Systemic Lupus Erythematosus: A Companion to Rheumatology

Other companion titles in the Rheumatology series: Ankylosing Spondylitis and the Spondyloarthropathies Psoriatic and ...

Author: George C. Tsokos | Caroline Gordon | Josef S. Smolen MD

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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

8

7

6

5

4

3

2

1

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

vi

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

vii


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

viii

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


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

ix


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

x

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


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




Medical recordsc

Medical records and death certificates

Medical recordsc

Practice plan medical recordsb



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


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


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


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


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


Renal and lung involvement more frequent in African Americans

Ward and Studenski112

1983–1989

258

United States


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


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


Renal involvement more frequent in non-Caucasians

Alarcón et al.8

1989–2000

568

United States


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


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


Hispanics accrue damage more rapidly

Rivest et al.147

1990’s

200

United States


African Americans exhibit more renal damage

INTRODUCTION

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

Damage

Continued

9


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


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


Lower socioeconomic status (not ethnicity) predicts mortality

Alarcón et al.23

1994–1999

288

United States


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

Fernández et al.141

1994–2004

578

United States


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


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


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.

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14

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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.


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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22

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23


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


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

—

—


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


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


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


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

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49. 50.

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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


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


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


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


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

41


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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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

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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

49


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.

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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.

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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,

55

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.

57


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

59


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.

61


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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with systemic lupus erythematosus. J.Clin Invest 2005;115: 3193-3204. Nagy G, Koncz A, Perl A. T- and B-cell abnormalities in systemic lupus erythematosus. Crit Rev Immunol 2005;25:123-140. Ramanujam M, Wang X, Huang W, Liu Z, Schiffer L, Tao H, et al. Similarities and differences between selective and nonselective BAFF blockade in murine SLE. J Clin Invest 2006;116:724-734. Kyttaris VC, JuangYT, Tsokos GC. Immune cells and cytokines in systemic lupus erythematosus: an update. Curr Opin Rheumatol 2005;17:518-522. Tsokos GC, Boumpas DT, Smith PL, Djeu JY, Balow JE, Rook AH. Deficient gamma-interferon production in patients with systemic lupus erythematosus. Arthritis Rheum 1986;29: 1210-1215. 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. 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;50:3958-3967. Nagafuchi H, Suzuki N, Mizushima Y, Sakane T. Constitutive expression of IL-6 receptors and their role in the excessive B cell function in patients with systemic lupus erythematosus. J Immunol 1993;151:6525-6534. Palucka AK, Banchereau J, Blanco P, Pascual V. The interplay of dendritic cell subsets in systemic lupus erythematosus. Immunol Cell Biol 2002;80:484-8. Jorgensen TN, Gubbels MR, Kotzin BL. New insights into disease pathogenesis from mouse lupus genetics. Curr Opin Immunol 2004;16:787-793. Wu J, Metz C, Xu X, Abe R, Gibson AW, Edberg JC, et al. A novel polymorphic CAAT/enhancer-binding protein beta element in the FasL gene promoter alters Fas ligand expression: a candidate background gene in African American systemic lupus erythematosus patients. J Immunol 2003;170:132-138. Horiuchi T, Nishizaka H, Yasunaga S, Higuchi M, Tsukamoto H, Hayashi K, et al. Association of Fas/APO-1 gene polymorphism with systemic lupus erythematosus in Japanese. Rheumatology (Oxford) 1999;38:516-520.

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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

63

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.

DRUGS

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


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


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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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.

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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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PATHOGENESIS

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

THE GENETICS OF LUPUS

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, p90%) 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.

