Hcv Infection and Cryoglobulinemia

HCV Infection and Cryoglobulinemia

Franco Dammacco Editor

HCV Infection and Cryoglobulinemia Foreword by Jay H. Hoofnagle

Editor Franco Dammacco Department of Internal Medicine and Clinical Oncology University of Bari Medical School Bari, Italy

ISBN 978-88-470-1704-7 e-ISBN 978-88-470-1705-4 DOI 10.1007/978-88-470-1705-4 Springer Milan Heidelberg Dordrecht London New York Library of Congress Control Number: 2011929637 © Springer-Verlag Italia 2012 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the Italian Copyright Law in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the Italian Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

To my wife Tecla, lifetime partner. Without her unfailing inspiration, I would have been unable to read even a single page of the endless, puzzling and fascinating Book of Nature.

Foreword

Major breakthroughs in biomedical research are often followed by paradigm shifts in our understanding of diseases. This was particularly true after the landmark discovery of hepatitis C virus (HCV). The identification of a small but viral-specific RNA sequence in the serum of patients with non-A, non-B hepatitis led directly to the development of tests for antibody and viral RNA. Moreover, it fostered recognition of several facts: (1) that hepatitis C is the most common cause of chronic liver disease, cirrhosis, and liver cancer in most countries of the world; (2) that post-transfusion hepatitis can be prevented by screening for antibody; (3) that the implementation of simple public health measures would markedly decrease the rate of new infections with this virus; and (4) that a therapy with beneficial effects on the disease could actually cure the infection and permanently eradicate the virus. Another paradigm shift, perhaps less well known but just as ground-breaking, was the recognition of hepatitis C as the major cause of the uncommon and poorly understood “autoimmune” disease known as essential mixed cryoglobulinemia. Immediately obvious was that the name “essential mixed cryoglobulinemia” was no longer appropriate. The syndrome was not “essential” but instead due to hepatitis C, and it was not always “mixed.” Importantly, cryogloblins were detectable in low amounts in a large proportion of patients with chronic hepatitis C, not all of whom had vasculitis. Perhaps a better term for the condition is HCV-related cryoglobulinemic vasculitis. This change in terminology points out that the mere presence of cryoglobulins is not adequate; rather, the diagnosis also requires clinical signs and symptoms of vasculitis. What is the nature of the cryoglobulins found in HCV-related vasculitis? They appear to be circulating immune complexes and to consist of intact hepatitis C virions bound by IgG anti-HCV. These immune complexes are then aggregated into large macromolecular complexes by pentameric rheumatoid factor, that is, IgM antibody to IgG. The ability of the large viral-IgG-IgM complexes to precipitate in the cold (thus “cryo”globulins) is well known but they can also precipitate in tissues, such as skin, joints, kidneys, lung, intestine and nerves, in response to cold or to other, less well defined stresses (perhaps including mechanical pressure, hypoxia, minor tissue damage, local immune activation, or immunoglobulin receptors). Precipitation of these viral-antibody complexes gives rise to the clinical signs and symptoms of the disease, which most commonly presents as an episodic cutaneous vasculitis over the lower extremities that is often painful and pruritic and may be accompanied by local edema, joint or muscle aches, and fatigue. More serious forms of cryoglobulinemia result in injury to the lungs (interstitial pneumonitis), intestine (intestinal infarction or perforation), kidney (glomerulonephritis), and peripheral nerves (neuropathy), probably as a result of local vascular injury. These complications can be severe, vii

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Foreword

disabling, or indeed fatal. Fortunately, the clinical syndrome of cryoglobulinemic vasculitis is rare; but its infrequency does not help the unfortunate affected individuals. Therapies directed at HCV eradication can result in remission of the clinical syndrome, but not all patients respond to the current antiviral regimens, and others respond but have an incomplete remission of the vasculitis and its complications. HCV-related cryoglobulinemic vasculitis itself represents a special paradigm for understanding complex diseases. It is basically an uncommon complication of a common disease. What do we know of its pathogenesis? It is more frequently seen in women than men with hepatitis C and it arises during the chronic phase of the illness, after years if not decades of infection. The severity of injury correlates only roughly with the levels of serum cryoglobulins and rheumatoid factor and with the degree of complement activation. The production of cryoglobulins apparently derives from an overactivation of B cells, resulting in the production of clones that secrete anti-HCV and rheumatoid factor. The prolonged stimulation and activation of B cells can give rise to genetic alterations that may cause unregulated, self-sustaining clonal B cell proliferation and even B cell lymphoma. Thus, a common chronic viral infection of the liver appears to be a cause of both an autoimmune disease and cancer. Further elucidation of the pathogenesis of HCV-related cryoglobulinemic vasculitis may thus also lead to fundamental discoveries regarding the pathogenesis of other autoimmune diseases (rare complications of common infections?) and cancer (unregulated cell growth caused by chronic stimulation by microbial antigens or toxins?). Recently, important inroads have been made in understanding the natural history of HCV-related cryoglobulinemic vasculitis as well as its treatment. Antiviral therapies that reduce or eradicate HCV also improve the vasculitis, with sustained viral clearance usually followed by long-term remission of the cryoglobulinemia and the disappearance of serum cryoglobulins. As new therapies for hepatitis C become available (particularly the new direct-acting antiviral agents), those for cryoglobulinemia will likely become more effective. In patients in whom eradication of hepatitis C is not possible, therapies directed at B cell overactivity (particularly rituximab) may nonetheless be effective, at least in the short-term. Combined approaches of anti-B cell followed by potent antiviral therapy may represent the best therapeutic strategy for patients with advanced or resistant disease. The current monograph, “Hepatitis C Virus Infection and Cryoglobulinemia,” brings together an international group of investigators from the fields of basic virology, clinical medicine, rheumatology, hematology, nephrology, oncology, immunology, and genetics to focus on perhaps the most unusual manifestation of this chronic viral infection. The editor and authors should be congratulated for this most welcomed and combined effort at understanding and improving the management of HCV-related cryoglobulinemic vasculitis. A broader and more complete understanding of this complex disease is likely to bring further paradigm shifts in our understanding of how the intricate interactions between an infectious agent and susceptible host are responsible for clinical disease. Bethesda, MD, USA

Jay H. Hoofnagle Director, Liver Disease Research Branch National Institutes of Health

Contents

1

Introductory Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Franco Dammacco and Domenico Sansonno

Part I 2

1

Hepatitis C Virus Infection and the Role of the Immune System

Natural History, Pathogenesis, and Prevention of HCV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Edgar D. Charles, Lynn B. Dustin, and Charles M. Rice

11

3

Immune Control of HCV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lynn B. Dustin

4

B Cell Activation: General to HCV-Specific Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vito Racanelli and Claudia Brunetti

37

Organ-Specific Autoimmunity in HCV-Positive Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corrado Betterle and Fabio Presotto

43

5

Part II 6

Cellular Compartments of HCV Infection (and Replication)

HCV and Blood Cells: How Can We Distinguish Infection from Association? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lynn B. Dustin and Charles M. Rice

7

Mechanisms of Cell Entry of Hepatitis C Virus . . . . . . . . . . . . . . . . . . . Franco Dammacco and Vito Racanelli

8

HCV Infection of Hematopoietic and Immune Cell Subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tram N.Q. Pham and Tomasz I. Michalak

Part III 9

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55 63

69

Cryoglobulinemia and the Complement System

Cryoglobulinemia and Chronic HCV Infection: An Evolving Story . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jürg A. Schifferli and Marten Trendelenburg The Complement System in Cryoglobulinemia . . . . . . . . . . . . . . . . . . . Marten Trendelenburg

79 85

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Contents

The Pivotal Role of C1qR in Mixed Cryoglobulinemia . . . . . . . . . . . . . Domenico Sansonno, Loredana Sansonno, and Franco Dammacco

Part IV

12

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Structural and Genetic Features, Cytokines and Chemokines in Cryoglobulinemia

Mixed Cryoglobulinemia (MC) Cross-Reactive Idiotypes (CRI): Structural and Clinical Significance . . . . . . . . . . . . . Peter D. Gorevic

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13

Molecular Insights into the Disease Mechanisms of Type II Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Valli De Re and Marica Garziera

14

The Role of VCAM-1 in the Pathogenesis of Hepatitis-C-Associated Mixed Cryoglobulinemia Vasculitis . . . . . . 113 Gilles Kaplanski

15

Up-Regulation of B-Lymphocyte Stimulator (BLyS) in Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . 119 Martina Fabris and Salvatore De Vita

16

Role of B-Cell-Attracting Chemokine-1 in HCV-Related Cryoglobulinemic Vasculitis . . . . . . . . . . . . . . . . . . . . 127 Sabino Russi, Silvia Sansonno, Gianfranco Lauletta, Domenico E. Sansonno, and Franco Dammacco

17

Serum a-Chemokine CXCL10 and b-Chemokine CCL2 Levels in HCV-Positive Cryoglobulinemia . . . . . . . . . . . . . . . . . 137 Alessandro Antonelli, Clodoveo Ferri, Silvia Martina Ferrari, Michele Colaci, IIaria Ruffilli, Caterina Mancusi, Ele Ferrannini, and Poupak Fallahi

Part V

Clinical Manifestations of Cryoglobulinemia

18

Experimental Models of Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . 145 Charles E. Alpers, Tomasz A. Wietecha, and Kelly L. Hudkins

19

The Expanding Spectrum of Clinical Features in HCV-Related Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . 155 Clodoveo Ferri, Alessandro Antonelli, Marco Sebastiani, Michele Colaci, and Anna Linda Zignego

20

Classification of Cryoglobulinemic Vasculitis. . . . . . . . . . . . . . . . . . . . . 163 Salvatore De Vita and Luca Quartuccio

21

Demographic and Survival Studies of Cryoglobulinemic Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Giuseppe Monti, Francesco Saccardo, and Laura Castelnovo

22

HCV-Associated Membranoproliferative Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Christos P. Argyropoulos, Sheldon Bastacky, and John Prentiss Johnson

Contents

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23

Rheumatologic Symptoms in Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Gianfranco Ferraccioli, Francesca Faustini, and Elisa Gremese

24

Endocrine Manifestations of HCV-Positive Cryoglobulinemia . . . . . . 191 Alessandro Antonelli, Clodoveo Ferri, Silvia Martina Ferrari, Michele Colaci, Alda Corrado, Andrea Di Domenicantonio, and Poupak Fallahi

25

Cutaneous Cryoglobulinemic Vasculitis . . . . . . . . . . . . . . . . . . . . . . . . . 195 Konstantinos Linos, Bernard Cribier, and J. Andrew Carlson

26

Peripheral Neuropathy and Central Nervous System Involvement in Cryoglobulinemia . . . . . . . . . . . . . . . . 209 Salvatore Monaco, Sara Mariotto, and Sergio Ferrari

27

Long-Term Course of Patients with Mixed Cryoglobulinemia . . . . . . . 219 Damien Sene and Patrice P. Cacoub

28

HBV/HCV Co-infection and Mixed Cryoglobulinemia . . . . . . . . . . . . . 227 Massimo Galli and Salvatore Sollima

29

Clinical and Immunological Features of HCV/HIV Co-infected Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . 233 David Saadoun and Patrice P. Cacoub

30

HCV-Negative Mixed Cryoglobulinemia: Facts and Fancies . . . . . . . . 239 Massimo Galli, Salvatore Sollima, and Giuseppe Monti

31

Cryoglobulinemia in HCV-Positive Renal Transplant and Liver Transplant Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Lionel Rostaing, Hugo Weclawiak, and Nassim Kamar

Part VI

HCV Infection, Cryoglobulinemia and Non-Hodgkin’s Lymphomas

32

Chromosome Abnormalities in HCV-Related Lymphoproliferation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Cristina Mecucci, Gianluca Barba, and Caterina Matteucci

33

Molecular Features of Lymphoproliferation in Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Valli De Re and Maria Paola Simula

34

The Higher Prevalence of B-Cell Non-Hodgkin’s Lymphoma in HCV-Positive Patients with and Without Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Franco Dammacco and Domenico Sansonno

35

Incidence and Characteristics of Non-Hodgkin’s Lymphomas in HCV-Positive Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Pietro Enrico Pioltelli, Giuseppe Monti, Maurizio Pietrogrande, and Massimo Galli

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Contents

Waldenström’s Macroglobulinemia Associated with Cryoglobulinemia: Pathogenetic, Clinical, and Therapeutic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Meletios A. Dimopoulos and Efstathios Kastritis

Part VII

Therapy of Cryoglobulinemia

37

Should HCV-Positive Asymptomatic Patients with Mixed Cryoglobulinemia Be Treated with Combined Antiviral Therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 José Luis Calleja Panero, Juan de la Revilla Negro, and Fernando Pons Renedo

38

The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 Francesco Zaja, Stefano Volpetti, Stefano De Luca, and Renato Fanin

39

Rituximab in Cryoglobulinemic Vasculitis: First- or Second-Line Therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Peter Lamprecht and Paul Klenerman

40

PIRR Therapy in HCV-Related Mixed Cryoglobulinemia . . . . . . . . . . 315 Franco Dammacco and Domenico Sansonno

41

Antiviral Therapy in HCV-Positive Non-Hodgkin’s Lymphoma: Pathogenetic Implications . . . . . . . . . . . . 325 Franco Dammacco, Cinzia Conteduca, and Domenico Sansonno

42

Active or Indolent Cutaneous Ulcers in Cryoglobulinemia: How Should They Be Treated? . . . . . . . . . . . . . . 335 Maurizio Pietrogrande

43

Double Filtration Plasmapheresis: An Effective Treatment of Cryoglobulinemia. . . . . . . . . . . . . . . . . . . . . 337 Alfonso Ramunni and Paola Brescia

44

Emergency in Cryoglobulinemia: Clinical and Therapeutic Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Francesco Saccardo, Laura Castelnovo, and Giuseppe Monti

45

Novel Therapeutic Approaches to Cryoglobulinemia: Imatinib, Infliximab, Bortezomib, and Beyond . . . . . . . . . . . . . . . . . . . 349 Giampaolo Talamo and Maurizio Zangari

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Contributors

Charles E. Alpers Department of Pathology, University of Washington, Seattle, WA, USA Division of Nephrology, Department of Medicine, University of Washington, Seattle, WA, USA Alessandro Antonelli Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Christos P. Argyropoulos Renal and Electrolyte Division, Department of Internal Medicine, University of Pittsburgh, Pittsburgh, PA, USA Gianluca Barba Hematology and Clinical Immunology Unit, Clinical and Experimental Medicine, University of Perugia, Perugia, Italy Sheldon Bastacky Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA Corrado Betterle Unit of Endocrinology, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy Paola Brescia Division of Nephrology, Department of Internal and Public Medicine, University of Bari, Bari, Italy Claudia Brunetti Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Patrice P. Cacoub UMR 7211 (UPMC/CNRS), U 959 (INSERM), Université Pierre Marie Curie, Paris, France Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, Paris, France José Luis Calleja Panero Gastroenterology and Hepatology Department, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain J. Andrew Carlson Division of Dermatology and Dermatopathology, Department of Pathology, Albany Medical College, Albany, NY, USA Laura Castelnovo Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy Edgar D. Charles Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA Michele Colaci Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Medical School, Modena, Italy xiii

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Cinzia Conteduca Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Alda Corrado Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Bernard Cribier Dermatologique Clinique, Les Hopitaux Universtaires de Strasbourg, Strasbourg, France Franco Dammacco Department of Biomedical Sciences and Clinical Oncology, University of Bari Medical School, Bari, Italy Stefano De Luca Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Valli De Re Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy Salvatore De Vita Clinic of Rheumatology, Department of Medical and Biological Sciences, Azienda Ospedaliero – Universitaria of Udine, Udine, Italy Andrea Di Domenicantonio Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Meletios A. Dimopoulos Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece Lynn B. Dustin Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA Martina Fabris Clinical Pathology and Clinic of Rheumatology, Azienda Ospedaliero – Universitaria of Udine, Udine, Italy Poupak Fallahi Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Renato Fanin Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Francesca Faustini Division of Rheumatology, Institute of Rheumatology and Affine Sciences(IRSA), CIC – Catholic University of the Sacred Heart, Rome, Italy Gianfranco Ferraccioli Division of Rheumatology, Institute of Rheumatology and Affine Sciences(IRSA), CIC – Catholic University of the Sacred Heart, Rome, Italy Ele Ferrannini Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Silvia Martina Ferrari Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Sergio Ferrari Department of Neuroscience, University of Verona, Verona, Italy Clodoveo Ferri Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Medical School, Modena, Italy

Contributors

Contributors

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Massimo Galli Department of Clinical Sciences “Luigi Sacco”, Section of Infectious and Tropical Diseases, Università di Milano, Milan, Italy Marica Garziera Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy Peter D. Gorevic Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA Elisa Gremese Division of Rheumatology, Institute of Rheumatology and Affine Sciences(IRSA), School of Medicine, CIC – Catholic University of the Sacred Heart, Rome, Italy Kelly L. Hudkins Department of Pathology, University of Washington, Seattle, WA, USA John Prentiss Johnson Renal and Electrolyte Division, Department of Internal Medicine, University of Pittsburgh, Pittsburgh, PA, USA Nassim Kamar Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, Toulouse, France INSERM U858/I2MR, Equipe 10, CHU Rangueil, Toulouse, France Gilles Kaplanski Service de Médecine Interne, Hôpital de la Conception, Marseille, France Efstathios Kastritis Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece Paul Klenerman Peter Medawar Building for Pathogen Research and National Institute for Health Research Biomedical Research Centre, University of Oxford, Oxford, UK Peter Lamprecht Department of Rheumatology, Vasculitis Center UKSH & Clinical Center Bad Bramstedt, University of Lübeck, Lübeck, Germany Gianfranco Lauletta Department of Internal Medicine and Human Oncology, University of Bari Medical School, Bari, Italy Konstantinos Linos Department of Pathology, Albany Medical College, Albany, NY, USA Caterina Mancusi Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Sara Mariotto Department of Neuroscience, University of Verona, Verona, Italy Caterina Matteucci Hematology and Clinical Immunology Unit, Clinical and Experimental Medicine, University of Perugia, Perugia, Italy Cristina Mecucci Hematology and Clinical Immunology Unit, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy Tomasz I. Michalak Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Center, Memorial University, St. John’s, NL, Canada

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Salvatore Monaco Department of Neuroscience, University of Verona, Verona, Italy Giuseppe Monti Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy Tram N.Q. Pham Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Center, Memorial University, St. John’s, NL, Canada Maurizio Pietrogrande Internal Medicine Unit, Policlinico San Marco, Osio Sotto, University of Milan, Milan, Italy Pietro Enrico Pioltelli Hematology Unit, Ospedale San Gerardo, Monza, Italy Fabio Presotto Department of Medical and Surgical Sciences, Medical University of Padua, Padua, Italy Unit of Internal Medicine, General Hospital of Este (Padua), Padua, Italy Luca Quartuccio Clinical Pathology and Clinic of Rheumatology, Azienda Ospedaliero – Universitaria of Udine, Udine, Italy Vito Racanelli Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Alfonso Ramunni Division of Nephrology, Department of Internal and Public Medicine, University of Bari, Bari, Italy Fernando Pons Renedo Department of Gastroenterology and Hepatology, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain Juan de la Revilla Negro Gastroenterology and Hepatology Department, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain Charles M. Rice Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA Lionel Rostaing Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, Toulouse, France INSERM U563, IFR 30, CHU Purpan, Toulouse, France Ilaria Ruffilli Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Sabino Russi Department of Internal Medicine and Human Oncology, University of Bari Medical School, Bari, Italy David Saadoun Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, Paris, France Francesco Saccardo Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy Domenico E. Sansonno Liver Unit, Department of Biomedical Sciences and Clinical Oncology, University of Bari Medical School, Bari, Italy

Contributors

Contributors

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Loredana Sansonno Laboratory of Genetics, Department of Biomedical Sciences, University of Foggia Medical School, Foggia, Italy Silvia Sansonno Clinical and Experimental Pharmacology – DOMERT, Centro di Riferimento Oncologico, Aviano, Italy Jürg A. Schifferli Division of Internal Medicine, Department of Medicine, University Hospital Basel, Basel, Switzerland Marco Sebastiani Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Medical School, Modena, Italy Damien Sene Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, Paris, France Maria Paola Simula Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy Salvatore Sollima Department of Clinical Sciences “Luigi Sacco”, Section of Infectious and Tropical Diseases, University of Milan, Milan, Italy Giampaolo Talamo Division of Hematology-Oncology, Penn State Hershey Cancer Institute, Hershey, PA, USA Marten Trendelenburg Clinic for Internal Medicine and Laboratory for Clinical Immunology, University Hospital Basel, Basel, Switzerland Stefano Volpetti Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Hugo Weclawiak Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, Toulouse, France Tomasz A. Wietecha Department of Pathology, University of Washington, Seattle, WA, USA Francesco Zaja Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Maurizio Zangari Division of Hematology, Blood/Marrow Transplant and Myeloma Program, University of Utah, Salt Lake City, UT, USA Anna Linda Zignego Department of Internal Medicine, University of Florence Medical School, Florence, Italy

Part I Hepatitis C Virus Infection and the Role of the Immune System

1

Introductory Remarks Franco Dammacco and Domenico Sansonno

The Strength of the Truth: Where there is truth there is also true knowledge. And where there is no truth there cannot be true knowledge. This is why the word knowledge is associated to the name of God. And where there is true knowledge there is always happiness. Mohandas Karamchand (‘Mahatma’) Gandhi

The first observation of cold-induced precipitation of serum proteins dates back to 1933, when Prof. Maxwell Myer Wintrobe, the legendary physicianscientist who laid the foundations of modern hematology, described, in collaboration with Dr. M. V. Buell, an unusual case of multiple myeloma in a woman whose serum reversibly precipitated at cold temperatures [1]. Fourteen years later, Lerner and Watson [2] showed cold-precipitable proteins to be gammaglobulins and named them “cryoglobulins.” They also coined the term “cryoglobulinemia” to indicate the corresponding clinical condition. At that time, cryoglobulins were thought to be structurally formed by a single protein. In 1962, testing isolated and purified cryoglobulins by anion-exchange chromatography, Lospalluto et  al. [3] used analytical ultracentrifugation to demonstrate that solubilized cryoproteins indeed contained two components, ­designated at that time as 7S and 19S on the basis of their sedimentation coefficients. These fractions ­corresponded to the immunoglobulins that we now call IgG and IgM, respectively. They were also able to show that a ­number of positive reactions for ­rheumatoid factor (RF) were associated with the 19S fraction and that, while the 7S (IgG) fraction could indifferently F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_1, © Springer-Verlag Italia 2012

belong to the patient’s serum or derive from pooled normal donors (Cohn’s faction II), replacement of the patient’s 19S (IgM) with a ­normal  IgM counterpart resulted in the disappearance of the ­cryoprecipitating properties. Thus, the 19S ­gammaglobulin component behaved as an incomplete cryoglobulin. Meltzer and Franklin [4] and Meltzer et  al. [5], in 1966, provided an accurate description of the typical clinical symptoms associated with cryoglobulinemia. They also confirmed that cryoglobulins consisted of two different globulin components and were consistently endowed with RF activity. Due to ignorance regarding its etiology, they called this clinical condition “essential” mixed cryoglobulinemia (MC). In the following years, many investigators carried out immunochemical studies of a number of isolated cryoglobulins and demonstrated their structural heterogeneity. Based on these studies and on a review of 86 patients, Brouet et al. [6] classified cryoglobulinemia into three main types, a classification that is still largely accepted. Type I cryoglobulins are single monoclonal immunoglobulins, typically IgM or IgG, but rarely also IgA and even Bence Jones proteins. Types II and III cryoglobulins are characterized by polyclonal IgG associated with monoclonal (type II) or polyclonal (type III) IgM. Since RF activity is regularly associated with the IgM fraction, immune complexes are formed through the binding of IgM-RF to autologous IgG. The immunochemical characterization of ­different cryoglobulins and their possible significance as ­inducers of tissue lesions were emphasized 1

2

F. Dammacco and D. Sansonno

Table 1.1  Important chronological advances (milestones) in the study of cryoglobulinemia Year 1933 1947 1962 1966 1968–1970 1973…

1974 1977… 1987 1990–1994 1994… 2003

2004 2005 2002–2005

2010

2010 2011…

Observations A cold-precipitable protein is discovered in the serum of a patient with multiple myeloma The terms “cryoglobulin” and “cryoglobulinemia” are proposed and the phenomenon is observed in various diseases Chromatographic separation of cryoglobulins shows their mixed (7 + 19S) nature and ­rheumatoid factor (RF) activity is found to be associated to the 19S fraction The clinical picture and the structural heterogeneity of so-called “essential” mixed ­cryoglobulinemia (MC) are clearly defined Different cryoglobulins are immunochemically characterized and their significance as inducers of tissue lesions is emphasized A number of IgMk RFs from patients with MC are shown to display a major cross-reactive idiotype (CRI) designated WA. The prevalence of CRIs has subsequently been the object of an extensive review aimed to assess the relationship of MC to rheumatic and lymphoproliferative disorders Cryoglobulins are classified into three main types The possible etiological role of hepatitis B virus in “essential” MC is claimed, though not confirmed in later studies Therapy with recombinant interferon-a (IFN-a) is shown to be effective in patients with “essential” MC The large majority of patients with MC are found to be hepatitis C virus (HCV)-infected Potential progression from MC to overt non-Hodgkin’s lymphoma (NHL) is reported by different groups, though with wide geographic variations Rituximab, a chimeric anti-CD20 monoclonal antibody, is found to be clinically effective in patients with relapsing or refractory MC, but it often results in an increase in the serum HCV RNA levels A B-cell clonal expansion involving RF-secreting cells is demonstrated to be the biological hallmark of MC By analogy with non-cryoglobulinemic chronic HCV infection, pegylated IFN-a plus ribavirin is proposed as the standard of care in HCV-positive MC IFN-a (plus RBV) anti-viral therapy is demonstrated to induce a ³75% complete remission of low-grade NHL in HCV-positive MC patients and similar results are reported in a later systematic review In HCV-infected asymptomatic patients, WA B-cells bearing the WA cross-idiotype are defined as a marker for the development of cryoglobulinemic vasculitis and associated B cell malignancies, and should therefore represent a specific therapeutic target The combination of pegylated IFN-a, ribavirin and rituximab (PIRR therapy) is shown to provide a high percentage of long-term responses Administration of new protease inhibitors and new anti-CD20 monoclonal antibodies (Ofatumumab) is likely to result in better results than PIRR therapy

Referencesa [1] [2] [3] [4, 5] [7, 8] [9, 10]

[6] [12–14] [45] [15–23] [35–40] [47, 48]

[25, 26] [49] [41, 42]

[44]

[50, 51] Clinical trials in progress

 eferences are an accurate selection of the oldest pertinent publications dealing with each observation, but do not necessarily reflect R strict chronological priority

a

by Wager et al. [7] and Barnett et al. [8]. In  ­addition, ­several IgMk RFs purified from MC patients were shown to have private idiotypes, hence a unique primary amino acid sequence and two major cross-reactive idiotypes (CRIs), although the best characterized of them were Wa-positive RFs [9, 10]. These historical data in the developing story of cryoglobulinemia and a number of subsequent advances up to the present time are summarized in Table 1.1. It soon became evident that to put forward a wellgrounded clinical suspicion of cryoglobulinemia was

not a difficult task in patients complaining of: (a) recurrent episodes of palpable purpura, usually confined to the lower limbs and often to the buttocks as well, which frequently result in chronic dyschromia, namely, a brownish pigmentation over the legs and the ankles secondary to hemosiderin deposits in the sites previously affected with the purpuric eruptions; leg ulcers and cold urticaria may also be seen, though much less often; (b) arthralgias affecting the small and/or large joints to a variable extent; (c) intense asthenia and chronic fatigability. The occurrence of these symptoms

1  Introductory Remarks

is indeed so frequent that the triad purpura/asthenia/ arthralgia is considered a practically unfailing stigma of this clinical condition [11]. However, since MC is a systemic vasculitis, along with liver, skin and joint involvement, there may be additional manifestations, including nephropathy and sensory-motor neuropathy, the latter being particularly resistant to treatment. As already shown by previous studies [3–5], the occurrence in the serum of RF and the low circulating levels of the complement fractions C3 and even more strikingly C4 strongly argue that cryoglobulinemia should be considered an immune complex-mediated vasculitis. However, the inciting agent capable of inducing immune-complex formation has remained unknown for a long time. In 1977, Levo et  al. [12] claimed they had found a correlation between cryoglobulinemia and hepatitis B virus (HBV) on the basis of clinical and serological features, which drew attention to the ­relatively frequent liver involvement in MC patients and the electron microscopy evidence of structures resembling the spheres, tubules and Dane particles ­characteristic of HBV infection in isolated and purified cryoprecipitates. Although the same authors hypothesized that other, at that time undetermined viruses were also involved, evidence of HBV infection in most of the patients with MC was not confirmed by other groups [13, 14], suggesting that this presumed correlation indeed reflected the prevalence of HBV infection in the geographic area in which the research was carried out. When at the beginning of the 1990s, in step with the availability of new reagents, it became possible to detect the serum occurrence of antibodies to the hepatitis C virus (HCV) and shortly after of HCV RNA, many groups in Europe [15–21] and the USA [22, 23] consistently reported in the space of 90% of these patients have HCV [59–61]. Non-HCV causes of MC include infectious agents (e.g., HIV, HBV) and autoimmune disorders (e.g., systemic lupus erythematosis, Sjögren’s syndrome, and systemic sclerosis) [62]. A shared feature of these disorders is chronic inflammation in the setting of high antigenic load, suggesting that antigendriven B cell dysregulation is a prerequisite for the development of MC [63]. Only a minority of patients develops the classical symptoms of palpable purpura, joint pains, and fatigue. Cryoglobulins are classified as type I (monoclonal immunoglobulin (Ig) only), type II (mixture of monoclonal Ig, usually IgM RF, and polyclonal IgG) and type III (mixture of polyclonal Ig, usually IgM, and polyclonal IgG) [64]. HCV is primarily associated with type II (which typically has an IgMk RF with anti-idiotypic activity [65]), and to a lesser extent, with type III MC. Type I MC is rarely seen in HCV. Serum levels of RF are often increased in the setting of HCV MC, while levels of complement, particularly C4, may be profoundly decreased. It has been hypothesized that C1q bound to IgM RF engages C1q receptors on vascular endothelium, leading to neutrophil recruitment and vasculitis [66]. HCV MC vasculitis primarily affects the small and medium-sized vessels of the skin, kidneys, and peripheral nerves. Histology typically reveals a leukocytoclastic vasculitis, with deposition of IgM RF, IgG, C3, and neutrophils in the vessel wall. A necrotizing vasculitis, with fibrinoid necrosis of the intima and inflammation of the entire vessel wall and perivascular space, may also occur. Palpable purpura, primarily of the lower legs, is seen in >90% of patients with symptomatic HCV MC, is frequently intermittent, and is often the cardinal manifestation of HCV MC [61]. These purpuric lesions may occasionally progress to chronic ulcers and frank gangrene. 2.3.3.3 Membranoproliferative Glomerulonephritis (MPGN) The renal involvement in HCV-MC is usually type I MPGN [67], and it frequently heralds a poor clinical course. Manifestations range from isolated proteinuria

16

to overt nephritic or nephritic syndrome with variable progression towards chronic renal insufficiency. The MPGN is characterized by endocapillary mesangial cell proliferation, monocytic infiltration, double contour membranes, glomerular IgM, IgG, and C3 deposition, eosinophilic PAS-positive intraluminal deposits, and vasculitis of the small and medium-sized renal arteries [68, 69].

2.3.3.4 Peripheral Neuropathy The incidence of neurological involvement is variable. Sensorimotor neuropathy arises from cryoglobulin deposition in the vasa vasorum. Painful paresthesias and concomitant weakness, particularly in the lower limbs may occur [70], as may isolated mononeuritis, manifested by foot or wrist drop. 2.3.3.5 Other Extrahepatic Manifestations Infection with HCV has been associated with thyroid disease (hyper- or hypothyroidism) that in turn is often associated with interferon therapy and pre-existing anti-thyroid peroxidase antibodies. In addition, there is increasing recognition of a relationship between HCV and steatosis, insulin resistance, and overt type 2 diabetes mellitus [71]. The relationship between HCV and lichen planus is controversial; meta-analysis of primarily case-control studies suggests a significant positive association with wide geographical variation [72]. HCV is also associated with increases in nonorgan-specific autoantibodies [73], the clinical significance of which is unclear.

2.4 Prevention of HCV Infection 2.4.1 Primary and Secondary Prevention 2.4.1.1 Identification of HCV-Infected Individuals As HCV infection is usually asymptomatic, a large reservoir of infected individuals is at risk of unwittingly transmitting HCV to others. A major public health goal is to screen those at high risk for HCV. Screening programs should prioritize those people who have a history of intravenous drug use, a history of blood-product transfusion or organ transplantation before 1992, and individuals infected with HIV or HBV. Screening should also be carried out among the newly and chronically incarcerated, given the high prevalence of HCV infection among prisoners [74, 75]

E.D. Charles et al.

and the risk of transmission among inmates via injection drug use or tattooing.

2.4.1.2 Secondary Prevention Once HCV-infected individuals are identified, they should be counseled to not donate blood, semen, or tissues, and to avoid the sharing of personal items that may be exposed to blood. It is not necessary to avoid close contact with family members or to avoid sharing meals or utensils. Given the low rate of heterosexual transmission, there is no need for people in monogamous long-term heterosexual relationships to change sexual practices [76]. However, as HCV may be transmitted sexually among men who have sex with men, condom usage is advisable in such cases. HCV-infected individuals should be referred to medical practitioners for assessment of liver function, ­detection of viremia by HCV RNA, and treatment consideration. A discussion of HCV treatment is beyond the scope of this chapter. Currently, the standard of care is combination therapy with an HCV-specific protease inhibitor, pegylated interferon and ribavirin, with duration of treatment dependent upon HCV genotype and HCV RNA response [77]. Treatment options are expected to dramatically expand as additional HCVspecific protease inhibitors, as well as polymerase inhibitors, and perhaps other medications come to market [78].

2.4.2 Management of Percutaneous or Mucosal Exposure to HCV Individuals who have percutaneous or mucosal exposure to blood at high risk for HCV viremia should have baseline anti-HCV and ALT measurements. HCV RNA may be measured at 4–6  weeks; follow-up anti-HCV and ALT should be done at 4–6 months. Immu­noglobulins and antiviral agents are not recommended for post-­ exposure prophylaxis [79]. As antiviral therapy may be beneficial in patients with acute HCV, symptomatic patients and patients with positive HCV RNA should be referred to a physician for treatment consideration.

2.4.3 Children Born to HCV-Positive Women Studies have failed to demonstrate a reduced risk after cesarean delivery compared to vaginal ­delivery.

2  Natural History, Pathogenesis, and Prevention of HCV Infection

Immunoglobulin and antiviral agents are not recommended for neonatal post-exposure prophylaxis. Breastfeeding is not contraindicated as there is no ­documented ­transmission by this route [76], although caution should be exercised in the setting of chafed nipples with skin breakdown. Testing for HCV RNA may be performed 2–3  months after delivery, and ­anti-HCV should be performed no sooner than 12 months. Children with either positive anti-HCV or HCV RNA should be referred to a physician for further management.

2.5 Concluding Remarks HCV is a major cause of worldwide morbidity and mortality. As infection is usually asymptomatic, there is a large reservoir of people who are unaware that they harbor virus. Complications of cirrhosis and extrahepatic disease occur in a minority of individuals, and often take decades to develop. Consequently, estimates of the natural history of disease vary widely and it is difficult to predict the clinical course following infection. It is clear that HCV exerts deleterious effects on organs beyond the liver, and a major goal of continuing research is to clarify such relationships from the epidemiologic to the cellular level.

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8. Ohto H, Terazawa S, Sasaki N, Hino K et  al (1994) Transmission of hepatitis C virus from mothers to infants. The Vertical Transmission of Hepatitis C Virus Collaborative Study Group. N Engl J Med 330:744–750 9. Mast EE, Hwang LY, Seto DS, Nolte FS et al (2005) Risk factors for perinatal transmission of hepatitis C virus (HCV) and the natural history of HCV infection acquired in infancy. J Infect Dis 192:1880–1889 10. Mast EE, Alter MJ, Margolis HS (1999) Strategies to prevent and control hepatitis B and C virus infections: a global perspective. Vaccine 17:1730–1733 11. (1999) Hepatitis C – global prevalence (update). Wkly Epidemiol Rec 74:425–427 12. Simmonds P (1999) Viral heterogeneity of the hepatitis C virus. J Hepatol 31(Suppl 1):54–60 13. Simmonds P, Bukh J, Combet C et al (2005) Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42:962–973 14. Bouchardeau F, Cantaloube JF, Chevaliez S et  al (2007) Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay. J Clin Microbiol 45:1140–1145 15. Liu CH, Chen BF, Chen SC et al (2006) Selective transmission of hepatitis C virus quasi species through a needlestick accident in acute resolving hepatitis. Clin Infect Dis 42:1254–1259 16. Quer J, Esteban JI, Cos J et al (2005) Effect of bottlenecking on evolution of the nonstructural protein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection. J Virol 79:15131–15141 17. Ray SC, Fanning L, Wang XH et al (2005) Divergent and convergent evolution after a common-source outbreak of hepatitis C virus. J Exp Med 201:1753–1759 18. Fan X, Mao Q, Zhou D et al (2009) High diversity of hepatitis C viral quasispecies is associated with early virological response in patients undergoing antiviral therapy. Hepatology 50:1765–1772 19. Goodman ZD (2007) Grading and staging systems for inflammation and fibrosis in chronic liver diseases. J Hepatol 47:598–607 20. Farci P, Alter HJ, Wong D et al (1991) A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 325:98–104 21. Courouce AM, Le Marrec N, Girault A et al (1994) Antihepatitis C virus (anti-HCV) seroconversion in patients undergoing hemodialysis: comparison of second- and thirdgeneration anti-HCV assays. Transfusion 34:790–795 22. Sulkowski MS, Ray SC, Thomas DL (2002) Needlestick transmission of hepatitis C. JAMA 287:2406–2413 23. Netski DM, Mosbruger T, Depla E et  al (2005) Humoral immune response in acute hepatitis C virus infection. Clin Infect Dis 41:667–675 24. Osburn WO, Fisher BE, Dowd KA et al (2009) Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection. Gastroenterology 138: 315–324 25. Page K, Hahn JA, Evans J et  al (2009) Acute hepatitis C virus infection in young adult injection drug users: a prospective study of incident infection, resolution, and reinfection. J Infect Dis 200:1216–1226 26. Thomas DL, Thio CL, Martin MP et al (2009) Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 461:798–801

18 27. Ge D, Fellay J, Thompson AJ et al (2009) Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461:399–401 28. Fattovich G, Giustina G, Degos F et al (1997) Morbidity and mortality in compensated cirrhosis type C: a retrospective follow-up study of 384 patients. Gastroenterology 112:463–472 29. Seeff LB (2002) Natural history of chronic hepatitis C. Hepatology 36:S35–S46 30. Kenny-Walsh E (1999) Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. Irish Hepatology Research Group. N Engl J Med 340: 1228–1233 31. Wiese M, Berr F, Lafrenz M et al (2000) Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study. Hepatology 32:91–96 32. Vogt M, Lang T, Frosner G et  al (1999) Prevalence and clinical outcome of hepatitis C infection in children who underwent cardiac surgery before the implementation of blood-donor screening. N Engl J Med 341:866–870 33. Seeff LB, Miller RN, Rabkin CS et al (2000) 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann Intern Med 132:105–111 34. Poynard T, Bedossa P, Opolon P (1997) Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 349:825–832 35. Kleter B, Brouwer JT, Nevens F et  al (1998) Hepatitis C virus genotypes: epidemiological and clinical associations. Benelux Study Group on Treatment of Chronic Hepatitis C. Liver 18:32–38 36. Poynard T, Ratziu V, Charlotte F et al (2001) Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis c. J Hepatol 34:730–739 37. Graham CS, Baden LR, Yu E et  al (2001) Influence of human immunodeficiency virus infection on the course of hepatitis C virus infection: a meta-analysis. Clin Infect Dis 33:562–569 38. Macias J, Berenguer J, Japon MA et al (2009) Fast fibrosis progression between repeated liver biopsies in patients coinfected with human immunodeficiency virus/hepatitis C virus. Hepatology 50:1056–1063 39. Qurishi N, Kreuzberg C, Luchters G et al (2003) Effect of antiretroviral therapy on liver-related mortality in patients with HIV and hepatitis C virus coinfection. Lancet 362: 1708–1713 40. Zarski JP, Bohn B, Bastie A et al (1998) Characteristics of patients with dual infection by hepatitis B and C viruses. J Hepatol 28:27–33 41. Cacciola I, Pollicino T, Squadrito G et al (1999) Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med 341:22–26 42. Leandro G, Mangia A, Hui J et  al (2006) Relationship between steatosis, inflammation, and fibrosis in chronic hepatitis C: a meta-analysis of individual patient data. Gastroenterology 130:1636–1642 43. Gumber SC, Chopra S (1995) Hepatitis C: a multifaceted disease. Review of extrahepatic manifestations. Ann Intern Med 123:615–620 44. Zignego AL, Ferri C, Pileri SA et  al (2007) Extrahepatic manifestations of hepatitis C virus infection: a general over-

E.D. Charles et al. view and guidelines for a clinical approach. Dig Liver Dis 39:2–17 45. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297:2010–2017 46. Ferri C, Caracciolo F, Zignego AL et al (1994) Hepatitis C virus infection in patients with non-Hodgkin’s lymphoma. Br J Haematol 88:392–394 47. Zuckerman E, Zuckerman T, Levine AM et  al (1997) Hepatitis C virus infection in patients with B-cell nonHodgkin lymphoma. Ann Intern Med 127:423–428 48. Talamini R, Montella M, Crovatto M et  al (2004) NonHodgkin’s lymphoma and hepatitis C virus: a case-control study from northern and southern Italy. Int J Cancer 110: 380–385 49. Matsuo K, Kusano A, Sugumar A et  al (2004) Effect of hepatitis C virus infection on the risk of non-Hodgkin’s lymphoma: a meta-analysis of epidemiological studies. Cancer Sci 95:745–752 50. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 43:94–102 51. De Vita S, Sacco C, Sansonno D et al (1997) Characterization of overt B-cell lymphomas in patients with hepatitis C virus infection. Blood 90:776–782 52. Misiani R, Bellavita P, Fenili D et al (1994) Interferon alfa2a therapy in cryoglobulinemia associated with hepatitis C virus. N Engl J Med 330:751–756 53. Hermine O, Lefrere F, Bronowicki JP et al (2002) Regression of splenic lymphoma with villous lymphocytes after ­treatment of hepatitis C virus infection. N Engl J Med 347:89–94 54. Zuckerman E, Zuckerman T, Sahar D et al (2001) bcl-2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection. Br J Haematol 112:364–369 55. Zignego AL, Ferri C, Giannelli F et al (2002) Prevalence of bcl-2 rearrangement in patients with hepatitis C virus-related mixed cryoglobulinemia with or without B-cell lymphomas. Ann Intern Med 137:571–580 56. Libra M, Gloghini A, Malaponte G et al (2008) Association of t(14;18) translocation with HCV infection in gastrointestinal MALT lymphomas. J Hepatol 49:170–174 57. Limpens J, Stad R, Vos C et al (1995) Lymphoma-associated translocation t(14;18) in blood B cells of normal individuals. Blood 85:2528–2536 58. Libra M, De Re V, De Vita S et al (2003) Low frequency of bcl-2 rearrangement in HCV-associated non-Hodgkin’s lymphoma tissue. Leukemia 17:1433–1436 59. Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327:1490–1495 60. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 33:355–374 61. Dammacco F, Sansonno D, Piccoli C et al (2001) The cryoglobulins: an overview. Eur J Clin Invest 31:628–638 62. Gorevic PD (1995) Cryopathies: cryoglobulins and cryofibrinogenimia. In: Frank MM, Austen KF, Claman HN (eds)

2  Natural History, Pathogenesis, and Prevention of HCV Infection Samter’s immunologic diseases. Little Brown, Boston, p 951 63. Charles ED, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 76:818–824 64. Brouet JC, Clauvel JP, Danon F et  al (1974) Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 57:775–788 65. Agnello V (1997) The etiology and pathophysiology of mixed cryoglobulinemia secondary to hepatitis C virus infection. Springer Semin Immunopathol 19:111–129 66. Sansonno D, Dammacco F (2005) Hepatitis C virus, cryoglobulinaemia, and vasculitis: immune complex relations. Lancet Infect Dis 5:227–236 67. Saadoun D, Landau DA, Calabrese LH, Cacoub PP (2007) Hepatitis C-associated mixed cryoglobulinaemia: a ­crossroad between autoimmunity and lymphoproliferation. Rheumatology (Oxford) 46:1234–1242 68. Johnson RJ, Gretch DR, Yamabe H et  al (1993) Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. N Engl J Med 328:465–470 69. D’Amico G (1998) Renal involvement in hepatitis C ­infection: cryoglobulinemic glomerulonephritis. Kidney Int 54:650–671 70. Authier FJ, Pawlotsky JM, Viard JP et al (1993) High incidence of hepatitis C virus infection in patients with cryoglobulinemic neuropathy. Ann Neurol 34:749–750 71. Lonardo A, Adinolfi LE, Loria P et al (2004) Steatosis and hepatitis C virus: mechanisms and significance for hepatic and extrahepatic disease. Gastroenterology 126:586–597

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72. Shengyuan L, Songpo Y, Wen W et  al (2009) Hepatitis C virus and lichen planus: a reciprocal association determined by a meta-analysis. Arch Dermatol 145:1040–1047 73. Cacoub P, Renou C, Rosenthal E et al (2000) Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Groupe d’Etude et de Recherche en Medecine Interne et Maladies Infectieuses sur le Virus de l’Hepatite C. Medicine (Baltimore) 79:47–56 74. Hennessey KA, Kim AA, Griffin V et al (2009) Prevalence of infection with hepatitis B and C viruses and co-infection with HIV in three jails: a case for viral hepatitis prevention in jails in the United States. J Urban Health 86:93–105 75. McGovern BH, Wurcel A, Kim AY et al (2006) Acute hepatitis C virus infection in incarcerated injection drug users. Clin Infect Dis 42:1663–1670 76. (1998) Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. Centers for disease control and prevention. MMWR Recomm Rep 47:1–39 77. Heathcote EJ (2007) Antiviral therapy: chronic hepatitis C. J Viral Hepat 14(Suppl 1):82–88 78. Meier V, Ramadori G (2009) Hepatitis C virus virology and new treatment targets. Expert Rev Anti Infect Ther 7: 329–350 79. U.S. Public Health Service (2001) Updated U.S. public health service guidelines for the management of ­occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 50:1–52

3

Immune Control of HCV Infection Lynn B. Dustin

3.1

Introduction

The purpose of this chapter is to summarize our current understanding of the ways the immune system fights the hepatitis C virus (HCV), and how the virus avoids elimination in the majority of those who contract it. According to the most recent World Health Organization estimates, between 130 and 170 million people worldwide are persistently infected with HCV [1, 2]. Long-term sequelae of HCV infection can include cirrhosis, liver failure, and hepatocellular carcinoma [3]; these conditions make HCV a leading indication for liver transplantation [4]. Despite recognition by innate antiviral pathways, and despite stimulating adaptive immune responses, HCV establishes persistent infection in the majority of those who contract it [5, 6]. While there have been encouraging developments, prophylactic and therapeutic vaccines are not yet available [7, 8]. Antiviral regimens based on pegylated interferon (IFN)-a2 and ribavirin produce sustained control of HCV infection in about one-half of those treated [9]; thus, new drugs are actively sought to expand coverage [10, 11]. In this chapter, we discuss our current understanding of the battle between HCV and innate and adaptive immune responses: how these responses target HCV and how HCV manages to persist in a majority of those who become infected.

L.B. Dustin Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_3, © Springer-Verlag Italia 2012

3.2

Hepatitis C Virus

HCV is an enveloped RNA virus in the family Flaviviridae. Its genomic structure and replication pathways have been reviewed [12–15]. The HCV genome is a single, positive-sense, 9.6-kb RNA encoding a polyprotein of about 3,000 amino acids. Host and virus-encoded proteases process the polyprotein to release the ten individual proteins making up the viral particle and replication machinery. One or more additional proteins may be produced by translational frame-shifting; however, the functions of these proteins are poorly understood. HCV replication and assembly take place in association with membranous structures and lipid droplets in the cytosol of infected cells [15]. The structural proteins (core (capsid), E1 and E2) are at the amino terminus of the polyprotein. It is believed that HCV RNA associates with multiple copies of the core protein to form the nucleocapsid, which is packaged in a lipid envelope bearing the E1 and E2 glycoproteins. The infectious form of the virus in vivo is still incompletely understood, but it is believed that HCV associates with low- or very lowdensity lipid particles. The nonstructural proteins, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, are present in infected cells but are probably not required components of the viral particles. The known roles of these proteins in the HCV lifecycle are reviewed elsewhere [11, 14, 15]. Briefly, p7 is a membrane-spanning protein possessing cation-channel activity; it is required for productive virus infection but not for HCV RNA replication. NS2 is a cysteine protease that plays an essential, but poorly understood, role in virus assembly. NS3 has NTPase/helicase and serine protease activities, the 21

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latter dependent on association with NS4A. NS3/4A catalyzes the release of the four downstream proteins (NS4A, NS4B, NS5A and NS5B) from the HCV polyprotein and, as discussed below, targets cellular proteins. NS4B may serve as a scaffold for the organization of the replication complex and causes rearrangement of cellular membranes. NS5A’s phosphorylation status may act as a switch regulating RNA replication and virus assembly. NS5B is the RNA-dependent RNA polymerase, required for synthesis of negative-sense (minus strand) and positive-sense RNA from an RNA template. HCV can evolve rapidly as its replication yields an impressive 1012 HCV particles per day in the infected liver. Newly produced HCV particles have an estimated serum half-life of 3 h [16, 17]. HCV’s NS5B RNA-dependent RNA polymerase lacks proofreading capacity. The high rate of virus production coupled with this error-prone replication mechanism permits rapid evolution under selection by the immune system and antiviral drugs. Within the infected host, HCV exists as a quasispecies, or swarm of related viral sequences.

3.3

HCV and Innate Antiviral Responses

Interferon response pathways are induced in the liver early in infection regardless of the outcome of infection [18–21]. Type I IFNs produced in response to HCV may induce an antiviral state in the surrounding cells. However, specific HCV gene products have been reported to target different steps in IFN induction or response. This may enable the virus to persist in the face of ongoing recognition by pathogen-associated molecular pattern receptors – and, indeed, in the face of IFN-based antiviral therapy. Acute HCV infection has been studied in experimentally infected chimpanzees. During the first 1–2 weeks of HCV infection, serum viral loads increase rapidly, with a mean doubling time half-life of 0.5 days [22, 23]. Based on measurements of viral RNA during acute infection, one study estimated that up to 10% of hepatocytes support HCV replication [18]. The rate of viral increase then slows abruptly to a mean doubling time half-life of 7.5 days, coinciding with evidence of an IFN response in the liver. It is believed that an initial IFN response establishes an antiviral state in most cells

of the liver, and that reduction in the number of susceptible cells slows the rate of virus production [22, 23]. The cellular source responsible for IFN production has not been identified. Autocrine production by infected hepatocytes may activate subsequent paracrine production by other cells. Alternatively, immune cells including plasmacytoid dendritic cells may produce the first IFNs after recognizing the presence of HCV-infected hepatocytes. Innate mechanisms, both within the infected cell and in patrolling immune cells, recognize RNA structures associated with viral infections such as HCV (reviewed in [24]). HCV targets innate virus-sensing mechanisms. HCV RNA replicates in the cytoplasm, and this process generates molecular patterns detected by the cytoplasmic DEx/D/H-Box RNA helicases, RIG-I and MDA-5. RIG-I may recognize HCV RNA due to HCV’s 5¢ triphosphate group [25] and a conserved uridine-rich sequence near the 3¢ end of HCV genomic RNA [26]. MDA-5 may recognize higher-order RNA structures generated during replication [27]. RNAactivated RIG-I and MDA-5 interact via their caspase activation and recruitment domains (CARDs) with the CARD of the mitochondrial membrane-tethered molecule, IPS-1 (reviewed in [28]; also known as VISA, MAVS, and Cardif). This binding induces IPS-1 to signal through at least two kinase complexes to stimulate the activation and translocation of latent IFN regulatory factor-3 (IRF-3) and other transcription factors (reviewed in [24, 29, 30]). These activate transcription of IFNb [28] as well as IFNl [31, 32] and other targets [33]. By binding to the IFNa receptor, IFNb activates a positive feedback loop in which IRF-3 and IRF-7 activate multiple IFNa genes. Similarly, IFNl1 (IL29) activates transcription of IFNl2 (IL28A) and IFNl3 (IL28B) genes [31, 32]. In a hepatoma line, HCV infection induces rapid – but transient – IRF-3 activation [34]. However, HCV can specifically inactivate the RIG-I-IPS-1 pathway of IFN induction. Thus, the NS3/4A protease was found to block RIG-I-mediated activation and translocation of IRF-3 [35]. NS3/4A specifically cleaves IPS-1 at a cysteine near the carboxy terminus of the protein [36, 37], releasing it from the mitochondrion and preventing its function [38]. In liver biopsies from patients with chronic HCV infection, HCV-infected cells (identified by NS3 immunostaining) show aberrant IPS-1 localization [34], supporting the importance of this observation in vivo. On a cautionary note, however, the

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Immune Control of HCV Infection

level of cleaved IPS-1 was not found to correlate with the level of HCV RNA in biopsy samples, suggesting that other mechanisms also mediate IPS-1 cleavage in the infected liver [39]; importantly, IPS-1 is targeted by cellular caspases [40].

23

strongly associated with genetic polymorphisms in the IL28B locus, suggesting a role for IL28B in control of HCV infection (reviewed in [53]).

3.3.3 3.3.1

Does HCV Interfere with the Detection of Extracellular Virus?

Extracellular HCV RNA, in the form of viral particles or debris from infected cells, may activate Toll-like receptors (TLRs) [41]. TLR signal transduction can activate the production of IFNs and thereby inhibit HCV replication in infected cells [42]. TLR3 detects double-stranded RNA in endosomes, for example after capture of viral RNA by endocytic uptake of debris from infected cells. Hepatocytes express TLR3 [43]; dendritic cell (DC) subsets may express TLR3 (mDCs) or TLR7/8 (pDCs). Signal transduction through TLR3 is dependent on the adaptor, TRIF, which is cleaved and inactivated by NS3/4A [44]. Thus, cells expressing NS3/4A may be impaired in their ability to detect extracellular viral RNA through TLR3. If mDCs were infected with HCV, this mechanism could impair their ability to produce IFNs; however, as discussed below, there is little strong evidence for widespread HCV infection in DCs. Signal transduction by TLR7 and TLR8, which detect single-stranded RNA in endosomal compartments, does not depend on TRIF. pDCs may detect infected hepatocytes by a mechanism dependent on TLR7, with consequent production of IFNa and IFNb [45].

3.3.2

Type III IFNs

Recent evidence from genome-wide association studies of HCV patients highlights the importance of the l or type III IFNs (IL29 or IFNl1, IL28A or IFNl2, and IL28B or IFNl3) in control of HCV infection [46–50]. IFNl expression can be induced by viral infections [31]. Hepatocytes and many other cell types express the IFNl receptor, which is related to the IL10 receptor. The IFNl receptor activates a signal transduction cascade qualitatively similar to that of the type I IFN receptor [51]. In a cell culture model, IFNl was shown to inhibit HCV replication [52]. Spontaneous and treatment-induced clearance of HCV infection are both

HCV May Target IFN-Stimulated Antiviral Programs

While a number of mechanisms have been proposed for the impairment of IFNa/b induction in HCV infected cells, those observations do not explain how HCV persists in the liver in the presence of a robust IFN response – and during IFNa-based antiviral therapy. HCV proteins are reported to subvert type I IFN receptor signal transduction and the function of downstream IFN effector pathways [54, 55]. Type I IFNs signal through the IFNa receptor to activate the kinases Tyk2 and Jak1, which phosphorylate STAT1 and STAT2. These translocate to the nucleus and associate with IRF-9, forming the transcription factor ISGF-3 (reviewed in [56, 57]). Type III IFNs signal through the IFNl receptor, a heterodimer of the IL10 receptor b chain and the IL28 receptor a chain [51, 58]. Activation of the IFNl receptor also stimulates the Jak1 and Tyk2 kinases, leading to phosphorylation of STATs 1 and 2 [59]. While IFNa/b and IFNl stimulate transcription of a similar set of genes in liver cells [60], gene activation in response to type I IFNs is more rapid and less sustained than that observed in response to type III IFNs [52]. HCV’s core protein was reported to activate the expression of suppressors of cytokine signaling (SOCS) proteins [61], which limit JAK-STAT signaling downstream of IFN receptors, in a study of core-transfected cells. However, this result should be interpreted with caution as it has not been reproduced in cells supporting HCV infection. ISGF-3 stimulates the transcription of 300 or more IFN-stimulated genes (ISGs), many with direct antiviral activities. ISGs with direct antiviral activity include 2¢, 5¢ oligoadenylate synthetase (OAS), RNAse L, protein kinase R (PKR), and ISG56 [56, 57]. Several reports suggest that HCV gene products inhibit the function of some ISGs. However, not all of these effects have been confirmed with HCV-infected cells. 2¢, 5¢OAS polymerizes ATP into 2¢, 5¢-linked oligoadenylates, which in turn activate RNAse L to degrade viral RNA. NS5A was reported to inhibit 2¢, 5¢OAS activity [62]. IFNa-based antiviral treatments may select for RNAse L-resistant HCV sequences [63].

