Current Topics in Complement (Advances in Experimental Medicine and Biology Vol 586)

CURRENT TOPICS IN COMPLEMENT

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Editorial Board: NATHAN BACK, State University of New York at Buffalo IRUN R. COHEN, The Weizmann Institute of Science DAVID KRITCHEVSKY, Wistar Institute ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research RODOLFO PAOLETTI, University of Milan

Recent Volumes in this Series Volume 576 N-ACETYLASPARTATE: A UNIQUE NEURONAL MOLECULE IN THE CENTRAL NERVOUS SYSTEM Edited by John R. Moffett, Suzannah B. Tieman, Daniel R. Weinberger, Joseph T. Coyle and Aryan M.A. Namboodiri

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Volume 586 CURRENT TOPICS IN COMPLEMENT Edited by John D. Lambris A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher.

CURRENT TOPICS IN COMPLEMENT Edited by

John D. Lambris

Editor: John D. Lambris, Ph.D. Professor of Pathology & Laboratory Medicine University of Pennsylvania Philadelphia, PA 19107 [email protected]

Library of Congress Control Number: 2006925169 Printed on acid-free paper. ISBN 10: 0-387-32231-0 ISBN-13: 978-0387-32231-5

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Preface

Complement has long been regarded as a pivotal effector arm of the innate immune response, eliciting important immunoregulatory functions in the context of inflammation and also serving as a vital link between the innate and adaptive immune response. In the post-genomic era, our knowledge of the innate immune system is enriched by findings that point to novel functions that do not strictly correlate with immunological defense and surveillance, immune modulation or inflammation. Several studies indicate that complement proteins exert functions that are either more complex than previously thought, or go well beyond the innate immune character of the system. The advent of high-throughput platforms for genome and proteome-wide profiling, together with the enormous amount of raw genetic information that has accumulated in the databases, have stirred new expectations in biomedical research. They have led complementologists to revisit established biological systems, such as the complement system, from a global and integrative perspective. Complement research is now faced with the challenge of trying to integrate isolated biochemical pathways into complex gene and protein regulatory circuits. In this respect, scientists from around the world convened at the Third Aegean Conferences Workshop on Complement Associated Diseases, Animal Models, and Therapeutics (June 5–10, 2005), to discuss recent advances in this fast evolving field. This volume represents a collection of topics on the "novel" functions of complement, pathophysiology, protein structures, design of complement inhibitors, and complement assays discussed during the conference. I am grateful to the contributing authors for the time and effort they have devoted to writing, what I consider exceptionally informative chapters in a book that will have a significant impact on the complement field. I am grateful to Rodanthi Lambris for her assistance in formatting the text. I also gratefully acknowledge the generous help provided by Dimitrios Lambris in managing the organization of this meeting. Finally, I also thank Andrea Macaluso of Springer Publishers for her supervision in this book's production. John D. Lambris, PhD

v

Contents

List of Contributors .......................................................................................... xvii 1.

Cross-Disciplinary Research Stirs New Challenges into the Study of the Structure, Function and Systems Biology of Complement

Dimitrios Mastellos and John D. Lambris 1. Introduction.................................................................................................. 2. Biophysical Approaches in Elucidating Complement Structure and Binding Energetics ................................................................................ 3. Thermodynamics of Complement Protein Binding ...................................... 4. Probing Conformational Changes of Complement Proteins with Hydrogen/Deuterium Exchange and Mass Spectrometry..................... 5. Combinatorial and in Silico Protein Design: In Search for More Potent C3 Inhibitors............................................................................ 6. Defining the Structural Determinants of Viral Immune Evasion: The C3b/SPICE/VCP Interaction ................................................................. 7. A "Systems Biology" Perspective of Innate Immunity: Newly Identified "Crosstalks" between Complement and Divergent Biological Networks.................................................................... 7.1. Complement Intercepts Cytokine-Driven Regenerative Networks in the Liver ......................................................................... 7.2. A Complement–Chemokine "Crosstalk" Regulates Hematopoietic Stem Cell Engraftment ............................................... 7.3. Complement Modulates Coagulation Processes ................................. 8. Future Perspectives ...................................................................................... 9. Acknowledgments........................................................................................ 10. References ....................................................................................................

2.

1 2 3 4 4 6

7 9 10 10 11 12 12

Liver Regeneration: A Link to Inflammation through Complement Robert A. DeAngelis, Maciej M. Markiewski, and John D. Lambris 1. Introduction.................................................................................................. 2. Liver Regeneration and Inflammatory Mediators ........................................ 2.1. Cytokines and Transcription Factors .................................................. 2.2. Growth Factors, Metalloproteases, Adhesion Molecules, and Acute Phase Proteins.................................................................... 2.3. Natural Killer T (NKT) Cells.............................................................. 3. The Role of Complement in Liver Regeneration.......................................... 4. Conclusion ................................................................................................... 5. Acknowledgments........................................................................................ 6. References .................................................................................................... vii

17 18 19 20 22 23 23 26 26

viii

3.