77


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


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


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 10, 95% CI=2.53–infinity, pM 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


F>M

50

B6.Sle1.Sle2.Sle3/5

C57BL/6

1, 4, 7


F>M

50

B6.Sle1.Sle3/5

C57BL/6

1, 7


F>M

49, 50

B6.Sle2.Sle3/5

C57BL/6

4, 7

ANA, GN

F>M

50

B6.Sle1.Yaa

C57BL/6

1, Y


F>M

50

B6.Sle1.Fasl

C57BL/6

1, 19


F>M

52

B6.Lmb3.Faslpr

C57BL/6

7, 19


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


75, 76

BlyS (BAFF, transgene)

C57BL/6


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


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


110

PECAM-1(CD31) (−/−)

C57BL/6

11

ANA, GN

111

PKCδ (−/−)

C57BL/6

14


112, 113

PTEN

+/−

16

References

ANA, GN,

64


101

ANA, GN

63


105

10

ANA, GN

106

C57BL/6

6

ANA, GN

C57BL/6

4


107-109

17


80

1

81

C57BL6 x 129

19


114

Sap (−/−)

129 x C57BL/6

1

ANA, GN

62

TSAd (−/−)

C57BL/6

3


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.

REFERENCES

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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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,

171

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

173


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


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

177


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

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.

185


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

187


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


●

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


Cryoglobulinemia, Type II

C4 may be decreased more than C3

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


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


PATIENT NO. 10

PATIENT NO. 23



TABLE 19.2 RENAL DISEASES ASSOCIATED WITH HYPOCOMPLEMENTEMIA

Date


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


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


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


192

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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


C8

150 (3-chain, αβγ, complex)


C9

70


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


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


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.

201


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


TABLE 20.3 SELECT STUDIES OF COMPLEMENT MEASURES IN SLEa

203 Continued


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.


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


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.

207


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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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.

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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


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


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.


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.


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

233

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

235


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

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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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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)


40

25%

50%

Wilson (1979) (4)

Neuroblastoma cell line Neuro-2a (mouse)


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)


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%)


-

-

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


Antiribosomal P antibodies

38 Kd surface human neuroblastoma cell line

Koren (1992) (65)

Antiphospholipid antibodies

Rat brain

Kent (2000) (69)

243


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.

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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


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.


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.

IN VIVO EVIDENCE SUPPORTING APOPTOSIS IN THE CASCADE TO CARDIAC INJURY 252

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 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 (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 (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

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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.

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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


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


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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316

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7. Gross WL, Trabandt A, Reinhold-Keller E. Diagnosis and evaluation of vasculitis. Rheumatology (Oxford) 2000;39:245-252. 8. Lhote F, Cohen P, Guillevin L. Polyarteritis nodosa, microscopic polyangiitis and Churg-Strauss syndrome. Lupus 1998; 7:238-258. 9. D’Cruz D. Vasculitis in systemic lupus erythematosus. Lupus 1998;7:270-274. 10. Takeuchi T, Abe T. Role of adhesion molecules in vasculitis syndrome. Intern Med 2002;41:41-44. 11. Calamia KT, Balabanova M. Vasculitis in systemic lupus erythematosis. Clin Dermatol 2004;22:148-156. 12. Erdogan O, Oner A, Demircin G, Bulbul M, Memis L, Uner C, et al. A boy with consecutive development of SLE and Wegener granulomatosis. Pediatr Nephrol 2004;19:438-441.