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PKR is activated by double-stranded RNA; activated PKR can phosphorylate and inactivate the eukaryotic translation initiation factor, eIF2-a, thus inhibiting protein synthesis [56]. It was reported that by activating PKR to phosphorylate eIF2-a, HCV may block the translation of mRNA encoding other IFN-stimulated genes [64]. In contrast, both NS5A and the envelope glycoprotein, E2, were reported to bind and inhibit PKR [65–67]. E2 binding of PKR presents a topological problem, since the E2 domain required for such an interaction is not believed to localize to the cytosol. Although IFNa receptor signal transduction is reportedly impaired in cells expressing HCV core or the complete HCV polyprotein (reviewed in [54, 55]), IFNs can abolish HCV replication in cell lines. Furthermore, ISG expression is readily detected in the liver of chronically infected patients and in chimpanzees with acute HCV infection. Interestingly, elevated hepatic expression of ISGs in patients with chronic HCV infection is associated with failure of subsequent IFN-based antiviral therapy [68, 69]. High levels of the IFN-induced chemokine CXCL10 (IP10) in the blood of chronic HCV patients before treatment presage treatment failure [70–74].

3.4

Innate Immune Cells and HCV Clearance

The liver is rich in innate immune cells including natural killer (NK) cells, NKT cells, and myeloid subsets including Kupffer cells (liver-resident macrophages) and dendritic cells (DCs) [75, 76]. Little information is available about the roles played by NKT cells in resolution or persistence of HCV infection, although some studies indicate that their levels or activation state are altered in the liver [77] or in the peripheral blood [78] during persistent infection.

3.4.1

Natural Killer Cells

Natural killer cells, abundant in the liver [76], are key early responders in viral infection. They may mediate lysis of infected cells, produce IFNg to directly control viral replication [79], and promote local accumulation of lymphoid and inflammatory cells [80]. Activated NK cells stimulate dendritic cell (DC) maturation, in part through TNFa and IFNg, and thus provide a direct link between innate and acquired immunity [81].

One study reported that the NK cells of HCV patients had decreased ability to promote DC maturation [82]. NK cell cytokine production has the potential to impact HCV replication in larger numbers of cells [83, 84]. However, NK cells from HCV patients may be biased toward cytotoxic rather than cytokine-mediated mechanisms [85], which would limit their antiviral efficacy and at the same time promote tissue damage. The importance of NK cells in the resolution of HCV infection is underscored by observations that genetic polymorphisms in HLA and NK cell inhibitory/activating receptors, which affect NK cell activation thresholds [84], are associated with the outcome (spontaneous clearance vs. chronicity) of HCV infection [86, 87]. Some studies have demonstrated a reduced frequency of circulating NK cells as well as alterations in their cytotoxic and cytokine secretion activities (reviewed in [83]), possibly resulting in a lower antiviral activity overall for NK cells in HCV patients than in healthy controls. Some groups have reported that recombinant HCV envelope proteins can alter the function of NK cells in vitro [88, 89]. However, this result was not reproducible using infectious HCV particles [90] unless the particles were immobilized on a solid support [91]. The latter observation underscores the need for caution in interpretation of studies based on single recombinant HCV proteins; it is not certain whether NK cells encounter significant levels of immobilized HCV particles in vivo. Recombinant purified NS5A was reported to inhibit NK cell activation by activating IL10 and, indirectly, TGFb production [92]; however, it is not known whether such a mechanism operates in vivo. Furthermore, NK cells are activated during acute HCV infection regardless of the outcome of infection [93, 94].

3.4.2

Dendritic Cells

There is some controversy regarding the effects of HCV infection on DC function (reviewed in [95, 96]). Differences in purification or maturation protocols may account for some differences between studies. Two functionally distinct DC populations must be considered: Plasmacytoid dendritic cells (pDCs) are a significant source of type I (a/b) IFNs. Viral infection is detected through the endosomal TLRs 7 and 9, and this detection is followed rapidly by tremendous up-regulation of type I IFN expression [97]. While HCV itself

3

Immune Control of HCV Infection

is a poor inducer of IFNs by pDCs [98, 99], HCVinfected hepatoma strongly induce IFNa [45]. Type I IFNs can activate antiviral programs, summarized above, in other cells. They promote increased expression of major histocompatibility complex antigen ligands for NK and CD8+ T cells. Type I IFNs also stimulate another DC subset, the conventional or myeloid DCs, to produce IL12 – an essential factor for the maturation of Th1 cells [100]. In addition, type I IFNs directly promote NK cell activation and the proliferation of antigen-specific effector CD8+ T cells [101]. pDCs can directly control HCV replication in infected liver-derived cells by secreting IFNs [45]. Some authors have reported that HCV partially blocks pDC function [99, 102], perhaps by down-modulating IRF-7 [99]. However, HCV patients do not demonstrate a major increase in susceptibility to other viral diseases, arguing against a significant degree of pDC impairment [96]. Myeloid dendritic cells (mDCs) develop from monocyte precursors. These cells are potent antigen presenters [103] and are major producers of IL12 and l IFNs [97, 104]. In the liver, mDCs steer the adaptive immune response through antigen presentation and the production of soluble mediators [105]. The mediators include cytokines such as the anti-inflammatory IL10 [106], and IL12, which promotes differentiation of T and NK cells primed for IFNg production [100]. Another important DC-derived mediator is the tryptophan catabolic enzyme, indoleamine 2, 3-dioxygenase, which blocks proliferation of T cells as well as many pathogens [103, 107]. While some investigators have reported reduced immunostimulatory function in DCs during chronic HCV infection [108–110], others have found that these cells are phenotypically and functionally normal [111–115]. We would expect nonspecific impairment in mDC function to have a devastating impact on immune function, yet there is little evidence for global immune dysfunction in HCV patients. Indeed, mDCs isolated from HCV patient livers had enhanced antigen presentation ability, and specifically secreted less of the inhibitory cytokine IL10 than was the case in mDCs from non-infected livers [116].

3.4.3

Does HCV Infect DCs?

Some groups, finding HCV genetic material associated with DC subsets, have proposed that infection of DCs could lead to impaired function [110, 117]. When any

25

quantitation is reported, the level of HCV RNA associated with DCs is far below one copy per cell, indicating that infected DCs must be rare if they exist [95, 114]. In some reports, but not others, expression or uptake of HCV gene products hindered DC functions [95, 118–120]. It is not clear how HCV would infect DCs, which do not express all of the entry factors required for HCV infection of hepatocytes [98]. Laboratory strains of HCV do not replicate in either mDCs or pDCs [98, 102]. The fact that most HCV patients do not suffer from global immunological dysfunction argues against a general defect in antigenpresenting or cytokine-production functions of patient DCs. However, it is possible that infection or impairment is limited to local populations of DCs in the liver or in the draining lymph nodes.

3.5

Adaptive Immunity and HCV

Since the development of methods to screen blood and blood products for HCV in the early 1990s, most new HCV infections have occurred away from medical care. Thus, our knowledge of immunological mechanisms that clear acute HCV infection comes from chimpanzee studies and from specific populations with ready access: healthcare workers who have suffered needle-stick injuries, the rather unusual individuals who develop symptoms of acute hepatitis, and people at risk of exposure who are followed prospectively. In addition, some retrospective studies have examined patients infected in single-source outbreaks.

3.5.1

Cell-Mediated Immune Responses to HCV

Although innate antiviral responses are observed early after HCV infection, resolution of infection is dependent on adaptive immunity. Spontaneous clearance of HCV infection is correlated with T cell responses that target multiple HCV epitopes early in infection (reviewed in [6, 55]). Both CD4+ and CD8+ T cells target diverse sets of epitopes in patients who effectively control HCV [121–123]. In contrast, infections are more likely to become persistent when the acute immune response is narrowly focused [124]. It is widely assumed that broadly focused, sustained immune responses to HCV benefit the patient by increasing the number of HCV epitopes that must be

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changed by mutation in order to escape immune recognition. However, this hypothesis is not yet formally proven. An alternative interpretation is that early control of infection prevents T cell exhaustion and permits the maintenance of a broadly focused HCV-specific immune response. Furthermore, at least transient HCV-specific T cell activation can be detected even as HCV establishes persistent infection. In most patients, cell-mediated immunity fails to eradicate HCV infection. Even after spontaneous clearance of HCV, re-infections by homologous and heterologous HCV strains are possible [125]. Protective immunity reduces the duration and the level of viremia in repeated infection [126–128], but sterilizing immunity has been difficult to prove. Protection from recurrent HCV infection is dependent on both CD4+ [129] and CD8+ [130] memory T cells.

3.5.2

T Cell Responses in Acute Infection

Acute HCV infection – approximately the first 6 months after onset of viremia – is usually asymptomatic. Symptomatic acute infection occurs in 80% of those who complete treatment. With the addition of novel STAT-C (specifically-targeted antiviral therapy for HCV) drugs currently in clinical trials [10, 11], there is hope many more patients can achieve SVR with shorter treatment regimens. However, HCV has already demonstrated the ability to evolve resistance to several STAT-C drugs. Thus, STAT-C drugs will likely be used mainly in combination with IFNa-based treatment. While a review of established and novel antiviral therapies is beyond the scope of this chapter, some antiviral therapies may impact innate and adaptive immunity to HCV. In IFNa-based therapeutic regimens, viral loads decline in two distinct phases [16, 17, 221]. The first phase lasts approximately 24 h and is attributed to a decline in virus production by infected cells – a direct effect of IFNa and innate antiviral mechanisms [17, 221]. A slower, second phase decline occurs over weeks to months and is attributed to the death of infected cells [16, 17, 221]. The role of adaptive immunity in this second-phase decline is controversial (reviewed in [5, 6]). Responsiveness to IFNa-based HCV therapies is affected by polymorphisms in the IL28B locus (reviewed in [53]), suggesting that the l IFNs play a significant role in IFNa-stimulated antiviral responses. STAT-C drugs directed at HCV’s NS3/4A protease may, in addition to blocking processing of the HCV polyprotein, restore some innate responses by impairing NS3/4A-mediated cleavage of IPS-1 and TRIF.

30 Acknowledgments The author’s research is supported by the National Institutes of Health (AI60561).

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CD8+ T cells results in antigen-specific T-cell tolerance. Nat Med 6:1348–1354 Bowen DG, Zen M, Holz L et al (2004) The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. J Clin Invest 114:701–712 Semmo N, Lucas M, Krashias G et al (2006) Maintenance of HCV-specific T-cell responses in antibody-deficient patients a decade after early therapy. Blood 107:4570–4571 Logvinoff C, Major ME, Oldach D et al (2004) Neutralizing antibody response during acute and chronic hepatitis C virus infection. Proc Natl Acad Sci USA 101:10149–10154 Netski DM, Mosbruger T, Depla E et al (2005) Humoral immune response in acute hepatitis C virus infection. Clin Infect Dis 41:667–675 Rehermann B, Nascimbeni M (2005) Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 5:215–229 Bassett SE, Thomas DL, Brasky KM et al (1999) Viral persistence, antibody to E1 and E2, and hypervariable region 1 sequence stability in hepatitis C virus-inoculated chimpanzees. J Virol 73:1118–1126 Pestka JM, Zeisel MB, Blaser E et al (2007) Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C. Proc Natl Acad Sci USA 104:6025–6030 Takaki A, Wiese M, Maertens G et al (2000) Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C. Nat Med 6:578–582 Hsu M, Zhang J, Cheng-Mayer C et al (2003) Hepatitis C virus glycoproteins mediate pH-dependent fusion and cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA 100:7271–7276 Bartosch B, Bukh J, Meunier JC et al (2003) In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proc Natl Acad Sci USA 100:14199–14204 Meunier J-C, Engle RE, Faulk K et al (2005) Evidence for cross-genotype neutralization of hepatitis C virus pseudoparticles and enhancement of infectivity by apolipoprotein C1. Proc Natl Acad Sci USA 102:4560–4565 Timpe JM, Stamataki Z, Jennings A et al (2008) Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 47:17–24 Liu L, Fisher BE, Dowd KA et al (2010) Acceleration of hepatitis C virus envelope evolution in humans is consistent with progressive humoral immune selection during the transition from acute to chronic infection. J Virol 84: 5067–5077 von Hahn T, Yoon JC, Alter H et al (2007) Hepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in vivo. Gastroenterology 132:667–678 Bresee JS, Mast EE, Coleman PJ et al (1996) Hepatitis C virus infection associated with administration of intravenous immune globulin. A cohort study JAMA 276:1563–1567 M-yW Yu, Bartosch B, Zhang P et al (2004) Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma. Proc Natl Acad Sci USA 101:7705–7710

36 214. Farci P, Alter HJ, Wong DC et al (1994) Prevention of hepatitis C virus infection in chimpanzee after antibodymediated in vitro neutralization. Proc Natl Acad Sci USA 91:7792–7796 215. Farci P, Shimoda A, Wong D et al (1996) Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci USA 93:15394–15399 216. Law M, Maruyama T, Lewis J et al (2007) Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 14:25–27 217. Burton DR (2002) Antibodies, viruses and vaccines. Nat Rev Immunol 2:706–713

L.B. Dustin 218. McKeating JA, Zhang LQ, Logvinoff C et al (2004) Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81 dependent manner. J Virol 78: 8496–8505 219. Dienstag JL, McHutchison JG (2006) American Gastroenterological Association technical review on the management of hepatitis C. Gastroenterology 130:231–264 220. Heathcote J, Main J (2005) Treatment of hepatitis C. J Viral Hepat 12:223–235 221. Layden-Almer JE, Ribeiro RM, Wiley T et al (2003) Viral dynamics and response differences in HCV-infected African American and white patients treated with IFN and ribavirin. Hepatology 37:1343–1350

4

B Cell Activation: General to HCV-Specific Considerations Vito Racanelli and Claudia Brunetti

4.1

Human B Cell Response in Physiology

B cell differentiation into antibody-secreting cells (ASC) provides the basis of the humoral adaptive immune system. All stages of this process require strict homeostatic controls in order to keep the cell pool size constant and to avoid the emergence of aberrant or self-reactive B cell populations. Indeed, naïve and memory B cells are governed by independent homeostatic mechanisms and different activation requirements [1–3]. In the context of a T cell-dependent (TD) B cell response, naïve B cells proliferate and differentiate into memory B cells and long-lived plasma cells only after the integration of three signals, namely: (i) B cell receptor (BCR) triggering by the antigen; (ii) CD4+ T cell help via CD40, following cognate interaction; and (iii) activation of Toll-like receptors (TLRs), whose expression is induced after BCR cross-linking [4]. By contrast, memory B cells can be effectively triggered in the absence of BCR engagement via polyclonal stimuli, such as bystander T cell help, provided in a non-cognate fashion by CD4+ T cells activated by a third-party antigen, and microbial products acting on TLRs, which are constitutively expressed by these cells [4]. In the context of thymus-independent (TI) B cell responses, naïve B cells exhibit the same differential requirements as memory B cells, since both BCR cross-linking and TLR stimulation are required. V. Racanelli (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_4, © Springer-Verlag Italia 2012

However, this response might also require surrogate T cell help, such as that provided by C4BP or BAFF [4]. Intriguingly, TLRs agonists are required both in the TD and TI responses, irrespective of the nature of T cell help, suggesting a pivotal role in B cell compartment activation. Naïve B cells do not express TLRs constitutively and cannot respond directly to microbial products; however, their capacity to rapidly up-regulate TLRs upon BCR stimulation endows them with high specificity, in that only naïve B cells stimulated by an antigen will focus on innate signals. In contrast, memory B cells constitutively express TLRs and can readily respond to a variety of environmental stimuli [5]. It has been speculated that TLR activation plays a distinct role in B cell response to viruses. The dual BCR-TLR engagement model of B cell activation perfectly fits with virus recognition by B cells, as viral particles expose an abundance of foreign antigens and contain non-self nucleic acids, a combination that should result in effective BCR and TLR triggering, followed by B cell activation and differentiation [6].

4.2

Human B Cell Response in Pathology

Several infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and Helicobacter pylori have been reported to play an etiological role in B cell lymphoproliferative disorders. Direct infection of B lymphocytes, generalized deregulation of the immune response, and chronic activation of specific cell-surface receptors are the main pathogenetic mechanisms by which these viruses act. Abnormal antibody production (hypergammaglobulinemia) and 37

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V. Racanelli and C. Brunetti

b

HCV BCR

HCV

c

HCV

BCR

HCV proteins and RNA

CD81

d

e

IgM RF

Immunecomplexed HCV

Self lgG HCV antigen

BCR

BCR CD81

E2-specific BCR

CD81

CD81

Endocytosis

HCV ssRNA

TLR7

Fig. 4.1 Possible mechanisms of B cell activation during chronic HCV infection. (a) After HCV entry and replication in B cells, viral proteins could exert their oncogenic potential by altering essential cell activation pathways. Consequently, infected B cells might lose proliferation control. (b) The ability of HCV-E2 to bind CD81 could determine a strong and sustained polyclonal stimulation of the B-cell compartment. (c) Dual engagement of CD81 and specific BCR by HCV-E2 could

effectively activate B cells, leading to their clonal expansion. (d) Molecular mimicry between an HCV antigen and a self-IgG could explain the selection of RF-producing B cell clones. (e) Immune-complexed HCV could effectively activate specific B cells through BCR. After immune complex internalization and HCV disruption in the endosomal/lysosomal compartment, viral RNA could reach TLR7 and start molecular pathways leading to B cell aberrant activation

B cell lymphoproliferative diseases, namely mixed cryoglobulinemia (MC) and B cell non-Hodgkin’s lymphoma (B-NHL) [5, 7, 8], are frequently observed during the chronic course of HCV infection. Several models have been proposed to explain how HCV, a predominantly hepatotropic virus, induces dysfunction of the B cell compartment.

the pathogenesis of HCV-related MC, which is characterized by a generalized B cell compartment expansion, even if the emergence of a specific rheumatoid-factorpositive (RF+) B cell clone suggests that HCV tropism is restricted to a B cell subset. According to this model, treatment of MC patients with rituximab should reduce HCV replication, but the increased serum HCV RNA levels often detected following its administration argues against the hypothesis of a B cell reservoir of infection [9]. Detectable levels of both HCV RNA and proteins [10, 11] have been reported in association with B cells and other blood cell subsets of infected individuals. In some reports [11], although not in others [12], HCV RNA was associated with peripheral blood

4.3

Direct Infection of B Cells

A number of studies hypothesized that direct infection of B cells leads to their malignant transformation (Fig. 4.1a). HCV lymphotropism may be involved in

4

B Cell Activation: General to HCV-Specific Considerations

39

mononuclear cells (PBMCs) even after antiviral therapy. Also, blood cells have been successfully infected in vitro [11, 13, 14]. However, no HCV replication was detected when PBMCs were treated with the HCV replicase inhibitor 2’C-methyl adenosine (2’-CMA), which allows viral replication to be discriminated from viral persistence [15]. In addition, the absence of important HCV entry factors, including scavenger receptor BI (SR-BI) and the tight-junction protein claudin-1 (CLDN1), on B cells impairs their proneness to infection [15]. Cumulative evidence suggests that viral RNA is bound or taken-up by cells without undergoing a complete infectious cycle. For instance, CD81, a known HCV entry factor, is expressed on B cells, thus mediating HCV capture from the environment. Surface anti-HCV antibodies may enable HCV-specific B cells to bind HCV, while Fc receptors, complement receptors, or RF on B cell membranes might be engaged by circulating virus-antibody complexes. Thus, viral particles may be detected in association with B cells, but HCV rarely infects them.

to be directed against E2 [20], suggesting specific BCR stimulation by HCV-E2. Moreover, the BCR cloned from an HCV-associated NHL was shown to specifically bind E2, although the RF activity of this E2 specific antibody has not been demonstrated [21]. Therefore, a modified model for HCV-E2 mediated B cell stimulation has been proposed. According to this model, the simultaneous engagement of BCR and CD81 by E2 would effectively reduce the B cell stimulation threshold, leading to extensive proliferation of E2-specific B cell clones [21] (Fig. 4.1c).

4.4

Polyclonal Stimulation of B Cells by HCV Antigens

The second portion of the HCV envelope glycoprotein E2 binds with high affinity to CD81, a tetraspanin expressed on several cell types [16]. Since on B cells CD81 is a component of the B cell co-receptor signaling complex [17], it has been proposed that CD81 engagement by E2 lowers the activation threshold of B cells, thus facilitating their proliferation and differentiation [18] (Fig. 4.1b). In agreement with this model, recombinant E2 protein, combined with crosslinking anti-CD81 monoclonal antibodies, polyclonally stimulates B cells in vitro [18]. However, the non-physiologically high concentration of E2 protein used in this study makes understanding of the events that occur in vivo difficult. It has also been claimed that E2/CD81 interaction triggers activation-induced (cytidine) deaminase (AID) expression and double-stranded breaks, resulting in stochastic immunoglobulin (Ig) hypermutation [19], but this model does not explain the emergence of clonal RF+ B cells and the presence of poorly hypermutated self-reacting antibodies, both features of MC. Intriguingly, an efficient HCVneutralizing monoclonal antibody, cloned from an asymptomatic HCV-infected patient, was demonstrated

4.5

Chronic Antigenic Stimulation by Specific HCV Proteins

The alternative scenario proposed for microbial species-associated B cell aberrations is chronic antigenic stimulation. This model has emerged with the description of several lymphomas developing in the context of chronic antigen-dependent immune stimulation, such as gastric mucosa-associated lymphoid tissue (MALT) positive for Helicobacter pylori [22], but it can also be transposed to other B cell abnormalities. According to this model, the pathogen is neither intrinsically transforming nor oncogenic, but its ability to persist in the host offers a chronic source of antigens to specific B cell clones [23]. Depending on the length of infection, additional oncogenic events may occur, leading the B cell proliferation to become independent of antigen stimulation [24]. According to this multistep mechanism of lymphomagenesis, t(14;18) and bcl-2 overexpression in lymphoid cells have been proposed to be the leading events for the pathogenesis of HCV-related lymphoproliferative disorders [25, 26]. Nonetheless, experimental data are still controversial [27], and t(14;18)-positive cells have also been detected in healthy individuals [28]. Of note, HCV-associated MC is characterized by monoclonal or oligoclonal expansion of specific B cell subsets both in the liver [29] and in the peripheral blood [30]. It can therefore be envisaged that an antigen-driven process is involved in B cell growth and clonal evolution. Molecular analysis of the IgH VDJ region in expanded B cell clones supports this hypothesis, showing restricted VH gene usage shared by the anti-HCV antibody response, e.g., directed against E2 (HCV/E2) [31–33]. Moreover, antibodies to E2 encode the VH/VL gene pair VH1-69 and VK3-23, which is also

40

utilized in the synthesis of WA cross-idiotype monoclonal RF in patients with MC [20, 34]. These findings suggest that RF secretion in part occurs as an antibody response to HCV antigens, such as E2. Molecular mimicry, a phenomenon in which microbial pathogens share structural similarities with self motifs, is an immune-evasion strategy possibly adopted by HCV in order to persist. As a secondary effect, molecular mimicry could confer distinctive characteristics to certain HCV antigens, making them more prone to evoke a strong autoimmune response. It has indeed been reported that the structural and antigenic homologies between the N-terminal region of the HCV-E2 protein and the human Ig variable domains (IgV) are responsible for the strong association between HCV and detection of RF [35]. Alternatively, it has been demonstrated that in MC patients the IgM RF cross-reacts with IgG Fc and HCV NS3, due to the molecular mimicry between these two proteins [36] (Fig. 4.1d). Though fascinating, these data account only for a minority of HCV MC patients and they are not sufficient to explain the general mechanisms underlying the pathogenesis of the disease. Recently, a new model of chronic antigenic stimulation has been proposed, in which the antigen can also stimulate pattern recognition receptors (PRR) receptors in B cells. According to this hypothesis, during chronic HCV infection, immune-complexed HCV stimulates the expansion of self-reactive B cells, acting through the dual ligation of BCR and TLR7 by IgG and HCV RNA, respectively [9] (Fig. 4.1e). In explorations of the pathogenetic mechanisms driving the self-reactive B cell clone expansion, the human leukocyte antigen (HLA) system has generated increasing interest. The DR5/DQ3 HLA combination [37] and a higher frequency of the DR11 HLA phenotype [38] have been proposed as predictive factors associated with an increased risk for HCV-related MC development. Although the mechanisms by which HLA polymorphysms might predispose to autoimmunity are still unknown, it has been proposed that continuous presentation of HCV-derived epitopes, through a limited set of HLA molecules, result, on the one hand, in low/ineffective viral clearance and, on the other, in positive selection of self-reactive B cell clones, sustained by T-helper lymphocytes [39]. In conclusion, studies of the human B cell response, both at steady state and during chronic HCV infection,

V. Racanelli and C. Brunetti

could help in unravelling HCV/B cell interactions in addition to providing relevant information with the potential to be promptly translated into new therapies both for autoimmune and infectious diseases.

References 1. Lanzavecchia A (1983) One out of five peripheral blood B lymphocytes is activated to high-rate Ig production by human alloreactive T cell clones. Eur J Immunol 13(10):820–824 2. Bernasconi NL, Traggiai E, Lanzavecchia A (2002) Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298(5601):2199–2202 3. Bernasconi NL, Onai N, Lanzavecchia A (2003) A role for toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells. Blood 101(11):4500–4504 4. Ruprecht CR, Lanzavecchia A (2006) Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur J Immunol 36(4):810–816 5. Lanzavecchia A, Bernasconi N, Traggiai E et al (2006) Understanding and making use of human memory B cells. Immunol Rev 211:303–309 6. Lanzavecchia A, Sallusto F (2007) Toll-like receptors and innate immunity in B-cell activation and antibody responses. Curr Opin Immunol 19(3):268–274 7. El-Serag HB, Hampel H, Yeh C et al (2002) Extrahepatic manifestations of hepatitis C among United States male veterans. Hepatology 36(6):1439–1445 8. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297(18):2010–2017 9. Charles ED, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 76(8):818–824 10. Zehender G, Meroni L, De Maddalena C et al (1997) Detection of hepatitis C virus RNA in CD19 peripheral blood mononuclear cells of chronically infected patients. J Infect Dis 176(5):1209–1214 11. Navas MC, Fuchs A, Schvoerer E et al (2002) Dendritic cell susceptibility to hepatitis C virus genotype 1 infection. J Med Virol 67(2):152–161 12. Bernardin F, Tobler L, Walsh I et al (2008) Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia. Hepatology 47(5):1446–1452 13. Sung VM, Shimodaira S, Doughty AL et al (2003) Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol 77(3):2134–2146 14. Kondo Y, Sung VM, Machida K et al (2007) Hepatitis C virus infects T cells and affects interferon-gamma signaling in T cell lines. Virology 361(1):161–173 15. Marukian S, Jones CT, Andrus L et al (2008) Cell cultureproduced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 48(6):1843–1850 16. Pileri P, Uematsu Y, Campagnoli S et al (1998) Binding of hepatitis C virus to CD81. Science 282(5390):938–941

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17. Bradbury LE, Kansas GS, Levy S et al (1992) The CD19/ CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu13 molecules. J Immunol 149(9):2841–2850 18. Rosa D, Saletti G, De Gregorio E et al (2005) Activation of naive B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc Natl Acad Sci USA 102(51):18544–18549 19. Machida K, Cheng KT, Pavio N et al (2005) Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells. J Virol 79(13):8079–8089 20. Keck ZY, Xia J, Cai Z et al (2007) Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions. J Virol 81(2):1043–1047 21. Quinn ER, Chan CH, Hadlock KG et al (2001) The B-cell receptor of a hepatitis C virus (HCV)-associated non-Hodgkin lymphoma binds the viral E2 envelope protein, implicating HCV in lymphomagenesis. Blood 98(13):3745–3749 22. Cavalli F, Isaacson PG, Gascoyne RD et al (2001) MALT lymphomas. Hematology Am Soc Hematol Educ Program: 241–258 23. Suarez F, Lortholary O, Hermine O et al (2006) Infectionassociated lymphomas derived from marginal zone B cells: a model of antigen-driven lymphoproliferation. Blood 107(8):3034–3044 24. Mayo MJ (2003) Extrahepatic manifestations of hepatitis C infection. Am J Med Sci 325(3):135–148 25. Zignego AL, Giannelli F, Marrocchi ME et al (2000) T(14;18) translocation in chronic hepatitis C virus infection. Hepatology 31(2):474–479 26. Zuckerman E, Zuckerman T, Sahar D et al (2001) bcl-2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection. Br J Haematol 112(2):364–369 27. Sansonno D, Tucci FA, De Re V et al (2005) HCV-associated B cell clonalities in the liver do not carry the t(14;18) chromosomal translocation. Hepatology 42:1019–1027 28. Roulland S, Lebailly P, Lecluse Y et al (2006) Long-term clonal persistence and evolution of t(14;18)-bearing B cells in healthy individuals. Leukemia 20(1):158–162 29. Racanelli V, Sansonno D, Piccoli C et al (2001) Molecular characterization of B cell clonal expansions in the liver of chronically hepatitis C virus-infected patients. J Immunol 167(1):21–29

30. Charles ED, Green RM, Marukian S et al (2008) Clonal expansion of immunoglobulin M+CD27+ B cells in HCV-associated mixed cryoglobulinemia. Blood 111(3):1344–1356 31. Carbonari M, Caprini E, Tedesco T et al (2005) Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis. J Immunol 174(10):6532–6539 32. Chan CH, Hadlock KG, Foung SK et al (2001) V(H)1-69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen. Blood 97(4):1023–1026 33. De Re V, De Vita S, Marzotto A et al (2000) Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 96(10):3578–3584 34. Machida K, Kondo Y, Huang JY et al (2008) Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein. J Virol 82(13):6711–6720 35. Hu YW, Rocheleau L, Larke B et al (2005) Immunoglobulin mimicry by hepatitis C virus envelope protein E2. Virology 332(2):538–549 36. De Re V, Sansonno D, Simula MP et al (2006) HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations. Leukemia 20(6):1145–1154 37. De Re V, Caggiari L, Monti G et al (2010) HLA DR-DQ combination associated with the increased risk of developing human HCV positive non-Hodgkin’s lymphoma is related to the type II mixed cryoglobulinemia. Tissue Antigens 75(2): 127–135 38. Cacoub P, Renou C, Kerr G et al (2001) Influence of HLA-DR phenotype on the risk of hepatitis C virusassociated mixed cryoglobulinemia. Arthritis Rheum 44(9): 2118–2124 39. De Re V, Caggiari L, De Vita S et al (2007) Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus. Dig Liver Dis 39 (Suppl 1):S65–S71

5

Organ-Specific Autoimmunity in HCV-Positive Patients Corrado Betterle and Fabio Presotto

5.1

Introduction

Hepatitis C virus (HCV) is a RNA virus that chronically infects more the 170 million individuals around the world. In the majority of cases, the acute infection develops undiagnosed such that HCV becomes a persistent infection that gives rise to chronic hepatitis, fibrosis, and even cirrhosis in about 20% of affected patients. The related severe complications and death are due to decompensated cirrhosis, end-stage liver disease, and hepatocellular carcinoma [1]. In the course of the disease, according to various studies, 40–80% of HCV-infected patients may develop at least one extrahepatic manifestation, which can be the first or only clinical sign of chronic infection with the virus [2]. Increasing attention is currently being focused on the pathogenic role of chronic HCV infection in triggering autoantibody production, autoimmune disease, and lymphoproliferative disorders [3].

5.2

Extrahepatic Manifestations

5.2.1

HCV-Associated Mixed Cryoglobulinemia

Cryoglobulins are antibody complexes that precipitate when serum is cooled and dissolve on rewarming [4]. Mixed cryoglobulinemia (MC), or type II

cryoglobulinemia, is the disease most commonly associated with chronic HCV infection. Cryoprecipitates usually contain large amounts of HCV antigens and/ or antibodies against HCV, and they can precipitate in the walls of small and medium-sized vessels, leading to activation of the complement cascade and manifestations of systemic vasculitis. The classical MC syndrome clinically presents as a triad of purpura, weakness, and arthralgias. The skin, kidney, nerves, and joints are the most frequent target organs affected by cryoglobulins. Although more than 50% of patients with chronic HCV infection have circulating serum cryoglobulins (cryoglobulinemia), the majority do not develop clinical signs or symptoms or need specific treatment [5, 6]. MC is extensively discussed in other chapters of this volume.

5.2.2

The most frequent and clinically important extrahepatic manifestations of HCV-related chronic infection involving organ-specific autoimmunity are thyroid disorders and diabetes mellitus. In the majority of cases, autoimmune endocrine disorders develop after treatment with interferons (IFNs) [7–9].

5.2.3 C. Betterle (*) Unit of Endocrinology, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_5, © Springer-Verlag Italia 2012

HCV-Related Organ-Specific Autoimmune Disorders

Pathogenesis of Extrahepatic Autoimmune Manifestations

The mechanisms that link HCV infection with autoimmunity are unknown. Moreover, it is unimportant to establish whether the presence of extrahepatic autoim43

44

C. Betterle and F. Presotto

mune manifestations (EHAMs) is a direct effect of HCV infection or the consequence of its treatment. In fact, antiviral therapy with IFN-a has been accused of triggering autoimmunity [10]. Both B cell and T cell lines of the immune system are thought to be involved in the generation of autoimmunity during chronic HCV infection. A non-specific activation of the immune system triggered by HCV infection seems to be responsible for the production of autoantibodies directed against extrahepatic antigens, such as the non-organ-specific autoantibodies (NOSA), but molecular mimicry also appears to be involved in their appearance [4, 11]. The specific binding of the HCV-related E2 protein to the CD81 molecule of B cells prompted the hypothesis of a role for HCV in stimulating a chronic polyclonal B cell response to viral antigens that facilitates the onset of a lymphoproliferative dysregulation [12]. The binding of HCV to B cells may also support viral persistence and drive the immune response towards a Th2-associated profile with an increased humoral response and autoantibody production [13]. As for the cell-mediated immune response, it has been reported that CD81 receptors are expressed on various target organ cells able to bind the HCV envelope glycoprotein E2. This binding may induce several signaling cascades and cause the release of both cytotoxic and proinflammatory mediators, such as tumor necrosis factor or interleukin (IL)-8, which in turn can lead to the bystander killing of neighboring (uninfected) cells and activation of an autoimmune response [14].

5.3

Autoimmune Thyroid Diseases

Two main and distinct entities of autoimmune thyroid disease (AITD) are recognized: chronic thyroiditis (CT) and Graves’ disease (GD) [15, 16]. CT is probably the most common autoimmune disease in the world, presenting with or without goiter. CT with goiter can be associated with frank hypothyroidism, subclinical hypothyroidism, or normal thyroid function. The non-goitrous type includes two main subtypes, i.e. primary myxedema and symptomless autoimmune thyroiditis. Early genetic studies in white people showed that HLA-B8 and HLA-DR3 haplotypes were associated with symptomless autoimmune thyroiditis and HLA-DR5 with goitrous autoimmune thyroiditis, thus suggesting that the two disorders

have different genetic backgrounds [15]. CT is also associated with polymorphisms of the gene encoding cytotoxic T-lymphocyte antigen 4 (CTLA-4) [16, 17]. Thyroglobulin and/or thyroid peroxidase autoantibodies are a common finding when the different variants of CT are diagnosed, being detectable in 80–99% of cases. The reported prevalence of CT varies considerably, depending on the diagnostic criteria employed and the age and characteristics of the patient considered (e.g., gender, genetic differences, geographical origin, and iodine intake) [15, 18]. In autopsy studies performed in the UK and the USA, CT was identified in 40–45% of women and 20% of men if any degree of focal thyroiditis was considered, while its prevalence dropped to 5–15% in women and 1–5% in men when only cases of severe thyroiditis were taken into account [15]. The prevalence of subclinical hypothyroidism is estimated to be 1–10%, and the highest age- and sexspecific rate is seen in women over 60 years of age, in whom it approaches 20% [18]. In community surveys to screen the apparently normal population, 10–13% of women and 2–3% of men tested positive for thyroid autoantibodies [15, 19]. The prevalence of thyroid autoantibodies increases with age, becoming as high as 33% in women age ³ 70 years, but it otherwise peaks in the fifth and sixth decades of life. The presence of circulating thyroid autoantibodies is related to lymphocytic infiltration in the thyroid gland [20]. Graves’ disease is an AITD classically presenting with hyperthyroidism (clinical or subclinical), with or without goiter, ophthalmopathy, or dermopathy. The clinical manifestations of GD are strictly related to its severity, the duration of hyperthyroidism, and the patient’s age [16]. In patients with hyperthyroidism, high serum levels of thyroid peroxidase autoantibodies (detectable in 75% of patients) and/ or thyroid-stimulating autoantibodies (detectable in 80–95% of patients), and/or the demonstration by radionuclide thyroid scan of a diffuse increased uptake are evidence of GD. Thyroid-stimulating autoantibodies cause both thyroid hyperfunction and thyroid follicle hypertrophy/hyperplasia, resulting in a characteristic diffuse goiter. There is a well-established association between GD and certain class II HLA alleles, which varies among different racial groups. In whites, HLA-DR3 and HLA-DQA1*0501 are positively associated with GD, whereas HLA-DRB1*0701 protects against the disease [16]. GD is also associated with CTLA-4 polymorphisms in several racial groups

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Table 5.1 Frequency of thyroid autoimmunity in patients with chronic HCV infection before interferon therapy and controls Author Tran Pateron Watanabe Boadas Carella Preziati Roti Loviselli Metcalfe Floreani Fernandez-Soto Ganne-Carrie Betterle Carella Antonelli Zusinaite Gehring All studies

Reference [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [7] [33] [9] [34] [35]

Year 1993 1993 1994 1995 1995 1995 1996 1997 1997 1998 1998 2000 2000 2002 2004 2004 2006

Thyroid autoantibodies Patients (n) (%) 72 12.5 66 13.5 109 9.2 96 5.2 75 10.7 78 35.9 32 9.4 86 9.3 111 4.5 47 12.7 134 20.0 97 13.4 70 5.7 147 10.2 630 21.0 90 2.2 123 1.5 2,066 Range 1.5–21

Controls (n) 60a nt nt 96 nt nt nt 1147 99 nt 41 97 100 nt 657 nt nt 2,297

Thyroid autoantibodies (%) 1.5 nt nt 12.0 nt nt nt 17.0 11.1 nt 5.0 3.1 3.0 nt 11.2 nt nt Range 1.5–17

P value 0.02 – – ns – – – ns ns – 0.02 0.02 ns – 0.001 – ns

nt not tested, ns not significant Patients with chronic HBV infection

a

[16]. The annual incidence of GD is around 0.14 per 1,000 [18], with the highest risk of onset between the ages of 40 and 60 years. GD is five to ten times more common in women than in men and is unusual in children [16].

5.3.1

Autoimmune Thyroid Diseases and HCV Before Interferon Therapy

The etiology of AITD remains unknown. Genetic predisposition is important, but non-genetic factors also play a major part. HCV infection seems to be one of the non-genetic factors involved. Many studies have evaluated the prevalence of thyroid autoantibodies (TA) or AITD in patients with chronic HCV infection, with conflicting and inconclusive results. Table 5.1 summarizes the studies performed, showing the frequency of AITD in patients and controls. An association between HCV infection and AITD was initially suggested by Tran [21], who studied 72 patients with chronic HCV infection (43 men and 29 women) and found TA in nine cases (12%), all women (31%). Two of these nine women also had hypothyroidism. Only 1.5% of 60 patients with chronic HBV infection (used as controls) had low titers of TA.

Further studies on these patients are listed in Table 5.1. Considering the cited publications as a whole, in a cohort of 2,066 patients with chronic HCV infection TA were detected in 1.5–21% of cases as opposed to 1.5–17% in controls. Only four studies reported a significant association between HCV infection and TA [9, 21, 31, 32]. However, five studies found no such association [7, 24, 28, 29], and another eight studies provided no data on matched controls [22, 23, 25–27, 30, 33, 34] (Table 5.1). Disparities in the prevalence of TA may be related to the different methods used to detect TA, the female/male ratio, or the ages of the patients examined. The majority of the studies also failed to provide data on matched controls. To ascertain whether a correlation exists between HCV infection and AITD, other studies investigated the frequency of HCV infection in patients with AITD. In 1993, Quaranta et al. studied 147 patients with AITD and identified HCV infection in 10 (6.8%), a frequency significantly higher than in controls (0.7%) [36]. By contrast, another study found that none of 30 patients with CT had HCV infection [37], and another no significant association between HCV genotypes and the development of TA [34]. We investigated the prevalence of HCV infection and TA in a general population of 697 adult Italians and found that 10.1% were

46

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Table 5.2 Occurrence of thyroid antibodies after interferon therapy in patients with chronic HCV infection

Author Baudin Watanabe Imagawa Carella Preziati Marazuela Roti Carella Gehring All studies

Reference [40] [23] [41] [25] [26] [42] [27] [43] [35]

Year 1993 1994 1995 1995 1995 1996 1996 2001 2006

Occurrence of thyroid Patients (n) antibodies (%) 68 5.9 109 1.9 58 8.9 75 34.6 78 40.0 144 4.9 32 12.5 114 31.6 123 15.5 801 Range 1.9–40

positive for HCV and 6.1% for TA, but no significant association emerged between those testing positive for HCV and for TA [38]; therefore, these findings failed to demonstrate any clear relationship between chronic HCV infection and AITD.

relative risk than men of developing thyroid disease; (f) while on IFN therapy, several patients develop thyroid dysfunction in the absence of TA, suggesting a direct toxic effect of IFN on the thyrocytes (so-called destructive thyroiditis); (g) in most cases of destructive thyroiditis, thyroid dysfunction is mainly subclinical and may regress spontaneously [44–46]. In conclusion, while receiving IFN therapy, patients with chronic HCV infection may develop either AITD (CT or GD), or even TA with no gland dysfunction, or non-AITD conditions (destructive thyroiditis). All patients with chronic HCV infection should therefore be screened for thyroid diseases by means of baseline TSH and TA assays before starting IFN therapy, and then every 2–3 months during treatment. If TSH levels are normal but TA are positive, the patient risks developing thyroid dysfunction. If thyroid dysfunction sets in and TA are negative, it may be advisable to test for TSH receptor antibodies and perform thyroid ultrasound [45].

5.4 5.3.2

Autoimmune Thyroid Diseases and HCV During or After Interferon Therapy

In 1992, Marcellin et al. first reported on two patients with chronic HCV hepatitis who developed hypothyroidism while on IFN therapy [39]. Many other authors subsequently confirmed this observation (Table 5.2). Judging from these studies, the prevalence of IFNinduced thyroid autoimmunity ranges from 1.9% to 40%, possibly depending on the dosage and duration of the medical therapy, the patients’ characteristics, and the definition of thyroid dysfunction. An analysis of the main data collected after IFN therapy suggests that: (a) patients already positive for TA may experience an increase in their antibody titers and develop a clinical or subclinical thyroid dysfunction; (b) initially negative patients acquire TA in 1.9–40% of cases, and a proportion of them will develop clinical or subclinical thyroid dysfunction; (c) serum thyroid-stimulating hormone (TSH) and anti-thyroperoxidase antibody (TPO) status before IFN treatment predict progression to AITD; (d) thyroid autoimmunity is reversible in some cases, but permanent in others, although the percentage of the latter cannot be predicted from the available data; (e) women have a two-fold higher

Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (DM) is a chronic autoimmune disorder with variable degrees of insulin deficiency resulting from an immune-mediated destruction of the pancreatic beta cells [47]. The disease is characterized by T lymphocyte infiltration of the pancreatic islets, with the disappearance of the beta cells. A genetic predisposition to type 1 DM has been mapped to the HLA region on chromosome 6, but environmental factors have also been implicated in the pathogenesis of this disorder. Islet cell autoantibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), and antibodies to the second islet antigen (IA-2A/ICA512) are the main serological markers of DM. One or more of these autoantibodies are detectable when diabetes is diagnosed in 90% of the cases, and they often appear even before the clinical onset of the disease [47].

5.4.1

Type 1 Diabetes Mellitus and HCV Chronic Infection Before Interferon Therapy

Epidemiological studies have shown a relationship between HCV infection and DM, suggesting that this disease is another extrahepatic manifestation. However,

5

Organ-Specific Autoimmunity in HCV-Positive Patients

47

Table 5.3 Autoantibodies to endocrine pancreas in patients with HCV chronic hepatitis before and after interferon (IFN) therapy

Authors Imagawa Di Cesare Imagawa Hieronimus Floreani Betterle Piquer Wesche Wasmuth All studies

Year 1995 1996 1996 1997 1998 2000 2001 2001 2001

Reference [41] [52] [53] [54] [30] [7] [55] [56] [57]

Patients (n) 58 48 40 47 47 70 277 75 56 718

Pancreatic autoantibodies before IFN (%) 0 0 0 2 4.2 2.8 1.4 4.0 0 Range 0–4.2

Pancreatic autoantibodies during IFN (%) 0 0 2.5 nt 12.7 nt 0 10.6 5.3 Range 0–12.7

Development of diabetes mellitus (%) 0 0 0 nr 2 nr 0 0 0 0–2

nt not tested, nr not reported

these studies found a greater association of HCV infection with type 2 diabetes, not type 1 [48–51]. The lack of association between HCV chronic infection and pancreatic autoimmunity before IFN therapy has also been demonstrated in many studies (Table 5.3). A meta-analysis was performed on 718 patients with chronic HCV infection. Pancreatic autoantibodies were detected in 0–4.2% before IFN therapy, a frequency that did not differ significantly from the situation in controls [58, 59] and suggesting that HCV infection is not linked to pancreatic autoimmunity.

5.4.2

Type 1 Diabetes Mellitus and Chronic HCV Infection During or After Interferon Therapy

The situation is different in patients with chronic HCV hepatitis treated with IFN. The first case of autoimmune diabetes occurring after IFNa treatment was described in 1992, in a male patient with chronic HCV infection found to have pancreatic autoantibodies in a serum sample collected before IFNa treatment. This prompted the speculation that IFNa triggers the onset of overt type 1 diabetes in patients with islet cell antibodies [60]. A study performed in Italy on more than 11,000 patients with chronic HCV infection treated with IFNa reported ten new cases of DM (0.08%), nine of them requiring permanent insulin therapy [61]. Another study, performed in Japan on 667 HCVpositive patients with chronic hepatitis, reported five new cases of type 1 diabetes (0.7%) after IFN therapy

[62]. In both studies, the prevalence of diabetes was higher than among controls. In the Japanese study, the majority of the cases of hyperglycemia regressed, albeit incompletely, after therapy was withdrawn. These studies did not consider the presence of pancreatic autoantibodies, however, which could have discriminated between patients with and without the autoimmune form of DM. From 1995 to 2001, other reports were published on the prevalence of autoantibodies to the endocrine pancreas after IFNa treatment (Table 5.3). These studies showed that, while on IFNa therapy, patients already positive for such autoantibodies had an increase in their antibody titers, and that up to 12.7% of patients initially negative acquired pancreatic antibodies de novo and then developed autoimmune type 1 diabetes [58, 59]. Following the first description, in 1992, of a case of autoimmune DM induced by IFN, by 2004 another 31 patients had reportedly developed type 1 DM during, or soon after, IFN therapy, as reviewed by Fabris et al. [58] and Davendra et al. [59]. An analysis of these cases showed that: (a) there were 25 males and 6 females, with a mean age at the onset of DM of 46.7 years (range 23–66 years); (b) 25 patients were treated with IFN for chronic HCV hepatitis, 3 for HBV hepatitis, and 3 for cancer; (c) nine patients were treated with IFNa and ribavirin, 1 with IFNa and IL-2, 1 with both IFNa and IFNb, 1 with IFNb, and the remainder with IFNa; the total dose of IFN before DM developed was 65–1350 U; (d) nine of the 25 cases evaluated had a family history of DM (type 1 in 3 cases, type 2 in 6); (e) the latency period from starting

48

C. Betterle and F. Presotto

IFN therapy to DM varied from 10 days to 4 years; (f) one or more pancreatic antibodies were positive in 9 of 18 patients tested before any IFN treatment, but in 23 of 30 (77%) evaluated at the onset of DM; (g) in 16 of 18 (89%) patients evaluated, an HLA conferring a susceptibility to type 1 DM was detected; (h) in the majority of patients, the clinical onset of DM was acute and the disease persisted even after IFN therapy was withdrawn, but the diabetes did regress spontaneously in some cases [58, 59]. Other cases of patients developing autoimmune DM after IFN therapy have since been reported [63–67]. The considerable number of patients developing IFN-related DM has been recently pointed out in a literature review investigating the Japanese population from 1992 to 2009 [68]. Out of the 143 collected cases, 104 were type 1 DM and 39 were nonautoimmune type-2-like DM. Patients with IFN-related type 1 DM had a HLA type similar to Japanese type 1 diabetic patients, and a high positive rate of GAD antibodies [68] These data confirm our initial hypothesis, that IFNa triggers the onset of overt type 1 diabetes in patients with pancreatic autoantibodies, or modifies the natural history of the disease in genetically predisposed individuals [59]. Patients with chronic HCV hepatitis who are candidates for IFN therapy are therefore considered at risk of autoimmune DM. Accordingly, they should be screened for pancreatic autoimmunity and glycemia before and during IFN therapy in order to identify DM or the risk of it developing [58].

5.5

Autoimmune Gastritis

Autoimmune gastritis is a disease affecting the body and fundus of the stomach, with lymphocytic infiltration and autoantibodies to parietal cells (PCA). Patients with chronic HCV infection test positive for PCA in 1.6–5% of cases, and 5% have high serum gastrin levels. After IFNa treatment, however, 13% of patients become PCA-positive and 16% develop hypergastrinemia [69]. In 22 patients, PCA and hypergastrinemia developed during IFN treatment; endoscopic investigation revealed chronic atrophic gastritis in 59%. Gastric autoimmunity correlated closely with the presence of thyroid autoimmunity (thyrogastric syndrome) [69]. Cases of pernicious anemia developing in patients with antibodies to intrinsic factor have also been described after IFNa therapy [70].

5.6

Celiac Disease

Autoantibodies against tissue transglutaminase or endomysium, which are recognized as serological markers of celiac disease, have been found in 1.3–2.0% of patients with HCV-positive chronic hepatitis, compared with 0.16–0.4% of controls [71, 72]. During IFNa treatment, six of seven patients (86%) with transglutaminase autoantibodies developed moderate-severe symptoms of celiac disease, which improved after therapy was withdrawn [71]. Based on these findings, transglutaminase autoantibodies should be assayed if gastrointestinal disorders occur during IFNa therapy.

5.7

Mechanisms of Interferon-Related Damage to Thyrocytes and Pancreatic Beta Cells

Although it remains to be determined exactly how IFN triggers or exacerbate autoimmune diseases such as CT, GD, autoimmune DM, autoimmune gastritis, or celiac disease, according to experimental data summarized by Mandac et al. [45], IFNa can: (a) increase the expression of class I HLA antigens and induce the expression of class II HLA on thyrocytes; this aberrant expression is then associated with cytotoxic T lymphocyte activation by a Th1-oriented response; (b) stimulate, in vitro, thyrocyte transcription of different genes encoding cytokines and adhesion molecules and significantly increase the expression of ICAM-1 and B7 molecules; this increased expression of adhesion molecules favors the presentation of thyroid autoantigens; (c) increase the activity of lymphocytes, macrophages, natural killer cells, neutrophils, and monocytes; (d) increase the production of cytokines such as IL-6 (IL-6 receptors on thyrocytes reduce TSH-induced iodine uptake and the release of thyroid hormones); and (e) reduce T regulatory lymphocyte function. A possible pathogenic mechanism behind autoimmune thyroiditis is therefore that IFNa polarizes the immune reaction towards a Th1 response, which in turn induces the release of IFNg and IL-2, both of which are potent proinflammatory cytokines [45]. IFNa may also be implicated in the onset or acceleration of autoimmune processes against pancreatic beta cells, thus triggering the onset of type 1 DM. Experimental data summarized by Devendra et al. [59]

5

Organ-Specific Autoimmunity in HCV-Positive Patients

indicate that: (a) high levels of serum IFNa are detectable in the majority of patients at the onset of type 1 diabetes; (b) the beta cells involved in insulitis produce IFNa; (c) high levels of IFNa are detectable in enterovirus-associated type 1 diabetes; (d) Coxsackie B4 virus stimulates IFNa production by beta cells in vitro; (e) transgenic mice carrying beta cells that produce IFNa develop autoimmune diabetes with insulitis, and IFNa inhibition with monoclonal antibodies protects these animals against the occurrence of diabetes; (f) IFNa expression is associated with the increased expression of HLA class I molecules on pancreatic beta cells.

5.8

Non-Organ-Specific Autoimmune Diseases or Systemic Autoimmune Diseases

Although nearly 40% of unselected patients with HCV have at least one extrahepatic manifestation, the prevalence of patients fulfilling the criteria for nonorgan-specific autoimmune diseases (NOSAD) is much lower (2–6%). In the HISPAMEC Registry (HispanoAmerican Study Group of Autoimmune Manifestations Associated with Hepatitis C Virus), the most commonly associated NOSAD was Sjögren’s syndrome, which accounted for 47.5% of all NOSADs, followed by rheumatoid arthritis (14.7%), systemic lupus erythematosus (12.6%), polyarteritis nodosa (7.6%), antiphospholipid syndrome (5.8%), and inflammatory myopathies (3.8%) [73]. The strong association between HCV infection and Sjögren’s syndrome is probably related to the sialotropism of HCV [74]. This syndrome is the NOSAD with the highest prevalence of chronic HCV infection, which was found in 151 (18%) of 858 of these patients who were tested for the virus [73]. Antinuclear antibodies, in addition to rheumatoid factor, anticardiolipin antibodies, and smooth muscle, liver/kidney, and microsomal antibodies, are detectable in 40–65% of patients with HCV infection [38, 75, 76]. The characteristic lymphotropism of HCV is probably behind the increased production of autoantibodies. The prevalence and titer of these autoantibodies remain substantially unchanged after IFNa therapy [71]. In a cohort of 963 treatment-naïve HCV patients tested for anti-nuclear and anti-smooth muscle antibodies, 172 (17.9%) had at least one autoantibody: anti-smooth muscle in 104 patients (10.8%), anti-nuclear in 54 (5.6%), and both in 14 (1.5%).

49

5.9

Conclusions

Hepatitis C virus infection appears to be primarily, and in most patients almost exclusively, confined to the liver, but there is a wide variety of extrahepatic manifestations that seem to be associated with the infection, especially autoimmune disorders. A common hypothesis is that non-hepatic diseases are caused by the widespread tropisms of HCV, and particularly lymphotropism, which may explain the production of autoantibodies and/or the activation of self-reactive lymphocytes. In genetically susceptible individuals at least, this may give rise to clinically overt autoimmune disorders. The link between chronic HCV infection and NOSADs has been clearly documented, whereas the links between chronic HCV infection and AITD or type 1 diabetes mellitus are less clear, but IFN treatment may have a crucial role in triggering latent autoimmune reactions in these patients and in inducing the development of the related clinical diseases.

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51 63. Cozzolongo R, Betterle C, Fabris P et al (2006) Onset of type 1 diabetes mellitus during peginterferon alpha-2b plus ribavirin treatment for chronic hepatitis C. Eur J Gastroenterol Hepatol 18:689–692 64. Soultati A, Dourakis S, Alexopoulou A et al (2007) Simultaneous development of diabetic ketoacidosis and hashitoxicosis in a patient treated with pegylated interferon-alpha for chronic hepatitis C. World J Gastroenterol 13:1292–1294 65. Tanaka J, Sugimoto K, Shiraki K et al (2008) Type 1 diabetes mellitus provoked by peginterferon a-2b plus ribavirin treatment for chronic hepatitis C. Intern Med 47:747–749 66. Ogihara T, Katagiri H, Yamada T et al (2009) Peginterferon (PEG-IFN) plus ribavirin combination therapy, but neither interferon nor PGE-IFN alone, induced type 1 diabetes in a patient with chronic hepatitis C. Intern Med 48: 1387–1390 67. Yamazaki M, Sato A, Takeda T, Komatsu M (2010) Distinct clinical courses in type 1 diabetes mellitus induced by peginterferon-alpha treatment for chronic hepatitis C. Intern Med 49:403–407 68. Muraishi K, Sasaki Y, Kato T et al (2011) Classification and characterization of interferon-related diabetes mellitus in Japan. Hepatol Res 41:184–188 69. Fabbri C, Jaboli F, Giovanelli S et al (2003) Gastric autoimmune disorders in patients with chronic hepatitis C before, during and after interferon-alpha therapy. World J Gastroenterol 9:1487–1490 70. Andrès E, Loukili NH, Ben Abdelghani M, Noel E (2004) Pernicious anemia associated with interferon-alpha therapy and chronic hepatitis C infection. Clin Gastroenterol 38: 382–383 71. Durante-Mangoni E, Iardino P, Resse M et al (2004) Silent celiac disease in chronic hepatitis C – impact of interferon treatment on the disease onset and clinical outcome. J Clin Gastroenterol 38:901–905 72. Ruggeri C, La Masa AT, Rudi S et al (2008) Celiac disease and non-organ-specific autoantibodies in patients with chronic hepatitis C virus infection. Dig Dis Sci 53:2151–2155 73. Ramos-Casals M, Muñoz S, Medina F et al (2009) HISPAMEC Study Group. Systemic autoimmune diseases in patients with hepatitis C virus infection: characterization of 1020 cases (The HISPAMEC Registry). J Rheumatol 36: 1442–1448 74. Arrieta JJ, Rodriguez-Inigo E, Ortiz-Movilla N et al (2001) In situ detection of hepatitis C virus RNA in salivary glands. Am J Pathol 158:259–264 75. Agnello V, De Rosa FG (2004) Extra-hepatic disease manifestations of HCV infection: some current issues. J Hepatol 40:341–352 76. Williams MJ, Lawson A, Neal KR, on behalf of the Trent HCV Group et al (2009) Autoantibodies in chronic hepatitis C virus infection and their association with disease profile. J Viral Hepat 16:325–333

Part II Cellular Compartments of HCV Infection (and Replication)

6

HCV and Blood Cells: How Can We Distinguish Infection from Association? Lynn B. Dustin and Charles M. Rice

6.1

Introduction

While the primary target of hepatitis C virus (HCV) infection is the liver, the frequent occurrence of extrahepatic manifestations such as mixed cryoglobulinemia (MC) has led investigators to examine the possibility that HCV replicates in other cells and tissues. A number of groups have reported association of HCV RNA with blood cells in patients with ongoing or even resolved HCV infection. These reports raise the possibility that HCV infection of peripheral blood mononuclear cells (PBMCs) affects their function, contributing to the pathogenesis of MC, B cell nonHodgkin lymphoma, or ineffective anti-HCV immune responses. It may be tempting to conclude that MC is a consequence of B cell infection by HCV. However, there are reasons to be skeptical [1, 2]. B cells, monocytes, and dendritic cells are all specialized to capture exogenous material for antigen presentation. Patients with chronic HCV infection, and certainly those with MC, have abundant viral material circulating in the form of immune complexes—a form likely to enhance capture by these cell types. Claims of HCV infection in lymphocytes should be subjected to the same rigorous scrutiny that has been applied to studies of HCV infection in hepatocytes. There is no reason a priori to assume that HCV replication proceeds by different pathways in lymphocytes than in liver cells. Thus, we expect that in any

L.B. Dustin (*) Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_6, © Springer-Verlag Italia 2012

susceptible cell HCV replication would involve a measurable increase in viral RNA over time, a measurable increase in HCV protein levels over time, and, importantly, a demonstration that the increased RNA and protein levels are sensitive to specific antiviral drugs. If these criteria are not met, then the study fails to distinguish HCV infection from simple association of HCV RNA and/or proteins. Studies lacking these features do not meet the criteria that have been adopted as proof of HCV infection in liver cells.