CONTENTS

The Role of Third Complement Component (C3) in Homing of Hematopoietic Stem/Progenitor Cells into Bone Marrow

Ryan Reca, Marcin Wysoczynski, Jun Yan, John D. Lambris, and Mariusz Z. Ratajczak 1. Introduction.................................................................................................. 2. The Function of CXCR4 Receptor Depends on Lipid Raft Formation......... 3. Complement Is Activated in BM during Myeloablative Conditioning for Hematopoietic Transplantation............................................................... 4. The Role of Complement in Regulating the Biology of HSPC .................... 5. Hematopoiesis in C3-Deficient Mice under Normal Steady-State and Stress Situations................................................................ 6. Molecular Explanation of the Defect in Homing/Engraftment of HSPC in C3-Deficient Mice......................................................................... 7. Conclusions.................................................................................................. 8. References ....................................................................................................

4.

41 42 46 46

53 54 54 54 55 55 56 57 58

To Regeneration ... With Complement Panagiotis A. Tsonis, John D. Lambris, and Katia Del Rio-Tsonis 1. 2. 3. 4. 5.

6.

37 39

Complement System and the Eye Purushottam Jha, Puran S. Bora, Jeong-Hyeon Sohn, Henry J. Kaplan, and Nalini S. Bora 1. Introduction.................................................................................................. 2. Complement and Ocular Protection ............................................................. 3. Complement and Ocular Diseases................................................................ 3.1. Complement and Corneal Diseases..................................................... 3.2. Complement and Autoimmune Uveitis............................................... 3.3. Complement and Age-Related Macular Degeneration........................ 4. Complement and Ocular Tolerance.............................................................. 5. Conclusions.................................................................................................. 6. References ....................................................................................................

5.

35 36

Regenerative Abilities in Vertebrates........................................................... Limb Regeneration....................................................................................... Lens Regeneration........................................................................................ The Complement System ............................................................................. References ....................................................................................................

63 64 65 66 68

Self, Non-Self and Danger: A Complementary View

Jörg Köhl 1. Introduction.................................................................................................. 2. Complement as a "Master Alarm System" of Innate Immunity ................... 3. Complement-Derived Danger-Transmitters Shape Innate and Adaptive Immune Responses following Physiological and Pathological Threats..................................................................................... 3.1. Danger Transmission through C1q Receptors .................................... 3.2. Danger Transmission through C3 Cleavage Fragments......................

71 72

74 75 75

CONTENTS

4. Danger Transmission Mediated through the Anaphylatoxic Peptides C3a and C5a................................................................................... 4.1. Anaphylatoxin Receptor-Dependent and -Independent Effects .......... 5. Anaphylatoxin-Mediated Danger Transmission in Non-Myeloid Cells ....... 6. C5a Receptor Signaling in Pulmonary Dendritic Cells Regulates Inhalation Tolerance..................................................................................... 7. C5a Receptor Signaling on APCs Impacts Danger Transmission through TLRs ............................................................................................... 8. Summary ...................................................................................................... 9. Acknowledgments........................................................................................ 10. References ....................................................................................................

7.

ix

78 79 81 82 84 85 86 86

gC1qR/p33 Serves as a Molecular Bridge between the Complement and Contact Activation Systems and Is an Important Catalyst in Inflammation

Berhane Ghebrehiwet, Claudia Cebada-Mora, Lee Tantral, Jolyon Jesty, and Ellinor I. B. Peerschke 1. Abstract ........................................................................................................ 95 2. Introduction.................................................................................................. 96 3. Materials and Methods ................................................................................. 97 3.1. Chemicals and Reagents ..................................................................... 97 3.2. Proteins and Antibodies ...................................................................... 97 3.3. Biotinylation of Proteins..................................................................... 97 3.4. Expression of Recombinant gC1qR.................................................... 98 3.5. Collection of Normal Human Serum .................................................. 98 3.6. Hemolytic Assay................................................................................. 98 3.7. Microplate Assay for Complement Activation ................................... 99 4. Results.......................................................................................................... 99 4.1. Inhibition of Hemolytic Activity by gC1qR ....................................... 99 4.2. Soluble gC1qR but not 74–96 gC1qR Can Activate the Classical Pathway ......................................................................... 100 5. Discussion .................................................................................................... 101 6. Acknowledgments........................................................................................ 103 7. References .................................................................................................... 104

8.

Possible Immunoprotective and Angiogenesis-Promoting Roles for Malignant Cell-Derived Prostasomes: A New Paradigm for Prostatic Cancer?

Kristina Nilsson Ekdahl, Gunnar Ronquist, Bo Nilsson, and Adil A. Babiker 1. Introduction.................................................................................................. 2. Hypothesis: Malignant Cell-Derived Prostasomes Provide Cancer Cells with a Zone of Innate Immune Privilege............................................. 3. Complement Activation and Expression of Complement Regulatory Proteins by Malignant Cells ......................................................................... 4. CD59 Transfer by Prostasomes Results in Protection against Complement-Mediated Lysis ....................................................................... 5. Extracellular Phosphorylation of Plasma Proteins........................................

