells in SLE.56-58 The cognate interaction between B and T cells is potentated via the CD40 ligand (on T cells)–CD40 (on B cells) pair of co-stimulatory molecules. Studies in lupus-prone mice showed that anti-CD40L treatment can lead to significant decreases in pathogenic autoantibody titers and improvement in nephritis and survival of the treated mice.56-58 Two different antibodies against CD40L were tried in humans.59 The first one (BG9588) led to a decrease in anti-dsDNA antibody production, an increase in C3 levels, and decreased hematuria in treated patients.60 Despite these preliminary encouraging results, the trial ended prematurely because of increased thromboembolic events (myocardial infarctions) in the treated patients; binding of the anti-CD40L to the CD40Lexpressing platelets may be responsible for this side effect. The second antibody (IDEC-131) proved ineffective in preliminary trials in SLE,61 and was also linked to a thromboembolic event in a Crohn’s patient,59 leading to a halt in further clinical trials with both antiCD40L antibodies.

CTLA-4 Ig Another co-stimulatory pair that is important for the initiation of the immune response is CD28 (on T cells)–CD80/86 (on antigen-presenting cells). CTLA4 is a molecule that is up-regulated on the surface of T cells following activation, and has a much higher affinity to CD80/86 than CD28. It therefore disrupts the CD28–CD80/86 interaction, and effectively ends T-cell activation. In order to disrupt co-stimulation, a fusion molecule of CTLA4 with Ig was constructed. The rationale behind the manufacturing of such a drug is that soluble CTLA-4 with its high affinity for CD80/86 on antigen-presenting cells will prevent the CD28–CD80/86 interaction and thus prevent T-cell activation. This fusion molecule was injected in NZBxNZW F1 mice and proved to be efficacious in blocking autoantibody production and prolonging survival.19 A human analogue of the CTLA4-Ig fusion molecule (called abatacept) was shown to be effective in the treatment of rheumatoid arthritis.62 Abatacept is currently in phase I/IIA trial in SLE patients.

CORRECTING INTRACELLULAR SIGNALING ABERRATIONS 520

The T cells play an important role in the dysregulated immune response that characterizes SLE. T cells in SLE exhibit increased spontaneous apoptosis, and at the same

time impaired activation and activation-induced cell death (AICD).34,63 This distinct lupus T-cell phenotype leads to a predisposition to viral infections,64 while facilitating the production of immunoglobulin by B cells.65 These properties of T cells in SLE are directly linked to an array of signaling aberrations; in particular, the lupus T cell exhibits an “overexcitable” phenotype of early and heightened calcium response and tyrosine phosphorylation upon activation, partly due to the substitution of FcR for chain on the T-cell receptor (TCR).66-68 These abnormally active early events in the stimulation of the T cell do not translate though to the production of the signature T-cell cytokine IL-2, a pivotal cytokine for T-cell growth, proliferation, and eventual death. An imbalanced mobilization of transcription factors to the promoter of the IL-2 gene is central in this paradox.69,70 T cells from patients with SLE but not from normal individuals express in the nucleus the calcium/calmodulin-dependent kinase IV (CaMKIV) that helps recruit the transcriptional repressor c-AMP response element modulator (CREM) to the promoter of the IL-2 gene.71 CREM binding to the IL-2 promoter leads to decreased expression of IL-2.72 CREM also limits the transcription of c-fos, a component of the activator protein-1 (AP-1, a c-fos–c-jun heterodimer), a transcriptional enhancer of IL-2.70 Ex vivo experiments have shown that targeting these well-described signaling abnormalities using gene therapy techniques can indeed restore the production of IL-2 by lupus T cells. Initially, T cells from patients with SLE were transfected with a plasmid encoding the TCR chain. The transfected lupus T cells expressed nearly normal levels of ζ chain, down-regulated the FcRγ chain, decreased calcium responses, and interestingly, higher production of IL-2 upon activation.73 Furthermore, the same technique was used to transfect lupus T cells with a plasmid encoding a dominant negative form of CaMKIV that effectively blocks the endogenous enzyme; this led to decreased CREM binding to the IL-2 promoter and increased IL-2 production.71 In a different approach, CREM translation in lupus T cells was blocked using an antisense CREM vector. This resulted in increased production of IL-2 following activation of the cell.74,75 These experiments demonstrate the feasibility of ex vivo use of sense, antisense, and dominant negative forms of key signaling molecules to alter their function and correct their downstream effects. One can speculate that upon using existent leukapheresis protocols, T cells from patients with SLE could be ex vivo transfected and re-infused in the donor patient, resulting in improved regulation of the immune response.

ENGINEERED CELLS Regulatory T cells (Treg) have received much attention in recent years because of their ability to suppress

CONCLUSIONS At the present time, nonspecific immunosuppression remains the treatment of choice for the induction of remission in SLE, while antimalarials and low-dose immunosuppressives are used to maintain remission. New medications are already in the pipeline that will provide options for the treatment of lupus. One can speculate that targeting B cells, T- and B-cell interaction, and/or co-stimulatory pathways will prove efficacious in the induction of remission of lupus. At the same time, targeting BLyS-mediated B-cell activation or efforts to reinstate tolerance in the T-cell compartment by using small molecules and/or gene therapy techniques may help maintain remission. Finally, certain manifestations of SLE such as refractory skin disease and nephritis may be successfully treated with medications such as TNA-a antagonists and anti–IL-6R antibody, respectively. In conclusion, it is apparent that although no new drugs have been approved for SLE for over three decades, uncovering pathophysiologic pathways and designing novel approaches to correct them will eventually lead to the development of safe and effective medications that will revolutionize treatment of SLE.

REFERENCES

the immune response in an antigen-specific manner. In theory, autoantigen-specific Treg engineered in vitro could be used to suppress the immune response against this particular autoantigen without being globally immunosuppressive. In the case of SLE, this approach could use autoantigens of nuclear origin that are thought to be involved in the expression of the disease. This approach was proven feasible and effective in the NZBxNZW F1 murine lupus model.76 Initially, murine CD4+ T cells were engineered to encoded alpha and beta chains of TCR specific for nucleosomal autoantigens (AN3 cells). A gene encoding CTLA4-Ig was co-transfected with the nucleosome-specific TCR, creating AN3 cells that were specific for nucleosome autoantigens and expressed the immunomodulatory CTLA4 molecule on their surface. These AN3/CTLA4 cells acted as functional Treg analogues by specifically blocking the presentation of nucleosomal autoantigens by antigen-presenting cells through the action of CTLA4, and thus preventing the antinucleosomal immune response. When injected in NZBxNZW F1, these AN3/CTLA4 cells led to a significant amelioration of clinical nephritis and a nonstatistically significant decrease in antinucleosomal autoantibodies. This preliminary study paves the way for further testing of Treg as a therapeutic tool in human SLE.

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47. Tokunaga M, Fujii K, Saito K, et al. Down-regulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab. Rheumatology (Oxford) 2005;44:176-182. 48. Saito K, Nawata M, Nakayamada S, et al. Successful treatment with anti-CD20 monoclonal antibody (rituximab) of life-threatening refractory systemic lupus erythematosus with renal and central nervous system involvement. Lupus 2003;12:798-800. 49. Gottenberg JE, Guillevin L, Lambotte O, et al. Tolerance and short term efficacy of rituximab in 43 patients with systemic autoimmune diseases. Ann Rheum Dis 2005;64:913-920. 50. Looney RJ, Anolik JH, Campbell D, et al. B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/II doseescalation trial of rituximab. Arthritis Rheum 2004;50:2580-2589. 51. Lajaunias F, Ida A, Kikuchi S, et al. Differential control of CD22 ligand expression on B and T lymphocytes, and enhanced expression in murine systemic lupus. Arthritis Rheum 2003;48:1612-1621. 52. Boumpas DT, Austin HA, III, Fessler BJ, et al. Systemic lupus erythematosus. emerging concepts. Part 1. Renal, neuropsychiatric, cardiovascular, pulmonary, and hematologic disease. Ann Intern Med 1995;122:940-950. 53. Wallace DJ, Tumlin JA. LJP 394 (abetimus sodium, Riquent) in the management of systemic lupus erythematosus. Lupus 2004;13:323-327. 54. Strand V, Aranow C, Cardiel MH, et al. Improvement in healthrelated quality of life in systemic lupus erythematosus patients enrolled in a randomized clinical trial comparing LJP 394 treatment with placebo. Lupus 2003;12:677-686. 55. Alarcon-Segovia D, Tumlin JA, Furie RA, et al. LJP 394 for the prevention of renal flare in patients with systemic lupus erythematosus. results from a randomized, double-blind, placebo-controlled study. Arthritis Rheum 2003;48:442-454. 56. Kalled SL, Cutler AH, Datta SK, Thomas DW. Anti-CD40 ligand antibody treatment of SNF1 mice with established nephritis. Preservation of kidney function. J Immunol 1998;160:2158-2165. 57. Daikh DI, Finck BK, Linsley PS, et al. Long-term inhibition of murine lupus by brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways. J Immunol 1997;159:3104-3108. 58. Early GS, Zhao W, Burns CM. Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand black x New Zealand white mice. Response correlates with the absence of an anti-antibody response. J Immunol 1996;157:3159-3164. 59. Sidiropoulos PI, Boumpas DT. Lessons learned from anti-CD40L treatment in systemic lupus erythematosus patients. Lupus 2004;13:391-397. 60. Boumpas DT, Furie R, Manzi S, et al. A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum 2003;48:719-727. 61. Kalunian KC, Davis JC Jr, Merrill JT, et al. Treatment of systemic lupus erythematosus by inhibition of T cell costimulation with anti-CD154. a randomized, double-blind, placebo-controlled trial. Arthritis Rheum 2002;46:3251-3258. 62. Genovese MC, Becker JC, Schiff M, et al. Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition. N Engl J Med 2005;353:1114-1123. 63. Emlen W, Niebur J, Kadera R. Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J Immunol 1994;152:3685-3692. 64. Iliopoulos AG, Tsokos GC. Immunopathogenesis and spectrum of infections in systemic lupus erythematosus. Semin Arthritis Rheum 1996;25:318-336. 65. Inghirami G, Simon J, Balow JE, Tsokos GC. Activated T lymphocytes in the perirpheral blood of patients with systemic lupus erythematosus induce B cells to produce immunoglobulin. Clin Exp Rheumatol 1988;6:269-276. 66. Liossis SN, Ding XZ, Dennis GJ, Tsokos GC. Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus. Deficient expression of the T cell receptor zeta chain. J Clin Invest 1998;101:1448-1457. 67. Liossis SN, Sfikakis PP, Tsokos GC. Immune cell signaling aberrations in human lupus. Immunol Res 1998;18:27-39.

73. Nambiar MP, Fisher CU, Warke VG, et al. Reconstitution of deficient T cell receptor zeta chain restores T cell signaling and augments T cell receptor/CD3-induced interleukin-2 production in patients with systemic lupus erythematosus. Arthritis Rheum 2003;48:1948-1955. 74. Tenbrock K, Juang YT, Tolnay M, Tsokos GC. The cyclic adenosine 5’-monophosphate response element modulator suppresses IL-2 production in stimulated T cells by a chromatin-dependent mechanism. J Immunol 2003;170:2971-2976. 75. Tenbrock K, Juang YT, Gourley MF, et al. Antisense cyclic adenosine 5’-monophosphate response element modulator up-regulates IL-2 in T cells from patients with systemic lupus erythematosus. J Immunol 2002;169:4147-4152. 76. Fujio K, Okamoto A, Tahara H, et al. Nucleosome-specific regulatory T cells engineered by triple gene transfer suppress a systemic autoimmune disease. J Immunol 2004;173:2118-2125.

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APPENDIX A

SYSTEMIC LUPUS ERYTHEMATOSUS DISEASE ACTIVITY INDEX SLEDAI-2000 FORM Name/ID: _________________________ Date of Birth: ____________ Date of Assessment: ____________ (Circle in “SLEDAI Score” column if descriptor is present at the time of the visit or in the preceding 10 days.) SLEDAI Score

Descriptor

Definition

8

Seizure

Recent onset. Exclude metabolic, infectious, or drug causes.

8

Psychosis

Altered ability to function in normal activity due to severe disturbance in the perception of reality. Include hallucinations, incoherence, marked loose associations, impoverished thought content, marked illogical thinking, and bizarre, disorganized, or catatonic behavior. Exclude uremia and drug causes.

8

Organic brain syndrome

Altered mental function with impaired orientation, memory, or other intellectual function (with rapid onset and fluctuating clinical features), inability to sustain attention to environment, and at least two (2) of the following: perceptual disturbance, incoherent speech, insomnia or daytime drowsiness, and increased or decreased psychomotor activity. Exclude metabolic, infectious, or drug causes.

8

Visual disturbance

Retinal changes of SLE. Include cytoid bodies, retinal hemorrhages, serous exudates or hemorrhages in the choroid, and optic neuritis. Exclude hypertensive, infectious, or drug causes.

8

Cranial nerve disorder

New onset of sensory or motor neuropathy involving cranial nerves.

8

Lupus headache

Severe, persistent headache. May be migrainous, but must be nonresponsive to narcotic analgesia.

8

CVA

New onset cerebrovascular accident(s). Exclude arteriosclerosis.

8

Vasculitis

Ulceration, gangrene, tender finger nodules, periungual infarction, splinter hemorrhages or biopsy, and angiogram proof of vasculitis.

4

Arthritis

≥2 joints with pain and signs of inflammation (i.e., tenderness, swelling, or effusion).

4

Myositis

Proximal muscle aching/weakness associated with elevated creatinine phosphokinase (CK)/aldolase or EMG changes or a biopsy showing myositis.

4

Urinary casts

Heme-granular or RBC casts.

4

Hematuria

>5 RBC/high-power field. Exclude stone, infection, or other cause.

4

Proteinuria

>0.5 gram/24 hours.

4

Pyuria

>5 WBC/high-power field. Exclude infection.

2

Rash

Inflammatory-type rash.

2

Alopecia

Abnormal, patchy, or diffuse loss of hair.

2

Mucosal ulcers

Oral or nasal ulcerations.

2

Pleurisy

Pleuritic chest pain with pleural rub or effusion, or pleural thickening.

2

Pericarditis

Pericardial pain with at least one (1) of the following: rub, effusion, or ECG or echocardiogram confirmation.

2

Low complement

Decrease in CH50, C3, or C4 below lower limit of normal for testing laboratory.

2

Increased DNA binding

Increased DNA binding above normal range for testing laboratory.

1

Fever

>38° C. Exclude infectious cause.

1

Thrombocytopenia

<100 x 109 platelets/L. Exclude drug causes.

1

Leukopenia

<3 x 109 WBC/L. Exclude drug causes.

524

TOTAL SCORE:

PHYSICIAN’S GLOBAL ASSESSMENT

SELENA SLEDAI

0

1

2

3

None

Mild

Moderate

Severe

APPENDIX B

SELENA-SLEDAI FLARE TOOL

MODIFIED SLEDAI SCORE FORM FOR SELENA-SLEDAI FLARE TOOL Weight

X

Descriptor

Definition

8

❒

Seizure

8

❒

Psychosis

8

❒

Organic brain syndrome

8

❒

Visual disturbance

8

❒

8

❒

8

❒

Cranial nerve disorder Lupus headache CVA

8

❒

Vasculitis

4

❒

Arthritis

4

❒

Myositis

4 4 4 4 2 2 2 2

❒ ❒ ❒ ❒ ❒ ❒ ❒ ❒

Urinary Casts Hematuria Proteinuria Pyuria Rash Alopecia Mucosal ulcers Pleurisy

2 2

❒ ❒

2 1 1

❒ ❒ ❒

1

❒

Pericarditis Low complement Anti-dsDNA Fever Thrombocytopenia Leukopenia

Recent onset (last 10 days). Exclude metabolic, infectious, or drug causes, or seizure due to past irreversible CNS damage. Altered ability to function in normal activity due to severe disturbance in the perception of reality. Include hallucinations, incoherence, marked loose associations, impoverished thought content, marked illogical thinking, and bizarre, disorganized, or catatonic behavior. Exclude uremia and drug causes. Altered mental function with impaired orientation, memory, or other intellectual function, with rapid onset and fluctuating clinical features. Include clouding of consciousness with reduced capacity to focus and inability to sustain attention to environment, plus at least two of the following: perceptual disturbance, incoherent speech, insomnia or daytime drowsiness, and increased or decreased psychomotor activity. Exclude metabolic, infectious, or drug causes. Retinal and eye changes of SLE. Include cytoid bodies, retinal hemorrhages, serous exudates, or hemorrhages in the choroid, optic neuritis, scleritis, or episcleritis. Exclude hypertension, infectious, or drug causes. New onset of sensory or motor neuropathy involving cranial nerves. Include vertigo due to lupus. Severe, persistent headache (may be migrainous, but must be nonresponsive to narcotic analgesia). New onset cerebrovascular accident(s). Exclude arteriosclerosis or hypertensive causes. Ulceration, gangrene, tender finger nodules, periungual infarction, splinter hemorrhages, or biopsy or angiogram proof of vasculitis. >2 joints with pain plus signs of inflammation (tenderness, swelling, or effusion). Proximal muscle aching/weakness associated with elevated CPK or EMG changes, or a biopsy showing myositis. Heme-granular or red blood cell casts. >5 RBCs/HPF. Exclude stone, infectious, or other causes. New onset or recent increase of more than 0.5 gm/24 h. >5 WBC/HPF. Exclude infection. Ongoing inflammatory lupus rash. Ongoing abnormal, patchy, or diffuse loss of hair due to active lupus. Ongoing oral or nasal ulcerations due to active lupus. Classic and severe pleuritic chest pain or plural rub or effusion, or new pleural thickening due to lupus. Classic and severe pericardial pain, rub or effusion, or EKG confirmation. Decrease in CH50, C3, or C4 below the lower limit of normal of the testing lab. >25% binding by Farr assay or above normal range for testing lab. >38° C. Exclude infectious cause. <100,000 platelets/ml.

FLARE INDEX Mild/Moderate Flare SLEDAI change 3 or more pts ❒ New/Worse: ❒ Discoid ❒ Photosensitive ❒ Profundus ❒ Cutaneous vasculitis ❒ Bullous lupus ❒ Nasopharyngeal ulcers ❒ Pleuritis ❒ Pericarditis ❒ Arthritis ❒ Fever (SLE) ❒ Increase in prednisone (but not > (0.5 mg/kg/day) ❒ Added NSAID or Plaquenil (for disease activity) ❒ ≥ 1.0 increase in PGA (but not >2.5) Severe Flare Change in SLEDAI to >12

❒ New/worse: ❒ CNS-SLE ❒ Vasculitis ❒ Nephritis ❒ Myositis ❒ Plt < 60,000 ❒ Heme anemia Hg <7 or decrease in Hg > 3%

❒ Requiring: ❒ Double prednisone ❒ Prednisone > 0.5 mg/kg/day

❒ Hospitilization ❒ Prednisone

> 0.5 mg/kg/day

❒ New Cytoxan,

Azathioprine, or Methotrexate ❒ Hospitilization (SLE) ❒ Increase in PGA to > 2.5

<3,000 white blood cells/ml. Exclude drug causes.

TOTAL SCORE: COMMENTS:

525

APPENDIX C

Guidelines for Use of SELENA-SLEDAI Flare Tool to Assess Disease Activity

GENERAL GUIDELINES FOR FILLING OUT THE SLEDAI ●

● ●

●

●

●

526

The main principle to keep in mind is that this instrument is intended to evaluate current lupus activity and not chronic damage. Severity is accounted for in part by the “weightedness” of the scale. Points are given exactly as defined. A descriptor is either scored the exact points allotted or not scored (i.e., given a zero). Descriptors are scored only if they are present at the time of the physician encounter or within the preceding 10 days. The descriptor must be documented by the notes written in the physician encounter form and generally applies to the clinical data and not to the laboratory data. The laboratory data is strictly defined per cutoffs, and documentation is provided by the reports from the commercial laboratory. Descriptors do not have to be new, but can be. They can be ongoing, recurrent, or initial events. Each would be scored the same way. An example would be a malar rash or mucosal ulcer. In these situations, a malar rash observed at the initial visit that remains unchanged for the next six months, irrespective of any treatment, is scored 2 points each time the SLEDAI is completed. Because the nature of lupus is that manifestations are not usually fleeting, it would be rare for descriptors to be present 10 days before and not at the time of the encounter. This is discussed in more detail for each descriptor but is especially relevant for the neurologic, pulmonary, and cutaneous manifestations. In some descriptors the exclusions written may not be exhaustive. The intent of the SLEDAI is that the descriptor be attributed to SLE. If the physician does not attribute the descriptor to SLE it should not be scored, but full documentation must be provided.

Written in italics in the following is the definition for each descriptor precisely as provided in the SELENASLEDAI Score form.

SEIZURE Definition: Recent onset (last 10 days). Exclude metabolic, infectious or drug cause, or seizure due to past irreversible CNS damage. This descriptor is scored if the patient has had a witnessed seizure or convincing description (such as tongue biting or incontinence) within 10 days of the current encounter. The patient need not have a positive EEG, CT scan, PET scan, QEEG, or MRI. The CSF may be totally normal. A seizure is also not counted: 1. If a metabolic cause is determined. 2. In the presence of a proven infectious meningitis, brain abscess, or fungal foci. 3. If there is a history of recent head trauma. 4. In the presence of an offending drug. 5. In the presence of severe hyperthermia or hypothermia. 6. If the patient has stopped taking anticonvulsant medication. 7. If the patient has a documented sub-therapeutic anticonvulsant drug level.

PSYCHOSIS Definition: Altered ability to function in normal activity due to severe disturbance in the perception of reality. Include hallucinations, incoherence, marked loose associations, impoverished thought content, marked illogical thinking, and bizarre, disorganized, or catatonic behavior. Exclude uremia and drug causes. This descriptor is scored if any of the criteria previously cited are met. With regard to drug causes, the most problematic situation is glucocorticoids. If the treating physician attributes the psychosis to glucocorticoids, this descriptor should not be counted.

Definition: Altered mental function with impaired orientation, memory, or other intellectual function (with rapid onset and fluctuating clinical features). Include clouding of consciousness with reduced capacity to focus, inability to sustain attention to environment, and at least two (2) of the following: perceptual disturbance, incoherent speech, insomnia or daytime drowsiness, and increased or decreased psychomotor activity. Exclude metabolic, infectious, or drug causes. This descriptor is scored in the following situations: a) Reduced capacity to focus as exemplified by new inability to perform everyday mathematical computations or disorientation to person, place, time, or purpose OR b) Inability to carry on a conversation OR c) Reduction in short-term memory PLUS: Documented abnormality on “neuropsychiatric” testing Neuropsychiatric testing may take the form of a mini–mental status exam or formal neuropsychiatric examination. The important aspect for scoring OBS is that it be reversible. Consideration should be given to the improvement of OBS after institution of glucocorticoids. This descriptor is not scored in the presence of a metabolic, infectious, or drug cause. If the problem is chronic, this descriptor is not scored in SLEDAI but is scored on the damage index.

For this descriptor to be counted, the headache must be present for greater than 24 hours and must not be responsive to narcotic analgesia. Objective documentation need not be present, although it is expected that such a complaint (given the severity) would prompt formal testing such as MRI, CT, LP, and so on. Furthermore, the headache should be of sufficient severity to warrant the initiation of glucocorticoids or additional immunosuppressive agents. Scoring of this descriptor means attribution of the headache to CNS lupus.

CVA Definition: New onset of cerebrovascular accident(s). Exclude arteriosclerosis or hypertensive causes. This descriptor is scored if the patient has had a CVA within 10 days of the current encounter. A patient recovering from a CVA that was documented more than 10 days prior to the current encounter is not given points for this descriptor. A patient may have had a previous CVA but to be scored the current CVA must be new. This descriptor is scored in the presence or absence of anti-phospholipid antibodies (i.e., the precise pathophysiologic mechanism need not be known). The CVA is scored even in the presence of a normal CT or MRI. A TIA is also scored if the patient gives a convincing history. To exclude atherosclerosis, the patient has to have a normal carotid and/or vertebral Doppler and cannot have uncontrolled hypertension.

VISUAL DISTURBANCE

VASCULITIS

Definition: Retinal and eye changes of SLE. Include cytoid bodies, retinal hemorrhages, serous exudate or hemorrhages in the choroid, optic neuritis, scleritis, and episcleritis. Exclude hypertensive, infectious, or drug causes. This is scored exactly as defined, with the understanding that it must be supported by objective evidence.

Definition: Ulceration, gangrene, tender finger nodules, periungual infarction, splinter hemorrhages, or biopsy or angiogram proof of vasculitis. To score this descriptor, the previously cited indications must be present. For example, erythematous lesions on the hands or feet that may be characteristically considered “leukocytoclastic vasculitis” but do not fulfill at least one of the previously cited indications and are not biopsied are not counted. Similarly, livedo reticularis is not counted. Healed ulcers with residual scar are not to be counted (but be sure to count these in the damage index). A lesion consistent with erythema nodosum should be counted whether it is biopsied or not. Purpura in the presence of a normal platelet count should be counted whether it has been biopsied or not.

CRANIAL NERVE DISORDER Definition: New onset of sensory or motor neuropathy involving cranial nerves. Include vertigo due to lupus. This is scored exactly as defined, with the understanding that it must be supported by objective evidence. However, it should be noted that hydroxychloroquine can affect the eighth cranial nerve.

APPENDIX C

ORGANIC BRAIN SYNDROME

ARTHRITIS LUPUS HEADACHE Definition: Severe persistent headache that may be migrainous but must be nonresponsive to narcotic analgesia.

Definition: More than two joints with pain and signs of inflammation (i.e., tenderness, swelling, or effusion). Arthritis is scored if it is ongoing. It need not be new or recurrent.

527

APPENDIX C

Arthritis is scored only if more than two joints manifest signs of inflammation. For example, if only the right second and left third PIPs are involved (or only both wrists) points for this descriptor are not given. Inflammation is strictly defined in this activity index as the presence of tenderness (the patient complains of pain upon palpating the joint or upon going through range of motion) PLUS any one of the following: 1. Swelling 2. Effusion 3. Warmth 4. Erythema (must exclude overlying cellulitis) The presence of tenderness alone is not sufficient. A patient’s complaints of pain in specific joints without objective findings are not sufficient. An exception would be arthritis of the hip, in which case pain in the groin on range of motion accompanied by decreased range of motion in the absence of swelling, warmth, or erythema would be counted. Inflammation of the tendons, ligaments, bursae, and other periarticular structures are not scored. For example, subacromial bursitis and trochanteric bursitis are not scored. If further evaluation reveals osteonecrosis or osteoarthritis, this descriptor is not counted.

MYOSITIS Definition: Proximal muscle aching/weakness associated with elevated creatine phosphokinase/aldolase or electromyogram changes, or a biopsy showing myositis. The patient complains of muscle aching and/or weakness in the proximal muscles (PLUS one of the following must be present): 1. Elevated serum creatine phosphokinase and/or aldolase 2. Abnormalities upon electromyogram consistent with myositis 3. Biopsy-proven myositis

URINARY CASTS Definition: Heme-granular or red blood cell casts. This is scored if red blood cell casts are seen, even if it is only one. Pigmented casts are counted, but nonpigmented granular casts, hyaline, or waxy casts are not counted.

HEMATURIA

528

Definition: >5 red blood cells per high-power field. Exclude stone, infection, or other cause. With regard to this descriptor, every attempt should be made to see patients when they are not menstruating. If this is not possible, the urinalysis should be deferred until the next visit.

This descriptor is not scored if there is documented renal calculi or infection. The latter must be confirmed by a positive urinary culture. However, it is acknowledged that associated conditions such as chlamydia or urethral irritation may result in mild hematuria and the physician’s best judgment is warranted. The important point is attribution: there must be other evidence of nephritis and other causes of hematuria must be excluded. In the complete absence of proteinuria, attribution of hematuria to active nephritis would be very unlikely unless pathology is limited to the mesangium.

PROTEINURIA Definition: New onset or recent increase of more than 0.5 gm/24 hours. The following strict guidelines must be used: a) If the baseline 24-hour urine is <500 mg/24 h, proteinuria is counted ONLY when there is a 500-mg increase from the baseline measurement. Once it is documented as proteinuria, it would not be counted again until the 24-hour urine increased by 500 mg or more from that point. Example: Baseline: 100 mg/24 h 1 Month: 400 mg/24 h 2 Months: 500 mg/24 h 3 Months: 600 mg/24 h (proteinuria is now counted) 4 Months: 800 mg/24 h 5 Months: 1150 mg/42 h (proteinuria is now counted) b) If the baseline 24-hour urine is abnormal (>500 mg/24 h), proteinuria is counted when there is a 500-mg increase from the baseline measurement. Once >500 mg/24 h is observed, thereafter the increment must be >500 mg/24 h. If the 24-hour urine decreases during the course of the study, proteinuria is then counted again when the 24-hour urine increases by 500 mg or more from the point at which it decreased. Example: Baseline: 500 mg/24 h 1 Month: 650 mg/24 h 2 Months: 850 mg/24 h 3 Months: 1300 mg/24 h (proteinuria is now counted) 4 Months: 1400 mg/24 h 5 Months: 600 mg/24 h 6 Months: 800 mg/24 h 7 Months: 1200 mg/24 h (proteinuria is now counted)

PYURIA Definition: >5 white blood cells per high-power field. Exclude infection.

MUCOSAL ULCERS

RASH

Definition: Classic and severe pleuritic chest pain or pleural rub or effusion or new pleural thickening due to lupus. This descriptor is scored if the patient complains of pleuritic chest pain lasting greater than 12 hours. The pain should be classic (i.e., exacerbated by inspiration) to help distinguish it from musculoskeletal conditions such as costochondritis, which could be confused with pleurisy. The symptom does not have to be accompanied by any objective findings. The presence of objective findings such as pleural rub or pleural effusions (in the absence of infection, congestive heart failure, malignancy, or nephrosis) is counted, even if not accompanied by symptoms. New pleural thickening should be counted only if other causes as described previously are absent.