6.2

HCV Genome and Protein Products

The genome of HCV is a single, positive-sense RNA approximately 9,600 bases in length [3]. HCV RNA is highly structured, with stem-loop and pseudoknot structures that play essential roles in RNA translation and replication. An internal ribosomal entry site, near the 5¢ end of the genome, directs the ribosome to translate HCV RNA. When a susceptible cell is infected, HCV RNA is translated as a single polyprotein of about 3,000 amino acids. The polyprotein is cleaved during and after translation by host and virus-encoded proteases, yielding the structural proteins core, E1, E2, and p7, and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The viral particle is believed to include HCV RNA in complex with core protein, surrounded by a lipid bilayer decorated with the envelope glycoproteins E1 and E2. In vivo, much of the extracellular HCV RNA is associated with lipoprotein particles. The NS proteins are present in the infected cell, where they modulate cellular functions and contribute to viral replication and assembly. Cells cannot be said to be productively infected simply because they 55

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are associated with HCV RNA and proteins. Capture or association of viral material, including debris from infected cells, by B cells or other cell types could allow the detection of HCV proteins and RNA even if these cells are not sites of active viral replication.

6.3

ranged from a high of less than one HCV genome per 30 B cells to a low of one HCV genome per 100,000 PBMCs. An exception is the study in [12]. Since a cell cannot be infected by a fraction of an HCV genome, these results strongly suggest that the number of infected cells cannot be high.

Which Cell Types Are Infected? 6.5

The association of HCV RNA with B cells has been reported by a number of groups [4–13]. One group reported that a B lymphoma cell line produced infectious HCV in culture [14]. In some of these studies HCV RNA was quantified but many others have used semi-quantitative assays, or simply reported that the reaction was positive. Other groups have found that T cells, monocytes, neutrophils, or dendritic cells contain HCV RNA and/or proteins [5, 6, 13, 15–18]. Of note, a recent report demonstrated the presence of HCV RNA and a NS protein exclusively in blood cells expressing the phagocytic Fc receptor, FcgRIIIA or CD16 [19]. This observation is consistent with the possibility that viral material is taken up from the blood in the form of immune complexes, or from sites of infection by phagocytosis of dying infected cells.

6.4

How Much HCV RNA Is There in Lymphocytes?

Published reports and our own data indicate that the level of HCV RNA associated with PBMCs in patients’ blood samples is actually very small. In most studies that have provided quantitative measurements of HCV RNA, the number of HCV genomes has been far below one copy per cell. As summarized in Table 6.1, reported levels of HCV RNA in B cells and in PBMCs have

Is It Really Replicating?

While some groups have shown that HCV RNA increases in B cells following activation in vitro, these studies have not included rigorous quantitation. In some, the amount of HCV protein demonstrated by immunofluorescence seems not to correlate with the level of viral RNA, suggesting that staining artifacts or uptake of material from infected tissues contributed to the observed signal. Others, using quantitative methods, found that residual HCV RNA declines rapidly when patient B cells are cultured in vitro with or without mitogens [2, 20].

6.6

Challenges in the Detection and Quantitation of HCV RNA Replication

The genetic material of HCV is a single positive-sense RNA. In susceptible cells, HCV RNA is translated to produce replicase enzymes, which use genomic RNA as a template for the production of a replicative intermediate, i.e., negative sense or minus strand RNA. This strand is then used as a template for the synthesis of more positive sense genomes. Measurement of the minus strand is challenging. In infected liver-derived cell lines, the minus strand is present at approximately tenfold lower levels than the genomic plus strand RNA.

Table 6.1 Measurements of HCV RNA levels in B cells and PBMCsa PBMCs 1.5 × 10−4a (median) Not reported

B cells Notes 2.6 × 10−3 (median) < 2.5 × 10−4 to 4.9 × 10−3 (range) Replicative intermediate detected only in liver except for one case in dendritic cells and one case in plasma 1 × 10−4 to 0.01 (range) 1 × 10−5 to 1 × 10−3 (range) Calculations assume one mg RNA/106 cells 0.033 (mean) Replicative intermediate detected only in liver 2.5 × 10−3 (mean) cells “Sustained virologic responders” 1.7 × 10−4 to 5.6 × 10−4 (range) Not reported 2.2 × 10−2 (mean) Calculations assume one mg RNA/106 cells a

Copies per cell

Reference [4] [6]

[13] [20] [21] [22]

6

HCV and Blood Cells: How Can We Distinguish Infection from Association?

Given the excess levels of plus strand relative to minus strand RNA, it is quite possible that the minus strand exists largely as a duplex with the plus strand, or may form a duplex when infected cells are lysed. Therefore, such duplexes should be rigorously denatured—or data obtained to prove that this is not necessary—before the minus strand is isolated. In addition, the HCV genome is extensively structured [23–25]. Most of the 5¢ noncoding region typically targeted for RT-PCR amplification is involved in stem-loop structures [25] that may be prone to self-priming, and the 3¢ non-translated region forms a stem-loop structure that acts as a primer in vitro. Thus, it is advisable to target less structured regions of the genome. Artifacts such as false priming and mispriming can result in detection of the plus strand under conditions thought to allow detection of only the minus strand [26]. Finally, levels of HCV RNA associated with blood cells are very low, and therefore the number of PCR cycles needed to detect either RNA strand is very high. In real-time PCR, background signals increase with each PCR cycle. We need to critically consider the possibility that PCR signals may be influenced by contamination, background noise, or falsepositive reactions. Controls that should be shown in each experiment include reactions without reverse transcriptase, no-template reactions, and wrong-strand controls. A standard curve should also be provided to document assay sensitivity—and specificity—using the exact conditions being tested (that is, in the presence of B cell RNA).

6.7

Measurement of HCV Protein Expression

Productively infected cells should express both structural and NS proteins. In order to demonstrate HCV protein expression, investigators must document the specificity of all antibodies used. Some published studies have depended on immunostaining with a single monoclonal antibody (mAb) to support the hypothesis that HCV replication occurs in lymphoid cells. However, certain commercially available mAbs produce peculiar immunostaining patterns suggesting protein localization in cellular locations—such as the nucleus—that do not correspond to known sites of HCV replication. In cells actively engaged in HCV replication, NS proteins are concentrated in punctate cytoplasmic structures [27–30]. Studies using flow cytometry would be expected to demonstrate a distinct

57

subpopulation of NS protein-expressing cells, since the number of HCV RNA copies is less than one per cell and therefore not all cells are expected to be infected. Data indicating a fluorescence shift of the entire lymphocyte population are inconsistent with the presence of far less than one HCV genome per cell. Studies reporting de novo HCV infection of B cells or PBMCs should document quantitation of HCV NS proteins over time, demonstrating increased levels of these proteins during the course of the experiment. More than one mAb should be used to establish the presence of HCV NS proteins.

6.8

Do Antiviral Drugs Affect the Level of HCV RNA and Protein?

In infected hepatoma cells, HCV replication is sensitive to interferons (IFNs) and to a number of antiviral drugs. Type I IFNs block HCV infection in cell culture [31] and are the backbone of the only currently approved therapeutic regimen for HCV infection [32, 33]. B cells and other blood cells express the type I IFN receptor and respond to IFN stimulation; therefore, it seems reasonable to expect that HCV replication in these cells is equally sensitive to IFNs, but this remains to be tested. Several small molecules targeting specific HCV enzyme activities are now in development [33, 34]. These drugs have demonstrated effectiveness against HCV replication and infection in vitro and in human HCV patients. Initial studies of the HCVcc (cell culture derived HCV) system used sensitivity to potent, specific antiviral drugs active against HCV as one criterion for bona fide HCV replication [31]. The use of these drugs in cell culture can help investigators distinguish HCV RNA persistence from HCV RNA replication [2]. In hepatoma cells as well as primary hepatocytes infected with HCVcc, HCV RNA and protein levels increase during the course of infection. Drugs that target HCV’s RNA-dependent RNA polymerase or NS3-4A protease block such increases in HCV RNA and protein. We have tested the same antiviral drugs on HCV RNA and protein levels in B cells, T cells, monocytes, macrophages, dendritic cells, and unfractionated PBMCs cultured with HCVcc. These cells, unlike hepatoma cells or primary hepatocytes, showed no increase in HCV RNA or protein levels over time. Even though HCV RNA persisted in these cells for varying lengths of time, there was no evidence

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B cell

APO-E/B HCV HCV (LVP)

HSPG SR-BI

CD81

CLDN1

LDL-R

OCLN

Hepatocyte

Fig. 6.1 HCV entry factors and mechanisms. Infection of target cells by HCV or HCV lipoviral particles (LVP) is a multistep process [35, 36]. Viral particles are first captured by heparin sulfate proteoglycans (HSPG), scavenger receptors (SR-BI) and/or the low-density lipoprotein receptor (LDL-R). This capture step may permit HCV binding to CD81. It is hypothesized that CD81-bound virus is actively transferred to intercellular tight junctions [37]. HCV entry is dependent on the presence of the

tight junction proteins CLDN1 and OCLN. It is not clear whether these proteins play roles in endocytosis, fusion, or other steps in infection. HCV is taken up by clathrin-dependent endocytosis. After acidification of the endocytic vesicle, the envelope glycoproteins E1 and E2 mediate fusion of the viral envelope with the vesicle membrane. This step releases HCV RNA into the cytoplasm. B cells express CD81 and a splice variant of SR-BI, but do not express appreciable levels of CLDN1 or OCLN

for any increase in HCV RNA levels during culture. Furthermore, such persistence was completely unaffected by antiviral drugs [2].

(Fig. 6.1). Not all of these proteins are expressed by B cells or by other cells in the blood. To study HCV entry, researchers use systems including HCVcc and HCV pseudoparticles (HCVpp) [3]. HCVpp bear HCV envelope proteins but deliver a reporter gene rather than the HCV genome; thus, it is even possible to study HCV entry using cells that may not permit a complete infectious cycle. HCVpp bearing the envelope proteins E1 and E2 from a large variety of HCV isolates have been prepared and extensively studied; the entry requirements of different HCV

6.9

How Can HCV Enter Blood Cells?

Investigators reporting B cell infection must evaluate how the virus was able to enter these cells. A number of proteins are now known to play essential roles in HCV glycoprotein-mediated entry into liver cells

6

HCV and Blood Cells: How Can We Distinguish Infection from Association?

genotypes are known to be very similar. As for HCVpp, it is now possible to prepare HCVcc chimeric viruses using structural-protein sequences from a variety of different HCV genotypes [38]. HCVcc can recapitulate the entire HCV infectious cycle in hepatoma cells, primary human hepatocytes, mice bearing human hepatocyte grafts, and chimpanzees. Studies using these systems and others have shown that CD81 expression, while necessary, is not sufficient for HCV infection. Therefore, although CD81 is widely expressed by blood cells (and by nearly every cell type), it cannot by itself mediate their infection with HCV. Susceptible cells may capture viral particles by means of heparin sulfated proteoglycans, the scavenger receptor SR-BI, and/or the low-density lipoprotein (LDL) receptor (reviewed in [35, 36]). Such capture may facilitate E2 interaction with CD81. It is now evident that HCV entry to susceptible cells is dependent on the tight junction proteins claudin-1 (CLDN1) [39] and occludin (OCLN) [40] in addition to SR-BI and CD81. Expression of all four of these factors together (SR-BI, CD81, CLDN1, and OCLN) conferred on mouse cells the ability to permit HCVpp entry [40]. B cells express CD81 and a splice variant of SR-BI, but do not express appreciable levels of the other entry factors [2]; (Marukian and Dustin, unpublished data). Of note, a subpopulation of B cells expressing the lectin DC-SIGN may capture and internalize HCV, but rather than becoming infected these B cells may release the captured virus to susceptible liver cells [41]. While it has been reported that CLDN6 and CLDN9 may also support HCV entry [42], these are also not expressed at high levels in B cells [2]. We and others have been unable to demonstrate B cell infection by HCVcc or HCVpp that bear envelopes representing a variety of isolates [2, 43, 44]. A B cell line engineered to express high levels of several entry factors remained resistant to infection with HCVcc or HCVpp [2].

6.10

Do Blood Cells Provide the Necessary Cofactors for Productive HCV Infection?

In liver cells, emerging evidence shows that HCV makes use of cellular machinery during RNA translation, replication, and virus assembly. HCV replication is dramatically enhanced by the liver-specific

59

microRNA miR-122, which binds to a pair of seed sites in the 5¢ non-translated region of the HCV genome [45–47]. Indeed, HCV does not replicate well in cells deficient in microRNA processing pathways [46]. HCV-infected chimpanzees treated with a miR122 antagonist demonstrate a dramatic reduction in serum HCV levels [48]. Despite its abundance in liver cells, miR-122 levels are limiting even in susceptible liver cell lines [49]. Also, miR-122 expression has not been reported in B cells. Mounting evidence indicates that production of infectious HCV virions is intimately tied in with the assembly of low and very-low-density lipoproteins (LDL and VLDL), particularly the latter [50]. HCV viral particles in the blood are associated with LDL and VLDL, and are termed “lipoviral particles” or LVP [50–53]. HCV isolated from the liver has a similar low density, and is associated with apolipoproteins B and E [53]. LDL and VLDL particles are produced only in the liver [54]. HCV is replicated and assembled in subcellular compartments associated with lipid droplets [30] and lipoprotein assembly [55, 56]. Inhibition of VLDL assembly—whether by drugs inhibiting microsomal transfer protein [55, 57] or by siRNA targeting of apolipoprotein E [58, 59], apolipoprotein B [55], or enzymes involved in VLDL biosynthesis [60]—strongly blocks HCV virus production in cell culture. Lower-density, lipoprotein-associated HCV is significantly more infectious than higher-density HCV lacking associated lipoproteins [30, 61]. There is, as yet, no published evidence that HCV-infected blood cells can provide the lipoprotein biosynthetic pathway components apparently required and used by the virus in hepatocytes.

6.11

Is There Evidence for B Cell-Specific HCV Quasispecies?

While there are reports of differential association of HCV RNA sequences with different compartments, researchers have not yet identified any sequences that are consistently associated with B cells as opposed to other cell types [8, 62]. B cell-associated HCV sequences differ from one report to another. Furthermore, it may be difficult to accurately characterize HCV quasispecies uniquely associated with B cells because the amounts of viral RNA that can be recovered from patient B cells are typically very low—and include peripherally associated

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viral RNA from the serum. HCV RNA diversity is analyzed, by necessity, by reverse transcription (RT) and PCR amplification followed by cloning and sequencing; less than perfect efficiency at the beginning of this process will result in the loss of some templates and therefore of some sequence variants. Low template copy numbers in the material under study, i.e. B cells, thus present a risk of under-sampling artifacts leading to incorrect conclusions of limited HCV sequence diversity in this compartment. At this time, the field does not enforce a standard for demonstrating that adequate sampling has taken place. Sequencing multiple clones from a single RT-PCR experiment does not address the undersampling problem. To reduce the likelihood of an undersampling artifact, it is important to analyze more than one RNA sample, verify the integrity of the RNA, and perform the entire RT-PCR amplification process more than once for each sample.

6.12

Summary

It is widely accepted that HCV RNA and even some viral proteins can be associated with B cells in HCV patients with and without MC. The level of HCV RNA, when measurements are reported, is very low. The frequency of infected cells, if any, must be correspondingly low. It is not yet clear how the possible infection of a small subset of B cells could lead to the functional changes associated with MC. Scientists and clinicians studying the potential role of B cell infection by HCV in the pathogenesis of MC would do well to apply state-of-the-art virological tools to the study of this important area. Quantitative assays must be used to document changes in HCV RNA and protein over time. Controls for the specificity of both RNA and protein measurements must be presented. In order to distinguish replicating virus from viral material that is adsorbed to the cell or is taken up by phagocytosis or endocytosis, it will be useful to determine the effects of specific antiviral drugs on the levels and rate of change of viral RNA and protein. If these assays are carried out and strong evidence is found to support the conclusion that B cells are sites of productive HCV infection, many new questions arise. We must understand how the virus is able to enter cells, given that B cells do not express the full complement of known HCV entry factors. We must also understand how HCV replicates and produces infectious viral particles in the

setting of a lymphoid cell, which lacks many of the features required for efficient HCV replication in liver cells. These studies will be important in order to understand how HCV infection of blood cells could possibly contribute to MC and the other immunological abnormalities seen in HCV patients.

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33. Zeuzem S (2008) Interferon-based therapy for chronic hepatitis C: current and future perspectives. Nat Clin Pract Gastroenterol Hepatol 5:610–622 34. Manns MP, Foster GR, Rockstroh JK et al (2007) The way forward in HCV treatment – finding the right path. Nat Rev Drug Discov 6:991–1000 35. Burlone ME, Budkowska A (2009) Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen Virol 90:1055–1070 36. Bartosch B, Cosset FL (2006) Cell entry of hepatitis C virus. Virology 348:1–12 37. Brazzoli M, Bianchi A, Filippini S et al (2008) CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes. J Virol 82:8316–8329 38. Gottwein JM, Scheel TK, Jensen TB et al (2009) Development and characterization of hepatitis C virus genotype 1–7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 49: 364–377 39. Evans MJ, von Hahn T, Tscherne DM et al (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446:801–805 40. Ploss A, Evans MJ, Gaysinskaya VA et al (2009) Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457:882–886 41. Stamataki Z, Shannon-Lowe C, Shaw J et al (2009) Hepatitis C virus association with peripheral blood B lymphocytes potentiates viral infection of liver-derived hepatoma cells. Blood 113: 585–593 42. Zheng A, Yuan F, Li Y et al (2007) Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus. J Virol 81:12465–12471 43. McKeating JA, Zhang LQ, Logvinoff C et al (2004) Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81 dependent manner. J Virol 78:8496–8505 44. Bartosch B, Dubuisson J, Cosset F-L (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633–642 45. Jopling CL, Yi M, Lancaster AM et al (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309:1577–1581 46. Randall G, Panis M, Cooper JD et al (2007) Cellular cofactors affecting hepatitis C virus infection and replication. Proc Natl Acad Sci USA 104:12884–12889 47. Chang J, Guo JT, Jiang D et al (2008) Liver-specific microRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells. J Virol 82:8215–8223 48. Lanford RE, Hildebrandt-Eriksen ES, Petri A et al (2010) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327(5962): 198–201, Epub 2009 Dec 3 49. Jopling CL, Norman KL, Sarnow P (2006) Positive and negative modulation of viral and cellular mRNAs by liverspecific microRNA miR-122. Cold Spring Harb Symp Quant Biol 71:369–376 50. Syed G, Amako Y, Siddiqui A (2010) Hepatitis C virus hijacks host lipid metabolism. Trends Endocrinol Metab 21(1):33–40, Epub 2009 Oct 23 51. Andre P, Komurian-Pradel F, Deforges S et al (2002) Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 76:6919–6928

62 52. Nielsen SU, Bassendine MF, Burt AD et al (2006) Association between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients. J Virol 80:2418–2428 53. Nielsen SU, Bassendine MF, Martin C et al (2008) Characterization of hepatitis C RNA-containing particles from human liver by density and size. J Gen Virol 89:2507–2517 54. Gibbons GF, Wiggins D, Brown AM et al (2004) Synthesis and function of hepatic very-low-density lipoprotein. Biochem Soc Trans 32:59–64 55. Huang H, Sun F, Owen DM et al (2007) Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins. Proc Natl Acad Sci USA 104:5848–5853 56. Benga WJ, Krieger SE, Dimitrova M et al (2010) Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles. Hepatology 51:43–53 57. Gastaminza P, Cheng G, Wieland S et al (2008) Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. J Virol 82:2120–2129

L.B. Dustin and C.M. Rice 58. Chang KS, Jiang J, Cai Z et al (2007) Human apolipoprotein E is required for infectivity and production of hepatitis C virus in cell culture. J Virol 81:13783–13793 59. Jiang J, Luo G (2009) Apolipoprotein E but not B is required for the formation of infectious hepatitis C virus particles. J Virol 83:12680–12691 60. Yao H, Ye J (2008) Long chain acyl-CoA synthetase 3-mediated phosphatidylcholine synthesis is required for assembly of very low density lipoproteins in human hepatoma Huh7 cells. J Biol Chem 283:849–854 61. Lindenbach BD, Meuleman P, Ploss A et al (2006) Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci USA 103: 3805–3809 62. Zehender G, De Maddalena C, Bernini F et al (2005) Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome. J Virol 79:9145–9156

7

Mechanisms of Cell Entry of Hepatitis C Virus Franco Dammacco and Vito Racanelli

7.1

Introduction

Hepatitis C virus (HCV) is a positive-strand RNA belonging to the Flaviviridae family. Its identification required long and painstaking research, involving the use of a number of molecular and serological methods [1], which finally led to the recognition of this third (in addition to HAV and HBV) etiological agent of infectious hepatitis. The existence of HCV had long been suspected and it was even provisionally named as hepatitis nonA-nonB virus, which emphasized its nosographic differentiation from the other two viruses [2]. Final confirmation of the structural uniqueness of HCV came in 1989, with the determination of its complete amino acid sequence [3]. In spite of the early and often remarkable production of neutralizing antibodies in the acute phase of HCV infection, the large majority of patients are unable to clear the virus and become chronically infected. One of the most intriguing and fascinating aspects of the molecular biology of HCV is the complex and so far largely unexplained strategy it has developed to escape host immune response and gain entry into target cells. Indeed, the HCV internalization pathway may be considered the forefront of viral-entry research. Deeper insight into the precise mechanisms whereby HCV evades (avoids) antibody-mediated neutralization and spreads from cell to cell would not only contribute to the comprehension of infection chronicity, but would also provide invaluable help in F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_7, © Springer-Verlag Italia 2012

devising novel therapeutic compounds targeting entry. Here, we summarize the main advances that have been accomplished in this field in the last decade.

7.2

HCV: The Hepatic Environment and Model Systems of Cell Entry

The molecular features of HCV have been described in several papers, including recent reviews [4, 5]. It is an enveloped virus that bears two surface glycoproteins, E1 and E2, and shows a primary tropism for human liver cells, with a less obvious and still controversial lymphotropism. A peculiar feature of hepatocytes is their polarized structure, in that basolateral and apical poles, through tight-junction separation, face the blood and the bile, respectively. Circulating HCV, linked to lipoproteins, wanders in the liver microenvironment and, when it comes into contact with the basolateral membranes of liver cells, interacts via entry molecules. In 1998, it was shown that protein E2 binds human CD81, a tetraspanin expressed on hepatocytes, B lymphocytes, and other cell types [6]. More precisely, the ability to bind E2 was ascribed to the major extracellular loop of CD81. It soon became evident that HCV cell entry is a much more complex and multi-step process, whose clarification has been largely hampered by the lack of suitable animal models and the limitations of in vitro systems based on the reaction of HCV particles with cell cultures. An important contribution to the advancement of knowledge in this field came from the establishment of new methods in molecular virology and, in terms of HCV research, a replicon system [7] with applications to basic as well as clinical research [8]. 63

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Several model systems, discussed in an excellent review [9], have been employed for the study of HCV cell entry. They include: (a) plasma-derived HCV, an older procedure [10] largely abandoned because of its many limitations [11]; (b) recombinant E2 glycoprotein, which allowed identification of the already-mentioned tetraspanin CD81 and of the human scavenger receptor class B (SR-BI) [12]; (c) HCV-like particles (HCV-LPs [13]), whose main limitation is their retention in intracellular vesicles rather than their secretion; (d) HCV pseudoparticles (HCVpp [14]), namely, the production of lentiviral particles able to incorporate HCV glycoproteins into the lipid envelope, although such pseudoparticles are not associated with lipoproteins; (e) cell-culture-produced HCV (HCVcc [15, 16]), an in vitro model that has marked a true milestone as it allows the reproduction of both early and late steps of the viral replication cycle and supports the production of virus particles with infectious properties both in vitro and in vivo; (f) a recently described smallanimal model, namely, the tree shrew or tupaia (Tupaia belangeri), which resembles a squirrel in its external appearance and habits. Primary tupaia hepatocytes have been shown to support the complete HCV infection cycle and are endowed with all of the receptors or co-receptors required for HCV entry; thus, it promises to become an important animal model in HCV-related research [17].

7.3

Receptors and Co-receptors for HCV Cell Entry

Following the identification of tetraspanin CD81 [6], several additional cell-surface molecules were demonstrated to play an important role as virus receptors on host cells. We briefly describe their properties, but refer the reader to specific reviews [9, 11, 18, 19] for a more detailed analysis of this probably still incomplete set of entry molecules.

7.3.1

Tetraspanin CD81

As a non-glycosylated protein, this tetraspanin is expressed on many cell types, acts as an HCV receptor molecule, and binds the virus surface glycoprotein E2 [6]. Its role is strongly suggested by studies showing

that anti-CD81 monoclonal antibodies are able to inhibit the infectivity of HCVpp and HCVcc in vitro [14, 16]. Growing evidence indicates that CD81 is a post-binding entry molecule, with the absence in liver cells of a natural inhibitor of CD81, EWI-2wint, probably accounting for the selective hepatotropism of HCV [20]. Of interest, CD81 also seems capable of modulating the adaptive immune response through its interaction with HCV on T and B lymphocytes. If a polyclonal B cell activation ensues, extrahepatic manifestations (including mixed cryoglobulinemia) of chronic HCV infection may occur [21].

7.3.2

Scavenger Receptor Class B (SR-BI)

Like tetraspanin CD81, SR-BI is a glycoprotein that also acts as a post-binding receptor. It is largely expressed by hepatocytes but to a lesser extent also by many other mammalian cells [22]. At least two mRNA splice variants are produced by the SR-BI gene, SR-BI and SR-BII, which differ in their carboxy-terminal ends [23]. SR-BI binds to high-, low- and very-lowdensity lipoproteins, but chemically modified lipoproteins can also be bound. There seems to be a close cooperation between SR-BI and CD81 in the process of HCV cell entry [22] in that SR-BI acts as a major cholesterol provider and controls the organization of CD81 at the level of plasma membrane. SR-BI is also able to bind serum amyloid A (SAA) protein, an acutephase reactant induced by a number of viral and bacterial infections [24]. SAA reduces HCV infectivity in cultured cells when added during HCV infection, but not after viral entry. Finally, interferon-a seems to exert its antiviral effects through a cell surface decrease in the expression of SR-BI, resulting in reduced HCV attachment and entry into liver cells [25].

7.3.3

Tight-Junction Proteins

These include four different components, but only claudins and occludins will be mentioned here as they play a crucial role in the cell-to-cell adhesion mechanism and in the separation of the apical from the basolateral membrane. Claudin-1 is expressed by all epithelial tissues and especially liver cells. Although it is not known whether

7

Mechanisms of Cell Entry of Hepatitis C Virus

claudin-1 directly interacts with HCV, it is probably involved in a later step of infection, after the virus binds to CD81 and SR-BI [26]. It has been shown that HCV infection of Huh-7 hepatoma cells down-regulates the expression of claudin-1 (and occludin as well), thus preventing superinfection [27]. Claudin-6 and claudin-9, which are additional members of the claudin family, are also involved in HCV entry but their roles in this process are still poorly defined. Occludin is a trans-membrane member of the tightjunction complex and is structurally related to the claudins. It is equally essential in HCV cell entry and in the activation of a productive HCV infection. Experimental data indicate that occludin, by direct interaction with glycoprotein E2, is capable of enhancing viral entry through the tight junctions of liver cells. In addition, based on the observed down-regulation of claudin-1 and occludin expression following HCV infection, this reduction of tight-junction proteins not only prevents, as already stated, superinfection of infected cells by HCVpp, but also affects the polarity of liver cells [27].

7.3.4

Glycosaminoglycans

These polysaccharides are largely expressed on the cell membrane and are able to bind many types of viruses, including HCV. They act as initial, low-affinity receptors in the early step of viral attachment, well before the virus is bound to higher-affinity receptors. Among glycosaminoglycans, the highly sulfated heparan sulfate seems to be particularly active. Lipoprotein lipase possibly mediates HCV cell entry by a mechanism resembling hepatic clearance of triglyceride-rich lipoproteins from the blood, thus favoring non-productive viral uptake [28].

7.3.5

Lectins

Two membrane proteins belonging to the C-type lectin family, DC-SIGN (dendritic-cell-specific intercellular adhesion molecule-3 grabbing non-integrin) and its homolog L-SIGN, have been found to take part in the binding, internalization, and clearance of several viruses, including HCV. Both are able to bind glycoprotein sE2 as well as natural HCV. DC-SIGN has

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been demonstrated on Kupffer cells, dendritic cells, and B cells, whereas L-SIGN is largely expressed on endothelial cells of liver sinusoids. Thus, both lectins likely act as capture receptors that, by binding and transmitting the virus to permissive cells, effectively start the infectious process [29].

7.4

Steps of Cell Entry

Through a multi-step, intricate, and finely regulated interplay between viral receptors and lipoprotein components, the virus is able to escape host immune responses [30]. Despite the large body of evidence on the mechanism(s) of cell entry, elucidated following advances in molecular virology, such as HCVpp and HCVcc [14–16], the precise sequence of events, the role played by each receptor or co-receptor, the cellular signaling network, and the overall coordination underlying “one virus/many receptors” still present pitfalls and knowledge gaps. It also seems reasonable to assume that the different circulating forms of HCV activate different pathways of cell entry such that infection proceeds by different modalities [9]. As shown in Fig. 7.1, largely inspired by models proposed by other authors [9, 11], the starting step of binding and internalization of ApoB-associated HCV requires the interplay of virus-associated VLDL and SR-BI. LDL receptors and glycosaminoglycans are possibly involved in this early phase, thus emphasizing the crucial role of lipoproteins, and in particular LDL receptors, in virus cell entry, as has been proposed since 1999 [31]. In the next step, the virus binds to the SR-BI/ CD81 complex, which results in its subsequent transfer to the tight-junction proteins claudin-1 and occludin. Following clathrin-mediated endocytosis, the virus enters the cell from the tight junction and undergoes a process of fusion that involves viral envelope glycoproteins and the endosomal membrane. In turn, this allows release of the viral RNA genome into the cytosol.

7.5

HCV Entry Inhibitors as Novel Therapeutic Agents

Precise recognition of the biomolecular mechanisms underlying cellular entry of HCV has obvious therapeutic implications, through the identification of

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novel therapeutic compounds targeting entry. At the moment, this approach includes inhibitors of glycosylation that target envelope glycans or cyanovirin-N, a lectin able to inhibit binding of the virus to cell-surface receptors [32]. The same therapeutic potential should be achievable using antibodies capable of downregulating the cell-surface expression of HCV receptors or co-receptors. Using a human liver-uPA-SCID mice system, a new chimeric mouse model permissive for HBV and HCV infection [33], it has been shown

a

that prophylactic treatment with anti-CD81 antibodies completely protected human liver-uPA-SCID mice from a subsequent challenge with HCV consensus strains of different genotypes, whereas the administration of anti-CD81 antibodies after viral challenge had no effect [34]. This observation suggests that it should be possible to prevent allograft reinfection after orthotopic liver transplantation in chronically infected HCV patients [35]. The same model is also being used in the development of new anti-viral compounds.

VLDL, LDL, ox LDL ↓ HDL ↑

LPL ↓↑ ?

SAA? ↓ ?

Claudin-1 SR-BI

Occludin

CD81

Clathrincoated pit

GAGs LDLR Early endosome H+

Fusion and genome release

Fig. 7.1 (a) A hypothetical model of cell entry of natural ApoB-associated HCV. The model implies that envelope glycoproteins directly interact with co-receptors. The SR-BI/CD81 complex binds the virus, which is then transferred to tight junctions where it interacts with claudin-1 and occludin. In the following step, HCV enters the cell by clathrin-dependent

endocytosis, followed by fusion of the viral envelope glycoproteins with the membrane of an early endosome. This results in the release of viral nucleocapsid into the cytoplasm. (b) Schematic representation of the localization of HCV entry molecules in the liver epithelium

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Mechanisms of Cell Entry of Hepatitis C Virus

b

CD81 DC-SIGN/L-SIGN

67

SR-BI

HCV

Claudin-1

Sinusoidal space

Blood (basolateral side)

Endothelium Space of Disse

Tight junction Hepatocytes Lumen of bile canaliculus (apical side)

Fig. 7.1 (continued)

References 1. Houghton M (2009) The long and winding road leading to the identification of the hepatitis C virus. J Hepatol 51:939–948 2. Alter MJ (1989) Non-A, non-B hepatitis: sorting through a diagnosis of exclusion. Ann Intern Med 110:583–585 3. Choo QL, Kuo G, Weiner AJ et al (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359–362 4. Fraser CS, Doudna J (2007) Structural and mechanistic insights into hepatitis C viral translation initiation. Nat Rev Microbiol 5:29–38 5. Poenisch M, Bartenschlager R (2010) New insights into structure and replication of the hepatitis C virus and clinical implications. Semin Liver Dis 30:333–347 6. Pileri P, Uematsu Y, Campagnoli S et al (1998) Binding of hepatitis C virus to CD81. Science 282:938–941 7. Lohmann V, Körner F, Koch J et al (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113 8. Bartenschlager R (2005) The hepatitis C virus replicon system: from basic research to clinical application. J Hepatol 43:210–216

9. Burlone ME, Budkowska A (2009) Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen Virol 90:1055–1070 10. Shimizu YK, Iwamoto A, Hijikata M et al (1992) Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line. Proc Natl Acad Sci USA 89:5477–5481 11. von Hahn T, Rice CM (2008) Hepatitis C virus entry. J Biol Chem 283:3689–3693 12. Scarselli E, Ansuini H, Cerino R et al (2002) The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 21:5017–5025 13. Barth H, Liang TJ, Baumert TF (2006) Hepatitis C virus entry: molecular biology and clinical implications. Hepatology 44:527–535 14. Bartosch B, Dubuisson J, Cosset FL (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633–642 15. Lindenbach BD, Evans MJ, Syder AJ et al (2005) Complete replication of hepatitis C virus in cell culture. Science 309: 623–626 16. Wakita T, Pietschmann T, Kato T et al (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796

68 17. Tong Y, Zhu Y, Xia X et al (2011) Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection. J Virol 85:2793–2802 18. Brass V, Moradpour D, Blum HE (2006) Molecular virology of hepatitis C virus (HCV): 2006 update. Int J Med Sci 3: 29–34 19. Zeisel MB, Fofana I, Fafi-Kremer S et al (2011) Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies. J Hepatol 54:566–576 20. Rocha-Perugini V, Montpellier C, Delgrange D et al (2008) The CD81 partner EWI-2wint inhibits hepatitis C virus entry. PLoS One 3:e1866 21. Machida K, Cheng KT, Pavio N et al (2005) Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells. J Virol 79:8079–8089 22. Zeisel MB, Koutsoudakis G, Schnober EK et al (2007) Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology 46:1722–1731 23. Grove J, Huby T, Stamataki Z et al (2007) Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity. J Virol 81:3162–3169 24. Cai Z, Cai L, Jiang J et al (2007) Human serum amyloid A protein inhibits hepatitis C virus entry into cells. J Virol 81:6128–6133 25. Murao K, Imachi H, Yu X et al (2008) Interferon alpha decreases expression of human scavenger receptor class BI, a possible HCV receptor in hepatocytes. Gut 57:664–667 26. Evans MJ, von Hahn T, Tscherne DM et al (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446:801–805

F. Dammacco and V. Racanelli 27. Liu S, Yang W, Shen L et al (2009) Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J Virol 83:2011–2014 28. Andréo U, Maillard P, Kalinina O et al (2007) Lipoprotein lipase mediates hepatitis C virus (HCV) cell entry and inhibits HCV infection. Cell Microbiol 9:2445–2456 29. Gardner JP, Durso RJ, Arrigale RR et al (2003) L-SIGN (CD 209 L) is a liver-specific capture receptor for hepatitis C virus. Proc Natl Acad Sci USA 100:4498–4503 30. Perrault M, Pécheur EI (2009) The hepatitis C virus and its hepatic environment: a toxic but finely tuned partnership. Biochem J 423:303–314 31. Agnello V, Abel G, Elfahal M et al (1999) Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci USA 96: 12766–12771 32. Steinmann E, Whitfield T, Kallis S et al (2007) Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus. Hepatology 46:330–338 33. Meuleman P, Leroux-Roels G (2008) The human liver-uPASCID mouse: a model for the evaluation of antiviral compounds against HBV and HCV. Antiviral Res 80:231–238 34. Meuleman P, Hesselgesser J, Paulson M et al (2008) Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo. Hepatology 48:1761–1768 35. Fafi-Kremer S, Fofana I, Soulier E et al (2010) Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation. J Exp Med 207:2019–2031

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HCV Infection of Hematopoietic and Immune Cell Subsets Tram N.Q. Pham and Tomasz I. Michalak

8.1

Introduction

Hepatitis C virus (HCV) is an important human pathogen that causes chronic hepatitis, with 170 million people infected with the virus world-wide. The 9600-base-pair genome of this single-stranded RNA virus encodes a number of structural (core, envelope) and non-structural (NS2–NS5) proteins that determine the morphology of the virus and are essential to its replication, respectively. Although hepatocytes are considered to be the primary targets of HCV, a large body of clinical and experimental evidence implies that this pathogen also invades and replicates in cells of other organs, particularly those of the immune system [1, 2]. It is conceivable that by setting up these extrahepatic sanctuaries of replication, HCV is able to establish persistence through evasion or modulation of host’s immune responses—a typical propensity of viruses capable of causing chronic infections [3]. In this respect, there are also studies that do not support the notion of HCV lymphotropism. The goal of this chapter is to provide an overview of our current understanding of the lymphoid cell compartment’s involvement in HCV infection in highly viremic patients with chronic hepatitis C (CHC) and in those with low-level, occult HCV infection (OCI). Regarding the latter, we summarize what is known to date on the occurrence of HCV in immune cell subsets of those individuals who either have or lack a past history of acute or chronic

T.I. Michalak (*) Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Center, Memorial University, St. John’s, NL, Canada e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_8, © Springer-Verlag Italia 2012

hepatitis C. Finally, the chapter reviews the data addressing the issue of HCV infection in cultured immune cells.

8.2

HCV Replication in Hematopoietic and Immune Cells of Patients with Acute or Chronic Hepatitis C

Over the past two decades, HCV lymphotropism has been the subject of numerous investigations. These works have reported the identification of HCV RNA positive and negative (replicative) strands as well as viral proteins in peripheral blood mononuclear cells (PBMCs) of patients with acute or chronic hepatitis C (AHC, CHC, respectively) [1, 4, 5]. Interestingly, the levels of the virus’ RNA replicative strand were shown to be significantly higher in the PBMCs of patients with CHC than in those of patients with AHC, suggesting that HCV infection of the immune system has a role in sustaining chronic infection [4, 5]. As alluded to earlier, by targeting the immune cells, the virus may likely acquire the ability to evade antiviral responses very early on, thereby establishing a stronghold within the infected host. Studies from different groups have addressed the question whether HCV displays a preferential tropism for specific PBMC subsets or infects different subtypes of circulating immune cells. Under certain conditions, HCV has been shown to compartmentalize much more frequently, if not exclusively, in unique cell subsets, such as CD19+ B cells [6–8]. However, in other studies, the detection of HCV RNA positive and negative strands was more widespread among the various immune cells, including CD4+ and CD8+ T cells, monocytes, dendritic cells and B cells; 69

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although different cell subtypes might be infected to various degrees in different patients [9–12]. Clonal sequencing and single-stranded conformational polymorphism (SSCP) analyses of HCV variants residing in PBMCs revealed interesting features that argued against the possibility of either a carry-over from plasma-derived free HCV RNA or non-specific viral adsorption to the cell surface [10, 11, 13–16]. First, HCV variants from lymphoid cells were genetically related but highly distinct from those encountered in serum or liver. In certain cases, HCV quasi-species identifiable in lymphoid cells were detectable in serum but not in hepatic tissue, supporting their extrahepatic origin. Second, HCV quasi-species from one cellular compartment, for example CD8+ T cells, were statistically more genetically similar to one another than they were to variants found in other immune cell subsets, such as CD4+ T cells. In one relevant study, HCV RNA sequences in CD8+ T lymphocytes phylogenetically clustered with each other but not with sequences detected in CD4+ T cells or CD19+ B cells [11]. In terms of functionality, certain sequence polymorphisms within the internal ribosomal entry site (IRES) of the 5¢-untranslated region (5¢-UTR) of the HCV genome isolated from lymphoid cells were found to coincide with an IRES translational activity unlike that in other infected cell types which would support HCV replication in cells carrying those variations but not in cells that do not [17, 18]. Indeed, a very recent study showed that within liver-derived hepatoma Huh7 cells, HCV IRES variants originating from plasma displayed a significantly higher translational activity than those from HCV residing in B cells [19]. Conversely, IRES variants of the virus replicating in B cells were found to have a similarly low translational efficiency in B cell lines, such as Raji and Daudi, and in hepatoma Huh7 cells, suggesting not only their extrahepatic origin but also an overall low capacity of HCV replication in B cells [19]. The concept of HCV lymphotropism is consistent with the meaningfully greater occurrence of lymphoproliferative disorders, such as non-Hodgkin’s lymphoma (NHL) [20, 21] and type II mixed cryoglobulinemia [22], in patients with CHC, as discussed elsewhere in this book. Suffice it to mention that in patients with markers of NHL, HCV RNA has been found at significantly higher frequencies in B-cell NHL than in non-B-cell NHL [23, 24]. Along this line, HCV infection of B cells also appears to be predominant in patients with lymphoproliferative disorders

T.N.Q. Pham and T.I. Michalak

compared to those without [2, 9]. Furthermore, there is evidence implying that the progression of these diseases is associated with high HCV viremia and, conversely, that their remission is correlated with a reduction in viral load following antiviral treatment [2, 25]. Taken together, these findings point towards an etiologic role not only for HCV in general, but also for immune cell infection in particular in the pathogenesis of these disorders. It should be mentioned that in patients with CHC, the identification of replicating HCV in lymphoid cells is not limited to only those cells in the circulation. In fact, HCV genomes and/or proteins have also been found in lymph nodes (LN), bone marrow (BM), and even the brain [8, 26–28]. In LNs, replicating HCV genomes as well as HCV core and NS3 proteins have been identified within B-cell-rich lymphoid follicles of biopsy specimens from patients with end-stage CHC [8]. Remarkably, not only were B cells the primary site of HCV infection in this secondary lymphoid tissue, but clonal sequencing analyses also indirectly revealed that, in some patients, HCV residing in LN-derived B cells contributed up to 40% of the total level of viremia [8]. Similarly, HCV RNA sequences found in the cerebrospinal fluid of patients co-infected with human immunodeficiency virus (HIV) were shown to be more similar to those in PBMCs and LNs than to those in plasma, suggesting that cells of the monocyte/macrophage lineage carry HCV into the brain and that resident microglial cells independently maintain viral replication [26, 27]. Lastly, HCV RNA positive and negative strands, as well as HCV structural and nonstructural proteins, were readily detectable in CD34+ hematopoietic progenitor cells in the BM of patients with CHC [28], lending further support to the concept of extrahepatic HCV replication. Although in the same study there was no evidence that primary CD34+ cells from healthy individuals supported de novo HCV infection, CD34+ cells from CHC patients were shown to release HCV RNA into culture supernatants, linking the development of CD34+ cells to their susceptibility to HCV infection. In an elegant study reported more than 10 years ago by the Brechot group [29], PBMCs from patients with CHC were injected intraperitoneally into severe combined immunodeficiency mice (SCID), with the animals subsequently monitored for HCV RNA persistence in both serum and PBMC for several months. Intriguingly, the authors documented the presence of HCV RNA in both compartments and the identification

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of replicating HCV in circulating PBMCs. Furthermore, this was the case only in animals inoculated with HCV RNA-positive PBMCs and not in those injected with HCV RNA-positive sera, sheep PBMCs preincubated with HCV, or human fibroblasts preincubated with HCV [29].

further support for HCV lymphotropism as well as its existence independent of whether infection is symptomatic or silent. Furthermore, the cytokine expression profiles and HCV-specific T cell responses of circulating immune cells were investigated in individuals with clinical SVR and compared to those of patients with CHC or healthy controls. PBMCs from individuals with SVR: (1) exhibited a cytokine expression pattern clearly distinct from that of healthy individuals or patients with CHC; (2) mounted a stronger virus-specific proliferative response; and (3) produced a greater abundance of antiviral cytokines following exposure to recombinant HCV antigens than was the case with PBMCs from patients with CHC [40–43]. Of note, these responses were documented long after the achievement of SVR and coincided, in many cases, with HCV detection in PBMCs. This would imply that low-level persistent infection engaging lymphoid cells is unlikely to be an immunologically neutral event. In fact, OCI may even be beneficial to the host in keeping the virus under immunological control. Regarding whether HCV RNA detectable in secondary OCI reflects authentic, replication-competent virions or defective virus, it was documented that, at least in some affected individuals, the virus is capable of inducing de novo productive infection in primary T cells derived from healthy donors [44]. Specifically, primary T lymphocytes exposed to the virus were able to generate HCV RNA negative strands, produce viral NS5A protein, and release HCV virion particles into the culture supernatant, thus recapitulating the entire viral life cycle. As mentioned, other studies reported the failure to detect virus persisting as OCI after clinical resolution of hepatitis C [35–38]. The reasons behind these very different results and whether they can be reconciled have been addressed elsewhere [39]. However, in the following, we provide a succinct look at the most important arguments and offer possible explanations for the discrepancies.

8.3

HCV Persistence in Lymphoid Cells of Individuals with Clinically Resolved Hepatitis C

It was previously thought that resolution of hepatitis C, as evidenced by normalization of liver function tests and disappearance of serum HCV RNA identifiable by clinical laboratory assays for at least 6 months upon completion of antiviral therapy, was a reflection of a complete eradication of HCV. This state of clinically apparent HCV clearance was referred to as a sustained virological response (SVR). However, as early as 2004, several groups independently reported the detection of residual HCV infection, namely, occult HCV infection, in plasma, PBMCs, and/or liver tissue of individuals with clinical resolved hepatitis C. These results were obtained by testing the samples using highly sensitive nucleic acid amplification assays, either alone or combined with hybridization of the amplified signals with HCV-specific probes [30–33]. We refer to this form of HCV infection as secondary OCI [34]. Meanwhile, there were also studies that refuted the existence of this form of HCV infection [35–38]. To this end, although OCI remains relatively controversial, there are factors that may reconcile these discordant findings [39]. The most important elements are briefly summarized below. It is worthwhile noting that in studies documenting the existence of secondary OCI, viral loads reported were generally very low, ranging between 100 and 200 virus genome equivalents (vge)/mL plasma, and between 10 and 100 vge/mg total RNA in PBMCs and hepatic tissue [10, 31]. As in the case of CHC, different immune cell subsets were found to be infected with HCV and in certain situations the major, if not exclusive reservoir of replicating HCV resided within a particular immune cell subtype [2, 10]. Similar to investigations in patients with CHC, the use of clonal sequencing and highly sensitive SSCP enabled the identification of HCV variants unique to immune cells within the 5¢-UTR IRES or the hypervariable region of E2 envelope glycoprotein [10, 13, 15]. This provided

8.4

Occult HCV Infection in Immune Cells of Patients with No History of Hepatitis C

In parallel with the identification of secondary OCI, which normally occurs against a background of detectable antibodies against HCV (anti-HCV) and normal liver function tests, OCI has also been described in

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anti-HCV non-reactive patients with persistently elevated liver enzymes [45–47]. In contrast to the usual situation, the etiology of the anti-HCV non-reactive OCI is not clear; thus, we refer to this form as cryptogenic OCI [34, 39]. In such patients, Schmidt et al. [46] reported that HCV RNA was repeatedly detectable in the whole blood of up to 67% of infected individuals versus in about 30% in whom HCV RNA was only intermittently identifiable when plasma was screened instead, implying a potentially significant contribution of hematopoietic cells to the overall viral load. Similarly, Carreno’s group [45] described the presence of HCV RNA in PBMCs of nearly 40% of individuals who were negative for both anti-HCV antibody and serum HCV RNA by standard clinical testing. Of note, nearly 60% of patients in the Carreno study [45] were also found to be reactive for replicating HCV genomes in liver tissues. Interestingly, in cryptogenic OCI as in secondary OCI discussed above, HCV-specific CD4+ and CD8+ T cell responses were detected. These were postulated to be inversely correlated with the extent of hepatic OCI infection [48]. However, the opposite scenario was also reported, in which patients with cryptogenic liver disease were not found to carry detectable HCV RNA in PBMCs; but whether the patients investigated were positive for HCV RNA in the liver was not addressed [49]. Again, explanations for the apparently contradictory data might be similar to those applicable to secondary OCI, as considered below.

8.5

Reconciling the Inconsistencies in the Detection of Occult HCV Infection

As discussed in the previously cited publication [39], the contradictory results regarding the existence of OCI reported by different groups may be due to variations in: (1) the collection and preparation of plasma and liver samples, and in particular PBMCs; (2) the method used for RNA extraction and the amount of material used in the experiments; (3) the number of time points at which the samples were analyzed (i.e., singular or serial samples); (4) the amount of template RNA ultimately tested for HCV genome expression; and (5) the sensitivity of the HCV RNA detection assay used. Furthermore, as mentioned above, there is ample evidence from independent groups that HCV is

compartmentalized to different degrees in different immune cell subsets [6–8, 10–12]. Consequently, a negative finding of HCV RNA in PBMCs does not always translate as the absence of HCV infection in individual lymphoid cell subsets. Also, it seems that ex vivo stimulation of immune cells with mitogens enhances viral replication, allowing for more sensitive virus detection [30, 50], as demonstrated even when a single PBMC sample was tested. This rate of positivity could increase up to 15% when serial PBMC samples are examined.

8.6

In Vitro Propagation of Wild-Type HCV Isolates in Lymphoid Cells

Support for the inherent propensity of HCV to infect and propagate in cells of the immune system also comes from studies in which lymphocytic cells from established human B cell lines (e.g., Rajii and Daudi) and T cell lines (e.g., Molt-4 and Jurkat) were exposed to HCV either in plasma or derived from hematopoeitic cells of CHC patients with type II mixed cryoglobulinemia [51, 52]. In addition, other groups have demonstrated the ability of primary lymphoid cells from healthy individuals, including total PBMCs [53], T cells [54], purified CD45RA+ CD45RO− CD4+ T cells [51], and monocytes/macrophages [55, 56], to support productive HCV infection. It should be stated that the infection of various immune cell subsets with HCV, despite a relatively low efficiency, could still lead to modulation of the host’s immune responses by: (1) negatively affecting interferon (IFN)-g gamma signaling [51]; (2) inhibiting T cell proliferation while enhancing CD95 (Fas)mediated apoptosis [52]; and (3) triggering the release of inflammatory cytokines [56]. For the most part, researchers have employed several experimental approaches in attempts to show that wild-type HCV is authentically replicated in their respective lymphoid cell cultures. These involve documentation of: (1) the presence of HCV RNA replicative (negative) strands in infected cells; (2) detection of viral proteins within the cytoplasm of de novo infected cells; (3) the susceptibility of virally infected cells to antiviral treatments, including, but not limited to, recombinant IFN-a; (4) the ability of cell-free supernatant from infected cell cultures to transmit infection to virus-naive cells, (5) viral particles released from

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infected cells possessing structural and physical properties of complete HCV virions; (6) visualization of HCV-like virions released by cultured immune cells via immunoelectron microscopy, and (7) evolution of HCV variants over the course of de novo infection. In contrast to the overall message conveyed by the above studies, that HCV can replicate in cells of the immune system, a recent work by Marukian et al. [57] reported that no immune cell subsets within PBMCs were capable of supporting de novo HCV infection. In their study, HCV particles generated by the transfection of a laboratory-adapted HCV chimera J6/JFH construct into hepatoma Huh7 cell line were used as inoculum. This, in essence, was a fundamental difference from all previous studies, in which clinical HCV isolates obtained directly from plasma or from the infected cells of patients with CHC or OCI were used as the source of virus. Since the ability of the JFH-1 strain to establish HCV replication in hepatoma Huh7 cells but not in lymphoid cell lines had been previously confirmed [58], the inability of the J6/JFH chimera to infect peripheral immune cells was not surprising. However, it is generally acknowledged that the levels of HCV are lower in immune cells than in hepatocytes and that HCV replication in the former is much less robust than in JFH-1/Huh7 cells and related systems. As such, the methods commonly used to detect and quantify HCV replication in hepatoma cells might be insufficiently sensitive to identify viral replication in lymphoid cells. With respect to the postulated apparent lack on lymphoid cells of receptors shown to be required for HCV entry into Huh7 cells [57], it cannot be ruled out that HCV uses entirely different receptors to gain access to cells of the immune system. In this context, measles virus (MeV), another single-stranded RNA virus, normally uses a molecule known as signaling lymphocyte activation molecule (SLAM) [59], whose expression is restricted to hematopoietic cells, to gain entry to its primary targets, i.e., lymphocytes and macrophages. However, very recent data revealed that MeV apparently uses CD147 as a functional entry receptor on other, less-recognized target cells, such as epithelial cells, which coincidentally do not express SLAM [60]. In this scenario, the flexibility of using another entry receptor allows viruses (such as MeV) to expand their cell target repertoire and therefore to induce a more readily persistent infection. With respect to HCV, CD5, a glycoprotein belonging to the scavenger receptor cysteine-rich family and specifically

expressed on T and B cells, appears to be essential for HCV entry into primary T cells or Molt4 and Jurkat T cell lines, all of which are susceptible to infection with wild-type virus (Sarhan et al., manuscript submitted) [61]. Importantly, hepatoma Huh7, Huh7.5, and HepG2 cell lines do not display CD5 (Sarhan et al., manuscript submitted).

8.7

Concluding Remarks

For nearly two decades, the issue of HCV lymphotropism has been investigated. The body of data accumulated through studies using clinical samples or cell culture infection models has demonstrated the propensity of HCV to target cells of the immune system. Nonetheless, acceptance of the notion that HCV invades sites beyond the liver is by no means unanimous. That HCV does not infect or replicate in immune cells to the same degree as it does in hepatocytes together with the essentially asymptomatic nature of immune-system infection in the majority of cases is probably one of the most important reasons behind the lack of agreement. In addition, because of the basic properties of HCV propagation in immune cells, the sensitivity of HCV RNA detection assays is an important factor in determining whether or not HCV lymphotropism is observed. Certainly, further molecular characterization of the mechanisms underlying immune cell susceptibility to HCV and governing viral replication in different immune cell subtypes, along with recognition of the immunological consequences of HCV infection of immune cells would be prudent in broadening our understanding of the role of the lymphatic system in the pathogenesis and natural progression of HCV infection. Moreover, it will be important to determine whether elimination of the virus within the lymphoid cell compartment contributes to the overall success of current or future antiviral therapies for hepatitis C, and if so, then how this occurs.