107 109 110 111 113

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CONTENTS

6. 7. 8. 9.

9.

Mapping of Protein Kinases on Prostasomes ............................................... C3 and other Substrates for Prostasomal PKs .............................................. Conclusions.................................................................................................. References ....................................................................................................

114 115 116 116

Diversified Components of the Bony Fish Complement System: More Genes for Robuster Innate Defense?

Miki Nakao, Yoko Kato-Unoki, Makiko Nakahara, Junichi Mutsuro, and Tomonori Somamoto 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction.................................................................................................. 121 Classical Pathway Components.................................................................... 123 Lectin Pathway Components........................................................................ 125 Alternative Pathway Components ................................................................ 126 Lytic Pathway Components.......................................................................... 131 Complement Receptors ................................................................................ 131 Regulatory Factors ....................................................................................... 132 Concluding Remarks and Future Directions................................................. 133 Acknowledgments........................................................................................ 134 References .................................................................................................... 134

10. C5b-9 Complement Complex in Autoimmune Demyelination: Dual Role in Neuroinflammation and Neuroprotection

Horea Rus, Cornelia Cudrici, and Florin Niculescu 1. Introduction.................................................................................................. 139 2. Role of C5b-9 in Neuroinflammation........................................................... 140 2.1. In Vitro Demyelination by C5b-9 ....................................................... 140 2.2. Role of C5b-9 in Demyelination during EAE..................................... 141 3. Role of C5b-9 in Neuroprotection................................................................ 142 3.1. Inhibition of Oligodendrocyte Apoptosis by Sublytic C5b-9.............. 142 3.2. Contribution of Complement C5 to Neuroprotection in EAE............. 145 4. Does C5b-9 Protect Oligodendrocytes from Apoptosis in Multiple Sclerosis?....................................................................................... 146 5. Acknowledgments........................................................................................ 148 6. References .................................................................................................... 148

11. The Double-Edged Flower: Roles of Complement Protein C1q in Neurodegenerative Diseases

Andrea J. Tenner and Maria I. Fonseca 1. 2. 3. 4. 5. 6. 7. 8.

Introduction.................................................................................................. 153 Complement in the Brain ............................................................................. 155 Murine Models of Alzheimer's Disease........................................................ 157 Potential Protective Roles of Complement in the CNS ................................ 162 Potential Complement-Based Therapeutics.................................................. 165 Summary ...................................................................................................... 166 Acknowledgments........................................................................................ 166 References .................................................................................................... 167

CONTENTS

xi

12. The Role of the Complement System in the Pathogenesis of Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

Nóra Terényi, József Prechl, and Anna Erdei 1. Introduction.................................................................................................. 2. Local Production as a Complement Source in the CNS ............................... 3. The Role of Complement Deposition in Myelin Damage ............................ 3.1. Decomplementation by CVF .............................................................. 3.2. C1 ....................................................................................................... 3.3. C3 ....................................................................................................... 3.4. C4 ....................................................................................................... 3.5. C5 ....................................................................................................... 3.6. C6-C9, MAC ...................................................................................... 4. Anaphylatoxin Effects in Demyelinization .................................................. 5. Complement Regulation in the CNS ............................................................ 6. Complement and Therapy of EAE ............................................................... 7. Acknowledgments........................................................................................ 8. References ....................................................................................................

177 180 180 180 182 182 182 183 183 183 185 185 186 186

13. The Complement System: A Potential Target for Stroke Therapy

J. Mocco, Michael E. Sughrue, Andrew F. Ducruet, Ricardo J. Komotar, Sergei A. Sosunov, and E. Sander Connolly Jr. 1. Introduction.................................................................................................. 2. Rationale for Blocking Complement Activation to Treat Stroke.................. 2.1. Inflammation Is Deleterious in Stroke, and Complement Is Activated in Stroke ............................................................................. 2.2. Complement Activation Exacerbates Ischemic Injury in Other Organs....................................................................................... 2.3. Complement Activation Causes Injury in Other Nervous System Diseases.................................................................................. 2.4. Neurons Seem to Be Unusually Susceptible to Complement Activation...................................................................... 2.5. In Vivo Evidence Suggests that Complement Is Involved in Cerebral I/R Pathogenesis............................................................... 3. Potential Negatives of Complement Blockade following Stroke ................. 3.1. Complement May Be Needed to Opsonize Cellular Debris after Stroke.............................................................................. 3.2. Complement Aids Tissue Recovery/Repair in Other Organs.............. 3.3. Complement Activation Products May Be Neuroprotective............... 4. Conclusion ................................................................................................... 5. References ....................................................................................................