Definition: Ongoing inflammatory lupus rash. A rash is scored if it is ongoing, new, or recurrent. Even if it is identical in terms of distribution and character to that observed on the last visit and the intensity is improved, it is counted. Therefore, despite improvement in a rash if it is still ongoing it represents disease activity. The rash must be attributable to SLE. A description of the rash must appear in the physical examination and should include distribution, characteristics such as macular or papular, and size. The following should not be scored: 1. Chronic scarred discoid plaques in any location 2. Transient malar flush (i.e., it is not raised and is evanescent) A common problem one may encounter is the differentiation between scoring a lesion as rash and/or as vasculitis. If a lesion meets the descriptive criteria of the latter, it should not also be counted as rash (i.e., the score would be 8 points, not 10 points). If a separate rash characteristic of SLE is present, only then would “rash” also be scored.

ALOPECIA Definition: Ongoing abnormal, patchy, or diffuse loss of hair due to active lupus. This should be scored if any of the following conditions are present: 1. There is temporal thinning that is newly present for less than six months (if temporal alopecia is present for more than six months with no change, it should not be counted) 2. Areas of scalp with total bald spots if present for less than six months (does not need to have accompanying discoid lesion or follicular plugging) 3. The presence of “lupus frizz” (i.e., short of strands of unruly hair in the frontal or temporal area) If a patient complains of hair loss and there is nothing apparent upon examination, this descriptor is not scored.

Definition: Ongoing oral or nasal ulcerations due to active lupus. An ulcer is scored if it is ongoing. It need not be new or recurrent. Ulcers can be present in either the nose or oral cavity. Erythema alone without frank ulceration is not sufficient to be scored, even if the erythema is present on the upper palate. Ulcers on the buccal mucosa and tongue are counted. Mucosal ulcers are not counted as vasculitis.

APPENDIX C

This descriptor is not scored if there is evidence of vaginal contamination (presence of any squamous epithelial cells) or a documented infection. The latter must be confirmed by a positive urinary culture. However, it is acknowledged that associated conditions such as chlamydia, trichomonas, or urethral irritation may result in mild pyuria and the physician’s best judgment is warranted. The important point is attribution. There must be other evidence of nephritis, and other causes of pyuria should be excluded. In the complete absence of proteinuria, attribution of hematuria to active nephritis would be very unlikely unless pathology were limited to the interstitium.

PLEURISY

PERICARDITIS Definition: Classic and severe pericardial pain or rub or effusion, or electrocardiogram confirmation. The symptom does not have to be accompanied by any objective findings.

LOW COMPLEMENT Definition: Decrease in CH50, C3, or C4 below the lower limit of normal for testing laboratory. Exclude a low C4 or CH50 in patients with known inherited deficiency of C4.

INCREASED DNA BINDING Definition: >25% binding by Farr assay or above normal range for testing laboratory.

FEVER Definition: >38°C. Exclude infectious cause. This would be scored if one of the following conditions were present: 1. A documented temperature elevation >100.4° F or >38° C at the time of the visit.

529

APPENDIX C

2. A convincing history from the patient that she/he has been febrile within the preceding 10 days prior to the visit without any signs or symptoms suggestive of infection. Febrile is defined as previously and not simply that the patient felt feverish. In this case, the patient need not be febrile at the time of the visit for a score of 2 to be given. As stated in the SLEDAI, fever secondary to infection is not to be scored, although it is acknowledged that concomitant lupus activity and infection can occur. Fever in the presence of infection should only be scored on the SLEDAI if other evidence of lupus activity is present.

THROMBOCYTOPENIA Definition: <100,000 platelets/mm3.

LEUKOPENIA Definition: <3,000 white blood cells/mm3. Exclude drug causes.

530

This is exactly as described (WBC <3,000/mm3). The presence of an absolute lymphopenia does not count in the SLEDAI. A note of caution: Do not confuse this WBC with that used to satisfy the ACR criteria for SLE, which is WBC <3,500/mm3. With regard to current use of possible offending drugs, the following guidelines are to be considered: 1. The nadir after cyclophosphamide (i.e., low WBC at 10 days after receiving cyclophosphamide) in a patient known to have a WBC ≥ 3,000 at the time of receiving cyclophosphamide should not be counted. 2. Do not score leukopenia appearing after initiation of a new medication known to be associated with leukopenia, such as azathioprine or sulfa drugs. If the patient develops a WBC <3,000 while taking drugs that may cause leukopenia, score this only if the dosage of medication is unchanged since the last WBC determination.

APPENDIX D

BRITISH ISLES LUPUS ASSESSMENT GROUP BILAG INDEX Centre:

Date:

Initials/Hospital No:

All features must be attributable to SLE and refer to last 4 weeks compared with prior disease activity. For instructions and glossary see Appendix F. Indicate features present:

1) Improving 2) Same 3) Worse 4) New or Y/N or value (where indicated) (default is 0 = not present)

43. 44. 45. 46.

Mild chronic myositis Arthralgia Myalgia Tendon contractures and fixed deformity 47. Aseptic necrosis

GENERAL 1. 2. 3. 4. 5.

( ( ( ( (

) ) ) ) )

MUCOCUTANEOUS

Y/N Y/N Y/N Y/N

( (

) )

(

)

( ( ( ( ( ( ( ( ( ( ( ( ( ( (

) ) ) ) ) ) ) ) ) ) ) ) ) ) )

NEUROLOGIC 24. Deteriorating level of consciousness 25. Acute psychosis or delirium or confusional state 26. Seizures 27. Stroke or stroke syndrome 28. Aseptic meningitis 29. Mononeuritis multiplex 30. Ascending or transverse myelitis 31. Peripheral or cranial neuropathy 32. Disc swelling/cytoid bodies 33. Chorea 34. Cerebellar ataxia 35. Headache severe, unremitting 36. Organic depressive illness 37. Organic brain syndrome including pseudotumor cerebri 38. Episodic migrainous headaches

48. Pleuropericardial pain 49. Dyspnea 50. Cardiac failure 51. Friction rub 52. Effusion (pericardial or pleural) 53. Mild or intermittent chest pain 54. Progressive cxr changes - lung fields 55. Progressive cxr changes - heart size 56. ECG evidence of pericarditis or myocarditis 57. Cardiac arrhythmias including tachycardia >100 in absence of fever 58. Pulmonary function fall by >20% 59. Cytohistologic evidence of inflammatory lung disease

( (

) )

Y/N Y/N

( ( ( ( ( ( ( (

) ) ) ) ) ) ) )

Y/N

(

)

Y/N Y/N

(

)

Y/N

(

)

( ( (

) ) )

( ( (

) ) )

(

)

Y/N

(

)

value value Y/N value

( ( ( (

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value Y/N Y/N value value Y/N

( ( ( ( ( (

) ) ) ) ) )

Y/N

(

)

value value value value value Y/N Y/N Y/N

( ( ( ( ( ( ( (

) ) ) ) ) ) ) )

Y/N Y/N

VASCULITIS 60. Major cutaneous vasculitis incl.ulcers 61. Major abdominal crisis due to vasculitis 62. Recurrent thromboembolism (excluding strokes) 63. Raynaud’s 64. Livido reticularis 65. Superficial phlebitis 66. Minor cutaneous vasculitis (nailfold vasculitis, digital vasculitis, purpura, urticaria) 67. Thromboembolism (excl. stroke) - 1st episode

(

)

RENAL

( ( ( ( ( ( ( ( ( ( ( (

) ) ) ) ) ) ) ) ) ) ) )

68. Systolic blood pressure (mmHg) 69. Diastolic blood pressure (mmHg) 70. Accelerated hypertension 71. Urine dipstick protein (+ = 1, ++ = 2, +++ = 3) 72. 24-hour urinary protein (g) 73. Newly documented proteinuria >1g/24h 74. Nephrotic syndrome 75. Creatinine (plasma/serum) 76. Creatinine clearance/GFR ml/min 77. Active urinary sediment 78. Histologic evidence of active nephritis - within 3 months

( (

) )

(

)

( ( (

) ) )

MUSCULOSKELETAL 39. Definite myositis (Bohan and Peter) 40. Severe polyarthritis - with loss of function 41. Arthritis 42. Tendonitis

) ) )

CARDIOVASCULAR AND RESPIRATORY

Pyrexia (documented) Weight loss - unintentional >5% Lymphadenopathy/splenomegaly Fatigue/malaise/lethargy Anorexia/nausea/vomiting

6. Maculopapular eruption - severe, active (or discoid/bullous) 7. Maculopapular eruption - mild 8. Active discoid lesions - generalized or extensive 9. Active discoid lesions - localized including lupus profundus 10. Alopecia (severe, active) 11. Alopecia (mild) 12. Panniculitis (severe) 13. Angio-edema 14. Extensive mucosal ulceration 15. Small mucosal ulcers 16. Malar erythema 17. Subcutaneous nodules 18. Perniotic skin lesions 19. Peri-ungual erythema 20. Swollen fingers 21. Sclerodactyly 22. Calcinosis 23. Telangiectasia

( ( (

HEMATOLOGY 79. 80. 81. 82. 83. 84. 85. 86.

Hemoglobin g/dl Total white cell count x 109/l Neutrophils x 109/L Lymphocytes x 109/L Platelets x 109/L Evidence of active hemolysis Coomb’s test positive Evidence of circulating anticoagulant

531

APPENDIX E

BILAG Scoring by System

GENERAL: CATEGORY SCORING

Category A Pyrexia recorded as 2 (Same), 3 (Worse), or 4 (New) Plus 2 others recorded as 2 (Same), 3 (Worse), or 4 (New)

Category B Pyrexia recorded as 2 (Same), 3 (Worse), or 4 (New) Or 2 others recorded as 2 (Same), 3 (Worse), or 4 (New)

Category C

Category C Any Category A or Category B criteria recorded as 1 (Improving) or Any one of the following recorded as 1 (Improving), 2 (Same), 3 (Worse), 4 (New), or Yes: ● Peri-ungual erythema ● Sclerodactyly ● Swollen fingers ● Mild alopecia ● Calcinosis ● Small mucosal ulceration ● Telangiectasia

Any one criterion recorded as 1 (Improving), 2 (Same), 3 (Worse), or 4 (New)

Category D

Category D

Category E

Previous mucocutaneous involvement, currently inactive

Previous General System involvement, currently active

No mucocutaneous involvement

Category E

NEUROLOGIC: CATEGORY GRADING

No previous General System involvement

Category A MUCOCUTANEOUS: CATEGORY GRADING

Category A Any one of the following recorded as 2 (Same), 3 (Worse), or 4 (New): ● Severe maculopapular, discoid, or bullous eruption [i.e., active facial and/or extensive (>2/9), scarring] ● Angio-edema ● Extensive mucosal ulceration

Category B

532

Any one of the following recorded as 2 (Same), 3 (Worse), or 4 (New): ● Malar erythema ● Severe active alopecia ● Mild maculopapular eruption ● Subcutaneous nodules ● Panniculitis ● Perniotic skin lesions

Any one of the following recorded as 3 (Worse) or 4 (New): ● Impaired level of consciousness ● Psychosis, delirium, or confusional state ● Grand mal seizure ● Stroke or stroke syndrome ● Aseptic meningitis ● Mononeuritis multiplex ● Ascending or transverse myelitis ● Peripheral or cranial neuropathy ● Chorea ● Cerebellar ataxia

Category B Any one of the following recorded as 3 (Worse) or 4 (New): ● Headache (severe unremitting) ● Organic depressive illness ● Chronic brain syndrome, including pseudotumor cerebri ● Disc swelling or cytoid bodies

Category C Episodic migrainous headaches recorded 1 (Improving), 2 (Same), 3 (Worse), or 4 (New):

as

Or any one of the following recorded as 1 (Improving) or 2 (Same): ● Stroke or stroke syndrome ● Aseptic meningitis ● Mononeuritis multiplex ● Ascending or transverse myelitis ● Peripheral or cranial neuropathy ● Choreacerebellar ataxia ● Headache (severe unremitting) ● Organic depressive illness ● Chronic brain syndrome, including pseudotumor cerebri ● Disc swelling or cytoid bodies

Category D Previous CNS disease, currently inactive

Category E No previous CNS disease

MUSCULOSKELETAL: CATEGORY GRADING

Category A Any one of the following recorded as 2 (Same), 3 (Worse), or 4 (New): ● Definite myositis (Bohan and Peter) ● Severe polyarthritis with loss of function

Category B Any one of the following recorded as 2 (Same), 3 (Worse), or 4 (New): ● Arthritis (definite synovitis) ● Tendonitis

Category C Any Category A or Category B criterion recorded as 1 (Improving) Or One or more of the following recorded as 1 (Improving), 2 (Same), 3 (Worse), 4 (New), or Yes: ● Arthralgia ● Myalgia ● Tendon contractures and fixed deformity ● Aseptic necrosis ● Mild chronic myositis

Category D Previous musculoskeletal involvement, currently inactive

APPENDIX E

Or Any of the following recorded as 1 (Improving) or 2 (Same): ● Impaired level of consciousness ● Pychosis, delirium, or convulsional state ● Grand mal seizure

Category E No previous musculoskeletal involvement

CARDIORESPIRATORY: CATEGORY GRADING

Category A Cardiac failure recorded as 2 (Same), 3 (Worse), or 4 (New) plus two other criteria (listed in the following) recorded as 2 (Same), 3 (Worse), 4 (New), or Yes Or Symptomatic effusion recorded as 2 (Same), 3 (Worse), or 4 (New) plus two other criteria (listed in the following) recorded as 2 (Same), 3 (Worse), 4 (New), or Yes Or Four of the criteria listed in the following, each recorded as 2 (Same), 3 (Worse), 4 (New), or Yes: ● Pleuropericardial pain ● Dyspnea ● Friction rub ● Progressive chest X-ray changes – lung fields ● Progressive chest X-ray changes – heart size ● ECG evidence of pericarditis or myocarditis ● Cardiac arrhythmias, including tachycardia ● >100 in absence of fever ● Deteriorating lung function: <20% of expected or >20% fall ● Cytohistologic evidence of inflammatory lung disease

Category B Two of the criteria listed in the following, each recorded as 2 (Same), 3 (Worse), 4 (New), or Yes: ● Pleuropericardial pain ● Dyspnea ● Friction Rub ● Progressive chest X-ray changes – lung fields ● Progressive chest X-ray changes – heart size ● ECG evidence of pericarditis or myocarditis ● Cardiac arrhythmias, including tachycardia >100 in absence of fever ● Deteriorating lung function: <20% of expected or >20% fall ● Cytohistologic evidence of inflammatory lung disease

Category C One of the criteria listed in the following, each recorded as 1 (Improving), 2 (Same), 3 (Worse), 4 (New), or Yes: ● Mild intermittent chest pain ● Pleuropericardial pain

533

APPENDIX E

● ● ● ● ● ●

●

●

Dyspnea Friction Rub Progressive chest X-ray changes – lung fields Progressive chest X-ray changes – heart size ECG evidence of pericarditis or myocarditis Cardiac arrhythmias, including tachycardia >100 in absence of fever Deteriorating lung function: <20% of expected or >20% fall Cytohistological evidence of inflammatory lung disease

Category D Previous cardiovascular/respiratory disease involvement, currently inactive

Category E No previous involvement

cardiovascular/respiratory

disease

VASCULITIS: CATEGORY GRADING

Category A Major cutaneous vasculitis (including ulcers), accompanied by infarction occurring in the past 4 weeks, recorded as 2 (Same), 3 (Worse), or 4 (New) Or Major abdominal crisis due to vasculitis, recorded as 2 (Same), 3 (Worse), or 4 (New) Or Recurrent thromboembolism (excluding strokes), recorded as 2 (Same), 3 (Worse), or 4 (New)

Category B Minor cutaneous vasculitis, recorded as 2 (Same), 3 (Worse), or 4 (New) Or Superficial phlebitis, recorded as 2 (Same), 3 (Worse), or 4 (New) Or Thromboembolism (excluding strokes), first episode, recorded as Yes

Category C Any Category A or Category B criterion recorded as 1 (Improving) Or Raynaud’s Phenomenon, recorded as 1 (Improving) 2 (Same), 3 (Worse), or 4 (New) Or Livedo reticularis, recorded as 1 (Improving) 2 (Same), 3 (Worse), or 4 (New) 534

Category D Previous vasculitis involvement, currently inactive

Category E No previous vasculitis involvement

RENAL: CATEGORY GRADING

Category A Two or more of the following, provided one of 1, 4, or 5 is included: 1. Proteinuria defined as: a) Urinary dipstick increased by 2 or more levels, or b) 24-hour urine protein rising from <0.20 g to >1 g, or c) 24-hour urine protein rising from >1 g by 100%, or d) Newly documented proteinuria of >1 g 2. Accelerated hypertension 3. Deteriorating renal function, defined as: a) Plasma creatinine ≥130 μmol/l and having risen to >130% of previous value, or b) Creatinine clearance having fallen to <67% of revious value, or c) Creatinine clearance <50 ml/min, and last time was ≥50 ml/min or was not measured 4. Active urinary sediment (on uncentrifuged specimen): pyuria (>5 wc/hpf), hematuria (>5 rbc/hpf), or red cell casts in the absence of infection or other cause 5. Histologic evidence of active nephritis by WHO criteria within last 3 months (or since previous assessment if seen less than 3 months ago; sclerosis without inflammation not counted)

Category B One of the following: One of the Category A criteria (see previous) (a) 1) Urine dipstick that has risen by 1+ or more to at least 2+, or 2) 24-hour urinary protein rising from >1 by >50% but <100% OR (b) Plasma creatinine >130 μmol/l and having risen to 115% of previous value

Category C One of the following: (a) 24-hour urine protein >0.25 g (b) Urine dipstick 1+ or more (c) Rising blood pressure, defined as (a) systolic rise of ≥30mm and (b) diastolic rise of ≥15 mm (provided the recorded values are >140/90)

Previous renal disease, currently inactive

Category E No evidence of any renal disease ever

HEMATOLOGIC: CATEGORY GRADING

Category A One of the following: ● Hemoglobin <8 ● White cell count <1000 ● Platelet count <25

Category B One of the following: ● Hemoglobin <11 ● White cell count <2500 ● Platelet count <100

●

Coomb’s test positive and evidence of active hemolysis (e.g., raised bilirubin ± increased reticulocyte count)

Category C

APPENDIX E

Category D

One of the following: ● White cell count <4000 ● Platelet count <150 ● Lymphocyte count <1500 ● Coomb’s test positive (no active hemolysis) ● Evidence of circulating lupus anticoagulant or other antiphospholipid antibody

Category D Previous hematologic disease involvement, currently inactive

Category E No previous hematologic disease involvement

535

APPENDIX F

BILAG Index of SLE Diseases Activity

(3) Worse Refers to manifestations that have deteriorated in the last 4 weeks compared to the previous 4 weeks.

INTRODUCTION

●

INSTRUCTIONS ●

●

●

●

●

Only record features attributable to SLE disease activity and not due to damage, infection, or other conditions. Assessment refers to manifestations occurring in the last 4 weeks compared with the previous 4 weeks. Activity refers to disease process that is reversible, whereas damage refers to permanent process/scarring (irreversible). Damage due to SLE should be considered a cause of features that are fixed/persistent (SLICC/ACR damage index uses persistence ≥6 months to define MOST damage items). In some manifestations, it may be difficult to differentiate SLE from other conditions because there may not be any specific test and the decision would then lie with the physician’s judgment on the balance of probabilities.

GUIDANCE FOR RECORDING ITEMS (4) New ● Manifestations are recorded as new when it is a new episode occurring in the last 4 weeks (compared to the previous 4 weeks) that has not improved, including new episodes (recurrence) of old manifestations. ● New episode occurring in the last 4 weeks but also satisfying the criteria for improvement (following) would be classified as improving instead of new.

(2) Same Refers to manifestations that have been present for the last 4 weeks and the previous 4 weeks without significant improvement or deterioration (from the previous 4 weeks). ● Also applies to manifestations that have improved over the last 4 weeks compared to the previous 4 weeks but do not meet the criteria for improvement. ●

(1) Improving Definition of improvement: (a) Amount of improvement is sufficient for consideration of reduction in therapy and would not justify escalation in therapy. (b) Improvement must be present currently and =2 weeks of the last 4 weeks.

●

(0) Not present

ADDITIONAL COMMENTS AND GLOSSARY If a lupus manifestation can be recorded as a mild or severe item (depending on glossary definition) and is recorded as the severe item, the corresponding mild item should be recorded as well. When the feature improves and changes to mild, just the mild item is recorded (but it will not be new; it will be improving). If a mild item deteriorates and becomes severe, the severe item is new and the mild item is recorded as worse.

General System: Item

Definition

1. Pyrexia

Temperature of <37.5° C documented (infection excluded).

2. Weight loss

Unintentional weight loss >5% in one month (due to lupus, not diet or co-morbid disease).

3. Lymphadenopathy

Palpable lymph nodes <1 cm in diameter usually vary in size with time.

4. Fatigue/malaise/weakness

Sufficiently severe to affect normal activities and tends to fluctuate with time (in contrast to chronic fatigue syndrome or fatigue of fibromyalgia, which tends to be constant and present all the time).

5. Anorexia/nausea/vomiting

Lupus-related (excludes symptoms due to drug side effects, infection, and so on).

536

Item

Definition

6. Maculopapular eruption – severe

Active maculopapular or bullous eruption. This must be extensive [>2/9 (18%) of body surface area], scarring, or causing disability (rule of 9s – see following).

7. Maculopapular eruption – mild

Limited to ≤2/9 (18%) or less of body surface area, non-scarring, non-disabling.

8. Active discoid lesions – generalized or extensive

>2/9 (18%) of body surface area.

9. Active discoid lesions – localized

APPENDIX F

Mucocutaneous System:

Limited to ≤2/9 or less of body surface area, and includes lupus profundus.

10. Alopecia–severe, active

Abnormal diffuse hair loss that is clinically detectable with scalp inflammation.

11. Alopecia–mild

Limited, relatively inactive; abnormal diffuse hair loss with little or no detectable scalp inflammation.

12. Panniculitis

Extensive, painful, erythematous subcutaneous nodules associated with an overlying discoid skin lesion.

13. Angio-edema

Potentially life threatening (e.g., stridor); NOT SUITABLE AS Entry Criterion for trial requiring A level disease as usually short-lived and requires emergency treatment.

14. Extensive mucosal ulceration

Severe, deep, and extensive disabling ulcers.

15. Small mucosal ulcers

More than 1 aphthous ulcer, painful or painless.

16. Malar erythema

Classical “butterfly” type erythematosus rash due to lupus but may only be small areas and may occur on other parts of the face as well as cheeks and nose, but spares naso-labial folds.

17. Subcutaneous nodules

As in rheumatoid arthritis.

18. Perniotic skin lesions

Also called chilblain lupus: red-purple patches and plaques on toes, fingers, heels, calves, elbows, knees, nose with or without fissuring, often in response to cold and may be associated with discoid lesions.

19. Peri-ungual erythema 20. Swollen fingers

Do not record if damage.

21. Scerodactyly

Do not record if damage.

22. Calcinosis

Do not record if damage.

23. Telangiectasia

Do not record if damage.

9% RULE OF NINES The body surface is divided into areas representing 9% or multiples

Anterior 18% 9%

Posterior 18% 1% 18% 18%

9%

The patient’s palm represents 1% of his or her body surface

537

APPENDIX F

Neurologic System: Item

Definition

24. Impaired level of consciousness

Acute deteriorating level of consciousness by any accepted clinical criteria (exclude drugs, infection, co-morbid disease).

25. Acute psychosis or delirium or confusional state

Acute severe disturbance in the perception or reality characterized by delusions, hallucinations, incoherence, marked illogical thinking, bizarre or catatonic behavior (exclude drugs, substance abuse, primary psychotic disorder).

26. Seizures

Independent description of seizure by reliable witness.

27. Stroke or stroke syndrome

Attributable to acute lupus inflammation; exclude atherosclerosis, emboli, hypoglycaemia, cerebral sinus thrombosis, vascular malformation, tumour, abscess.

28. Aseptic meningitis

Criteria: acute/subacute onset, headache, with fever and abnormal CSF (raised protein, lymphocytes predominant but negative cultures; i.e., without evidence of infection or bleed) ± photophobia, neck stiffness, signs of meningeal irritation.

29. Mononeuritis multiplex

Multiple (>1) nerves affected by inflammatory process.

30. Ascending or transverse myelitis

Acute onset of rapidly evolving paraparesis or quadriparesis and/or sensory level (exclude intramedullary and extramedullary space occupying lesion).

31. Peripheral or cranial neuropathy

Acute symmetrical distal peripheral or cranial sensory and/or motor deficit

32. Disc swelling/cytoid bodies

(exclude diabetic retinopathy, and so on).

33. Chorea

Exclude drug-induced.

34. Cerebellar ataxia

In isolation of other CNS features (not brain stem stroke); usually subacute presentation.

35. Headache, severe and unremitting

Continuous disabling headache lasting ≥3 days, not relieved by narcotic analgesia (exclude intracranial space occupying lesion and CNS infection).

36. Organic depressive illness

Attributable to lupus and associated with somatic symptoms and severe enough to merit treatment with antidepressive medication.

37. Organic brain syndrome

Impaired orientation, memory, or other intellectual function in the absence of metabolic, psychiatric, or pharmacologic causes. ●

Clinical features develop over a short period (usually hours to days) and tend to fluctuate over the course of the day: a) Clouding of consciousness with reduced capacity to focus and sustain attention to environment b) I. Perceptual disturbance; illusions, or hallucinations. II. Incoherent speech III. Insomnia or daytime drowsiness IV. Increased or decreased psychomotor activity c) Disorientation and recent memory impairment

38. Episodic migrainous headaches

Recurrent lupus-related headaches lasting 4 - 72 hours, may be preceded by neurologic aura (lasting up to 1 hour).

Musculoskeletal System:

538

39. Myositis

At least 3 of the following Bohan and Peter criteria: acute proximal muscle weakness, elevated muscle enzymes (CK), positive biopsy, and abnormal EMG.

40. Polyarthritis with loss of function

Active joint inflammation in at least 2 joints with clinically significant loss of the functional range of movement of the involved joints.

41. Arthritis

Active joint inflammation in 1 or more joints (tenderness, warmth, or swelling without loss of functional range of motion).

42. Tendonitis

Inflammatory not mechanical.

43. Mild chronic myositis

2 or 3 Bohan and Peter criteria or sub-acute (not damage).

Item

Definition

44. Arthralgia

Inflammatory joint pain with morning stiffness on history and with no signs of inflammation (exclude OA, mechanical problems, fibromyalgia).

45. Myalgia

Inflammatory muscle pain without weakness or elevated CPK (exclude fibromyalgia).

46. Tendon contractures and fixed deformity

Damage, so should not be recorded.

47. Aseptic necrosis

Damage, so should not be recorded.

APPENDIX F

Musculoskeletal System—Cont’d

Cardiorespiratory System: 48. Pleuropericardial pain

Localized sharp or dull pain in the chest aggravated by respiration (upon inspiration) without chest wall tenderness.

49. Dyspnea

Upon exertion (but not orthopnea alone, and exclude angina, infection, asthma, chronic bronchitis, and so on).

50. Cardiac failure

Cardiac failure due to lupus myocarditis or non-infective inflammation of endocardium or cardiac valves (endocarditis); exclude other causes.

51. Friction rub

Pleural or pericardial (exclude chronic fibrosis).

52. Effusion (pericardial or pleural)

Clinically detectable.

53. Mild intermittent chest pain

Non-specific (not clearly pleuritic, pericardial, musculoskeletal, or angina).

54. Progressive CXR changes – lungs

Due to lupus.

55. Progressive CXR changes – heart

Due to lupus.

56. ECG evidence of pericarditis or myocarditis 57. Cardiac arrhythmias including tachycardia >100 in absence of fever

Due to lupus.

58. Pulmonary function fall >20%

>20% fall since last assessment or >20% below lower limit of normal if not measured before in lung volumes and/or transfer factor corrected for lung volumes (must be associated with change in symptoms to suggest acute inflammatory disease not co-morbid lung conditions or infection).

59. Cytohistologic evidence of inflammatory lung disease

Due to lupus.

Vasculopathy (Vasculitis) System:

60. Major cutaneous vasculitis, including ulcers

Extensive gangrene and/or ulceration.

61. Major abdominal crisis due to vasculitis

Small or large bowel, gall bladder, and so on with supportive imaging and/or biopsy findings.

62. Recurrent thromboembolism (excluding stroke) 63. Raynaud’s phenomenon 64. Livedo reticularis 65. Superficial phlebitis 66. Minor cutaneous vasculitis

Includes nailfold vasculitis, digital vasculitis, purpura, ulcers, leukocytoclastic/ hypersensitivity, vasculitis.

67. Thromboembolism (excluding stroke) 1st episode

539

APPENDIX F

Renal System (original, see following for 2004 revision): Item

Definition

68. Systolic BP mmHg 69. Diastolic BP mmHg

5th phase.

70. Accelerated hypertension

BP rising to >170/110 (5th phase) within 1 month, if accompanied by grade IV retinal changes (i.e., hemorrhage, exudates).

71. Dipstick (+ = 1, ++ = 2, +++ = 3)

Proteinuria (exclude infection if positive).

72. 24 h urine protein (g) 73. New documented proteinuria of >1 g/24 h 74. Nephrotic syndrome

Heavy proteinuria (>50 mg/kg/day or >3.5 g/day), hypoalbuminemia, and edema.

75. Creatinine (plasma/serum)

Record value and units.