References 1. Blackard JT, Kemmer N, Sherman KE (2006) Extrahepatic replication of HCV: insights into clinical manifestations and biological consequences. Hepatology 44:15–22 2. Zignego AL, Giannini C, Monti M, Gragnani L (2007) Hepatitis C virus lymphotropism: lessons from a decade of studies. Dig Liver Dis 39(Suppl 1):S38–S45

74 3. Oldstone MB (1996) Virus-lymphoid cell interactions. Proc Natl Acad Sci USA 93:12756–12758 4. Chang TT, Young KC, Yang YJ et al (1996) Hepatitis C virus RNA in peripheral blood mononuclear cells: comparing acute and chronic hepatitis C virus infection. Hepatology 23:977–981 5. Gong GZ, Lai LY, Jiang YF et al (2003) HCV replication in PBMC and its influence on interferon therapy. World J Gastroenterol 9:291–294 6. Lerat H, Rumin S, Habersetzer F et al (1998) In vivo tropism of hepatitis C virus genomic sequences in hematopoietic cells: influence of viral load, viral genotype, and cell phenotype. Blood 91:3841–3849 7. Morsica G, Tambussi G, Sitia G et al (1999) Replication of hepatitis C virus in B lymphocytes (CD19+). Blood 94: 1138–1139 8. Pal S, Sullivan DG, Kim S et al (2006) Productive replication of hepatitis C virus in perihepatic lymph nodes in vivo: implications of HCV lymphotropism. Gastroenterology 130:1107–1116 9. Inokuchi M, Ito T, Uchikoshi M et al (2009) Infection of B cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C. J Med Virol 81:619–627 10. Pham TN, King D, MacParland SA et al (2008) Hepatitis C virus replicates in the same immune cell subsets in chronic hepatitis C and occult infection. Gastroenterology 134:812–822 11. Roque-Afonso AM, Ducoulombier D, Di Liberto G et al (2005) Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells. J Virol 79:6349–6357 12. Zignego AL, Macchia D, Monti M et al (1992) Infection of peripheral mononuclear blood cells by hepatitis C virus. J Hepatol 15:382–386 13. Di Liberto G, Roque-Afonso AM, Kara R et al (2006) Clinical and therapeutic implications of hepatitis C virus compartmentalization. Gastroenterology 131:76–84 14. Ducoulombier D, Roque-Afonso AM, Di Liberto G et al (2004) Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes. Hepatology 39:817–825 15. Navas S, Martin J, Quiroga JA, Castillo I et al (1998) Genetic diversity and tissue compartmentalization of the hepatitis C virus genome in blood mononuclear cells, liver, and serum from chronic hepatitis C patients. J Virol 72:1640–1646 16. Vera-Otarola J, Barria MI, Leon U et al (2009) Hepatitis C virus quasispecies in plasma and peripheral blood mononuclear cells of treatment naive chronically infected patients. J Viral Hepat 16:633–643 17. Forton DM, Karayiannis P, Mahmud N et al (2004) Identification of unique hepatitis C virus quasispecies in the central nervous system and comparative analysis of internal translational efficiency of brain, liver, and serum variants. J Virol 78:5170–5183 18. Laporte J, Bain C, Maurel P et al (2003) Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells. Blood 101:52–57 19. Durand T, Di Liberto G, Colman H et al (2010) Occult infection of peripheral B cells by hepatitis C variants which have

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36. George SL, Bacon BR, Brunt EM et al (2009) Clinical, virologic, histologic, and biochemical outcomes after successful HCV therapy: a 5-year follow-up of 150 patients. Hepatology 49:729–738 37. Marcellin P, Boyer N, Gervais A et al (1997) Long-term histologic improvement and loss of detectable intrahepatic HCV RNA in patients with chronic hepatitis C and sustained response to interferon-alpha therapy. Ann Intern Med 127:875–881 38. Maylin S, Martinot-Peignoux M, Ripault MP et al (2009) Sustained virological response is associated with clearance of hepatitis C virus RNA and a decrease in hepatitis C virus antibody. Liver Int 29:511–517 39. Pham TN, Coffin CS, Michalak TI (2010) Occult hepatitis C virus infection: what does it mean? Liver Int 30:502–511 40. Casanovas-Taltavull T, Ercilla MG et al (2004) Long-term immune response after liver transplantation in patients with spontaneous or post-treatment HCV-RNA clearance. Liver Transpl 10:584–594 41. Pillai V, Lee WM, Thiele DL, Karandikar NJ (2007) Clinical responders to antiviral therapy of chronic HCV infection show elevated antiviral CD4+ and CD8+ T-cell responses. J Viral Hepat 14:318–329 42. Quiroga JA, Llorente S, Castillo I et al (2006) Virus-specific T-cell responses associated with hepatitis C virus (HCV) persistence in the liver after apparent recovery from HCV infection. J Med Virol 78:1190–1197 43. Pham TNQ, Mercer SE, Michalak TI (2009) Chronic hepatitis C and persistent occult hepatitis C virus infection are characterized by distinct immune cell cytokine expression profiles. J Viral Hepat 16:547–556 44. MacParland SA, Pham TN, Guy CS, Michalak TI (2009) Hepatitis C virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro. Hepatology 49:1431–1441 45. Castillo I, Pardo M, Bartolome J et al (2004) Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown. J Infect Dis 189:7–14 46. Schmidt WN, Wu P, Cederna J et al (1997) Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests. J Infect Dis 176:27–33 47. Stapleton JT, Schmidt WN, Katz L (2004) Seronegative hepatitis C virus infection, not just RNA detection. J Infect Dis 190:651–652

48. Quiroga JA, Llorente S, Castillo I et al (2006) Cellular immune responses associated with occult hepatitis C virus infection of the liver. J Virol 80:10972–10979 49. Halfon P, Bourliere M, Ouzan D et al (2008) Occult hepatitis C virus infection revisited with ultrasensitive real-time PCR assay. J Clin Microbiol 46:2106–2108 50. Pham TN, MacParland SA, Coffin CS et al (2005) Mitogeninduced upregulation of hepatitis C virus expression in human lymphoid cells. J Gen Virol 86:657–666 51. Kondo Y, Sung VM, Machida K et al (2007) Hepatitis C virus infects T cells and affects interferon-gamma signaling in T cell lines. Virology 361:161–173 52. Kondo Y, Machida K, Liu HM et al (2009) Hepatitis C virus infection of T cells inhibits proliferation and enhances Fasmediated apoptosis by down-regulating the expression of CD44 splicing variant 6. J Infect Dis 199:726–736 53. Cribier B, Schmitt C, Bingen A et al (1995) In vitro infection of peripheral blood mononuclear cells by hepatitis C virus. J Gen Virol 76:2485–2491 54. MacParland SA, Pham TN, Gujar SA, Michalak TI (2006) De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro. J Gen Virol 87: 3577–3586 55. Laskus T, Radkowski M, Jablonska J et al (2004) Human immunodeficiency virus facilitates infection/replication of hepatitis C virus in native human macrophages. Blood 103: 3854–3859 56. Radkowski M, Bednarska A, Horban A et al (2004) Infection of primary human macrophages with hepatitis C virus in vitro: induction of tumour necrosis factor-alpha and interleukin 8. J Gen Virol 85:47–59 57. Marukian S, Jones CT, Andrus L et al (2008) Cell cultureproduced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 48:1843–1850 58. Murakami K, Kimura T, Osaki M et al (2008) Virological characterization of the hepatitis C virus JFH-1 strain in lymphocytic cell lines. J Gen Virol 89:1587–1592 59. Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893–897 60. Watanabe A, Yoneda M, Ikeda F et al (2010) CD147/ EMMPRIN acts as a functional entry receptor for measles virus on epithelial cells. J Virol 84:4183–4193 61. Sarhan MA, Michalak TI (2009) CD5-Mediated susceptibility of human T lymphocytes to wild-type hepatitis C virus. Hepatology 50:350A

Part III Cryoglobulinemia and the Complement System

9

Cryoglobulinemia and Chronic HCV Infection: An Evolving Story Jürg A. Schifferli and Marten Trendelenburg

The observation of protein precipitation at low ­temperature is an old one. Heidelberger and Kendall, already in 1929, described the precipitation of immune complexes in the cold after an immune response to pneumococcal polysaccharides [1]. In 1933, Wintrobe and Buell described the same phenomenon in the plasma of a patient with multiple myeloma [2]. A more detailed analysis of different types of cryoglobulins, however, began later, after it became clear that some of the cryoprecipitates contained different types of immunoglobulins. Mixed cryoglobulins were first described in 1962, following the observation that the cold-­ precipitable proteins in the serum of a patient with renal tubular acidosis were complexes consisting of 7S gamma globulins and 19S rheumatoid factors (RF), with 7S and 19S indicating what would later be designated as monomeric IgG and IgM, respectively [3]. The IgM and IgG present in these complexes differed from monoclonal cryoproteins, which precipitate on their own in the cold. Indeed, each of the two components, IgG and IgM, was an “incomplete” cryoglobulin, referring to the ability to precipitate in the cold only when mixed with the other, thus explaining the terminology of “mixed cryoglobulin.” Since the IgM had rheumatoid factor (RF) activity, it was immediately considered that the reaction of IgM with its antigen (IgG) was the  necessary requirement for cryoprecipitation. The ­corresponding clinical syndrome of mixed cryoglobulinemia (palpable purpura,

J.A. Schifferli (*) Division of Internal Medicine, Department of Medicine, University Hospital Basel, Basel, Switzerland e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_9, © Springer-Verlag Italia 2012

arthralgias, and weakness) was first described by Meltzer and Franklin [4], who ­confirmed the mixed IgG-IgM composition of the cryoglobulins and found that the IgM-RF was polyclonal in some patients and monoclonal in others. These authors and their colleagues also provided the first evidence for a role of mixed cryoglobulins in the pathogenesis of renal lesions associated with the disease. Immunofluorescence studies demonstrated that both IgG and IgM were deposited intraluminally and along the basement membranes of glomeruli [5]. Neither the clinical nor the pathological features in 9 of the 11 originally described patients with mixed cryoglobulins were typical of any known disease entity, and the term essential mixed cryoglobulinemia (EMC) was therefore introduced. The immunochemical classification of cryoglobulins that is now widely used describes three types of cryoglobulins [6, 7]. Those in type I consist of a single monoclonal immunoglobulin and are predominantly associated with malignancies of the immune system. Types II and type III are mixed cryoglobulins. Whereas in type II the cryoglobulins consist of polyclonal IgG and monoclonal IgM RFs, in type III both the IgG and the RF are polyclonal. The RF seen in type II and type III cryoglobulinemia usually consists of IgM but other classes of immunoglobulins are possible as well. More recently, Tissot described the presence of a few dominant oligoclonal IgM RFs in the serum of some patients with mixed cryoglobulinemia, suggesting a transition phase from type III (polyclonal IgM RF) to type II (monoclonal IgM RF) [8]. It soon also became evident that many of the RFs of type II cryoglobulinemia were genetically related, since they expressed the common idiotype Wa, as if the events preceding the emergence of the RF had some common features [9]. 79

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As noted above, most of the type II and type III mixed cryoglobulinemias are classified as essential, reflecting the fact that they occur in the absence of any apparent underlying disease. Secondary mixed cryoglobulins, predominantly type III, are seen in infectious, autoimmune, and chronic liver diseases whereas the secondary type II mixed cryoglobulins having a monoclonal component are often found in association with malignancies of the immune system. The prominent role of chronic viral hepatitis in the pathogenesis of cryoglobulins was not obvious in initial reports [10], although Levo et al. [11] had suggested a major role for hepatitis B virus (HBV) in EMC based on their detection of hepatitis B antigen or antibody in 13 of 53 patients (25%) with the disease. However, 14% of their control groups, made up of systemic lupus erythematosus and rheumatoid arthritis patients were infected with HBV, which was a very high percentage. In addition, this association was not confirmed by others. In 1990, Pascual presented ­evidence of hepatitis C virus (HCV) infection in three out of ten patients with EMC based on the results of a ­first-generation assay for anti-HCV antibodies [12]. Subsequently and very rapidly, evidence for a strong association between HCV and EMC was provided by different groups using newer anti-HCV antibody assays [13–24]. The prevalence of HCV antibodies in the sera of patients with type II and type III mixed cryoglobulinemia ranged from 30% to 98%. It is worth emphasizing that in most of the initial studies on mixed cryoglobulinemia, the test results were obtained using different generations of enzymelinked immunosorbent and recombinant immunoblot assay (ELISAs and RIBAs, respectively), leading to conflicting results on the prevalence of anti-HCV antibodies [13, 14, 21–23, 25]. However, quantitative studies of HCV antibodies from dissociated cryoglobulins showed that these antibodies were concentrated in the cryoglobulin. The results suggested that in non-dissociated cryoglobulins the anti-HCV antibodies are blocked by antigen and therefore unavailable for detection. Over time, assays for the detection of anti-HCV antibodies became more sensitive such that nowadays there are few patients with cryoglobulinemia and HCV hepatitis who do not have detectable antibodies in the blood circulation. Moreover, the detection of HCV RNA is a more direct indication of infection. Advances in polymerase chain reaction (PCR) methodology have allowed this method to become a practical technique for detecting and measuring hepatitis C viremia. It is,

J.A. Schifferli and M. Trendelenburg

Fig. 9.1  Serum obtained from a patient with cryoglubulinemia type II and HCV. After 24 h at 4°C, a cryoprecipitate is clearly visible

however, worthwhile to emphasize that in some patients with mixed cryoglobulinemia (5% approximately) HCV is not detectable; instead, other viruses are thought to trigger the emergence of the polyclonal/ monoclonal RF, such as HBV or HIV. Alternatively, these patients may have a clinical syndrome that includes some autoimmune features. These isolated observations enhance the notion that while HCV is indeed an excellent trigger of mixed cryoglobulinemia, the cascade of events induced by the virus corresponds somehow to “a stereotyped response” of the immune system in certain individuals. Interestingly, evidence for a direct pathogenic effect of cryoglobulins is lacking although their pathogenicity appears to be obvious. Cryoprecipitation of immunoglobulins, as it is used for diagnostic purposes (Fig. 9.1), is primarily an in vitro phenomenon the relevance of which has not been clearly shown in vivo. The concentration of cryoglobulins was, from the start, shown not to correlate well with the clinical syndrome. In spite of this knowledge, it is intriguing to see that the measurements of cryoglobulins in many labs continues to be based on the cryocrit, which is performed very differently from one lab to another and regardless of the method used will fail to detect low levels of cryoglobulins. Efforts to standardize these measurements or to convert them into units of mg/l, as defined by protein determination of the precipitate, have not yet been successful and in part explain the large differences in reports of the prevalence of cryoglobulins in HCV infection. In contrast, the correlation of cryoglobulins with skin lesions is clinically well established in many patients, as illustrated by the local application

9  Cryoglobulinemia and Chronic HCV Infection: An Evolving Story

Fig. 9.2  Patient with HCV and a cryoglobulin type II. She applied ice cubes to her “burning” skin, with resulting necrosis

of ice cubes to the skin of patients suffering from type II cryoglobulinemia (Fig.  9.2). These observations strongly support the idea that the formation of cryoprecipitates is not only an in vitro phenomenon but is of importance in vivo as well. The physicochemical properties of cryoglobulins might play a major role here. Following a unique case of a cryoglobulin precipitating only in the presence of calcium, it was possible to analyze the mechanisms of temperature-dependent precipitation [26]. Cryoprecipitation was shown to be a two-step event: (1) the reaction between antibody (IgM) and antigen (i.e., the Fc portion of IgG) reaction, which is not significantly influenced by temperature and (2) an IgM- and temperature-dependent aggregation. These and other data indicate clearly that IgG-IgM complexes circulate in vivo. However, unlike immune complexes in which IgG is the antibody, they are not cleared but instead circulate with a half life of weeks, comparable to monomeric IgG [27]. In addition, although they activate complement, they do not fix enough C3b to allow clearance by the complement/ CR1 pathway [28, 29]. This “non-clearance” might lead to the accumulation of IgM (RF)-IgG complexes in blood vessels and their subsequent deposition when the temperature drops or “saturation” is achieved. Immune complex deposition would then lead to local inflammation mediated by complement activation, with local amplification of C3 deposition and the release of C5a. The latter is the most powerful chemotactic factor for polymorphonuclear leukocytes and is responsible for the initiation of local tissue damage [30]. Izui’s group suggested that the combination of RF and cryoprecipitability is responsible for the

81

v­ asculitis seen in their mouse model of cryoglobulinemia. Interestingly, the temperature at which the mice are kept determines the presence of vasculitis and glomerulonephritis [31]. Whereas mice kept at room temperature develop glomerular depositions of cryoglobulins, glomerulonephritis is not seen in mice maintained in a warm environment (37°C). In line with the above-mentioned experimental findings, the most likely hypothesis explaining this result is that the large cryoglobulinemic aggregates formed in superficial blood vessels at temperatures below 37°C might not dissociate fast enough before arriving in the kidney or other organs. Despite the detection of HCV or proteins thereof in immune deposits, it remains unresolved whether their presence is directly involved in the local deposition of cryoglobulins in tissues such as the kidney and skin [32, 33]. Indeed, any antigen trapped in the cryoglobulin will be found at the site of immune aggregation. Furthermore, tissue deposition of type II cryoglobulins in Sjögren’s syndrome without HCV involvement occurs in the same organs and is indistinguishable from that of HCV-associated cryoglobulinemia. It is also worthwhile to think back about the treatment options that were at our disposal before we ­realized that HCV was the major cause of EMC. Immunosuppression and plasma exchange were efficient in many cases and, ironically, might correspond to the current use of rituximab [34], since both are aimed at inhibiting the synthesis of RF. Interestingly, interferon (IFN)-a was used in the treatment of cryoglobulinemia with some success by Bonomo in 1987, before HCV was identified as the cause of the disease [35]. Nowadays, IFN-a is the basis of anti-HCV therapy. It is also of interest that liver disease identical to that seen in many patients with mixed cryoglobulinemia was described in several reports long before 1990. The type of infiltration was reminiscent of the histology of “nonA-nonB” hepatitis, and in fact, as already mentioned, many authors had looked for viruses including HBV, Epstein-Barr virus, and cytomegalovirus even in the absence of convincing evidence for a viral origin to the pathology. The slow progression of many of these patients also suggested that EMC was more of a lowgrade lymphoma particularly, when histological images of even the kidney suggested tumor infiltration. This was the basis of the trial performed by Bonomo [35]. Most likely the future will teach us to recognize similar, apparently obvious relationships, since in many patients

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with HCV-related cryoglobulinemia the expansion of B cells across organs corresponds to the development of low-grade lymphoma. The history of essential cryoglobulinemia is fascinating since it demonstrates the slow progression of knowledge from one generation of research groups to the next, beginning with the initial emphasis on the immune reactants discovered to the realization that behind the deregulation of the immune system lay an infectious organism. Similar observations have been made for other viruses (Epstein-Barr virus) or bacteria (Helicobacter pylori), which have been shown to trigger a lymphoproliferation that starts as a benign process but becomes aggressive with time. The relation between HCV, the immune response, and induced deregulation are, however, far from being understood. While the present volume provides a synthesis of our current knowledge, there is much that remains to be understood: What are the steps between this specific viral trigger and the response of the human immune system? How can we better inhibit this response? Is the immune response genetically defined, and if so by which genes? Do other lymphoproliferative phenomena have similar triggers? Let’s hope that the readers of this book will be inspired to write the next chapters of the history of mixed HCV- and nonHCV-related cryoglobulinemia.

References 1. Heidelberger M, Kendall FE (1929) A quantitative study of the precipitin reaction between type III pneumococcus polysaccharide and purified homologous antibody. J Exp Med 50:809–823 2. Wintrobe MM, Buell MV (1933) Hypoproteinemia associated with multiple myeloma. Bull Johns Hopkins Hosp 52: 156–165 3. Lospalluto J, Dorward B, Miller W Jr, Ziff M (1962) Cryoglobulinemia based on interaction between a gamma macroglobulin and 7s gamma globulin. Am J Med 32:142–147 4. Meltzer M, Franklin EC (1966) Cryoglobulinemia: a study of twentynine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med 40:828–836 5. Meltzer M, Franklin EC, Elias K, McCluskey RT, Cooper N (1966) Cryoglobulinemia: a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity. Am J Med 40:837–856 6. Brouet JC, Clauvel JP, Danon F et  al (1974) Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 57:775–788

J.A. Schifferli and M. Trendelenburg 7. Gorevic PD (1986) Mixed cryoglobulinemia: an update of recent clinical experience. In: Ponticelli C, Minetti L, D’Amico G (eds) Antiglobulins, cryoglobulins and glomerulonephritis. Martinus Nijhoff, Dordrecht 8. Tissot JD, Schifferli JA, Hochstrasser DF, Pasquali C, Spertini F, Clément F, Frutiger S, Paquet N, Hugues GJ, Schneider P (1994) Two-dimentional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide. J Immunol Methods 173: 63–75 9. Agnello V, Barnes JL (1986) Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains. J Exp Med 164:1809–1814 10. Brouet JC (1983) Les cryoglobulinémies. Presse Med 12: 2991–2996 11. Levo Y, Gorevic PD, Kassab HJ, Tobias H, Franklin EC (1977) Liver involvement in the syndrome of mixed cryoglobulinemia. Ann Intern Med 87:287–292 12. Pascual M, Perrin L, Giostra E, Schifferli JA (1990) Hepatitis C virus in patients with cryoglobulinemia type II (letter). J Infect Dis 162:569–570 13. Ferri C, Greco F, Longombardo G, Palla P, Moretti A, Marzo E, Fosella PV, Pasero G, Bombardieri S (1991) Antibodies to hepatitis C virus in patients with mixed cryoglobulinemia. Arthritis Rheum 34:1606–1610 14. Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II mixed cryoglobulinemia. N Engl J Med 327:1490–1495 15. Alter HE (1992) New kit on the block: evaluation of second generation assays for detection of antibody to the hepatitis C virus. Hepatology 15:350–353 16. Ferri C, Palla P, Greco F, Marzo E, Longombardo G, Moretti A (1991) Hepatitis C virus antibodies in mixed cryoglobulinemia (letter). Clin Exp Rheumatol 9:95–96 17. Bambara LM, Carletto A, Biasi D, Pacor ML, Caramaschi P (1991) Cryoglobulinemia and hepatitis C virus (HCV) infection (letter). Clin Exp Rheumatol 9:96–97 18. Durand JM, Lefevre P, Harle JR, Boucrat J, Vitvitski L, Soubeyrand J (1991) Cutaneous vasditis and cryoglobulinemia type II associated with hepatitis C virus infection (letter). Lancet 337:499–500 19. Casato M, Taliani G, Pucillo LP, Goffredo F, Lagana B, Bonomo L (1991) Cryoglobulinemia and hepatitis C virus (letter). Lancet 337:1047–1048 20. Arribas JR, Barbado FJ, Zapico R, Sendino A, Gonzalez I, Vazquez JJ (1991) Association between hepatitis C virus and mixed cryoglobulinemia. Rev Infect Dis 13:770–771 21. Disdier P, Harle JR, Weiller PJ (1991) Cryoglobulinemia and hepatitis C infection (letter). Lancet 338:1151–1152 22. Pechère-Bertschi A, Perrin L, de Saussure P, Widmann JJ, Giostra E, Schifferli JA (1992) Hepatitis C: a new etiology for cryoglobulinemia type II. Clin Exp Immunol 89: 419–422 23. Ferri C, Greco F, Longombando G, Palla P, Moretti A, Marzo E, Mazzoni A, Pasero G, Bombardieri S, Highfield P, Corbishley T (1991) Association between hepatitis C virus and mixed cryoglobulinemia. Clin Exp Rheumatol 9: 621–624

9  Cryoglobulinemia and Chronic HCV Infection: An Evolving Story 24. Misiani R, Bellavita P, Fenili D, Borelli G, Marchesi D, Massazza M, Vedramin G, Comotti B, Tanzi E, Scudeller G, Zanetti A (1992) Hepatitis C virus infection in patients with essential mixed cryoglobulinemia. Ann Intern Med 117: 573–577 25. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R (1990) Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet 335:754–757 26. Qi M, Steiger G, Schifferli JA (1992) A calcium dependent cryoglobulin IgM kappa/polyclonal IgG. J Immunol 149:2345–2351 27. Schifferli JA, Amos N, Pusey CD, Sissons JGP, Peters DK (1983) Metabolism of autologous and homologous IgG in patients with mixed essential cryoglobulinemia type II. Absence of fast elimination of IgG. Clin Exp Immunol 51: 305–316 28. Schifferli JA, Ng YC, Estreicher J, Walport MJ (1988) The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans: complementand erythrocyte CR1-dependent mechanisms. J Immunol 140:899–904 29. Madi N, Steiger G, Estreicher J, Schifferli JA (1991) Defective immune adherence and elimination of hepatitis B

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surface Ag/Ab complexes in patients with mixed essential cryoglobulinaemia type II. J Immunol 147:495–502 30. Schifferli JA, Steiger G, Polla L, Didierjean L, Saurat JH (1985) Activation of the alternative pathway of complement by skin immune deposits. J Invest Dermatol 85:407–411 31. Fulpius T, Berney T, Lemoine R et al (1994) Glomerulopathy induced by IgG3 anti-trinitrophenyl monoclonal cryoglobulins derived from non-autoimmune mice. Kidney Int 45:962–971 32. Sansonno D, Gesualdo L, Manno C, Schena FP, Dammacco F (1997) Hepatitis C virus-related proteins in kidney tissue from hepatitis C virus-infected patients with cryoglobulinemic membranoproliferative glomerulonephritis. Hepatology 25:1237–1244 33. Agnello V, Abel G (1997) Localization of hepatitis C virus in cutaneous vasculitic lesions in patients with type II cryoglobulinemia. Arthritis Rheum 40:2007–2015 34. Terrier B, Saadoun D, Sène D et al (2009) Efficacy and tolerability of rituximab with or without PEGylated interferon alfa-2b plus ribavirin in severe hepatitis C virus-related vasculitis: a long-term followup study of thirty-two patients. Arthritis Rheum 30:2531–2540 35. Bonomo L, Casato M, Afeltra A, Caccavo D (1987) Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 83:726–730

The Complement System in Cryoglobulinemia

10

Marten Trendelenburg

10.1 The Complement Cascade Complement is part of the innate immune system and underlies one of the main effector mechanisms of antibody-mediated immunity. It has three major physiologic activities: defending against pyogenic bacterial infection, bridging innate and adaptive immunity, and disposing of immune complexes and the products of inflammatory injury. There are three pathways of activation of the complement system: the classical, mannose-binding lectin, and alternative pathways (Fig. 10.1). These pathways leading to the cleavage of C3 are triggered enzyme cascades, analogous to the coagulation, fibrinolysis, and kinin pathways. The terminal complement pathway, resulting in the formation of the membrane-attack complex, is a unique system that builds up a lipophilic complex in cell membranes from several plasma proteins (C5b-9). Complement is a system comprising more than 30 proteins in plasma and on cell surfaces. Complement proteins in plasma are present in concentrations of > 3 g/l and they constitute approximately 15% of the globulin fraction. The nomenclature of complement follows the historical order of discovery and as a consequence might appear confusing. The first complement pathway that was discovered, the classical pathway, begins with the formation of immune complexes, e.g., when antibody binds to a cell surface, and ends with lysis of the cell. The proteins of this pathway

M. Trendelenburg  Clinic for Internal Medicine and Laboratory for Clinical Immunology, University Hospital Basel, Basel, Switzerland e-mail: [email protected]

are designated C1–C9. It was subsequently discovered that the numbering of the proteins did not quite correspond with the order of the reaction, since C1 is followed in succession by C4, C2, C3, and C5, with the numerical order being restored from C6 through C9. Proteins of the second pathway to be discovered, the alternative pathway, are called factors and are followed by a letter, such as factor B. The alternative pathway is spontaneously and constantly activated on biological surfaces in plasma and in most or all other body fluids. This spontaneous activation readily initiates amplification by the “amplification loop” and requires regulatory proteins protecting cells from complement attack. Therefore, complement proteins on cell membranes are not only receptors for activated complement proteins but also proteins that regulate complement. The third but evolutionarily perhaps the oldest pathway of complement activation is the mannose-binding lectin (MBL) pathway. As a multimeric complex, complement MBL is able to recognize carbohydrate pathogen-associated molecular patterns on a wide range of microorganisms, leading to complement activation in an antibody- and C1q-independent manner. Besides these three well-known pathways of complement activation, further pathways seem to exist, such as the direct cleavage of complement C5 by thrombin. Several complement proteins are cleaved during activation of the system, and the fragments are designated with lowercase suffixes; for example, C3 is cleaved into two fragments, C3a and C3b. With the exception of C2 for historical reasons, the large fragment is designated “b” and the small fragment “a.” The regulatory mechanisms of complement are finely balanced so that, on the one hand, the activation of complement is focused on the surface of invading microorganisms and, on the

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86 Fig. 10.1  Overview of the main components and activation pathways of complement

M. Trendelenburg Classical pathway - Immune complexes

Alternative pathway - Activating surfaces MBL pathway - carbohydrates (mannose)

C1 C4

MASP C2

C4 C2

C3b Factor B Factor D

C3 convertase C3a C5 convertase

Thrombin pathway - thrombin

C5a

C5b-9 Membrane-attack complex (MAC)

other hand, the deposition of complement on normal cells and tissues is limited. When the mechanisms that regulate this delicate balance go awry, the complement system may cause injury. Thus, complement may be friend or foe, depending on the circumstances. Under physiologic conditions, complement promotes the clearance of immune complexes, an important means of eliminating, e.g., antibody-coated bacteria. If, however, immune complexes cannot be eliminated, then complement becomes chronically activated and can incite inflammation. In addition, chronic infections can perpetuate the formation of immune complexes, which in hepatitis C infection and bacterial endocarditis cause relentless activation and consumption of complement [1–5].

10.2 Complement Consumption and Deposition in Cryoglobulinemia Low levels of complement suggesting ongoing complement activation and consumption are a well-known phenomenon in patients with cryoglobulinemia. It was first reported by Riethmüller et  al. in 1966 [6], who described seven patients with mixed cryoglobulins and very low levels of complement C2, suggesting activation and consumption of complement via the classical pathway. Further studies confirmed the predominant

consumption of components of the classical pathway [7–9]. In addition, it became apparent that some cryoglobulins are also able to directly activate the alternative pathway of complement [10]. However, whereas patients with cryoglobulinemia usually have low levels of C1, C4, and C2, concentrations of C3 and of the components of the terminal pathway often remain within the normal range, suggesting that activation of  complement beyond C3 is inefficient although not  absent [11, 12]. In a more recent analysis, cryoglobulins were found to also activate the MBL pathway of complement. All 16 cryoprecipitates of patients with mixed cryoglobulinemia were found to contain MBL [13]. In line with these data, it can frequently be observed that consumption of C4 is more pronounced than the relative consumption of C1q, suggesting that, in addition to activation of the classical pathway, complement activation by cryoglobulins occurs via the MBL pathway. The consumption of complement components is paralleled by the deposition of complement in affected tissues such as the kidney [13–15]. Whereas the predominant immunoreactants in mixed cryoglobulinemia are IgG and IgM, these deposits are usually accompanied by the glomerular deposition of MBL, C4 (C4d), C3, and, less frequently, C1q, with a distribution very similar to that of immunoglobulins. Complement deposition seems to be the consequence of both the local and ongoing activation of ­complement

10  The Complement System in Cryoglobulinemia

after trapping of pre-formed cryo-precipitates as well as of secondary deposition of complement components that are already present in the trapped complexes.

10.3 Interaction of Complement with Cryo-Immune Complexes Cryoglobulins can be considered as immune complexes consisting not only of immunoglobulins but also of trapped antigen, such as hepatitis C virus particles, and other serum components, such as C-reactive protein and complement [9, 16]. Complement participates in the elimination of immune complexes in many circumstances and is likely to be involved in the clearance of cryoglobulins as well. When immune complexes first form in the circulation, complement inhibits their aggregation because binding of components of the classical pathway as well as the covalent binding of C3b to the immune complexes modifies their biophysical properties [17]. This modification by the binding of complement facilitates the retention of immune complexes in solution, a process called “inhibition of immune precipitation.” In addition, complement has the capacity to solubilize immune precipitates that have already formed [18]. However, solubilization by complement is much less efficient, requiring a considerable amount of complement activation and thus bearing the risk of inflammatory tissue damage [19]. Once they are opsonized (C3b coated), immune complexes attach to cells bearing C3b receptors (complement receptor 1, CR1) in the circulation, in particular to erythrocytes, since in humans 85–90% of CR1 in the blood is located on these cells. This immune adherence binding reaction is a physiological system that allows immune complexes to be transported through the circulation to the fixed macrophages of the mononuclear phagocyte system, where they are safely eliminated. The deposition of circulating complement-fixing immune complexes in various organs such as the kidney may be considered as a failure of this transport system. This is apparent in complement-deficient and -depleted states as well as in non-complement-fixing immune complexes such as those of IgA. The formation of insoluble immune complexes (by definition, immune deposits found in human pathology are insoluble) produces complement activation and inflammation at the site of the immune aggregate. Type II

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cryoglobulins were shown to activate complement in vitro but rapid clearance in vivo was not observed [20]. Most likely, on the one hand, the IgM rheumatoid factor (RF) covered the Fc fragments of IgG, thus altering their interference with Fc receptors. On the other hand, despite efficient complement activation, the cryo-immune complexes were not able to bind ­sufficient C3b and thus to be cleared via the erythrocyte-CR1 transport system [6, 21–23]. The insufficient binding of C3b perhaps explains the consumption of early components of the complement cascade that is usually not accompanied by similarly low C3 levels or the depletion of components of the terminal pathway, as outlined above. In addition, “non-clearance” might concur with the accumulation of IgM (RF)-IgG complexes in blood vessels that deposit when the temperature drops or “saturation” is achieved. Immune complex deposition then leads to local inflammation mediated by complement activation, with local amplification of C3 deposition and the release of C5a, which is the most powerful chemotactic factor for polymorphonuclear leukocytes and responsible for the initiation of local tissue damage [24].

10.4 Pathogenic Role of Complement Activation by Cryoglobulins In Vivo The abundant occurrence of IgG in cryoprecipitates and thus the large number of potentially available Fc fragments suggests that Fc gamma receptors play a major role in the pathogenic mechanisms of cryoglobulinemia. However, in  vivo experiments using mice that develop cryoglobulinemia could not confirm a major pathogenic role for Fc gamma receptors in this entity [25, 26]. These rather surprising results may be related to the fact that Fc fragments are no longer accessible after the binding of RFs, as occurs in mixed cryoglobulins. In contrast, there is some evidence that complement activation is a major factor explaining the inflammatory organ damage seen in cryoglobulinemia in  vivo. In our study, the role of complement was investigated in a mouse model of induced cryoglobulinemic glomerulonephritis. Depending on the genetic background of the mice, the cryoprecipitate consisted of a murine monoclonal IgG3 cryoglobulin only (type I cryoglobulinemia) or of murine monoclonal IgG3 RF–polyclonal IgG2a complexes (type II cryoglobulinemia). Several complement-deficient mice with

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either genetic background were investigated and ­compared to strain-matched, wild-type controls. The ­survival of mice was not affected by complement deficiency but glomerular influx of neutrophils was ­significantly less in C3-, factor-B-, and C5-deficient mice than in wild-type and C1q-deficient mice. The influx of neutrophils did not correlate with the C3 deposition mediated by activation of the classical and alternative pathways, but did correlate with the amount of C6 deposited. In addition, deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Taken together, these results suggested that: (i) the generation of C5a was critical for the neutrophil influx observed in this model of cryoglobulinemia, and (ii) in spite of additional complement activation via the classical pathway, the alternative pathway played a prominent role in the cleavage of C5 [27]. However, data on the role of complement in ­cryoglobulinemic glomerulonephritis is conflicting. Transgenic mice that overexpress thymic stromal lymphopoietin (TSLP) and as a consequence develop mixed cryoglobulinemia with renal disease resembling human cryoglobulin-associated membranoproliferative glomerulonephritis. In these mice, no anti-inflammatory effect of the additional overexpression of the murine complement regulator Crry could be detected [28]. In contrast, factor B deficiency, which blocks the alternate pathway of complement, either alone or in addition to Crry overexpression did not alleviate, but instead ­aggravated the renal lesions in TSLP transgenic mice [29, 30]. However, it might be important to note that C1q staining in these mice was absent and that C3 deposition was much less than IgG and IgM deposition. Therefore, more studies are required to clarify the role of complement in cryoglobulinemia.

References 1. Fujita T (2002) Evolution of the lectin-complement pathway and its role in innate immunity. Nat Rev Immunol 2:346–353 2. Zipfel PF, Skerka C (2009) Complement regulators and inhibitory proteins. Nat Rev Immunol 9:729–740 3. Walport MJ (2001) Complement. First of two parts. N Engl J Med 344:1058–1066

M. Trendelenburg 4. Walport MJ (2001) Complement. Second of two parts. N Engl J Med 344:1140–1144 5. Huber-Lang M, Sarma JV, Zetoune FS et  al (2006) Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med 12:682–687 6. Riethmüller G, Meltzer M, Franklin E, Miescher PA (1966) Serum complement levels in patients with mixed (IgM-IgG) cryoglobulinaemia. Clin Exp Immunol 1:337–339 7. Linscott WD, Kane JP (1975) The complement system in cryoglobulinaemia. Interaction with immunoglobulins and lipoproteins. Clin Exp Immunol 21:510–519 8. Tanimoto K, Cooper NR, Johnsons JS, Vaughan JH (1975) Complement fixation by rheumatoid factor. J Clin Invest 55:437–445 9. Wilson MR, Arroyave CM, Miles L, Tan EM (1977) Immune reactants in cryoproteins. Relationship to complement activation. Ann Rheum Dis 36:540–548 10. Poskitt TR, Poskitt PK (1979) Temperature dependent activation of the alternate complement pathway by an IgG cryoglobulin. Am J Hematol 7:147–154 11. Tarantino A, Anelli A, Costantino A et  al (1978) Serum complement pattern in essential mixed cryoglobulinaemia. Clin Exp Immunol 32:77–85 12. Greenstein JD, Peake PW, Charlesworth JA (1996) The metabolism of C9 in normal sbjects and in patients with autoimmune disease. Clin Exp Immunol 104:160–166 13. Ohsawa I, Ohi H, Tamano M et al (2001) Cryoprecipitate of patients with cryoglobulinemic glomerulonephritis contains molecules of the lectin complement pathway. Clin Immunol 101:59–66 14. D’Amico G, Colasanti G, Ferrario F, Sinico RA (1989) Renal involvement in essential mixed cryoglobulinemia. Kidney Int 35:1004–1014 15. Beddhu S, Bastacky S, Johnson JP (2002) The clinical and morphologic spectrum of renal cryoglobulinemia. Medicine (Baltimore) 81:398–409 16. Weiner SM, Prasauskas V, Lebrecht D et  al (2001) Occurrence of C-reactive protein in cryoglobulins. Clin Exp Immunol 125:316–322 17. Schifferli JA, Taylor RP (1989) Physiological and pathological aspects of circulating immune complexes. Kidney Int 35:993–1003 18. Miller GW, Nussenzweig V (1975) A new complement function: solubilization of antigen-antibody aggregates. Proc Natl Acad Sci USA 72:418–422 19. Takahshi M, Tack BF, Nussenzweig V (1977) Requirements for the solubilization of immune aggregates by complement; assembly of a factor B-dependent C3-convertase on the immune complexes. J Exp Med 145:86–100 20. Schifferli JA, Amos N, Pusey CD et al (1983) Metabolism of autologous and homologous IgG in patients with mixed essential cryoglobulinemia type II. Absence of fast elimination of IgG. Clin Exp Immunol 51:305–316 21. Ng YC, Peters DK, Walport MJ (1988) Monoclonal rheumatoid factor-IgG immune complexes: poor fixation of opsonic C4 and C3 despite efficient complement activation. Arthritis Rheum 31:99–107 22. Schifferli JA, Ng YC, Estreicher J, Walport MJ (1988) The clearance of tetanus toxoid- anti tetanus toxoid immune complexes from the circulation of humans: ­complement- and

10  The Complement System in Cryoglobulinemia erythrocyte CR1-dependent mechanisms. J Immunol 140: 899–904 23. Madi N, Steiger G, Estreicher J, Schifferli JA (1991) Defective immune adherence and elimination of hepatitis B surface Ag/Ab complexes in patients with mixed essential cryoglobulinaemia type II. J Immunol 147:495–502 24. Schifferli JA, Steiger G, Polla L et al (1985) Activation of the alternative pathway of complement by skin immune deposits. J Invest Dermatol 85:407–411 25. Watanabe N, Akikusa B, Park SY et  al (1999) Mast cells induce autoantibody-mediated vasculitis syndrome through tumor necrosis factor production upon triggering Fcgamma receptors. Blood 94:3855–3863 26. Guo S, Mühlfeld AS, Wietecha TA et al (2009) Deletion of activating Fcgamma receptors does not confer protection in murine cryoglobulinemia-associated membranoproliferative glomerulonephritis. Am J Pathol 175:107–118

89 27. Trendelenburg M, Fossati-Jimack L, Cortes-Hernandez J et  al (2005) The role of complement in cryoglobulininduced immune complex glomerulonephritis. J Immunol 175:6909–6914 28. Muhlfeld AS, Segerer S, Hudkins K et  al (2004) Overexpression of complement inhibitor Crry does not prevent cryoglobulin-associated membranoproliferative glomerulonephritis. Kidney Int 65:1214–1223 29. Wietecha TA, Hudkins KL, Iyoda M et al (2006) Deletion of murine factor B in thymic stromal lymphopoietin mice aggravates cryoglobulin-associated membranoproliferative glomerulonephritis. J Am Soc Nephrol 17:F-PO820 30. Wietecha TA, Hudkins KL, Iyoda M et al (2007) Inhibition of complement pathways of the murine protein crry and deletion of factor B in thymic stromal lymphopoietin mice aggravates cryoglobulin-associated membranoproliferative glomerulonephritis. J Am Soc Nephrol 18:SA-PO317

The Pivotal Role of C1qR in Mixed Cryoglobulinemia

11

Domenico Sansonno, Loredana Sansonno, and Franco Dammacco

11.1

Introduction

Cryoglobulinemic tissue damage is likely the consequence of a pathogenetic noxa that acts upon the host’s immune system, resulting in an altered regulation of the peripheral immune response [1]. Recent data have clearly established that the structural composition of cold-precipitating immune complexes (ICs) includes HCV core protein as the relevant ligand [2]. HCV nucleocapsid, devoid of enveloped proteins, has been detected in the bloodstream of HCV-infected patients and is a good indicator of circulating viral load, possibly reflecting overproduction during virogenesis. Non-enveloped HCV core protein was shown to be secreted by transfected hepatoma cell lines in culture and in HCV transgenic mice [3]. The core protein has been detected in the serum of most HCV chronic carriers with active liver disease and almost half of those with inactive disease. In addition, serum levels of HCV core protein change following antiviral therapy and become undetectable in responsive patients [4]. In HCV-related cryoglobulinemia, HCV core protein is cold-precipitated in the context of ICs and its colddependent insolubility seems to be the result of IgM rheumatoid factor (RF), which acts as an incomplete cryoglobulin [2]. The complement system is highly activated in patients with HCV-related cryoglobulinemia [5].

D. Sansonno (*) Department of Biomedical Sciences and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

Normal mean levels of C3 and C4 in the soluble phase correspond to very low amounts (if any) in the cryoprecipitate, suggesting the existence of two virtually distinct microenvironments in which complement is differentially activated. Complement is known to be a major interdependent regulator of IC size and composition and its binding to nascent ICs may decrease their size and maintain them in solution [6]. Compared with supernatant, significant differences in C1q and C1qbinding activity have been shown in insolubilized ICs [2]. Efficient engagement of C1q protein by cryoglobulins may be an important pathogenetic mechanism involved in the cryoglobulin-related pathway. HCV core protein has been shown to interact directly with the globular domain of the C1q receptor (gC1qR) [7]. This HCV core–gC1qR interaction is assumed to play a critical role in modulating the T-cell immune response [8]. HCV core-induced inhibition of T cell responsiveness may underlie the pathogenetic process that blocks the suppression of B-cell clones producing RF autoantibodies, generated by chronic antigenic challenge in HCV-related type II cryoglobulinemia [9]. Nonetheless, engagement of circulating HCV core protein with gC1qR displayed on the surface of B lymphocytes [10] can provide the virus with a direct means of alterin host immunity. The wide expression of gC1qR on the surface of both circulating blood cells and endothelial cells [11] favors their specific binding to HCV core-protein-containing ICs. HCV core deposition has indeed been reported in the skin [12] and kidney [13] of cryoglobulinemic patients, suggesting that HCV core-gC1qR interactions play a relevant role in cryoglobulin-related damage and in IC formation and cryoprecipitation.

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11.2

D. Sansonno et al.

gC1qR-HCV Core Interaction

The 33-kDa gC1qR is an acidic protein expressed on somatic cells. It binds to the globular heads of C1q and modulates complement activation [14]. Apart from its specific interaction with C1q, gC1qR binds to numerous cell-surface proteins, such as kininogen [15], vitronectin [16], nucleus-related like TFII B [17], lamin B receptor [18], splicing factor-2 (SF2) [19], mitochondrial-related cytochrome b2 [20], and BH3 only protein Hrk [21]. In addition to cellular proteins, gC1qR interacts with several bacterial and viral pathogenic proteins, such as adenovirus core protein [22] and HIV rev [23], suggesting that it is a part of the system that imports proteins to the cell. gC1qR is highly expressed on endothelial cells, implying a major role in cryoglobulinemic vascular damage. The binding of kininogen to gC1qR on the endothelial cell surface has been shown to serve as a platform for the assembly and activation of the intrinsic coagulation cascade that leads to the generation of bradykinin and thus to infiltration of vascular tissue by proinflammatory cells [24]. It was previously determined that HCV core protein binds the gC1qR region spanning amino acids 188– 259, while the interaction site on the core protein encompasses residues 26–124 [7]. Additionally, C1q examer, a highly positively charged molecule, binds, via its globular head complex, the NH2-terminal portion of gC1qR, spanning amino acids 74–95 [25]. Potential insights into the structural basis of these interactions may derive from the reported crystal structure of gC1qR, which revealed that three gC1qR molecules form a doughnut-shaped quaternary structure, with a sizable central channel and an asymmetric charge distribution on the surface, including exposed acidic residues in the COOH-terminal portion of the molecule and in the NH2-terminal a-helical domain. These negatively charged residues represent the binding site for the positively charged HCV core protein [26]. Data obtained from experiments performed in our laboratory indicate that the amount of gC1qR shed in the plasma as bioactive molecules is significantly higher in cryoglobulinemic patients than in HCVinfected patients without cryoglobulinemia or in healthy subjects. It has also been noted that there is a positive correlation between circulating gC1qR, plasma levels of IgM with RF activity, and C1q concentration in patients with mixed cryoglobulinemia

HCV CORE PROTEIN gC1qR C1q PROTEIN

IgM - RF

Fig. 11.1 Model of circulating immune complexes comprising gC1qR bound to HCV core protein and C1q protein, which in turn binds IgM with RF activity. These complexes possibly represent a portion of the spectrum of the cryoprecipitating immune complexes in MC patients

(MC). By contrast, no relation has been observed with circulating viral load, levels of non-enveloped HCV core protein, liver histology activity index, grade of liver fibrosis, and serum alanine aminotransferase levels. Soluble gC1qR circulates as a complexed form containing both C1q and HCV core proteins simultaneously bound to IgM molecules with RF activity. The nature and target of IgM binding have not been ascertained. However, according to preliminary data, IgM RF is not complexed with IgG molecules, suggesting that it is not likely directed to the Fc portion of IgGcontaining ICs. Instead, it can be inferred that IgM RF interacts directly with C1q, based on reports that the C1q domain interacts with IgM via the top of the molecule [27]. C1q has a rigid structure and is composed mainly of b-sheets. Interaction with IgM seems to be mediated by the globular head module B, due to its equatorial position [28]. A model of circulating ICs containing C1q, HCV core protein, and soluble gC1qR bound to IgM molecules is depicted in Fig. 11.1. In HCV-infected patients without MC, it was found that gC1qR simultaneously binds C1q and HCV core proteins, but not IgM molecules. These observations provide valuable information for understanding and dissecting the functions of gC1qR and related proteins, and point to a major impact of IgM RF in the activation of the complement system [29]. It has been proven that single IgM molecules activate the complement classical pathway by binding and activating the C1q fraction [30].

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The Pivotal Role of C1qR in Mixed Cryoglobulinemia

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Fig. 11.2 Localization of HCV core (a) and C4d protein (b) in a skin biopsy from an HCV-positive MC patient. Note that core protein is mainly located within the vessel lumen, whereas C4d is found along the vessel walls

11.3

gC1qR-HCV Core-Mediated Complement Activation

Binding to C1q protein is the primary requisite for the activation of complement via the classical pathway; this can be incontrovertibly demonstrated by measuring complement fragments generated during proteolytic cleavage of the C4 fraction. Indeed, C4d fragment is regarded as a reliable marker of complement activation [31] and C4d levels have been found in the sera of most patients with rheumatoid arthritis, an immune complex disease in which complement is activated via the classical pathway [32]. Median serum C4d levels are significantly higher in healthy controls than in MC patients but lower than in HCV-infected patients without MC. Lower levels of circulating C4d fragment in MC patients may indeed reflect its diversion near the sites of C4 activation. Thus, it seems possible that C4d fragment is detectable in different biological compartments, where C4 is involved in immune complex-mediated reactions. To test this hypothesis, skin biopsy samples from patients with cryoglobulinemic vasculitis were explored for the presence of HCV core and C4d deposits by indirect immunohistochemistry [33]. As shown in Fig. 11.2, both HCV core protein and C4d fragment were demonstrated in vascular structures, specifically, the vessel lumen, endothelium, and lamina propria. HCV core engagement of gC1qR on lymphocytes limits the induction of Th1 responses and may contribute to viral persistence [34]. In vitro effects of HCV core protein on lymphocytes from patients with and without

MC and from healthy subjects were shown to include the extensive inhibition of lymphocyte proliferation, ranging from 50% to 90%. Surprisingly, in the same samples, gC1qR concentrations are greatly increased in the supernatants of HCV-related MC patients, whereas a very limited increment was demonstrable in the nonMC group and no significant variations were found in healthy controls. These findings strongly support the contention that there is an intrinsic difference in the regulation and secretion of lymphocyte-derived gC1qR by HCV core proteins in MC patients. In line with higher gC1qR expression on lymphocytes of HCV-related MC patients, quantitative realtime RT-PCR confirmed the occurrence of greater amounts of gC1qR mRNA in the cells of these patients. Specific gC1qR mRNA expression was at least 6- to 10-fold higher in cells from MC patients than in those from the non-MC group and healthy controls. In vitro experiments showed that gC1qR mRNA expression progressed in a dose-dependent manner to the HCV core-induced suppressive effect of cell proliferation, raising the possibility that the magnitude of gC1qR expression is predisposed by an intrinsic difference in gC1qR gene regulation in patients with MC.

11.4

Conclusions

Soluble gC1qR may have a deep impact on the clinical features in patients with MC for several reasons, including the fact that it acts as a bridging molecule responsible for linking apoptotic cells is able to activate

94 Fig. 11.3 Pathogenetic model of cryoglobulinemic tissue damage. Complement regulates the size and composition of immune complexes, reducing their size and maintaining them in solution. Complement consumption resulting from C4 diversion contributes to vascular cryoglobulininduced damage by deposition of C1q-dependent immune complexes

D. Sansonno et al.

C4- HYPOCOMPLEMENTEMIA

C4 DIVERSION

CIRCULATING IMMUNE COMPLEXES

SKIN TISSUE DEPOSITS

HCV CORE PROTEIN

C4d gC1qR C1q PROTEIN

IgM

INSOLUBILITY OF CRYOPROTEINS

endothelial cells and platelets, and initiates the intrinsic blood coagulation and kinin-generating pathway [35]. These findings shed new light on our comprehension of the pathogenetic mechanisms underlying cryoglobulin-induced vascular damage. For example, soluble gC1qR may contribute to the vasculitic process by modulating complement activation and by enrichment of tissue deposits of HCV core protein. Measurement of circulating C4d has provided evidence of lower amounts in MC patients. Indeed, the exposure of a thioester group by split C4d leads to the formation of a covalent bond near the site of C4 activation [36]. Examination of skin biopsy tissues shows that C4d deposits are detectable in almost all MC patients, but in none of those without MC. Thus, low levels of circulating C4d in MC patients are likely the result of sequestered fragments. Furthermore, regression of cryoglobulinemic vasculitis, which has been observed following a successful therapeutic response, is characterized by a significant reduction of soluble gC1qR and a parallel increment of serum C4d fragment, strongly suggesting a disengagement of entangled C4 and inhibition of the C1 activation pathway. In this context, it seems reasonable to hypothesize a pathogenetic model whereby the dysregulated shedding of C1qR molecules modulates complement activation, which in turn leads to the consumption and deposition of C4d fragment-containing ICs within tissues, as summarized in Fig. 11.3.

Interestingly, HCV core protein generates C4d in serum depleted of C1q protein, indicating that it directly activates the complement cascade, likely via the mannan-binding lectin pathway. Thus, in the presence of high levels of circulating gC1qR, the HCV core protein can exacerbate the inflammatory condition by combined and simultaneous activation of both complement pathways. Under these conditions, endothelial cells are then activated with consequent initiation of a local inflammatory response. It has been shown that HCV core protein suppresses the host immune response by engaging gC1qR on the surface of immune cells. Core protein is readily detected in the plasma of HCV-infected patients with or without MC, and extensive inhibition of mitogenstimulated proliferation of peripheral blood lymphocytes can be demonstrated. In vitro experiments have indeed defined a unique property of MC patients, in that large amounts of soluble gC1qR are released in culture supernatants in step with HCV core inhibition of the lymphocyte proliferative response. Very limited increments were noted in non-MC patients and no changes from basal levels were demonstrated in healthy subjects. This strongly indicates that the mechanisms underlying gC1qR synthesis and release from lymphocytes are HCV core-mediated and negatively regulated by cell proliferation. No direct relation was found between the levels of non-enveloped HCV core protein and those of soluble gC1qR in patients

11

The Pivotal Role of C1qR in Mixed Cryoglobulinemia

with or without MC, indicating that the circulating amount of viral protein is not a critical factor. Why gC1qR expression levels differ in patients with as compared to those without MC is as yet unknown, but it might be the result of transcriptional control of the gC1qR gene and a genetic difference in the host gene regulation. Indeed, higher levels of soluble gC1qR are demonstrable in HCV-positive and HCV-negative MC patients. Quantitative real-time RT-PCR assay for specific gC1qR mRNA has shown that expression is 6- to 14-fold higher in cells from MC than in those from non-MC patients. These results parallel those obtained from analyses of gC1qR protein expression on lymphocytes. The percentages of cells expressing gC1qR in MC patients are 3- to 4-fold higher than those found in non-MC patients, suggesting that synthesis and release of gC1qR protein in cells is dependent on upregulation of the gC1qR gene. At variance from previous data showing that agents or proteins inducing cell proliferation are able to increase the amount of gC1qR in the surrounding milieu [37], these observations indicate that release of soluble gC1qR is regulated by core-mediated inhibition of cell proliferation. Moreover, they suggest that a novel mechanism capable of modulating gC1qR expression is operative. However, further investigations are needed to determine the precise activating conditions which result in its HCV core-mediated release. Once released, this receptor regulates complement activation and consumption of the C4 fraction in vascular bed and deposition of C4d fragment-containing ICs within tissues. In our proposed model, gC1qR initiates kinin generation as well as clotting on the endothelial surface via the contact system, resulting in damage to the vessel walls [38]. A genetic polymorphism of the gC1qR gene could explain dysregulation of its soluble levels in MC patients. However, thus far there are no such data and whether a gC1qR high-producer allele is associated with MC remains to be ascertained.