189 190 190 191 191 191 192 193 194 194 195 195 195

14. Observations on Complement Activity in the Two-Stage Inflammatory/Hemostatic Response in the Baboon and Human Models of E. Coli Sepsis and Endotoxemia

Fletcher B. Taylor Jr., Eric Hack, and Florea Lupu 1. Introduction..................................................................................................

203

xii

CONTENTS

2. Description of the Baboon and Human Models of E. Coli Sepsis and Endotoxemia.......................................................................................... 2.1. Baboon E. Coli Sepsis Model ............................................................. 2.2. Human Endotoxin Model.................................................................... 3. Results.......................................................................................................... 3.1. Activation Parameters of the Complement System in Baboons after Lethal and Sublethal E. Coli Challenge ....................... 3.2. Activation Parameters of Cytokine Complement and Hemostatic Systems in Humans after Endotoxin Challenge: Evidence Establishing Two Distinct Sequential Pathophysiologic Events ................................................... 3.3. Evidence Suggesting that There Is a Unique Counterpart to the Second Stage of the Compensated Response to E. Coli that Is Distinct from the Lethal Counterpart to the First Stage.................................... 4. Conclusions.................................................................................................. 5. References ....................................................................................................

204 204 204 206 206

208

210 210 215

15. Complement Activation during Sepsis in Humans

Heike Schreiber, Daniel Rittirsch, Michael Flierl, Uwe Brueckner, Marion Schneider, Manfred Weiss, Florian Gebhard, and Markus Huber-Lang 1. Introduction.................................................................................................. 2. Material and Methods................................................................................... 2.1. Reagents ............................................................................................. 2.2. Patient Selection ................................................................................. 2.3. Measurement of Serum Concentrations of C3a, C5a, and MAC............................................................................ 2.4. Hemolytic Complement Assay ........................................................... 2.5. Neutrophil Isolation ............................................................................ 2.6. Analysis of C5aR Content on Neutrophils.......................................... 2.7. Statistical Analysis.............................................................................. 3. Results.......................................................................................................... 3.1. Epidemiological Assessments............................................................. 3.2. Sepsis-Induced Complement Activation during Septic Shock in Humans ..................................................................... 3.3. Sepsis-Induced Impairment of Complement Function during Septic Shock in Humans.......................................................... 3.4. Loss of C5aR on Neutrophils Is Associated with a Lethal Outcome during Sepsis in Humans.......................................... 4. Discussion .................................................................................................... 5. Acknowledgments........................................................................................ 6. References ....................................................................................................

217 218 218 218 219 219 219 219 220 220 220 220 221 221 222 224 225

CONTENTS

xiii

16. Three Distinct Profiles of Serum Complement C4 Proteins in Pediatric Systemic Lupus Erythematosus (SLE) Patients: Tight Associations of Complement C4 and C3 Protein Levels in SLE but not in Healthy Subjects Yee-Ling Wu, Gloria C. Higgins, Robert M. Rennebohm, Erwin K. Chung, Yan Yang, Bi Zhou, Haikady N. Nagaraja, Dan J. Birmingham, Brad H. Rovin, Lee A. Hebert, and C. Yung Yu 1. Abstract ........................................................................................................ 227 2. Introduction.................................................................................................. 228 3. Materials and Methods ................................................................................. 229 3.1. Study Populations ............................................................................... 229 3.2. Preparation of EDTA-Plasma and Genomic DNAs ............................ 229 3.3. C4 Phenotyping and Genotyping ........................................................ 229 3.4. Mutations of Complement C4 and C2 Genes...................................... 230 3.5. Clinical Information............................................................................ 230 3.6. Statistics.............................................................................................. 230 4. Results.......................................................................................................... 230 4.1. Demographics and Clinical Features of the Pediatric SLE Study Population......................................................................... 231 4.2. Three Types of C3–C4 Protein Profiles in SLE Patients .................... 233 4.3. Tight Correlation between Serum C3 and C4 Concentrations in SLE Patients but not in Healthy Subjects ....................................... 233 4.4. Serum C3 and C4 Levels Both Correlated BMI but BMI Alone Could not Account for the Tight Association between Serum C3 and C4 Levels .................................. 238 4.5. C4 Genotypic and Phenotypic Variations in Pediatric SLE................ 239 4.6. C4 Gene Dosage Is a Determinant of the Maximum Serum C4 Concentrations in Pediatric SLE ........................................ 241 5. Discussion .................................................................................................... 242 6. Acknowledgments........................................................................................ 244 7. References .................................................................................................... 244

17. A Minimum CR2 Binding Domain of C3d Enhances Immunity following Vaccination

Joseph F. Bower and Ted M. Ross 1. Abstract ........................................................................................................ 2. Introduction.................................................................................................. 3. Materials and Methods ................................................................................. 3.1. Plasmid DNA...................................................................................... 3.2. Purification of Recombinant Protein Antigens ................................... 3.3. Protein Expression .............................................................................. 3.4. Animals and Immunizations ............................................................... 3.5. ELISA................................................................................................. 3.6. ELISpot............................................................................................... 3.7. Statistical Analysis.............................................................................. 4. Results.......................................................................................................... 4.1. Expression of Vaccine Plasmids .........................................................