76. Creatinine clearance/GFR (ml/min)

Record value.

77. Active urinary sediment

Uncentrifuged specimen with pyuria (>5 wbc/hpf), hematuria (>5 rbc/hpf), or red cell casts in the absence of infection or any other cause.

78. Histologic evidence of active nephritis (within 3 months)

Histologic evidence of active nephritis according to WHO criteria. Sclerosis alone (without inflammation) will not be regarded as evidence of active nephritis.

Hematologic System:

540

79. Hemoglobin (g/dL)

Exclude if abnormal value not due to lupus (e.g., iron deficiency anemia, drugs, and so on).

80. Total white cell count x 109/L

Exclude if abnormal value not due to lupus (e.g., iron deficiency anemia, drugs, and so on).

81. Neutrophils x 109/L

Exclude if abnormal value not due to lupus (e.g., iron deficiency anemia, drugs, and so on).

82. Lymphocytes x 109/L

Exclude if abnormal value not due to lupus (e.g., iron deficiency anemia, drugs, and so on).

83. Platelets x 109/L

Exclude if abnormal value not due to lupus (e.g., iron deficiency anemia, drugs, and so on).

84. Evidence of active hemolysis

Positive Coomb’s test and evidence of hemolysis (raised bilirubin or raised reticulocyte count or reduced haptoglobulins).

85. Coombs test positive

Isolated without evidence of hemolysis (exclude infection).

86. Evidence of circulating anticoagulant

Evidence of circulating lupus anticoagulant, anticardiolipin, or other antiphospholipid antibody.

APPENDIX G

BRITISH ISLES LUPUS ASSESSMENT GROUP BILAG2004 INDEX Centre:

Date:

Initials/Hosp No:

Only record items due to SLE Disease Activity & assessment refers to manifestations occuring in the last 4 weeks (Compared with the previous 4 weeks). ◆◆

TO BE USED WITH THE GLOSSARY ◆◆ CARDIORESPIRATORY

Scoring: ND Not done 1 Improving 2 Same 3 Worse 4 New Yes/No OR Value (where indicated) ❒ Indicate if not due to SLE activity (default is 0 = not present)

CONSTITUTIONAL 1. 2. 3. 4.

Pyrexia - documented > 37.5° C Weight loss - unintentional >5% Lymphadenopathy/splenomegaly Anorexia

( ( ( (

) ) ) )

( ( ( ( ( ( ( ( ( ( ( ( (

) ) ) ) ) ) ) ) ) ) ) ) )

(

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(

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MUCOCUTANEOUS 5. Skin eruption - severe

6. Skin eruption - mild 7. Angio-edema - severe 8. Angio-edema - mild 9. Mucosal ulceration - severe 10. Mucosal ulceration - mild 11. Panniculitis/bullous lupus - severe 12. Panniculitis/bullous lupus - mild 13. Major cutaneous vasculitis/thrombosis 14. Digital infarcts or nodular vasculitis 15. Alopecia - severe 16. Alopecia - mild 17. Peri-ungual erythema/chilblains 18. Splinter hemorrhages

NEUROPSYCHIATRIC 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

Aseptic meningitis Cerebral vasculitis Demyelinating syndrome Myelopathy Acute confusional state Psychosis Acute inflammatory demyelinating polyradiculoneuropathy Mononeuropathy (single/multiplex) Cranial neuropathy Plexopathy Polyneuropathy Seizure disorder Status epilepticus Cerebrovascular disease (not due to vasculitis) Cognitive dysfunction Movement disorder Autonomic disorder Cerebellar ataxia (isolated) Lupus headache - severe unremitting Headache from IC hypertension

MUSCULOSKELETAL 39. 40. 41. 42. 43.

Myositis - severe Myositis - mild Arthritis (severe) Arthritis (moderate)/tendonitis/tenosynovitis Arthritis (mild)/arthralgia/myalgia

Weight (kg): Serum urea (mmol/l): African ancestry: Yes/No Serum albumin (g/l):

44. Myocarditis - mild 45. Myocarditis/endocarditis + cardiac failure 46. Arrhythmia 47. New valvular dysfunction 48. Pleurisy/pericarditis 49. Cardiac tamponade 50. Pleural effusion with dyspnea 51. Pulmonary hemorrhage/vasculitis 52. Interstitial alveolitis/pneumonitis 53. Shrinking lung syndrome 54. Aortitis 55. Coronary vasculitis

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value value Yes/No (+=1, ++=2, +++=3) mg/mmol mg/mmol value Yes/No mmol/l ml/min/1.73 m2 Yes/No Yes/No

( ( ( ( ( ( ( ( ( ( (

)❒ )❒ ) )❒ )❒ )❒ )❒ )❒ ) )❒ )❒

value value value value value Yes/No

( ( ( ( ( ( (

)❒ )❒ )❒ )❒ )❒ ) )

97. Coombs’ test positive (isolated) Yes/No

(

)

GASTROINTESTINAL 56. 57. 58. 59. 60. 61. 62. 63. 64.

Lupus peritonitis Abdominal serositis or ascites Lupus enteritis/colitis Malabsorption Protein losing enteropathy Intestinal pseudo-obstruction Lupus hepatitis Acute lupus cholecystitis Acute lupus pancreatitis

OPHTHALMIC 65. Orbital inflammation/myositis/proptosis 66. Keratitis - severe 67. Keratitis - mild 68. Anterior uveitis 69. Posterior uveitis/retinal vasculitis - severe 70. Posterior uveitis/retinal vasculitis - mild 71. Episcleritis 72. Scleritis - severe 73. Scleritis - mild 74. Retinal/choroidal vaso-occlusive disease 75. Isolated cotton-wool spots (cytoid bodies) 76. Optic neuritis 77. Anterior ischemic optic neuropathy

RENAL 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89.

Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Accelerated hypertension Urine dipstick protein Urine albumin-creatinine ratio Urine protein-creatinine ratio 24-hour urine protein (g) Nephrotic syndrome Creatinine (plasma/serum) GFR (calculated) Active urinary sediment Active nephritis

(

HAEMATOLOGY 90. 91. 92. 93. 94. 95. 96.

Hemoglobin (g/dl) Total white cell count (x 109/l) Neutrophils (x 109/l) Lymphocytes (x 109/l) Platelets (x 109/l) TTP Evidence of active hemolysis

)

541

APPENDIX H

BILAG2004 Index Scoring

●

Scoring based on the principle of physician’s intention to treat.

Category

Definition

A

Severe disease activity requiring any of the following treatment: 1. Systemic high-dose oral glucocorticoids (equivalent to prednisolone >20 mg/day) 2. Intravenous pulse glucocorticoids (equivalent to pulse methylprednisolone ≥500 mg) 3. Systemic immunomodulators (include biologicals, immunoglobulins, and plasmapheresis) 4. Therapeutic high-dose anticoagulation in the presence of high-dose steroids or immunomodulators (e.g., warfarin with target INR 3 - 4)

B

Moderate disease activity requiring any of the following treatment: 1. Systemic low-dose oral glucocorticoids (equivalent to prednisolone ≤20 mg/day) 2. Intramuscular or intra-articular or soft-tissue glucocorticoids Injection (equivalent to methylprednisolone <500 mg) 3. Topical glucocorticoids 4. Topical immunomodulators 5. Antimalarials or thalidomide or prasterone or acitretin 6. Symptomatic therapy (e.g., NSAIDs for inflammatory arthritis)

C

Stable mild disease

D

Inactive disease but previously affected

E

System never involved

CONSTITUTIONAL

Category A Pyrexia recorded as 2 (same), 3 (worse), or 4 (new) AND Any 2 or more of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Weight loss ● Lymphadenopathy/splenomegaly ● Anorexia

Category B 542

Pyrexia recorded as 2 (same), 3 (worse), or 4 (new) OR

Any 2 or more of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Weight loss ● Lymphadenopathy/splenomegaly ● Anorexia BUT do not fulfill criteria for Category A.

Category C Pyrexia recorded as 1 (improving) OR One or more of the following recorded as > 0: ● Weight loss ● Lymphadenopathy/Splenomegaly ● Anorexia BUT does not fulfill criteria for Category A or Category B.

●

Previous involvement.

●

Category E

●

No previous involvement.

● ●

MUCOCUTANEOUS

Category A Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Skin eruption—severe ● Angio-edema—severe ● Mucosal ulceration—severe ● Panniculitis/bullous lupus—severe ● Major cutaneous vasculitis/thrombosis

Category B Any Category A features recorded as 1 (improving) OR Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Skin eruption—mild ● Panniculitis/bullous lupus—mild ● Digital infarcts or nodular vasculitis ● Alopecia—severe

Category C Any Category B features recorded as 1 (improving) OR Any of the following recorded as >0: ● Angio-edema—mild ● Mucosal ulceration—mild ● Alopecia—mild ● Periungual erythema/chilblains ● Splinter hemorrhages

Category D

● ● ●

Psychosis Acute inflammatory demyelinating polyradiculoneuropathy Mononeuropathy (single/multiplex) Cranial neuropathy Plexopathy Polyneuropathy Status epilepticus Cerebellar ataxia

APPENDIX H

Category D

Category B Any Category A features recorded as 1 (improving) OR Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Seizure disorder ● Cerebrovascular disease (not due to vasculitis) ● Cognitive dysfunction ● Movement disorder ● Autonomic disorder ● Headache severe unremitting ● Headache due to raised intracranial hypertension

Category C Any Category B features recorded as 1 (improving)

Category D Previous involvement.

Category E No previous involvement.

MUSCULOSKELETAL

Category A Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Myositis—severe ● Arthritis—severe

Previous involvement.

Category B Category E

Any Category A features recorded as 1 (improving) OR

No previous involvement.

Any of the following recorded as 2 (same), 3 (worse), or 4 (new): Myositis—mild Arthritis/tendonitis/tenosynovitis—moderate

NEUROLOGIC

Category A Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Aseptic meningitis ● Cerebral vasculitis ● Demyelinating syndrome ● Myelopathy ● Acute confusional state

Category C Any Category B features recorded as 1 (improving) OR Any of the following recorded as > 0: Arthritis/arthralgia/myalgia—mild

Category D Previous involvement. 543

APPENDIX H

Category E

Category C

No previous involvement.

Any Category B features recorded as 1 (improving).

CARDIORESPIRATORY

Category A Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Myocarditis/endocarditis + cardiac failure ● Arrhythmia ● New valvular dysfunction ● Cardiac tamponade ● Pleural effusion with dyspnoea ● Pulmonary hemorrhage/vasculitis ● Interstitial alveolitis/pneumonitis ● Shrinking lung syndrome ● Aortitis ● Coronary vasculitis

Category B Any Category A features recorded as 1 (improving) OR Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Pleurisy/pericarditis ● Myocarditis—mild

Category C Any Category B features recorded as 1 (improving).

Category D Previous involvement.

Category E No previous involvement.

GASTROINTESTINAL

Category A Any of the following recorded as 2 (same), 3 (worse) or 4 (new): ● Peritonitis ● Lupus enteritis/colitis ● Intestinal pseudo-obstruction ● Acute lupus cholecystitis ● Acute lupus pancreatitis

Category B

544

Any Category A feature recorded as 1 (improving) OR Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Abdominal serositis and/or ascites ● Malabsorption ● Protein losing enteropathy ● Lupus hepatitis

Category D Previous involvement.

Category E No previous involvement.

OPHTHALMIC

Category A Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Orbital inflammation/myositis/proptosis ● Keratitis—severe ● Posterior uveitis/retinal vasculitis—severe ● Scleritis—severe ● Retinal/choroidal vaso-occlusive disease ● Optic neuritis ● Anterior ischemic optic neuropathy

Category B Any Category A features recorded as 1 (improving) OR Any of the following recorded as 2 (same), 3 (worse), or 4 (new): ● Keratitis—mild ● Anterior uveitis ● Posterior uveitis/retinal vasculitis—mild ● Scleritis—mild

Category C Any Category B features recorded as 1 (improving) OR Any of the following recorded as >0: ● Episcleritis ● Isolated cotton-wool spots (cytoid bodies)

Category D Previous involvement.

Category E No previous involvement.

RENAL

Category A Two or more of the following, provided 1, 4, or 5 is included: 1. Deteriorating proteinuria (severe) defined as: (a) Urine dipstick increased by ≥2 levels; or (b) 24-hour urine protein >1 g that has not decreased (improved) by ≥50%; or

4. 5. 6.

Category B One of the following: 1. One of the Category A features. 2. Urine dipstick that has risen by 1 level to at least 2+. 3. Plasma creatinine >130 μmol/l and having risen to ≥115% but ≤130% of previous value.

Category C One of the following: 1. Mild/stable proteinuria defined as: (a) Urine dipstick ≥1+ but has not fulfilled criteria for categories A and B; or (b) 24-hour urine protein >0.25 g but has not fulfilled criteria for categories A and B; or (c) Urine protein-creatinine ratio >25 mg/mmol but has not fulfilled criteria for categories A and B; or (d) Urine albumin-creatinine ratio >25 mg/mmol but has not fulfilled criteria for categories A and B 2. Rising blood pressure (provided the recorded values are <140/90 mmHg) that has not fulfilled criteria for categories A and B, defined as: (a) Systolic rise of ≥30 mmHg; and (b) Diastolic rise of ≥15 mmHg

Category D Previous involvement.

Category E

APPENDIX H

2. 3.

(c) Urine protein-creatinine ratio >100 mg/mmol that has not decreased (improved) by ≥50%; or (d) Urine albumin-creatinine ratio >100 mg/mmol that has not decreased (improved) by ≥50% Accelerated hypertension. Deteriorating renal function (severe) defined as: (a) Plasma creatinine >130 μmol/l and having risen to >130% of previous value; or (b) GFR having fallen to <67% of previous value; or (c) GFR <50 ml/min per 1.73 m2, and last time was >50 ml/min per 1.73 m2 or was not measured Active urinary sediment. Histologic evidence of active nephritis within last 3 months. Nephrotic syndrome.

No previous involvement. Note: Although albumin-creatinine ratio and proteincreatinine ratio are different, we use the same cut off values for this index.

HEMATOLOGY

Category A TTP recorded as 2 (same), 3 (worse), or 4 (new) OR Any of the following: ● Hemoglobin <8 g/dl ● White cell count <1.0 x 109/l ● Neutrophil count <0.5 x 109/l ● Platelet count <25 x 109/l

Category B TTP recorded as 1 (improving) OR Any of the following: ● Hemoglobin 8 - 8.9 g/dl ● White cell count 1 - 1.9 x 109/l ● Neutrophil count 0.5 - 0.9 x 109/l ● Platelet count 25 - 49 x 109/l ● Evidence of active hemolysis

Category C Any of the following: ● Hemoglobin 9 - 10.9 g/dl ● White cell count 2 - 3.9 x 109/l ● Neutrophil count 1 - 1.9 x 109/l ● Lymphocyte count <1.0 x 109/L ● Platelet count 50 - 149 x 109/l ● Isolated Coombs’ test positive

Category D Previous involvement.

Category E No previous involvement.

545

APPENDIX I

BILAG2004 Index Glossary

INSTRUCTIONS ●

●

●

●

●

●

●

Only record features attributable to SLE disease activity and not due to damage, infection, thrombosis (in absence of inflammatory process) or other conditions. Assessment refers to manifestations occurring in the last 4 weeks compared with the previous 4 weeks. Activity refers to disease process that is reversible, whereas damage refers to permanent process/scarring (irreversible). Damage due to SLE should be considered a cause of features that are fixed/persistent (SLICC/ACR damage index uses persistence ≥6 months to define damage). In some manifestations, it may be difficult to differentiate SLE from other conditions because there may not be any specific test and the decision would then lie with the physician’s judgment on the balance of probabilities. Ophthalmic manifestations would usually need to be assessed by ophthalmologist (using the pro forma included with this glossary) and these items would need to be recorded retrospectively after receiving the response from ophthalmologist. Guidance for scoring:

(4) New Manifestations are recorded as new when it is a new episode occurring in the last 4 weeks (compared to the previous 4 weeks) that has not improved, including new episodes (recurrence) of old manifestations. ● New episode occurring in the last 4 weeks but also satisfying the criteria for improvement (following) would be classified as improving instead of new. ●

(3) Worse Refers to manifestations that have deteriorated in the last 4 weeks compared to the previous 4 weeks.

●

(2) Same Refers to manifestations that have been present for the last 4 weeks and the previous 4 weeks without significant improvement or deterioration (from the previous 4 weeks).

●

546

●

Also applies to manifestations that have improved over the last 4 weeks compared to the previous 4 weeks but do not meet the criteria for improvement.

(1) Improving Definition of improvement: (a) The amount of improvement is sufficient for consideration of reduction in therapy and would not justify escalation in therapy AND (b) Improvement must be present currently and for at least 2 weeks out of the last 4 weeks OR Manifestation that has completely resolved and remained absent over the whole of last 1 week

●

(0) Not present (ND) Not done ● It is important to indicate if a test has not been performed (particularly laboratory investigations) so that this will be recorded as such in the database and not as normal or absent (which is the default).

❑ INDICATE (TICK) IF NOT DUE TO SLE ACTIVITY ● For descriptors based on measurements (in renal and hematology systems), it is important to indicate if these are not due to lupus disease activity (for consideration of scoring) because they are usually recorded routinely into a database. CHANGE IN SEVERITY CATEGORY There are several items in the index that have been divided into categories of mild and severe (depending on definition). ● If a mild item deteriorates and fulfills the definition of severe category, the severe item should then be scored as new (4) and the mild item scored as worsening (3). ● If a severe item improves to the extent that it no longer fulfills definition of severe category (i.e., change to mild category), the mild item should then be scored as improving (1) and the severe item scored as not present (0). ●

1. 2. 3. 4.

Pyrexia Unintentional weight loss >5% Lymphadenopathy Anorexia

MUCOCUTANEOUS 5. Severe eruption

6. Mild eruption

7. Severe angio-edema

8. Mild angio-edema 9. Severe mucosal ulceration 10. Mild mucosal ulceration 11. Severe panniculitis or bullous lupus

12. Mild panniculitis or bullous lupus 13. Major cutaneous vasculitis/ thrombosis 14. Digital infarct or nodular vasculitis 15. Severe alopecia 16. Mild alopecia

Temperature >37.5° C documented. Lymph node more than 1 cm diameter (exclude infection).

APPENDIX I

CONSTITUTIONAL

>18% body surface area. Any lupus rash except panniculitis, bullous lesion, and angio-edema. Body surface area (BSA) is estimated using the rules of nines (used to assess extent of burns) as follows: Palm (excluding fingers) = 1% BSA Each lower limb = 18% BSA Each upper limb = 9% BSA Torso (front) = 18% BSA Torso (back) = 18% BSA Head = 9% BSA Genital (male) = 1% BSA ≤18% body surface area. Any lupus rash except panniculitis, bullous lesion, and angio-edema. Malar rash must be observed by a physician and has to be present continuously (persistent) for at least 1 week to be considered significant (to be recorded). Potentially life threatening (e.g., stridor). Angio-edema is a variant form of urticaria that affects the subcutaneous, submucosal, and deep dermal tissues. Not life threatening. Disabling (significantly interfering with oral intake), and extensive and deep ulceration. Must have been observed by a physician. Localized and/or non-disabling ulceration. Any one: ● >9% body surface area ● Facial panniculitis ● Panniculitis that is beginning to ulcerate ● Panniculitis that threatens integrity of subcutaneous tissue (beginning to cause surface depression) on >9% body surface area Panniculitis presents as a palpable and tender subcutaneous induration/nodule. Note that established surface depression and atrophy alone is likely to be due to damage. ≤9% body surface area does not fulfill any criteria for severe panniculitis (for panniculitis). Resulting in extensive gangrene or ulceration or skin infarction. Localized single or multiple infarct(s) over digit(s) or tender erythematous nodule(s). Clinically detectable (diffuse or patchy) hair loss with scalp inflammation (redness over scalp). Diffuse or patchy hair loss without scalp inflammation (clinically detectable or by history).

547

APPENDIX I

17. Peri-ungual erythema or chilblains

Chilblains are localized inflammatory lesions (may ulcerate) precipitated by exposure to cold.

18. Splinter hemorrhages

NEUROPSYCHIATRIC 19. Aseptic meningitis

20. Cerebral vasculitis 21. Demyelinating syndrome

22. Myelopathy

23. Acute confusional state

24. Psychosis

25. Acute inflammatory demyelinating polyradiculoneuropathy

26. Mononeuropathy (single/multiplex) 27. Cranial neuropathy 28. Plexopathy

29. Polyneuropathy 30. Seizure disorder 31. Status epilepticus 32. Cerebrovascular disease 548

Criteria (all): ● Acute/subacute onset ● Headache ● Fever ● Abnormal CSF (raised protein and/or lymphocyte predominance) but negative cultures. Preferably photophobia, neck stiffness, and meningeal irritation should be present as well (but these are not essential for diagnosis). Exclude CNS/meningeal infection and intracranial hemorrhage. Should be present with features of vasculitis in another system. Supportive imaging and/or biopsy findings. Discrete white matter lesion with associated neurologic deficit not recorded elsewhere. Ideally there should have been at least one previously recorded event. Supportive imaging required. Exclude multiple sclerosis. Acute onset of rapidly evolving paraparesis or quadriparesis and/or sensory level. Exclude intramedullary and extramedullary space occupying lesion. Acute disturbance of consciousness or level of arousal with reduced ability to focus, maintain, or shift attention. Includes hypo- and hyperaroused states and encompasses the spectrum from delirium to coma. Delusion or hallucinations.Does not occur exclusively during course of a delirium. Exclude drugs, substance abuse, and primary psychotic disorder. Criteria: ● Progressive polyradiculoneuropathy ● Loss of reflexes ● Symmetrical involvement ● Increased CSF protein without pleocytosis ● Supportive electrophysiology study Supportive electrophysiology study required. Except optic neuropathy, which is classified under ophthalmic system. Disorder of brachial or lumbosacral plexus resulting in neurologic deficit not corresponding to territory of single root or nerve. Supportive electrophysiology study required. Acute symmetric distal sensory and/or motor deficit. Supportive electrophysiology study required. Independent description of seizure by reliable witness. A seizure or series of seizures lasting ≥30 minutes without full recovery to baseline. Any one with supporting imaging: (not due to vasculitis) ● Stroke syndrome ● Transient ischemic attack

33.

34. 35.

36. 37.

38.

APPENDIX I

Intracranial hemorrhage. Exclude hypoglycemia, cerebral sinus thrombosis, vascular malformation, tumour, and abscess. Cerebral sinus thrombosis not included as definite thrombosis and not considered part of lupus activity. Cognitive dysfunction Significant deficits in any cognitive functions: ● Simple attention (ability to register and maintain information) complex attention ● Memory (ability to register, recall, and recognize information [e.g., learning, recall]) ● Visual-spatial processing (ability to analyze, synthesize, and manipulate visual-spatial information) ● Language (ability to comprehend, repeat, and produce oral/written material [e.g., verbal fluency, naming]) ● Reasoning/problem solving (ability to reason and abstract) ● Psychomotor speed ● Executive functions (e.g., planning, organizing, sequencing) In absence of disturbance of consciousness or level of arousal. Sufficiently severe to interfere with daily activities. Neuropsychologic testing should be done or corroborating history from third party if possible. Exclude substance abuse. Movement disorder Exclude drugs. Autonomic disorder Any one: ● Fall in blood pressure to standing >30/15 mmHg (systolic/ diastolic) ● Increase in heart rate to standing ≥30 bpm ● Loss of heart rate variation with respiration (max – min <15 bpm, expiration: inspiration ratio <1.2, Valsalva ratio <1.4) ● Loss of sweating over body and limbs (anhidrosis) by sweat test Exclude drugs and diabetes mellitus. Cerebellar ataxia Cerebellar ataxia in isolation of other CNS features. Usually subacute presentation. Severe lupus headache (unremitting) Disabling headache unresponsive to narcotic analgesia and lasting ≥3 days. Exclude intracranial space occupying lesion and CNS infection. Headache from IC hypertension Exclude cerebral sinus thrombosis. ●

MUSCULOSKELETAL 39. Severe myositis

40. Mild myositis

41. Severe arthritis

42. Moderate arthritis, tendonitis

Significantly elevated serum muscle enzymes with significant muscle weakness. Exclude endocrine causes and drug-induced myopathy. Significantly elevated serum muscle enzymes with myalgia but without significant muscle weakness. Asymptomatic elevated serum muscle enzymes not included. Exclude endocrine causes and drug-induced myopathy. Observed active synovitis ≥2 joints, with marked loss of functional range of movements and significant impairment of activities of daily living, that has been present on several days (cummulatively) over the last 4 weeks. Tendonitis/tenosynovitis or active synovitis ≥1 or tenosynovitis joint (observed or through history), with some loss of functional range of movements, that has been present on several days over the last 4 weeks.

549

APPENDIX I

43. Mild arthritis or arthralgia or myalgia

Inflammatory type of pain (worse in the morning with stiffness, usually improves with activity, and not brought on by activity) over joints/muscle. Inflammatory arthritis that does not fulfill the previously cited criteria for moderate or severe arthritis.

CARDIORESPIRATORY 44. Mild myocarditis

45. Cardiac failure

46. Arrhythmia

47. New valvular dysfunction

48. Pleurisy/pericarditis

49. Cardiac tamponade 50. Pleural effusion with dyspnea 51. Pulmonary haemorrhage/vasculitis

52. Interstitial alveolitis/pneumonitis

53. Shrinking lung syndrome

54. Aortitis

55. Coronary vasculitis 550

Inflammation of myocardium with raised cardiac enzymes and/or ECG changes and without resulting cardiac failure, arrhythmia, or valvular dysfunction. Cardiac failure due to myocarditis or non-infective inflammation of endocardium or cardiac valves (endocarditis). Cardiac failure due to myocarditis is defined by left ventricular ejection fraction ≤40% and pulmonary edema or peripheral edema. Cardiac failure due to acute valvular regurgitation (from endocarditis) can be associated with normal left ventricular ejection fraction. Diastolic heart failure is not included. Arrhythmia (except sinus tachycardia) due to myocarditis or non-infective inflammation of endocardium or cardiac valves (endocarditis). Confirmation by electrocardiogram required (history of palpitations alone inadequate). New cardiac valvular dysfunction due to myocarditis or noninfective inflammation of endocardium or cardiac valves (endocarditis). Supportive imaging required Convincing history/physical findings you would consider treating. In absence of cardiac tamponade or pleural effusion with dyspnea. Do not score if you are unsure whether or not it is pleurisy/pericarditis. Supportive imaging required. Supportive imaging required. Inflammation of pulmonary vasculature with hemoptysis and/or dyspnea and/or pulmonary hypertension. Supportive imaging and/or histologic diagnosis required. Radiologic features of alveolar infiltration not due to infection or hemorrhage required for diagnosis. Corrected gas transfer Kco reduced to <70% normal or fall of >20% if previously abnormal ongoing activity would be determined by clinical findings and lung function tests, and repeated imaging may be required in those with deterioration (clinically or lung function tests) or failure to respond to therapy. Acute reduction (>20% if previous measurement available) in lung volumes (to <70% predicted) in the presence of normal corrected gas transfer (Kco) and dysfunctional diaphragmatic movements. Inflammation of aorta (with or without dissection) with supportive imaging abnormalities. Accompanied by >10 mmHg difference in BP between arms and/or claudication of extremities and/or vascular bruits. Repeated imaging would be required to determine ongoing activity in those with clinical deterioration or failure to respond to therapy. Inflammation of coronary vessels with radiographic evidence of non-atheromatous narrowing, obstruction, or aneurysmal changes.

56. Lupus peritonitis 57. Serositis 58. Lupus enteritis or colitis 59. Malabsorption

60. Protein-losing enteropathy

61. Intestinal pseudo-obstruction 62. Lupus hepatitis

63. Acute lupus cholecystitis 64. Acute lupus pancreatitis

Serositis presenting as acute abdomen with rebound/guarding. Not presenting as acute abdomen. Vasculitis or inflammation of small or large bowel with supportive imaging and/or biopsy findings. Diarrhea with abnormal D-xylose absorption test or increased fecal fat excretion after exclusion of celiac disease (poor response to gluten-free diet) and gut vasculitis. Diarrhea with hypoalbuminemia or increased fecal excretion of IV radiolabeled albumin after exclusion of gut vasculitis and malabsorption. Subacute intestinal obstruction due to intestinal hypomotility. Raised transaminases. Absence of autoantibodies specific to autoimmune hepatitis (e.g., anti-smooth muscle, anti-liver cytosol 1) and/or biopsy appearance of chronic active hepatitis. Hepatitis typically lobular with no piecemeal necrosis. After exclusion of gallstones and infection. Usually associated multisystem involvement.

APPENDIX I

GASTROINTESTINAL

OPHTHALMIC 65. Orbital inflammation 66. Severe keratitis 67. Mild keratitis 68. Anterior uveitis 69. Severe posterior uveitis and/ or retinal 70. Mild posterior uveitis and/or retinal 71. Episcleritis 72. Severe scleritis

73. Mild scleritis 74. Retinal/choroidal vaso-occlusive

75. Isolated cotton-wool spots 76. Optic neuritis 77. Anterior ischemic optic neuropathy

Orbital inflammation with myositis and/or extra-ocular muscle swelling and/or proptosis. Supportive imaging required. Sight threatening. Includes corneal melt and peripheral ulcerative keratitis. Not sight threatening. Sight-threatening and/or retinal vasculitis vasculitis not due to vaso-occlusive disease. Not sight threatening. Vasculitis not due to vaso-occlusive disease. Necrotising anterior scleritis. Anterior and/or posterior scleritis requiring systemic steroids/immunosuppression and/or not responding to NSAIDs. Anterior and/or posterior scleritis not requiring systemic steroids. Excludes necrotising anterior scleritis. Includes: retinal arterial and venous occlusion disease, serous retinal and/or retinal pigment, and epithelial detachments secondary to choroidal vasculopathy. Also known as cytoid bodies. Excludes anterior ischemic optic neuropathy. Visual loss with pale swollen optic disc due to occlusion of posterior ciliary arteries.