References 1. Saadoun D, Rosenzwajg M, Landau D et al (2008) Restoration of peripheral immune homeostasis after rituximab in mixed cryoglobulinemia vasculitis. Blood 111(11):5334–5341 2. Sansonno D, Lauletta G, Nisi L et al (2003) Non-enveloped HCV core protein as constitutive antigen of cold-precipitable immune complexes in type II mixed cryoglobulinaemia. Clin Exp Immunol 133(2):275–282

95 3. Sabile A, Perlemuter G, Bono F et al (1999) Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates. Hepatology 30:1064–1076 4. Kurtz JB, Boxall F, Qusir N et al (2001) The diagnostic significance of an assay for ‘total’ hepatitis C core antigen. J Virol Methods 96:127–132 5. Gorevic PD, Frangione B (1991) Mixed cryoglobulinemia cross-reactive idiotypes. Implications for the relationship of MC to rheumatic and lymphoproliferative diseases. Semin Hematol 28:79–94 6. Lindahl G, Sjobring U, Johnsson E (2000) Human complement regulators: a major target for pathogenic microorganisms. Curr Opin Immunol 12:44–51 7. Kittlesen DJ, Chianese-Bullock KA, Yao ZQ et al (2000) Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation. J Clin Invest 106:1239–1249 8. Yao ZQ, Nguyen DT, Hiotellis AI et al (2001) Hepatitis C core protein inhibits human T lymphocyte responses by a complement-dependent regulatory pathway. J Immunol 167: 5264–5272 9. Dammacco F, Sansonno D, Piccoli C et al (2000) The lymphoid system in hepatitis C virus infection: autoimmunity, mixed cryoglobulinaemia, and overt B-cell malignancy. Semin Liver Dis 20:143–145 10. Yao Z-Q, Prayter D, Trabue C et al (2008) Differential regulation of SOCS-1 sognalling in B and T lymphocytes by hepatitis C virus core protein. Immunology 2:197–207 11. Lim BL, Reid KBM, Ghebrehiwet B et al (1996) The binding for globular heads of complement C1q, gC1qR. Functional expression and characterization as a novel vitronectin binding factor. J Biol Chem 271:26739–26744 12. Sansonno D, Cornacchiulo V, Iacobelli AR et al (1995) Localization of hepatitis C virus antigens in liver and skin tissues of chronic hepatitis C virus-infected patients with mixed cryoglobulinaemia. Hepatology 21:305–312 13. Sansonno D, Gesualdo L, Monno C et al (1997) Hepatitis C virus-related proteins in kidney tissue from hepatitic C virusinfected patients with cryoglobulinemic membranoproliferative glomerulonephritis. Hepatology 25:1237–1244 14. Ghebrehiwet B, Lim B-L, Kumar L et al (2001) gC1q-R/ p33: a member of a new class of multifunctional and multicompartimental cellular proteinsis involved in inflammation and infection. Immunol Rev 180:65–77 15. Joseph K, Ghebrehiwet B, Peerschke EI et al (1996) Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular “heads” of C1q (gC1q-R). Proc Natl Acad Sci U S A 93: 8552–8557 16. Lim BL, Reid KBM, Ghebrehiwet B et al (1996) The binding protein for globular heads of complement C1q, gC1qR. Functional expression and characterization as a novel vitronectin binding factor. J Biol Chem 271:26739–26744 17. Paul DB, Kuhns MC, McNamara AL et al (1995) Short-term stability of HIV provirus levels in the peripheral blood of HIV-infected individuals. J Med Virol 47:292–297 18. Simos G, Georgatos SD (1994) The lamin B receptor-associated protein p34 shares sequence homology and antigenic determinants with the splicing factor 2-associated protein p32 [letter]. FEBS 346:225–228

96 19. Krainer AR, Mayeda A, Kozak D et al (1991) Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70 K, and drosophila splicing regulators. Cell 66:383–394 20. Seytter T, Lottspeich F, Neupert W et al (1998) Mam33p, an oligomeric, acidic protein in the mitochondrial matrix of Saccharomyces cerevisiae is related to the human complement receptor gC1q-R. Yeast 14(4):303–310 21. Sunayama J, Ando Y, Itoh N et al (2004) Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore forming protein p32. Cell Death Differ 11:771–781 22. Matthews DA, Russell WC (1998) Adenovirus core protein V interacts with p32 a protein which is associated with both the mitochondria and the nucleus. J Gen Virol 79: 1677–1685 23. Luo Y, Yu H, Peterlin BM (1994) Cellular protein modulates effects of human immunodeficiency virus type I Rev. J Virol 68:3850–3856 24. Joseph K, Ghebrehiwet B, Kaplan A-P (2001) Activation of the kininforming cascade on the surface of endothelial cells. Biol Chem 382:71 25. Peerschke EI, Ghebrehiwet B (1988) Identification and partial characterization of human platelet C1q binding sites. J Immunol 141(10):3505–3511 26. Jiang J, Zhang Y, Krainer AR et al (1999) Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein. Proc Natl Acad Sci USA 96(7):3572–3577 27. Zlatarova AS, Rouseva M, Roumenina LT et al (2006) Existence of different but overlapping IgG- and IgM-binding sites on the globular domain of human C1q. Biochemistry 45(33):9979–9988 28. Gaboriaud C, Jaunhuix J, Gruez A et al (2003) The crystal structure of globular head of complement protein C1q pro-

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Part IV Structural and Genetic Features, Cytokines and Chemokines in Cryoglobulinemia

Mixed Cryoglobulinemia (MC) Cross-Reactive Idiotypes (CRI): Structural and Clinical Significance

12

Peter D. Gorevic

12.1

Introduction

The early history of mixed cryoglobulinemia (MC) included recognition of a bias toward oligoclonality or monoclonality apparent in either the IgM or IgG constituents of mixed cryoglobulins. In particular, IgM rheumatoid factor (RF) was found to be strikingly (>95%) skewed toward the utilization of k light chains, leading to the designation “type 2 cryoglobulinemia.” The percent contribution of type 2 cryoglobulinemia has increased in large series as the use of immunofixation has become routine in most clinical laboratories and with the recognition of the increased incidence of clonal IgMk RF associated with hepatitis C virus (HCV) infection and with primary SS. Intermediate forms of type 2 cryoglobulins have been described, spanning the spectrum between polyclonality and monoclonality [1], but are not necessarily part of a continuum that represents a temporal evolution of the immune reaction to HCV antigens [2]. The clonality of IgMk was confirmed by sequence analysis of Vk at the protein level, demonstrating conservation of hypervariable (CDR) and framework (FR) sequences among cryoglobulin IgMk proteins isolated from unrelated individuals. These studies focused on the light chain because of the relative ease of carrying out protein

P.D. Gorevic Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA e-mail: [email protected]

sequence analysis of k light chains whereas there is considerably less information regarding m VH protein sequences. Conservation of Vk and VH amino acid sequences provided, in part, a basis for the recognition of cross-reactive idiotypes (CRIs) among cryoglobulin IgMRF, as had been established serologically with polyclonal antibodies (Wa, Po, and later Bla). The latter were utilized to define broad subsets of cross-reactivity among type 2 cryoglobulins and correlated predominantly (but not exclusively) with light or heavy chain antigenic determinants. Further refinement of these CRIs came from the development of monoclonal antibodies (MAbs) to MC CRIs (17.109; 6B6.6; G6; Lc1; JG-B1; B6 and G8) and the use of synthetic peptides corresponding to conserved CDR2 (PLS2) and CDR3 (PSL3) sequences of mixed cryoglobulins to generate additional polyclonal reagents. The composite value of these antibodies for the definition of MC CRIs was to establish that: (a) cross-idiotypy may be a function of structural determinants of the variable region of IgMk, which may encompass the light and/or heavy chain, FR and CDR sequences, and even the antigen-binding site, and (b) MCI CRIs are significantly shared with primary SS and with specific lymphoproliferative diseases, some of which may be associated with MC or SS. The purpose of this chapter is to update an earlier review of MC CRI [3] to include the association of MC and certain lymphoproliferative diseases with HCV infection/exposure and to incorporate nomenclature for V-region genes that has been made possible by full sequencing of the VH and Vk gene loci [4].

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_12, © Springer-Verlag Italia 2012

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100

12.2

P.D. Gorevic

Further Studies of IgM RF Cross-Reactive Idiotypes

The polyclonal antibody Wa recognizes ~80% of monoclonal (m)IgMk mixed cryoglobulins. Its specificity is predominantly directed to the heavy chain but is also dependent on pairing with specific VkIII light-chain products. The VH specificity is directed to either VH1 (V1-69/DP-10/51p1) or VH3 (V3-7/ DP-53) heavy chains, the former coexpressed with V3-20 and the latter with V3-15k V-region genes; that is, there is a skewing of the combinatorial pairing of specific heavy- and light-chain V-region genes that is characteristic of type 2 cryo-mIgMk. Both Vk3 genes utilize a Jk1 joining segment for expression. These VH1 genes preferentially make use of the D3-22 diversity gene segments, with VH1-69 rearranging to JH4 and D region consensus 1, and V3-7 rearranging to JH3 or JH4 gene segments and D region consensus 2 [1]. B6 is an IgG1k MAb raised against a human IgM RF paraprotein; it is reactive with 24% of mRF (k) and 15% of non-RF mIgM, with predominantly H-chain specificity. Further analysis indicated that this MC CRI is encoded in a set of closely related (V3-11/DP-35/22-2B, V3-7, V3-30/DP-49,1.9III/ hv3005, V3-30.3/DP-46/56p1/GL-SJ2 and V3-33/ DP-50/3019b9) genes from the VH3 family, with crucial involvement of the lysine at position 57 of CDR2. Increased expression of the CRI was identified in primary SS but not in chronic lymphocytic leukemia (CLL) [3, 5]. G6 and G8 are MAbs produced in response to an IgMk RF. They are reactive with 35% of mRF (k) and 5% and 0% of non-RF mIgM, respectively, both with predominantly H-chain specificity. Further analysis indicated that these MC CRIs are encoded by the VH169 multiallelic gene locus and are directed to conformational epitopes requiring pairing with kIIIb (A27/ humkv325/DPK22) light chains. G6 and G8 bind distinct and overlapping allelic variants comprising the gene segment 51p1 (VH783) at the VH1-69 locus, with major idiotopes mapping to CDR2 but also involving FR1 and FR3, which are topographically related to the antigen-binding site. Increased expression of the CRIs for both these MAbs was identified in marginal B cells of lymphoid follicles and primary SS as well as in 20–22% patients with CLL and 13% of patients with lymphoma [3, 6].

12.3

Mixed Cryoglobulinemia Cross-Reactive Idiotypes and HCV Infection

In most series, 60–80% patients with MC are productively infected by HCV, and up to 60% of patients with HCV have cryoprecipitable and non-cryoprecipitable immune complexes, with both the laboratory phenomenon and overlapping syndromes of vasculitis, glomerulonephritis, arthropathy, and neuropathy correlating with disease that is more severe and of longer duration. In addition, a direct role for HCV, as well the immune response to virus and nucleocapsid, is indicated by the concentration of antibodies to HCV antigens, notably including core antigen and viral nucleic acid binding predominantly to the IgG fraction of mixed cryoglobulins [7]. Cc1 and Lc1 are MAbs produced to different mRFs that react with H chain determinants associated with VH1 and VH4 gene products, respectively. Cc1 is reactive with ~70% of mIgMk RF and Lc1 with ~25%. Both CRIs are significantly expressed in patients with primary SS [8] and among patients infected by HCV. These MAbs are reactive with MC CRIs expressed on the surface of B cells and (particularly Cc1) have been used to purify subsets in which HCV RNA could be demonstrated by RT-PCR [9].

12.4

Clonal Populations of B Cells in Liver, Peripheral Blood, and Bone Marrow in HCV Infection

In situ hybridization and sequencing of CDRH3 gene segments showed that oligoclonal or monoclonal expansions of B cells accounted for ~50% of the lymphoid follicles isolated by microdissection of the livers of HCV-infected patients. In addition, the presence of clonal RF-producing cells could be demonstrated by analysis of gene product. Similar expansions were also identified in bone marrow (6%) and peripheral blood (26%) [10, 11]. Three types of monoclonal B-cell lymphocytosis (MBL) have been identified by FACS analysis in the peripheral blood of ~30% patients infected by HCV: “atypical” CLL MBL (CD5+CD20bright), CLL-like (CD5bright, CD20dim), and CD5-MBL (0.85 ng/mL, black bars) was significantly more frequent both in MCsn patients (*p < 0.0001) and in HCV-positive patients without MCsn (**p = 0.0026). BLOQ Below the level of quantitation (0.85 ng/mL) for BLyS and two of them (arrows) had “high” BLyS levels (>2.82 ng/mL, i.e. mean + 2 SD of the MCsn patients’ distribution). (From [44], with the permission of Rheumatology, Oxford University Press)

*

**

MCsn (66)

HCV-positive w/o MCsn (33)

121

* **

80 60 40 20 0

BLOQ (%)

b

BLyS (ng/ml)

15

HBDs (48)

>0.85 ng/ml (%)

*** 12 11 10 9 8 7 6 5 4 3 2 1 0

2.82 ng/ml 0.85 ng/ml MCsn (66)

HCV-positive w/o MCsn (33)

indicating that high BLyS serum levels at the onset of AHC are a predictor of disease evolution to chronic infection [46]. BLyS serum levels higher than in the general population were detected in a percentage of both MCsn patients and HCV-infected patients without MCsn (with or without serum cryoglobulins). Why such an increase is limited to a fraction of MCsn and HCV-positive cases is unknown. Similar observations were previously reported in RA, SLE, and SS, thus suggesting individual genetic predisposition [47]. Several polymorphisms in the BLyS gene have been described [48, 49], some of which influence BLyS expression. Accordingly, infection (by HCV or other infectious agents in HCV-negative MCsn, as well as in other autoimmune diseases) in a subset of genetically predisposed individuals would induce BLyS expression, possibly favoring the subsequent development of autoimmune and lymphoproliferative features in some of them.

HBDs (48)

While the etiologic role of chronic infection is well established in MCsn, such information is lacking in SLE, RA, and SS, in which the role of a putative infectious trigger may be transient, leading to a secondary, fully self-perpetuating autoimmune response. Seen in this light, BLyS up-regulation in HCV-positive MCsn, and in HCV infection overall, serves as a general model linking viral infection, B cell proliferation, and autoimmune disease [50]. Of note, BLyS expression is also elevated in SS, the leading autoimmune disease associated with HCVnegative mixed cryoglobulinemia (with or without MCsn). Furthermore, the B-cell clones proliferating in SS express immunoglobulin gene sequences with remarkable similarities to those expanded in HCVrelated MCsn [51]. T cells were shown to abnormally produce BLyS in SS salivary lesions, in which monoclonal RFs with the cryoglobulin idiotype are seen [52]. Epithelial salivary cells may also secrete

122

BLyS in SS [9]. Thus, BLyS expression in SS-related MALT (mucosa-associated lymphoid tissue) lesions might be linked to local B-cell expansion and cryoglobulin production [53, 54]. BLyS is significantly elevated in the course of several human hematological diseases, such as Hodgkin’s and non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, and multiple myeloma [55–58]. In our series [44], MCsn patients with an overt lymphoproliferative disease had high BLyS levels more frequently than patients with MCsn but without lymphoproliferative disease (p = 0.04), consistent with the reported association between BLyS and lymphoproliferation. In contrast, BLyS was not associated with any of the specific organ manifestations that characterize MCsn (neuropathy, nephritis, skin ulcers, etc.). These data were confirmed by French authors [45], who showed that BLyS serum levels were positively associated with markers of HCV-associated B-cell lymphoproliferation. In addition, serum levels of BLyS were higher in HCVinfected patients with type II MC, high MC levels, positive RF, and associated systemic vasculitis. Finally, serum BLyS concentrations were higher (two-fold increase) in HCV-related MC vasculitis than in either healthy controls or patients with chronic HCV infection without MC vasculitis, and HCV-related B-NHL was associated with an even greater increase (threefold) in serum BLyS [59]. The immunochemical types of MC may vary during the disease course [60]. Type III MC can evolve to an oligoclonal form and finally to type II MC, thus evidencing a monoclonal component [61, 62]. BLyS serum levels may follow this course, increasing as B-cell monoclonality (type II MC) appears [45, 59]. The relationship between serum BLyS and MC levels suggests a role for BLyS in sustaining a high level of immunoglobulin secretion by B cells, as demonstrated in mice overexpressing BLyS [24–26]. Regarding HCV-related MCsn, HCV infection could be the earlier, critical event that leads to BLyS elevation, rather than a triggering by downstream biological events, when serum cryoglobulins appear (detected in up to 40% of HCV-positive subjects), or coincident with the appearance of overt MC syndrome (occurring in 1–5% of HCV-positive subjects). In this scenario, BLyS would play a role as an early, chronic “background” stimulus for B-cell autoimmunity and lymphoproliferation in a subgroup of HCV-infected individuals [44, 50].

M. Fabris and S. De Vita

15.4

BLyS, HCV, and B-Cell Lymphomagenesis

Autocrine production of BLyS has been documented in cases of neoplastic B-cell proliferations [63]. A very recent study [64] demonstrated that both the canonical and alternative NF-kB pathways are constitutively activated in diffuse large B-cell lymphoma, in which NIK kinase aberrantly accumulates in the neoplastic cells due to constitutive activation of BAFF-R, through interaction with autochthonous BLyS ligand, finally leading to autonomous lymphoma cell growth and survival. These results are a very important contribution to elucidating the mechanisms involved in abnormal NF-kB activation in neoplastic B-cells and to better defining new therapeutic approaches for patients with lymphoma, and possibly also those with pre-lymphomatous diseases. A critical step could be the acquisition of autocrine BLyS expression capacity, with intervention at this point perhaps essential to reducing the risk of subsequent, fully irreversible neoplastic transformation. B-cell lymphoma is a relevant risk in MCsn patients [65]. This is consistent with the hypothesis that HCV initiates a multistep process of lymphomagenesis in which additional factors (i.e., genetic, environmental, immunological) are involved [44, 47, 48, 66]. The regression of established HCVassociated B-cell lymphoma in MCsn patients after antiviral treatment reflects the role of HCV in lymphomagenesis. However, in 14 patients with HCV-related non-Hodgkin’s lymphoma (splenic lymphomas) who received antiviral therapy, long-term clinico-pathologic and molecular follow-up showed clinical and pathological disease regression after successful antiviral treatment but also persistence of the lymphomatous clone. In fact, monoclonal B-cell expansion was still detectable in the peripheral blood lymphocytes of all the patients, demonstrating an absence of a molecular regression and the persistence of an antigen-driven B-cell proliferation, despite effective clearance of the HCV infection [67]. Therefore, HCV infection may represent the initiation step, which is followed by a perpetuation step in which autoantigens, infectious triggers (including HCV itself), cell deregulation at the molecular level, anti-apoptotic factors, and B-cell growth factors, such as BLyS, may sustain, to differing degrees, autoimmunity and B-cell lymphoproliferation [44, 68, 69].

15

Up-Regulation of B-Lymphocyte Stimulator (BLyS) in Patients with Mixed Cryoglobulinemia

INFECTION + AUTOIMMUNITY

HCV targeting

AUTOIMMUNE DISEASE

Direct B cell targeting

Indirect B cell targeting

Fig. 15.2 HCV targeting, direct and indirect B cell targeting. HCV infection and autoreactivity may play different roles in sustaining B-cell autoimmunity or autoimmune disease and lymphoproliferation in different cases of MCsn. There is a strong rationale for therapy directed against the infectious triggering antigen or for an approach based on direct or indirect B-cell targeting, depending on the specific case and the disease stage. Sequential or combination approaches also merit investigation

15.5

BLyS and HCV Infection: Implications for Treatment Strategies in MCsn

Consistent with the latter statement, direct targeting of the infectious trigger or the B-cell autoimmune and lymphoproliferative disorder (e.g., by means of anti-CD20 treatment) or indirect B-cell targeting (e.g., by drugs blocking B-cell growth/anti-apoptotic factors) may prove effective in HCV-related MCsn [50] (Fig. 15.2). Antiviral therapy against HCV has been shown to significantly increase BLyS levels in CHC patients. IFN-a, rather than ribavirin, may lead to increased BLyS, as previously demonstrated by in vitro studies [4]. This effect was independent from the concomitant HCV-RNA clearance, although two studies noted a trend toward BlyS normalization after suspension of antiviral treatment only in patients who achieved a virological response [44, 46]. Those studies reported that the up-regulation of BLyS levels during antiviral therapy appeared to be reversible [44, 46]. IFN therapy can be effective in HCV-related MCsn, mainly in patients who become HCV-RNA-negative [70]. However, MCsn patients may also show persistently active disease despite viral RNA negativization [71], with persistent positivity of RF and cryoglobulins, i.e. the autoimmune and lymphoproliferative disorder persists. Finally, some MCsn manifestations, such as neuropathy, nephritis, and skin ulcers, may worsen

123

[72]; indeed, the onset of MCsn was reported after IFN therapy effective against HCV infection [73, 74]. Therefore, it cannot be ruled out that IFN-a related BLyS up-regulation in part contributes to these effects in predisposed individuals. BLyS may favor the survival of RF-positive B-cells, which may undergo antigen stimulation by different immune complexes also in the absence of the original HCV trigger [50, 75]. Anti-CD20 therapy similarly up-regulates the BLyS system. An increase in BLyS soon after B-cell depletion therapy with rituximab (RTX) was reported in SLE [76], RA [77], and SS [54] patients, as well as in MCsn patients [59] (Fabris M, personal communication). Both antiviral therapy and RTX may then also favor biological mechanisms implicated in the persistence of B-cell expansion, through BLyS up-regulation. Accordingly, a therapeutic strategy specifically targeting BLyS may be appropriate in MCsn [50]. Anti-BLyS therapy with belimumab appears to be effective and safe in SLE [78, 79] and was partially effective in RA. A trial with belimumab in the treatment of SS patients is ongoing in our institution. Other factors or mechanisms sustaining B-cell expansion, e.g., IL-6, the CD40/40 L system, or the B7/CD28 system, may also be successful targets. Finally, the sequential or combined use of the three different treatment strategies (anti-viral, anti-B cell direct, anti-B cell indirect) also deserves attention [80].

15.6

Conclusion

The discovery of BLyS and thus its possible role in HCV infection, B-cell proliferation, and autoimmunity, including the development, persistence, and evolution of MCsn, is currently a topic of intensive research. Strikingly, these observations provide the basis for a model with which to better dissect the link between infection, autoimmunity, and lymphoproliferation in other autoimmune and lymphoproliferative diseases of unknown etiology. Furthermore, determinations of BLyS levels and genetics in patients with MCsn and other autoimmune diseases may allow better classification of these patients in addition to providing predictors of response. Finally, different anti-BLyS treatment options are available, with phase III trials successfully completed. The application of these treatments, as for other biologics, should result in a better understanding of disease biology.

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Role of B-Cell-Attracting Chemokine-1 in HCV-Related Cryoglobulinemic Vasculitis

16

Sabino Russi, Silvia Sansonno, Gianfranco Lauletta, Domenico E. Sansonno, and Franco Dammacco

16.1 Introduction Chronic active liver disease is an inflammatory disorder in which several distinct etiologies and pathogenetic mechanisms have been recognized [1]. Within the inflamed liver, there is an accumulation of lymphoid and myeloid cells, including T and B cells [2]. Local activation of these cells is thought to play an essential role in perpetuating the chronic inflammatory process and enhancing liver damage [3]. T and B cells frequently accumulate in the portal tracts, where they become part of follicle-like structures with features of germinal centers [4]. At these sites, both local differentiation of follicular dendritic cells and plasma cells and antibody production may occur [5]. Moreover, the development of these germinal-center-like structures appears to contribute to the pathogenesis of the disease, as evidenced by functional and molecular analyses showing that they are characterized by B-cell oligoclonal/monoclonal expansions [6, 7]. These B-cell clonotypes are of B-cell origin, indicating that IgH VDJ mutational activity is up-regulated in the liver microenvironment [8]. It has also been shown that distinct B-cell expansions contribute to the formation of intraportal follicle-like structures that possibly represent peculiar features of hepatitis C virus (HCV) infection [9]. In this context, sequence analyses of IgH CDR-3 gene segments have revealed a wide range of variations likely to be the result of an antigen-driven response [10]. S. Russi (*) Department of Internal Medicine and Human Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

The occurrence of B-cell clonal expansions in the liver of HCV-infected patients has been found to deeply influence the clinical picture, in that they are strictly related to mixed cryoglobulinemia (MC) and, in general, to lymphoproliferative features, including the unexplained high serum levels of rheumatoid factor (RF) and a monoclonal gammopathy of undetermined significance [8]. The relationships between the emergence and persistence of intrahepatic or circulating B-cell clonotypes and HCV infection are not known. Accumulating evidence indicates that certain chemokines are critical to providing the appropriate environment for the activation and expansion of naïve lymphocytes in response to signals delivered by antigen-presenting cells [11]. B-cell-attracting chemokine-1 (BCA-1, CXCL13), also referred to as B-lymphocyte chemoattractant, is a member of the CXC subtype of the chemokine superfamily [12]. It is essential for secondary lymphoid tissue development and the attraction of lymphocytes within microenvironments [13]. The BCA-1 gene maps to chromosome segment 4q21 and encodes a putative protein of 109 amino acids [12]. Within the human BCA-1 sequence, an arginine residue separates two of the four conserved cysteine residues that are peculiar to CXC chemokines [12, 13]. The primary BCA-1 receptor is CXCR5, a seven-transmembrane G-protein expressed by B lymphocytes [14], follicular helper T cells [15], osteoblasts [16], podocytes [17], and skinderived dendritic cells [18]. Interestingly, BCA-1 and CXCR5 knockout mice exhibit similar abnormalities, including a deficient development of peripheral lymphoid organs, impaired B-cell translocation to the B/T cell boundary, and reduction of the antibody response [19, 20]. BCA-1 is constitutively expressed in the

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_16, © Springer-Verlag Italia 2012

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128

300 Serum BCA-1 (pg/mL)

B-cell follicles of secondary lymphoid organs [12], in pleural and peritoneal cavities [19], and in ectopic lymphoid follicles within the synovium of patients with rheumatoid arthritis [21]. BCA-1 protein and the transcription of its gene have been determined in MC patients, leading to the hypothesis that BCA-1 contributes to the pathogenesis of cryoglobulinemic vasculitis by B-cell deregulation.

S. Russi et al.

p < 0.02

p < 0.007

200 p < 0.01 100

16.2 BCA-1 Levels in the Serum of MC Patients With MC

16.3 BCA-1 Expression in Tissues BCA-1 protein, as studied by indirect immunofluorescence in skin biopsy samples of MC patients, is mostly expressed along the collagen bundles and involves the superficial dermis, with variable mild/ deep dermal extension in patients with active vasculitis (Fig.  16.3). By contrast, BCA-1-protein deposition was not detected in skin biopsy tissues from healthy subjects or from HCV-infected patients with-

Healthy Controls

Fig. 16.1  BCA-1 serum levels in HCV-infected patients with and without MC and in healthy controls Cryoglobulinemic Vasculitis 900

Active

Non-active

800 Serum BCA-1 (pg/mL)

Serum measurements of BCA-1 are reported in Fig. 16.1. Compared with healthy blood donors, mean serum BCA-1 levels were higher in patients with MC than in those without the disease. It was also shown that BCA-1 serum levels were significantly higher in MC patients than in patients without MC. No correlation has been established between BCA-1 concentration and circulating viral load, liver histology activity index, grade of liver fibrosis, ALT activities, cryocrit percentages, serum IgM concentration, RF activity, or C4 levels. We stratified MC patients according to the occurrence of active or non-active cutaneous vasculitis. Although not reaching significance, the average cryocrit value was lower in patients with active vasculitis, whereas type III MC occurred more frequently in those with non-active vasculitis. Serum IgM levels, RF activity, complement C4 concentration, and the frequency of nephropathy and peripheral neuropathy were not significantly different in the two subgroups. Conversely, a significantly higher (p 90%)

Primary SS salivary gland inv. anti-SSA/SSB+, RF+ HCV+ rare

Autoimm. hepatitis ALT+, ASMA+/ANA+, RF+/– autoimm. systemic symptoms HCV +/–

Rheumatoid Arthr. symmetrical erosive polyarthritis, anti-CCP+, RF+/–, rare HCV+

Fig. 19.2 The potential overlap between mixed cryoglobulinemia (MC) syndrome and some autoimmune-lymphoproliferative disorders, such as primary Sjögren’s syndrome (pSS), rheumatoid arthritis (RA), autoimmune hepatitis (AIH), and B-cell non-Hodgkin’s lymphoma (B-NHL). Each disease may exhibit, during its clinical course, one or more autoimmune features, such as vasculitic purpura, sicca syndrome, arthralgias/arthritis, and thyroiditis, with the expression of serum RF and other autoantibodies. In addition, HCV infection, detectable in >90% of MC patients, may be involved in a significant percentage of B-NHLs and in a small percentage of other diseases. Given the presence of these common clinico-serological and virological findings, it is difficult to correctly classify individual patients. For the differential diagnosis, certain important parameters may be usefully employed: in addition to the histopathological characteristics, severity of salivary gland involvement and specific autoantibodies (anti-RoSSA/LaSSB) are rarely found in MC patients; conversely, cutaneous leukocytoclastic vasculitis,

visceral organ involvement (MPGM: membranoproliferative glomerulonephritis, hepatitis), low C4, and HCV infection, typically seen in MC, are seldom in primary SS. In the setting of HCV infection, there may be B-NHL, but this malignancy can also be a late complication of MC syndrome and pSS. In these cases, the patient’s previous clinical history and serological markers may facilitate the diagnosis. Arthralgias and arthritis are frequent manifestations of different autoimmune diseases, but erosive symmetrical polyarthritis and serum anti-cyclic citrullinated peptide antibodies (anti-CCP) are specific findings of classical RA. Finally, AIH may share various extrahepatic symptoms with other disorders; however, the activity/severity of hepatitis along with the presence of specific serological markers may help to differentiate AIH from other conditions, mainly HCV+MC patients. The overlapping territory involving these disorders frequently represents a gray zone in which the diagnosis in single patient may remain doubtful

A small proportion of MC patients develop widespread vasculitis involving small to medium-sized arteries, capillaries, and venules, with multiple organ involvement, namely, the skin, kidneys, lungs, central nervous system, and gastrointestinal tract [1–3, 11, 12]. In rare cases, intestinal vasculitis, typically

presenting as pain simulating an acute abdomen, may suddenly complicate the disease, often in patients with renal and/or liver involvement. A timely diagnosis and aggressive steroid treatment are necessary to intervene in this life-threatening complication [1, 2].

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Clinically overt interstitial lung involvement has been anecdotally observed in MC syndrome as well as in patients with isolated HCV infection [1, 2]. More often, lung involvement in MC is characterized by subclinical alveolitis, as demonstrated by means of broncho-alveolar lavage in unselected patient series [1, 2]. Hyperviscosity syndrome, due to high levels of serum cryoglobulins, is another rare clinical manifestation of MC, while hemorheological alterations may contribute to clinical symptoms such as orthostatic purpura, skin ulcers, and renal involvement [1, 2]. The severity of clinical symptoms generally does not correlate with serum cryoglobulin levels and/or hemolytic complement consumption [1, 2]. The latter is characterized by a typical pattern that is independent of disease activity: low or undetectable C4 with normal or slightly reduced C3 serum levels (Table 19.1). In some cases, sudden variations of C4, increasing from very low to abnormally high levels, may be the presenting symptom of B-cell lymphoma complicating MC [1, 2]. Some endocrinological diseases are significantly more frequent in MC patients than in the general population, including thyroid disorders, diabetes, and gonadal dysfunction [1, 2, 13, 14]. The most common thyroid disorders are autoimmune thyroiditis, subclinical hypothyroidism, and thyroid cancer; overt hyperthyroidism is less frequent and may occur as a reversible complication of IFN treatment. In addition, the incidence of diabetes mellitus type 2 in HCV-positive patients with and without MC syndrome was shown to be a statistically higher than in the general population [14]. Finally, HCV-positive males with or without cryoglobulinemic vasculitis may develop erectile dysfunction, attributable to hormonal and/or neurovascular alterations [1, 2]. B-cell lymphoma is one of the most frequent malignancies complicating the clinical course of MC syndrome, often as a late disease manifestation [1, 2, 5, 15] (Figs. 19.1 and 19.2). This complication may be related to peripheral B-lymphocyte expansion as well as to the lymphoid infiltrates, which represent the pathological substrate of the disease and are observed in the liver and bone marrow [1, 2, 5, 8]. Indeed, these infiltrates are regarded by some authors as “early lymphomas,” since they are sustained by lymphoid components indistinguishable from those of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL) and immunocytoma

C. Ferri et al.

(Ic) [1, 2, 5]. However, unlike frank malignant lymphomas, they tend to remain unmodified for years or even decades and are followed by overt lymphoid tumors only in about 10% of patients [1, 2, 5]. These characteristics justify the proposed term “monotypic lymphoproliferative disorder of undetermined significance” (MLDUS) [1, 2, 5, 8]. Interestingly enough, type II MC-related MLDUS has its highest incidence in the same geographic areas where about 30% of patients with “idiopathic” B-cell lymphomas also display HCV positivity and where an increased prevalence of HCV genotype 2a/c has been observed in both MC and lymphomas [1, 2, 5, 8]. Type II MC-associated MLDUS presents two main pathological patterns: B-CLL-like and Ic-like [1, 2, 5]. In clinical practice, MC patients with malignant B-cell lymphomas with mild clinical course are not atypical and are sometimes diagnosed unexpectedly during a routine evaluation. There may be a sudden decrease or disappearance of serum cryoglobulins and RF, sometimes associated with abnormally high levels of C4; indeed, these serological variations may be the presenting findings of complicating B-cell malignancy [1, 2]. Among the neoplastic manifestations of MC, hepatocellular carcinoma occurs less frequently than noncryoglobulinemic type C hepatitis whereas papillary thyroid cancer is a rare complication [1, 2, 5, 16]. On the whole, since MC syndrome can be regarded as a pre-neoplastic disorder, careful clinical monitoring of these patients is recommended, even in the presence of a mild clinical course [1, 2, 5, 8].

19.5

Cryoglobulinemic Vasculitis and HCV Syndrome

Mixed cryoglobulinemia can represent a crossroads between certain autoimmune diseases (autoimmune hepatitis, Sjögren’s syndrome, polyarthritis, glomerulonephritis, thyroiditis, type 2 diabetes, etc.) and malignancies (B-cell lymphomas, hepatocellular carcinoma) [1–3, 5, 10–17] (Figs. 19.1 and 19.2). Consistent with the striking association between MC and HCV infection, the clinical history of the disease mirrors the natural course of this viral infection. It is not rare for patients to experience a slow disease progression: from mild HCV-associated hepatitis to various extrahepatic manifestations (arthralgias, sicca syndrome, Raynaud’s

19 The Expanding Spectrum of Clinical Features in HCV-Related Mixed Cryoglobulinemia

phenomenon, RF positivity, etc.), and ultimately to overt MC syndrome with typical clinico-serological manifestations. In a minority of these patients, a malignancy may develop, generally after a prolonged follow-up [1, 2]. Since the early 1990s, after the discovery of the causative role of HCV in MC [4, 7], the involvement of HCV in other extrahepatic disorders has gained increasing acceptance [5, 8, 17]. A correlation between several immune-mediated, organ- and non-organ specific disorders and HCV infection has been established. One of these, porphyria cutanea tarda (PCT) [5, 8], has been investigated in studies performed worldwide, reporting a wide range of associations [5, 8]. The pathogenesis of HCV-related PCT may be related to metabolic factors, in particular altered genes involved in iron metabolism, and host vs. HCV antigen cross-reactivity [5]. HCV-related lichen planus, i.e., orally located disease, is another possible association with variable geographic prevalence [5, 17] as well as several muco-cutaneous manifestations, as noted in a few reports or anecdotal observations [5, 17]. Peripheral neuropathy is a common complication of HCV infection, mainly in cryoglobulinemic vasculitis [1, 2, 5, 17], whereas central nervous system involvement is less common and more often affects patients with overt cryoglobulinemic vasculitis [5]. Some cardiovascular manifestations, mainly cardiomyopathy, during HCV infection have been reported in patient populations from Asian countries [1, 5, 18]. While a possible etiopathogenetic role of HCV in autoimmune hepatitis has been proposed, it is as yet controversial [1, 5]. A significant number of patients with autoimmune hepatitis may present with mixed cryoglobulins, HCV infection, and extrahepatic manifestations, such as thyroiditis, sicca syndrome, arthritis; conversely, in patients with HCV infection, one or more non-organspecific auto-antibodies are typically detected. The antigenic target specificity of HCV-related autoantibodies generally differs only quantitatively from that of autoantibodies associated with “primary” autoimmune hepatitis [5, 17]. Of interest, HCV-associated autoimmune hepatitis shows a clear heterogeneous geographic distribution, suggesting the involvement of various pathogenetic cofactors. In this context, HCV might trigger a peculiar clinico-serological subset of autoimmune hepatitis, mainly in specific geographic areas. The strength of the association with HCV as well as the pathogenic role of the virus varies largely among the different diseases and for a given disease

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among patient series from different countries [1, 5, 8]. Each disease can be regarded as a syndrome that includes distinct clinico-serological subsets, which in turn are the resulting phenotypes of multiple (genetic, environmental, infectious) pathogenetic cofactors. In this scenario, HCV-related clinical syndromes may trigger distinct autoimmune or neoplastic disease subsets, with additional important pathogenetic contributions. HCV-related disorders, or the HCV syndrome, represent a continuum, as suggested by the clinical history of some patients, in whom the entire spectrum may be displayed [5, 17]. In this setting, the expanding spectrum of HCV-related MC clinical features frequently reproduces the entire symptom complex of HCV syndrome.

References 1. Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 16(3):25, http://www.ojrd.com/content/3/1/25 2. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 33:355–374 3. Dammacco F, Sansonno D (1997) Mixed cryoglobulinemia as a model of systemic vasculitis. Clin Rev Allergy Immunol 15:97–119 4. Ferri C, Monti M, La Civita L et al (1994) Hepatitis C virus infection in non-Hodgkin’s B-cell lymphoma complicating mixed cryoglobulinaemia. Eur J Clin Invest 24:781–784 5. Ferri C, Antonelli A, Mascia MT et al (2007) HCV-related autoimmune and neoplastic disorders: the HCV syndrome. Dig Liver Dis 39:S13–S21 6. Zignego AL, Macchia D, Monti M et al (1992) Infection of peripheral mononuclear blood cells by hepatitis C virus. J Hepatol 15:382–386 7. Ferri C, Monti M, La Civita L et al (1993) Infection of peripheral blood mononuclear cells by hepatitis C virus in mixed cryoglobulinemia. Blood 82:3701–3704 8. Zignego AL, Ferri C, Pileri SA, et al for the Italian Association of the Study of Liver (AISF) (2007) Commission on Extrahepatic Manifestations of HCV infection. Extrahepatic manifestations of hepatitis C virus infection: a general overview and guidelines for a clinical approach. Dig Liver Dis 39:2–17 9. Zignego AL, Ferri C, Giannelli F et al (2002) Prevalence of Bcl-2 rearrangement in hepatitis C virus-related mixed cryoglobulinemia with or without complicating B-cell lymphoma. Ann Intern Med 137:571–580 10. Ramos-Casals M, De Vita S, Tzioufas A (2005) Hepatitis C virus, Sjogren’s syndrome and B-cell lymphoma: linking infection, autoimmunity and cancer. Autoimmun Rev 4: 8–15 11. Agnello V, De Rosa FG (2004) Extrahepatic disease manifestations of HCV infection: some current issues. J Hepatol 40:341–352

162 12. Dammacco F, Lauletta G, Montrone M, Sansonno D (2007) Mixed cryoglobulinemia: a model of virus-related disease in internal medicine. Dig Liver Dis 39:S8–S12 13. Antonelli A, Ferri C, Fallahi R et al (2004) Thyroid involvement in patients with overt HCV-related mixed cryoglobulinaemia. QJM 97:499–506 14. Antonelli A, Ferri C, Ferrari SM et al (2008) Immunopathogenesis of HCV-related endocrine manifestations in chronic hepatitis and mixed cryoglobuliemia. Autoimmun Rev 8:18–23 15. Monti G, Pioltelli R, Saccardo E et al (2005) Incidence and characteristics of non-Hodgkin lymphomas in a multicenter

C. Ferri et al. case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias. Arch Intern Med 165: 101–105 16. Antonelli A, Ferri C, Fallahi R (1999) Thyroid cancer in patients with hepatitis C infection. JAMA 281:1588 17. Ferri C, Mascia MT, Saadoun D, Cacoub P (2009) Cryoglobulinemia and systemic manifestations of hepatitis C virus. EULAR compendium on rheumatic diseases, Ed. BMJ Publishing Group Ltd, London. Chap. 42a accessed on May 1 2009 18. Matsumori A (2005) Hepatitis C virus infection and cardiomyopathies. Circ Res 96:144–147

Classification of Cryoglobulinemic Vasculitis

20

Salvatore De Vita and Luca Quartuccio

20.1

Introduction

Cryoglobulinemic syndrome or cryoglobulinemic vasculitis (CV) is a systemic vasculitis associated with serum positive cryoglobulins, usually linked to non-malignant B-cell lymphoproliferation [1–3] and often triggered by chronic hepatitis C virus (HCV) infection [4, 5]. Classification criteria developed according to an accepted methodology are presently lacking for CV. However, they are essential for epidemiologic studies and research, and in turn for clinical practice [6, 7]. Recent recommendations for the management of CV [8], for instance, cannot be well applied given the absence of a patient classification system.

20.2

Pre-existing Classification Criteria and the Need for New Criteria

Earlier classification criteria of CV were elaborated by individual experts or by a panel of experts [9–12]. The latter was the case for the 1995 criteria of the GISC (Italian Study Group on Cryoglobulinemia) [9], which required positive cryoglobulinemia (³1% cryocrit for at least 6 months), the presence of at least two of the three clinical manifestations of purpura, fatigue, and arthralgias, plus positive laboratory features including positive RF or low C4 (10 days, no cause) Fever (>38°C, no cause) Fibromyalgia Arthralgias • Articular involvement Arthritis •

Vascular involvement

•

Neurologic involvement

Raynaud’s phenomenon Purpura Necrotizing vasculitis Skin ulcers Hyperviscosity syndrome Peripheral neuropathy Cranial nerve involvement Vasculitic CNS involvement

iii. Laboratory item: at least 2 out of the following 3 (present) • • •

Reduced serum C4 Positive serum rheumatoid factor Positive serum M component

Fig. 20.1 Preliminary classification criteria for cryoglobulinemic vasculitis

and using a standardized methodology in a large number of real cases. They now need formal validation, possibly involving experts from a larger number of countries. In addition, they should be further tested in HCV-related vs. HCV-unrelated CV. These studies as well as more descriptive analyses and sub-analyses are ongoing or have been planned.

References 1. Meltzer M, Franklin EC (1966) Cryoglobulinemia – a study of twenty-nine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med 40(6): 828–836 2. Gorevic PD, Frangione B (1991) Mixed cryoglobulinemia cross-reactive idiotypes: implications for the relationship of MC to rheumatic and lymphoproliferative diseases. Semin Hematol 28(2):79–94 3. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do

4.

5.

6.

7.

8.

9.

10.

not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 43(1):94–102 Ferri C, Greco F, Longombardo G et al (1991) Antibodies to hepatitis C virus in patients with mixed cryoglobulinemia. Arthritis Rheum 34:1606–1610 Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327(21):1490–1495 Johnson SR, Goek ON, Singh-Grewal D et al (2007) Classification criteria in rheumatic diseases: a review of methodologic properties. Arthritis Rheum 57:1119–1133 Dougados M, Gossec L (2007) Classification criteria for rheumatic diseases: why and how? Arthritis Rheum 57: 1112–1115 Mukhtyar C, Guillevin L, Cid MC et al (2009) European Vasculitis Study Group. EULAR recommendations for the management of primary small and medium vessel vasculitis. Ann Rheum Dis 68:310–317 Invernizzi F, Pietrogrande M, Sagramoso B (1995) Classification of the cryoglobulinemic syndrome. Clin Exp Rheumatol 13(Suppl 13):S123–S128 Vitali C, Bombardieri S, Moutsopoulos HM et al (1993) Preliminary criteria for the classification of Sjögren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 36:340–347

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Classification of Cryoglobulinemic Vasculitis

11. Vitali C, Bombardieri S, Moutsopoulos HM et al (1996) Assessment of the European classification criteria for Sjögren’s syndrome in a series of clinically defined cases: results of a prospective multicentre study. The European Study Group on Diagnostic Criteria for Sjögren’s Syndrome. Ann Rheum Dis 55:116–121 12. Vitali C, Bombardieri S, Jonsson R et al (2002) European Study Group on Classification Criteria for Sjögren’s Syndrome. Classification criteria for Sjögren’s syndrome: a revised version of the European criteria proposed by the AmericanEuropean Consensus Group. Ann Rheum Dis 61:554–558 13. Lamprecht P (2004) The cryoglobulinemic syndrome – report from the workshop on classification and on the 10th

167 conference of the Italian Society for the Treatment of Cryoglobulinemia, Modena, 29 Sept, 2003. Z Rheumatol 63:235–238 14. De Vita S, Soldano F, Isola M et al (2010) Preliminary classification criteria for the cryoglobulinemic syndrome. Ann Rheum Dis 69(Suppl 3):77 15. De Vita S, Soldano F, Isola M et al (2010) Preliminary classification criteria for the cryoglobulinemic syndrome. Arthritis Rheum 62(Suppl 10):S851–S852 16. De Vita S, Soldano F, Isola M et al (2011) Preliminary classification criteria for the cryoglobulinemic vasculitis. Ann Rheum Dis 70(7):1183–1190

Demographic and Survival Studies of Cryoglobulinemic Patients

21

Giuseppe Monti, Francesco Saccardo, and Laura Castelnovo

21.1

Introduction

Cryoglobulinemic syndrome (CS) is highly heterogeneous in terms of its clinical presentation, the extent and severity of organ involvement, immunological abnormalities, and clinical course. It refers to the presence in the serum of immunoglobulins (Ig) that precipitate at temperatures below 37°C. Type I cryoglobulins are usually associated with lymphoproliferative disorders, formerly referred to as Waldenstrom disease, and with multiple myeloma, while mixed cryoglobulinemias (MCs), involving either type II or type III cryoglobulins, may be associated with connective tissue diseases, lymphoproliferative disorders, and chronic infections. A related and typical clinical manifestation of CS is vasculitis of the small and medium-size vessels. As CS is considered to be a relatively rare disorder, there are as yet no epidemiological studies regarding its overall prevalence, but according to the EURORDIS definition it is 20%, has the highest frequency) [6]. Moreover, the WHO has calculated that each year there are between three and four million new cases of hepatitis C [7]. Over the past 20 years, the incidence of HCV infection in Western countries has decreased due to greater safety in blood transfusions and improvements in health conditions; however, increased drug abuse and the immigration of people from areas with high distribution of the virus pose a challenge to preventing renewed increases in infection. In Northern Europe, the overall prevalence is between 0.1% and 1%. In central Europe, it is intermediate, ranging from 0.2% in the Netherlands to 1.2% in France, and in southern Europe, including Italy, the prevalence varies between 2.5% and 3.5% [6]. Given the worldwide presence of HCV infection, the overall prevalence of cryoglobulinemia is likely to be under-reported; moreover, a higher incidence of HCV-related mixed cryoglobulinemia can be expected,

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especially in underdeveloped countries. If measured with the appropriate methods, cryoglobulins are positive in about 47–73% of HCV patients. Viganò et al., in 2007 [8], detected cryoglobulins in 47% of their patients. In Ramos Casals’ study [9], 73% of patients had asymptomatic cryoglobulinemia while 27% presented with cryoglobulinemic symptoms. Our unpublished data show a prevalence of cryoglobulins in HCV patients of about 70%. Some studies report a small increased incidence of type II mixed cryoglobulinemia [10] whereas Viganò, in his 10-year prospective study, demonstrated a type III predominance (80%) [8]. Two studies [9, 11] reported a prevalence of HCV genotype 1, which was not confirmed in other reports [12–14]. Frank symptomatic MC is uncommon, probably occurring in 90% in more recent studies [7] (70% for patients with type III cryglobulins). The majority (>50%) of HCV-positive patients with MPGN will also have cryoglobulins in their serum [13, 14] and 30% of patients with mixed cryoglobulinemia will have some form of renal involvement [3, 7, 12]. Host immunogenetics appear to play a role in the development of cryoglobulinemia. In two studies, from Spain [15] and Italy [14], the presence of HLA-DRB1*11 was associated with 2.5-fold elevated risk, while HLA-DRB1*07 decreases this risk by 67%. However the interpretation of such associations as causal is complicated by the higher prevalence of HLA-DRB1*11 in HCV (+) patients [16] with normal ALT levels and milder hepatic injury. It is thus possible that the aforementioned associations between HLA alleles and cryoglobulinemia reflect a length-time bias; patients with milder hepatic injury survive longer, allowing for polyclonal activation of B cells followed by the emergence of a dominant clone after many years of antigenic stimulation. These epidemiologic observations suggest that the association between HCV infection and MPGN is mediated by the development of cryoglobulinemia. The latter reflects the presence of one or more immunoglobulins that precipitate at temperatures below

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Fig. 22.1 Peripheral blood smear in a patient with chronic hepatitis C virus (HCV) infection and renal cryoglobulinemia, showing aggregates of cryoglobulin precipitate. Some of these precipitates were counted by an automated blood cell counter as white blood cells, erroneously elevating the automated white blood cell count (Wright-Giemsa stain; 400×). The renal biopsy shown in Fig. 21.5 evidenced numerous cryoglobulin thrombi in glomerular capillaries

37°C but resolve upon rewarming (see Fig. 1 in [17]), Cryoglobulins are occasionally observed in peripheral blood smears (Fig. 22.1); they are classified pathologically in three groups depending on the type and clonality of the cryoglobulins. Type I cryoglobulins are monoclonal IgG, IgM, or IgA that self-aggregate through the Fc portion of the immunoglobulin molecule. Type II and III cryoglobulins are of mixed type, composed of monoclonal (IgMk) or oligoclonal IgMs respectively, with intrinsic rheumatoid factor activity which are complexed with antigens and polyclonal IgGs. Non-HCV conditions associated with cryoglobulins include other infections (hepatitis B, bacterial endocarditis, infectious mononucleosis, post-streptococcal glomerulonephritis, chronic parasitic infections), neoplasms (chronic lymphocytic leukemia, lymphomas), and autoimmune conditions (systemic lupus erythematosus, rheumatoid arthritis). In chronic hepatitis C infection, it is thought that the development of cryoglobulinemia is the result of the activation and proliferation of multiple clones of rheumatoid-factorproducing B cells infected by the HCV virus through CD81 receptors. After many years of stimulation, a dominant pre-malignant clone emerges, transforming the cryoglobulinemia from type III to type II. This orderly progression is supported by a number of epidemiologic observations, including a 30–200%

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increase in the risk of non-Hodgkin lymphoma and a 3-fold increased risk for Waldenström’s macroglobulinemia in HCV(+) patients [6, 18]. Approximately 70% of cryoglobulins causing renal disease are type II and 24% are type III [14]. Cryoglobulins are thought to mediate renal injury by a typical immune complex mechanism. Polymeric IgGs (i.e., in type III cryoglobulinemia) [19] and the IgMk rheumatoid factor of type II [20] have an affinity for fibronectin, which is found in the mesangium matrix. After localization to this area of the kidney, local activation of the classical complement pathway leads to a typical immune-complex glomerulonephritis. Other mechanisms have also been implicated in the intense cellular proliferative response seen in patient biopsies. Viral proteins and RNA are intrinsic components of the immune complexes seen in HCV-associated MPGN [21–23] and renal parenchymal cells express receptors (e.g., CD81) for such proteins. Activation of the pattern-recognition Tolllike receptors (TLR) in monocytes could trigger the innate immune system response; activation of TLR3 seems to differentiate HCV-associated from primary MPGN [24].

22.3

Pathology

Type I MPGN is the most common hepatitis-C-associated glomerular pattern of injury, with both cryoglobulinemic (majority) and non-cryoglobulinemic forms occurring. Type I MPGN is characterized by diffuse global endocapillary proliferation, with lobular accentuation, mesangial matrix expansion, glomerular capillary endothelial cell swelling, and glomerular capillary wall thickening (Fig. 22.2). Thickening of the glomerular capillary wall is due to mesangial interposition, characterized by ingrowth of mesangial cells and matrix into the contiguous subendothelial space of the glomerular basement membrane (GBM) and the formation of a new layer of subendothelial GBM, producing a double contour (tram tracking), as highlighted by silver stain (Fig. 22.3). In the cryoglobulinemic form of type I MPGN, there is frequently prominent infiltration of glomerular capillaries by circulating monocytes (Fig. 22.4), and PAS-positive hyaline thrombi are often present in glomerular capillary lumina (Fig. 22.5). Occasionally, cryoglobulin thrombi will occlude arterioles and small arteries (Fig. 22.6), sometimes producing microinfarcts. Rare patients develop

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HCV-Associated Membranoproliferative Glomerulonephritis

Fig. 22.2 Glomerulus from a patient with chronic HCV infection and type II cryoglobulinemia, who had type I membranoproliferative glomerulonephritis. Note the prominent lobular endocapillary proliferation (H&E; 400×)

Fig. 22.3 Portion of a glomerulus in a patient with type I membranoproliferative glomerulonephritis. Note the prominent glomerular basement membrane double contours (tram tracking) corresponding to mesangial interposition (methenamine silver; 1,000× oil immersion)

necrotizing vasculitis and/or crescents (Fig. 22.7) [7–9]. In the chronic phase of MPGN, the cellularity diminishes and there is increased mesangial sclerosis. Immunofluorescence microscopy reveals granular GBM and, less commonly, granular mesangial staining for IgG, IgM, and C3 (Fig. 22.8); approximately 50% of biopsies are also positive for C1q. In patients with cryoglobulinemia, hyaline thrombi with an identical staining profile may be present. Ultrastructural examination demonstrates electron-dense deposits in the interposed mesangium within the lamina rara interna of the GBM,

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Fig. 22.4 Portion of a glomerulus from the patient described in Fig. 22.2. Note the abundant circulating monocytes in the glomerular capillaries (methenamine silver; 600×)

Fig. 22.5 Glomerulus from the patient described in Fig. 22.1. Note the abundant PAS-positive cryoglobulin thrombi within the glomerular capillaries (PAS; 400×)

surrounded by a new layer of subendothelial lamina densa (Fig. 22.9). Mesangial electron-dense deposits are frequently but inconsistently present. Scattered subepithelial electron-dense complex deposits occur in a small subset of patients, ranging from large deposits resembling post-infectious humps through more numerous, smaller deposits resembling superimposed membranous glomerulopathy (type III MPGN, Burkholder variant). In cryoglobulinemic MPGN, the deposits often have microtubular/annular, fibrillar, and/ or tactoid/paracrystalline substructures (Fig. 22.10), although a minority of cryoglobulinemic deposits have no substructure. Intravascular cryoglobulin thrombi with similar substructures are also observed [25].

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Fig. 22.6 Glomerulus from a HCV-seronegative patient with a history of type III cryoglobulin dating back to 1996. The initial renal biopsy in 1996 and a more recent renal biopsy from 2009 show cryoglobulin thrombi in glomerular arterioles and small arteries and veins, with associated microinfarcts in the initial biopsy (not depicted). Of note, the serum cryoglobulin screen concurrent with the 2009 biopsy was negative (PAS; 400×)

Fig. 22.7 Necrotizing granulomatous vasculitis in a renal pericapsular small blood vessel in a patient with chronic hepatitis C virus infection and type II cryoglobulinemia. Note the fibrinoid necrosis (orange) (methenamine silver; 400×)

22.4

Clinical and Laboratory Manifestations

Patients with HCV-associated MPGN may present with the following: • Proteinuria (>0.5 g/day) with microscopic hematuria is the most common presenting syndrome seen in MPNG patients (41–55%) [14, 26]. Such patients may have abnormal kidney function upon presenta-

Fig. 22.8 Immunofluorescence stain of a glomerulus from a patient with type III cryoglobulinemia and chronic HCV infection acquired following a liver transplant for end-stage chronic autoimmune hepatitis. Note the granular, predominantly glomerular basement membrane staining for IgM. C3 and IgG stains were also positive in a similar distribution (IgM immunofluorescence; 400×)

tion and in up to 20% of patients with glomerulonephritis the proteinuria will be in the nephrotic range. • Acute nephritic syndrome, consisting of microscopic hematuria, proteinuria, hypertension and worsening renal function with or without oliguria, is seen in approximately 14–25% of patients. It is also associated with extrarenal manifestations of cryoglobulinemic vasculitis (rash, arthralgias, sensorimotor neuropathy, mononeuritis multiplex). Signs of systemic vasculitis occur in 10% of patients. • Isolated urinary abnormalities will be seen in a small percentage of patients (20%). Such patients will have a milder histologic picture of segmental mesangial proliferation. • Other, less frequent renal manifestations include acute (9%) and chronic renal insufficiency without urine abnormalities (12%) [14]. Most patients present between the fifth and sixth decades of life. The median time between the first symptoms of mixed cryoglobulinemia and renal involvement is 48 months. Hypertension (>140/95 mmHg) is almost invariably present (48–86%) even in non-nephritic patients [27]. Creatinine elevations are documented in 47% of patients referred for renal biopsy [26]; however, this frequency is likely to be overstated due to confounding by indication. The most common extrarenal clinical manifestations upon presentation are: purpura

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Fig. 22.9 Electron photomicrograph of an isolated glomerular capillary with mesangial interposition and electron-dense cryoglobulin deposits in a patient with chronic HCV infection and type II cryoglobulinemia (5,600×)

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New GBM

Cryoglobulin

Mesangial Interposition

Fig. 22.10 Electron photomicrograph from a patient with chronic HCV infection and type II cryoglobulinemia. Note the electron-dense deposits with a fibrillar substructure in the interposed mesangium with the subendothelial glomerular basement membrane (13,000×)

(70%), hepatomegaly (49%), arthralgias (46%), splenomegaly (36%), peripheral neuropathy (22%) and fever of unknown origin (19%). The prevalence of such manifestations increases with the duration of followup, so that arthralgias and organomegaly will be seen in 80–90% during their lifetime. Purpura is an invariable feature of HCV-associated MPGN; in a large series

EM 1-1

from Italy, only 6% of patients never developed this symptom [26]. The classic Meltzer triad of generalized weakness, purpura, and arthralgia will be present in one-third of patients. Pulmonary involvement occurs in 2–5% of cases and can be dramatic, with nodular infiltrates, pulmonary hypertension, and congestive heart failure. Other cutaneous manifestations include ulcers and Raynaud’s phenomenon, with an unadjusted prevalence of 8 and 4% of cases, respectively [14]. Hepatic involvement (organomegaly, stigmata of liver disease) will be observed in 50–70% of cases and these patients will test positive for hepatitis C (antibody or PCR test). Patients will have detectable cryoglobulins (most likely type II), with rheumatoid factor activity in their serum but the percentage (cryocrit) will vary from patient to patient and from time to time. Serum complement levels will be depressed (C4 more than C3), and the early component of the complement cascade (C1q) will be depressed as well. In contrast, total complement hemolytic activity (CH50) will be normal. The prevalence of abnormal C3 and C4 levels varies according to the severity of the renal lesions, ranging from 20%/60% (mesangial proliferative pattern) to 30%/60% (focal MPGN). In patients with diffuse MPGN depressed C3, C4 levels are seen in 52 and 93% of cases, respectively.