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13. Drenkard C, Villa AR, Reyes E, Abello M, Alarcon-Segovia D. Vasculitis in systemic lupus erythematosus. Lupus 1997;6:235-242. 14. Golan TD. Lupus vasculitis: Differential diagnosis with antiphospholipid syndrome. Curr Rheumatol Rep 2002;4:18-24. 15. el-Gamal YM, Heshmat NM, el-Kerdany TH, Fawzy AF. Serum thrombomodulin in systemic lupus erythematosus and juvenile idiopathic arthritis. Pediatr Allergy Immunol 2004;15:270-277. 16. Molnar K, Kovacs L, Kiss M, Husz S, Dobozy A, Pokorny G. Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus. Clin Exp Dermatol 2002;27:59-61. 17. D’Cruz DP, Houssiau FA, Ramirez G, Baguley E, McCutcheon J, Vianna J, et al. Antibodies to endothelial cells in systemic lupus erythematosus: A potential marker for nephritis and vasculitis. Clin Exp Immunol 1991;85:254-261. 18. Johnson PA, Alexander HD, McMillan SA, Maxwell AP. Up-regulation of the endothelial cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) by autoantibodies in autoimmune vasculitis. Clinical & Experimental Immunology 1997;108:234-242. 19. Renaudineau Y, Dugue C, Dueymes M, Youinou P. Antiendothelial cell antibodies in systemic lupus erythematosus. Autoimmun Rev 2002;1:365-372. 20. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 1994; 76:301-314. 21. Argenbright LW, Barton RW. The Shwartzman response: a model of ICAM-1 dependent vasculitis. Agents & Actions 1991; 34:208-210. 22. van Vollenhoven RF. Adhesion molecules, sex steroids, and the pathogenesis of vasculitis syndromes. Current Opinion in Rheumatology 1995;7:4-10. 23. Tervaert JW, Kallenberg CG. Cell adhesion molecules in vasculitis. Current Opinion in Rheumatology 1997;9:16-25. 24. Kevil CG, Bullard DC. Roles of leukocyte/endothelial cell adhesion molecules in the pathogenesis of vasculitis. American Journal of Medicine 1999;106:677-687. 25. Pober JS, Cotran RS. The role of endothelial cells in inflammation. Transplantation (Baltimore) 1990;50:537-544. 26. Bevilacqua MP, Nelson RM, Mannori G, Cecconi O. Endothelialleukocyte adhesion molecules in human diseases. Ann Rev Med 1994;45:361-378. 27. Takeuchi T, Amano K, Sekine H, Koide J, Abe T. Upregulated expression and function of integrin adhesive receptors in systemic lupus erythematosus patients with vasculitis. Journal of Clinical Investigation 1993;92:3008-3016. 28. Takeuchi T, Aoki K, Koide J, Sekine H, Abe T. Systemic lupus erythematosus with necrotizing vasculitis and upregulated expression of VLA-4 antigen. Clinical Rheumatol 1995;14:370-374. 29. Amano K, Sekine H, Itoh T, Hosono OJK, Takeuchi T, Abe T. An usual variant of Wegener’s granulomatosis and the role of integrin receptors for pulmonary lesion. Jap J Rheum 1994; 5:217-226. 30. Chakravorty SJ, Howie AJ, Cockwell P, Adu D, Savage CO. T lymphocyte adhesion mechanisms within inflamed human kidney: studies with a Stamper-Woodruff assay. American Journal of Pathology 1999;154:503-514. 31. Belmont HM, Buyon J, Giorno R, Abramson S. Upregulation of endothelial cell adhesion molecules characterizes disease activity

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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

319

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

321

MECHANISMS OF SKIN DAMAGE

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.

PATHOGENESIS

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.

323


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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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

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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

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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


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


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.

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30.

31. 32.

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.

REFERENCES

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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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 (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


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

339

KIDNEY INVOLVEMENT IN SLE

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

340

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

●


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.

341

KIDNEY INVOLVEMENT IN SLE

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.)

342

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 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.

369

HEART AND SLE

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.

Treatment

370

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.