RENAL 78. Systolic blood pressure 79. Diastolic blood pressure 80. Accelerated hypertension

Blood pressure rising to >170/110 mmHg within 1 month with grade 3 or 4 Keith-Wagener-Barker retinal changes (flame-shaped hemorrhages or cotton-wool spots or papilloedema).

551

APPENDIX I

81. 82. 83. 84. 85.

Urine dipstick Urine albumin-creatinine ratio Urine protein-creatinine ratio 24 hour urine protein Nephrotic syndrome

86. Plasma/serum creatinine 87. GFR

88. Active urinary sediment

89. Histology of active nephritis

On freshly voided urine sample. On freshly voided urine sample. Criteria: ● Heavy proteinuria (≥3.5 g/day or protein-creatinine ratio ≥300 mg/mmol or albumin-creatinine ratio ≥300 mg/mmol) ● Hypoalbuminemia ● Edema MDRD formula: GFR = 170 x [serum creatinine (mg/dl)]−0.999 x [age]−0.176 x [serum urea (mg/dl]−0.17 x [serum albumin (g/dl)]0.318 x [0.762 if female] x [1.180 if African ancestry] ● Units = ml/min per 1.73 m2 ● Normal: male = 130 ± 40, female = 120 ± 40 Conversion: ● Serum creatinine - mg/dl = (μmol/l)/88.5 ● Serum urea - mg/dl = (mmol/l) x 2.8 ● Serum albumin - g/dl = (g/l)/10. Creatinine clearance not recommended because it is not reliable. Pyuria [>5 WCC/hpf or >10 WCC/mm3 (μl)] OR hematuria [>5 RBC/hpf or >10 RBC/mm3 (μl)] OR red cell casts OR white cell casts. In absence of other causes (especially infection, vaginal bleed, calculi). WHO Classification (1995; any one): ● Class III – (a) or (b) subtypes ● Class IV – (a), (b), or (c) subtypes ● Class V – (a), (b), (c), or (d) subtypes ● Vasculitis OR ISN/RPS Classification (2003; any one): ● Class III – (A) or (A/C) subtypes ● Class IV – (A) or (A/C) subtypes ● Class V ● Vasculitis within last 3 months. Glomerular sclerosis without inflammation not included.

HEMATOLOGY

552

90. 91. 92. 93. 94.

Hemoglobin White cell count Neutrophil count Lymphocyte count Platelet count

Exclude dietary deficiency and GI blood loss.

Exclude thrombocytopenia of antiphospholipid syndrome.

96. Evidence of active hemolysis

Thrombotic thrombocytopaenic purpura. Clinical syndrome of micro-angiopathic hemolytic anemia and thrombocytopenia in absence of any other identifiable cause. Positive Coomb’s test and evidence of hemolysis (raised bilirubin or raised reticulocyte count or reduced haptoglobulins).

APPENDIX I

95. TTP

97. Isolated positive Coomb’s test

ADDITIONAL ITEMS These items are required mainly for calculation of GFR: i. Weight ii. African ancestry iii. Serum urea iv. Serum albumin

553

APPENDIX J

EUROPEAN CONSENSUS LUPUS ACTIVITY MEASUREMENT INDEX Generalized manifestations

Fever, fatigue

0.5

Articular manifestations

Arthritis, evolving arthralgia

1

Active mucocutaneous manifestations

Malar rash, generalized rash, discoid rash, skin vasculitis, oral ulcers

0.5

Evolving mucocutaneous manifestationsa

1

Myositisb

2

Pericarditis

1

Intestinal manifestations

Intestinal vasculitis, sterile peritonitis

2

Pulmonary manifestations

Pleurisy, pneumonitis, ingravescent dyspnea

1

Evolving neuropsychiatric manifestationsb

Headache/migraine, seizures, stroke, organic

2

brain disease, psychosis Renal manifestationsb,c

Proteinuria, urinary casts, hematuria, raised

0.5

serum creatinine or reduced creatinine clearance Evolving renal manifestationsd Hematologic features

2d Nonhemolytic anemia, hemolytic anemia leukopenia

1

(or lymphopenia), thrombocytopenia

Erythrocyte sedimentation rate

Raised ESR

1

Hypocomplementemia

C3, CH50

1

Evolving hypocomplementemia observation a

1

Further details about the items to be recorded can be found in: Griffilles B., Mosca M., Gordon C. Assessment of patients with systemic lupus erythematosus and the use of lupus disease activity indices. Best Practice & Research Clinical Rheumatology 2005; 19 (5) 685-708.

554

b

If any of the above mucocutaneous manifestations are new or have worsened since the last observation, add 1 point.

c

If this system (or manifestation) is the only involvement present from among items 1 through 10, add 2 points.

d

Excluding patients with end-stage chronic renal disease.

e

If any of the previous renal manifestations are new or have worsened since the last 2 observations, add 2 points.

Clinical Variables

Points

Laboratory Variables

Points

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

1 1 1 1 1 2 1 2 1 3 3 1 1 1 2 3 2 1 1 3 1 33

22. ESR 25-50 mm/h ESR >50 mm/h 23. DNA binding <50% DNA binding >50% 24. Mild hypocomplementemia f (CH50 80-150 U/ml) Severe hypocomplementemia f (CH50 <80 U/ml) 25. CPK >100, aldolase >10U/ml 26. LE anticoagulant 27. Proteinuria <1.5 g/24 h e Proteinuria >1.5 g/24 h e 28. 5-15 RBC or 1-3 casts/HPF >15 RBC or >3 casts/HPF 29. Hemolytic anemia (>8 g Hb) Hemolytic anemia (<8 g Hb) 30. Thrombocytopenia (40-100,000) Thrombocytopenia (<40,000) 31. Neutropenia (<3,000) 32. Lymphopenia (<1,000)

1 2 1 2

2 2 1 1 2 1 2 1 2 1 2 1 1

Maximum

19

Fatigue Temperature >38°C Arthralgia Arthritis (joint effusion) Myalgia Muscle weakness Serositis (pain) Serositis (frict. rub/X-ray/sonogr.) Vasculitis (minor a) Vasculitis (major b) Bulluous skin lesions Active SLE rash Active alopecia Mucosal ulcers CNS (minor c) CNS (major d, e) Cranial nerve palsy Blood pressure >150/90 Lymphadenopathy Noninfectious lung infiltratey Active thromboembolic event Maximum Total SIS:............ (maximum: 52) Physician’s assessment of activity: 0 __________________________ 100 mm None Most severe

APPENDIX K

SLE ACTIVITY INDEX SCORE (SIS) FORM

1

Categories: SIS 0-4: Inactive disease SIS 4-8: Mildly active disease (+)e SIS 9-12: Moderate activity (++)e SIS 13-15: Active disease (+++) SIS >15: Very active disease (++++)

REFERENCES: 1. Smolen JS. Clinical and serological features: Incidence and diagnostic approach. In JS Smolen, CC Zielinsky (eds.), Systemic Lupus Erythematosus. Berlin: Springer 1987:170-196. 2. Bencivelli W, Vitali C, Isenberg DA, et al. Disease activity in systemic lupus erythematosus. Report of the Consensus Study Group of the European Workshop for Rheumatology Research, III: Development of a computerised clinical chart and its application to the comparison of different indices of disease activity [review]. Clin Exp Rheumatol 1992;10:549-554.

Key: a. b. c. d. e.

Raynaud’s phenomenon, periungual infarcts, purpura. Ulcerations, cytoid bodies, mononeuritis. Confusion, depression, organic brain syndrome. Stupor, coma, seizures, CVA. Usually there will be no very active manifestations in vital organs without concomitant involvement of other organ systems and/or abnormal laboratory findings. f. Consider possibility of inborn C4 deficiency.

555

APPENDIX L

SYSTEMIC LUPUS ACTIVITY MEASURE—REVISED FORM Patient’s Initials:

Hospital No:

Date of Assessment:

Manifestations should be attributable to SLE and should be present for significant period of time or frequency during the MONTH prior to assessment. Description

Scoring System

Absent

Mild

Moderate

Severe

CONSTITUTIONAL 1. Weight Loss 2. Fatigue

3. Fever

Score 1: Mild to moderate ≤0% body weight Score 3: Severe >10% body weight Score 1: Does not limit normal activity, only with recreational Score 2: Limits ADL Score 1: 37.5 to 38.5°C Score 2: >38.5°C

0

1

3

0

1

2

0

1

2

Score 1: Present

0

1

Score 1: Hair loss with brushing or spontaneous Score 2: Alopecia observed Score 1: <20% total body surface Score 2: 20 to 50% total body surface Score 3: >50% total body surface Score 1: <20% total body surface Score 2: 20 to 50% total body surface Score 3: >50% Total body surface or necrosis

0

1

2

0

1

2

3

0

1

2

3

0

1

3

0

1

3

0

1

3

0

1

2

0

1

2

0

1

2

3

0 0

1 1

2

3

2

3

2

3

INTEGUMENT 4. Oral or nasal ulcers, or periungual erythema, or malar rash, or photosensitive rash, or nailfold infarct 5. Alopecia 6. Erythematous maculopapular rash, or discoid lupus, or lupus profundus, or bullous lesions 7. Vasculitis (leukocytoclastic vasculitis, urticaria, palpable purpura, livedo reticularis, ulcer, or panniculitis)

EYE 8. Cytoid bodies 9. Hemorrhages (retinal or choroidal) or episcleritis 10. Papillitis or pseudotumor cerebri

Score 1: Present Score 3: Visual acuity <20/200 Score 1: Present Score 3: Visual acuity <20/200 Score 1: Present Score 3: Visual acuity <20/200 or field cut

RETICULOENDOTHELIAL 11. Lymphadenopathy 12. Hepatomegaly or splenomegaly

Score 1: Shotty Score 2: Diffuse or nodes >1.5 cm x 1 cm Score 1: Palpable only with inspiration Score 2: Palpable without inspiration

PULMONARY 13. Pulmonary

Score 1: Shortness of breath or pain, exam normal Score 2: Shortness of breath or pain with exercise or abnormal lung exam Score 3: Shortness of breath or pain at rest or abnormal lung exam

14. Raynaud’s 15. Hypertension

Score 1: Present Score 1: Diastolic 90 - 105 Score 2: Diastolic 105 -115 Score 3: Diastolic >115 Score 2: Chest pain or arrhythmia Score 3: Myocarditis with hemodynamic compromise and/or arrhythmia

CARDIOVASCULAR

16. Carditis

0

GASTROINTESTINAL 17. Abdominal pain (serositis, pancreatitis, ischemic bowel, etc.)

556

Score 1: Complaint Score 2: Limiting pain Score 3: Peritoneal signs/ascites

0

1

Scoring System

Absent

Mild

Moderate

Severe

NEUROMOTOR 18. Stroke syndrome [includes mononeuritis multiplex, reversible neurological deficit (RND), cerebrovascular accident (CVA), retinal vascular thrombosis] 19. Seizure 20. Cortical dysfunction

21. Headache (including migraine equivalents and aseptic meningitis) 22. Myalgia/Myositis

Score 2: RND, or mononeuritis multiplex, or cranial neuropathy, or chorea Score 3: CVA/myelitis, retinal vascular occlusion

0

2

3

Score 2: One or more per month Score 3: Status epilepticus Score 1: Mild depression/personality disorder or cognitive deficit Score 2: Change in sensorium or severe depression or limiting cognitive impairment Score 3: Psychosis or dementia or coma Score 1: Symptoms or transient neuro-deficit Score 2: Interferes with normal activities/aseptic meningitis Score 1: Complaint Score 2: Limits some activity Score 3: Incapacitating

0

2

3 3

0

1

2

0

1

2

0

1

2

3

0

1

2

3

0

1

2

3

0

1

2

3

0

1

2

3

0

1

2

3

0

1

2

3

0

1

2

3

0

1

2

3

APPENDIX L

Description

JOINTS 23. Joint pain

Score 1: Arthralgia only Score 2: Objective inflammation Score 3: Limited function

24. Hematocrit

Score 1: 30 - 35 Score 2: 25 - 29.9 Score 3: <25 Score 1: 2000 - 3500 Score 2: 1000 - 2000 Score 3: <1000 Score 1: 1000 -1499 Score 2: 500 - 999 Score 3: <500 Score 1: 100 - 150 Score 2: 50 - 99 Score 3: <50 Score 1: 25 - 50 Score 2: 51 - 75 Score 3: >75 Score 1: SCr 120 – 180 μmol/l or 60 - 79% CrCl Score 2: SCr 181 – 350 μmol/l or 30 - 59% CrCl Score 3: SCr >350μmol/l or <30% CrCl Score 1: >5 RBC and/or WBC/hpf, and/or 1 - 3 granular, and/or non-RBC casts/hpf, and/or 1+ proteinuria, and/or <500 mg 24-h urine protein Score 2: >10 RBC, and/or WBC/hpf or >3 granular, and/or non-RBC casts/hpf, and/or 2+ - 3+ proteinuria, and/or 0.5 - 3.5 24-h urine protein Score 3: >25 RBC, and/or WBC/hpf, and/or RBC casts, and/or 4+ proteinuria, and/or >3.5 g 24-h urine problem

LABORATORY

25. WBC

26. Lymphocyte count

27. Platelet count (x1,000)

28. ESR (Westergren)

29. Serum creatinine or creatinine clearance 30. Urine sediment

557

APPENDIX M

SYSTEMIC LUPUS ERYTHEMATOSUS QUALITY OF LIFE QUESTIONNAIRE (SLEQOL) Use this scale to answer the following question: 1 = not difficult at all, 2 = hardly difficult, 3 = somewhat difficult, 4 = moderately difficult, 5 = quite difficult, 6 = very difficult, 7 = extremely difficult. How difficult has each of these activities been in the last week as a result of your SLE? 1 Walking outdoors on level ground

1..2..3..4..5..6..7

2 Shopping

1..2..3..4..5..6..7

3 Turning taps on and off

1..2..3..4..5..6..7

4 Going to the market

1..2..3..4..5..6..7

5 Bathing and drying yourself

1..2..3..4..5..6..7

6 Walking 3 kilometers

1..2..3..4..5..6..7

Use this scale to answer the next two questions: 1 = not at all, 2 = hardly troubled, 3 = somewhat troubled, 4 = moderately troubled, 5 = quite troubled, 6 = very troubled, 7 = extremely troubled. How troubled have you been in the last week by each of these social or occupational activities as a result of your SLE? 7 Work and school performance

1..2..3..4..5..6..7

8 Interference with career or education

1..2..3..4..5..6..7

9 Missing work or school

1..2..3..4..5..6..7

10 Relationship with friends and relatives

1..2..3..4..5..6..7

11 Taking part in sports

1..2..3..4..5..6..7

12 Sex

1..2..3..4..5..6..7

13 Taking part in social activities

1..2..3..4..5..6..7

14 Unable to go out under the sun

1..2..3..4..5..6..7

15 Making less money because I have SLE

1..2..3..4..5..6..7

How troubled have you been by each of these symptoms in the last week as a result of your SLE? 16 Poor memory

1..2..3..4..5..6..7

17 Loss of appetite

1..2..3..4..5..6..7

18 Fatigue

1..2..3..4..5..6..7

19 Poor concentration

1..2..3..4..5..6..7

20 Itchy skin

1..2..3..4..5..6..7

21 Sore mouth

1..2..3..4..5..6..7

22 Sore, painful or stinging skin

1..2..3..4..5..6..7

23 Joint pain and swelling

1..2..3..4..5..6..7

Use this scale to answer the next question: 1 = not at all, 2 = hardly troubled, 3 = somewhat troubled, 4 = moderately troubled, 5 = quite troubled, 6 = very troubled, 7 = extremely troubled. How troubled have you been by each of these problems related to medical treatment in the last week as a result of your SLE? 24 Fear of needles

1..2..3..4..5..6..7

25 Dietary restrictions

1..2..3..4..5..6..7

26 Inconvenience of daily medication

1..2..3..4..5..6..7

27 Inconvenience of frequent clinic visits

1..2..3..4..5..6..7

Use this scale to answer the next two questions: 1 = not at all, 2 = hardly ever, 3 = somewhat often, 4 = moderately often, 5 = quite often, 6 = very often, 7 = extremely often. How often during the last week have you been troubled by these emotions as a result of your SLE?

558

28 Self-consciousness

1..2..3..4..5..6..7

29 Feeling low

1..2..3..4..5..6..7

30 Depression

1..2..3..4..5..6..7

31 Anxiety

1..2..3..4..5..6..7

32 I wish other people did not know I have SLE

1..2..3..4..5..6..7

33 Being made fun of by my friends and colleagues

1..2..3..4..5..6..7

34 Low self-esteem

1..2..3..4..5..6..7

35 Embarrassment about my SLE

1..2..3..4..5..6..7

36 Concern about the financial burden to my family

1..2..3..4..5..6..7

37 Concern that medicines do not work

1..2..3..4..5..6..7

38 Concern about side effects of medicines

1..2..3..4..5..6..7

39 Fear of receiving bad news from doctors

1..2..3..4..5..6..7

40 Consuming more alcohol or tobacco

1..2..3..4..5..6..7

APPENDIX M

How often in the last week have you been troubled by these feelings as a result of your SLE?

Data from Leong KP, Kong KO, Thong BY et al. Development and preliminary validation of a systemic lupus erythematosusspecific quality-of-life instrument (SLEQOL). Rheumatology (Oxford) 2005;44:1267.

559

APPENDIX N

ENGLISH VERSION OF THE SLE SYMPTOM CHECKLIST (SSC) No (0)

Yes (1)

Painful joints

❒

❒

Painful muscles

❒

❒

Headache

❒

❒

Fatigue

❒

❒

Ulcers in mouth or throat

❒

❒

Hair loss

❒

❒

Skin rash

❒

❒

Red and painful eyes

❒

❒

Pain while breathing

❒

❒

Shortness of breath

❒

❒

‘White’ fingers in cold weather

❒

❒

Itch

❒

❒

Ankle edema

❒

❒

Chubby cheeks/face

❒

❒

More appetite

❒

❒

Less appetite

❒

❒

Pimples

❒

❒

Facial hair growth

❒

❒

Blue/purple stretch marks on the skin

❒

❒

Spontaneous bruises

❒

❒

Poor wound healing

❒

❒

Muscle weakness

❒

❒

Blurred vision

❒

❒

Nightmares

❒

❒

Mood changes

❒

❒

Nausea/vomiting

❒

❒

Stomach complaints

❒

❒

Sensitivity to sunlight

❒

❒

Sensitivity to artificial light

❒

❒

Fits

❒

❒

Fainting

❒

❒

Genital sores

❒

❒

Chest pain

❒

❒

Loss of concentration

❒

❒

Muscle cramps

❒

❒

Vulnerable skin

❒

❒

If yes, how burdensome Not (1) A little (2) Quite (3)

Extremely (4)

Did you, in the past month, have…

Disturbed memory

❒

❒

Weight gain

❒

❒

j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j

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Do you have other symptoms? If yes, please specify. Data from Grootscholten C, Ligtenberg G, Derksen RHWM et al. Health-related quality of life in patients with systemic lupus erythematosus: Development and validation of a lupus specific symptom checklist. Qual Life Res 2003;12:635.

560

Index

Note: Page numbers followed by f, t, and b indicate figures, tables, and boxed text, respectively.

A Abatacept, 517t, 520 for lupus nephritis, 346 Abdominal pain differential diagnosis of, 389-390, 389t in intestinal pseudo-obstruction, 385 in mesenteric insufficiency, 384 in mesenteric/intestinal vasculitis, 384 Abetimus, for lupus nephritis, 346 aCCP ELISA, 270-271 Acebutol, in drug-induced lupus, 65t ACE inhibitors, for lupus nephritis, 344-345 Acetaminophen, in pregnancy, 453-454, 454t Acquired immunodeficiency syndrome, 151-152 ACTH, corticosteroid effects on, 495 α-Actinin, anti-dsDNA antibody binding to, 159 Activated protein C, in antiphospholipid syndrome, 237, 411 Acute confusional state, 417-418. See also Cognitive dysfunction Acute pancreatitis, 388-389 Acute pneumonitis, 374, 375t Acute reversible hypoxemic syndrome, 378 Acute toxic epidermal necrolysis–like cutaneous lupus erythematosus, 318, 319, 352, 353 Adenosine triphosphate (ATP) in apoptosis, 295 in necrosis, 296-297 Adhesion molecules. See Cell adhesion molecules Adnz1 susceptibility locus, in murine models, 176t, 178 Adrenocorticotropic hormone (ACTH), corticosteroid effects on, 495 AEOL-10113, 306-307 AEOL-10150, 306-307 Aerobic exercise, for fatigue, 331 African Americans. See also Racial/ethnic differences nitric oxide in, 305 Age at onset, 6 prognosis and, 29, 29t Aicardi-Goutiéres syndrome, with coexistent SLE, 70 AIDS, 151-152 Aire gene, 101 Airways disease, 376-377. See also Pulmonary disease Alfalfa, disease flares due to, 56, 70 Alopecia nonscarring, 358 scarring, 355, 355f Alveolar hemorrhage, 374-375, 375f, 375t

American College of Rheumatology (ACR) classification criteria of, 1, 24-25, 473, 474t for cutaneous disease, 474t incomplete lupus and, 473-475, 477t Disease Damage Index of, 26-27 Aminoguanidine, 305t, 306 Aminotransferase, elevated, in neonatal lupus, 470 Amsler grid, 447 Anakinra, for lupus nephritis, 347 Androgens gene targets of, 88t immunologic effects of, 87-88, 87t therapeutic use of, 484-485 Anemia, 408-409 of chronic disease, 408 fatigue and, 330 hemolytic, 408 corticosteroids for, 492 in incomplete lupus, 475, 478t-479t iron deficiency, 408 pernicious, 383-384 in pregnancy, 452 pure red cell aplasia and, 409 Anesthesia, epidural obstetric, 456 Angina pectoris, 362t, 368-370 Angiography fluorescein in lupus choroidopathy, 445 in retinal disease, 443, 443f, 445 indocyanine green, in choroidal disease, 445 magnetic resonance, in neuropsychiatric disease, 421 Angiotensin-converting enzyme (ACE) inhibitors, for lupus nephritis, 344-345 Angiotensin-receptor blockers, for lupus nephritis, 344-345 Animal models. See Murine models Annexin, in antiphospholipid syndrome, 237, 238 Anorexia, 333 Antianxiety agents, for neuropsychiatric disease, 423 Anti-β2GPI in antiphospholipid syndrome, 234-238 lupus anticoagulant and, 234 Antibiotic prophylaxis, for bacterial endocarditis, 507 Anti–B-lymphocyte stimulator (BLyS), 516-518, 517t Anti-BLyS monoclonal antibody, for lupus nephritis, 346-347 Antibodies. See also Autoantibodies; Immunoglobulin(s) abnormalities of, opportunistic infections and, 396 lymphocytotoxic, in neuropsychiatric disease, 242-243, 242t monoclonal, 516-521, 517t

561

INDEX

562

Antibodies (Continued) anticomplement, 207 for autoantibody studies, 157-158 for severe/life-threatening disease, 507 structure of, 156-157, 156f Anti-C1q, as lupus nephritis biomarker, 51 Anti-C1q assay, for immune complexes, 221-222 Anti-C3 assay, for immune complexes, 222 Anti-C5 monoclonal antibodies, 207, 517t, 519 Anticardiolipin antibodies, 156. See also Antiphospholipid antibodies in antiphospholipid syndrome, 410. See also Antiphospholipid syndrome autoimmune hemolytic anemia and, 408 cognitive dysfunction and, 420 ELISA for, 233-234 in immune complexes, 221 in mixed connective tissue disease, 433-434 pathogenicity of, 158 Anticardiolipin antibody test, for antiphospholipid antibodies, 409 in antiphospholipid syndrome, 411 Anticardiolipin ELISA, for antiphospholipid syndrome, 233-234 Anti-CD20 monoclonal antibody, 517t, 519 for lupus nephritis, 290 Anti-CD22 monoclonal antibody, 517t, 519 Anti-CD40L monoclonal antibody, 517t, 520 for lupus nephritis, 346 Anti-chromatin antibodies, 258-262. See also Anti-histone antibodies Anticoagulation for antiphospholipid syndrome, 237, 368, 378, 411-412, 511-513, 513t in pregnancy, 456, 513-514, 514t for neuropsychiatric disease, 423 in pregnancy, 454, 454t adverse effects of, 514 in antiphospholipid syndrome, 456, 513-514, 514t for pulmonary embolism, 378 for thromboembolic disease, 378 for valvular heart disease, 368 Anti-complement antibodies, 189 therapeutic use of, 206-207, 207f Anticyclic citrullinated peptides ELISA, 270-271 Antidepressants, for neuropsychiatric disease, 423 Anti-DNA antibodies, 156, 225-230. See also Autoantibodies anti-dsDNA, 225-226, 225t in lupus nephritis, 287-288 in phagocytosis, 281, 283 in pregnancy, 452 antigenic DNA and, 226 antigenic drive and, 228-229 anti-NR2 glutamate receptor, 243 anti-ssDNA, 156, 225-226, 225t in apoptotic cell clearance, 281 assays for, 225-226 avidity of, 226 as biomarkers, 48t, 49, 225, 226-227 clinical expression of, 226-227 in cytokine production, 229-230 in disease activity, 226-227 in immune complex production, 229-230, 281 induction of, 228-229 in lupus nephritis, 158-160, 229, 287-288 in disease monitoring, 338-339 in normal immunity, 227 in pathogenesis, 157-160, 229-230, 281 in phagocytosis, 281 in pregnancy, 452 properties of, 225-226, 225t specificity of, 226

Anti–DNA-histone antibodies, 259, 259f Anti-dsDNA antibodies, 225-226, 225t. See also Anti-DNA antibodies in lupus nephritis, 287-288 in phagocytosis, 281, 283 in pregnancy, 452 Antiemetics, with cyclophosphamide, 501b Anti–endothelial cell antibody, in vasculitis, 311-312 Antiganglioside antibodies, in neuropsychiatric disease, 242-243 Antigen(s) dendritic capture of, 121-122, 122f DNA as, 226 in immune complexes, 221 Antigen-antibody complexes. See Immune complexes Antigen-presenting cells dendritic cells as, 121-122, 122f T cells and, 100 Anti-histone antibodies, 156, 258-262 against histone-DNA complexes, 259-260, 259f against histone-histone complexes, 259-260, 259f historical perspective on, 258-259 against nucleosomes, 261-262, 262t against single histones, 259-260, 259f, 260f target antigens for, 259-261, 259f Anti-5-HT antibodies, in congenital heart block, 249-250 Antihypertensives, for lupus nephritis, 344-345 Anti-interferon antibody, 517t, 518 Anti–interleukin-6 monoclonal antibody, 517t, 518 Anti–Jo-1 antibody, 429t, 431 Anti-Ku antibody, in overlap syndromes, 429t, 431 Anti-La antibodies, as biomarkers, 49 Anti-La/SS antibodies. See Anti-SSA/Ro–SSB/La antibodies Anti–LMS complex, 277-278 Anti-LSM4 antibodies, 277-278 Antimalarials, 483-484. See also Chloroquine; Hydroxychloroquine; Quinacrine cardiotoxicity of, 365-366 for constitutional features, 334 for cutaneous LE, 358 disease course and, 14 for incomplete lupus, 479, 480t ocular toxicity of, 447, 484 Antineuronal antibodies, 241-245, 415-416 antigenic specificity of, 242-243, 243t assays for, 241, 241t clinical associations of, 242-245, 242t in neuropsychiatric disease, 242-245, 242t prevalence of, 241, 241t Anti-neutrophil antibodies, in lupus neutropenia, 284 Anti–neutrophil cytoplasmic antibody testing, in vasculitis, 269t, 271 Anti-NR2 glutamate receptor antibodies, in neuropsychiatric disease, 243, 416 Anti-nRNP antibodies, 276-277, 278 Antinuclear antibody (ANA) as biomarkers, 48t, 49 in classification, 474 in parvovirus B19 infection, 151 as precursors of clinical disease, 103, 134 Antinuclear antibody–negative disease, 24 Antinuclear antibody test, 24 Antinuclear ribonucleoprotein, as biomarker, 49 Anti-nucleosome antibodies, 156, 261-262, 262t Anti-P antibodies, in neuropsychiatric disease, 242t, 243, 416 Antiphospholipid antibodies, 233-238 anticardiolipin ELISA for, 233-234 in antiphospholipid syndrome, 410. See also Antiphospholipid syndrome atherosclerosis and, 306, 344, 369 in autoimmune hemolytic anemia, 408