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Table 22.1 Differential diagnosis of HCV-associated membranoproliferative glomerulonephritis (MPGN) pattern of injury on biopsy Infections (usually MPGN I) Hepatitis C (non-cryoglobulinemic associated with MPGN I and III), hepatitis B Infection (MPGN I in adult HBsAg (+) patients), schistosomiasis (class III associated with S. mansoni, class VI (cryoglobulinemic) seen in S. mansoni/HCV co-infection), chronic bacterial infections (atrioventricular shunt nephritis, osteomyelitis, intra-abdominal, pelvic, pleural infections, cyanotic heart disease), leprosy, parasitic infections (quartan malaria, onchocerciasis, trichinosis, elephantiasis) Autoimmune diseases Systemic lupus erythematosus (type I or IV MPGN), mixed connective tissue disease Complement disorders Hereditary complement deficiency (C1q, C2,C3,C4 : MPGN I), acquired complement deficiency (C4 nephritic factor: MPGN I, C3 nephritic factor : MPGN II), congenital or acquired factor H deficiency Monoclonal immune deposition disease Light chain deposition disease (MPGN II), heavy chain deposition disease, type I cryoglobulinemia Chronic thrombotic microangiopathies Hemolytic uremic syndrome, drug induced Chronic liver disease (non-viral) Alpha 1 antitrypsin deficiency, schistosomiasis Malignancies (without cryoglobulinemia) Chronic lymphocytic leukemia, lymphoma, thymoma, renal cell carcinoma

22.5

Differential Diagnosis

The differential diagnosis depends on the presenting clinical (nephritic or nephrotic) renal syndrome. HCVassociated cryoglobulinemic MPGN rarely gives rise to these two clinical syndromes and the diagnosis will usually be considered during the routine diagnostic investigation for secondary causes (e.g., positive hepatitis C and cryoglobulin tests). In adults, the two most common causes of nephrotic syndrome are membranous nephropathy and focal segmental glomerulonephritis, both of which are associated with hypertension and microhematuria (in 30% of cases) and have been described in patients with chronic HCV infection without cryoglobulinemia or rheumatoid factor positivity [28, 29].The differential diagnosis of the nephritic syndrome is very wide and includes both primary (e.g., IgA nephropathy) and secondary (e.g., systemic lupus erythematosus) conditions. A secondary form of IgA nephropathy has been reported in chronic liver disease, including in patients with chronic hepatitis C viral infection [30]. The pathogenesis is thought to be the overabundance of circulating IgA-containing immune complexes due to increased IgA production in the gastrointestinal mucosa coupled with impaired hepatic clearance. For patients presenting acutely with systemic symptoms and purpura, other small-vessel vasculitis syndromes will need to be considered in the differential. Henoch Schönlein purpura is character-

ized by dominant IgA deposits in the vascular walls of purpuric lesions. A positive anti-neutrophil cytoplasmic antibody (ANCA) test and minimal immunoglobulin deposits suggest one of the ANCA-positive vasculitides (Wegener’s, microscopic polyangiitis or Churg-Strauss syndrome). Depressed complement levels and renal dysfunction characterize a number of conditions, including athero-embolic renal disease (depressed C3 and eosinophilia), thrombotic microangiopathies, systemic lupus erythematosus (simultaneous depression of C3 and C4), post-streptococcal glomerulonephritis, shunt nephritis, and endocarditis (low C3, type III cryoglobulins). Complement levels may also be low in significant hepatic dysfunction due to depressed synthesis; in case of combined liver and renal dysfunction, the hepatorenal syndrome should be considered. Many other conditions will be associated with the MPGN pattern of renal injury and they should be included in the differential (Table 22.1). Fibrillary and immunotactoid glomerulonephritis have been reported [31] to occur in a small number of patients with chronic HCV infection, with light microscopy showing a spectrum ranging from mild mesangial proliferation with mesangial matrix expansion and glomerular capillary wall thickening through MPGN (types I and III), with some patients having cellular or fibrous crescents. Congo red staining is negative for amyloid, but amyloid P protein and fibronectin have been demonstrated in the protein deposits. Electron

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microscopy reveals haphazardly oriented fibrils 16–24 nm in diameter in fibrillary glomerulonephritis, and microtubule-like structures with a mean diameter of 33–40 nm in immunotactoid glomerulonephritis. Serum complement levels are depressed in some patients, but cryoglobulin precipitates are not identified by routine laboratory serum testing.

22.6

Prognosis and Course

The clinical course is variable, characterized by exacerbations alternating with quiescence. Renal flares can be seen in up to 53% of patients and are often multiple (range 1–4 over 10 years). Remissions can be either spontaneous or following therapy in 15% of cases [27]. In a large Italian study, progression to end-stage renal disease (ESRD) was noted in 15% of patients over a 10-year period. In a more recent study, the five year cumulative incidence of chronic kidney disease stage 5 (estimated glomerular filtration rate 50), nephrotic range proteinuria, nephritic presentation, elevated creatinine at the time of biopsy (>1.5 mg/l in the two large Italian studies). The number of flare ups is an independent risk factor for the development of renal dysfunction.

22.7

Therapy and Outcomes

Therapy of MPGN associated with HCV may be considered in at least three differing manners: therapy directed at proteinuria and hypertension common to progressive renal diseases; therapy directed at cryoglobulins and their effects; and therapy directed against HCV. These are not, of course, mutually exclusive and in some cases, as shall be seen, all three approaches have been combined. Several other factors complicate the discussion of treatment of HCVassociated MPGN. Most important among such them is the stage of renal and liver disease. Patients with chronic stable illness are often approached differently

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than those with more aggressive and active renal disease. Furthermore, when patients with very advanced renal disease near or even on dialysis are considered for treatment, the actual object of therapy changes. Therapy is, in such cases, not designed to treat the MPGN but rather to eliminate or minimize the HCV status of the patient prior to liver transplantation. This discussion will focus on treatment of the renal disease and only briefly consider treatment schemes in dialysis or transplant patients. MPGN is characterized by proteinuria, hypertension, and variable progression of renal insufficiency. There is general agreement that all patients should be managed with standard conservative approaches to progressive glomerular disease, including strict control of blood pressure centered around the use of angiotensin converting enzyme inhibitors or angiotensin receptor blockers (or both), diuretics, and lipid-lowering agents. Treating hypertension in this context is not an easy task; even with multiple (2–3) agents a large proportion of patients will have abnormal office blood pressure readings. Although many approaches have been tried, there is no clear evidence-based therapy that is generally efficacious for primary or idiopathic MPGN [32]. Therapy of HCV-associated MPGN is therefore directed against either HCV or the cryoglobulins associated with HCV and the associated MPGN and systemic vasculitis. Therapy directed against HCV per se initially centered around the use of interferon a (IFN-a) and showed modest effects on proteinuria and renal function (reviewed in [33, 34]). However, IFN-a in a variety of dosing schedules was, unfortunately, associated with a low level of sustained virologic remission (SVR) and a relatively high rate of relapse with viremia [35]. Rates of SVR became substantially higher with the advent of a long-acting IFN-a preparation, pegylated interferon (PEG-IFN), which is used in combination with the antiviral nucleoside antimetabolite ribavirin (RBV). PEG-IFN is given subcutaneously weekly and RBV daily by mouth for periods from 24 weeks to 1 year, depending, to some extent on the genotype of the HCV [35]. This has become the standard therapy for both HCV liver and renal disease, including MPGN [34, 36]. With this approach, the SVR may be as high as 90% [33], although rates are lower with genotype 1 virus [37]. RBV and its metabolites are principally excreted by kidney, so dose adjustments must be made for glomerular filtration rate (GFR) 84% of RA and 8.8% of HCV carriers [11, 12]. In some

patients, even though the ACR criteria for RA are often satisfied, nodules and erosions may be lacking. The best possible explanation for this form of arthritis is an IC-related arthropathy. Since RA can include ACPA and erosions, it becomes very important to define whether in HCV chronic infection the occurrence of arthritis is truly MC arthritis and not RA in a patient with chronic HCV infection. While RA that is seropositive either for RF and/or ACPA needs to be aggressively treated with methotrexate (MTX) and/or a tumor necrosis factor (TNF)-a blocker, irrespective of HCV infection, cryoglobulinemic arthropathy requires a much less aggressive approach, with antimalarials and/ or MTX, sometimes with small doses of glucocorticoids, in short courses. The biologic evidence is that TNF-a and the imbalance between Th1 and Th2 responses contributes to make HCV patients refractory to interferon (IFN). All these issues are very important from a clinical perspective since MTX, as well as nonsteroidal anti-inflammatory drugs (NSAIDs), can be hepatotoxic, while glucocorticoids may favor viral replication [13]. Therefore, one has to consider the cost-benefit ratio of any therapeutic choice in these patients. Furthermore, IFN therapy can lead to the occurrence of autoimmunity and to a relapse of the arthritis [14]. According to these statements, the key messages are: 1. All patients with recent onset of arthritis should be routinely tested for HCV (and other pre-existing infections if previously transfused), before starting disease modifying anti-rheumatic drugs (DMARDs) therapy. 2. In case of HCV antibodies positivity, the viral RNA should be investigated and genotyped. 3. RF and ACPA should be determined simultaneously in order to differentiate between RA and HCV-related arthropathy. 4. Erosions should be looked-for radiographically (X-rays) and ultrasonographically in order to confirm or exclude their presence. These data will allow HCV-related arthropathy to be differentiated from RF-positive RA and thus for the disease to be treated accordingly. Data obtained from several studies indicate that HCV-infected RA patients tolerate TNF-a blockers along with MTX, avoiding viral replication and liver damage [15]. This, of course, is very reassuring for the many patients in Mediterranean countries who carry an occult HCV infection. Patients poorly responsive to TNF-a blockers plus MTX can be

23  Rheumatologic Symptoms in Patients with Mixed Cryoglobulinemia

treated with B-cell-depleting biologics [16] or the antiIL-6 biologic tocilizumab (a monoclonal antibody directed against the IL-6 receptor), thus allowing control of both the RA autoimmune inflammation and the B-cell activation occurring in HCV chronic infection. If patients, on the other hand, are infected with continuously replicating viral species, a possible option is treatment with cyclosporine A (CsA). Experimental evidence has shown that cyclophylin-B, which is a target of CsA, is capable of regulating the viral protein NS5B, an HCV RNA-polymerase that plays a major role in HCV replication. Successful clinical use of CsA has been reported in such cases [17].

23.2.2 Sicca Syndrome and Sjögren’s Syndrome In 1994, we demonstrated the possible involvement of HCV in the development of chronic sialoadenitis [18]. In 1997, we showed that a lymphocytic infiltration mimicking clear-cut Sjögren’s syndrome (SS) was frequent in patients with HCV chronic infection [19]. In the same year, salivary and lacrimal gland inflammation strongly similar to Sjögren’s sialoadenitis was replicated in HCV transgenic mice, suggesting that HCV is not only lymphotropic but also sialotropic [20]. The presence of HCV RNA in the salivary glands of patients with SS was later confirmed in two clinical studies [21, 22]. This led us to hypothesize that HCV plays a direct role in driving autoimmune inflammation in the salivary glands, truly mimicking SS. In a monocentric analysis of our SS patients, we were able to show that HCV-infected patients with SS diagnosed according to the European-ACR criteria had several immunological features in common with primary SS patients [23, 24]. This was confirmed in a multicenter study in which 137 cases were collected and analyzed. It was shown that 58% of patients had diverse extraglandular manifestations, with the most frequent phenotypic presentations being arthritis-arthralgias (44%), vasculitis (20%), and neuropathy (16%). Of interest, parotidomegaly was observed in 16% of the case series, with labial histology in agreement with the diagnosis in 73% of the analyzed specimens. ANA were observed in 65%, low complement levels in 51%, and cryoglobulins were strongly associated with vasculitis and the presence of RF. Positive anti-Ro or anti-La auto-antibodies were detected in 23%. Splenomegaly and hepatomegaly

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were often seen (78%), whereas mild increases of ALT were noted in 68%; 42% of patients had sicca syndrome only. When the authors compared patients who were Ro- and/or La-positive with those who were negative for these antibodies, positive patients had a higher frequency of parotidomegaly (28% vs. 12%) and more arthritis (62% vs. 38%), but less frequent liver function test abnormalities (50% vs. 86%). Moreover, monoclonal bands were demonstrated in 43% of the patients, which is clearly higher than in primary SS, in which 20% of patients have only a monoclonal gammopathy [25]. Of interest, an association of HLA-DQ1*02 with sicca syndrome and with viral persistence was seen. All these findings together with the clinical and histopathological features, almost indistinguishable from primary SS, indicate that HCV-related SS is the first known infectious-related autoimmune disease.

23.2.3 Myalgias/Fibromyalgia Fibromyalgia syndrome (FS) is a clinical entity characterized by widespread muscular pain, several (>11 sites) tender points, and diffuse tenderness that is very sensitive to meteorological changes. In particular, windy weather and low barometric pressure are generally associated with increased tenderness all over the body, along with poor, unsatisfactory, non-relaxing sleep. FS was examined in patients with HCV chronic infection and found to be strongly increased in nonHCV related cirrhosis and in the main controls [26]. In particular, fatigue, which is a cardinal feature of FS, is a characterizing clinical hallmark of HCV-infected patients [27]. Studies have shown a general prevalence of FS of 17% (range 16–18.9) vs. 3% in controls (range 0–5). Since thyroid disease with anti-thyroid peroxidase and anti-thyroid microsomes auto-antibodies is present in 2–48% of chronic HCV carriers and subclinical hypothyroidism has been demonstrated in 2–9% [28], a multifactorial cause as the basis of the myalgiafibromyalgia syndrome has to be postulated. The obvious consequence is that therapy must address all the underlying possible mechanisms of these symptoms.

23.2.4 Mono/Multineuropathy Leukocytoclastic vasculitis is a prototypic vasculitis of the small vessels that can affect even the vasa ­nervorum.

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This vasculitis is the classical peripheral neuropathy, occurring in 70–80% of MC patients [29]. In rare cases, anti-neutrophil cytoplasmic auto-antibodies (ANCA) are detected. Subjective symptoms have been reported in up to 91% of patients. Sensory fibers are more commonly affected than motor fibers, with pure motor neuropathy in approximately 5% of patients. Since a significant increase of plasma anti-neuronal (anti-GM1, anti-sulfatide) auto-antibodies has been detected in MC, the vasa nervorum vasculitis, the thrombophilia sometimes associated with anti-cardiolipin auto-antibodies, and the anti-neuronal autoimmunity could together explain why neuropathy is frequent in MC and why plasma-exchange may be a therapeutic alternative to immunosuppressive therapies, especially in some acute cases [30].

G. Ferraccioli et al.

should raise the suspicion of a lymphoplasmacellular disorder or of a Sjögren’s-like evolution of the chronic infection [36–44].

23.2.7 Interstitial Lung Disease Italian patients with interstitial lung disease (ILD) have shown an increased prevalence of HCV infection [45]. On the other hand, MC patients often present with ILD [46, 47], which remains stable over time and is not accompanied by a decline of lung function [48]. Considering that MC can present as SS or as ANCApositive vasculitis and the great majority of such cases as an ongoing immune-complex vasculitis, all patients presenting with systemic complaints in the course of MC should be screened clinically and functionally (or though imaging techniques) for ILD.

23.2.5 Anti-phospholipid Syndrome Anti-cardiolipin auto-antibodies are detected in 20–27% of HCV-infected patients, without the occurrence of either anti-b2-glicoprotein I auto-antibodies or arterial or venous thrombosis. Moreover, ischemic lesions in the CNS were not higher in HCV patients with autoantibodies than in those without anti-cardiolipin, leading to the conclusion that the latter are the likely cause of the B-cell polyclonal activation [31, 32].

23.2.6 Acrocyanosis-Raynaud’s Increased plasma viscosity leading to impaired vascular supply and impaired vasal motility is observed mainly in type I cryoglobulinemia, associated with lymphoplasmacellular dyscrasias (myeloma, macroglobulinemia, lymphoma), but can also be observed in renal complications of MC, manifesting as an acute renal failure due to an acute nephritis. This scenario is seen in up to 31% of patients and can severely impact both the quality of life and survival [33, 34]. In these cases, manifestations include acrocyanosis, livedo reticularis, retinal hemorrhage, and severe Raynaud’s phenomenon with digital ulceration, purpura, and arterial thrombosis. Eventually ulcerations may occur, as commonly evidenced by nail-fold capillary abnormalities such as dilation, capillary shortening, and neo-angiogenesis [35]. The occurrence of these features in patients with type II and type III disease

23.2.8 Osteosclerosis Some patients with HCV chronic infection present with diffuse, persistent, sometimes throbbing bone pain. Twelve cases of bone osteosclerosis have been described. In these patients, cortical bone sclerosis and thickening of periosteal surfaces in the long bones are seen on X-ray. Bone turnover markers show an increased turnover; in some cases, anti-resorptive agents have been of clinical benefit. An imbalance between RANK-L/osteoprotegerin, with the latter prevailing, is the current interpretation of these still obscure manifestations [49, 50].

23.3 Conclusion and Discussion Rheumatic manifestations are common in MC and can present as a local manifestation of an ongoing IC disease (arthritis, ILD) or as a systemic autoimmuneinflammatory disease (SS, systemic vasculitis, mono/ multineuropathy, acrocyanosis, myalgia/myositis). The therapeutic approach depends upon the severity of the manifestations. According to current opinion, eradication of the virus should be the main goal in any case [51]. The minor clinical presentations (arthralgias, myalgias, sicca syndrome) can benefit from antimalarials and/or small daily doses of glucocorticoids. Once there is an association between erosive and

23  Rheumatologic Symptoms in Patients with Mixed Cryoglobulinemia

s­ eropositive RA and HCV infection, MTX can be used in combination with TNF-a blockers. The most severe manifestations (acute renal failure in nephritis, resistant skin ulcers) may require a prompt lavage of the IC-plugged vessel, using plasma exchange associated with immunosuppressants. Persistent inflammatory conditions (extracapillary nephritis, severe neuropathies, severe ulcers, systemic vasculitis) may require higher doses of glucocorticoids along with B-cell depletion through monoclonal antibodies [18] or more targeted biologics therapy, such as anti-IL6.

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14. Nissen MJ, Fontanges E, Allam Y et al (2005) Rheumatological manifestations of hepatitis C: incidence in a rheumatology and non-rheumatology setting and the effect of methotrexate and interferon. Rheumatology 44:1016–1020 15. Ferri C, Ferraccioli G, Ferrari D, GISEA Group et al (2008) Safety of anti-tumor necrosis factor-alpha therapy in patients with rheumatoid arthritis and chronic hepatitis C virus infection. J Rheumatol 35:1944–1949 16. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101: 3827–3834 17. Galeazzi M, Bellisai F, Manganelli S et  al (2006) Cyclosporine A for the treatment of autoimmune disorders in HCV infected patients. Autoimmun Rev 5:493–498 18. Pirisi M, Scott C, Fabris C et al (1994) Mild sialoadenitis: a common finding in patients with hepatitis C virus infection. Scand J Gastroenterol 29:940–942 19. Scott CA, Avellini C, Desinan L et al (1997) Chronic lymphocytic sialoadenitis in HCV-related chronic liver disease: comparison with Sjogren’s syndrome. Histopathology 30: 41–48 20. Koike K, Moriya K, Ishibashi K et  al (1997) Sialadenitis histologically resembling Sjogren syndrome in mice transgenic for hepatitis C virus envelope genes. Proc Natl Acad Sci USA 94:233–236 21. Toussirot E, Le Huédé G, Mougin C et al (2002) Presence of hepatitis C virus RNA in the salivary glands of patients with Sjögren’s syndrome and hepatitis C virus infection. J Rheumatol 29:2382–2385 22. Arrieta JJ, Rodríguez-Iñigo E, Ortiz-Movilla N et al (2001) In situ detection of hepatitis C virus RNA in salivary glands. Am J Pathol 158:259–264 23. De Vita S, Damato R, De Marchi G et al (2002) True primary Sjögren’s syndrome in a subset of patients with hepatitis C infection: a model linking chronic infection to chronic sialadenitis. Isr Med Assoc J 4:1101–1105 24. Vitali C, Bombardieri S, Jonsson R, European Study Group on Classification Criteria for Sjögren’s Syndrome et al (2002) Classification criteria for Sjogren’s syndrome: a revised version of the European criteria proposed by the AmericanEuropean Consensus Group. Ann Rheum Dis 61:554–558 25. Ramos-Casals M, Loustaud-Ratti V, De Vita S, SS-HCV Study Group et al (2005) Sjögren syndrome associated with hepatitis C virus: a multicenter analysis of 137 cases. Medicine 84:81–89 26. Buskila D, Shnaider A, Neumann L et al (1997) Fibromyalgia in hepatitis C virus infection. Another infectious disease relationship. Arch Intern Med 157:2497–2500 27. Kozanoglu E, Canataroglu A, Abayli B et  al (2003) Fibromyalgia syndrome in patients with hepatitis C infection. Rheumatol Int 23:248–251 28. Rocco A, Gargano S, Provenzano A et al (2001) Incidence of autoimmune thyroiditis in interferon alpha treated and untreated patients with chronic hepatitis C virus infection. Neuro Endocrinol Lett 22:39–44 29. Gemignani F, Pavesi G, Fiocchi A et  al (1992) Peripheral neuropathy in essential mixed cryoglobulinaemia. J Neurol Neurosurg Psychiatry 55:116–120 30. Alpa M, Ferrero B, Cavallo R et al (2008) Antineuronal antibodies in patients with HCV related mixed cryoglobulinemia. Autoimmun Rev 8:56–58

190 31. Ramos-Casals M, Cervera R, Lagrutta M, Hispanoamerican Study Group of Autoimmune Manifestations of Chronic Viral Disease (HISPAMEC) et  al (2004) Clinical features related to antiphospholipid syndrome in patients with chronic viral infections (hepatitis C virus/HIV infection): description of 82 cases. Clin Infect Dis 38:1009–1016 32. Sène D, Limal N, Cacoub P (2004) Hepatitis C virus-associated extrahepatic manifestations: a review. Metab Brain Dis 19:357–381 33. Ferri C, Moriconi L, Gremignai G et al (1986) Treatment of the renal involvement in mixed cryoglobulinemia with prolonged plasma exchange. Nephron 43:246–253 34. Madore F, Lazarus JM, Brady HR (1996) Therapeutic plasma exchange in renal diseases. J Am Soc Nephrol 7:367–386 35. Rossi D, Mansouri M, Baldovino S et  al (2004) Nail fold videocapillaroscopy in mixed cryoglobulinemia. Nephrol Dial Transplant 19:2245–2249 36. Inokuchi M, Ito T, Uchikoshi M et  al (2009) Infection of B  cells with hepatitis C virus for the development of ­lymphoproliferative disorders in patients with chronic ­hepatitis C. J Med Virol 81:619–627 37. Silvestri F, Pipan C, Barillari G et al (1996) Prevalence of hepatitis C virus infection in patients with lymphoproliferative disorders. Blood 87:4296–4301 38. de Sanjose S, Benavente Y, Vajdic CM et al (2008) Hepatitis C and non-Hodgkin lymphoma among 4784 cases and 6269 controls from the International Lymphoma Epidemiology Consortium. Clin Gastroenterol Hepatol 6:451–458 39. Charles ED, Green RM, Marukian S et  al (2008) Clonal expansion of immunoglobulin M+CD27+ B cells in HCVassociated mixed cryoglobulinemia. Blood 111:1344–1356 40. Zignego AL, Giannini C, Ferri C (2007) Hepatitis C virusrelated lymphoproliferative disorders: an overview. World J Gastroenterol 13:2467–2478

G. Ferraccioli et al. 41. De Vita S, Sacco C, Sansonno D et al (1997) Characterization of overt B-cell lymphomas in patients with hepatitis C virus infection. Blood 90:776–782 42. Ferri C, Caracciolo F, La Civita L et al (1994) Hepatitis C virus infection and B-cell lymphomas. Eur J Cancer 30A:1591–1592 43. Ferri C, Monti M, La Civita L et al (1994) Hepatitis C virus infection in non-Hodgkin’s B-cell lymphoma complicating mixed cryoglobulinaemia. Eur J Clin Invest 24:781–784 44. Zignego AL, Giannelli F, Marrocchi ME et  al (2000) T (14;18) translocation in chronic hepatitis C virus infection. Hepatology 31:474–479 45. Meliconi R, Andreone P, Fasano L et al (1996) Incidence of hepatitis C virus infection in Italian patients with idiopathic pulmonary fibrosis. Thorax 51(3):315–317 46. Bombardieri S, Paoletti P, Ferri C et al (1979) Lung involvement in essential mixed cryoglobulinemia. Am J Med 66:748–756 47. Bertorelli G, Pesci A, Manganelli P et al (1991) Subclinical pulmonary involvement in essential mixed cryoglobulinemia assessed by bronchoalveolar lavage. Chest 100:1478–1479 48. Viegi G, Fornai E, Ferri C et  al (2005) Lung function in essential mixed cryoglobulinemia: a short-term follow-up. Clin Rheumatol 8:331–338 49. Manganelli P, Giuliani N, Fietta P et al (2005) OPG/RANKL system imbalance in a case of hepatitis C-associated osteosclerosis: the pathogenetic key? Clin Rheumatol 24:296–300 50. Fiore CE, Riccobene S, Mangiafico R et al (2005) Hepatitis C associated osteosclerosis (HCAO): report of a new case with involvement of the OPG/RANKL system. Osteoporos Int 16:2180–2184 51. Saadoun D, Resche-Rigon M, Thibault V et  al (2006) Antiviral therapy for hepatitis C virus – associated mixed cryoglobulinemia vasculitis: a long-term follow-up study. Arthritis Rheum 54:3696–3706

Endocrine Manifestations of HCV-Positive Cryoglobulinemia

24

Alessandro Antonelli, Clodoveo Ferri, Silvia Martina Ferrari, Michele Colaci, Alda Corrado, Andrea Di Domenicantonio, and Poupak Fallahi

24.1

Introduction

Hepatitis C virus (HCV) is known to be responsible for both hepatic and extrahepatic diseases. Among the systemic HCV-related extrahepatic diseases (HCVEHDs), mixed cryoglobulinemia (MC) has been extensively studied by clinico-epidemiological, immunological, and virological approaches [1–4]. However, recently, an increased prevalence of endocrine disorders has been observed in large series of HCV-infected patients and in those with MC. The most frequent and clinically important endocrine disorders of hepatitis C chronic infection are thyroid disorders and type 2 diabetes mellitus.

24.2

Autoimmune Thyroid Disorders

Many studies have examined the prevalence of autoimmune thyroid disorders (AITD) in HCV-positive (HCV+) patients. From a meta-analysis of the literature, a significant association between HCV infection and AITD has been reported [5]. The frequency of high levels of anti-thyroid antibodies in HCV+ patients ranges from 8% to 48% in different studies [6], while that of hypothyroidism ranges from 2% to 13%.

A. Antonelli (*) Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy e-mail: [email protected]

Recently [6], the prevalence of thyroid disorders was investigated in 630 consecutive HCV+ patients with chronic hepatitis. Patients with chronic hepatitis C (CH) were more likely to have hypothyroidism (13%), anti-thyroglobulin antibodies (AbTg) (17%), and antithyroperoxidase antibodies (AbTPO) (21%) compared to the control groups. Thyroid autoimmunity was also investigated in 93 MC patients [7], matched by sex and age to 93 patients with CH without MC and 93 healthy (HCV-negative) controls. The following thyroid abnormalities were significantly more frequent in MC patients than in HCV-negative controls: serum AbTPO (28% vs. 9%, p = 0.001); serum AbTPO and/ or AbTg (31% vs. 12%, p = 0.004); subclinical hypothyroidism (11% vs. 2%, p = 0.038); thyroid autoimmunity (35% vs. 16%, p = 0.006). Serum AbTPO were also significantly more frequent in MC patients than in CH controls (28% vs. 14%, p = 0.035). This study therefore demonstrated an increased prevalence of thyroid disorders in patients with HCV-related MC. These results are in agreement with those of a recent retrospective cohort study of users of the US Veterans Affairs health care facilities from 1997 to 2004, which included 146,394 patients infected with HCV. These patients had a significantly increased risk of thyroiditis [8]. Both differences in genetic variability and environmental co-factors, such as iodine intake or infectious agents other than HCV, could play an important role in the development of AITD [9]. Female gender is a risk factor for AITD development, while major risk factors for the development of hypothyroidism are again female gender in addition to the presence of AbTPO [9, 10]. HCV RNA has been detected in the thyroid of chronically infected patients [11, 12] but the possible

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_24, © Springer-Verlag Italia 2012

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consequences of HCV thyroid infection on thyrocyte function, vitality, and immunogenicity remain to be clarified. In the thyroid disorders observed in HCV infection and in MC, there are increased circulating levels of AbTPO, with the risk of hypothyroidism being higher in AbTPO-positive individuals [5]. A similar pattern was observed in patients treated with interferon (IFN)a [13]. Furthermore, it has been shown that viral NS5A and core proteins, alone or by the synergistic effect of cytokines [IFNg and tumor necrosis factor(TNF)a], are capable of up-regulating chemokine (C-X-C motif) ligand 10 (CXCL10) expression and secretion in cultured human hepatocyte-derived cells [14]. This suggests a key role for the CXCL10 produced by HCV-infected hepatocytes in regulating T-cell trafficking in the liver during chronic HCV infection, by recruiting Th1 lymphocytes, which secrete IFNg and TNFa, inducing CXCL10 secretion by hepatocytes, thus perpetuating the immune cascade [14]. Recently, high levels of CXCL10 were demonstrated in patients with autoimmune thyroiditis (AT) and overall in the presence of hypothyroidism [15], with a Th1 immune response shown to be involved in the induction of AT [16, 17]. Accordingly, a scenario can be proposed in which HCV thyroid infection up-regulates CXCL10 gene expression and secretion in thyrocytes (as previously shown in human hepatocytes), thus recruiting Th1 lymphocytes, which in turn secrete IFNg and TNFa, thus inducing CXCL10 secretion by thyrocytes and perpetuating the immune cascade, finally leading to the appearance of AITD in genetically predisposed subjects. A recent study [18] evaluated CXCL10 serum levels in HCV+ patients associated with MC, in the presence or absence of AT: in 50 MC patients without AT; in 40 MC patients with AT (MC+AT); in two genderand age-matched control groups [50 healthy controls (without HCV or AT; control); 40 controls with AT (without HCV and MC; control+AT)]. CXCL10 was significantly higher: (1) in control+AT than in control; (2) in MC patients than in control; (3) in MC+AT patients than in control or control+AT, or in MC. This study therefore evidenced high serum levels of CXCL10 in MC and showed that CXCL10 levels in MC+AT patients are significantly higher than in MC patients [18, 19].

A. Antonelli et al.

24.3

Thyroid Cancer, HCV Infection, and MC

A preliminary study reported a high prevalence of thyroid cancer complicating HCV-related hepatitis [20]. Subsequently, the prevalence of thyroid cancer in a series of unselected patients with HCV-related MC was investigated and compared with that of a control group [21]. Among 94 consecutive patients with MC, two had papillary thyroid cancer, while no such case was determined among controls (n = 2,401; p = 0.0019). In contrast, the prevalence of thyroid nodules was higher, although not significantly, in controls than in MC patients (65.3% vs. 54.8%). These data were subsequently confirmed in case–control studies, which concluded that HCV infection was associated with a high risk for thyroid cancer [5, 22]. However, a recent retrospective cohort study of users of US Veterans Affairs health care facilities, which included patients infected with HCV, failed to confirm an increased risk of thyroid cancer in these patients [8]. The exact mechanisms that transduce the HCV carcinogenic potential in thyroid cancer remain to be investigated; however, chronic AT is regarded as a preneoplastic condition [23]. In our studies in HCVinfected [22] and MC (MC-HCV+) [21] patients, features of AITD were seen significantly more often in patients with thyroid papillary cancer than in the other HCV+ patients. These findings suggest that AITD is a predisposing condition for thyroid papillary cancer.

24.4

HCV and Type 2 Diabetes

Several clinical epidemiologic studies [24] have reported an association between HCV infection and diabetes. However, almost all these studies consisted of HCV patients with and without cirrhosis [24], and it is well known that cirrhosis, of whatever etiology, is a risk factor for type 2 diabetes mellitus (T2D). Nonetheless, we have recently shown an association between HCV infection (in patients without cirrhosis), and T2D, both in HCV-related chronic liver [25] disease and in MC [26]. In our study, 229 consecutively recruited MC-HCV+ patients were compared with 217 sex- and age-matched controls without HCV infection [26]. The prevalence of T2D was significantly higher in MC-HCV+ patients than in controls (14.4% vs. 6.9%, p < 0.01). Diabetic MC-HCV+ patients were leaner than diabetic patients without MC-HCV

24

Endocrine Manifestations of HCV-Positive Cryoglobulinemia

(p < 0.0001) and had significantly lower total and low-density lipoprotein cholesterol levels (p < 0.001) and lower systolic (p = 0.01) and diastolic (p = 0.005) blood pressure. Non-organ-specific autoantibodies were present more frequently in MC-HCV+ diabetic patients than in non-diabetic MC-HCV+ patients (34% vs. 18%, p = 0.032). A population study (National Health and Nutrition Examination Survey-NHANES III 1988–1994) showed an adjusted odds ratio of 3.8 for T2D for individuals who were age >40 years and HCV+ [27] and an increased incidence of T2D [28]. There have also been a few reports in which IFN treatment of HCV infection was shown to improve glucose tolerance [26, 29] following the eradication of HCV infection; however, another study did not confirm these results [30]. In conclusion, these data indicate that HCV chronic infection is a risk factor for developing T2D in MC, although the mechanisms involved in the association between HCV infection and diabetes are not well clarified. It is speculated that insulin resistance (IR) (as a consequence of hepatic steatosis, which is present in about 50% HCV–infected individuals) [24], and/or elevated expression of TNFa (strongly correlated with the degree of liver diseases and the level of IR) [24] lead to the development of T2D [24]. Moreover, direct islet cell destruction by HCV was hypothesized by Masini et al. [31], in a study showing a direct cytopathic effect of HCV on islet beta cells. Indeed, virus-like particles were observed in islet cells from HCV+ pancreases, mainly close to the membranes of the Golgi apparatus, which was hyperplastic and dilated. These morphological changes were accompanied by reduced in vitro glucose-stimulated insulin release; however, apoptosis was not increased. More recently, the autoimmune induction of diabetes by HCV has been hypothesized; but, in fact, the type of diabetes manifested by patients with HCV chronic infection is not the typical form of T2D. Three studies reported [25, 26, 32] that HCV+ patients with T2D were leaner than T2D controls and showed significantly lower LDL-cholesterol, systolic and diastolic blood pressure. Furthermore, non-organspecific-autoantibodies were more frequently detected in MC-HCV+ patients with T2D than in non-diabetic MC-HCV+ patients (34% vs. 18%) [26]. We have postulated that MC-HCV+ diabetes has an immune-mediated pathogenesis [26]. In fact, HCV is able to infect islet cells [31] and may up-regulate expression of the CXCL10 gene. As discussed above,

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CXCL10 secretion by islet cells results in the recruitment of Th1 lymphocytes, which produce IFNg and TNFa. In the pancreas, this in turn induces CXCL10 secretion by pancreatic islet cells, thus perpetuating the immune cascade and leading to the appearance of islet cells dysfunction. We recently confirmed this hypothesis: in that CXCL10 serum levels were found to be higher in HCV+ patients with T2D than in a gender- and age-matched control group of T2D without HCV infection [4, 9].

24.5

Research Perspectives

Many aspects remain to be clarified regarding the immunopathogenesis of these disorders. An increasing number of studies have linked a Th1 immune response with HCV infection, MC, AITD, and diabetes. These data suggest a common immunological Th1 pattern as the pathophysiological basis of the association; however, this remains to be confirmed in further studies.

References 1. Ferri C, Antonelli A, Mascia MT et al (2007) B-cells and mixed cryoglobulinemia. Autoimmun Rev 7:114–120 2. Sansonno D, Carbone A, De Re V et al (2007) Hepatitis C virus infection, cryoglobulinaemia, and beyond. Rheumatology (Oxford) 46:572–578 3. Antonelli A, Ferri C, Galeazzi M et al (2008) HCV infection: pathogenesis, clinical manifestations and therapy. Clin Exp Rheumatol 26:S39–S47 4. Antonelli A, Ferri C, Ferrari SM et al (2008) Immunopathogenesis of HCV-related endocrine manifestations in chronic hepatitis and mixed cryoglobulinemia. Autoimmun Rev 8:18–23 5. Antonelli A, Ferri C, Fallahi P et al (2006) Thyroid disorders in chronic hepatitis C virus infection. Thyroid 16:563–572 6. Antonelli A, Ferri C, Pampana A et al (2004) Thyroid disorders in chronic hepatitis C. Am J Med 117:10–13 7. Antonelli A, Ferri C, Fallahi P et al (2004) Thyroid involvement in patients with overt HCV-related mixed cryoglobulinaemia. QJM 97:499–506 8. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297:2010–2017 9. Antonelli A, Ferri C, Ferrari SM et al (2009) Endocrine manifestations of hepatitis C virus infection. Nat Clin Pract Endocrinol Metab 5:26–34 10. Antonelli A, Ferri C, Fallahi P (2009) Hepatitis C: thyroid dysfunction in patients with hepatitis C on IFN-alpha therapy. Nat Rev Gastroenterol Hepatol 6:633–635

194 11. Gowans EJ (2000) Distribution of markers of hepatitis C virus infection throughout the body. Semin Liver Dis 20:85–102 12. Bartolomé J, Rodríguez-Iñigo E, Quadros P et al (2008) Detection of hepatitis C virus in thyroid tissue from patients with chronic HCV infection. J Med Virol 80:1588–1594 13. Prummel MF, Laurberg P (2003) Interferon-alpha and autoimmune thyroid disease. Thyroid 13:547–551 14. Apolinario A, Majano PL, Lorente R et al (2005) Gene expression profile of T-cell-specific chemokines in human hepatocyte-derived cells: evidence for a synergistic inducer effect of cytokines and hepatitis C virus proteins. J Viral Hepat 12:27–37 15. Antonelli A, Rotondi M, Fallahi P et al (2004) High levels of circulating CXCL10 are associated with chronic autoimmune thyroiditis and hypothyroidism. J Clin Endocrinol Metab 89:5496–5499 16. Antonelli A, Rotondi M, Fallahi P et al (2005) Increase of interferon-gamma inducible alpha chemokine CXCL10 but not beta chemokine CCL2 serum levels in chronic autoimmune thyroiditis. Eur J Endocrinol 152:171–177 17. Antonelli A, Rotondi M, Ferrari SM et al (2006) Interferongamma-inducible alpha-chemokine CXCL10 involvement in Graves’ ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. J Clin Endocrinol Metab 9:614–620 18. Antonelli A, Ferri C, Fallahi P et al (2008) High values of CXCL10 serum levels in patients with hepatitis C associated mixed cryoglobulinemia in presence or absence of autoimmune thyroiditis. Cytokine 42:137–143 19. Antonelli A, Ferri C, Fallahi P et al (2008) Alpha-chemokine CXCL10 and beta-chemokine CCL2 serum levels in patients with hepatitis C-associated cryoglobulinemia in the presence or absence of autoimmune thyroiditis. Metabolism 57:1270–1277 20. Antonelli A, Ferri C, Fallahi P (1999) Thyroid cancer in patients with hepatitis C infection. JAMA 281:1588 21. Antonelli A, Ferri C, Fallahi P et al (2002) Thyroid cancer in HCV-related mixed cryoglobulinemia patients. Clin Exp Rheumatol 20:693–696

A. Antonelli et al. 22. Antonelli A, Ferri C, Fallahi P et al (2007) Thyroid cancer in HCV-related chronic hepatitis patients: a case-control study. Thyroid 17:447–451 23. Okayasu I, Fujiwara M, Hara Y et al (1995) Association of chronic lymphocytic thyroiditis and thyroid papillary carcinoma: a study of surgical cases among Japanese, and white and African Americans. Cancer 76:2313–2318 24. Noto H, Raskin P (2006) Hepatitis C infection and diabetes. J Diabetes Complications 20:113–120 25. Antonelli A, Ferri C, Fallahi P et al (2005) Hepatitis C virus infection: evidence for an association with type 2 diabetes. Diabetes Care 28:2548–2550 26. Antonelli A, Ferri C, Fallahi P et al (2004) Type 2 diabetes in hepatitis C-related mixed cryoglobulinaemia patients. Rheumatology (Oxford) 43:238–240 27. Mehta SH, Brancati FL, Sulkowski MS et al (2000) Prevalence of type 2 diabetes mellitus among persons with hepatitis C virus infection in the United States. Ann Intern Med 133:592–599 28. Mehta SH, Brancati FL, Strathdee SA et al (2003) Hepatitis C virus infection and incident type 2 diabetes. Hepatology 38:50–56 29. Tanaka H, Shiota G, Kawasaki H (1997) Changes in glucose tolerance after interferon-alpha therapy in patients with chronic hepatitis C. J Med 28:335–346 30. Giordanino C, Bugianesi E, Smedile A et al (2008) Incidence of type 2 diabetes mellitus and glucose abnormalities in patients with chronic hepatitis c infection by response to treatment: results of a cohort study. Am J Gastroenterol 103: 2481–2487 31. Masini M, Campani D, Boggi U et al (2005) Hepatitis C virus infection and human pancreatic beta-cell dysfunction. Diabetes Care 28:940–941 32. Skowronski M, Zozulinska D, Juszczyk J et al (2006) Hepatitis C virus infection: evidence for an association with type 2 diabetes. Diabetes Care 29:750

Cutaneous Cryoglobulinemic Vasculitis

25

Konstantinos Linos, Bernard Cribier, and J. Andrew Carlson

25.1

Introduction

The modern era of cryoglobulinemia started in 1966, when Meltzer et al. described nine patients with serum cryoglobulins [1]. Cryoglobulins are coldprecipitating immunoglobulins that persist in the serum and resolubilize when rewarmed [2, 3]. Mixed cryoglobulins, composed of different immunoglobulins, with a monoclonal IgM rheumatoid factor component and polyclonal IgG in type II, and polyclonal immunoglobulins IgM (with rheumatoid factor activity) and IgG in type III, are associated with connective tissue disease, hematologic malignancies, and/or infectious diseases, chiefly hepatitis C infection virus (HCV) [4–6]. Type I cryoglobulins are monoclonal and typically produce hyaline thrombi rather than vasculitis. Mixed cryoglobulinemia (MC) is associated with a broad range of clinical manifestations, ranging from asymptomatic MC to life-threatening vasculitis. The commonest manifestation of MC is cutaneous vasculitis, denoted by palpable purpura [7, 8]. Few diseases cause as much diagnostic and therapeutic consternation as vasculitis, an inflammatory process directed primarily at the vasculature that results in destruction of the vessel walls, leading to hemorrhage, ischemia, and/or infarction. The skin, in part due to its large vascular bed, exposure to cold temperatures, and frequent circulatory presence of stasis,

J.A. Carlson (*) Division of Dermatology and Dermatopathology, Department of Pathology, Albany Medical College, Albany, NY, USA e-mail: [email protected]

is involved in many distinct as well as un-named vasculitic syndromes, that vary from localized and self-limited to generalized and life-threatening with multi-organ disease [9]. Cryoglobulinemic vasculitis (CV) exemplifies the range of clinical manifestations, degrees of severity, and diverse outcomes for patients affected by vasculitis. CV is defined as “vasculitis, with cryoglobulin immune deposits, affecting small vessels (i.e. capillaries, venules, or arterioles), and associated with cryoglobulins in the serum”according to the Chapel Hill Consensus criteria [10]. However, this pathologic-serologic definition falls short of capturing all cases of CV and does not acknowledge the overlap with thrombotic factors in the pathogenesis of clinical findings. Specifically, many of the cutaneous manifestations of CV are caused by muscular-vessel vasculitis and/or thrombosis, the latter of which can resemble hyaline thrombi of type 1 cryoglobulinemia. This chapter reviews the epidemiology, etiology, pathogenesis, clinical presentations, pathologic and laboratory findings, prognosis, and management of CV, with emphasis on its cutaneous presentations.

25.2

Epidemiology

The incidence of biopsy-proven, cutaneous vasculitis ranges from 39.6 to 59.8 cases per million per year [9]. Cutaneous vasculitis affects people of all ages, slightly fewer males than females, and adults more often than children, with 90% of the latter having HenochSchonlein purpura [9]. For most patients, the onset of cutaneous vasculitis can be associated with exposure to a trigger, such as a drug or infection, and will represent a single, acute, self-limited episode. However, for

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_25, © Springer-Verlag Italia 2012

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a minority of patients, relapsing or chronic unremitting disease will ensue, and in this population of patients, MC is a common etiologic factor [9]. The prevalence of CV in patients presenting with cutaneous vasculitis ranges from 2.5% to 8% [9, 11], a range that mirrors the seropositivity rate for HCV in the population studied [9, 12]. For example, CV is more common in Southern Europe (4.8 per million in Lugo, Spain [13]) than in Northern Europe or North America (0.6 per million, Capital District of New York (personal observation, [9]) [14]. In underdeveloped countries, where HCV infection is becoming increasingly prevalent, the incidence of HCV-related CV is expected to rise [15]. Notably, females are more frequently affected than males, by a ratio of 3:1.

25.3

Etiology and Pathogenesis

Table 25.1 lists the disorders associated with MC/CV, which include infections (mostly hepatotropic viruses), connective tissue diseases (commonly Sjögren’s syndrome), and hematolymphoid proliferations (mostly B-cell proliferations). The term “essential” cryoglobulinemia is reserved for the small number of cases with no overt infectious, neoplastic, or immuno-rheumatologic disorders [19]. The causative role of hepatotropic viruses in MC/CV has long been hypothesized based on the frequent association of liver involvement in patients with CV [20, 21]. Since Pascual et al. [22] noted the strong association between HCV infection and what was previously called “essential cryoglobulinemia,” the causative role of HCV infection in CV has been widely acknowledged, based on many studies [23–26]. Depending on the region of the world, HCV accounts for 42–98% of MC/CV cases [16] whereas hepatitis-B-related CV represents only a minority (~2%) [25]. HCV infection not only causes chronic hepatic inflammation, but also an array of extrahepatic manifestations, which include MC/CV, diverse systemic autoimmune diseases, such as Sjögren’s syndrome, rheumatoid arthritis, systemic lupus erythematosus, and polyarteritis nodosa, and non-Hodgkin lymphomas [27, 28]. Immunologic problems common to these patients are reflected in the presence of anti-nuclear antibody (ANA), rheumatoid factor (RF), and cryoglobulins. Indeed, the frequency of cryoglobulins increases with the duration of HCV infection, and type

Table 25.1 Disorders and infections associated with cyroglobulinemia Association Disorders Infections Viral infections Hepatitis C virus, 42–98% of cases [16] Hepatitis A and B viruses, human immunodeficiency virus, Herpes viridae, Epstein-Barr virus, varicella zoster virus, cytomegalovirus, parvovirus B19, human T cell lymphotropic virus type I, influenza virus; rubella virus; hanta virus Bacterial Bacterial endocarditis, syphilis, rickettsial infections infections, Q fever, leprosy, Lyme disease, post streptococcal nephritis Fungal Coccidioidomycosis infections Parasitic Toxoplasmosis, echinococciasis, malaria, infections leishmaniasis (kala-azar), schistosomiasis Autoimmune Sjögren’s syndrome diseases Systemic lupus erythematosus Rheumatoid arthritis Dermatomyositis/polymyositis Systemic sclerosis, endomyocardial fibrosis, pulmonary fibrosis Inflammatory bowel disease Systemic vasculitis: giant cell (temporal) arteritis, Henoch-Schönlein purpura Sarcoidosis Autoimmune thyroiditis Primary antiphospholipid syndrome Pemphigus vulgaris Biliary cirrhosis Myelo/ B cell non-Hodgkin lymphoma lymphoprolifChronic lymphocytic leukemia erative diseases Multiple myeloma Waldenström’s macroglobulinemia Angioimmunoblastic lymphoma Chronic myeloid leukemia Castleman disease Cold agglutinin disease Myelodysplasia and chronic myeloid leukemia Thrombocytopenic thrombotic purpura Adapted from [7, 17, 18]

III and oligoclonal MC are considered an intermediate stage in the progression to type II MC [8, 29, 30]. Up to 70% of patients with HCV have MC; however, CV develops only in a minority of them [28, 30–35], with a prevalence of 2–13% [36, 37]. Factors associated with the development of MC include female sex, alcohol consumption above 50 g/day, HCV genotype 2 or

25

Cutaneous Cryoglobulinemic Vasculitis

3, and extensive liver fibrosis [38]. Persistent HCV infection is believed to act as a chronic stimulator of the immune system, resulting in clonal B-lymphocyte proliferations and the production of cryoglobulins, which lead to circulating immune complexes (onethird of which become insoluble when exposed to cold temperatures) [3, 8, 39]. Indeed, the major difference between HCV patients with and without MC is the presence of functional derangements of B cells and restrictions of the humoral response promoted by the expansion of clonal B-cell proliferations [40, 41]. Expansion of RF-synthesizing B lymphocytes (the hallmark of MC) in combination with RF activity and cryoprecipitability is believed to be responsible for the pathogenesis of CV, an immune complex (antigen-antibody) mediated systemic vasculitis (Coombs Gel type III reaction) [8, 9]. In HCV-related CV, cold-insoluble immune complexes are formed by IgM with RF activity linked to IgG, which also bind HCV particles and non-enveloped nucleocapsid proteins, in which the viral proteins confer peculiar physical and chemical properties on cryoimmunoglobulins [5]. Indeed, in support of the immune complex pathogenesis of CV are electron microscopy findings of the typical crystalline structures of cryoglobulins in vessels [42–44]. In addition, IgM and IgG vascular deposits are detected by direct immunofluorescence exam, immunohistochemistry, and in situ hybridization localizing HCV to vessel endothelium affected by vasculitis [45–47]. The deposition of immune complexes (IgM-RF-IgG-HCV) induced by cold exposure, stasis, or another trigger, such as local inflammation, activates complement, which in turn attracts neutrophils, setting off an inflammatory cascade that disrupts the integrity of the vessel wall resulting in the clinical manifestations of vasculitis [9, 45, 48]. While HCV is the principal antigen driving immune-complex formation, in some patients either a sustained anti-viral response does not lead to remission of CV or CV relapses shortly afterwards [49, 50]. These phenomena implicate other, unknown antigens and progression of the aberrant immune response by epitope spreading [51] and/or the development of autonomous B-cell proliferations [5]. Cellmediated cytotoxicity may also play a role in the pathogenesis of CV, as there may be numerous CD8+ lymphocytes in cutaneous lesions of HCV-related CV [46], and lymphocytic vasculitis has been reported in rare cases of CV [47, 52].

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Lastly, non-inflammatory small- and/or muscularvessel thrombosis is an under-recognized pathogenic mechanism contributing to the cutaneous manifestations of MC/CV, mostly ulceration. About a third of CV patients exhibit thrombosis [6, 37, 42, 47, 53–62]. These CV-related vaso-occlusive thrombi can be found either concurrently with vasculitis or independently [42, 53, 54, 59, 61, 63]. In general, abnormal coagulation, blood flow (stasis), chronic inflammation, and endothelial cell activation all contribute to the development of individual lesions of vasculitis [9]. Moreover, hypercoagulable states (e.g., factor V Leiden, protein C or S deficiency) are significantly more frequent in patients with ulcerative cutaneous vasculitis [64, 65]. In connective tissue disease vasculitis, the coexistence of prothombotic antiphospholipid antibodies contributes to the rapid evolution of vascular insufficiency, with progressive tissue ischemia and infarction [66, 67]. Recurrent episodes of CV and its corresponding tissue damage likely lead to abnormal blood flow, producing a prothrombotic environment and the subsequent formation of vaso-occlusive thrombi, in some cases type I cryoglobulin thrombi.

25.4

Clinical Findings

Table 25.2 lists the demographics and clinical manifestations found in MC/CV. Most of these clinical symptoms, signs, and complications of CV significantly increase with disease duration [25] The disease expression is variable, ranging from mild clinical symptoms to life-threatening complications [8], but this is dependent on disease activity and duration [25] The most frequent target organs in patients with MC are the skin, joints, nerves, and kidney. Up to 80% of patients present with the typical clinical CV triad of purpura, arthralgias, and weakness [25]. Palpable purpura, the clinical hallmark of cutaneous vasculitis, is found in almost all patients at some point in their disease and usually affects the lower extremities, as venous stasis and environmental exposure favor the precipitation of cryoglobulins [24, 52, 69]. Extension of palpable purpura above the waistline is a clue to the presence of systemic disease in patients presenting with cutaneous vasculitis [9] and is a correspondingly frequent occurrence in CV [4]. In decreasing order of frequency, the lower extremities, the upper extremities, trunk, head and neck, oral and nasal mucosa are involved by CV [61]. Typically, cutaneous CV

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Table 25.2 Evolution of clinical findings in mixed cryoglobulinemia/cryoglobulinemic vasculitis Clinical findings Female/male ratio Mean age at onset Mean duration of disease Cutaneous manifestations (any) Arthralgias Arthritis Weakness (asthenia) Raynaud’s phenomenon Neurologic involvement (peripheral and CNS) Peripheral neuropathy Central nervous system Renal involvement Sicca syndrome Skin ulcers Lung disease Liver disease (abnormal liver functions, hepatomegaly) Hyperviscosity syndrome Systemic (diffuse) vasculitis Cancer (B-cell lymphoma, hepatocellular carcinoma, thyroid cancer) Gastrointestinal disease

Onset/beginning of follow-upa 3:1 53 ± 12 years

End of follow-upa

11 ± 8 years 56%, (9–100%)b

98% (purpura)

39%, (17–72%)b 8% 72% (9–80%)b 24% (5–50%)b

91% 7% 98% 48%

24% (8–52%)b

58%

81%

2%c

2% c

20% (2–40%)b 13% (2–29%)b 11% 0 35% (0–58%)b

30% 53% 22% 2% 77%

0

0.5%

0

6.2%

0.5%

16%

0.2%c

1%c

digital gangrene (Table 25.3 and Figs. 25.1–25.3). The ulcers of CV are distinctive. They are located distally on the legs, are painful and bilateral, show a fibrinous or necrotic bed, and have well circumscribed margins surrounded by purpuric and pigmented skin [55] (Fig. 25.3). As many of these clinical findings can be due to nonvasculitic disorders, skin biopsy is crucial in documenting the presence of vasculitis. This histologic finding coupled with direct immunofluorescence data, serologic studies, and review of the body’s organ systems is the most effective method at arriving at a specific diagnosis of a vasculitic syndrome and excluding vasculitis mimics [9, 89, 90]. Notably, cryocrit levels correlate with cutaneous findings of CV: purpuric outbreaks are frequently observed in the late afternoon, when the cryocrit levels are highest [25, 91]. In patients with CV, palpable purpura is accompanied less frequently by systemic symptoms, such as fever (>70%), arthralgias (70%), membranoproliferative glomerulonephritis type I (55%), neuropathy (> C4) vascular deposits Basement membrane zone immunoreactants (Lupus band) Cutaneous HCV studies Immunohistochemistry anti-HCV In situ hybridization HCV RNA

Mean frequencya 74% 34% 34% 15% 11% 10% 8% 7% 4% 3% 70 years with renal failure and receiving corticosteroids who may develop severe infections [43].

27.3.3 Summary Both HCV-related mixed cryoglobulinemia and the associated symptoms can be alleviated following appropriate treatment. A complete immunological response, defined as the disappearance of MC and associated symptoms, can be achieved using: (1) the best antiviral treatment combination, i.e., PEG-IFN-a + ribavirin, which induces viral clearance in up to 60% of patients; (2) B-cell depleting rituximab, shown to achieve immunological and clinical responses in up to 70% of patients; or (3) both strategies, particularly in patients with severe forms of the disease that include kidney involvement.

27.3.4 Role of IgMk MC in RituximabAssociated Systemic Reactions We recently reported six cases of severe, often lifethreatening, systemic side effects after rituximab infu-

sions in patients with HCV-related MC vasculitis, demonstrating an important relationship between IgMk type II MC and the dose of rituximab infusion [39]. These side effects included four life-threatening flares of MC vasculitis and two cases of typical serum sickness syndrome. In this study, systemic side effects occurring after rituximab infusions in a cohort of 22 prospectively followed HCV-infected patients with biopsy-proven MC vasculitis were analyzed. Eighteen patients received the low-dose rituximab protocol, consisting of 375 mg/m2/week (RTX-375) for 4 consecutive weeks, and four patients the high-dose protocol of 1,000 mg rituximab on days 1 and 15 (RTX-1000). Six out of the 22 (27.8%) patients experienced severe systemic drug reactions after rituximab infusions. Four patients with type II IgMk MC developed a severe flare-up of MC vasculitis, including cardiac (n = 2), renal (2), digestive (3), and neurological (1) involvement, 1–2 days after rituximab infusion (Fig. 27.2). These patients successfully responded to methylprednisolone pulses (n = 4) and plasma exchanges (n = 2). Two other patients developed typical serum sickness syndrome 7 and 9 days after the first RTX-1000 infusion, with a spontaneous recovery. Compared to the absence of rituximab-associated drug reactions, the most striking finding was an association between the occurrence of rituximab-associated drug reactions and higher MC (1.4 ± 0.8 vs. 0.71 ± 0.77 g/L; p = 0.0475) and lower C4 (0.02 ± 0.006 vs. 0.073 ± 0.07 g/L;

224 Fig. 27.3 (a) In vitro cryoprecipitation experiments after rituximab addition (RTX+) to serum containing type II MC with an IgMk component. Within 1.00 g/L) and C4 levels low (£0.03 g/L). In these situations, rituximab is better administered at low doses (375 mg/m2 weekly for four consecutive weeks or even lower doses [40]), preferentially preceded by plasma exchanges to lower MC serum levels.