249 250 251 251 251 252 252 253 253 254 254 254

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CONTENTS

4.2. Cell-Mediated Immune Responses Elicited by Env-mC3d3 ................ 255 4.3. Anti-Env Antibody Responses............................................................ 256 4.4. Mucosal Immunizations...................................................................... 256 5. Discussion .................................................................................................... 258 6. Acknowledgments........................................................................................ 260 7. References .................................................................................................... 260

18. Structure and Function of Ficolins

Yuichi Endo, Yu Liu, and Teizo Fujita 1. 2. 3. 4. 5.

6. 7. 8. 9.

Introduction.................................................................................................. 265 Structure of Ficolin ...................................................................................... 266 Tissue and Cell Type Expressing Ficolin ..................................................... 268 Phylogeny of the Ficolin Family .................................................................. 269 Function of Ficolin ....................................................................................... 271 5.1. Carbohydrate Binding of Ficolin ........................................................ 271 5.2. Binding of Ficolin to Bacteria............................................................. 271 5.3. Ficolin as a Recognition Molecule in the Lectin Pathway .................. 274 Polymorphisms of the Ficolin Gene ............................................................. 275 Conclusions.................................................................................................. 275 Acknowledgments........................................................................................ 276 References .................................................................................................... 276

19. Role of Mannose-Binding Lectin (MBL2) Genotyping in Predicting the Risk of Recurrent Otitis Media (rOM)

Lieve Nuytinck, Els De Meester, Martine Van Thielen, and Paul Govaerts 1. Introduction.................................................................................................. 281 2. MBL2 Gene and Polymorphisms.................................................................. 283 3. Materials and Methods ................................................................................. 284 3.1. Patients and Controls .......................................................................... 284 3.2. MBL2 Genotyping .............................................................................. 285 4. Results.......................................................................................................... 285 5. Discussion .................................................................................................... 286 6. References .................................................................................................... 289

20. Conformational Complexity of Complement Component C3

Bert J.C. Janssen and Piet Gros 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction.................................................................................................. 291 Structural Organization of C3 ...................................................................... 292 Convertase Formation .................................................................................. 296 Decay Acceleration ...................................................................................... 299 Cofactor Activity.......................................................................................... 301 Signaling Roles of C3B Fragments .............................................................. 302 Concluding Remarks .................................................................................... 303 Acknowledgments........................................................................................ 304 References .................................................................................................... 304

CONTENTS

xv

21. Disease-Associated Sequence Variations in Factor H: A Structural Biology Approach

Andrew P. Herbert, Dinesh C. Soares, Michael K. Pangburn, and Paul N. Barlow 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction.................................................................................................. Regulation of the Complement System ........................................................ Factor H ....................................................................................................... Functional Sites of Factor H......................................................................... Atypical Hemolytic Uremic Syndrome ........................................................ Age-Related Macular Degeneration ............................................................. Modeled Modules of Factor H ..................................................................... Predicted Structural Consequences of Amino-Acid Substitutions................ References ....................................................................................................

313 314 315 317 318 318 319 321 323

22. Transdermal Pharmacology of Small Molecule Cyclic C5a Antagonists

Lavinia M. Proctor, Trent M. Woodruff, Prakirti Sharma, Ian A. Shiels, and Stephen M. Taylor 1. Abstract ........................................................................................................ 2. Introduction.................................................................................................. 3. Material and Methods................................................................................... 3.1. Materials ............................................................................................. 3.2. Isolation of Polymorphonuclear Leukocytes....................................... 3.3. Receptor Binding Assay ..................................................................... 3.4 Myeloperoxidase Release from PMNs................................................ 3.5. In Vivo Studies ................................................................................... 3.6. Statistical Analysis.............................................................................. 4. Results.......................................................................................................... 4.1. In Vitro Activity of PMX Compounds................................................ 4.2. Transdermal Pharmacokinetics of Cyclic C5a Receptor Antagonists.......................................................................... 4.3. Effect of Administration of C5a Antagonists on LPS-Induced Neutropenia and Hypotension....................................... 5. Discussion .................................................................................................... 6. References ....................................................................................................

329 330 332 332 332 333 333 333 334 335 335 335 337 341 342

23. Inactivation of Complement by Recombinant Human C3 Derivatives

Edzard Spillner, Johanna Kölln, and Reinhard Bredehorst 1. 2. 3. 4. 5. 6. 7.

Introduction.................................................................................................. Generation of CVF Chimeras and C3 Derivatives ....................................... Functional Characteristics of the C3 Derivatives ......................................... The C345C Domain in Complement ............................................................ Therapeutical Implications ........................................................................... Conclusions.................................................................................................. References ....................................................................................................