Anti-RNP antibodies, in mixed connective tissue disease, 433, 436 Anti-Ro52 antibodies, in congenital heart block, 249 Anti-Ro antibodies, 156 as biomarker, 49 Anti-Ro/SSA antibodies. See Anti-SSA/Ro–SSB/La antibodies Anti-Sm antibodies, 274-276, 275f, 278-279 as biomarkers, 49 in mixed connective tissue disease, 432, 433 Anti-spliceosomal antibodies, 274-279 anti–LMS complex, 277-278 anti-LSM4, 277-278 anti-Sm. See Anti-Sm antibodies anti–SR protein, 278 anti–U1 RNP, 276-277 anti–U2 RNP, 277 assays for, 278-279 rare, 278 Anti–SR protein antibody, 278 Anti-SSA/Ro–SSB/La antibodies arrhythmias and, 467 as biomarkers, 49 in breast milk, 470-471 in congenital heart block, 248-254, 256f, 363, 466-469, 471 apoptosis and, 250-253 histopathology and, 250 murine model of, 253-254 target autoantigens and, 248-250 in cutaneous lupus, 353, 354 in lupus neutropenia, 284 in neonatal lupus, 248-254, 354, 363, 466 in overlap syndromes, 429t, 431-432 in pregnancy, 451-452 in Rowell’s syndrome, 354 Anti-ssDNA antibodies, 156, 225-226, 225t. See also Anti-DNA antibodies Antithrombotic therapy. See also Anticoagulation for antiphospholipid syndrome, 411-412 Anti-tRNA synthetase syndrome, 429t, 431 Anti–tumor necrosis factor agents, for overlap syndromes, 434-436 Anti–U1-RNP antibody, 276-277 in mixed connective tissue disease, 433, 436 in overlap syndromes, 429t, 431f, 432 Anti–U2 RNP antibody, 277 Anti–VLA-4 antibodies, as therapeutic target, in vasculitis, 316 Anxiety, 36-37, 417. See also Neuropsychiatric disease treatment of, 423 APH50 assay, 201 Aphthous stomatitis, 353, 353f, 382-383 treatment of, 390 Apoptosis, 134-140 anti-DNA antibodies in, 281 in antigen presentation and processing, 99-100 in antiphospholipid syndrome, 236 ATP in, 295 autoantigen modification in, 136-137 biochemical pathways in, 134-135 C1q deficiency and, 135, 177, 199-200 cell clearance in. See also Phagocytosis impaired, 135-136, 138-139, 199-200, 283 cell clustering in blebs in, 134-135, 137, 251 in congenital heart block anti-SSA/Ro–SSB/La antibodies and, 251-253 murine model of, 253-254 in cutaneous LE, 319 defective, 62 definition of, 236 LE cell and, 259, 281 of neutrophils, 281-284, 283t nitric oxide in, 302, 304

INDEX

Antiphospholipid antibodies (Continued) cognitive dysfunction and, 420 in complement activation, 189 in immune complexes, 221 in mixed connective tissue disease, 433-434 in neuropsychiatric disease, 242t, 243, 416 in parvovirus B19 infection, 151 pathogenicity of, 158, 411 as precursors of clinical disease, 103, 134 in pregnancy, 452 pulmonary embolism and, 378 pulmonary hypertension and, 378 in retinal vasculitis, 443-444, 444f in thrombocytopenia, 409 valvular heart disease and, 367, 368 in vasculitis, 311-312 Antiphospholipid syndrome, 233-238, 409-412 anti-β2GPI in, 233-235, 234 anticoagulation for, 237, 368, 378, 411-412, 511-513, 512t, 513t in pregnancy, 456, 513-514, 514t anti-prothrombin in, 234 apoptosis in, 236 aspirin for, 511 assisted reproductive technologies in, 463-464 autoantibody production in, 236 B cells in, 236 catastrophic, 410, 511 treatment of, 411, 513 clinical features of, 233, 410 complement in, 236-237 corticosteroids for, 513 definition of, 233 dendritic cells in, 236 diagnosis of, 233-234, 411 endothelial cell receptors in, 238 historical perspective on, 409-410 hydroxychloroquine for, 511 immunosuppressive therapy for, 513 lupus anticoagulant in, 233-234, 409-410, 411 molecular mimicry in, 236 nonthrombotic complications of, 410 pathogenesis of, 411 pathophysiology of, 236-238, 409-410 platelet receptors in, 237-238 pregnancy in, 233, 236-238, 409, 410, 411-412, 450, 456, 513-514, 514t fetal loss in, 233, 236-238, 409, 410, 449-450, 513-514. See also Fetal loss fetal monitoring in, 514 treatment considerations in, 456 prognosis in, 511 in renal disease, 344 retinal vasculitis in, 443-444, 444f T cells in, 236 thrombocytopenia in, 409, 410 treatment of, 513 thrombosis in, 158, 233, 236-238 prevention of, 368, 378, 411-412, 511-513, 513t recurrent, 511-512 treatment of, 411-412, 511-521 valvular heart disease in, 367, 368, 410 Antiplatelet antibodies, 409 Anti-PM/Scl antibody, in overlap syndromes, 429t, 431 Anti–poly ADP ribose antibody, 156 Anti-prothrombin antibody in antiphospholipid syndrome, 234, 235, 236 lupus anticoagulant and, 234 Antipsychotics, for neuropsychiatric disease, 423 Anti–ribosomal P antibodies, in neuropsychiatric disease, 242t, 243, 416

563

INDEX

564

Apoptosis (Continued) opsonization in, 138-139 in pathogenesis, 56, 62, 135-140, 199-200, 283 in disease initiation, 136 in disease propagation, 137-139, 139f signaling in, 294-295 in T cells, 294-295 tolerance induction by, 135 Toll-like receptors in, 136 in UV-induced lupus, 68-69 in viral infections, 137 Apoptotic bodies, 251 Appetite loss, 333 Approach to patient, 25b Arginine, in nitric oxide synthesis, 302 Arrhythmias, 362t, 363-364. See also Cardiac disease antimalarials and, 365-366 anti-SSA/Ro–SSB/La antibodies and, 467 in congenital heart block, 250. See also Congenital heart block in pericardial disease, 362, 362t Arteriolar occlusion, retinal, 444 Arteritis, coronary, 370 Arthritis/arthralgia corticosteroids for, 491t in incomplete lupus, 475 in mixed connective tissue disease/overlap syndromes, 432, 435t rheumatoid, in overlap syndromes, 429, 430-432, 430t Arthritis Impact Measurement Scale, 34t, 35 Artificial tears, 333, 446 Ascites, 386 Aspergillosis, 402 Aspirin, 483 for antiphospholipid syndrome, 411-412, 511 in pregnancy, 456, 513-514, 514t for thrombosis prophylaxis, 511 in pregnancy, 453-454, 454t, 456, 456t in antiphospholipid syndrome, 456, 513-514, 514t Asplenia, functional, opportunistic infections and, 396 Assessment disease activity scales for, 19-22, 20t, 25-26. See also Disease activity scales economic, 36-43 quality of life, 32-36, 34t. See also Quality of life, measures of Assisted reproductive technologies, 462-464, 462t, 463t Association studies, 74-75 Atherosclerosis antiphospholipid antibodies and, 306, 344, 369 cerebrovascular disease in, 417 coronary artery disease in, 362t, 368-370, 369f free radicals and, 306 in lupus nephritis, 306, 344, 369 mesenteric insufficiency in, 384 opportunistic infections in, 396-397 pathogenesis of, 306, 369 Autoantibodies, 156-167. See also specific types in apoptotic opsonization, 138-139 B-cell production of, 103 autoantibody production by, 95, 103 T cells in, 95, 104-105 two-signal hypothesis for, 104-105 as biomarkers, 48t, 49 cross-reactivity with self-antigens, 143-144, 144f, 144t monoclonal, 157-158 natural, 103 in neuropsychiatric disease, 415-416 origin of, 157 in overlap syndromes, 429, 429t, 430-432 overview of, 156 pathogenicity of, 157-160

Autoantibodies (Continued) structure and, 158-160, 159f as precursors of clinical disease, 103, 134 structure of, 156-157, 156f in vasculitis, 311-312 Autoantibody testing, 265-272 for anti-spliceosome antibodies, 278-279 in clinical laboratory, 266-267 clinical practice guidelines for, 268, 269t commercial kits for, 265-266 validation and adoption of, 266 cost-benefit analysis for, 268 ELISA in, 265-266, 279 evidence-based approach to, 268 indications for, 268, 269t quality assurance for, 269-270, 271t-272t reference sera for, 269, 270t regulation of, 269-270 results of, interpretation of, 267-268 standardization of, 265, 269-271, 279 improvement of, 269-270, 271t-272t paradigm for, 270-271 technology platforms for, 265 Autoantigen-reactive T cells, 97-98, 97t, 98t Autoantigens, apoptotic modification of, 136-137 Autoimmune disease estrogen in, 87-92. See also Estrogen; Hormonal factors molecular mechanisms of, 91, 92b protective effects of, 89 in overlap syndromes, 429-432, 429t Autoimmune hemolytic anemia, 408 corticosteroids for, 492 fatigue and, 330 Autoimmune hepatitis, 387-388, 391 Autoimmunity bystander effects in, 144-145, 145f DNA methylation in, 65-68, 66f gender differences in, 87-88. See also Gender differences immune complexes in, 215-216, 216f molecular mimicry in, 143-144, 144f, 236 Autoreactive antibodies. See Autoantibodies Autoreactivity, induction of. See Pathogenesis Avascular necrosis, steroid-related, 493 5-Azacytidine DNA methylation and, 65-66 in T-cell regulation, 65-66, 67 Azathioprine, 498-499, 500t, 506t adverse effects of, 499, 500t clinical uses of, 499, 499t, 505, 506t dosage of, 499, 500t drug interactions with, 499 for incomplete lupus, 479, 480t for lupus nephritis, 340-343, 341t, 342-343 monitoring for, 500t for neuropsychiatric disease, 423 opportunistic infections and, 397-398, 397t pharmacokinetics of, 498-499 in pregnancy, 454t, 455, 456, 456t, 508 for protein-losing gastroenteropathy, 391 warfarin and, 512 B B7 co-stimulatory molecules, 105 Bacterial DNA, immune properties of, 227 Bacterial endocarditis, 507 Bacterial infections opportunistic, 152, 398, 399-400, 499t. See also Infections, opportunistic in pathogenesis, 69 BBS2, 305t, 306

Blindness, 442b. See also Ocular disease B-lymphocyte stimulator (BLyS), 107, 346-347, 516-518, 517t Bone avascular necrosis of, steroid-related, 493 infection of, in complement deficiency, 185f Branch retinal arteriole occlusion, 444, 444f Breast-feeding maternal drug therapy and, 508 in neonatal lupus, 470-471, 508 British Isles Lupus Assessment Group (BILAG) scale, 19-20, 20t, 329, 332, 531-536, 531-553 Bronchiolitis obliterans organizing pneumonia (BOOP), 377 Budd-Chiari syndrome, 388 Bullous lupus erythematosus, 318, 319, 352, 353, 357 dapsone for, 484 histopathology of, 319 Burned-out lupus, renal failure in, 345 Butterfly rash, 351-353, 352f. See also Cutaneous lupus erythematosus retinoids for, 484 Bxs3 susceptibility locus, in murine models, 175-177, 176t Bxs6 susceptibility locus, in murine models, 176t, 179 BXSB mice, 163-164, 164t, 171. See also Murine models Bystander effects, in autoimmunity, 144-145, 145f C C1 inhibitor, 197t deficiency of, 188 C1q, 196t in complement activation, 194-195, 195f deficiency of, 184-185, 185f defective apoptosis in, 135, 177, 199-200 C1q antibodies immune complexes and, 221-222 in lupus nephritis, 220 C1q receptors, 197 C1qRP, 197t C1r, 196t in complement activation, 195, 195f deficiency of, 185 C1s, 196t in complement activation, 195, 195f deficiency of, 185, 186-187 C2, 196t in complement activation, 195, 195f in cutaneous LE, 324 deficiency of, 187 genetic factors in, 76 C3, 196t as biomarker, 48t, 49 cleavage of, 195, 195f in complement activation, 195 deficiency of in CNS disease, 205 in disease activity, 202-204, 202t in hematologic disease, 205 in pathogenesis, 189, 190t in renal disease, 189, 190t, 205, 339 effector functions of, 195-196, 196t in flares, 189, 190t in lupus nephritis, 189, 190t, 339 measurement of in disease monitoring, 202-204, 202t drawbacks and problems with, 204-205 proteolytic fragments of, 195-196, 196t receptors for, 197-198, 197t C3 antibodies, immune complexes and, 222 C3a receptor, 197t C3b, 195, 195f C3 receptors, 197-198

INDEX

B cell(s), 103-107 abnormalities of, 58-59, 60 opportunistic infections and, 396 in antiphospholipid syndrome, 236 autoantibody production by, 95, 103 T cells in, 95, 104-105 two-signal hypothesis for, 104-105 autoantigen presentation by, 99, 105 autoreactive, 103-107 normal, 103 origin of, 103 as biomarkers, 48t, 51 cytokines and, 105 dendritic cells and, 126 in Epstein-Barr virus infection, 149-150 immunosuppressant effects on, 104 intrinsic defects in, 104-105 loss of tolerance in, 104-105 in lupus nephritis, 288, 289-290 in mixed connective tissue disease, 433 in pathogenesis, 103-107, 104f phenotypic changes in, 105, 106t regulation of, estrogen in, 89 in signaling, 106-107 subpopulations of, abnormalities of, 103-104 type 1, 103-104 type 2, 103, 104 B-cell activating factor (BAFF), in pathogenesis, 59, 60 B-cell receptor(s), 106-107 complement, 106-107 deficiency of, genetic factors in, 76, 77 in pathogenesis, 59 signaling defects and, 106. See also Signaling surface inhibitory, 107 surface stimulatory, 107 bcl2, in apoptosis, 295 Behavioral interventions, for improved quality of life, 37-38 Betamethasone, 488t for congenital heart block, 453 β2GPI, 234-235 antibodies to in antiphospholipid syndrome, 234-238 lupus anticoagulant and, 234 β2 integrins, neutrophils and, 282, 282t BILAG scale, 19-20, 20t, 329, 332, 531-536 Biliary cirrhosis, 388 Biliary disease, 383t, 388 in neonatal lupus, 469-470 Bioanalytical assessment, 47 Biomarkers, 46-52 in bioanalytical assessment, 47 cellular, 48t, 51 conventional, 48t, 49-50 definition of, 46 of endothelial activation, 48t, 51 in lupus nephritis, 48t, 51 false positive/negative, 47 of lupus nephritis, 48t, 51-52 of overall disease activity, 48t potential uses of, 48t promising candidate, 48t, 50-52 sensitivity and specificity of, 46-47 studies of, limitations of, 47-48 as surrogate endpoints, 46, 47, 47t. See also Surrogate endpoints validation of, 46-48, 47t Biopsy endomyocardial, 365, 365f, 366t renal, 337-338, 338f, 339t Bladder injury, from cyclophosphamide, 501b, 503, 507 Bleeding time, delayed, in antiphospholipid syndrome, 409, 411

565

INDEX

566

C4, 196t as biomarker, 48t, 49 in complement activation, 195, 195f in cutaneous lupus, 324 deficiency of, 185-186 in CNS disease, 205 disease activity and, 202-204, 202t genetic factors in, 76 in hematologic disease, 205 in pathogenesis, 189, 190t in renal disease, 205 in flares, 189, 190t in lupus nephritis, 339 measurement of in disease monitoring, 202-204, 202t drawbacks and problems with, 204-205 proteolytic fragments of, receptors for, 197-198, 197t C4A/B deficiency, 185-186 C4d as biomarker, 50, 206 erythrocyte-bound, 206 platelet-bound, 206 reticulocyte-bound, 206 C4 receptors, 197-198 C5, 196t deficiency of, 187 C5a receptor, 197t C5a receptor antagonists, 207 C6, 196t deficiency of, 187 C7, 196t deficiency of, 187 C8, 196t deficiency of, 187 C9, 196t deficiency of, 187 Calcineurin, 91 Calcineurin inhibitors, for cutaneous LE, 358 Calreticulin, 197t CaMKIV, 517t, 520 cAMP response element modulator (CREM), 520 in pathogenesis, 58, 58f Cancer, 26-27 bladder, cyclophosphamide-related, 501b, 503 fever in, 331-332 hematologic. See also Lymphoma cyclophosphamide-related, 502 lymphadenopathy in, 331-332 screening for, 508 subacute cutaneous lupus erythematosus in, 354 Candidiasis, 402 Captopril, in drug-induced lupus, 65t Carbamazepine, in drug-induced lupus, 65t Carboxypeptidase N, 197t Cardiac arrhythmias. See Arrhythmias Cardiac disease, 361-370, 362t ACR classification criteria for, 474t antimalarial-related, 365-366 in antiphospholipid syndrome, 367, 368 conduction system abnormalities, 362t, 363-364 congestive heart failure in, 368-370 coronary artery, 362t, 368-370, 369f corticosteroids for, 490-492, 491t endocardial, 362t, 366-368, 367f ischemic, 365 in mixed connective tissue disease/overlap syndromes, 435t myocardial, 364-366, 365f in neonatal lupus, 248-255, 363. See also Congenital heart block pericardial, 361-363, 362f, 362t valvular, 362t, 366-368 antibiotic prophylaxis in, 507

Cardiac disease (Continued) in antiphospholipid syndrome, 410 bacterial endocarditis in, 507 Cardiac pacemakers, for congenital heart block, 467 Cardiac tamponade, pericardial, 361 Cardiocytes, apoptosis of, in congenital heart block, 252 murine model of, 253-254 Cardiomyopathy, in neonatal lupus, 467 Cardiovascular disease, 26. See also Atherosclerosis; Cardiac disease; Hypertension in mixed connective tissue disease/overlap syndromes, 435t Caspase, in apoptosis, 136-137 Cataracts, steroid-related, 446-447 Catastrophic antiphospholipid syndrome, 410, 511, 513. See also Antiphospholipid syndrome treatment of, 411, 513 cC1qR, 197t, 198 CC-chemokine receptor 7 (CCR7), 123 CD4+ cells in cutaneous lupus, 320, 320f, 326-327 DNA methylation and, 65-66, 66f in pathogenesis, 100, 100t CD4+CD25+ cells, 124 CD8+ cells in cutaneous LE, 320, 320f, 326-327 in pathogenesis, 100, 100t CD11a. See LFA-1 CD21, deficiency of, genetic factors in, 76 CD22 in murine SLE, 178 in pathogenesis, 178 CD25+, 100, 100t CD27high plasma cells, 104 CD35, 196t CD40, 346 CD40 ligand, 105 CD46, 196t CD55, 196t CD59, 197t CD88, 197t CD95, in cutaneous lupus, 324 Celiac disease, 330 Cell adhesion molecules in cutaneous lupus, 326-327 in pathogenesis, 100 in T-cell co-stimulation, 100 in vasculitis, 312-315, 313f-315f, 314t Cell-bound complement activation products as biomarkers, 48t, 49-50, 191, 205-206 in flares, 189-191 measurement of, 48t, 49-50, 205-206 Cell death by apoptosis. See Apoptosis by necrosis, 296-297 Cell signaling abnormal, in pathogenesis, 57-58, 58f in apoptosis, 294-295 B-cell, 106-107 class I recognition receptors in, 126-127 in dendritic cell maturation, 123 in drug-induced lupus, 67, 67f estrogen receptors in, 90-91, 90f in pathogenesis, 57-58, 58f, 59, 67, 67f, 90-91, 98-99, 98t T-cell, 98-99, 293-298, 296t as therapeutic target, 520 in vasculitis, 315-316, 316f Cell surface receptors altered expression of, 59t, 60-61, 61f in pathogenesis, 60-61 Central nervous system disease. See Neuropsychiatric disease Central serous chorioretinopathy, 444-445

Complement activation of. See Complement activation antibodies against, 189 in antiphospholipid syndrome, 236-237 biology of, 194-198 as biomarker, 48t, 49, 201, 202-206 deficiency of. See Complement deficiency in disease activity, 189-191, 190f, 190t, 191f, 194, 198-199 in disease monitoring, 189, 190f in lupus nephritis, 338-340 effector functions of, 188, 195-196, 196t assessment of, 201 in flares, 201, 202, 203t-204t functional activity of, 188 measurement of, 201 in immune complex processing, 188-189, 191f, 195-196, 214-215, 216-218 in inflammation, 196 in lupus nephritis, 188-191, 190f, 190t, 336, 337t, 338-339. See also Complement deficiency of, 183-191, 336, 337t, 339. See also Complement deficiency in disease monitoring, 205, 336, 337t, 338-339 flares and, 189-191 measurement of, 205, 338-339 in pathogenesis, 188-191 measurement of, 201-206 as biomarker, 202-206 of cell-bound complement activation products, 205-206 clinical applications of, 202-206 drawbacks and problems with, 204-205 functional assays in, 201 in hematologic disease, 205 immunochemical assays in, 201-202 in lupus nephritis, 205 methods of, 201-202 nephelometry in, 202 organ-specific involvement and, 205 in opsonization, 196 in osmotic lysis of microorganisms, 196 in pathogenesis, 188-191, 198-201 in pregnancy, 452 reno-injurious effects of, 189-191, 190f, 190t in therapeutic monitoring, 189, 190f therapeutic targeting of, 517t, 519 types of, 196t Complement activation alternative pathway in, 195, 195f antiphospholipid antibodies in, 189 in antiphospholipid syndrome, 411 breakdown products of, in lupus nephritis, 339 classical pathway of, 194-195, 195f in disease activity, 189-191, 190f, 190t, 191f, 194, 198-199 in lupus nephritis, 339-340 immune complexes in, 189, 198-199, 218, 284 inhibitors of, therapeutic uses of, 207 lectin pathway in, 195, 195f in pregnancy, 452 proteinuria in, 189 regulation of, 196-197 reno-injurious effects of, 189 tissue damage from, 189-191, 190f, 190t, 191f, 194, 198-199 Complement activation products as biomarkers, 48t, 49-50, 191, 205-206 in flares, 189-191 measurement of, 48t, 49-50, 205-206 Complement breakdown products, in lupus nephritis, 339 Complement components, 196t-197t, 203t measurement of, 201-204

INDEX

Central tolerance. See also Immunologic tolerance defects in, 95, 97t, 101 Cerebrospinal fluid, in neuropsychiatric disease, 422-423 Cerebrovascular disease, 417. See also Vasculitis in antiphospholipid syndrome, prevention of, 368, 378, 411-412, 511-513, 513t C4d in, 206 Cervical cancer, screening for, 508 CH50 assay, 201 Chemicals, in pathogenesis, 56, 69-70, 69t Chemokine receptors, in cutaneous LE, 326 Chest pain, 362t, 368-370 Chilblain lupus erythematosus, 318, 319, 356-357, 356b, 356f Children. See Pediatric lupus Chloroquine, 483-484 cardiotoxicity of, 365-366 for cutaneous lupus, 358 ocular toxicity of, 447 Chlorpromazine, in drug-induced lupus, 65t Cholecystitis, 388 Cholesterol, elevated, 369-370. See also Atherosclerosis in lupus nephritis, 306, 344, 369 statins for, hepatotoxicity of, 388 steroid-related, 369-370 Chondrodysplasia punctata, with coexistent SLE, 70 Chorea, 417 Chorioretinitis, 444 Choroidopathy, 444-445, 445t Chromatin autoimmune response to, 258-262. See also Anti-histone antibodies structure of, 258f Chronic interstitial lung disease, 375t, 376, 376f in mixed connective tissue disease/overlap syndromes, 435t, 436 Chronic viral hepatitis, 387-388 treatment of, 391 Cigarette smoking, coronary artery disease and, 369, 370 Cirrhosis hepatic, 387 primary biliary, 388 Civatte bodies, 318, 319f Classification criteria, 1, 24-25, 473, 474t for cutaneous disease, 474t incomplete lupus and, 473-475, 477t Clinically active serologically quiescent disease, 25 Clinical manifestations, 25, 329-334 Clusterin, 197t Coagulation abnormalities, 409-412 in antiphospholipid syndrome, 409-412. See also Antiphospholipid syndrome in neonatal lupus, 470 in thrombocytopenia, 409-412 Cofactors, nuclear receptor, 90 Cognitive dysfunction, 418-420. See also Neuropsychiatric disease in acute confusional state, 417-418 assessment of, 418-419 clinical associations with, 419 differential diagnosis of, 418t evolution of, 419, 419f non-SLE causes of, 418, 418t risk factors for, 419-420 subclinical, 418 treatment of, 423-424 Cognitive rehabilitation, 423-424 Cognitive Systems Inventory, 418 Colitis, 386 Collagenous colitis, 387 Colonic disease, 383t, 386-387

567

INDEX

568

Complement deficiency, 183-191 C1 inhibitor, 188 C1q, 184-185, 185f defective apoptosis in, 135, 177 C1r, 185 C1s, 186-187 C2, 187 C4A/B, 185 C5, 187 C6, 187 C7, 187 C8, 187 C9, 187 clinical manifestations of, 184-188, 184t, 185f in CNS disease, 205 CR1, 188 in cutaneous LE, 324 in cutaneous lupus, 184, 185f in diagnosis, 49 disease associations in, 184, 184t early-onset disease and, 184 genetic factors in, 76-77, 177 in hematologic disease, 205 in lupus nephritis, 183-191, 336, 337t, 338-340 mannose-binding lectin, 78, 187-188 opportunistic infections and, 184-188, 185f, 396 overview of, 183-184 in pathogenesis, 56, 76-77, 199-201 in pregnancy, 452 in renal disease, 205 Complement inhibitors, 517t, 519 therapeutic use of, 191 Complement proteins, 196t-197t, 203t measurement of, 201-202 Complement receptors, 197t. See also specific types (e.g., CR1) B-cell, 106-107 as biomarkers, 50 deficiency of, 208 genetic factors in, 76, 77 immune complex deposition and, 217-218 neutrophilic, 281-282, 282t Complement split products, 196t-197t, 203t measurement of, 202-204 Complete blood count, in pregnancy, 452 Complete heart block, 363 Compstatin, anticomplement activity of, 207 Computed tomography in alveolar hemorrhage, 375, 375f in interstitial lung disease, 376, 376f in neuropsychiatric disease, 421 in pulmonary hypertension, 377 Congenic murine models, 164-165, 165t Congenital heart block, 248-255, 363, 466-469, 471 anti-SSA/Ro–SSB/La antibodies in, 248-254, 256f, 363, 466-469, 471 apoptosis and, 250-253 histopathology and, 250 murine model of, 253-254 target autoantigens and, 248-250 classification of, 466 complete vs. incomplete, 466 conduction abnormalities in, 250-251 endocardial fibroelastosis and, 467 fibrosis in, 250, 253, 255 histopathology of, 250 morbidity and mortality from, 466, 466t, 467 murine model of, 253-254 overview of, 466-467 pathogenesis of, 466-467, 471 anti-5-HT antibodies in, 249-250 anti-Ro52 antibodies in, 249

Congenital heart block (Continued) anti-SSA/Ro–SSB/La antibodies in. See Congenital heart block, anti-SSA/Ro–SSB/LA antibodies in apoptosis in, 251-253 fetal genetic factors in, 254-255 hypoxia in, 255 murine model of, 253-254 target autoantigens in, 248-249 in utero environmental factors in, 255 prenatal management of, 468-469, 469t prenatal monitoring of, 468-469, 469t prenatal screening for, 453, 468 prenatal treatment of, 453 progression of, 466 recurrence in subsequent pregnancies, 466 Congestive heart failure, 362t, 368-370 Connective tissue disease. See Mixed connective tissue disease; Overlap syndromes; Undifferentiated connective tissue disease Constitutional features, 25, 329-334 Constrictive pericarditis, 361 Corneal disorders, 333, 440 treatment of, 446 Coronary arteritis, 370 Coronary artery disease, 362t, 368-370, 369f. See also Atherosclerosis antiphospholipid antibodies and, 306 free radicals and, 306 Coronary artery spasm, 370 Corticosteroid reduction syndrome, 492-493 Corticosteroids, 487-495 adverse effects of, 493, 494t anti-inflammatory effects of, 488-489 for autoimmune hemolytic anemia, 408 for cardiac disease, 490-492 clinical trials of, 495 for cognitive dysfunction, 423 for constitutional features, 334 for cutaneous disease, 358, 490 with cyclophosphamide, 501b efficacy of, assessment tools for, 488 forms of, 488t, 489-490 future directions for, 495 for gastrointestinal disease, 390-391 half-life of, 488t for hematologic disease, 492 historical perspective on, 487 hyperlipidemia and, 369-370 hypothalamic-adrenal-pituitary axis suppression by, 495 immunizations and, 508 immunosuppressive effects of, 489 for incomplete lupus, 479, 480t indications for, 490-492 intralesional, 489 intramuscular, 490 intrasynovial, 489 intravenous, 490 for lupus nephritis, 340, 341t, 343, 492 mechanism of action of, 488-489 for mixed connective tissue disease/overlap syndromes, 434-436, 435t morbidity and mortality and, 14 for musculoskeletal disease, 490 for myocardial disease, 366 for myositis, 490 for neuropsychiatric disease, 423, 492 ocular toxicity of, 446-447 opportunistic infections and, 397-398, 397t, 507 for optic neuropathy, 446 oral, 489 for overlap syndromes, 434-436, 435t

Cutaneous lupus erythematosus (Continued) lupus erythematosus profundus, 318, 356, 356f lupus erythematosus telangiectodes, 356 oral, 382, 390 dapsone for, 484 discoid, 319, 354-356, 355f, 355t. See also Discoid lupus erythematosus drug-related, 352, 354 epidermal hyperkeratosis in, 319, 319f genetic factors in, 322-325, 323t histopathology of, 318-320, 319 epidermal cells in, 318-319, 319f inflammatory cells in, 319-320, 320f mucin deposition in, 320, 320f hypertrophic/verrucous, 355 immunopathology of, 320-322, 321f, 321t intermittent, 318, 320, 351, 352t, 356-357, 356f, 357f lesions in bullous, 318, 319, 352, 357 classification of, 351, 352t LE-specific, 351, 352t non–LE-specific, 351, 357-358, 357t lupus band test for, 321-322, 321f lupus erythematosus tumidus, 318, 320, 351, 356, 356f lymphocytes in, 320, 320f, 326-327 malar rash in, 351-353, 352f neonatal lupus and, 354 nonspecific, 351, 357-358, 357t oral, 382, 390 oral lesions in, 353, 353f, 382-383, 390 treatment of, 390 pathogenesis of, 322-327 pathology of, 318-322 photosensitivity in, 325-326, 325f, 352, 353-354, 353f, 353t, 355, 357, 358 retinoids for, 484 subacute, 318, 319, 351, 352t, 353-354, 353f, 353t C1q deficiency in, 185 with concurrent acute form, 352 subtypes of, 318, 351, 352t Cyclic AMP response element modulator (CREM), 520 in pathogenesis, 58, 58f Cyclooxygenase-2 inhibitors, 483 Cyclophosphamide, 499-503, 500t, 506t adverse effects of, 500t, 501-502, 501b clinical trials for, 502-503 dosage of, 500t, 501, 501b, 502-503, 504t for lupus nephritis, 340-343, 341t, 503 with stem cell transplant, 347, 347f with methylprednisolone, 502-503, 505 monitoring for, 500t, 501b for neuropsychiatric disease, 423 opportunistic infections and, 397-398, 397t for optic neuropathy, 446 oral, 502 pharmacokinetics of, 500 in pregnancy, 454t, 455, 456t, 457, 508 for pulmonary hypertension, in mixed connective tissue disease, 436 in pulse therapy, 500-501, 501b, 502-503 routes of administration for, 500-501 for severe/life-threatening disease, 507 therapeutic uses of, 502-503, 505, 506t Cyclosporine, 500t, 505-506 Cyproterone, 485 Cystitis, hemorrhagic, cyclophosphamide-related, 501b, 503, 507 Cytofectins, 228 Cytokine(s). See also specific types B-cell, 105 as biomarkers, 48t, 50-51 for lupus nephritis, 48t, 51-52