RTX+

RTX–

RTX+

27.3.5 Summary Up to one-fourth of HCV-infected patients with MC vasculitis may develop severe systemic reactions after rituximab infusion. These reactions are related to the “accelerated” formation of immune complexes between rituximab and type II IgMk MC exhibiting rheumatoid factor activity, and they occur in a rituximab dose- and serum-MC-level-dependent manner. Accordingly, rituximab should be prescribed cautiously in HCV-related MC vasculitis, with the low-dose rituximab protocol (375 mg/m2/week) and, in patients with high baseline MC levels, plasma exchanges prior to rituximab infusion as the recommended therapeutic strategy.

27.4

Mixed Cryoglobulinemia Relapse After a Sustained Virological Response

The relapse of mixed cryoglobulinemia and MC vasculitis is frequent in the absence of a sustained virological response after HCV treatment. Conversely, relapse of the disease, including its associated clinical

27

Long-Term Course of Patients with Mixed Cryoglobulinemia

manifestations, is rarely reported after successful HCV treatment. Levine et al. presented four patients in whom MC vasculitis relapse occurred during the first year after withdrawal of antiviral treatment, accompanied by rising MC levels and decreasing C4 levels. An exhaustive work-up excluded the presence of a BNHL [41]. Our group reported on eight patients who experienced a relapse of HCV-related MC vasculitis despite having achieved a sustained viral response after HCV treatment [42]. Relapse appeared early after the end of antiviral treatment (2.5 ± 3.5 months) and was associated with an increase in serum MC levels. In most patients, the relapse was brief and the MC vasculitis manifestations subsided. A search for HCV RNA by transcription-mediated amplification was negative, both in sera and in cryoprecipitates, in seven out of the eight patients tested. In three patients, MC vasculitis symptoms persisted and were associated with high serum MC levels. BNHL was finally diagnosed in two of these three patients. In summary, a relapse of MC vasculitis occurs in only a few patients with HCV-related MC vasculitis who have achieved a sustained viral response. In such patients, different underlying conditions should be considered, with a special emphasis on malignant B-cell lymphoproliferative disease.

27.5

Conclusion

The long-term course of mixed cryoglobulinemia during HCV infection depends on chronic HCV antigenic stimulation of B cells. As seen in many patients, the natural tendency of the disease is the evolution of type III MC to the oligoclonal type and finally to type II MC, which mostly exhibit rheumatoid factor activity and are frequently associated with vasculitis. The course of mixed cryoglobulinemia after treatment depends on the ability to achieve viral clearance. Clinical studies have shown that PEG-IFN-a + ribavirin is the most efficient treatment, inducing a complete immunological response in most patients, always associated with viral clearance. Clinical and immunological responses were achieved in most patients treated with rituximab, which specifically targets B cells and thus MC production; however, there was a high rate of post-treatment relapse due to viral persistence. More recently, a combined approach, consisting of rituximab

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and antiviral treatment, was shown as the best current therapeutic strategy in patients with HCV-related MC vasculitis, since both HCV clearance and B-cell depletion are targeted. However, rituximab must be used cautiously, preferably at a low dose and, in those patients with high serum MC levels, preceded by plasma exchanges. After a sustained virological response, MC and symptoms related to MC vasculitis may relapse in a very few patients, in which case an underlying BNHL should be ruled out.

References 1. Brouet JC, Clauvel JP, Danon F et al (1974) Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 57(5):775–788 2. Tridon A, Abergel A, Kuder P et al (1997) Mixed cryoglobulins and autoimmunity in hepatitis C. Pathol Biol (Paris) 45(4):291–297 3. Sène D, Ghillani-Dalbin P, Thibault V et al (2004) Longterm course of mixed cryoglobulinemia in patients infected with hepatitis C virus. J Rheumatol 31(11):2199–2206 4. Meltzer M, Franklin EC, Elias K et al (1966) Cryoglobulinemia – a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity. Am J Med 40(6):837–856 5. Cacoub P, Delluc A, Saadoun D et al (2008) Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemic vasculitis: where do we stand? Ann Rheum Dis 67(3):283–287 6. Schifferli JA, French LE, Tissot JD (1995) Hepatitis C virus infection, cryoglobulinemia, and glomerulonephritis. Adv Nephrol Necker Hosp 24:107–129 7. Musset L, Diemert MC, Taibi F et al (1992) Characterization of cryoglobulins by immunoblotting. Clin Chem 38(6):798–802 8. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 43(1):94–102 9. Sansonno D, De Vita S, Iacobelli AR et al (1998) Clonal analysis of intrahepatic B cells from HCV-infected patients with and without mixed cryoglobulinemia. J Immunol 160(7):3594–3601 10. Migliaresi S, Tirri G (1995) Interferon in the treatment of mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl 13):S175–S180 11. Misiani R, Bellavita P, Fenili D et al (1994) Interferon alfa2a therapy in cryoglobulinemia associated with hepatitis C virus. N Engl J Med 330(11):751–756 12. Cohen P, Nguyen QT, Deny P et al (1996) Treatment of mixed cryoglobulinemia with recombinant interferon alpha and adjuvant therapies. A prospective study on 20 patients. Ann Med Interne (Paris) 147(2):81–86 13. Adinolfi LE, Utili R, Zampino R et al (1997) Effects of longterm course of alpha-interferon in patients with chronic hepatitis C associated to mixed cryoglobulinaemia. Eur J Gastroenterol Hepatol 9(11):1067–1072

226 14. Polzien F, Schott P, Mihm S et al (1997) Interferon-alpha treatment of hepatitis C virus-associated mixed cryoglobulinemia. J Hepatol 27(1):63–71 15. Casato M, Agnello V, Pucillo LP et al (1997) Predictors of long-term response to high-dose interferon therapy in type II cryoglobulinemia associated with hepatitis C virus infection. Blood 90(10):3865–3873 16. Calleja JL, Albillos A, Moreno-Otero R et al (1999) Sustained response to interferon-alpha or to interferon-alpha plus ribavirin in hepatitis C virus-associated symptomatic mixed cryoglobulinaemia. Aliment Pharmacol Ther 13(9): 1179–1186 17. Cresta P, Musset L, Cacoub P et al (1999) Response to interferon alpha treatment and disappearance of cryoglobulinaemia in patients infected by hepatitis C virus. Gut 45(1):122–128 18. Naarendorp M, Kallemuchikkal U, Nuovo GJ et al (2001) Longterm efficacy of interferon-alpha for extrahepatic disease associated with hepatitis C virus infection. J Rheumatol 28(11):2466–2473 19. Cacoub P, Renou C, Kerr G et al (2001) Influence of HLA-DR phenotype on the risk of hepatitis C virus-associated mixed cryoglobulinemia. Arthritis Rheum 44(9):2118–2124 20. Cacoub P, Lidove O, Maisonobe T et al (2002) Interferonalpha and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 46(12): 3317–3326 21. Cacoub P, Ratziu V, Myers RP et al (2002) Impact of treatment on extra hepatic manifestations in patients with chronic hepatitis C. J Hepatol 36(6):812–818 22. Zuckerman E, Keren D, Slobodin G et al (2000) Treatment of refractory, symptomatic, hepatitis C virus related mixed cryoglobulinemia with ribavirin and interferon-alpha. J Rheumatol 27(9):2172–2178 23. Mazzaro C, Zorat F, Comar C et al (2003) Interferon plus ribavirin in patients with hepatitis C virus positive mixed cryoglobulinemia resistant to interferon. J Rheumatol 30(8):1775–1781 24. Manns MP, McHutchison JG, Gordon SC et al (2001) Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 358(9286):958–965 25. Fried MW, Shiffman ML, Reddy KR et al (2002) Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347(13):975–982 26. Saadoun D, Resche-Rigon M, Thibault V et al (2006) Antiviral therapy for hepatitis C virus–associated mixed cryoglobulinemia vasculitis: a long-term followup study. Arthritis Rheum 54(11):3696–3706 27. Cacoub P, Saadoun D, Limal N et al (2005) PEGylated interferon alfa-2b and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 52(3):911–915 28. Zaja F, Russo D, Fuga G et al (1999) Rituximab for the treatment of type II mixed cryoglobulinemia. Haematologica 84(12):1157–1158

D. Sene and P.P. Cacoub 29. Zaja F, De Vita S, Russo D et al (2002) Rituximab for the treatment of type II mixed cryoglobulinemia. Arthritis Rheum 46(8):2252–2254, author reply 2254–2255 30. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101(10):3827–3834 31. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101(10):3818–3826 32. Roccatello D, Baldovino S, Rossi D et al (2004) Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis. Nephrol Dial Transplant 19(12):3054–3061 33. Lamprecht P, Lerin-Lozano C, Merz H et al (2003) Rituximab induces remission in refractory HCV associated cryoglobulinaemic vasculitis. Ann Rheum Dis 62(12):1230–1233 34. Catuogno M, Rezai S, Priori R et al (2005) Serum sickness associated with rituximab in a patient with hepatitis C virusrelated mixed cryoglobulinaemia. Rheumatology (Oxford) 44(3):406 35. Basse G, Ribes D, Kamar N et al (2005) Rituximab therapy for de novo mixed cryoglobulinemia in renal transplant patients. Transplantation 80(11):1560–1564 36. Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with peg-interferon-ribavirin in refractory HCVassociated cryoglobulinemia vasculitis. Ann Rheum Dis 67(10):1431–1436 37. Saadoun D, Resche Rigon M, Sene D et al (2010) Rituximab plus Peg-interferon-alpha/ribavirin compared with Peginterferon-alpha/ribavirin in hepatitis C-related mixed cryoglobulinemia. Blood 116(3):326–334 38. Dammacco F, Tucci FA, Lauletta G et al (2010) Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a longterm study. Blood 116(3):343–353 39. Sène D, Ghillani-Dalbin P, Amoura Z et al (2009) Rituximab may form a complex with IgMkappa mixed cryoglobulin and induce severe systemic reactions in patients with hepatitis C virus-induced vasculitis. Arthritis Rheum 60(12): 3848–3855 40. Visentini M, Granata M, Veneziano ML et al (2007) Efficacy of low-dose rituximab for mixed cryoglobulinemia. Clin Immunol 125(1):30–33 41. Levine JW, Gota C, Fessler BJ et al (2005) Persistent cryoglobulinemic vasculitis following successful treatment of hepatitis C virus. J Rheumatol 32(6):1164–1167 42. Landau DA, Saadoun D, Halfon P et al (2008) Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response. Arthritis Rheum 58(2):604–611 43. Terrier B, Saadoun D, Sene D et al (2009) Efficacy and tolerability of rituximab with or without PEGylated interferon alfa-2b plus ribavirin in severe hepatitis C virus-related vasculitis: a long-term followup study of thirty-two patients. Arthritis Rheum 60(8):2531–2540

HBV/HCV Co-infection and Mixed Cryoglobulinemia

28

Massimo Galli and Salvatore Sollima

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major public health problems. It is estimated that there are more than 350 million chronic carriers of HBV, and 170 million people are chronically infected with HCV. Since the two viruses share routes of transmission, dual infections are a common occurrence, particularly in highly endemic areas. Co-infection is also frequently seen in individuals at risk for parenterally transmissible infections, such as injection drug users, patients on hemodialysis or undergoing organ transplantation, and patients with b-thalassemia, as well as in HIV-positive individuals [1–8]. Due to the lack of large-scale population-based studies, the worldwide prevalence of dual chronic infection with HBV and HCV remains undefined [1]. Among HBV-infected patients, anti-HCV positivity is estimated to be 10–15%, with wide geographic variation [6]. Moreover, limited data are available on the correlates of risk of co-infection. In a multicenter prospective study performed in Italy, age over 42 years, history of injection drug use and/or blood transfusion, and residence in the south of the country were independently related to HBV/HCV co-infection [9]. Compared with mono-infections, HBV/HCV coinfections are accompanied by more severe liver injury, with a higher probability of liver cirrhosis and hepatic failure, and a higher incidence of hepatocellular carcinoma [1, 4, 6, 10–15].

M. Galli (*) Department of Clinical Sciences “Luigi Sacco”, Section of Infectious and Tropical Diseases, Università di Milano, Milan, Italy e-mail: [email protected]

Clinical and laboratory studies have shown that HBV and HCV interact in chronically co-infected patients [5, 15]. In particular, in vitro studies showed that HCV core protein can exert an inhibitory effect on HBV replication [16–19]. In most co-infected patients, HCV viremia is detectable whereas serum HBV DNA is low, suggesting an interplay between the two viruses in which HBV replication is inhibited [3, 12, 20–22]. Nevertheless, clinical studies do not uniformly report a dominant role of HCV in HBV/ HCV co-infections; instead, some findings suggest a reciprocal interference, or even a dominant effect of HBV [2, 4, 6, 7, 23–26]. Zarski et al. found that HCV RNA levels were significantly lower in HBV/HCV coinfected patients with positive serum HBV DNA than in HBV DNA-negative patients [6]. Furthermore, in a multicenter longitudinal follow-up study, Raimondo et al. demonstrated wide fluctuations of HBV and HCV viremia levels over time, with both viruses showing alternating phases of activation and suppression [7]. These findings suggest that each virus exerts its own pathogenetic role. The resulting cumulative effects on the liver may explain the high grade of disease severity frequently observed in HBV/HCV coinfected patients [1, 7]. A further intriguing but poorly investigated issue regarding HBV/HCV interactions is the so-called occult HBV infection, defined as the presence of HBV genomes in the liver tissue and sometimes, at very low levels, in the serum of hepatitis B surface antigen (HBsAg)-negative individuals. Suspected since the early 1980s, this peculiar form of chronic viral infection has been demonstrated in the last 15 years, as highly sensitive molecular biology techniques have become available [27].

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_28, © Springer-Verlag Italia 2012

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Occult HBV status is due to the long-lasting persistence of HBV DNA as covalently closed circular DNA (cccDNA) in hepatocyte nuclei [28, 29]. It may be caused by mutant viruses either producing a modified HBV S protein undetectable by HBsAg assays [30–32] or unable to express the S gene [33]. More frequently, it is due to the strong suppression of viral replication and gene expression exerted by the host’s immune system or co-infecting agents, particularly HCV [27, 34–36]. In fact, the highest prevalence (up to 50% in some series) of occult HBV is seen in patients with HCV infection [27, 37], in whom HBV replication is often inhibited by HCV, as discussed above. Occult HBV infection is usually associated with the presence of anti-HBV antibodies (namely, anti-HBc and antiHBs), although more than 20% of occult carriers are negative for all serum markers of HBV infection [38]. This condition may impact transmission of the infection by blood transfusion or organ transplantation [39–41], with the potential for acute reactivation occurring against a background of immunosuppression [42–45]. Moreover, there is abundant evidence that occult HBV infection favors the progression of liver fibrosis and the development of cirrhosis and hepatocellular carcinoma [24, 38, 46–52]. These complex interactions between the two viruses raise many questions about their roles in causing cryoglobulinemias. It is well known that HBV can induce immuno-pathogenetic processes that are responsible for extrahepatic manifestations, such as polyarteritis nodosa (PAN) and glomerulonephritis [53, 54]. Even before the discovery of HCV, an association between liver disease and cryoglobulin production was recognized [55, 56]. Initially, HBV was investigated as the possible causative agent of so-called essential cryoglobulinemia [57]. However, the attribution of a causative role to HBV was confuted within a short time on the basis of epidemiological and serological data. In particular, HBsAg and HBV DNA were detected only in a small minority of patients with essential cryoglobulinemia, and the prevalence of antiHBV antibodies widely varied among different patient populations with mixed cryoglobulinemia (MC) and did not significantly differ from the prevalence found in age and sex-matched individuals living in the same areas and sharing the same degree of risk of parenteral exposure as cryoglobulinemic patients [58–61]. Nevertheless, an etiologic role of HBV in MC can be postulated in some patients, in the absence of other

M. Galli and S. Sollima

conditions known to be capable of inducing cryoglobulin production. In a study performed in the first half of the 1990s, we found that a role for HBV in causing MC could be supported in 3 g/dL However, depending on the properties of the monoclonal type I IgM cryoglobulin, the symptomatic threshold may be significantly lower [4, 28]. Manifestations indicating the presence of cryoglobulins may include purpuric lesions, acrocyanosis, Raynaud phenomena, dystrophic manifestations, and the formation of torpid ulcers and gangrene. Cryoglobulinemic neuropathy of type I cryoglobulinemia is characterized by a distal, sensory, symmetric polyneuropathy or with mononeuropathy multiplex, which is often axonal. Symptoms of peripheral neuropathy may be the presenting symptom and indicate the need for treatment initiation even when other symptoms (such as cytopenia or symptomatic organomegaly) are lacking [44–47]. Central nervous system involvement is rare, although hemorrhagic vasculopathic encephalopathy associated with a type I cryoglobulinemia has been reported [48]. Renal involvement in type I cryoglobulinemia is manifested mainly in the form of albuminuria. Since there is no inflammatory reaction within the glomeruli,

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there should be no active urine sediment. However, in the presence of proliferative glomerular damage, a nephritic picture with hematuria and red cell casts may be seen. In patients with significant albuminuria, the possibility of renal amyloidosis should be considered. The presence of rapidly progressive glomerulonephritis should raise suspicion of cryoglobulinemia with immune activation and proliferative glomerular damage [10, 37, 40, 41, 49–51]. There are also a variety of cutaneous manifestations that may occur in the context of type I cryoglobulinemia, including palpable purpura, ischemic necrosis, cutaneous ulcerations, livedo reticularis infarctions, cold urticaria, inflammatory macules and papules, Raynaud phenomenon, scarring of the nose, pinnae, fingertips, and toes, and hyperkeratotic spicules on acral surfaces [4, 24, 28]. The clinical syndrome of type II cryglobulinemia (mixed cryoglobulinemia syndrome) is characterized by a typical clinical triad of purpura, weakness, and arthralgias. However, multi-system organ involvement, including chronic hepatitis, membranoproliferative glomerulonephritis, and peripheral neuropathy due to leukocytoclastic vasculitis of small and medium-sized vessels is common. Different clinical patterns may be present at the initial diagnosis. In asymptomatic patients, isolated serum mixed cryoglobulins may be the only feature and probably expresses early-stage disease while other patients may present with a complete cryoglobulinemic syndrome characterized by the combination of serological findings and clinicopathological features. Mixed cryglobulinemia syndrome may initially appear as an incomplete form, gradually evolving during long-term follow-up into an overt syndrome. In some patients, the symptoms of WM may dominate, with mixed cryglobulinemia becoming overt during the course of the disease, while in others clinically overt type II cryoglobulinemia may not be associated with the additional features of WM, except the clinicopathological findings in the bone marrow. All WM patients with cold-sensitive symptoms should be tested for the presence of cryoglobulins.

36.4

Treatment Options

Plasmapheresis/plasma exchange is very effective for the immediate reduction of the amount of circulating cryoglobulins [4, 24, 36, 52]. Circulating

levels of mixed cryoglobulins and monoclonal IgM cryoglobulins are more easily reduced than those of IgG cryoglobulins [53]. A dramatic response may be seen even after just one plasmapheresis procedure, but the effects may be temporary if it is not repeated at scheduled intervals. For some patients with type I cryoglobulinemia and symptomatic hyperviscosity syndrome, normal saline should be used instead of albumin as replacement fluid. It is important that the albumin or saline is pre warmed to 37°C in order to avoid exacerbation of the symptoms due to a decrease in core temperature, thus leading to cryoprecipitation. Blood transfusions should be avoided if possible; commonly, the hematocrit is factitiously low due to the high IgM levels, which lead to blood volume expansion. In any case, any blood product should be pre-warmed to 37°C. Cascade filtration is as effective as plasma exchange, and both can remove 50% of plasma IgM and reduce plasma viscosity by 60% [54]. Although cascade filtration selectively removes macroglobulins, it is not more effective than plasma exchange [55]. On-line cryopheresis, using special filters with an average pore size of 0.2 mm, was previously used but the technique is hindered by quick plugging of the filter, thus requiring frequent backwashing or filter changes, and its use therefore cumbersome [56]. Cryofiltration apheresis using a high-capacity cryofilter is the most selective procedure to remove cryoglobulins and is specific for the treatment of cryoglobulinemia [57]. In some patients with WM and cryoglobulinemia, the tumor load at presentation is low, anemia and organomegaly are absent, and the main discomfort is caused by the cryoglobulins. In such cases, an intensive series of plasma exchange may rapidly reduce the monoclonal protein and provide immediate symptomatic improvement. However, plasma exchange alone does not usually lead to the prolonged remission of symptoms, and systemic treatment is needed in almost all of these patients. Recently, treatment recommendations for patients with symptomatic WM were formulated and published by a panel of experts. They provide specific recommendations for initial and salvage treatment of patients with symptomatic WM [52]. The number of patients who are diagnosed with asymptomatic WM is increasing. These patients have the typical features of WM on bone marrow biopsy and a monoclonal IgM that may be quite large; however,

36

Waldenström’s Macroglobulinemia Associated with Cryoglobulinemia

they do not have symptoms reflecting WM. Specifically, these patients do not have anemia or thrombocytopenia but may have lymphadenopathy or some splenomegaly, although neither is symptomatic. According to the consensus criteria, these patients should be followed without treatment; rather, initiation of treatment should only be considered for patients with symptoms due to WM and not on the basis of the amount of IgM per se [58]. However, in patients presenting with symptoms due to the cryoglobulinemic properties of the monoclonal IgM, treatment should be considered even when other features of the disease, such as cytopenia or lymphadenopathy, are absent or when the levels of monoclonal IgM are low. Treatments targeting the IgM-producing clone of WM may take weeks to effectively reduce the amount of circulating immunoglobulin. After the initiation of plasma exchange, anti-lymphoma treatment should be started as soon as possible. A highly effective regimen should be used preferably; single agent agents such as chlorambucil are not indicated. The single agent rituximab is also not recommended for patients suffering symptomatic type I cryoglobulinemia, due to the slow response, and an increase in IgM levels may follow its administration. This surge of IgM does not indicate treatment failure, but in patients with cryoglobulinemia and hyperviscosity it may exacerbate symptoms. Indeed, after initiation of rituximab, exacerbations of symptoms or complications related to cryoglobulinemia in patients without previous symptoms of the disease have been reported [59]. Thus, close follow-up is needed, at least during the first months after initiation of rituximab. Bortezomib has shown activity in WM patients with relapsed/refractory disease and in newly diagnosed patients. It has the advantage that it rapidly reduces the levels of IgM and thus may be a useful option for some patients with symptoms of hyperviscosity. However, bortezomib is neurotoxic and in patients with cryoglobulinemic neuropathy its use may be contraindicated. Nucleoside analogues are very effective and may improve symptomatic cryoglobulinemic neuropathy but these drugs are associated with immunosuppression; in younger patients who may be candidates for autologous stem cell transplantation, they should probably be deferred. According to recent recommendations, a combination of chemotherapy and immunotherapy based on the monoclonal antibody rituximab is the preferable regimen for newly diagnosed previously untreated

281

patients. CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) combined with rituximab or the combination of DRC (dexamethasone, rituximab, cyclophosphamide) may be used. Time to response varies between 1 and 4 months, and repeated plasmapheresis may be needed. Bortezomib, as a single agent, may be used in some cases due to its rapid reduction of IgM levels. Supportive measures are also very important for patients suffering from symptoms of cryoglobulinemia. Thus, both the avoidance of exposure to cold temperatures and the use of gloves, hats, etc., should be encouraged. A warm room, or warming blankets in some cases, could be useful in reducing symptoms and complications [4, 50]. In WM patients with type II cryoglobulinemia, manifestations of the disease are triggered by the immunologic properties of the IgM-IgG complex and the amounts of IgM are not as high as in type I cryoglobulinemia. Plasmapheresis may not be as effective since even small amounts of the IgM-IgG complex can trigger vasculitis. Rituximab has been used successfully to target the B-cell clone and to reduce the immunologic phenomena of type II cryoglobulinemia. Steroids with or without cyclophosphamide have also been used as an immunosuppressive adjunct. Patients with WM and symptomatic type II cryoglobulinemia with an underlying HCV infection may need special management. Rituximab targets the WM clone and has shown activity in refractory HCV-related type II cryoglobulinemia, although it is mostly effective in the treatment of skin features and less so in renal complications of the syndrome. A flare of HCV RNA load has been reported in some patients, without deterioration of liver biology [60–71]. However, there are limited data on the use of chemotherapy combinations with rituximab in patients with HCV and symptomatic WM with or without cryoglobulinemia [72].

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41. Meyrier A, Simon P, Mignon F et al (1984) Rapidly progressive (‘crescentic’) glomerulonephritis and monoclonal gammapathies. Nephron 38(3):156–162 42. Ropper AH, Gorson KC (1998) Neuropathies associated with paraproteinemia. N Engl J Med 338(22):1601–1607 43. Garcia-Bragado F, Fernandez JM, Navarro C et al (1988) Peripheral neuropathy in essential mixed cryoglobulinemia. Arch Neurol 45(11):1210–1214 44. Meier C (1985) Polyneuropathy in paraproteinaemia. J Neurol 232(4):204–214 45. Vital C, Vallat JM, Deminiere C et al (1982) Peripheral nerve damage during multiple myeloma and Waldenstrom’s macroglobulinemia: an ultrastructural and immunopathologic study. Cancer 50(8):1491–1497 46. Nobile-Orazio E (2004) IgM paraproteinaemic neuropathies. Curr Opin Neurol 17(5):599–605 47. Vital A, Vital C, Ragnaud JM et al (1991) IgM cryoglobulin deposits in the peripheral nerve. Virchows Arch 418(1): 83–85 48. Mazzola L, Antoine JC, Camdessanche JP et al (2003) Brain hemorrhage as a complication of type I cryoglobulinemia vasculopathy. J Neurol 250(11):1376–1378 49. Zlotnick A, Rosenmann E (1975) Renal pathologic findings associated with monoclonal gammopathies. Arch Intern Med 135(1):40–45 50. Shaikh A, Habermann TM, Fidler ME et al (2008) Acute renal failure secondary to severe type I cryoglobulinemia following rituximab therapy for Waldenstrom’s macroglobulinemia. Clin Exp Nephrol 12(4):292–295 51. Yonemura K, Suzuki T, Sano K et al (2000) A case with acute renal failure complicated by Waldenstrom’s macroglobulinemia and cryoglobulinemia. Ren Fail 22(4): 511–515 52. Dimopoulos MA, Gertz MA, Kastritis E et al (2009) Update on treatment recommendations from the Fourth International Workshop on Waldenstrom’s Macroglobulinemia. J Clin Oncol 27(1):120–126 53. Berkman EM, Orlin JB (1980) Use of plasmapheresis and partial plasma exchange in the management of patients with cryoglobulinemia. Transfusion 20(2):171–178 54. Valbonesi M, Montani F, Guzzini F et al (1985) Efficacy of discontinuous flow centrifugation compared with cascade filtration in Waldenstrom’s macroglobulinemia: a pilot study. Int J Artif Organs 8(3):165–168 55. Hoffkes HG, Heemann UW, Teschendorf C et al (1995) Hyperviscosity syndrome: efficacy and comparison of plasma exchange by plasma separation and cascade filtration in patients with immunocytoma of Waldenstrom’s type. Clin Nephrol 43(5):335–338 56. Yamashita M, Malchesky PS, Omokawa S et al (1990) Limitation of plasmapheresis in cryoglobulinemia with high levels of cryoglobulins. Prog Clin Biol Res 337:491–494 57. Siami GA, Siami FS (2001) Current topics on cryofiltration technologies. Ther Apher 5(4):283–286 58. Kyle RA, Treon SP, Alexanian R et al (2003) Prognostic markers and criteria to initiate therapy in Waldenstrom’s

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macroglobulinemia: consensus panel recommendations from the second international workshop on Waldenstrom’s macroglobulinemia. Semin Oncol 30(2):116–120 Treon SP (2009) How I treat Waldenstrom macroglobulinemia. Blood 114(12):2375–2385 Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101(10): 3818–3826 Saadoun D, Rosenzwajg M, Landau D et al (2008) Restoration of peripheral immune homeostasis after rituximab in mixed cryoglobulinemia vasculitis. Blood 111(11): 5334–5341 Roccatello D, Baldovino S, Rossi D et al (2008) Rituximab as a therapeutic tool in severe mixed cryoglobulinemia. Clin Rev Allergy Immunol 34(1):111–117 Sailler L (2008) Rituximab off label use for difficult-to-treat auto-immune diseases: reappraisal of benefits and risks. Clin Rev Allergy Immunol 34(1):103–110 Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinaemia vasculitis. Ann Rheum Dis 67(10):1431–1436 Cacoub P, Delluc A, Saadoun D et al (2008) Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemic vasculitis: where do we stand? Ann Rheum Dis 67(3):283–287 Visentini M, Granata M, Veneziano ML et al (2007) Efficacy of low-dose rituximab for mixed cryoglobulinemia. Clin Immunol 125(1):30–33 Quartuccio L, Soardo G, Romano G et al (2006) Rituximab treatment for glomerulonephritis in HCV-associated mixed cryoglobulinaemia: efficacy and safety in the absence of steroids. Rheumatology (Oxford) 45(7):842–846 Basse G, Ribes D, Kamar N et al (2005) Rituximab therapy for de novo mixed cryoglobulinemia in renal transplant patients. Transplantation 80(11):1560–1564 Ghobrial IM, Uslan DZ, Call TG et al (2004) Initial increase in the cryoglobulin level after rituximab therapy for type II cryoglobulinemia secondary to Waldenstrom macroglobulinemia does not indicate failure of response. Am J Hematol 77(4):329–330 Ghijsels E, Lerut E, Vanrenterghem Y, Kuypers D (2004) Anti-CD20 monoclonal antibody (rituximab) treatment for hepatitis C-negative therapy-resistant essential mixed cryoglobulinemia with renal and cardiac failure. Am J Kidney Dis 43(5):e34–e38 Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101(10):3827–3834 Tedeschi A, Miqueleiz S, Ricci F et al (2007) Fludarabine, cyclophosphamide and rituximab in Waldenstrom’s macroglobulinemia: an effective regimen requiring a new category of response criteria and a delayed assessment of results. ASH annual meeting abstracts, November 16, 2007. Blood 110(11):1290

Part VII Therapy of Cryoglobulinemia

Should HCV-Positive Asymptomatic Patients with Mixed Cryoglobulinemia Be Treated with Combined Antiviral Therapy?

37

José Luis Calleja Panero, Juan de la Revilla Negro, and Fernando Pons Renedo

37.1

Introduction

The term “cryoglobulinemia” refers to the presence of cryoglobulins in the serum. While cryoglobulinemia is frequently an asymptomatic condition, the deposition of circulating immune complexes, mainly cryoglobulins, and complement in the vascular lumen can cause a leukocytoclastic type of vasculitis called cryoglobulinemic vasculitis. Mixed cryoglobulinemia (MC) syndrome follows a benign clinical course in over 50% of patients, but a moderate-severe clinical course is observed in one-third of patients, with their prognosis severely affected by renal and/or liver failure. Before the discovery of HCV, MC was considered an essential disease. In 1990, three cases of HCVassociated MC were described [1]. This was followed by an increasing number of studies that ultimately established a crucial role for HCV in the pathogenesis of MC. Indeed, between 50% and 92% of patients with MC are HCV-positive. However, the incidence of detectable cryoglobulins in these patients is only 40–50% [2] and, within this group, only 5% will develop overt MC syndrome [3]. Some studies claim that the characteristics of patients with HCV-associated MC differ from those of HCV patients without cryoglobulinemia. The prevalence of female sex is higher, the average age at diagnosis is older, the duration of liver disease is longer,

J.L.C. Panero (*) Gastroenterology and Hepatology Department, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain e-mail: [email protected]

the serum IgM concentration is higher and rheumatoid factor activity is noted, in addition to a higher prevalence of cirrhosis [2, 4]. In patients with symptomatic HCV-associated MC, the most frequent extrahepatic manifestation is palpable purpura. Anywhere from 10% to 91% of cryoglobulinemic patients suffer from arthralgias. The incidence of renal involvement varies from 8% to 58%, and polyneuropathy from 40% to 70% [5–7].

37.2

Treatment of HCV-Associated Mixed Cryoglobulinemia

The sequelae of symptomatic MC cause significant morbidity and mortality; therefore, this complication of HCV may require treatment independent of the severity of the liver disease. Spontaneous resolution is seen only in rare cases. Before the recognition of viral hepatitis as an etiologic factor, corticosteroids with or without additional immunosuppressives, mainly cyclophosphamide, and sometimes with the addition of intravenous immune globulin or plasmapheresis, were considered the standard therapy [8]. However, in most patients these efforts were only marginally effective and long-term remission was not achieved. Following the discovery of HCV as the etiologic agent of MC, new therapeutic opportunities but also problems emerged. One of the persisting issues is the potentially adverse effects of immunosuppressive therapy consisting of glucocorticoids and cytotoxic drugs on the underlying chronic viral infection. Another concern involves the cornerstone of HCV antiviral therapy, interferon (IFN), which has the potential to exacerbate

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_37, © Springer-Verlag Italia 2012

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autoimmune disease states [9]. In addition, IFN may newly induce vasculitis and worsen certain complications, such as neuropathy. Following the main steps of the etiopathogenetic process, from HCV infection to B-lymphocyte proliferation and to cryoglobulinemic vasculitis, MC can be managed at different levels [3, 10]: • Etiologic treatment: eradication of the causative agent by means of antiviral therapy; this is recommended in all patients with HCV-associated MC. • Pathogenetic treatment: reversal of the immunologic derangement producing circulating immune complexes; this is achieved with immunosuppressive therapy. • Symptomatic therapies: amelioration of the clinical manifestations of the disease, with aid of immunomodulators/anti-inflammatory agents. • In this chapter we analyze the scientific evidence regarding antiviral therapy in HCV-associated MC. Therapeutic effectiveness can be classified in terms of: (a) virological response, based on the concept of sustained virological response (SVR), (b) clinical response, in which there is symptom resolution, and (c) immunologic response, with the reduction or even sustained disappearance of cryoglobulins.

37.2.1 Treatment of MC with IFN Monotherapy The first report of the use of IFN in the treatment of MC was published in 1987, i.e., before the recognition of HCV [11]. Seven patients with refractory essential MC were treated with recombinant IFN-a2a for 4–12 months. A conspicuous reduction of circulating cryoglobulins and a remarkable improvement of the clinical pattern were noted, which seemed to be consistent and prolonged in some patients. The good results obtained with IFN were attributed to its immune regulatory and antiproliferative effects. Another prospective study [12], conducted in 21 patients with severe essential MC unresponsive to immunosuppresive regimens, showed a 77% rate of clinical and immunological response after long-term treatment (from 8 to 30 months) with recombinant IFN-a or natural IFN-b. In that study, the disease in five patients (24%) remained in prolonged remission (18–40 months) after withdrawal of the drug. These studies confirmed that complete remission of symptoms could be achieved in

J.L.C. Panero et al.

some patients with the use of IFN, in contrast to conventional immunomodulatory regimens, which offered only temporary responses. Following evidence of an etiopathogenetic link of HCV with MC, Misiani et al. [13] published a prospective randomized controlled trial in HCV-associated MC patients treated with IFN-a. The authors concluded that the efficacy of the drug in this group of patients was closely related to its antiviral activity, by means of the virological response of HCV-RNA. Quickly, IFN became the therapy of choice for MC. Several studies have addressed IFN monotherapy in MC, but they are not easy to compare due to different IFN doses and types as well as length of treatment. Moreover, they employed less sophisticated virological laboratory procedures and quantitative methods in the determination of viral titers. Additionally, universal criteria for the end of treatment and SVR were not widely applied, which leads to uncertainties in interpretation of the data. The most frequently used IFN is recombinant IFN-a, although in some reports natural interferon or lymphoblastoid IFN-a were preferred. The usual dose of IFN is 3 MU, administered subcutaneously three times a week for 6–12 months. However, induction regimens with daily doses in the first 1–3 months of therapy have been explored. The main studies conducted in patients with HCVassociated MC treated with conventional IFN-a monotherapy are reported in Table 37.1. One of the first questions that the initial trials sought to answer was whether IFN was effective in the control of MC symptoms. In two open trials [16, 17], 44 patients were treated with IFN-a at a dose of 2–5 MU/3 times a week (ttw) for 6–12 months. At the end of treatment, 65–72% of the patients had clinical and immunological responses and in 50% HCV was negative. The clinical benefits used to define response were limited to cutaneous and articular manifestations; renal and neurologic involvement was less sensitive. However, these global beneficial effects were often transient, except for the small proportion of patients achieving a sustained clearance of HCV. In a randomized, crossover-controlled trial [20], 20 patients were assigned to one of two groups and after the first 6 months with or without IFN therapy the patients were crossed over to the other half of the study for an additional 6 months. While all the patients who completed treatment experienced a significant improvement of purpura,

37

Should HCV+ Asymptomatic Patients with MC Be Treated with Antiviral Therapy?

289

Table 37.1 Published studies in which HCV-associated MC treated with interferon monotherapy Number of Author (year) patients Dammacco (1994) [14] A: 15 B: 17

Casato (1997) [15]

C: 18 D: 15 31

Migliaresi (1995) [16] 24 Akriviadis (1997) [17] 18 Pellicano (1999) [18] 32 HCV MC+ 30 HCV MC− A: 18 Mazzaro (1995) [19] B: 18 23 HCV MC+ Polzien (1997) [4] 36 HCV MC− 20 Ferri (1993) [20]

Cresta (1999) [21] Misiani (1994) [13]

Calleja (1999) [22]

43 HCV MC+ 44 HCV MC− 27

18 HCV MC+ 195 HCV MC−

IFN regimen A: IFN 3 MU/3tw B: IFN 3 MU/3tw + PRED 16 mg/4tw C: PRED 16 mg/day D: No therapy IFN 3 MU/day 3 months + IFN 3 MU/3tw ³9 months IFN 3 MU/3tw IFN 3–5 MU/3tw IFN lymphoblastoid 3 MU/3tw IFN 3 MU/3tw IFN 3 MU/3tw IFN 2 MU/d 1 month + INF 2 MU/3tw 5 months IFN 3 MU/3tw IFN 1.5 MU/3tw 1 week + IFN 3MU/3tw 23 weeks IFN 3 MU/3tw

Duration of therapy 12 months

³12 months

SVR (%) A: 28 B: 28

CR to therapy (%)a A: 53.3 B: 52.9

Sustained CR (%)b A: 25 B: 33

C: 0 D: 0 16

C: 26.7 D: 6.7 62

C: 0 D: 0 48

46 100 –

12.5 11 –

12 months 6–12 months 6–12 months

8 5 HCV MC+: 22 HCV MC−: 20 A: 6 months A: 11 B: 12 months B: 22 6–14 months HCV MC+: 5 HCV MC−:22 6 months 10

A: 28 B: 39 –

–

100

0

6 months

43

21

60

0

55

27

6 months

12 months

HCV MC+: 14 HCV MC−: 18 0

HCV MC+: 27 HCV MC−: 19

–

SVR sustained virological response, IFN interferon, tw times a week, CR (complete response): improvement/reduction or disappearance of symptoms and/or cryoglobulins a CR to therapy: at the end of therapy b Sustained CR: after withdrawal of therapy (on follow-up)

transaminases, and cryoglobulins, a rebound phenomenon involving clinical and serological parameters was observed after IFN discontinuation. Some studies have asked whether the presence of circulating cryoglobulins affects antiviral therapy and whether treatment efficacy is similar in patients with symptomatic and asymptomatic MC [4, 18]. The data collected in these trials unanimously demonstrated that the antiviral effects of IFN were not altered by the presence of cryoglobulins, with SVR rates of 14–35% in the MC group vs. 18–22% in the control HCV group. Although there was a trend for a higher SVR in cryoglobulin-positive patients, this did not reach statistical significance. The influence of symptoms on the virological response was evaluated in only one trial [21]. No differences were detected in virological response but the disappearance of cryoglobulins occurred less frequently in symptomatic patients. Viral and host

predictive factors associated with response to therapy did not differ between the groups; in some studies, a low probability of response has been associated with high cryocrit levels [15, 19]. There have been several attempts to establish an optimal therapeutic regimen in HCV-associated MC. Two studies evaluated the duration of therapy, showing that a prolonged regimen (³1 year of therapy) seems to offer better efficacy, with higher long-term response off therapy, although associated with higher toxicity [15, 23]. For patients who relapse off drug and have a high risk of progressive MC vasculitic disease, longterm IFN maintenance therapy may be an option [24]. In summary, early studies demonstrated that treatment with IFN monotherapy in HCV-associated MC patients is as effective as in HCV patients without MC, with improvement of MC complications linked to viral clearance, normalization of alanine aminotransferase,

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Table 37.2 Published studies in which HCV-associated MC was treated with IFN plus ribavirin combination therapy

Author (year) Calleja (1999)c [22]

Number of patients 13

Zuckerman (2000)c [26] 9 Donada (1998)c [27] Parise (2007) [25] Cacoub (2002) [28]

Schmidt (2001) [29]

17 HCV MC+ 19 HCV MC− 31 HCV MC+ 71 HCV MC− 14

25 HCV MC+ 64 HCV MC−

IFN and ribavirin regimen IFN 3 MU/3tw + Riba 1,200 mg/day IFN 3 MU/3tw + Riba 1,000–1,200 mg/day IFN 6 MU/3tw 3 months + IFN 3

Duration of therapy 12 months

SVR (%) 54

CR to therapy Sustained CR (%)b (%)a 77 54

6 months

22

77

55

6 months

HCV MC+: 38.5d HCV MC−: 35.7 HCV MC+: 29 HCV MC−: 31 64

–

–

–

–

–

71

–

–

IFN 3 MU/3tw + Riba 1,000–1,250 mg/day

6–12 months

IFN 648 MU + Riba 895 ± 250 mge

IFN: 20 ± 14 months Riba: 14 ± 12 monthsf 6–12 months

IFN 3 MU/3tw + Riba 1,000–1,200 mg/day

HCV MC+: 52 HCV MC−: 18

SVR sustained virological response, IFN interferon, Riba ribavirin, tw times a week, CR (complete response): improvement/reduction or disappearance of symptoms and/or cryoglobulins a CR to therapy: at the end of therapy b Sustained CR: after withdrawal of therapy (on follow-up) c Includes only HCV non-responders to and patients with disease relapse after IFN monotherapy d Limited to patients with disease relapse e Median cumulative IFN dose and mean daily Riba dose f Mean duration of IFN and Riba therapy

and reduction of circulating cryoglobulins. The side effects and tolerance profile of IFN in patients with HCV-associated MC are similar to what is seen in the HCV general population, with a treatment discontinuation rate of 10–20% of patients.

37.2.2 MC Treatment with IFN Plus Ribavirin The discovery of the anti-inflammatory effect of ribavirin in HCV-infected patients led to a pilot study in which five patients with symptomatic MC who were HCVpositive but non-responders to a previous IFN course received oral ribavirin alone [25]. Ribavirin therapy resulted in clinical improvement in all patients and a decrease in alanine aminotransferase concentrations but without clearance of the virus. Clinical relapses occurred after drug withdrawal in all patients. Between 1998 and 2007, several studies on combination therapy with IFN and ribavirin in MC patients were published (Table 37.2). Almost all of them used the same doses of IFN-a (3 MU, 3 days weekly) and

weight-adjusted ribavirin doses (15 mg/kg or 1,000– 1,200 mg/day).All trials except one administered the drugs for 6–12 months. Four studies used combination antiviral therapy in naïve HCV-associated MC. Parise et al. [29] compared the virological response of 31 HCV MC patients (8 symptomatic) with that of 102 HCV patients without MC. SVR rates were similar (30%) in the two groups. No differences were found between symptomatic and asymptomatic patients with respect to virological response. In the majority of patients with SVR, cryoglobulins were reduced or eliminated. Another study of similar design obtained different results [28], reporting SVR in HCV-associated MC patients of 52% compared to only 28% in HCVinfected cryoglobulin-negative patients, with no significant differences in the distribution of viral genotypes. Of particular interest was the low virological response to IFN and ribavirin in the control group compared to the response reported in pivotal trials. A cohort of severely symptomatic patients with HCV and systemic vasculitis were treated with longterm IFN and ribavirin (average of 20 months) [30]. SVR was obtained in 66% of patients, with a significant

37

Should HCV+ Asymptomatic Patients with MC Be Treated with Antiviral Therapy?

improvement in difficult to treat symptoms, such as neurologic and renal involvement. This clinical response could have been related to the longer duration of therapy used in this trial. The immunological response of cryoglobulins to combination therapy was evaluated in another retrospective study of 25 asymptomatic HCV-associated MC patients who reached SVR [26]. Only one of the 25 patients had persistent cryoglobulins at the end of follow up. The results of patients unresponsive to previous courses of IFN therapy are different. As expected, patients without circulating cryoglobulins had a lower virological response, especially null non-responders. The virological response in previous non-responders ranged from 0% to 39%; however, the duration of therapy varied from 6 to 12 months. Two trials investigated IFN-a (3–6 MU/ttw) pus ribavirin (15 mg/kg/day) for 6 months. The first recruited nine severely symptomatic HCV MC patients refractory to previous therapies, including immunesuppressives and IFN [27]. Only 22% of the patients achieved virological, clinical, and immunological sustained responses. The second study compared 17 HCV MC asymptomatic patients with 19 HCV patients without circulating cryoglobulins [22]. Both groups received 3 months of induction therapy with 6 MU of IFN. None of the non-responders and 39% of the relapsers maintained the virological response off therapy. Response rates were very similar in patients with or without serum cryoglobulins. The results of trials employing a longer duration of combination therapy have been better. Calleja [31] treated 13 HCV-associated MC patients, either IFN non-responders or with disease relapse following IFN monotherapy, with 12 months of combination therapy. Seven achieved SVR (54%), demonstrating the superiority of the combination regimen over monotherapy (28%) in the same patient cohort. The response was higher in the relapse group than in the non-responders (80% vs. 38%). The conclusions drawn from this study can be extended to the HCV-associated MC combination therapy experience in general. The presence of cryoglobulins does not affect the response to antiviral treatment since the rate of SVR is similar in patients with or without cryoglobulins. These cold-precipitable proteins can therefore be considered as an epiphenomenon that does not modify the response of HCV to treatment. IFN, either alone or in combination with

291

ribavirin, is a safe and effective treatment for HCVassociated MC. Eradication of the virus is linked to an improvement or the disappearance of MC-associated clinical manifestations.

37.2.3 MC Treatment with Pegylated IFN-a Plus Ribavirin The evolution of HCV treatment modalities for HCV has been broadened to include HCV cryoglobulin-positive patients. However, data on the effectiveness of the recently introduced pegylated IFN combination therapy in this subgroup of patients are limited to small pilot studies. Mazzaro [32] treated 18 HCV-symptomatic MC-naïve patients with pegylated IFN-a2b (1 mg/kg/ week) plus ribavirin (1,000–1,200 mg/day) for 48 weeks, regardless of the viral genotype. At the end of therapy, HCV RNA was undetectable in 83% of patients. At the end of follow-up, SVR was determined in 44%. The same results were obtained for clinical (disappearance of purpura and arthralgias) and biochemical (aminotransferase normalization) responses. Only 33% of the patients obtained a complete immunological response (disappearance of circulating cryoglobulins). When genotypes were analyzed separately, higher rates of SVR were obtained with genotype 2 or 3 than with genotype 1 infections (71% vs. 27%). One of the major weaknesses of this study was a pegylated IFN dosage lower than that usually recommended in HCV therapeutic guidelines. A French group published the results of the largest study of combination therapy performed thus far [33, 34]. Their series consisted of 72 unselected HCV MC patients treated with either standard IFN-a2b (n = 32, 3 MU/ttw) or pegylated IFN-a2b (n = 40, 1.5 mg/kg/ week) combined with ribavirin (600–1,200 mg/day) for at least 6 months (mean 16.63 ± 7.8 months). Twenty patients had received prior first-line therapy with standard IFN-a2b, either alone or in combination with ribavirin. After a long-term follow up of 40 months (± 24 months) after discontinuation of antiviral therapy, SVR was determined in 58.3%. There was a higher rate of virological responses in patients receiving pegylated IFN plus ribavirin than in those treated with IFN plus ribavirin (62.5% vs. 53.1%), regardless of viral load or genotype. In addition, with pegylated IFN combination therapy there was a trend toward a

292

shorter duration of anti-HCV therapy (13.25 vs. 18.35 months), less frequent concomitant use of corticosteroids (35% vs. 47%), and a lower rate of death (5% vs. 18.8%). Clinical and virological responses were closely correlated, with a rate of complete clinical response of 62.5%. Although a significant decrease in proteinuria was observed in sustained virologic responders, serum creatinine levels did not significantly differ. In fact, renal insufficiency was a negative predictive factor of complete clinical response. In spite of the paucity of treatment data regarding pegylated IFN combination therapy in patients with MC, we can conclude that this regimen is safe and more effective than standard IFN plus ribavirin, with higher virological and clinical sustained responses. This evidence strongly suggests the combination of pegylated IFN and ribavirin as the therapy of choice in patients with HCV-associated MC.

37.2.4 Other Combinations with IFN-Based Therapies 37.2.4.1 MC Treatment with Standard IFN Plus Prednisone The largest source of information available on this topic comes from early publications, when monotherapy with IFN was the only antiviral therapy for HCV infection. Only two studies evaluated the effectiveness of IFN and prednisone combination therapy in HCVassociated MC [14, 35]. Although combination therapy with prednisolone resulted in a more prompt response and delayed relapse, the increasing viremia found with prednisolone and the marginal effects of combined therapy did not warrant the use of this protocol. 37.2.4.2 MC Treatment with Standard IFN Plus Plasmapheresis Plasma exchange has been used to treat life-threatening MC manifestations, such as progressive renal or central nervous system involvement or multivisceral vasculitis. This technique is usually performed three times weekly for several weeks, removing cryoglobulins from the circulation. Plasma exchange should be used in association with others therapies to avoid a rebound in cryoglobulins after the discontinuation of apheresis. It may be a way to buy time while awaiting HCV therapy efficacy. An investigation of the pharmacokinetics of IFN-a during plasma exchange sessions [36] showed increased IFN clearance between the initiation and

J.L.C. Panero et al.

termination of apheresis, which may decrease the pharmacological activity of the treatment. However, large amounts of newly produced virions were introduced into the vascular compartment just after the plasmapheresis-mediated drop in HCV viremia. Taken together, if plasma exchange is used in combination with anti-HCV treatment, IFN should be given after each apheresis session in order to maintain the virological effect of the drug.

37.2.4.3 MC Treatment with Pegylated IFN-Ribavirin Plus Rituximab Rituximab is a monoclonal antibody directed against CD20, a transmembrane protein expressed on pre-B lymphocytes and mature lymphocytes. As such, it interferes with monoclonal IgM production, cryoglobulin synthesis, and the deposition of immunocomplexes. Recently, Ahmed and Wong [37] reviewed the literature of rituximab in HCV MC patients. Most publications consisted of case series and case reports [38, 39], with no randomized controlled trials. Rituximab was used as rescue therapy in almost all patients despite previous treatments with IFN-a and/ or immunosuppressive therapies. In the majority of studies, four weekly doses of rituximab 375 mg/m2 were administered. The majority of patients achieved a complete clinical response with rituximab (73%), and some of the relapsing patients who were rechallenged with the antibody had a good response. A relatively small number of side effects were reported. One potential concern regarding this therapy is rituximab’s ability to increase HCV viremia. However, neither significant variation of serum transaminases nor deterioration of liver disease has been noted in most series [40]. In this setting, rituximab seems to be a suitable rescue therapy in recalcitrant HCVassociated MC but it cannot be considered a curative treatment as long as the viral antigen triggering the vasculitis remains. Based on the limitations of each therapy (antiviral and rituximab), with 20–30% of MC patients remaining symptomatic despite anti-CD20 monoclonal antibody or combined antiviral therapy, the association of rituximab with pegylated IFN-a plus ribavirin appears to be logical. Thus far, one single case and a small case series have been reported. The first publication was in 2003 [41]. A 45-year-old woman diagnosed with HCV MC and presenting with general symptoms, polyneuropathy and a low-grade lymphocytic NHL, was treated initially with standard IFN-a

37

Should HCV+ Asymptomatic Patients with MC Be Treated with Antiviral Therapy?

plus ribavirin, without clinical or virological response. Afterward, she received plasmapheresis combined with cyclophosphamide pulses, but with no control of vasculitis activity. Finally, she was treated with six weekly sessions of rituximab, which yielded an excellent clinical response (including NHL). HCV was eliminated with pegylated IFN-a2b combined with ribavirin for nearly 12 months. The second study [42], published in 2008, assessed 16 patients with severe HCV-MC who were resistant to or had disease relapse after combination treatment with standard or pegylated IFN-a2b plus ribavirin. Treatment consisted of four weekly doses of rituximab followed by pegylated IFN-a2b (1.5 mg/week) plus ribavirin (600–1,200 mg/day) for 12 consecutive months. SVR was achieved in 68.7%, with a complete clinical response in all patients. In 62.5% of the patients, serum cryoglobulins disappeared. After a mean follow-up of 19.4 months, only two patients (12.5%) experienced clinical relapse, associated with the simultaneous reappearance of HCV RNA and cryoglobulin. Peripheral neuropathy and nephropathy, the most challenging complications to treat in MC, achieved a complete response in 38.4% and 57.2% of cases, respectively. Treatment was well tolerated, with only two patients experiencing side effects linked to peg-IFN treatment that required discontinuation of antiviral therapy.

37.3

Conclusions

The eradication of HCV with antiviral therapy should be attempted in all cases of symptomatic HCVassociated MC, irrespective of the severity of liver disease, since even in the absence of SVR there may be marked symptomatic improvement. Although the response rate of the general and skin manifestations is high, the majority of reports describe only a partial improvement in neuropathy and glomerulonephritis. However, a recently published review of therapy in HCV cryoglobulinemic glomerulonephritis recommended antiviral therapy as the initial treatment except in the presence of nephritic-range proteinuria and/or rapidly progressive kidney failure [43]. The clinical response of HCV-associated MC is linked to the disappearance of HCV RNA, while a relapse of symptoms usually follows the reappearance of HCV viremia. However there are two reports in which cryoglobulinemic symptoms recurred despite

293

successful antiviral therapy. In some of those patients, recurrence was associated with the development of lymphoproliferative disorders [44, 45]. In HCV-infected patients with circulating cryoglobulins but without clinical involvement, the indications for antiviral therapy are the same as in HCV patients without these immune complexes. Virological responses and tolerance of antiviral therapy in HCVassociated MC patients and in HCV matched controls are similar. Although only scarce evidence is available regarding the use of pegylated IFN plus ribavirin in HCVassociated MC patients, the higher clinical and virological response rates reported thus far make this combination the therapy of choice. Immunosuppression should only be used in MC patients whose symptoms severely impact the quality of life or persist despite SVR [10]. The therapeutic strategies suggested [9] for HCVassociated MC are based on the severity of MC symptoms (Fig. 37.1). Patients with mild to moderate disease severity, i.e., the Meltzer triad, isolated urine analysis alterations, or polyneuropathy, should be treated with pegylated IFN-a plus ribavirin. The duration of therapy has not been rigorously determined, but at least 12 months seems to be mandatory irrespective of HCV genotype. Patients presenting with severe disease, i.e., extensive skin manifestations with deep ulcers or distal necrosis, worsening of renal function, or monopolyneuritis, require an induction phase of immunosuppression while awaiting the initial slow response of antiviral treatments. Traditionally, a combination of high-dose steroids (1.0–1.5 mg/kg/day) plus cyclophosphamide or azathioprine has been used. With the recent availability of biologic B-cell-directed therapy, combination therapy with rituximab followed by pegylated IFN plus ribavirin is a logical strategy, as these drugs target both the viral trigger and the downstream B-cell arm of autoimmunity. For patients with the most fulminant life-threatening presentations, including necrosis involving the extremities, rapidly progressive glomerulonephritis, or central nervous system/systemic organ vasculitis, plasma exchange can bring initial rapid control of the disease but it needs to be combined with immunosuppression (cytotoxic agents, steroids) to avoid a postapheresis rebound of MC. Combination treatment with pegylated IFN and ribavirin should be initiated after the critical phase.

294

J.L.C. Panero et al.

HCV associated MC

Asymptomatic

Symptomatic

Is there any indication for antiviral therapy based on hepatic disease?