347 349 351 353 355 356 356

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CONTENTS

24. Complement Analysis in Clinic and Research

Tom E. Mollnes and Michael Kirschfink 1. Introduction.................................................................................................. 1.1. The Complement System.................................................................... 2. Clinical Indications for Complement Analysis............................................. 2.1. Recurrent Infections............................................................................ 2.2. Autoimmune Diseases ........................................................................ 2.3. Membranoproliferative Glomerulonephritis (MPGN) and Hemolytic Uremic Syndrome (HUS) ........................................... 2.4. Hereditary Angioedema...................................................................... 2.5. Paroxysmal Nocturnal Hemoglobinuria (PNH) .................................. 3. Complement Tests........................................................................................ 3.1. Functional Assays............................................................................... 3.2. Protein Quantification of Individual Components .............................. 3.3. Genetic Analysis................................................................................. 3.4. Cell Surface Expression of Complement Proteins .............................. 3.5. Analysis of Complement Activation Products .................................... 4. Complement Analysis in Experimental Settings .......................................... 4.1. In Vitro Experiments with Human Serum and Blood ......................... 4.2. Animal Experiments ........................................................................... 5. Outlook ........................................................................................................ 6. References ....................................................................................................

361 361 363 364 365 365 366 366 366 366 370 370 370 370 372 372 373 375 375

25. Cell-Bound Complement Activation Products (CB-CAPs) as a Source of Lupus Biomarkers

Sarah J. Calano, Pei-an B. Shih, Chau-Ching Liu, Amy H. Kao, Jeannine S. Navratil, Susan Manzi, and Joseph M. Ahearn 1. Introduction.................................................................................................. 381 2. Measurement of Complement in SLE .......................................................... 382 2.1. Serum C3 and C4 and SLE Disease Activity...................................... 382 2.2. Issues Associated with Measuring Soluble ........................................ Complement Components................................................................... 382 2.3. Complement Activation Products and SLE Disease Activity ............. 384 3. Cell-Bound Complement Activation Products ............................................. 384 3.1. Erythrocyte-Bound C4d as a Diagnostic Assay for SLE..................... 385 3.2. Reticulocyte-Bound C4d as an "Instant Messenger" of Disease Activity in SLE...................................................................... 387 4. Summary ...................................................................................................... 387 5. Acknowledgments........................................................................................ 388 6. References .................................................................................................... 388

Author Index....................................................................................................... 391 Subject Index ...................................................................................................... 393

List of Contributors Joseph M. Ahearn Lupus Center of Excellence University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania, USA

Reinhard Bredehorst Institut für Biochemie und Lebensmittelchemie Universität Hamburg, Germany Uwe Brueckner Department of Traumatology, HandPlastic and Reconstructive Surgery Ulm, Germany

Adil A. Babiker Department of Oncology, Radiology, and Clinical Immunology University Hospital Uppsala, Sweden

Sarah J. Calano Department of Medicine University of Pittsburgh Pittsburgh, Pennsylvania, USA

Paul N. Barlow Joseph Black Chemistry Building University of Edinburgh Edinburgh, UK

Claudia Cebada-Mora Department of Medicine SUNY at Stony Brook Stony Brook, New York, USA

Dan J. Birmingham Department of Internal Medicine The Ohio State University Columbus, Ohio, USA

Erwin K. Chung Columbus Children's Research Institute The Ohio State University Columbus, Ohio, USA

Nalini S. Bora Department of Ophthalmology Jones Eye Institute University of Arkansas for Medical Sciences Little Rock, Arkansas, USA

E. Sander Connolly Jr. Department of Neurological Surgery Columbia University College of Physicians & Surgeons New York, New York, USA

Puran S. Bora Department of Ophthalmology Jones Eye Institute University of Arkansas for Medical Sciences Little Rock, Arkansas, USA

Cornelia Cudrici Department of Neurology University of Maryland School of Medicine Baltimore, Maryland, USA

Joseph F. Bower Department of Medicine Division of Infectious Diseases University of Pittsburgh Pittsburgh, Pennsylvania, USA

Robert A. DeAngelis Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia, Pennsylvania, USA xvii

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CONTRIBUTORS

Katia Del Rio-Tsonis Department of Zoology Miami University Oxford, Ohio, USA

Berhane Ghebrehiwet Department of Medicine SUNY at Stony Brook Stony Brook, New York, USA

Els De Meester Innogenetics NV Ghent, Belgium

Paul Govaerts The Eargroup Antwerpen-Deurne, Belgium

Andrew F. Ducruet Department of Neurological Surgery Columbia University College of Physicians & Surgeons New York, New York, USA

Piet Gros Department of Chemistry Utrecht University Utrecht, The Netherlands

Yuichi Endo Department of Immunology Fukushima Medical University School of Medicine Fukushima, Japan Anna Erdei Department of Immunology Eötvös Loránd University Budapest, Hungary Micheal Flierl Department of Traumatology, HandPlastic and Reconstructive Surgery Ulm, Germany