INDEX

Corticosteroids (Continued) overview of, 487 for pericarditis, 363 for pleuritis, 490-492 in pregnancy, 454-455, 454t, 456, 456t for congenital heart block, 468-469, 469t, 470f for protein-losing gastroenteropathy, 391 for pulmonary disease, 490-492 in pulse therapy, 489, 490 rationale for, 487-489 respiratory muscle dysfunction due to, 378 routes of administration for, 489-490 for scleritis, 446 for shrinking lung syndrome, 379 systemic, 489-490 tapering and withdrawal of, 492-493 therapeutic monitoring for, 488 topical, 489 for undifferentiated connective tissue disease, 434, 435t Cortisone, 488t Cost-benefit analysis, 39 Cost domains, 39-40 Cost-effectiveness studies, 36 Cost-minimization studies, 36 Cost of illness assessment, 36 Cost-utility studies, 36-37 Counterimmunoelectrophoresis, for anti-spliceosome autoantibodies, 278 Course. See Disease course; Prognostic factors COX-2 inhibitors, 483 CpG motif, 228 CR1, 197-198, 197t as biomarker, 50 deficiency of, 188, 200 genetic aspects of, 76 immune complex deposition and, 217-218 soluble, therapeutic applications of, 207 CR2, 197t, 198 B-cell, 106-107 deficiency of, 200-201 genetic aspects of, 76 in murine SLE, 177 CR3, 197t, 198 deficiency of, 201 CR4, 197t, 198 C-reactive protein complement and, 189 in flares vs. infections, 403 in pregnancy, 452 Crohn’s disease, 386-387 Cryoglobulin assay, for immune complexes, 222 Cryptococcosis, 402 Crystalline silica, in pathogenesis, 56, 69-70 CSCR3, in cutaneous LE, 326 CTLA-4, 78, 521 CTLA-4 Ig, for lupus nephritis, 346 CTLA-4 Ig fusion molecule, 517t, 520 C-type lectins, in dendritic cell maturation, 123 Cutaneous lupus erythematosus, 318, 319, 351-358 ACR classification criteria for, 474t acute, 351-353, 352f, 352t, 353f with concurrent subacute form, 352 acute toxic epidermal necrolysis–like, 318, 319, 352, 353 apoptosis in, 319 UV-induced, 325-326, 325f basement membrane thickening in, 319, 319f bullous, 318, 319, 352, 357 in cancer, 354 chronic, 351, 352t, 354-356. See also Discoid lupus erythematosus chilblain lupus erythematosus, 318, 319, 356-357, 356b, 356f hypertrophic/verrucous, 355

569

INDEX

570

Cytokine(s) (Continued) in congenital heart block, 253 in cutaneous disease, 324 in dendritic cell maturation, 123, 127-128, 128f in lupus nephritis, 48t, 51-52, 346-347 in neuropsychiatric disease, 416-417 in pathogenesis, 58, 59-60, 109-116. See also specific cytokines polymorphisms in, 115t production of, anti-DNA antibodies in, 229, 230f secretion of, apoptotic opsonization in, 138-139 in T-cell necrosis, 297 in vasculitis, 313-314, 314f Cytokine receptors, as biomarkers for disease activity, 48t, 50-51 for lupus nephritis, 48t, 51-52 Cytomegalovirus infection, 150-151, 152t, 401 Cytopenia, corticosteroids for, 492 Cytotoxic drugs, 498-508. See also specific drugs adverse effects of, 500t azathioprine, 498-499, 506t cyclophosphamide, 499-503, 506t cyclosporine, 500t, 505-506 dosage of, 500t, 506t guidelines for, 505, 506t methotrexate, 500t, 505 monitoring of, 500t, 501b mycophenolate mofetil, 500t, 503-505, 504t in pregnancy, 508 selection of, 505 for severe/life-threatening disease, 507 Cytotoxic T-lymphocyte antigen. See also CTLA-4 in pathogenesis, 56 D Danazol, 485 Dapsone for bullous lupus, 484 for Pneumocystis carinii pneumonia prophylaxis, 507 Death rates, 10-14, 11t-12t, 13f. See also Mortality Decision-analytic modeling, 39 Deep vein thrombosis. See Thrombosis Dehydroepiandrosterone (DHEA) gene targets of, 88t immunologic effects of, 87-88, 87t therapeutic use of, 484-485 Delirium, 417-418. See also Cognitive dysfunction Demyelination, 417 Dendritic cells, 121-128 abnormalities of, 60-61 antigen capture and presentation by, 99, 121-122, 122f in antiphospholipid syndrome, 236 B cells and, 126 biology of, 121-126 central tolerance and, 126 in cutaneous disease, 326 cytokines and, 123, 127-128, 128f Fcλ receptor and, 127 follicular, 215 functions of, 121 interferon-α and, 125-126, 127-128, 137-138 interstitial, 124 Langerhans cells, 123, 124, 125, 126 in cutaneous LE, 326 functional properties of, 125 loss of tolerance in, 124 in lymphocyte priming, 123-124 maturation of, 123-124 migration of, 122-123 myeloid, 125 in pathogenesis, 125-126, 127-128, 128f, 137-138, 215-216 peripheral tolerance and, 126-127

Dendritic cells (Continued) plasmacytoid, 124, 125-126 interferon-α production by, 125-126, 137-138, 215 precursor, 125 progenitor, 124-125 subsets of functional properties of, 125 T-cell response and, 124 types of, 124-126 in T-cell necrosis, 297 in T-cell response, 123-124 tolerance and, 126-128 Dental disease, 383 Depression, 36-37, 417. See also Neuropsychiatric disease fatigue and, 37, 331 Dexamethasone, 488t. See also Corticosteroids for congenital heart block prevention, 468-469 with cyclophosphamide, 501b DHEA. See Dehydroepiandrosterone (DHEA) Dialysis, 345 Diaphragmatic weakness, 378-379 DID assay, for anti-spliceosome antibodies, 274-275, 278 Diet, disease flares and, 56, 70 Diffuse alveolar hemorrhage, 374-375, 375f, 375t Diffusion-weighted imaging, in neuropsychiatric disease, 421-422 Digital edema, in mixed connective tissue disease, 432 Direct immunofluorescence, in cutaneous LE, 321-322, 321t Discoid lupus erythematosus, 319, 354-356, 355f, 355t. See also Cutaneous lupus erythematosus classification of, 318 clinical manifestations of, 351-357 complement deficiency and, 184, 184t, 185f corticosteroids for, 491t direct immunofluorescence in, 321-322, 321t hypertrophic/verrucous, 355 oral, 382 pathology and pathogenesis of, 318-327 Disease activity scales, 19-22, 20t, 25-26, 524-560 British Isles Lupus Assessment Group, 19-20, 20t, 531-556 European Consensus Lupus Activity Measurement, 20-21, 20t, 332, 557 fatigue in, 329 fever in, 331 Lupus Activity Index, 20t, 21-22 lymphadenopathy in, 332 Systemic Lupus Activity Measure, 20t, 21, 559-560 Systemic Lupus Erythematosus Disease Activity Index, 20t, 21, 332, 558 weight loss in, 333 Disease course, 24-29. See also Flares; Prognostic factors gender differences in, 6, 7t-8t racial/ethnic differences in, 6-10, 9t-10t Disease damage, 26-27 contributory factors in, 26-27, 26b quality of life and, 36 Disease flares. See Flares Diverticular disease, 387 DNA antibodies to. See Anti-DNA antibodies antigenic properties of, 226, 227-228 bacterial, immune properties of, 227 in immune complexes, 219, 221 immunogenic properties of, 227-228 DNA methylation definition of, 64 in drug-induced lupus, 65-68 5-azacytidine and, 65-66 drug mechanisms of action and, 66-68 induction of, 65 functions of, 64-65

E Ea gene, protective effects of, in murine SLE, 174-175 E-C4d, as biomarker, 48t, 50 Echocardiography in myocardial disease, 364 in pericardial disease, 362, 362t prenatal, for congenital heart block, 453, 468, 469 ECLAM (European Consensus Lupus Activity Measurement), 20-21, 20t, 332, 557 Eclampsia, 453 Economic assessment, 36-43 analytic perspective for, 39 cost-benefit, 39 cost domains in, 39-40 cost-effectiveness, 36 cost-minimization, 36 cost of illness, 36 cost-utility, 36-37 decision-analytic modeling in, 39 direct cost estimation in, 39-40 health service use measures in, 40 health service valuation in, 40 indirect cost estimation in, 40-41 productivity impairment in, 40-41 SLE-specific clinic-based studies in, 41-42 population-based studies in, 42 time horizon for, 39 E-CR1, as biomarker, 48t, 50 Eculizumab, 207

Edema digital, in mixed connective tissue disease, 432 optic disc, 445 periorbital, 441 pulmonary, 379 Electrocardiography in myocardial disease, 364 neonatal, for congenital heart block, 468 in pericardial disease, 362, 362t ELISA. See Enzyme-linked immunosorbent assay (ELISA) Embolism. See also Thrombosis pulmonary antiphospholipid antibodies and, 378, 379 pulmonary hypertension and, 377-378 Encephalopathy, 417-418. See also Cognitive dysfunction; Neuropsychiatric disease Endocardial disease, 362t, 366-368, 367f Endocardial fibroelastosis, 467-468 Endocarditis, bacterial, 507 Endocrine system. See Hormonal factors Endomyocardial biopsy, 365, 365f, 366f Endothelial activation, biomarkers of, 48t, 51 Endothelial cell–leukocyte adhesion, in vasculitis, 313-315, 313f-315f Endothelial cell receptors, in antiphospholipid syndrome, 238 End-stage renal disease. See also Lupus nephritis; Renal disease management of, 345 Engineered cells, 520-521 Enteritis, infective, 386 Environmental factors, 64-71 Enzyme deficiencies, with coexistent SLE, 79-80 Enzyme-linked immunosorbent assay (ELISA) for anti-β2GPI, 234 for anticardiolipin antibodies, 233-234 for anticyclic citrullinated peptides, 270-271 for anti-spliceosome antibodies, 278-279 in autoantibody testing, 266. See also Autoantibody testing standardization of, 270-271, 271t-272t in complement measurement, 202 Epidemiology, 1-14 age at onset and, 6 gender and. See Gender differences genetic, 74 geographic distribution and, 1 incidence rates and, 1, 2t-3t mortality rates in. See Mortality prevalence rates and, 1-5, 4t-5t racial/ethnicity and, 6-10 Epidermolysis bullosa acquisita, 357 Epidural anesthesia, obstetric, 456 Episcleritis, 441 treatment of, 446 Epitopes, T-cell, autoantigenicity and, 98, 99-100 Epratuzumab, for lupus nephritis, 346 Epstein-Barr virus infection, 146-150, 401 B-cell response in, 149-150 in pathogenesis, 56, 66, 67f, 69, 146-148, 147f, 401 evidence for, 147-148, 152t in SLE vs. controls, 148-150 T-cell response in, 149 viral load in, 148-149 Erythrocyte-bound C4d, as biomarker, 48t, 50, 206 Erythrocyte-bound CR1, as biomarker, 48t, 50 Erythrocyte complement receptor type 1. See CR1 Erythrocyte sedimentation rate, in pregnancy, 452 E-selectin in cutaneous disease, 326 in vasculitis, 313-314, 313f Esophageal disease, 383, 383t in mixed connective tissue disease, 433, 436 treatment of, 390

INDEX

DNA methylation (Continued) in idiopathic SLE, 68 of LFA-1, 100 in pathogenesis, 64-68, 100 in UV-induced lupus, 68 DNA methyltransferase inhibitors, in drug-induced lupus, 65-68, 65t, 66f, 67f DNA receptors, in immune complex clearance, 219 Double immunodiffusion (DID) assay, for anti-spliceosome antibodies, 274-275, 278 DR2, 76 DR3, 76 Drug-induced hepatotoxicity, 388 Drug-induced lupus, 55, 64-68 DNA methylation in, 64-68 drug mechanisms of action in, 66-68, 66f, 67f drugs most often implicated in, 65t hydralazine-related, 65t, 66-68, 66f, 67f procainamide-related, 66-68, 66f, 67f pulmonary involvement in, 379 Drug therapy. See also specific drugs and drug families breast-feeding and, 508 nonsteroid, 483-485 novel agents in, 516-521, 517t in pregnancy, 453-456, 455t, 508 for severe/life-threatening disease, 507 targeting cell-surface molecules/cell-cell interaction, 517t, 519-521 targeting soluble mediators, 516-519, 517t Dry eye, 333, 435t, 440 treatment of, 446 Dry mouth, 333, 382-383, 435t treatment of, 390 Duodenal ulcers, 383 D-xylose suppression test, 385 Dyes, hair, 70 Dysphagia, 383 Dyspnea, respiratory muscle dysfunction and, 378 Dysrhythmias. See Arrhythmias

571

INDEX

572

Estradiol, immunologic effects of, 89, 89f Estrogen in autoimmune disease, protective effects of, 89 in cutaneous disease, 324-325 gene targets of, 88t immunologic effects of, 87-92, 88t, 92b molecular mechanisms in SLE, 91, 92b in pathogenesis, 57, 91 in T-cell regulation, 89-90, 89f, 91 Estrogen receptor agonists/antagonists, 91, 92 Estrogen receptors, 90-91, 90f Ethnicity, 6-10, 9t-10t. See also Racial/ethnic differences Etiopathogenesis, 55-62. See also Pathogenesis European Consensus Lupus Activity Measurement (ECLAM), 20-21, 20t, 332, 557 European Quality of Life Scale, 34t, 35 Exercise therapy, 331 Extracellular signal-regulated kinase (ERK) pathway, in drug-induced lupus, 67, 67f Eye. See under Ocular F Factor B, 195, 195f, 196t deficiency of, in pathogenesis, 189, 190t, 202, 202t Factor D, 195, 195f, 196t Factor H, 197t Factor I, 197t Factor XI, β2GPI binding of, 235 False positive/negative biomarkers, 47 Familial systemic lupus erythematosus, 74. See also under Genetics/genetic factors Farr-type immunoprecipitation assay, 225 Fas, in cutaneous LE, 324 Fas/Fas ligand mutations, in murine SLE, 172-173 Faslpr haplotype, in murine models, 174-175 Fatigue, 37, 329-331 anemia and, 330 depression and, 37, 331 disease activity and, 329-330 exercise therapy for, 331 fibromyalgia and, 330 in incomplete LE, 475, 478t-479t Fcgr2b, in murine SLE, 176-177 Fcλ receptor dendritic cells and, 127 immune complexes and, 218, 220 in leukocytoclastic vasculitis, 284 in lupus nephritis, 159-160, 288-289 neutrophil–immune complex interactions and, 281-283, 282t in pathogenesis, 56, 57-58, 58t, 60, 77, 107, 282-283 FcλRIIA1, in immune complex clearance, 218 FcλRIIB1, 107 Female-to-male ratio. See Gender differences Fertility. See Infertility; Pregnancy Fetal echocardiography, for congenital heart block, 453, 468, 469 Fetal loss in antiphospholipid syndrome, 233, 236-238, 409, 410, 449-450 prevention of, 411-412, 513-514, 514t causes of, 460t classification of, 460t in congenital heart block, 466, 466t, 470t. See also Congenital heart block disease-independent, 460t, 461-462 evaluation of fetal/child risks from, 464 maternal risks from, 464 lupus-dependent, 462 by miscarriage, 449

Fetal loss (Continued) prevention of, 411-412 risk factors for, 460 by stillbirth, 449-450 Fetal monitoring, 453 in antiphospholipid syndrome, 514 Fever, 331-333 causes of, 331-332 in disease activity scales, 331 in opportunistic infections, 402-403 Fibromyalgia, 37, 330 fatigue and, 330 Flares after immunizations, 404 anti-DNA antibodies in, 229 anti-dsDNA antibodies in, 160 complement in, 188-191, 190f, 190t, 191f, 201, 202, 203t-204t in corticosteroid tapering/withdrawal, 493 fatigue in, 329-330 infections and, 393, 403 in lupus nephritis complement-related, 189-191 treatment of, 344 in pregnancy, 450 vs. infections, 403 Fludrocortisone, 488 Fluorescein angiography, 443, 443f, 445 in lupus choroidopathy, 445 in retinal disease, 443, 443f, 445 Flurbiprofen, for scleritis, 446 Focal adhesion kinase, in vasculitis, 315 Folic acid deficiency, anemia in, fatigue and, 330 Foxp3, 90 Free radicals. See also Nitric oxide; Reactive oxygen intermediate atherosclerosis and, 306 definition of, 301 in innate immunity, 301-302 in T cells apoptosis and, 293-296 necrosis and, 296-297 as therapeutic targets, 297-298 Fungal infections, opportunistic, 152. See also Infections, opportunistic G Gallbladder disease, 388 γ/δ T cells, dendritic cells and, 126 Ganisetron, with cyclophosphamide, 501b Gastric and vascular ectasia, 384 Gastric disease, 383-384, 383t Gastroenteropathy, protein-losing, 385-386, 386f, 391 Gastrointestinal disease, 382-391 ACR classification criteria for, 474t ascites in, 386 biliary, 388 in neonatal lupus, 469-470 colitis, 386 diverticular disease, 387 esophageal, 383, 383t, 390, 433, 436 gastric, 383-384, 383t hepatic, 387-388, 391 in neonatal lupus, 469-470 infective enteritis, 386 inflammatory bowel disease, 386-387 intestinal pseudo-obstruction, 384-385, 390-391 large intestinal, 383t, 386-387 malabsorption, 385-386, 391 mesenteric insufficiency, 384, 390 mesenteric vasculitis, 384, 390

Granulocyte colony-stimulating factor, in lupus neutropenia, 284 Granulomatous necrotizing lymphadenitis, 332 Granzyme B, in apoptosis, 136-137 Group psychotherapy, for improved quality of life, 37-38 GW273629, 306 GW274150, 305t, 306 H H1/H2 autoantibodies, 259-260. See also Anti-histone antibodies H2b haplotype, in murine models, 174-175 H2d/z haplotype, in murine models, 174 H2O2, in T cells in apoptosis, 293-296, 296t in necrosis, 296-297 H2 susceptibility locus, in murine models, 176t, 179 Hair, lupus, 358 Hair dyes, 70 Hands, swelling of, in mixed connective tissue disease, 432 Headache, 417 Healthcare costs. See Economic assessment Health-related quality of life. See Quality of life Health services usage measures for, 40 valuation of, 40 Heart block, 363 complete, 363-364 congenital, 248-255, 363. See also Congenital heart block Heart disease. See Cardiac disease; Cardiovascular disease Heat shock proteins, in cutaneous LE, 324 Heavy metals, in pathogenesis, 56, 69, 69t, 70 Helicobacter pylori infection, in pathogenesis, 69 HELLP syndrome, 453 Helper T cells, abnormalities of, 60 Hematologic disease ACR classification criteria for, 474t anemia, 330, 383-384, 408-409 anemia in. See Anemia complement measurement in, 205 in mixed connective tissue disease/overlap syndromes, 435t in pregnancy, 452 Hematologic malignancies, cyclophosphamide-related, 502 Hematuria, in lupus nephritis, 336, 337t Hemodialysis, 345 Hemolytic anemia, 408. See also Anemia corticosteroids for, 492 fatigue and, 330 Hemophilus influenzae immunization, 508 Hemorrhage, alveolar, 374-375, 375f, 375t Hemorrhagic cystitis, cyclophosphamide-related, 501b, 503, 507 Heparin. See also Anticoagulation anticomplement activity of, 207 for antiphospholipid syndrome, 411-412, 511-513, 513t in pregnancy, 456, 513-514, 514t in pregnancy, 454, 454t Hepatic disease, 383t, 387-388 in neonatal lupus, 469-470 Hepatic vein thrombosis, 388 Hepatitis, 387-388 chronic viral, 387-388 lupus, 387 treatment of, 391 Hepatotoxicity, of nonsteroidal antiinflammatory drugs, 483 Herpesvirus infections, 146-151. See also Cytomegalovirus infection; Epstein-Barr virus infection Herpes zoster, 400-401, 401t Heterogeneous nuclear autoantibody in mixed connective tissue disease, 433 in overlap syndromes, 429t, 431 High mobility group B1, in T cell necrosis, 297 Histones, in chromatin, 258, 258f

INDEX

Gastrointestinal disease (Continued) in mixed connective tissue disease/overlap syndromes, 435t in neonatal lupus, 469-470 oral, 333, 353, 353f, 382-383, 383t pancreatic, 383t, 388-389, 391 peritonitis, 386, 391 protein-losing gastroenteropathy, 385-386, 386f, 391 small intestinal, 383t, 384-386 treatment of, 390-391 Gastrointestinal toxicity of cyclophosphamide, 500t, 501b of mycophenolate mofetil, 500t, 503 gC1qR, 197t Gender differences, 6, 7t-8t in course and outcome, 6, 7t-8t hormonal factors in, 87-92. See also Estrogen; Hormonal factors in incidence and prevalence, 6 in late-onset lupus, 6 in pediatric lupus, 6 in survival, 29, 29t Gene knockout mice, 165-167, 166t Gene mapping, 82 Gene therapy, 520 Genetically engineered murine models, 165-167, 166t Genetic diseases, with coexistent SLE, 79-80 Genetic epidemiology, 74 Genetics/genetic factors candidate genes and, 79, 80t complement components and, 76-77 CTLA-4 and, 78, 521 Fcλ receptor genes and, 56, 57-58, 58t, 60, 77. See also Fcλ receptor inheritance patterns and, 74 interferons and, 79. See also Interferon(s) mannose-binding protein and, 78. See also Mannose-binding protein MHC genes and, 56, 75-76. See also MHC genes in murine SLE, 171-180, 180b nuclear receptors and, 90, 90f in pathogenesis, 56-57 PD-1 and, 78 phenotypic variation and, 75 PTPN22 and, 78-79 tumor necrosis factor and, 77-78. See also Tumor necrosis factor Genetic studies association, 74-75 gene mapping, 82 linkage analysis, 75, 80-82, 80t, 81f, 82t Geographic distribution, 1, 8-10. See also Racial/ethnic differences Glial fibrillary acidic protein, 422 Glomerular filtration rate, measurement of, 337, 338 Glomerulonephritis. See also Lupus nephritis; Renal disease complement in, 188-191, 190f, 190t Glomerulosclerosis. See also Lupus nephritis; Renal disease clinical manifestations of, 336, 337t Glucocorticoid receptor, 488 Glucocorticoids, 488. See also Corticosteroids Glutathione deficiency, 293, 295-296, 296t Glutathione S-transferase, in cutaneous LE, 324 β2-Glycoprotein I (β2GPI), in antiphospholipid syndrome, 410, 412 Gonadal toxicity, of cyclophosphamide, 501-502 Gonococcal infections, opportunistic, 399, 399t gp70, in lupus nephritis, 179 β2GPI, 234-235 antibodies to in antiphospholipid syndrome, 234-238 lupus anticoagulant and, 234

573

INDEX

574

HIV infection, 151-152 HLA genes, 56, 75-76. See also MHC genes in cutaneous LE, 323-324, 323f, 323t hnRNP A2, 98 Homocysteine, elevated, atherosclerosis and, 369 Hormonal factors, 57, 87-93. See also specific hormones in immune response, 87-88, 88t sexual dimorphism and, 87-88. See also Gender differences HSP70, in cutaneous LE, 324 Human immunodeficiency virus infection, 151-152, 401 Human leukocyte antigen (HLA) genes, 56, 75-76. See also MHC genes in cutaneous LE, 323-324, 323f, 323t Human papillomavirus infection, 401 Human T-cell lymphotropic virus infection, 151-152 Hydralazine in drug-induced lupus, 65t lupus induction by, 66-68. See also Drug-induced lupus in pathogenesis, 55 Hydrocortisone, 488, 488t Hydroxychloroquine, 483-484 for antiphospholipid syndrome, 511, 512 cardiotoxicity of, 365-366 for cutaneous lupus, 358 disease modifying effects of, 14 for fatigue, 330 lipid-lowering effects of, 369-370 for mixed connective tissue disease/overlap syndromes, 436 ocular toxicity of, 447 in pregnancy, 454t, 455, 456, 456t Hyperbilirubinemia, in neonatal lupus, 470 Hyperhomocysteinemia, atherosclerosis and, 369 Hyperlipidemia, 369-370. See also Atherosclerosis in lupus nephritis, 306, 344, 369 statins for, hepatotoxicity of, 388 steroid-related, 369-370 Hypertension, 369, 370 ocular, steroid-related, 447 in preeclampsia, 452-453, 456 pulmonary, 375t, 377-378, 377t antiphospholipid antibodies and, 378 in mixed connective tissue disease, 432-433, 435t, 436 pulmonary embolism and, 377-378 renal, treatment of, 344-345 Hypertensive nephropathy, 344 Hypertrophic lupus erythematosus, 318 Hypertrophic/verrucous discoid lupus erythematosus, 355 Hypocomplementemia. See Complement deficiency Hypopigmentation, in neonatal lupus, 469 Hypothalamic-adrenal-pituitary axis, steroid effects on, 495 Hypothyroidism, fatigue in, 330 Hypoxia, intrauterine, in congenital heart block, 255 I ICAM-1 in cutaneous LE, 326-327 in vasculitis, 313-315, 313f-315f IgG, structure of, 156-157, 156f IgG lymphocytotoxic antibodies, 242t, 243-244 IgM lymphocytotoxic antibodies, 242t, 243-244 Imh1 susceptibility locus, in murine models, 176t, 177 Immune complexes, 214-222 antibody isotopes and, 219 anticardiolipin antibodies and, 221 anti-DNA antibodies and, 229-230, 281 antigen in, 221 antiphospholipid antibodies and, 221 assays for, 221-222 in autoimmunity, 215-216, 216f C1q antibodies and, 221-222 c3 antibodies and, 222

Immune complexes (Continued) clearance of, 188-189, 196, 214-215, 216-218 DNA receptors in, 219 neutrophils in, 281-283 in complement activation, 189, 198-199, 218, 284 complement processing of, 188-189, 191f, 195-196, 214-215 condensation and rearrangement of, 219-220 in cryoglobulin, 222 in cutaneous lupus, 321-322, 321t dermoepidermal junction, 321-322, 321f epidermal, 322 vascular, 322 in disease activity, 215, 216f DNA in, 219 formation of anti-DNA antibodies in, 229-230 sites of, 218-219 in situ, 229 immunochemistry of, 214 immunomodulatory effects of, 220 in interferon production, 215-216 in kidney, 218-219 lattice structure of, 218-219 in leukocytoclastic vasculitis, 284 in lupus nephritis, 188-189, 218-219, 288-289 neutrophils and, 188-189, 281-282 nitric oxide and, 303 in pathogenesis, 188-191, 198-199, 214-216, 216f persistence of, 219-220 physiochemical composition of, 218-219 precipitin curve and, 214-215, 214f in retinal vasculitis, 443-444 size of, clearance and, 218 structure of, 214, 214f therapeutic implications of, 220-221 tissue damage from, 189-191, 190f, 190t, 191f, 194, 198-199, 215, 216f, 220 tissue localization of, 218-219 in vasculitis, 215, 311, 322, 443-444 Immune complex processing, 191f complement in, 188-189, 191f, 195-196, 214-215 Immune response, 59-62, 61f cell surface receptor abnormalities and, 59-62, 61f hormonal effects on, 87-92. See also Hormonal factors and specific hormones immune complexes and, 220 impaired, opportunistic infections and, 394-397, 394t. See also Infections, opportunistic Immune thrombocytopenia, 409 Immunizations, 403-404, 507-508 Immunodiffusion, in complement measurement, 202 Immunoglobulin(s). See also Antibodies abnormalities of, opportunistic infections and, 396 intravenous, 506-507 in pregnancy, 456, 456t, 508 in antiphospholipid syndrome, 514 for congenital heart block prevention, 468, 470f structure of, 156-157, 156f Immunoglobulin G, structure of, 156-157, 156f Immunoglobulin G lymphocytotoxic antibodies, 242t, 243-244 Immunoglobulin-like receptors, 126-127 Immunoglobulin M lymphocytotoxic antibodies, 242t, 243-244 Immunologic tolerance apoptotic induction of, 135 central, defects in, 95, 97t, 101 defects in, 95-97, 96f, 97t, 100-101 loss of in B cells, 104 in T cells, 95-97, 96f, 97t peripheral, defects in, 95, 96f, 97t, 100-101