1. Keefer EB, Felty AR. Acute disseminated lupus erythematosus. Bull Johns Hopkins Hosp 1924;35:294-304. 2. Cervera R, Khamashta MA, Font J, et al. Systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 1000 patients. Medicine 1993;72:113-124. 3. Jouhikainen T, Pohjola-Sintonen S, Stephansson E. Lupus anticoagulant and cardiac manifestations in systemic lupus erythematosus. Lupus 1994;3:167-172. 4. Houman MH, Smiti-Khanfir M, Ben Ghorbell I, et al. Systemic lupus erythematosus in Tunisia: demographic and clinical analysis of 100 patients. Lupus 2004;13:204-211. 5. Kahl LE. The spectrum of pericardial tamponade in systemic lupus erythematosus. Report of 10 patients. Arthritis Rheum 1992;35:1343-1349. 6. Crozier IG, Li E, Milne MJ. Cardiac involvement in systemic lupus erythematosus detected by echocardiography. Am J Cardiol 1990;65:1145-1148. 7. Nihoyannopoulos P, Gomez PM, Joshi J. Cardiac abnormalities in systemic lupus erythematosus. Association with raised anticardiolipin antibodies. Circulation 1990;82:369-375. 8. Bulkley BH, Roberts WC. The heart in systemic lupus erythematosus and the changes induced in it by corticosteroid therapy. A study of 36 necropsy patients. Am J Med 1975;58:243-264. 9. Libman E, Sacks B. A hitherto undescribed form of valvular and mural endocarditis. Arch Int Med 1924;33:701-734. 10. Gross L. Cardiac lesions in Libman-Sacks disease with consideration of it relationship to acute diffuse lupus erythematosus. Am J Pathol 1940;16:375-408. 11. Harvey AM, Shulman LE, Tumulty PA, et al. Systemic lupus erythematosus: a review of the literature and clinical analysis of 138 cases. Medicine 1954;33:291-437. 12. Doherty NE, Siegel RJ. Cardiovascular manifestations of systemic lupus erythematosus. Am J Heart 1985;110:1257-1265. 13. Costallat LT, Coimbra AM. Systemic Lupus Erythematosus: clinical and laboratory aspects related to age at disease onset. Clin Exp Rheumatol 1994;12:603-607. 14. Hoffman IE, Peene I, Meheus L, et al. Specific antinuclear antibodies are associated with clinical features in systemic lupus erythematosus. Ann Rheum Dis 2004;63:1155-1158. 15. Jacobson EJ, Reza MJ. Constrictive pericarditis in systemic lupus erythematosus. Demonstration of immunoglobulins in the pericardium. Arthritis Rheum 1978;21:972-974. 16. Quismorio FP Jr. Immune complexes in the pericardial fluid in systemic lupus erythematosus. Arch Intern Med 1980;140:112-114. 17. Klemperer P, Pollack AD, Baehr G. Pathology of disseminated lupus erythematosus. Arch Pathol 1941;32:569-631. 18. Estes D, Christian CL. The natural history of systemic lupus erythematosus by prospective analysis. Medicine 1971;50:85-95. 19. Man BL, Mok CC. Serositis related to systemic lupus erythematosus: prevalence and outcome. Lupus 2005;14:822-826. 20. Reiner JS, Furie RA. Cardiac tamponade as an initial manifestation of systemic lupus erythematosus. J Rheumatol 1989; 16:1127-1129. 21. Ghose MK. Pericardial tamponade. A presenting manifestation of procainamide-induced lupus erythematosus. Am J Med 1975; 58:581-585. 22. Greenberg JH, Lutcher CL. Drug-induced systemic lupus erythematosus. A case with life-threatening pericardial tamponade. JAMA 1972;222:191-193. 23. Carey RM, Coleman M, Feder A. Pericardial tamponade: a major presenting manifestation of hydralazine-induced lupus syndrome. Am J Heart 1973;54:84-87. 24. Verma SP, Yunis N, Lekos A, et al. Carbamazepine-induced systemic lupus erythematosus presenting as cardiac tamponade. Chest 2000;117:597-598. 25. Sunder SK, Shah A. Constrictive pericarditis in procainamideinduced lupus erythematosus syndrome. Am J Cardiol 1975; 36:960-962. 26. Knodell RG, Manders SJ. Staphylococcal pericarditis in a patient with systemic lupus erythematosus. Chest 1974;65:103-105. 27. Kaufman LD, Seifert FC, Eilbott DJ, et al. Candida pericarditis and tamponade in a patient with systemic lupus erythematosus. Arch Intern Med 1988;148:715-717.

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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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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

REFERENCES

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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3. Wiedermann HP, Matthay RA. Pulmonary manifestations of systemic lupus erythematosus. J Thorac Imaging 1992; 7:1-18. 4. Stoll T, Seifert B, Isenberg DA. SLICC/ACR Damage Index is valid, and renal and pulmonary organ scores are predictors of severe

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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.

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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.

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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.


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.


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


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


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.


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).

387


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)

389


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.