Eric Hack Department of Immunopathology Sanquin Research Amsterdam, The Netherlands Lee A. Hebert Department of Internal Medicine The Ohio State University Columbus, Ohio, USA Andrew P. Herbert School of Chemistry/Institute of Structural and Molecular Biology University of Edinburgh Edinburgh, UK

Maria I. Fonseca Departments of Molecular Biology and Biochemistry and Pathology University of California Irvine, California, USA

Gloria C. Higgins Columbus Children's Research Institute Department of Pediatrics The Ohio State University Columbus, Ohio, USA

Teizo Fujita Department of Immunology Fukushima Medical University School of Medicine Fukushima, Japan

Markus Huber-Lang Department of Traumatology, HandPlastic and Reconstructive Surgery Ulm, Germany

Florian Gebhard Department of Traumatology, Hand-Plastic and Reconstructive Surgery Ulm, Germany

Bert J.C. Janssen Department of Chemistry Utrecht University Utrecht, The Netherlands

CONTRIBUTORS

Jolyon Jesty Department of Medicine SUNY at Stony Brook Stony Brook, New York, USA Purushottam Jha Department of Ophthalmology Jones Eye Institute University of Arkansas for Medical Sciences Little Rock, Arkansas, USA

xix

John D. Lambris Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia, Pennsylvania, USA Chau-Ching Liu Department of Medicine University of Pittsburgh Pittsburgh, Pennsylvania, USA

Amy H. Kao Department of Medicine University of Pittsburgh Pittsburgh, Pennsylvania, USA

Yu Liu Department of Immunology Fukushima Medical University School of Medicine Fukushima, Japan

Henry J. Kaplan Department of Ophthalmology and Visual Science University of Louisville Louisville, Kentucky, USA

Florea Lupu Oklahoma Medical Research Foundation Oklahoma City, Oklahoma, USA

Yoko Kato-Unoki Department of Bioscience and Biotechnology Kyushu University Hakozaki, Fukuoka, Japan

Susan Manzi Department of Medicine University of Pittsburgh Pittsburgh, Pennsylvania, USA

Michael Kirschfink Institute of Immunology University of Heidelberg Heidelberg, Germany

Maciej M. Markiewski Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia, Pennsylvania, USA

Jörg Köhl Division of Molecular Immunology Cincinnati Children's Hospital Research Foundation Cincinnati, Ohio, USA

Dimitrios Mastellos National Center for Scientific Research "Demokritos" Athens, Greece

Johanna Kölln Institut für Biochemie und Lebensmittelchemie Universität Hamburg, Germany

J. Mocco Department of Neurological Surgery Columbia University College of Physicians & Surgeons New York, New York, USA

Ricardo J. Komotar Department of Neurological Surgery Columbia University New York, New York, USA

Tom E. Mollnes Institute of Immunology Rikshospitalet University Hospital University of Oslo, Norway

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CONTRIBUTORS

Junichi Mutsuro Department of Bioscience and Biotechnology Kyushu University Hakozaki, Fukuoka, Japan

Michael K. Pangburn Department of Biochemistry University of Texas Health Science Center Tyler, Texas, USA

Haikady N. Nagaraja Department of Statistics The Ohio State University Columbus, Ohio, USA

Ellinor I. B. Peerschke Department of Pathology Weill Medical College of Cornell University New York, New York, USA

Makiko Nakahara Department of Bioscience and Biotechnology Kyushu University Hakozaki, Fukuoka, Japan Miki Nakao Department of Bioscience and Biotechnology Kyushu University Hakozaki, Fukuoka, Japan Jeannine S. Navratil Department of Medicine University of Pittsburgh Pittsburgh, Pennsylvania, USA Florin Niculescu Department of Medicine University of Maryland Baltimore, Maryland, USA Bo Nilsson Department of Medical Sciences Uppsala University Hospital Uppsala, Sweden Kristina Nilsson-Ekdahl Department of Chemistry and Biomedical Sciences University of Kalmar, Sweden Lieve Nuytinck Innogenetics NV Ghent, Belgium

József Prechl Department of Immunology Eötvös Loránd University Pázmány P.s.1/C Budapest, Hungary Lavinia M. Proctor Promics Ltd. Brisbane, Queensland, Australia Mariusz Z. Ratajczak Stem Cell Biology Program University of Louisville Louisville, Kentucky, USA Ryan Reca Stem Cell Biology Program University of Louisville Louisville, Kentucky, USA Robert M. Rennebohm Columbus Children's Research Institute Department of Pediatrics The Ohio State University Columbus, Ohio, USA Daniel Rittirsch Department of Traumatology, HandPlastic and Reconstructive Surgery Ulm, Germany Gunnar Ronquist Department of Medical Sciences Uppsala University Hospital, Sweden