Infections (Continued) pathogenic, 26, 56, 66, 67f, 69, 75-76, 143-152 apoptosis in, 137 bacterial, 69 cytomegalovirus, 150-151 H. pylori, 69 MHC genes and, 75-76 parvovirus B 19, 151 retrovirus, 151-152 viral. See Viral infections Infective endocarditis, 507 Infective enteritis, 386 Infertility, 449, 450t, 460-464 age-related, 460 in autoimmune disease, 461 causes of, 460t, 461t cyclophosphamide-related, 501-502 definition of, 460 disease-related, 460-461 evaluation of, 462 treatment of, 462-464, 462t treatment-related, 461, 461t Inflammatory bowel disease, 386-387 Infliximab, for lupus nephritis, 347 Influenza immunization, 403-404, 507 Integrins, in vasculitis, 314-315 Interferon(s), 518 in pathogenesis, 59-60, 61, 61f, 79, 127-128, 137-138, 177, 215-216 polymorphisms in, 115t production of anti-DNA antibodies in, 229, 230f by immune complexes, 215-216 in T-cell necrosis, 297 therapy targeting, 517t, 518 Interferon-1 in pathogenesis, 215-216 reactive oxygen intermediate and, 294 Interferon-α as biomarker, 48t, 50 dendritic cells and, 125-126, 127-128, 137-138, 215 in pathogenesis, 59-60, 61, 61f, 79, 110-111, 115t, 127-128, 137-138, 215-216 Interferon-γ, in pathogenesis, 110, 115t Interferon inhibitors, 517t, 518 Interferon-R1, polymorphisms in, 115t Interferon signature, 59, 79, 229 Interleukin(s) B cells and, 105 in cutaneous lupus, 324 in lupus nephritis, 347 in neuropsychiatric disease, 416 reactive oxygen intermediate and, 294, 298 Interleukin-1 in lupus nephritis, 347 in pathogenesis, 113-114, 115t polymorphisms in, 115t Interleukin-2, in pathogenesis, 58, 58f, 59, 109 Interleukin-2 receptor, as biomarker, 48t, 50 of disease activity, 48t, 50 for lupus nephritis, 48t, 51 Interleukin-4 in pathogenesis, 60, 111-112, 115t polymorphisms in, 115t Interleukin-5, in pathogenesis, 112, 115t Interleukin-6 B cells and, 105 in lupus nephritis, 48t, 51, 347 monoclonal antibody to, 517t, 518 in pathogenesis, 59, 60, 112-113, 115t polymorphisms in, 115t

INDEX

Immunosuppressive therapy for antiphospholipid syndrome, 513 for constitutional features, 334 for cutaneous lupus, 358 for immune thrombocytopenia, 409 for lupus nephritis in flares, 341t, 343-344 in membranous disease, 341t, 343-344 in proliferative disease, 340-343, 341t with stem cell transplant, 347, 347f for neuropsychiatric disease, 423 non-Hodgkin lymphoma and, 27 for ocular disease, 446 opportunistic infections and, 397-398, 397t for optic neuropathy, 446 for vasculitis, 316 Incidence rates, 1, 2t-3t Incomplete lupus erythematosus, 24-25, 473-480. See also Overlap syndromes ACR classification criteria and, 473-475, 477t course of, 475-479, 478t-479t disease activity in, 475, 478t-480t epidemiology of, 474-475, 476t laboratory findings in, 475 overview of, 473-480 presentation of, 475 progression to SLE, 473-474, 475f, 478t-479t signs and symptoms of, 475, 478t-480t tissue damage in, 478, 478t-480t treatment of, 478-479, 480t as undifferentiated connective tissue disease, 430, 434-436, 435t-436t Increased intraocular pressure, steroid-related, 447 Indocyanine green angiography, in choroidal disease, 445 Infections opportunistic, 152, 393-404 bacterial, 399-400, 399t in complement deficiencies, 184-188, 184t, 185f. See also Complement deficiency cyclophosphamide-related, 501 diagnosis of, 402-403 disease burden in, 393 drug-related, 397-398, 397t fever in, 331 flares and, 393 fungal, 402 immunizations for, 403-404 lymphadenopathy due to, 333 mortality from, 393, 394t mycobacterial, 400, 400t, 403 parasitic, 401-402, 402t pathogenesis of, 394-397, 395t B-cell defects in, 396 complement defects in, 396 cytokine defects in, 396, 396t immunoglobulin defects in, 396 macrophage defects in, 395 natural killer cell defects in, 396 neutrophil defects in, 395-396, 395t spleen/reticuloendothelial defects in, 396 T-cell defects in, 396 vascular defects in, 396-397 prevention of, 403-404, 507 pulmonary, 379 risk factors for, 393, 394t signs and symptoms of, 403 steroid-related, 397-398, 397t, 507 types and sites of, 398, 398t viral, 400-401, 401t vs. flares, 403

575

INDEX

Interleukin-8 deficiency of, 284 in lupus nephritis, 48t, 51 Interleukin-10 B cells and, 105 in cutaneous lupus, 324 in pathogenesis, 59, 60, 113, 115t polymorphisms in, 115t reactive oxygen intermediate and, 294, 298 Interleukin-10 antagonists, for redox signaling dysfunction, 298 Interleukin-12, in pathogenesis, 111, 115t Internuclear ophthalmoplegia, 445 Interstitial lung disease, 375t, 376, 376f in mixed connective tissue disease/overlap syndromes, 435t, 436 Intestinal disease. See also Gastrointestinal disease large intestinal, 386-387 small intestinal, 384-386 Intestinal malabsorption, 385-386 Intestinal pseudo-obstruction, 384-385 treatment of, 390-391 Intestinal vasculitis, 384 Intraocular hypertension, steroid-related, 447 Intravenous immunoglobulin, 506-507 in pregnancy, 456, 456t, 508 in antiphospholipid syndrome, 514 for congenital heart block prevention, 468, 470f In vitro fertilization, 462-464, 462t, 463t Iron deficiency anemia, 408 Ischemic heart disease, 362t, 365, 368-370, 369f. See also Atherosclerosis; Cardiac disease; Coronary artery disease Isoniazid, in drug-induced lupus, 65t J Jaundice, in neonatal lupus, 470 K Keratitis superficial punctate, 440 treatment of, 446 Keratoconjunctivitis sicca, 333, 440 treatment of, 446 Kidney. See also under Lupus nephritis; Renal immune complexes in, 218-219 Kikuchi-Fujimoto syndrome, 332 Killer-cell immunoglobulin receptor, 126 Killer-cell lectin-like receptor, 126 Klinefelter’s syndrome, with coexistent SLE, 79 Knockout mice, 165-167, 166t

576

L Labor and delivery, epidural anesthesia in, 456 Langerhans cells, 123, 124. See also Dendritic cells in cutaneous lupus, 326 functional properties of, 125 Latent disease, 24-25. See also Incomplete lupus erythematosus Late-onset lupus, 6 female-to-male ratio in, 6 Lbw2 susceptibility locus, in murine models, 176t, 177 Lbw5 susceptibility locus, in murine models, 176t, 178 Lbw7 susceptibility locus, in murine models, 175-177, 176t Learning disabilities, maternal SLE and, 450 LE cell, 259, 281 Lectin(s) in complement activation, 195, 195f in dendritic cell maturation, 123 mannose-binding. See Mannose-binding protein Leukocyte–endothelial cell adhesion, in vasculitis, 313-315, 313f-315f Leukocyte-function-associated antigen. See LFA-1

Leukocyte immunoglobulin-like receptors, 126 Leukocytoclastic vasculitis, neutrophils in, 284 LFA-1 in drug-induced lupus, 65-68, 66f in T-cell co-stimulation, 100, 100t in vasculitis, 313-315, 313f, 315f Libman-Sacks endocarditis, 367-368, 367f Linkage analysis, 75, 80-82, 80t, 81f, 82t Lipid-lowering agents hepatotoxicity of, 388 for lupus nephritis, 344 Lipid rafts, in T cells, 57, 59t Listeriosis, opportunistic, 399t, 400 Liver, in immune complex clearance, 216-217 Liver disease, 387-388. See also Hepatitis in neonatal lupus, 470 Liver function tests, abnormal, 387 LJP-394, 517t, 519 for lupus nephritis, 346 Lmb3 susceptibility locus, in murine models, 176t, 178 L-NIL, 305t, 306 L-NMMA, 305t, 306 L-selectin, neutrophils and, 282, 282t Lung. See also under Pulmonary shrinking, 378-379, 379f Lung disease, 374-379, 508. See also Pleuropulmonary disease ACR classification criteria for, 474t corticosteroids for, 490-492 immunizations for, 403, 507 in mixed connective tissue disease/overlap syndromes, 432-433, 435t, 436 Lupus Activity Index, 20t, 21-22 Lupus anticoagulant, 409. See also Antiphospholipid antibodies in antiphospholipid syndrome, 233-234, 409-410, 411 assays for, 234 in neuropsychiatric disease, 416 in thrombocytopenia, 409 Lupus anticoagulant test, for antiphospholipid antibodies, 409 in antiphospholipid syndrome, 411 Lupus band test, 321-322, 321f Lupus choroidopathy, 444-445 Lupus colitis, 386 Lupus endocarditis, 362t, 366-368, 367f Lupus erythematosus (LE) cell, 259, 281 Lupus erythematosus phenomenon, 24 Lupus erythematosus profundus, 318, 356, 356f Lupus erythematosus telangiectodes, 356 Lupus erythematosus tumidus, 318, 351, 356-357, 357f mucin deposits in, 320 Lupus flare. See Flares Lupus hair, 358 Lupus hepatitis, 387 treatment of, 391 Lupus myocarditis, 362t, 364-365 Lupus nephritis, 26, 288-289. See also Renal disease ACR classification criteria for, 474t anti-DNA antibodies in, 226-227, 229 anti-nucleosome antibodies in, 262 atherosclerosis and, 306, 344, 369 biomarkers of, 48t, 51-52. See also Biomarkers C1q antibodies in, 220 classification of, 338, 339t clinical manifestations of, 336, 337t complement in, 188-191, 190f, 190t. See also Complement deficiency of, 183-191, 336, 337t, 339 in disease monitoring, 205, 338-339 flares and, 189-191 measurement of, 205, 338-339 in pathogenesis, 188-191 diagnosis of, 336-338 renal biopsy in, 337-338, 338f

Lupus neutropenia, 283-284, 283t Lupus pancreatitis, 388-389 Lupus panniculitis, 318, 356, 356f Lupus pericarditis, 361-363, 362f, 362t, 363f Lupus peritonitis, 386 treatment of, 391 Lupus pneumonitis, 374 corticosteroids for, 491t, 492 Lupus profundus, dapsone for, 484 Lupus psychosis, 417. See also Neuropsychiatric disease Lupus Quality of Life Scale, 34t, 36 Lupus retinopathy, 441-444 classic, 441-442, 442f, 443f, 444t peripheral pigmentary, 444 severe vaso-occlusive, 442-444, 443f, 444t Lymphadenitis, granulomatous necrotizing, 332 Lymphadenopathy, 332-333 in Kikuchi-Fujimoto syndrome, 332 Lymphatic system, dendritic cell migration through, 123 Lymphocytes. See also B cell(s); T cell(s) abnormalities of, 57-59, 58f, 59t as biomarkers, 48t, 51 in cutaneous lupus, 320, 320f, 326-327 Lymphocytic interstitial pneumonia, 376 Lymphocytotoxic antibodies, in neuropsychiatric disease, 242-243, 242t Lymphoma, 26-27, 508 cyclophosphamide-related, 502 fever in, 332 Lymphopenia, corticosteroids for, 492 LymphoStat-B, for lupus nephritis, 346-347 Lyn, 107 Lysosomal alpha-mannosidase deficiency, with coexistent SLE, 79-80 M Macrophages, defective, opportunistic infections and, 394t, 395 Maculopapular rash, 352-353. See also Cutaneous lupus erythematosus Magnetic resonance angiography, in neuropsychiatric disease, 421 Magnetic resonance imaging in myocardial disease, 365 in neuropsychiatric disease, 421 Magnetic transfer imaging, in neuropsychiatric disease, 421 Major histocompatibility genes. See MHC genes Malabsorption, 385-386 treatment of, 391 Malaria. See also Antimalarials nitric oxide and, 305 Malar rash, 351-353, 352f. See also Cutaneous lupus erythematosus retinoids for, 484 Male-to-female ratio. See Gender differences Mannose-binding protein, 196t as biomarker, 78 in complement activation, 195, 195f deficiency of, 78, 187-188 in pathogenesis, 219 Mannose-binding protein–associated serine proteases (MASPs), 195, 196t Mannosidase deficiency, with coexistent SLE, 79 Matrix-metalloproteinase-9, in neuropsychiatric disease, 416-417 Medical Outcomes Study 36-Item Short Form (SF-36), 33, 34t Megaloblastic anemia, fatigue and, 330 Membrane attack complex, 195 Mercury, in pathogenesis, 56, 70 Mer tyrosine kinase, deficiency of, defective apoptosis in, 135

INDEX

Lupus nephritis (Continued) renal function tests in, 337 urinalysis in, 336-337 diffuse alveolar hemorrhage and, 375, 376f disease monitoring in, 338-340 complement in, 205, 338-339 renal function tests in, 337 urinalysis in, 338 Fcλ receptor in, 159-160, 288-289 flares in complement-related, 189-191 treatment of, 344 genetic factors in, 52, 288 glomerular filtration rate in, 337, 338 hematuria in, 336, 337t histology of, 339t hyperlipidemia in, 306, 344, 369 immune complexes in, 188-189, 218-219, 288-289 incidence and prevalence of, 336 membranous, 336, 337t clinical presentation of, 336, 337t histology of, 339t treatment of, 343-344 mesangial clinical manifestations of, 336, 337t histology of, 339t molecular phenotyping in, 52 mortality in, 14 murine models of, 289-290 nephritic syndrome in, 336, 337t nitric oxide in, 305 nocturia in, 336, 337t opportunistic infections and, 397t, 398 pathogenesis of anti-dsDNA antibodies in, 158-160, 159f autoantibodies in, 287-289 B cells in, 288, 289-290 Fcλ receptor in, 159-160 immune complexes in, 180 T cells in, 289-290 in pediatric lupus, 6 in pregnancy, 450 prognosis in, 340 proliferative, 336, 337t clinical presentation of, 336, 337t histology of, 339t treatment of, 340-343 proteinuria in, 336, 337t, 338 rate of progression of, 345 renal cells in, 290 severity of, determinants of, 290 tissue damage in, 287-290 treatment of, 340-348 adjunctive, 344-345 B-cell depleting, 290 biologic therapies in, 345-348 corticosteroids in, 491t, 492 cyclophosphamide in, 503 cyclosporine in, 505-506 emerging therapies in, 345 in end-stage disease, 345-348 for flares, 344 for hyperlipidemia, 344 for hypertension, 344-345 immunosuppression in, 340-344 for membranous disease, 343-344 monoclonal antibodies in, 290, 345-348 mycophenolate mofetil in, 341t, 342-343, 503-505, 504t for proliferative disease, 340-343 recombinant cytokines in, 346-348 stem cell transplant in, 345-348

577

INDEX

578

Mer tyrosine kinase protooncogene (Mertk), 80 Mesenteric insufficiency, 384 treatment of, 390 Mesenteric vasculitis, 384 treatment of, 390 Mesna, with cyclophosphamide, 501b Metabolic enzyme deficiencies, with coexistent SLE, 79-80 Methotrexate, 500t, 505 opportunistic infections and, 397-398, 397t in pregnancy, 454t, 455 Methylprednisolone, 488t. See also Corticosteroids with cyclophosphamide, 502-503, 505, 506t for severe/life-threatening disease, 507 intravenous, 490 adverse effects of, 493 for lupus nephritis, 340, 341t, 343 with mycophenolate mofetil, 503 oral, 489 in pulse therapy, 490 for scleritis, 446 MFG-E8, deficiency of, defective apoptosis in, 135 MHC genes in cutaneous lupus erythematosus, 323-324, 323f, 323t dendritic cells and, 121-122, 122f in murine SLE, 174-175 in pathogenesis, 56, 75-76 in T-cell signaling, 98-99 Milk fat globule epidermal growth factor 8 (MFG-E8), deficiency of, defective apoptosis in, 135 Miller-Fisher syndrome, 445 Mineralocorticoids, 488. See also Corticosteroids Minocycline in drug-induced lupus, 65t hepatotoxicity of, 388 Miscarriage, 449. See also Fetal loss Mitochondrial hyperpolarization, in T cells in apoptosis, 293-296 in necrosis, 296-297 as therapeutic target, 297-298 Mitogen-activated protein kinase (MKAP), in T-cell signaling, 99 Mixed connective tissue disease, 429, 432-436 classification of, 429-430, 430t clinical features of, 432-433 course and prognosis of, 434, 436 course of, 434 definition of, 429 pathogenesis of, 433-434 serologic and immunologic features of, 433, 433t treatment of, 434-436, 435t-436t MN(III) tetrakis(N-ethylpyridinium-2yl)porphyrin, 306-307 Molecular mimicry in antiphospholipid syndrome, 236 in autoimmunity, 143-144, 144f Monoclonal antibodies, 516-521, 517t anticomplement, 207 for autoantibody studies, 157-158 for lupus nephritis, 345-346 for severe/life-threatening disease, 507 Monocyte chemoattractant protein-1, as biomarker, for lupus nephritis, 48t, 51 Mood disorders, 36-37, 417. See also Neuropsychiatric disease fatigue and, 37, 331 Mortality, 10-14, 11t-12t, 13f, 26-29, 27t, 29t. See also Prognostic factors causes of death and, 28 disease-related factors and, 28 gender differences in, 29 post-pregnancy maternal, 451 race/ethnicity and, 10-14, 11t-12t, 28-29 socioeconomic factors and, 13-14, 28-29

Mouse models. See Murine models Mouth dry, 333, 382-383, 390 ulcers of, 353, 353f, 382-383, 390 MRL/lpr mice, 163, 164t, 171-172. See also Murine models Mucin, dermal deposition of, 320, 320f Mucinosis, paponodular, 358 Mucocutaneous lesions, 353, 353f oral, 382-383 treatment of, 390 Multiple sclerosis, PTPN22 in, 78 Murine models, 57, 163-167, 171-180 congenic, 164-165, 165t of congenital heart block, 253-254 Ea protective effects in, 174-175 engineered, 165-167, 166t Fas/Fas ligand mutations in, 172-173 H2d/z heterozygosity in, 174 of lupus nephritis, 289-290 MHC genes in, 174-175 multigenic disease in, 172, 180b non–MHC-linked susceptibility loci in, 175-179 on chromosome 1, 175-177, 176t on chromosome 4, 176t, 177-178 on chromosome 7, 176t, 178 on chromosome 13, 176t, 179 on chromosome 17, 176t spontaneous, 163-164, 164t, 172-174 Yaa mutation in, 173-174 Musculoskeletal complications, 26 ACR classification criteria for, 474t corticosteroids for, 490, 491t Myasthenia gravis, 417 Mycobacterial infections opportunistic, 400, 400t prevention of, 403 Mycophenolate mofetil, 500t, 503-505, 504t, 506t adverse effects of, 500t, 503 clinical trials for, 503, 504t clinical uses of, 503-505, 504t, 506t dosage of, 500t, 503, 504t for lupus nephritis, 341t, 342-343, 503-505, 504t with methylprednisolone, 503 monitoring of, 500t opportunistic infections and, 397t, 398 pharmacokinetics of, 503 in pregnancy, 454t, 455, 508 for severe/life-threatening disease, 507 Myocardial disease, 362t, 364-366, 365f Myocardial infarction, 362t, 368-370 Myocarditis, corticosteroids for, 491t, 492 Myositis corticosteroids for, 490, 491t in mixed connective tissue disease, 432, 436 orbital, 441 N Nails, fungal infections of, 402 Natural history, 24-29 racial/ethnic differences in, 6-10, 9t-10t Natural killer cells defective, opportunistic infections and, 396 dendritic cells and, 126 in pathogenesis, 100, 100t Nausea and vomiting cyclophosphamide-related, 501b mycophenolate mofetil–related, 501b Nba1 susceptibility locus, in murine models, 176t, 177 Nba5 susceptibility locus, in murine models, 176t, 178

Neutropenia, 283-284, 283t corticosteroids for, 492 Neutrophils, 281-284 apoptosis of, 281-284, 283t defective, opportunistic infections and, 394t, 395 disease activity and, 284 functions of, 281-282 immune complexes and, 281-282 in innate immune response, 281-283 LE cell and, 281 in leukoclastic vasculitis, 284 in pathogenesis, 281-283 surface receptors of, 281-283, 282t Nitric oxide, 301-307 in apoptosis, 302, 304 atherosclerosis and, 306 cytotoxic effects of, 302 in innate immunity, 301-302, 302f in lupus nephritis, 305 in pathogenesis, 295, 298, 302-307 in African Americans, 305 murine models of, 302-305 synthesis of, 301, 302f in T-cell activation, 295, 298 as therapeutic target, 306-307, 306t Nitric oxide synthase endothelial, 301 inducible, 301 atherosclerosis and, 306 in lupus nephritis, 305 in nitric oxide synthesis, 301, 302f in pathogenesis, 301-307 as therapeutic target, 306-307, 306t isoforms of, 301 neuronal, 301 NMDA receptors, in neuropsychiatric disease, 243 Nocardiosis, 402 Nocturia, in lupus nephritis, 336, 337t Nodular regenerative hyperplasia, 388 Non-Hodgkin lymphoma, 26-27, 508 cyclophosphamide-related, 502 fever in, 332 Nonsteroidal antiinflammatory drugs, 483 adverse effects of, 483 in pregnancy, 453-454, 454t, 456, 456t Noonan syndrome, with coexistent SLE, 70 Nottingham Health Profile, 34, 34t Nuclear receptors, 90, 90f cofactors for, 90 NZB mice, 163, 164t, 171-172. See also Murine models NZW mice, 163, 164t, 171-172. See also Murine models O Ocular disease, 440-447 anterior segment, 440-441 arteriolar occlusion, 444 choroidal, 444-445 classification of, 440 clinical presentation of, 440-445 diagnosis of, 445 episcleritis, 441 evaluation of, 440 keratoconjunctivitis sicca, 333, 440, 446 lid involvement in, 441 ocular motility abnormalities, 445 ophthalmic examination in, 447 ophthalmoplegia, 445 optic nerve, 445 orbital, 441 posterior segment, 441-445 red eye in, differential diagnosis of, 441, 441b

INDEX

Necrosis proinflammatory effects of, 297 T-cell, 296-297 Necrotizing vasculitis, 310, 311t Neisseria infections, opportunistic, 399, 399t Neonatal lupus, 466-471 anti-SSA/Ro–SSB/La antibodies in, 248-254, 354, 363, 466 apoptosis in, 251 autoantibodies in, 248-254, 354, 363, 466-467 breast-feeding in, 470-471 classification of, 467 congenital heart block in, 248-255, 363, 466-469, 471. See also Congenital heart block hepatobiliary involvement in, 469-470 incidence of, 466, 466t overview of, 466-467 rash in, 354, 466, 469, 469t transient manifestations of, 466, 469-470 Nephelometry, 202 Nephritic syndrome, 336, 337t Neurofilament triplet protein, 422 Neuropathy, 417 Neuropsychiatric disease, 26, 36-37, 414-424 ACR classification criteria for, 474t acute confusional state in, 417-418 anti-dsDNA antibodies in, 159 antineuronal antibodies and, 242-245, 242t. See also Antineuronal antibodies in antiphospholipid syndrome, 410 anxiety in, 417 autoantibodies in, 415-416 biomarkers in, 422 cerebrovascular disease in, 417 chorea in, 417 classification of, 414, 424t clinical impact of, 420, 420f clinical manifestations of, 417-420 cognitive dysfunction in, 418-420 in acute confusional state, 417-418 assessment of, 418-419 clinical associations with, 419 differential diagnosis of, 418t evolution of, 419, 419f non-SLE causes of, 418, 418t risk factors for, 419-420 subclinical, 418 treatment of, 423-424 cognitive rehabilitation in, 423-424 corticosteroids for, 491t, 492 demyelination in, 417 diagnosis of, 421-423 differential diagnosis of, 414-415, 418t epidemiology of, 414-415, 415-416 fatigue in, 331 headache in, 417 imaging studies in, 421-422 inflammatory mediators in, 416-417 laboratory findings in, 422-423 in mixed connective tissue disease/overlap syndromes, 435t mood disorders in, 417 myasthenia gravis in, 417 neuropathy in, 417 in pediatric lupus, 6 prognosis of, 420-421, 420f psychosis in, 417 quality of life and, 36-37, 415f, 420, 420f seizures in, 417 transverse myelopathy in, 417 treatment of, 37-38, 423-424 vasculopathy in, 415

579

INDEX

580

Ocular disease (Continued) retinal, 441-445 chorioretinopathy, 444-445 classic lupus retinopathy, 441-442, 442f, 443f peripheral pigmentary retinopathy, 444 retinal detachment, 444-445 retinal necrosis, 444, 444f retinal vasculitis, 442-444, 443f retrochiasmal, 445 scleritis, 441 steroid-related, 446-447 superficial punctate keratitis, 440 treatment of, 445-447 venular occlusion, 444-445 vision loss in, 442b Ocular dryness, 333, 435t, 440, 446 Ocular hypertension, steroid-related, 447 Ocular motility disorders, 445 Ocular toxicity of antimalarials, 447, 484 of steroids, 446-447 Ondansetrone, with cyclophosphamide, 501b ONOO. See Peroxynitrite (ONOO) Onychomycosis, 402 Ophthalmic examination, 447 Ophthalmoplegia, 445 Opportunistic infections. See Infections, opportunistic Opsonization apoptotic, 138-139 complement in, 196 Optic neuropathy, 445 treatment of, 446 Oral dryness, 333, 382-383 treatment of, 390 Oral ulcers, 353, 353f, 382-383 treatment of, 390 Orbital disorders, 441 Organ damage, 26-27 contributory factors in, 26-27, 26b quality of life and, 36 Organic brain syndrome, 417-418. See also Cognitive dysfunction Osteomyelitis, in complement deficiency, 185f Osteonecrosis, steroid-related, 493 Ovarian failure, cyclophosphamide-related, 501-502 Overlap syndromes, 429-432, 429t. See also Incomplete lupus erythematosus classification of, 430-431 definition of, 429, 430 treatment of, 434-436, 435t-436t Ovulation induction, in infertility, 462-463, 462t, 463t Oxidative stress, in T cells, 293-298 P Pacemakers, for congenital heart block, 467 Pain abdominal differential diagnosis of, 389-390, 389t in intestinal pseudo-obstruction, 385 in mesenteric insufficiency, 384 in mesenteric/intestinal vasculitis, 384 chest, 362t, 368-370 Pancreatic disease, 383t, 388-389 treatment of, 391 Paponodular mucinosis, 358 Pap smears, 508 Paraneoplastic syndrome, subacute cutaneous lupus erythematosus as, 354 Parasitic infections, 401-402. See also Infections, opportunistic Parvovirus B 19 infection, 151, 152t

Passive hemagglutination assay (PHA), for anti-spliceosome antibodies, 278 Pathogenesis, 55-62 anti-DNA antibodies in, 229-230, 281 apoptosis in, 56, 62, 135-140 B cells in, 58-59, 103-107, 105f cell adhesion molecules in, 100 cell surface receptor alterations in, 60-62, 77 chemicals in, 56, 69-70 complement in, 56, 76-77, 199-201 CTLA-4 in, 78 cytokines in, 59-60, 77-78, 79, 109-116 dendritic cells in, 125-126, 127-128, 128f, 137-138, 215-216 diet in, 56 DNA methylation in, 64-68, 100 drugs in, 55, 64-68, 379 environmental factors in, 55-56, 64-71. See also Environmental factors Epstein-Barr virus in, 56 Fcλ receptor in, 56, 57-58, 58t, 60, 77, 107, 282-283 feed-forward loop in, 134 apoptosis in, 136, 138-139, 139f serum factors in, 139, 139f Toll-like receptors in, 136 genetic factors in, 56-57, 74-82. See also Genetics/genetic factors heavy metals in, 56, 69, 69t, 70 hormonal factors in, 57, 87-93 immune complexes in, 188-191, 198-199, 214-216, 216f immunizations in, 404 infections in, 13-15, 26, 56, 66, 67f, 69, 75-76, 143-152 interferons in, 59-60, 61, 61f, 78-79, 127-128, 137-138 loss of tolerance in, 95, 96f, 97t, 100-101 mannose-binding protein in, 78, 219 MHC genes in, 56, 75-76 neutrophils in, 281-284 nitric oxide in, 295, 298, 301-307 murine models of, 302-305 overview of, 55, 61f, 62 PD-1 in, 78 peroxynitrite (ONOO) in, 304-305, 304t PTPN22 in, 78-79 T cells in, 57-58, 58f, 59t, 60, 62, 95-101 Toll-like receptors in, 136, 137 tumor necrosis factor-α in, 59, 60, 77-78 two-step model of, 103, 134 ultraviolet light in, 55-56, 68-69 PD-1, 78 PDCD-1, in pathogenesis, 56 Pediatric lupus, 6. See also Neonatal lupus female-to-male ratio in, 6 gender and, 6 neuropsychiatric involvement in, 6 prognosis in, 6 renal involvement in, 6 Penicillamine, in drug-induced lupus, 65t Peptic ulcer disease, 383-384, 383t Pericardial disease, 361-363, 362f, 362t, 363f corticosteroids for, 490-492 Pericardial tamponade, 361 Periodontal disease, 383 Periorbital edema, 441 Peripheral neuropathy, 417 Peripheral tolerance. See also Immunologic tolerance defects in, 95-97, 96f, 97t, 100-101 Peritonitis, 386 treatment of, 391 Pernicious anemia, 383-384 fatigue and, 330