REFERENCES 1. Sultan SM, Ioannou Y, Isenberg DA. A review of gastrointestinal manifestations of systemic lupus erythematosus. Rheumatology (Oxford) 1999;38:917-932. 2. Burge SM, Frith PA, Juniper RP, et al. Mucosal involvement in systemic and chronic cutaneous lupus erythematosus. Br J Dermatol 1989;121:727-741. 3. Manoussakis MN, Georgopoulou C, Zintzaras E, et al. Sjogren’s syndrome associated with systemic lupus erythematosus: clinical and laboratory profiles and comparison with primary Sjogren’s syndrome. Arthritis Rheum 2004;50:882-891. 4. Meyer U, Kleinheinz J, Handschel J, et al. Oral findings in three different groups of immunocompromised patients. J Oral Pathol Med 2000;29:153-158. 5. Kobayashi T, Ito S, Yamamoto K, et al. Risk of periodontitis in systemic lupus erythematosus is associated with Fcgamma receptor polymorphisms. J Periodontol 2003;74:378-384. 6. Novo E, Garcia-MacGregor E, Viera N, et al. Periodontitis and antineutrophil cytoplasmic antibodies in systemic lupus erythematosus and rheumatoid arthritis: a comparative study. J Periodontol 1999;70:185-188. 7. Scannapieco FA, Bush RB, Paju S. Associations between periodontal disease and risk for atherosclerosis, cardiovascular disease, and stroke. A systematic review. Ann Periodontol 2003;8:38-53. 8. Gutierrez F, Valenzuela JE, Ehresmann GR, et al. Esophageal dysfunction in patients with mixed connective tissue diseases and systemic lupus erythematosus. Dig Dis Sci 1982; 27:592-597. 9. Lapadula G, Muolo P, Semeraro F, et al. Esophageal motility disorders in the rheumatic diseases: a review of 150 patients. Clin Exp Rheumatol 1994;12:515-521. 10. Mok CC. Investigations and management of gastrointestinal and hepatic manifestations of systemic lupus erythematosus. Best Pract Res Clin Rheumatol 2005;19:741-766.

11. Medina F, Ayala A, Jara LJ, et al. Acute abdomen in systemic lupus erythematosus: the importance of early laparotomy. Am J Med 1997;103:100-105. 12. al-Hakeem MS, McMillen MA. Evaluation of abdominal pain in systemic lupus erythematosus. Am J Surg 1998;176:291-294. 13. Junca J, Cuxart A, Olive A, et al. Anti-intrinsic factor antibodies in systemic lupus erythematosus. Lupus 1993;2:111-114. 14. Molad Y, Rachmilewitz B, Sidi Y, et al. Serum cobalamin and transcobalamin levels in systemic lupus erythematosus. Am J Med 1990;88:141-144. 15. Hallegua DS, Wallace DJ. Gastrointestinal and hepatic manifestations. In: Wallace DJ, Hahn BH, eds. Dubois Lupus Erythematosus, 6th ed. Baltimore: Williams & Wilkins, 2000. 16. Zizic TM, Classen JN, Stevens MB. Acute abdominal complications of systemic lupus erythematosus and polyarteritis nodosa. Am J Med 1982;73:525-531. 17. Lee CK, Ahn MS, Lee EY, et al. Acute abdominal pain in systemic lupus erythematosus: focus on lupus enteritis (gastrointestinal vasculitis). Ann Rheum Dis 2002;61:547-550. 18. Buck AC, Serebro LH, Quinet RJ. Subacute abdominal pain requiring hospitalization in a systemic lupus erythematosus patient: a retrospective analysis and review of the literature. Lupus 2001;10:491-495. 19. Passam FH, Diamantis ID, Perisinaki G, et al. Intestinal ischemia as the first manifestation of vasculitis. Semin Arthritis Rheum 2004;34:431-441. 20. Helliwell TR, Flook D, Whitworth J, et al. Arteritis and venulitis in systemic lupus erythematosus resulting in massive lower intestinal haemorrhage. Histopathology 1985;9:1103-1113. 21. Ko SF, Lee TY, Cheng TT, et al. CT findings at lupus mesenteric vasculitis. Acta Radiol 1997;38:115-120. 22. Grimbacher B, Huber M, von Kempis J, et al. Successful treatment of gastrointestinal vasculitis due to systemic lupus erythematosus

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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.


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

393

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


All infections

Active lupus nephritis, prednisone dose

Leukopenia

Nived et al., 1985 (20)

All infection

Disease activity

Leukopenia, low complement


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


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


Decrease

IL-2

Decreased production in certain patients

Decrease

IL-10

Increased production

Possible decrease

TNF


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


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.


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


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 (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

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