Mild-moderate disease

Life-threatening symptoms

Severe disease

PLASMA EXCHANGE YES

NO

INDUCTION Steroids ± Immunosuppressants ± Rituximab

RITUXIMAB FOLLOW UP PEGYLATED INTERFERON + RIBAVIRIN

2

nd

PHASE

PEGYLATED INTERFERON + RIBAVIRIN

Fig. 37.1 Proposed algorithm for management of patients with hepatitis C virus (HCV)-associated mixed cryoglobulinemia

References 1. Pascual M, Perrin L, Giostra E, Schifferli JA (1990) Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 162:569–570 2. Kayali Z, Buckwold VE, Zimmerman B, Schmidt WN (2002) Hepatitis C, cryoglobulinemia, and cirrhosis: a meta-analysis. Hepatology 36:978–985 3. Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 3:25–42 4. Polzien F, Schott P, Mihm S et al (1997) Interferon-a treatment of hepatitis C virus-associated mixed cryoglobulinemia. J Hepatol 27:63–71 5. Schott P, Hartmann H, Ramadori G (2001) Hepatitis C virusassociated mixed cryoglobulinemia. Clinical manifestations, histopathological changes, mechanisms of cryoprecipitation and options of treatment. Histol Histopathol 16:1275–1285 6. Braun GS, Horster S, Wagner KS et al (2007) Cryoglobulinemic vasculitis: classification and clinical and therapeutic aspects. Postgrad Med J 83:87–94 7. Sterling RK, Bralow SP (2006) Extrahepatic manifestations of hepatitis C virus. Curr Gastroenterol Rep 8:53–59 8. Tavoni A, Mosca M, Feri C et al (1995) Guidelines for the management of essential mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl):191S–195S 9. Saadoun D, Delluc A, Piette JC, Cacoub P (2008) Treatment of hepatitis C-associated mixed cryoglobulinemia vasculitis. Curr Opin Rheumatol 20:23–28

10. Kayali Z, LaBrecque DR, Schmidt WN (2006) Treatment of hepatitis C cryoglobulinemia: mission and challenges. Curr Treat Options Gastroenterol 9:497–507 11. Bonomo L, Casato M, Afeltra A, Caccavo D (1987) Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 83:726–730 12. Casato M, Lagana B, Antonelli G et al (1991) Long-term results of therapy with interferon-alpha for type II essential mixed cryoglobulinemia. Blood 78:3142–3147 13. Misiani R, Bellavita P, Fenili D et al (1994) Interferon alfa2a therapy in cryoglubulinemia associated with hepatitis C virus. N Engl J Med 330:751–756 14. Lauta VM, DeSangro MA (1995) Long-term results regarding the use of recombinant interferon alpha-2b in the treatment of II type mixed essential cryoglobulinemia. Med Oncol 12:223–230 15. Casato M, Agnello V, Pucillo LP et al (1997) Predictors of long-term response to high-dose interferon therapy in type II cryoglobulinemia associated with hepatitis C virus infection. Blood 90:3865–3873 16. Migliaresi S, Tirri G (1995) Interferon in the treatment of mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl):175S–180S 17. Akriviadis E, Xanthakis I, Navrozidou C, Papadopoulos A (1997) Prevalence of cryoglubulinemia in chronic hepatitis C virus infection and response to treatment with interferonalpha. J Clin Gastroenterol 25:612–618 18. Pellicano R, Marietti G, Leone N et al (1999) Mixed crioglobulinemia associated with hepatitis C virus infection: a

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19.

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Should HCV+ Asymptomatic Patients with MC Be Treated with Antiviral Therapy? predictor factor for treatment with interferon? J Gastroenterol Hepatol 14:1108–1111 Mazzaro C, Carniello GS, Colle R et al (1997) Interferon therapy in HCV-positive mixed cryoglobulinemia: viral and host factors contributing to efficacy of the therapy. Ital J Gastroenterol Hepatol 29:343–350 Ferri C, Marzo E, Longombardo G et al (1993) Interferonalpha in mixed cryoglobulinemia patients: a randomized, crossover-controlled trial. Blood 81:1132–1136 Cresta P, Musset L, Cacoub P et al (1999) Response to interferon a treatment and disappearance of cryoglobulinemia in patients infected by hepatitis C virus. Gut 45:122–128 Donada C, Crucitti A, Donadon V et al (1998) Interferon alpha and ribavirin combination therapy in patients with chronic hepatitis C and mixed cryoglobulinemia. Blood 92:2983–2984 Mazzaro C, Lacchin T, Moretti M et al (1995) Effects of two different alpha-interferon regimens on clinical and virological findings in mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl):181S–185S Naarendorp M, Kallemuchikkal U, Nuovo GJ, Gorevic PD (2001) Longterm efficacy of interferon-alpha for extrahepatic disease associated with hepatitis C virus infection. J Rheumatol 28:2466–2473 Durand JM, Cacoub P, Lunel-Fabiani F et al (1998) Ribavirin in hepatitis C related cryoglobulinemia. J Rheumatol 25: 1115–1117 D’Amico E, Chincoli C, Cacciatore P et al (2005) Effects of combined antiviral therapy on asymptomatic mixed cryoglobulinemia in naive patients with chronic hepatitis C virus infection: a preliminary study. Dig Dis Sci 50:2344–2347 Zuckerman E, Keren D, Slobodin G et al (2000) Treatment of refractory, symptomatic, hepatitis C virus related mixed cryoglobulinemia with ribavirin and interferon-a. J Rheumatol 27:2172–2178 Schmidt WN, LaBrecque DR, Voigt MD (2001) Combination antiviral therapy in patients with chronic hepatitis C and cryoglobulinemia. Hepatology 34:421A Parise ER, Oliveira AC, Ferraz ML et al (2007) Cryoglobulinemia in chronic hepatitis C: clinical aspects and response to treatment with interferon alpha and ribavirin. Rev Inst Med Trop Sao Paulo 49:67–72 Cacoub P, Lidove O, Maisonobe T et al (2002) Interferon-a and ribavirin treatment in patients with hepatitis C virusrelated systemic vasculitis. Arthritis Rheum 46:3317–3326 Calleja JL, Albillos A, Moreno-Otero R et al (1999) Sustained response to interferon-a or to interferon-a plus ribavirin in hepatitis C virus-associated symptomatic mixed cryoglobulinemia. Aliment Pharmacol Ther 13:1179–1186

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32. Mazzaro C, Zorat F, Caizzi M et al (2005) Treatment with peg-interferon alpha-2b and ribavirin of hepatitis C virus associated mixed cryoglobulinemia: a pilot study. J Hepatol 42:632–638 33. Cacoub P, Saadoun D, Limal N et al (2005) PEGylated interferon alfa-2b and ribavirin treatment in patients with hepatitis c virus-related systemic vasculitis. Arthritis Rheum 52:911–915 34. Saadoun D, Resche-Rigon M, Thibault V et al (2006) Antiviral therapy for hepatitis C virus-associated mixed cryoglobulinemia vasculitis. Arthritis Rheum 54:3696–3706 35. Dammacco F, Sansonno D, Han JH et al (1994) Natural interferon-alpha versus its combination with 6-methyl-prednisolone in the therapy of type II mixed cryoglobulinemia: a long-term, randomized, controlled study. Blood 84:3336–3343 36. Hausfater P, Cacoub P, Assogba U et al (2002) Plasma exchange and interferon-alpha pharmacokinetics in patients with hepatitis C virus-associated systemic vasculitis. Nephron 91:627–630 37. Ahmed MS, Wong CF (2007) Should rituximab be the rescue therapy for refractory mixed cryoglobulinemia associated with hepatitis C? J Nephrol 20:350–356 38. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101: 3827–3834 39. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon a with an anti-CD20. Blood 101:3818–3826 40. Sansonno D, Tucci FA, Montrone M et al (2007) B-cell depletion in the treatment of mixed cryoglobulinemia. Dig Liver Dis 39(Suppl):116S–121S 41. Lamprecht P, Lerin-Lozano C, Merz H et al (2003) Rituximab induces remission in refractory HCV associated cryoglobulinemic vasculitis. Ann Rheum Dis 62:1230–1233 42. Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinemia vasculitis. Ann Rheum Dis 67:1431–1436 43. Fabrizi F, Lunghi G, Messa P, Martin P (2008) Therapy of hepatitis C virus-associated glomerulonephritis: current approaches. J Nephrol 21:813–825 44. Levine JW, Gota C, Fessler BJ, Calabrese LH, Cooper SM et al (2005) Persistent cryoglobulinemic vasculitis following successful treatment of hepatitis C virus. J Rheumatol 32: 1164–1167 45. Landau DA, Saadoun D, Halfon P et al (2008) Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response. Arthritis Rheum 58:604–661

The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia

38

Francesco Zaja, Stefano Volpetti, Stefano De Luca, and Renato Fanin

38.1

Introduction

Type II mixed cryoglobulinemia (MC) is a systemic vasculitis largely mediated by immune complexes, usually associated with hepatitis C virus (HCV) infection, and characterized by the expansion of rheumatoid factor (RF)-positive B cell clones, leading to cryoglobulin production. It typically affects adult patients with a median age of 56 years [1] and a female prevalence. The link between HCV and lymphoproliferative disorders was clearly established based on epidemiological data as well as biological and pathological evidence. HCV is a lymphotropic virus [2–4] that is able to infect B cells and other components of the immune system, initiating an antigen-driven multistep lymphomagenic process. Chronic HCV infection is associated with a higher incidence of non-Hodgkin lymphoma [3, 4]. Probably the strongest association between lymphoproliferative disorders and HCV is the one linking viral hepatitis with MC: in 90–95% of patients with MC [1, 5] a concurrent or previous HCV infection is evidenced by serological tests; 10–70% of HCVpositive patients will develop MC [6]; and antiviral therapy may contrast lymphoid expansion with improved cryoglobulinemic manifestations. B-cell proliferation serves as the biological substrate of MC. It is generally represented by non-neoplastic oligoclonal B cell expansion, with pathological and phenotypic similarity to chronic lymphocytic leukemia F. Zaja (*) Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy e-mail: [email protected]

(CLL) and lymphoplasmocytic lymphoma. These B-lymphoid infiltrates are CD20-positive, present in the bone marrow, liver, and lymph nodes, and tend to remain unmodified for years or even decades. Based on these characteristics, the term “monotypic lymphoproliferative disorder of undetermined significance (MLDUS)” has been suggested. In a minority of patients, a typical picture of overt B-cell lymphoma is already documentable at diagnosis; in nearly 10% of patients, lymphoma will develop during the clinical history of MC. The occurrence of B-cell lymphoma represents the final event of a multistep and multifactorial process that usually requires several years to develop. Diffuse large B-cell lymphoma, observed in 40–50% of cases, is the most frequent subtype of lymphoma; in the remaining cases, marginal-zone lymphoma (extranodal, nodal or splenic) or, rarely, CLL and lymphoplasmacytic lymphoma are documented. Beyond the characteristic features secondary to HCV infections and lymphoid expansion, patients with MC may exhibit variable autoimmune manifestations, with impairment of single or multiple organs, such as skin, kidney, nerves, lungs, and joints. More frequently, these features are the expression of a small-vessel vasculitis secondary to cryoprecipitation. Other autoimmune hematological phenomena, such as hemolytic anemia, immune neutropenia, and thrombocytopenia, may also occur. Recent studies evidenced an increase in serum B cellactivating factor (BAFF) in patients affected by MC. BAFF is usually described in systemic lupus erythematosus, rheumatoid arthritis, and Sjögren syndrome. It seems to be required for B-cell survival and may prevent autoreactive B cells from undergoing apoptosis [7].

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_38, © Springer-Verlag Italia 2012

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The chimeric monoclonal antibody (IgG1/k) rituximab (RTX) is directed against the CD20 antigen, expressed on the surface of normal (from pre-B to mature B lymphocytes) and malignant B lymphocytes [8]. The action of this drug, while not yet fully understood, seems to be related to complement-dependent cytotoxicity (CDC) [9], antibody dependent cell mediated cytotoxicity (ADCC) [8, 9], and the induction of apoptosis [10, 11]. RTX proved to be highly effective in the therapy of lowgrade and high-grade CD20-positive B-cell lymphomas and its use has now been registered in combination with chemotherapy or as maintenance therapy. Initial studies in animals and, subsequently, in patients treated with RTX, showed the development of a marked (even transient) B-cell depletion from peripheral blood, bone marrow, and lymph nodes, but only mild changes in immunoglobulin and complement serum levels [12]. These results, along with the good tolerance and handling of the drug, have recently prompted its use in several autoimmune diseases. Encouraging results were obtained in immune thrombocytopenia, hemolytic anemia, thrombotic thrombocytopenic purpura, different rheumatological disorders, anti-MAG polyneuropathies, pemphigus vulgaris, and several other autoimmune disorders. The therapeutic effect of B-cell depletion in autoimmune phenomena is not yet clearly defined but it likely involves the inhibition of auto-antibody production and the prevention of immune complex formation, and thus stimulation of an inflammatory response. B cells may also act by producing specific cytokines and have antigen-processing activity. Moreover, a regulatory effect of B cells on T cells has been documented, suggesting a primary role of the former in the pathophysiology of some autoimmune disorders. There is a strong rationale for using RTX in the treatment of MC, since this disorder is sustained by a low-grade B lymphoproliferation exhibiting autoimmune features. Biological evidence of effective targeting of RF-positive B cell clones by RTX has been reported, both in preliminary clinical studies and in animal models [13]. In addition, RTX was demonstrated to be effective and safe in B-cell lymphoma as well as in autoimmune diseases. The first use of RTX in MC dates to 1999 [14], when a 58-year old man, unresponsive to interferon (IFN), cyclophosphamide, and steroids, achieved a clinically significant response after two doses of RTX, with a progressive improvement in all signs of disease, including the nearly complete disappearance of purpura and arthralgia and

F. Zaja et al.

a progressive reduction in RF level. The response lasted for 3 months, when the patient again complained of purpura and arthralgia. On the basis of this report and the preliminary results obtained in autoimmune diseases characterized by pathogenic RF [14–16], RTX was tested in a larger cohort of patients. The antibody was administered i.v. generally at the standard dose of 375 mg/m2 weekly for 4 weeks, according to the previous experience in B-cell lymphoma. More recently, a schedule consisting of two bi-weekly 1,000 mg doses was proposed. Here we review data from the literature regarding the role of RTX in MC. Our literature sources were drawn from the electronic databases of MEDLINE (from 1998), which were searched using the explore function for the Medical Subject Heading (MeSH) terms “rituximab,” “rituxan,” “mabthera,” “anti-CD20,” and “cryoglobulinemia.” Only those papers reporting data on at least one patient with MC who was treated with RTX and only those papers in which the response to RTX was reported according to specific organ involvement were taken into account.

38.2

Rituximab and Cutaneous Lesions in MC

The skin is a very frequent target of MC, involved in >90% of patients [1]. Cutaneous manifestations are characterized by the development of purpura, skin ulcers, and urticarial lesions. Two major studies [13, 17] confirmed the therapeutic activity of RTX on MC cutaneous manifestations. In our experience, 11 out of 12 patients achieved complete disappearance of purpura while one patient had a partial response. Response duration was 6 months or more in nine patients. Similarly, we documented complete ulcer healing in five out of five patients, with response duration of 6 months or more in all of them. One of these patients has maintained a response after 10 years. One single case of urticaria had a complete and long-lasting response upon RTX therapy. Similar results were documented by Sansonno et al. [17]; their study showed improvement of purpura in 12 out of 14, skin ulcers in three out of five, and urticaria in three out of three patients. Other favorable experiences, even if involving a small number of cases, have been reported by other authors [18, 19] (Table 38.1). Notably, Visentini et al. [26] reported a complete disappearance of purpura in

38

The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia

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Table 38.1 Results with rituximab (RTX) therapy for cutaneous lesions in mixed cryoglobulinemia References Zaja et al. [13]

Cutaneous lesions Purpura

Patients 12

Response OR: 12 CR: 11

Ulcers

5

OR: 5 CR: 5

Saadoun et al. [27]

Urticaria Purpura Ulcers Urticaria Ulcers Purpura Purpura Ulcers Purpura Purpura Purpura Ulcers Purpura

1 14 5 3 1 1 1 1 1 1 3 3 13

OR: 1 CR: 1 OR: 12 OR: 3 OR: 3 OR = 1 OR: 1 CR: 1 OR: 1 CR: 1 OR: 1 CR: 1 OR: 1 CR: 1 Brief improvement, then flare OR: 3 CR: 3 OR: 2 CR: 2 OR: 11

Roccatello et al. [18] Tallarita et al. [28]

Ulcers Ulcers Ulcers

2 3 1

OR: 2 OR: 3 OR: 1 CR: 1

Ulcers Purpura Ulcers Urticaria

1 46 22 4

OR: 1 CR: 1 OR: 41 (89%) OR: 19 (86%) OR: 4 (100%)

Sansonno et al. [17]

Arzoo et al. [20] Lamprecht et al. [21] Koukoulaki et al. [22] Ghobrial et al. [23] Ghijsels et al. [24] Cohen et al. [25] Visentini et al. [26]

Da Silva et al. [19] Total

Response duration < 6 months in 3 Response duration > 6 months in 9 Response duration < 6 months in 1 Response duration > 6 months in 4 Response duration > 6 months in 1

Low-dose RTX RTX in combination with PEG-IFN and ribavirin

RTX in combination with plasma exchange

OR overall response, CR complete response, IFN interferon

three out of three patients and complete ulcer healing in two out of three patients using a lower-dose RTX schedule (250 mg/m2 × 2). Taken together, these results highlight the effective therapeutic activity of RTX for the treatment of cutaneous lesions secondary to MC. Overall, the response rate to purpura, ulcer, and urticaria manifestations were 90%, 89% and 100%, respectively. In many cases, the response was complete and durable.

38.3

Rituximab and Renal Involvement in MC

Renal involvement occurs in nearly one-third of cases and represents the worst prognostic factor for patients with MC. In the study of Ferri et al. [1], renal involvement was clinically evident at diagnosis and during follow-up in 20% and 30% of patients, respectively, and was associated with significantly lower survival

rates than in the remaining patient population (10-year survival: 33.1% vs. 62.1% ). Cryoglobulinemic nephropathy is generally an expression of immune complex membranoproliferative glomerulonephritis and is associated with proteinuria and nephritis. The activity of RTX in MC nephritis has been described in nearly 40 patients, in most cases with clinical improvement (approximately 80% response rate; Table 38.2). The response duration was variable, with some patients showing medium to long periods of remission. Re-treatment was generally effective and in some patients a maintenance policy was favorably adopted [29] (Table 38.2). Remarkably, the group of Saadoun et al. [27] administered RTX in association with pegylated IFN plus ribavirin in seven patients with MC nephritis in order to simultaneously treat both the B-cell disorder and HCV. Treatment was generally well tolerated and was associated with renal improvement in four out of the seven patients. Basse et al. [30] treated seven patients with cryoglobulinemic nephritis that

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Table 38.2 Results with rituximab (RTX) therapy for renal involvement in mixed cryoglobulinemia (MC) References Zaja et al. [13] Sansonno et al. [17] Arzoo et al. [20] Ghijsels et al. [24] Koukoulaki et al. [22] Quartuccio et al. [29]

Patients 2 1 1 1 1 5

Response OR: 1 CR: 1 OR : 0 OR: 1 OR: 1 OR: 1 OR: 5

Basse et al. [30]

7

OR: 7

Visentini et al. [26] Saadoun et al. [27] Roccatello et al. [18]

1 7 7

OR: 1 CR: 1 OR : 4 OR: 7

Total

33

OR: 28 (87.5%)

Response duration > 6 months Response duration > 8 months Response duration > 24 months Two patients maintained response status after 15 and 21 months. Three patients relapsed after 5, 7, and 12 months, respectively. Retreatment with RTX was efficacious in 2 patients who subsequently underwent maintenance treatment. MC after renal transplant. Severe infectious complications in two patients

Good safety profile No increase in HCV load Response duration: 12–18 months

OR overall response, CR complete response

developed after renal transplant, documenting a sustained remission or an improvement of nephrotic syndrome in five patients and the disappearance of nephritic syndrome in one patient, with a sustained clearance of cryoglobulins in six. However, in two patients the clinical course was complicated by severe infections.

38.4

Rituximab and Neuropathy in MC

Peripheral neuropathy is a common manifestation of MC and is often refractory to standard treatment. Neuropathic inflammation is secondary to vasculitis of the vasa nervorum. It more frequently affects the nerve structures of the inferior limbs, impairing various levels of sensitivity and/or motility. In our study [13], seven out of seven patients unresponsive to conventional treatments improved after salvage therapy with RTX. A decrease of 50% or more in neuropathic pain was recorded in six out of the seven patients, with complete disappearance in one of them after 6 months of treatment. Similarly, a decrease in paresthesias in the lower limbs was noted in seven of seven patients, with complete disappearance in two of them after 6 months. Symptom improvement usually occurred during the second and third months, and it was still present at the end of the sixth month in all patients. Electromyography was consistent with axonal and myelinic damage and detected sensory and mild motor

involvement in all patients. In four out of four patients who agreed to repeat the test after 6 months, it remained unchanged with respect to baseline. In a similar study by Sansonno et al. [17], an improvement of neuropathic symptoms was observed in six out of 14 patients who had impaired nerve conduction velocity, in four out of 11 patients with paresthesias, and in two out of three patients with dysesthesia. In a more recent study, Cavallo et al. [31] treated 13 patients presenting with paresthesias (11 patients), burning feet (six patients), restless legs syndrome (one patient), and asthenia (12 patients). After six doses of RTX 375 mg/m2, a clinical improvement was evident in five out of 11 patients (45%) with paresthesias, in four out of six (67%) with burning feet, and in 10 out of 12 (83%) with asthenia. One patient who was unable to walk began to walk again following the third infusion. The mean clinical neuropathy disability score (CNDS) was 46.08 ± 7.62 before treatment and 50.23 ± 7.17 after RTX (p < 0.001). The electrophysiological data were also consistent with a beneficial effect of anti-CD20 therapy, showing an improvement in motor response and conduction. Moreover, two patients regained sensory response after therapy. In conclusion, RTX therapy seems to be effective in the majority of patients with neuropathy, even if the long-term efficacy of the drug is difficult to assess due to the lack of longer studies.

38

The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia

38.5

Rituximab and Arthritis in MC

Arthralgia is present in most patients and is generally caused by non-erosive lesions of the hands, ankles, knees, and elbows. Administration of RTX has been shown to improve MC arthritis. Seven different studies, collecting data from 59 patients, analyzed the effect of RTX on arthralgia: overall, 62% of patients experienced the disappearance or a significant reduction of arthralgias. In particular, in our series [13], all four patients rapidly responded after treatment and in two of them the response was maintained for 6 months or more. Our observations were confirmed by Sansonno et al. [17], in a study of 16 patients, eight of whom responded to treatment. Other studies, with fewer patients, reported higher overall response rates. Roccatello et al. [18] reported a clinical response in all patients treated (6/6, 100%) in terms of a >50% decrease in the Visual Analogue Scale. In the study of Saadoun et al. [27], which investigated the activity of combination therapy with RTX, pegylated IFN, and ribavirin, five out of six patients had clinical improvement upon RTX treatment. In conclusion, RTX seems to be highly active for arthralgia, either alone or together with antiviral therapy, although prospective trials are needed to confirm the safety profile and overall response of combination treatment.

38.6

Rituximab and Hyperviscosity Syndrome in MC

Hyperviscosity syndrome occurs in patients with systemic rheumatic diseases and other conditions associated with high titers of RF and large amounts of circulating immune complexes [32, 33]. In MC patients, hyperviscosity syndrome is relatively rare and present only in those with higher cryoglobulin levels. These patients are treated with plasma exchange, which is generally successful, even if only in the short term, in normalizing hemo-rheological parameters and controlling symptoms. At present, only a few cases of MC-related hyperviscosity syndrome treated with RTX have been described; one particularly impressive report was the case of a patient who had an extremely high cryocrit (78%) that became negative after treatment, as did the IgM monoclonal component (from 30.5 g/L to undetectable levels after therapy) [26].

301

These results matched the experience reported in patients with Waldenström macroglobulinemia (WM), suggesting that RTX is useful and safe in the treatment of hyperviscosity syndrome. Similar to what has been described in WM, in MC patients with higher cryoglobulins there may be a flare effect after RTX therapy. In particular, Sene et al. [34] reported four cases of life-threatening flare vasculitis that developed 1–2 days after the administration of RTX, particularly if given at high dose (1,000 mg). This phenomenon may be secondary to the possibility that RTX, almost immediately and especially at higher doses, enhances cryoprecipitation, by forming complexes with IgM/k that have rheumatoid activity. Sene and colleagues analyzed this hypothesis in in vitro experiments and found that the addition of RTX to serum containing an RF-positive IgM/k type MC was associated with visibly accelerated cryoprecipitation. For these reasons, in patients with high baseline levels of cryoglobulins RTX should be very cautiously prescribed, initially administering standard (375 mg/m2) or even lower doses after the removal of IgM/k by plasma exchange and accompanied by strict clinical and laboratory monitoring.

38.7

Modification of Specific Laboratory Parameters After Rituximab in MC

The specific laboratory findings of MC include cryoglobulins, IgM/k monoclonal gammopathy, RF positivity, and a decreased level of the complement component C4. The effect of RTX on these parameters has been investigated in different studies aimed at determining a correlation with clinical response. In our experience [13], a significant reduction in serum RF levels, cryoglobulins, and IgM was noted after treatment, while serum C4 levels increased significantly. Roccatello et al. [35] observed that in six patients with MC and nephritis, proteinuria, erythrocyte sedimentation rate, and cryocrit were significantly decreased at 2, 6, and 12 months. RF and IgM significantly decreased at 6 months whereas C4 significantly increased at 2 and 6 months and IgG remained stable. These findings were confirmed in three of the patients even after 18 months, although in one of them RF levels increased and C4 decreased at 19 months following the initiation of therapy. Another study [26] confirmed a reduction in serum cryoglobulins in four out of five evaluable patients, with an impressive negativization of the

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cryocrit in a woman who had a 78% cryocrit before treatment. Correlating blood tests and response, Sansonno et al. [36] demonstrated a decrease in the mean cryocrit value of patients who responded (16/20, 80%); in three patients (18.7%), cryoglobulins stably disappeared throughout the observation period. In terms of duration of response, 12 months after the discontinuation of RTX, the response maintenance rate was 75%. In conclusion, the results of these studies seem to demonstrate a significant improvement in several MC-specific laboratory parameters after treatment with RTX, in particular a decrease in serum cryoglobulin levels, RF, and IgM/k and an increase in C4 levels.

38.8

Effect of Rituximab on B-Cell Expansion in MC

A key pathological and molecular feature in MC is the B-cell oligo/monoclonal expansion in both peripheral blood and the bone marrow. These B-cell lymphoid infiltrates, which are CD20-positive, usually remain unmodified for years or even decades, but in a small percentage of patients (nearly 10%) they can evolve to lymphoma. Sansonno et al. [36] analyzed the dynamics of peripheral CD20+ B cells in 20 patients with cryoglobulinemia (13 patients with mixed type II and 7 with type III) who were treated with RTX. As expected, RTX reduced the number of peripheral blood CD20+ cells to 12 months), two cases of lymphoma and one case of breast cancer were noted. Recently, Sene et al. [34] reported six cases of systemic drug reactions after RTX: four patients developed a severe flare of MC vasculitis within 2 days after infusion while two patients developed serum sickness

RTX appears to be an active agent for the treatment of patients with MC. Based on its unique biologic activity, it acts specifically against B-cell lymphoproliferative disorders characterized by sustained cryoglobulin production and organ involvement. Almost all clinical manifestations may benefit from RTX treatment. Overall, RTX is well tolerated and safe; the possible increase of HCV viremia rarely causes hepatitis re-activation. However, caution should be exerted in patients with high cryoglobulin levels or hyperviscosity at baseline, because of possible flare syndrome. Prospective comparative trials are warranted in order to better evaluate the therapeutic impact of RTX and to define the best treatment approach, taking into consideration the other available therapies and in particular antiviral agents.

References 1. Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 3:25 2. Andreone P, Zignego AL, Cursaro C et al (1998) Prevalence of monoclonal gammopathies in patients with hepatitis C virus infection. Ann Intern Med 129:294–298

304 3. Mangia A, Clemente R, Musto P et al (1996) Hepatitis C virus infection and monoclonal gammopathies not associated with cryoglobulinemia. Leukemia 10:1209–1213 4. Pozzato G, Mazzaro C, Santini G et al (1996) Hepatitis C virus and non-Hodgkin’s lymphomas. Leuk Lymphoma 22: 53–60 5. Gordon SC (1996) Extrahepatic manifestations of hepatitis C. Dig Dis 14:157–168 6. Charles ED, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 76:818–824 7. Sène D, Limal N, Ghillani-Dalbin P et al (2007) Hepatitis C virus-associated B-cell proliferation–the role of serum B lymphocyte stimulator (BLyS/BAFF). Rheumatology (Oxford) 46:65–69 8. Reff ME, Carner K, Chambers KS et al (1995) Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 83:435–445 9. Golay J, Zaffaroni L, Vaccari T et al (2000) Biologic response of B-lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 95:3900–3908 10. Shan D, Ledbetter JA, Press OW (2000) Signaling events involved in anti-CD20 induced apoptosis of malignant humam B-cells. Cancer Immunol Immunother 48:673–683 11. Hofmeister JK, Cooney D, Coggeshall KM (2000) Clustered CD20-induced apoptosis: Src-family kinase, the proximal regulator of tyrosine phosporylation calcium influx and caspase 3-dependent apoptosis. Blood Cells Mol Dis 26: 133–143 12. Maloney DG, Grillo-Lopez AJ, White CA et al (1997) IDEC-C2B8 (rituximab) anti CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 90:2188–2195 13. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101:3827–3834 14. Zaja F, Russo D, Fuga G et al (1999) Rituximab for the treatment of type II mixed cryoglobulinemia. Haematologica 84: 1157–1158 15. De Vita S, Zaja F, Sacco S et al (2002) Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis: evidence for a pathogenetic role of B cells. Arthritis Rheum 46: 2029–2033 16. Zaja F, De Vita S, Russo D et al (2002) Rituximab for the treatment of type II mixed cryoglobulinemia. Arthritis Rheum 46:2252–2254 17. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101:3818–3826 18. Roccatello D, Baldovino S, Rossi D et al (2004) Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis. Nephrol Dial Transplant 19:3054–3061 19. da Silva Fucuta Pereira P, Lemos LB, de Oliveira Uehara SN et al (2010) Long-term efficacy of rituximab in hepatitis C virus-associated cryoglobulinemia. Rheumatol Int 30: 1515–1518 20. Arzoo K, Sadeghi S, Liebman HA (2002) Treatment of refractory antibody mediated autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab). Ann Rheum Dis 61:922–924

F. Zaja et al. 21. Lamprecht P, Lerin-Lozano C, Merz H et al (2003) Rituximab induces remission in refractory HCV associated cryoglobulinaemic vasculitis. Ann Rheum Dis 62:1230–1233 22. Koukoulaki M, Abeygunasekara SC, Smith KG et al (2005) Remission of refractory hepatitis C-negative cryoglobulinaemic vasculitis after rituximab and infliximab. Nephrol Dial Transplant 20:213–216 23. Ghobrial IM, Uslan DZ, Call TG et al (2004) Initial increase in the cryoglobulin level after rituximab therapy for type II cryoglobulinemia secondary to Waldenström macroglobulinemia does not indicate failure of response. Am J Hematol 77:329–330 24. Ghijsels E, Lerut E, Vanrenterghem Y et al (2004) Anti-CD20 monoclonal antibody (rituximab) treatment for hepatitis C-negative therapy-resistant essential mixed cryoglobulinemia with renal and cardiac failure. Am J Kidney Dis 43: 34–38 25. Cohen H, Green S, Jones S et al (2007) Lack of efficacy of Rituximab in a patient with essential mixed cryoglobulinaemia. Rheumatology (Oxford) 46:366–367 26. Visentini M, Granata M, Veneziano ML et al (2007) Efficacy of low-dose rituximab for mixed cryoglobulinemia. Clin Immunol 125:30–33 27. Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinaemia vasculitis. Ann Rheum Dis 67:1431–1436 28. Tallarita T, Gagliano M, Corona D et al (2009) Successful combination of Rituximab and plasma exchange in the treatment of cryoglobulinemic vasculitis with skin ulcers: a case report. Cases J 2:7859 29. Quartuccio L, Soardo G, Romano G et al (2006) Rituximab treatment for glomerulonephritis in HCV-associated mixed cryoglobulinaemia: efficacy and safety in the absence of steroids. Rheumatology (Oxford) 45:842–846 30. Basse G, Ribes D, Kamar N et al (2006) Rituximab therapy for mixed cryoglobulinemia in seven renal transplant patients. Transplant Proc 38:2308–2310 31. Cavallo R, Roccatello D, Menegatti E et al (2009) Rituximab in cryoglobulinemic peripheral neuropathy. J Neurol 256: 1076–1082 32. Monti G, Galli M, Invernizzi F et al (1995) Cryoglobulinaemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease. GISC. Italian Group for the Study of Cryoglobulinaemias. QJM 88: 115–126 33. Ghobrial IM, Gertz MA, Fonseca R (2003) Waldenstrom macroglobulinaemia. Lancet Oncol 4:679–685 34. Sène D, Ghillani-Dalbin P, Amoura Z et al (2009) Rituximab may form a complex with IgMkappa mixed cryoglobulin and induce severe systemic reactions in patients with hepatitis C virus-induced vasculitis. Arthritis Rheum 60:3848–3855 35. Roccatello D, Baldovino S, Rossi D et al (2008) Rituximab as a therapeutic tool in severe mixed cryoglobulinemia. Clin Rev Allergy Immunol 34:111–117 36. Sansonno D, Tucci FA, Montrone M et al (2007) B-cell depletion in the treatment of mixed cryoglobulinemia. Dig Liver Dis 39(Suppl 1):S116–S121 37. Quartuccio L, Salvin S, Fabris M et al (2008) Disappearance of bone marrow B cell clonal expansion in patients with type II hepatitis C virus-related cryoglobulinemic

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Rituximab in Cryoglobulinemic Vasculitis: First- or Second-Line Therapy?

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Peter Lamprecht and Paul Klenerman

39.1 Introduction: Cryoglobulinemic Vasculitis Cryoglobulinemic vasculitis is a potentially life-threatening systemic vasculitis resulting from the deposition of circulating immune complexes and complement, predominantly in small vessels (capillaries, venules, and/or arterioles) and, less frequently, in medium-sized vessels [1]. The major cause of cryoglobulinemic vasculitis is chronic hepatitis C virus (HCV) infection, with HCV-negative “idiopathic” disease and secondary forms in rheumatoid arthritis, connective tissue diseases, other chronic infections, non-Hodgkin lymphomas, and other hematologic disorders accounting for the remainder. The clinical spectrum of cryoglobulinemic vasculitis encompasses mild to moderate disease manifestations (e.g., Meltzer’s triad with purpura, arthralgia, and asthenia), severe disease (e.g., worsening renal function, polyneuropathy, mononeuritis ­multiplex, and skin ulcers, acral necrosis), and lifethreatening disease (e.g., rapidly deteriorating renal function, gastrointestinal vasculitis, central nervous system involvement, hyperviscosity syndrome). Purpura and arthralgia are the most frequent symptoms of cryoglobulinemic vasculitis, affecting nearly all patients (³95%), followed by polyneuropathy, renal involvement, and Raynaud’s phenomenon in ³50% of the patients [2].

P. Lamprecht (*) Department of Rheumatology, Vasculitis Center UKSH & Clinical Center Bad Bramstedt, University of Lübeck, Lübeck, Germany e-mail: [email protected]

39.2 Rationale for B-Cell-Depleting Therapy in Cryoglobulinemic Vasculitis Mixed cryoglobulinemia is present in 40–60% of the patients with chronic HCV infection, but symptomatic cryoglobulinemic vasculitis develops in 90% of patients), although very few individuals present at this stage. It is likely that such treatment reverses the excess risk of lymphoproliferation-related disease, although the time course for the induction of autonomous B-cell activity is not known [28]. Given HCV’s highly dynamic virion production, its escape mechanisms from cellular immunity, and its resistance to IFN, coupled with its inherent propensity for the promotion of lymphoproliferative disease, it seems highly desirable to eliminate the virus early, at the same time as the prevention of HCV-induced lymphoproliferative disease [20–22]. Relapses of HCV-associated cryoglobulinemic vasculitis in patients with chronic HCV who exhibit a SVR to combination therapy have been reported, underscoring the need for the control of clonal B-cell proliferation in addition to virus elimination. In some patients, the withdrawal of IFN-a caused a short-lived relapse of cryoglobulinemic vasculitis; in others, the underlying lymphoproliferative disease evolved to malignant B-cell NHL [29]. Treatment of HCV-associated cryoglobulinemic vasculitis with PEG-IFN-a and ribavirin results in sustained clinical and virological responses in up to 60%

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of patients. Clearance of mixed cryoglobulinemia is observed in about half of the patients [22]. Successful anti-viral therapy with IFN-a in HCV-associated cryoglobulinemic vasculitis is accompanied by a reversal of monoclonal B-cell proliferation [30]. Thus, antiviral treatment combined with PEG-IFN-a plus ribavirin is an appropriate first-line therapeutic option for patients with mild to moderate disease severity and activity (i.e., Meltzer’s triad). However, about 30% of the patients continue to have active disease while receiving anti-viral therapy. Thus, B-cell depleting therapy is warranted in patients lacking early virological and clinical responses and in patients with severe disease activity, in whom rapid clinical improvement is the goal and a delayed clinical response, as seen with anti-viral therapy, is not tolerable. While rituximab induces a complete clinical response in 60–70% of the patients with HCV-associated cryoglobulinemic vasculitis, reverses clonal VH 1-69+ B-cell expansion and concomitant Treg abnormalities, and evokes a cryoglobulin clearance in about 30% of the patients, viremia persists or even increases, as discussed above. Moreover, 30% of the patients are subject to disease relapse during peripheral blood B-cell recovery [13, 14, 22, 24–26]. Thus, the combination of rituximab and PEG-IFN-a plus ribavirin may well be a logical approach to successfully combine the anti-proliferative impact and anti-viral potencies of both treatment approaches [22]. Our group was the first to report the successful induction of remission with a combination therapy of rituximab and PEG-IFN-a2b plus ribavirin, in a patient with refractory HCV-associated cryoglobulinemic vasculitis. The patient suffered from Meltzer’s triad, severe polyneuropathy, and intestinal involvement. Leukocytoklastic vasculitis of the skin and small-vessel vasculitis of the colon were diagnosed from biopsies, and a low-grade lymphocytic NHL discovered upon further investigation. Interestingly, the patient showed neither a clinical nor a virological response to an initial treatment approach consisting of standard IFN-a2b and ribavirin. She was subsequently treated with prednisolone, cyclophosphamide, and plasmapheresis, but did not respond clinically. Clinical remission of the cryoglobulinemic vasculitis and complete remission of the NHL were ultimately induced with six thrice-weekly infusions of 500 mg rituximab. Remission was maintained and HCV successfully eliminated with PEG-IFN-a (which had meanwhile

39  Rituximab in Cryoglobulinemic Vasculitis: First- or Second-Line Therapy?

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Table 39.2  Summary of clinical outcome in a case report and uncontrolled case series using rituximab or combined rituximab and PEG-IFN-a2b plus ribavirin as first- or second-line therapy for the induction of remission in HCV-associated cryoglobulinemic vasculitis Author [reference] Lamprecht et al. [31] Saadoun et al. [32]

Number of patients 1 16

RTX as first- or second-line therapy Second line Second line

Terrier et al. [33]

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First- and second-line (two treatment arms: RTX alone vs. combined RTX and PEG-IFN-a2b plus ribavirin)

Clinical outcome CR CR 10/16 (63%) Re 2/16 (13%) CR 7/12 (58%) in RTX alone CR 16/20 (80%) in combined RTX and PEG-IFN-a2b plus ribavirin Re 7/32 (22%)

PEG-IFN-a Peglyated interferon-a, RTX rituximab, CR complete remission, Re relapse during follow-up

become available) plus ribavirin. The patient has remained in remission for over 6 years and is assumed to be cured [31]. More recently, a pilot study in 16 patients demonstrated the efficacy of combined rituximab and PEGIFN-a2b plus ribavirin in patients with severe HCV-associated cryoglobulinemic vasculitis who were either resistant to or experienced a relapse on previous combination treatment with standard IFN-a or PEGIFN-a2b plus ribavirin. The treatment schedule consisted of four weekly doses of 375  mg/m2 rituximab intravenously followed 1 month later (to avoid cumulative side effects) by PEG-IFN-a2b (1.5  mg/kg per week subcutaneously) plus ribavirin (600–1,200  mg/ day orally) for 12  months. During treatment, 15 patients (94%) showed a clinical response (improvement of rheumatologic and other symptoms) and 10 patients (63%) achieved a complete clinical response. HCV-RNA and cryoglobulins both became undetectable in the group of complete responders. Early virological response (within 3 months) to PEG-IFN-a plus ribavirin and vasculitis of shorter duration than the previous episode were associated with a complete response. Whereas skin manifestations (purpura and leg ulcers) were successfully treated, peripheral neuropathy and renal involvement responded less favorably. Three patients had a NHL; in two, complete remission of the NHL could be induced whereas in the third partial remission was achieved. Of note, peripheral blood B-cell reconstitution was delayed until the end of anti-viral treatment. During follow-up, the two complete responders experienced a relapse, with the simultaneous reappearance of HCV-RNA and cryoglobulins. Adverse events in two patients (worsening of polyneuropathy and flare of psoriasis) were attributed to PEG-IFN-a2b [32].

The results of the pilot study were confirmed in a larger study of 32 patients with severe HCV-associated cryoglobulinemic vasculitis who were treated with either rituximab alone or rituximab and PEG-IFN-a2b plus ribavirin according to the above-mentioned treatment schedule (plus steroids and/or plasmapheresis in patients with life-threatening vasculitis). A complete clinical response was determined in 7/12 (58%) and 16/20 (80%) patients treated with rituximab alone and rituximab and PEG-IFN-a2b plus ribavirin, respectively. Thus, the response was better in patients treated with the combined protocol. After a 23 ± 12  months (mean ± SD), seven patients (22%) experienced a clinical relapse, which was associated with the failure of virological control (either non-responders to PEGIFN-a2b plus ribavirin or patients treated with rituximab without anti-viral therapy) and recovery of peripheral blood B-cells. Retreatment with rituxmab successfully induced remission in these patients. In contrast, none of the patients with SVR and B-cell recovery experienced a relapse. The main side effects were attributable to rituximab and consisted of serum sickness in 6/32 (19%) and neutropenia in 2/32 (6%) patients [33]. Systemic reactions may be associated with higher doses of rituximab (1,000  mg) [34]. Table 39.2 summarizes the data on clinical outcomes in the three discussed studies [31–33].

39.5 Conclusion: Rituximab as First- or Second-Line Therapy in Different Clinical Settings The current clinical evidence, based on uncontrolled non-randomized trials in different clinical settings, suggests the use of PEG-IFN-a plus ribavirin as

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­ rst-line therapy for patients with mild to moderate fi disease severity and activity (i.e., Meltzer’s triad) in HCV-associated cryoglobulinemic vasculitis. In patients with active disease resistant to anti-viral treatment and those with severe manifestations and activity of cryoglobulinemic vasculitis (e.g., worsening of renal function, polyneuropathy, mononeuritis multiplex, skin ulcers, acral necrosis), combined treatment with rituximab and PEG-IFN-a plus ribavirin should be considered. In fulminant and life-threatening cryoglobulinemic vasculitis (e.g., rapidly deteriorating renal function, gastrointestinal vasculitis, central nervous system involvement, hyperviscosity syndrome), the initial therapy should combine plasmapheresis and immunosuppressive therapy (e.g., cyclophosphamide and steroids). Immunosuppressive treatment is continued until the clinical response is sustained and steroids can be safely tapered. Thereafter, PEG-IFN-a plus ribavirin, either alone or in combination with rituximab, may help to eliminate the HCV and terminate B-cell clonal proliferation. Relapses of HCV-associated cryoglobulinemic vasculitis can be treated with a second round of the same rituximab regimen [21, 22]. Since rituximab as a first- or second-line treatment option, alone or in combination with PEG-IFN-a plus ribavirin, induces a sustained clinical and virological remission in only about 60% of the patients with HCVassociated cryoglobulinemic vasculitis, further treatment options are needed to both eliminate HCV and revert clonal B-cell proliferation. Accordingly, randomized controlled trials are necessary to verify and extend our current treatment options in HCV-associated cryoglobulinemic vasculitis [21, 22, 35]. Fortunately, a number of strategies to improve the control of HCV replication using specific anti-viral drugs are now in advanced clinical trials [28, 35]. The most promising of these to date is telaprevir, a targeted inhibitor of HCV protease. In clinical trials, telaprevir was given together with combination therapy and led to improvement in SVR rates from 45% to 65%, even using shortened treatment regimens (6 vs. 12  months) [36, 37]. However, the drug is associated with substantial excess side effects, mainly rash, which may necessitate its withdrawal, but the above figures are cited from intention-to-treat analyses [38]. Unfortunately, telepravir and other targeted therapies in early-phase trials show rapid selection of HCV escape mutants during 1–2 weeks of therapy. Thus, these drugs will need to be given in combination with the current standard of

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care, until, potentially, a range of two or three highly active drugs (nucleoside inhibitors, non-nucleoside inhibitors, and protease inhibitors) can be given in combination, analogous to the strategy currently used to treat HIV patients [28, 35, 38]. An alternative strategy to improve treatment outcome for HCV is the use of immunotherapy. Therapeutic vaccine trials for HCV have been attempted, although the tested vaccines were not highly immunogenic and had only a modest impact on viral load in a subset of patients. Alternative strategies include blockades of interleukin-10 and the inhibitory molecule PD-1. However, with immunomodulators such as these there is the risk of exacerbating the autoimmune disease. Consequently, they may not be appropriate for patients with mixed cryoglobulinemia, even if they prove to be successful in the treatment of uncomplicated HCV infection [28, 35]. In HCV-negative “idiopathic” cryoglobulinemic vasculitis, rituximab represents a new treatment option, also in disease resistant to conventional immunosuppressive therapy [24, 25]. Moreover, IFN-a was able to induce remission in two patients with severe HCVnegative cryoglobulinemic vasculitis, presumably based on its anti-proliferative effect [39].

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25. Roccatello D, Baldovino S, Rossi D et al (2004) Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis. Nephrol Dial Transplant 19:3054–3061 26. Quartuccio L, Soardo G, Romano G et al (2006) Rituximab treatment for glomerulonephritis in HCV-associated mixed cryoglobulinaemia: efficacy and safety in the absence of ­steroids. Rheumatology (Oxford) 45:842–846 27. Koukoulaki M, Abeygunasekara SC, Smith KG, Jayne DR (2005) Remission of refractory hepatitis C-negative cryoglobulinaemic vasculitis after rituximab and infliximab. Nephrol Dial Transplant 20:213–216 28. Pereira AA, Jacobson IM (2009) New and experimental therapies for HCV. Nat Rev Gastroenterol Hepatol 6: 403–411 29. Landau DA, Saadoun D, Halfon P et al (2008) Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response. Arthritis Rheum 58:604–611 30. Casato M, Mecucci C, Agnello V et al (2002) Regression of lymphoproliferative disorder after treatment for hepatitis C virus infection in a patient with partial trisomy 3, Bcl-2 overexpression, and type II cryoglobulinemia. Blood 99: 2259–2261 31. Lamprecht P, Lerin-Lozano C, Merz H et al (2003) Rituximab induces remission in refractory HCV associated cryoglobulinaemic vasculitis. Ann Rheum Dis 62:1230–1233 32. Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinaemia vasculitis. Ann Rheum Dis 67:1431–1436 33. Terrier B, Saadoun D, Sène D et al (2009) Efficacy and tolerability of rituximab with or without PEGylated interferon alfa-2b plus ribavirin in severe hepatitis C virus-related vasculitis: a long-term followup study of thirty-two patients. Arthritis Rheum 60:2531–2540 34. Sène D, Ghillani-Dalbin P, Amoura Z et al (2009) Rituximab may form a complex with iGmkappa mixed cryoglobulin and induce severe systemic reactions in patients with hepatitis C virus-induced vasculitis. Arthritis Rheum 60:3848–3855 35. Webster DP, Klenerman P, Collier J, Jeffery KJM (2009) Development of novel treatments for hepatitis C. Lancet Infect Dis 9:108–117 36. McHutchison JG, Everson GT, Gordon SC et  al (2009) Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N Engl J Med 360:1827–1838 37. Hézode C, Forestier N, Dusheiko G (2009) Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med 360:1839–1850 38. Hoofnagle JH (2009) A step forward in therapy for hepatitis C. N Engl J Med 360:1899–1901 39. Casato M, Lagana B, Pucillo LP, Quinti I (1998) Interferon for hepatitis C virus-negative type II mixed cryoglobulinemia. N Engl J Med 338:1386–1387

PIRR Therapy in HCV-Related Mixed Cryoglobulinemia

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Franco Dammacco and Domenico Sansonno

40.1 HCV-Related Mixed Cryoglobulinemia and Therapeutic Implications For many years, the etiology of mixed cryoglobulinemia (MC) remained unknown, thus accounting for its designation as “essential” MC. Since the end of the 1980s, an overwhelming body of evidence has consistently shown that over 90% of cryoglobulinemic patients are infected with hepatitis C virus (HCV) [1– 5], suggesting that the virus plays a crucial role in the development of this intriguing immune-complexmediated vasculitis. With rare exceptions, patients are therefore more properly diagnosed as having HCVassociated, rather than essential MC. Interestingly, the use of an anti-viral drug such as interferon (IFN)-a in the therapy of MC was first proposed by Bonomo et al. [6] a few years before the association between MC and HCV infection was clearly established. These authors reasoned that if a chronic viral infection is involved in the etiopathogenesis of MC, then the IFN-mediated inhibition of viral replication could play an important role in its treatment. In addition, if one considers MC as a low-grade B-cell lymphoma-like disorder, IFN-a might prove effective because of its anti-proliferative action. And indeed, the use of recombinant IFN-a in seven patients diagnosed with type II idiopathic MC resulted in each case in a striking clinical improvement and a remarkable reduction of circulating cryoglobulins, with usually mild and transient side effects. F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

A number of subsequent reports [7–9] confirmed the efficacy and safety of this treatment strategy, although relapse occurred in a significant proportion of patients a few months after drug discontinuation. Since corticosteroids (CS) were widely prescribed for MC prior to the introduction of IFN-a, we asked whether a combination of CS plus IFN-a would induce a better response than obtained with the administration of IFN-a as a single drug [10]. Following a one-year treatment of 65 patients who were then monitored for 8–17  months after treatment discontinuation, a complete response was determined in approximately 53% of the patients receiving either IFN-a alone or the IFN-a plus 6-methyl-prednisolone (PDN) combination, with combined therapy resulting in a quicker (within 3  months) response. Partial responses were also largely comparable in the two groups. However, the probability of relapse within 3  months from the end of therapy was 75% in patients receiving IFN-a alone vs. none of the patients given IFN-a plus PDN. The pegylated (p) IFN-a/ribavirin (RBV) combination has now become the standard of care worldwide for HCV chronically infected patients [11–13]. This treatment induces a sustained virological response (SVR) rate of 45–50% in patients infected with HCV genotype 1 and 70–80% in those infected with HCV genotypes 2 and 3 [14, 15]. The same combined therapy has been administered to patients with HCV-positive MC, with the obvious aim of eradicating the underlying HCV infection. A remarkable improvement was observed [16] in up to 54% of patients unresponsive to IFN-a alone, as they achieved both clinical remission and an SVR. Even better results were reported in another study [17], in which >70% of the patients attained both an SVR and a complete clinical response.

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_40, © Springer-Verlag Italia 2012

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Although the achievement of an SVR in MC patients receiving pIFN-a/RBV combination therapy is usually associated with regression or remarkable improvement of vasculitis, little change, if any, occurs in terms of neuropathy and/or glomerulonephritis, strongly suggesting that factors other than viral infection are involved in their pathogenesis. In addition, more recent observations indicate that cryoglobulinemic vasculitis can persist in spite of the treatment-induced disappearance of viremia [18] or it may unexpectedly relapse in patients who had achieved an SVR [19]. A possible explanation of these phenomena stems from biomolecular studies, which have clearly shown that: (a) restriction of the humoral immune response occurs in MC patients, in whom B-cell clonal expansions may be detected in the liver, bone marrow, and circulation [20, 21], and (b) restriction of V-gene usage is invariably associated with extrahepatic manifestations of HCV infection [22]. At variance from these observations, B-cell clonal expansions are rarely detectable in HCV-infected patients without MC [21, 23].

40.2 Rituximab in Cryoglobulinemic Vasculitis Based on these observations, a therapeutic approach aimed at deleting expanded B-cell clones was attempted by two independent Italian groups [24, 25] using the chimeric monoclonal antibody, rituximab (RTX), which is directed against the transmembrane CD20 antigen expressed on pre-B and differentiated B lymphocytes. Treatment resulted in a striking improvement of cutaneous vasculitis, arthromyalgias, and weakness in approximately 80% of the 20 patients studied [24]. Parallel decreases of the cryocrit, serum IgM levels, and rheumatoid factor titers were also recorded, in step with improvements in C3 and C4 hypocomplementemia, sometimes including the restoration of normal values. A complete response was maintained in 75% of 16 responders throughout the follow-up. In addition, molecular monitoring of the B-cell response revealed disappearance of peripheral clones in the responders. RTX administration was found to be well tolerated and no remarkable adverse events were reported. However, a matter of concern was the demonstration that RTX increased HCV RNA to approximately twice the baseline levels in responders, whereas viremia remained mostly unchanged in non-responders.

F. Dammacco and D. Sansonno

The effectiveness and safety of RTX was confirmed in several studies thereafter, summarized by Cacoub et al. in a systematic review of the literature [26]. After excluding review papers, the authors selected 13 contributions reporting data on a total of 57 patients with MC, 75% of whom had HCV-related disease. Independent of the few variations in the treatment schedules among the different studies, RTX was found to positively affect the main signs of vasculitis and to induce a clinical response in 80–93% of the patients; however, a relapse was recorded in 39%. It should also be emphasized that the safety and efficacy of RTX were demonstrated in MC patients with advanced liver disease [27] and in those with non-viral cryoglobulinemic vasculitis [28].

40.3 PIRR Therapy as a New Therapeutic Approach to Mixed Cryoglobulinemia The occurrence of HCV infection in the large majority of patients with MC, chronic antigenic (viral) stimulation of the immune system, and expansion of B-cell clones have been established as crucial factors in the pathogenesis of cryoglobulinemia [29]. However, since enhanced viremia is a potentially harmful outcome of RTX administration and the ensuing B-cell depletion, it seems reasonable to combine this monoclonal antibody with antiviral drugs in order to achieve and stabilize the deletion of expanded B-cell clones, on the one hand, and induce an SVR or at least avoid an increase in the viral load, on the other. In line with these goals, a few trials have been carried out, which consistently indicated the effectiveness of the respective therapeutic measures.

40.3.1 Personal Experience We administered a combination of pIFN-a plus RBV plus RTX (PIRR) to 22 naïve HCV-positive patients with MC. Fifteen additional MC patients received pIFN-a plus RBV combined therapy without RTX [30]. All of the patients were included in a prospective, single-center, randomized study on the basis of the ­following criteria: (a) occurrence of anti-HCV antibodies and detectable HCV RNA in the serum; (b) cryocrit ³ 3% associated with the classical symptom triad of purpura, arthralgia, and asthenia; (c) recent (£ 3 months) liver biopsy showing chronic active hepatitis; (d) no

40  PIRR Therapy in HCV-Related Mixed Cryoglobulinemia Fig. 40.1  General design of pegylated interferon-a (pIFN-a) plus ribavirin plus rituximab (PIRR) therapy. RTX Rituximab, RBV Ribavirin, ST start of therapy, A-ETR assessment of end-of-treatment response, A-SR assessment of sustained response, A-LTR assessment of long-term response

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previous administration of IFNs or immunosuppressive factors; (e) lack of co-infection with HBV or HIV. Exclusion criteria were neuropsychiatric disorders, primary biliary cirrhosis, and cardiovascular, metabolic, neoplastic, and autoimmune diseases. Pregnant women and patients ingesting >40  g of alcohol per day were also excluded. The study design, which conformed to the Declaration of Helsinki and followed the Good Clinical Practice Guidelines, was approved by the Ethical Committee of the University of Bari. As shown in Fig.  40.1, patients who entered the PIRR arm received weekly subcutaneous injections of 180 mg pIFN-a2a or 1.5 mg/kg pIFN-a2b, in combination with 1,000 or 1,200  mg/day RBV according to their body weight (£75  kg or >75  kg, respectively). Both antiviral drugs were given for 48 weeks, independent of HCV genotype and viral load. In addition, RTX infusions (375 mg/m2) were administered to all PIRR patients once a week in the first month and then after 5 and 11 months. As already stated, patients assigned to the control arm received the same pIFN-a/RBV combination, without RTX. The patients were followed for up to 36 months from the end of treatment. Table  40.1 summarizes the main demographic, clinical, immunological, and virological features of the patients enrolled in the PIRR and control arms. All patients, regardless of the enrollment arm, completed the study and were examined for the entire duration of the follow-up. Definitions of clinical, immunological, virological, and molecular responses as well as the diagnostic criteria of complete response,

Table 40.1  Main clinical, immunological and virological features of 37 patients with HCV-related mixed cryoglobulinemia enrolled in two therapeutic arms Parameters Age/sex Purpura (%) Asthenia (%) Arthralgias (%) Chronic active hepatitis (%) Renal impairment (%) Peripheral neuropathy (%) Cryocrit (%; mean ± SD) Cryoglobulin type II/ type III (%) Rheumatoid factor (IU/mL) Complement fractions (mg/dL) C3 (mean ± SD) C4 (mean ± SD) Anti-HCV antibodies (%) HCV RNA (IU/mL × 106; mean ± SD) Genotype 1/genotype 2 (%)

PIRR therapy (22 patients) 63 (51–68)/ 15F-7M 100 95 74 91 22.7 27.2 6.4 ± 3.9 91/9

pIFN-a/RBV (15 patients) 59 (50–66)/ 10F-5M 100 87 73 93 26.6 20 5.5 ± 3.0 80/20

415.2 ± 138.55 482.5 ± 283.5 119.3 ± 40.7 116 ± 31 4.8 ± 3.0 3.7 ± 1.9

100 1.6 ± 0.5

100 1.9 ± 0.7

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partial response, and no response are reported in Table 40.2. Assessment of the end-of-treatment response indicated that 54.5% of the patients enrolled in the PIRR arm achieved a complete response, compared to 33.3% of those included in the control arm (p