Pneumococcal disease, immunization for, 404 Pneumocystis carinii pneumonia, 402, 402t prevention of, 403, 507 Pneumonia, 379 bronchiolitis obliterans organizing, 377 lymphocytic interstitial, 376 nonspecific interstitial, 376, 376f Pneumocystis carinii, 402, 402t, 403, 507 Pneumonitis, 374, 375t corticosteroids for, 491t, 492 Polyarteritis nodosa, 310 Polymorphonuclear leukocytes. See Neutrophils Polymyositis in mixed connective tissue disease, 429, 430t, 432-436 in overlap syndromes, 429, 430-432, 430t Positron emission tomography, in neuropsychiatric disease, 421 Postinfectious lupus. See Infections, pathogenic PPD testing, 507 Precipitin curve, 214-215, 214f Prednisolone, 488, 488t. See also Corticosteroids for constitutional features, 334 for incomplete lupus, 479, 480t for ocular disease, 446-447 opportunistic infections and, 397-398, 397t oral, 489 for protein-losing gastroenteropathy, 391 for scleritis, 446 Prednisone, 488, 488t. See also Corticosteroids for cognitive dysfunction, 423 dosage of, 489, 491t hyperlipidemia and, 369-370 indications for, 491t for lupus nephritis, 340, 341t, 343, 491t, 492 for mixed connective tissue disease/overlap syndromes, 436 for neuropsychiatric disease, 423, 491t, 492 opportunistic infections and, 397-398, 397t oral, 489 for pericarditis, 363 in pregnancy, 454-455, 454t Preeclampsia prevention of, aspirin for, 456 vs. SLE complications, 452-453 Pregnancy, 449-457. See also Infertility anticoagulation in adverse effects of, 514 in antiphospholipid syndrome, 456, 513-514, 514t in antiphospholipid syndrome, 233, 236-238, 409, 410, 411-412, 450, 456, 513-514, 514t fetal loss in, 233, 236-238, 409, 410, 449-450, 462, 513-514. See also Fetal loss fetal monitoring in, 514 treatment considerations in, 411-412, 456 antiphospholipid syndrome in, 233, 236-238, 409, 410, 411-412, 450, 456, 513-514, 514t fetal loss in, 233, 236-238, 409, 410, 411-412, 449-450, 513-514. See also Fetal loss fetal monitoring in, 514 treatment considerations in, 456 assisted reproductive technologies for, 462-464, 462t, 463t fetal monitoring in, 453 in antiphospholipid syndrome, 514 in mixed connective tissue disease/overlap syndromes, 435t SLE in anemia in, 452 anti-dsDNA antibodies in, 452 antiphospholipid antibodies in. See Pregnancy, antiphospholipid syndrome in anti-SSA/Ro antibodies in, 451-452

INDEX

Peroxynitrite (ONOO), 302 in apoptosis, 302, 304 cytotoxic effects of, 302 pathogenic effects of, 304-305, 304t as therapeutic target, 306-307, 306t PET (positron emission tomography), in neuropsychiatric disease, 421 Phagocytosis, 60, 62, 281-284. See also Apoptosis anti-DNA antibodies in, 281 apoptotic, 135-136, 138-139, 199-200 opsonization and, 138-139, 199-200 C1q deficiency and, 200 defective, opportunistic infections and, 396 of immune complexes, 188-189, 196, 214-215, 216-218 neutrophils in, 281-284 Phosphatidylserine in antiphospholipid syndrome, 235 in apoptotic opsonization, 138 β2GPI binding to, 235 prothrombin binding to, 235 Photosensitive lupus dermatitis, 352-353. See also Cutaneous lupus erythematosus Photosensitivity, in cutaneous lupus, 325-326, 325f, 352, 353-354, 353f, 353t, 355, 357, 358 Pigmentary retinopathy, 444 Pilocarpine, for Sjögren’s syndrome, 333 Pimecrolimus, for cutaneous lupus, 358 Placental thrombosis, in antiphospholipid syndrome, 410 Plasma cells, CD27high, 104 Plasmacytoid dendritic cells, 124, 125-126. See also Dendritic cells interferon-α production by, 125-126, 127-128, 137-138, 215 Plasmapheresis, 221 for congenital heart block prevention, 468 Plasmin, transforming growth factor-β and, 116 Platelet-bound C4d, as biomarker, 206 Platelet deficiencies, 409 in antiphospholipid syndrome, 409, 410 treatment of, 513 corticosteroids for, 491t, 492 danazol for, 485 in neonatal lupus, 470 in pregnancy, 452 Platelet receptors, 409 in antiphospholipid syndrome, 237-238 Pleuropulmonary disease, 374-379, 375f, 375t. See also under Pulmonary acute pneumonitis, 374 acute reversible hypoxemic syndrome, 378 bronchiolitis obliterans, 377 chronic interstitial lung disease, 375t, 376, 376f in mixed connective tissue disease/overlap syndromes, 435t, 436 corticosteroids for, 490-492, 491t, 492 diffuse alveolar hemorrhage, 374-375 in drug-induced lupus, 379 in mixed connective tissue disease/overlap syndromes, 432-433, 435t, 436 overview of, 374, 375t pleural disease, 374, 375f, 375t pneumonia, 379. See also Pneumonia pneumonitis, 374, 375t pulmonary edema, 379 pulmonary embolism, 377-378, 379 pulmonary fibrosis, 376 pulmonary hypertension, 375t, 377-378, 377t in mixed connective tissue disease, 432-433 respiratory muscle dysfunction, 378-379, 378f shrinking lung syndrome, 378-379, 378f small airways disease, 375t, 376-377 types of, 375t

581

INDEX

582

Pregnancy (Continued) complement in, 452 complete blood count in, 452 C-reactive protein in, 452 disease activity in, 450-451 drug therapy for, 453-457, 456t, 508 erythrocyte sedimentation rate in, 452 fetal loss in, 233, 236-238, 409, 410, 411-412, 449-450, 460t, 461-462, 464-465, 513-514. See also Fetal loss flares in, 450 hematologic abnormalities in, 452 laboratory testing in, 451-452 long-term effects on offspring and, 450 lupus effects in, 449-450 lupus nephritis in, 452 maternal death after, 450-451 maternal-fetal monitoring in, 451-453 neonatal lupus and, 466-471. See also Neonatal lupus placental damage in, 451 preterm birth in, 450 proteinuria in, 450 renal disease in, 450 renal function tests in, 452 risk factors in, 449-450 vs. preeclampsia, 452-453 Prematurity, 450 Prenatal screening, for congenital heart block, 453, 468 Preterm birth, 450 Prevalence rates, 1-5, 4t-5t Primary biliary cirrhosis, 388 PR interval, in fetal echocardiography, 468 Procainamide lupus induction by, 66-68. See also Drug-induced lupus in pathogenesis, 55 Productivity impairment, in economic assessment, 40-41 Progesterone gene targets of, 88t immunologic effects of, 87, 87t Prognostic factors, 13-14, 28-29. See also Mortality age at onset, 29, 29t disease-related, 28, 29t gender-related, 29t racial/ethnic, 13-14, 28-29, 29t socioeconomic, 13-14, 28-29, 29t Programmed cell death. See Apoptosis Programmed cell death-1, in pathogenesis, 56, 100, 100t Prolactin, 57, 88, 88t, 92 disease activity and, 57, 88 gene targets of, 88t immunologic functions of, 88t in pathogenesis, 57, 88 Prolidase deficiency, with coexistent SLE, 79 Properdin, 195, 195f, 196t Prostacyclin inhibitors, 483 Protamine sulfate, for heparin reversal, 456 Protease inhibitors, anticomplement activity of, 207 Protein A proliferation-inducing ligand (APRIL), 59 Protein C, in antiphospholipid syndrome, 237, 411 Protein-losing gastroenteropathy, 385-386, 386f treatment of, 391 Protein tyrosine phosphatase non-receptor type 22 gene, 78-79 Proteinuria in lupus nephritis, 336, 337t in pregnancy, 450, 452 in preeclampsia, 452-453 Protein Z, in antiphospholipid syndrome, 237 Prothrombin, 235 in antiphospholipid syndrome, 234, 235, 236 P-selectin, in vasculitis, 313-314, 313f, 315 Psychological disorders. See Neuropsychiatric disease

Psychosis, 417 Psychotherapy, for improved quality of life, 37-38 PTPN22, 78-79, 80 Pulmonary disease, 374-379, 508. See also Pleuropulmonary disease ACR classification criteria for, 474t corticosteroids for, 490-492 immunizations for, 403, 507 in mixed connective tissue disease/overlap syndromes, 432-433, 435t, 436 Pulmonary dysfunction, in mixed connective tissue disease, 432 Pulmonary edema, 379 Pulmonary embolism antiphospholipid antibodies and, 378, 379 pulmonary hypertension and, 377-378 Pulmonary fibrosis, 376 Pulmonary hypertension, 375t, 377-378, 377t antiphospholipid antibodies and, 378 in mixed connective tissue disease, 432-433, 435t, 436 pulmonary embolism and, 377-378 Pure red cell aplasia, 409 Q Quality of life, 32-38 behavioral interventions and, 37-38 disease damage and, 36 domains of, 33 fatigue and, 37, 329-331 fibromyalgia and, 37 measures of, 32-36. See also Disease activity scales Arthritis Impact Measurement Scale, 34t, 35 disease-specific, 33, 34-35, 34t European Quality of Life Scale, 34t, 35 generic, 33-35, 34t Lupus Quality of Life Scale, 34t, 36 Nottingham Health Profile, 34, 34t SF-36, 33-34, 34t, 330-331 Sickness Impact Profile, 34-35, 34t SLE-specific, 34t, 35-36 SLE Symptom Checklist, 34t, 36 Stanford Health Assessment Questionnaire Disability Index, 34t, 35 Systemic Lupus Erythematosus Quality of Life Questionnaire, 34t, 36, 561-563 validity of, 32-33 World Health Organization Quality of Life Scale, 34t, 35 in neuropsychiatric disease, 36-37, 415f, 420, 420f psychosocial aspects of, 36-37 Quinacrine, 483-484 Quinidine, in drug-induced lupus, 65t R Racial/ethnic differences, 6-10 in course and outcome, 6-10, 9t-10t in survival, 10-14, 11t-12t, 28-29 in incidence and prevalence, 8-10, 8t Radial immunodiffusion, in complement measurement, 202 Radiation, ultraviolet in pathogenesis, 55-56, 68-69 sensitivity to, in cutaneous lupus, 325-326, 325f, 352, 353-354, 353f, 353t, 355, 357, 358 Raji cell assay, for immune complexes, 221 Rash. See also Cutaneous lupus erythematosus in acute cutaneous lupus, 352-353, 352f eyelid, 441 maculopapular, 352-353 malar (butterfly), 351-353, 352f in mixed connective tissue disease, 432 in neonatal lupus, 354, 466, 469, 469t retinoids for, 484 in subacute cutaneous lupus, 353-354, 353f, 354f

S Salmonellosis, opportunistic, 399-400, 399t Scarring, in discoid lupus, 355, 355f sCD25, as biomarker, 48t, 50 Schirmer’s test, 440, 445 Schwartzman reaction, in vasculitis, 313-314 Scleritis, 441, 445 treatment of, 446 Sclerodactyly, in mixed connective tissue disease, 432

Scleroderma in mixed connective tissue disease, 429, 430t, 432-436 in overlap syndromes, 429, 430-432, 430t sCR1, therapeutic applications of, 207 Screening for cancer, 508 prenatal, for congenital heart block, 453, 468 for tuberculosis, 507 Seizures, 417 Selectins in cutaneous disease, 326 neutrophils and, 282, 282t in vasculitis, 313-314, 313f, 315 Self-antigens, cross-reactivity with autoantibodies, 143-144, 144f, 144t Sensitivity, of biomarkers, 46 Serine-arginine–rich (SR) proteins, autoantibodies to, 278 Serologically active clinically quiescent disease, 25 Serositis, corticosteroids for, 490-492, 491t Sex steroid receptors, 90-91, 90f Sexual dimorphism. See Gender differences SF-36 health survey, 33-34, 34t fatigue in, 330-331 Sgp3 susceptibility locus, in murine models, 176t, 179 Sgp4 susceptibility locus, in murine models, 176t, 177, 179 Shrinking lung syndrome, 378-379, 379f Sicca symptoms in mixed connective tissue disease/overlap syndromes, 435t ocular, 333, 440, 446 oral, 333, 382-383 treatment of, 390, 446 Sickness Impact Profile, 34-35, 34t Signaling abnormal, in pathogenesis, 57-58, 58f in apoptosis, 294-295 B-cell, 106-107 class I recognition receptors in, 126-127 in dendritic cell maturation, 123 in drug-induced lupus, 67, 67f estrogen receptors in, 90-91, 90f in pathogenesis, 57-58, 58f, 59, 67, 67f, 90-91, 98-99, 98t T-cell, 98-99, 293-298, 296t as therapeutic target, 520 in vasculitis, 315-316, 316f Signs and symptoms, 25, 329-334 sIL-2 receptor, as biomarker for disease activity, 48t, 50 for lupus nephritis, 48t, 51 Silica, in pathogenesis, 56, 69-70 Single-nucleotide-gene polymorphisms, in pathogenesis, 56-57 Single-photon emission computed tomography (SPECT), in neuropsychiatric disease, 421 Sjögren’s syndrome in overlap syndromes, 431-432 sicca symptoms in ocular, 333, 440, 446 oral, 333 treatment of, 390, 446 Skin lesions, 351-358. See also Cutaneous lupus erythematosus; Rash corticosteroids for, 491t in mixed connective tissue disease, 432 in neonatal lupus, 354, 466, 469, 469t, 470t retinoids for, 484 Sle1 susceptibility locus, in murine models, 175-177, 176t Sle2 susceptibility locus, in murine models, 176t, 177-178 Sle3 susceptibility locus, in murine models, 176t, 178 SLEDAI (Systemic Lupus Erythematosus Disease Activity Index), 20t, 21, 524-530 Sleep problems, 330-331

INDEX

Raynaud’s phenomenon, in mixed connective tissue disease/overlap syndromes, 432, 435t Reactive nitrogen intermediate, 301. See also Nitric oxide Reactive oxygen intermediate atherosclerosis and, 306 in T cells apoptosis and, 293-296 necrosis and, 296-297 as therapeutic target, 297-298 Red eye, differential diagnosis of, 441, 441b Redox signaling, in T cells, 293-298 Reference sera, for autoantibody testing, 269, 270t Rehabilitation, cognitive, 423-424 Renal biopsy, 337-338, 338f, 339t Renal disease. See also Lupus nephritis ACR classification criteria for, 474t antiphospholipid syndrome and, 344 atherosclerosis and, 344, 369 in burned-out lupus, 345 corticosteroids for, 491t hyperlipidemia in, 344, 369 hypertensive nephropathy in, 344 in incomplete lupus, 475 microangiopathic, 344 in mixed connective tissue disease/overlap syndromes, 436t renal vein thrombosis in, 344 Renal function tests, 337 in pregnancy, 452 Renal hypertension, treatment of, 344-345 Renal toxicity, of nonsteroidal antiinflammatory drugs, 483 Renal transplantation, 345 Renal vein thrombosis, 344, 410 Respiratory dysfunction, 374-379. See also Pleuropulmonary disease in mixed connective tissue disease/overlap syndromes, 432-433, 435t, 436 Respiratory muscle dysfunction, shrinking lung syndrome and, 378-379, 378f Reticulocyte-bound C4d, as biomarker, 206 Retinal detachment, 444-445 Retinal necrosis, 444, 444f Retinal vasculitis, 442-444, 443f Retinitis, 444 Retinitis pigmentosa, 70 Retinoids, 484 Retinopathy classic lupus, 441-442, 442f, 443f, 444t peripheral pigmentary, 444 severe vaso-occlusive, 442-444, 443f, 444t Retrochiasmal disease, 445 Retrovirus infections, 151-152, 152t in pathogenesis, 66, 69 Rheumatoid arthritis, in overlap syndromes, 429, 430-432, 430t Ritodrine, in pregnancy, for congenital heart block, 469 Rituximab for lupus nephritis, 345-346 for severe/life-threatening disease, 507 RNA polymerase, in overlap syndromes, 429t, 431 Rowell’s syndrome, 354 Rsl, in murine SLE, 179

583

INDEX

584

SLEQOL scale, 34t, 36, 561-563 SLE Symptom Checklist, 34t, 36 SLICC/ACR Damage Index, 26, 27 Small airways disease, 376-377 Small nuclear ribonucleoproteins (snRNPs). See snRNPs Smoking, coronary artery disease and, 369, 370 snRNPs (small nuclear ribonucleoproteins) anti-spliceosomal antibodies and, 274-279, 275f autoantibodies recognizing conformational structures in, 277 Social support, health status and, 37 Socioeconomic factors, in survival, 13-14, 28-29, 29t Soluble CD25, as biomarker, 48t, 50 Soluble CR1, therapeutic applications of, 207 Soluble interleukin-2 receptor, as biomarker for disease activity, 48t, 50 for lupus nephritis, 48t, 51 Soluble thrombomodulin, as biomarker for disease activity, 48t, 51 for lupus nephritis, 48t, 51 Soluble VCAM-1, as biomarker, 48t, 51 Specificity, of biomarkers, 46 SPECT, in neuropsychiatric disease, 421 Spinal anesthesia, obstetric, 456 Spleen disorders of, opportunistic infections and, 396 in immune complex clearance, 216-217 Splenectomy, for immune thrombocytopenia, 409 Spliceosome components of, 274, 275f snRNPs in, 274, 275f Spontaneous murine models, 163-164, 164t, 171-172 S protein, 197t SR proteins, autoantibodies to, 278 Stanford Health Assessment Questionnaire Disability Index, 34t, 35 Staphylococcal infections, opportunistic, 399, 399t Statins hepatotoxicity of, 388 for lupus nephritis, 344 Stem cell transplantation, for lupus nephritis, 347 Steroid reduction syndrome, 492-493 Steroids. See Corticosteroids Stillbirth, 449-450. See also Fetal loss sTM, as biomarker, 48t, 51 Stomach disorders, 383-384, 383t. See also Gastrointestinal disease Stomatitis, aphthous, 353, 353f, 382-383 treatment of, 390 Streptococcal infections, opportunistic, 399, 399t Stress, oxidative, in T cells, 293-298 Stroke, in antiphospholipid syndrome, prevention of, 368, 378, 411-412, 511-513, 513t Subclinical lupus. See Incomplete lupus erythematosus Sulfasalazine, in drug-induced lupus, 65t Sunlight. See Ultraviolet (UV) radiation Sun protection, for cutaneous LE, 358 Superficial punctate keratitis, 440 Surrogate endpoints, 46, 47t. See also Biomarkers definition of, 46 qualification of, 46, 47 validation of, 46-47, 47t Survival. See Mortality; Prognostic factors sVCAM-1, as biomarker, 48t, 51 Swelling. See also Edema of hands, in mixed connective tissue disease, 432 Syk kinase, in pathogenesis, 57, 58f Systemic Lupus Activity International Collaborating Clinics Damage Index, 26-27 Systemic Lupus Activity Measure, 20t, 21, 332, 559-560

Systemic lupus erythematosus ANA–negative, 24 asymptomatic, 103, 134 biomarkers for, 46-52 classification criteria for, 1, 24-25, 473-475, 474t, 477t clinically active serologically quiescent disease, 25 clinical manifestations, 25, 329-334 disease activity scales for, 19-22, 20t epidemiology of, 1-14 familial, 74. See also under Genetics/genetic factors flares in. See Flares incomplete lupus progression to, 473-474, 475-479, 478t-479t latent (incomplete), 24-25 murine models of, 163-167 natural history of, 24-29 neonatal, 466-471 opportunistic infections in, 152, 393-404 pathogenesis of, 55-62 pediatric, 6 postinfectious, 26, 56, 66, 67f, 69, 75-76, 143-152 quality of life in, 32-38 serologically active clinically quiescent, 25 Th1/Th2, 59, 109 Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), 20t, 21, 332, 524-530, 558 Systemic Lupus Erythematosus Quality of Life Questionnaire (SLEQOL), 34t, 36, 561-563 T Tachycardia, 363 Tacrolimus, for cutaneous LE, 358 Tamponade, pericardial, 361 Tanning booths, 69 T cell(s), 95-101, 520 in aberrant signaling, 520 abnormalities of, 57-58, 58f, 59t, 60, 62, 520 opportunistic infections and, 396 activation of, redox control in, 294-295 in antiphospholipid syndrome, 236 apoptosis in, 293, 294-295 autoantigen presentation to, 99-100 autoreactive, 97-98, 98t in B-cell autoantibody production, 95, 104-105 as biomarkers, 48t, 51 CD4+ in cutaneous lupus, 320, 320f, 326-327 DNA methylation and, 65-66, 66f in pathogenesis, 100, 100t CD4+CD25+, 124 CD8+ in cutaneous LE, 320, 320f, 326-327 in pathogenesis, 100, 100t in cutaneous lupus, 320, 320f, 326-327 dendritic cells and, 123-124, 126 engineered, 520-521 in Epstein-Barr virus infection, 149 γ/δ, dendritic cells and, 126 helper, abnormalities of, 60 lipid rafts in, 57 loss of tolerance in, 95-97, 96f, 97t in lupus nephritis, 289-290 mitochondrial hyperpolarization in, 293-298 in apoptosis, 293-296 in necrosis, 296-297 as therapeutic target, 297-298 in mixed connective tissue disease, 433 necrosis of, 296-297 oxidative stress in, 293-298 in pathogenesis, 57-58, 58f, 59t, 60, 62, 95-101 regulation of

Treatment. See also specific drugs and drug families androgens in, 484-485 antimalarial drugs in, 483-484 corticosteroids in, 487-495 cytotoxic drugs in, 498-508 dapsone in, 484 nonsteroidal antiinflammatory drugs in, 483 retinoids in, 484 thalidomide in, 484 T-regulatory cells deficiency of, 100, 100t dendritic cells and, 124 engineered, 520-521 Triamcinolone, 488t Trimethoprim-sulfamethoxazole, for Pneumocystis carinii pneumonia prophylaxis, 507 T-sign, in scleritis, 445 Tuberculosis, 400, 400t, 507 prevention of, 403, 507 Tumor necrosis factor as biomarker, 48t, 50-51 in cutaneous LE, 324 in lupus nephritis, 347 Tumor necrosis factor-α in congenital heart block, 253 in pathogenesis, 59, 60, 77-78, 114, 115t polymorphisms in, 115t therapeutic targeting of, 517t, 518-519 Tumor necrosis factor-α inhibitors, 517t, 518-519 Tumor necrosis factor inhibitors for lupus nephritis, 347 for overlap syndromes, 434-436 Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), in neutrophil apoptosis, 284 U U1 RNP, 275-276 autoantibodies to, 276-277 U-1 small nuclear ribonucleoproteins (snRNPs), 274, 275f U-2 small nuclear ribonucleoproteins (snRNPs), 274, 275f U-4 small nuclear ribonucleoproteins (snRNPs), 274, 275f U-5 small nuclear ribonucleoproteins (snRNPs), 274, 275f U-6 small nuclear ribonucleoproteins (snRNPs), 274, 275f uIL-6, as biomarker, for lupus nephritis, 48t, 51 uIL-8, as biomarker, for lupus nephritis, 48t, 51 Ulcer(s) corneal, 440 esophageal, 383 gastric, 383-384, 383t oral, 353, 353f, 382-383 treatment of, 390 Ulcerative colitis, 386-387 Ultrasonography in fetal monitoring, in antiphospholipid syndrome, 514 in posterior scleritis, 445 in pregnancy, 453 Ultraviolet (UV) radiation in pathogenesis, 55-56, 68-69 sensitivity to, in cutaneous lupus, 325-326, 325f, 352, 353-354, 353f, 353t, 355, 357, 358 uMCP-1, as biomarker, for lupus nephritis, 48t, 51 Undifferentiated connective tissue disease, 430, 434-436, 435t-436t. See also Overlap syndromes incomplete lupus as, 474, 475f, 479-480 Urinalysis, in lupus nephritis, 336, 337t Urinary interleukin-8, as biomarker, for lupus nephritis, 48t, 51 Urinary monocyte chemoattractant protein-1, as biomarker, for lupus nephritis, 48t, 51 UV radiation. See Ultraviolet (UV) radiation

INDEX

T cell(s) (Continued) CTLA-4 in, 78 DNA methylation in, 65-68, 66f, 67f estrogen in, 89-90, 89f, 91 PD-1 in, 78 redox signaling in, 294-296, 296t regulatory deficiency of, 100, 100t dendritic cells and, 124 engineered, 520-521 T-cell–antigen interactions, abnormalities in, 100 T-cell co-stimulation, abnormalities in, 100 T-cell epitopes, autoantigenicity and, 98, 99-100 T-cell receptor, 98-99, 98t cross-reactivity in, 98 in pathogenesis, 57-58, 58f T-cell signaling, defects in, 98-99, 98t Tear production, decreased, 333, 435t, 440, 446 Telangiectasia, in neonatal lupus, 469 Teratogenic drugs, 453-456, 455t Testicular failure, cyclophosphamide-related, 502 Tetanus immunization, 403, 404, 508 Th1/Th2 systemic lupus erythematosus, 59, 109 Thalidomide, 484 in pregnancy, 454t, 455-456 Therapeutic monitoring complement in, 189, 190f for corticosteroids, 488 for cytotoxic drugs, 500t, 501b Thrombocytopenia, 409 in antiphospholipid syndrome, 409, 410 treatment of, 513 corticosteroids for, 491t, 492 danazol for, 485 in neonatal lupus, 470 in pregnancy, 452 Thrombomodulin, as biomarker, 48t, 51 Thrombosis in antiphospholipid syndrome, 158, 233, 236-238. See also Antiphospholipid syndrome prevention of, 368, 378, 411-412, 511-513, 513t recurrent, 511-512 hepatic vein, 388 placental, 410 pulmonary embolism and antiphospholipid antibodies and, 378 pulmonary hypertension and, 377-378 renal vein, 344, 410 Thyroid disease, fatigue in, 330 Tissue damage, 26-27 contributory factors in, 26-27, 26b quality of life and, 36 Tocilizumab, for lupus nephritis, 347 Tolerance. See Immunologic tolerance Toll-like receptor(s) in apoptosis, 136 in autoantigen presentation and processing, 99, 99t bacterial DNA and, 228 dendritic cell expression of, 123 in pathogenesis, 136, 137, 215 Toll-like receptor-9, 60 Toxic epidermal necrolysis–like cutaneous lupus erythematosus, 318, 319, 352, 353 Toxoplasmosis, 401-402 Transforming growth factor-β in congenital heart block, 253, 254-255 in pathogenesis, 59, 60, 114-116, 115t Transplantation renal, 345 stem cell, for lupus nephritis, 347 Transverse myelopathy, 417

585

INDEX

586

V Vaccinations, 403-404, 507-508 Valvular heart disease, 362t, 366-368 antibiotic prophylaxis in, 507 in antiphospholipid syndrome, 410 bacterial endocarditis in, 507 Variant lupus. See Incomplete lupus erythematosus Vasculitis, 310-316, 357 anti–neutrophil cytoplasmic antibody testing in, 269t, 271 autoantibodies in, 311-312 cell adhesion molecules in, 312-315, 313f-315f, 314t cell wall injury mechanisms in, 315-316 classification of, 310, 311t clinical manifestations of, 310, 312t coronary artery, 370 cutaneous, corticosteroids for, 490, 491t definition of, 310 differential diagnosis of, 311 esophageal, 383 gastric, 383 immune complexes in, 215, 311, 322 incidence of, 310-311 intestinal, 384 leukocytoclastic, neutrophils in, 284 mesenteric, 384 treatment of, 390 necrotizing, 310, 311t in neuropsychiatric disease, 415 opportunistic infections and, 396-397 pathogenesis of, 311-316 primary, 310, 311t retinal, 442-444, 443f Schwartzman reaction in, 313-314 secondary, 310, 311t signaling in, 315-316, 316f treatment of, 316 vessel size and, 310, 311t, 312f VLA-4 in, 312-313, 313f VCAM-1 as biomarker, 48t, 51 in cutaneous lupus, 326-327 in vasculitis, 313-315, 313f-315f Ventricular pacing, for congenital heart block, 467 Venular occlusion, retinal, 444

Verrucae, 401 Verrucous chilblain lupus erythematosus, 318, 319 Verrucous endocarditis, 366-368 Viral hepatitis, chronic, 387-388 Viral infections apoptosis in, 137 autoimmunity in, 143-152 bystander effects in, 144-145, 145f molecular mimicry in, 143-144, 144f Epstein-Barr virus, 56, 66, 67f, 69, 146-150 etiologic agents in, 145-146 lymphadenopathy due to, 333 opportunistic, 152, 400-401. See also Infections, opportunistic in pathogenesis, 26, 56, 66, 67f, 69, 143-152 in SLE vs. normal controls, 148-150 Vision loss, 442, 442b. See also Ocular disease Vitamin B12 deficiency, fatigue and, 330 Vitamin supplements, in pregnancy, 453, 454t, 456t Vitronectin, 197t VLA-4, in vasculitis, 312-315, 313f Vomiting cyclophosphamide-related, 501b mycophenolate mofetil–related, 501b Von Willebrand factor, as biomarker, 51 W Warfarin. See also Anticoagulation for antiphospholipid syndrome, 411, 511-513, 513t azathioprine and, 512 teratogenicity of, 514 Warts, 401 Weight loss, 333 Western blotting, for anti-spliceosome antibodies, 274-275, 278 Women. See Gender differences World Health Organization Quality of Life Scale, 34t, 35 X Xerophthalmia, 333, 435t, 440, 446 Xerostomia, 333, 382-383, 390, 435t D-Xylose suppression test, 385 Y Yaa mutation, in murine SLE, 173-174