CONTRIBUTORS

Ted M. Ross Department of Medicine University of Pittsburgh Pittsburgh, Pennsylvania, USA Brad H. Rovin Department of Internal Medicine The Ohio State University Columbus, Ohio, USA Horea Rus Department of Neurology University of Maryland Baltimore, Maryland, USA Marion Schneider Department of Anesthesiology University of Ulm Medical School Ulm, Germany Heike Schreiber Department of Traumatology, HandPlastic and Reconstructive Surgery Ulm, Germany Prakirti Sharma School of Biomedical Sciences University of Queensland, Australia Ian A. Shiels School of Biomedical Sciences University of Queensland, Australia Pei-an B. Shih Department of Medicine University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania, USA

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Tomonori Somamoto Department of Bioscience and Biotechnology Kyushu University Fukuoka, Japan Sergei A. Sosunov Department of Neurological Surgery Columbia University College of Physicians & Surgeons New York, New York, USA Edzard Spillner Institut für Biochemie und Lebensmittelchemie Universität Hamburg, Germany Michael E. Sughrue Department of Neurological Surgery Columbia University College of Physicians & Surgeons New York, New York, USA Lee Tantral Department of Medicine SUNY at Stony Brook Stony Brook, New York, USA Fletcher B. Taylor Jr Oklahoma Medical Research Foundation Oklahoma City, Oklahoma, USA Stephen M. Taylor School of Biomedical Sciences University of Queensland, Australia

Dinesh C. Soares School of Chemistry/Institute of Structural and Molecular Biology University of Edinburgh Edinburgh, UK

Andrea J. Tenner Departments of Molecular Biology and Biochemistry and Pathology Center for Immunology University of California Irvine, California, USA

Jeong-Hyeon Sohn Department of Ophthalmology and Visual Science University of Louisville Louisville, Kentucky, USA

Nóra Terényi Department of Immunology Eötvös Loránd University Budapest, Hungary

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CONTRIBUTORS

Panagiotis A. Tsonis Department of Biology University of Dayton Dayton, Ohio, USA

Marcin Wysoczynski Stem Cell Biology Program University of Louisville Louisville, Kentucky, USA

Martine Van Thielen Innogenetics NV Ghent, Belgium

Jun Yan Stem Cell Biology Program University of Louisville Louisville, Kentucky, USA

Manfred Weiss Department of Anesthesiology University of Ulm Medical School Ulm, Germany Trent M. Woodruff Promics Ltd. Brisbane, Queensland, Australia Yee-Ling Wu Columbus Children's Research Institute Integrated Biomedical Science Graduate Program The Ohio State University Columbus, Ohio, USA

Yan Yang Columbus Children's Research Institute Columbus, Ohio, USA C. Yung Yu Columbus Children's Research Institute Department of Pediatrics The Ohio State University Columbus, Ohio, USA Bi Zhou Columbus Children's Research Institute Columbus, Ohio, USA

1 CROSS-DISCIPLINARY RESEARCH STIRS NEW CHALLENGES INTO THE STUDY OF THE STRUCTURE, FUNCTION AND SYSTEMS BIOLOGY OF COMPLEMENT Dimitrios Mastellos and John D. Lambris

1. INTRODUCTION Complement is a pivotal effector arm of the innate immune response that participates in various immunoregulatory circuits via a complex network of pro1 tein–protein interactions . The complement cascade is a dynamic network of interactions involving a wide array of soluble glycoproteins, membrane-bound 2 receptors, and fluid-phase or membrane-anchored regulatory proteins . Upon complement activation, a well-orchestrated sequence of protein–protein interactions is initiated that results in proteolytic cleavage of precursor molecules, release of bioactive peptides, and downstream activation of receptors that relay the appropriate signals to the intracellular molecular circuit of complement-targeted cells. In recent years complement pathobiology has been reiterated with the advent of proteomics and functional genomics, the use of high-throughput analytical approaches, transgenic mouse models, and the exponential growth of research data that implicate several components in processes that go beyond the 3 classical immunologic milieu . Complement components appear to modulate critical developmental processes by intercepting molecular circuits that control the cell cycle, cell migration and proliferation, and the homing of myeloid pro4 genitors into tissues . Furthermore, the need to contain the detrimental proinflammatory effects of complement activation, without eliminating its beneficial properties in host im-

Dimitrios Mastellos, National Center for Scientific Research “Demokritos,” Aghia Paraskevi Attikis, Athens 15310, Greece. John D. Lambris, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 401 Stellar-Chance Labs, 422 Curie Blvd., Philadelphia, PA 19104, USA. Web: www.complement.us.

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mune homeostasis, has led researchers to adopt multidisciplinary and highthroughput approaches in a systematic effort to develop rational drug-design platforms and more potent complement-based antiinflammatory therapeutics that might be amenable to clinical protocols. In this respect, emphasis has been placed on the elucidation of key structural elements that govern the dynamics 5 and energetics of protein interactions within the complement system . The integrated use of fine biophysical and in silico approaches in monitoring distinct conformational changes of complement proteins has thus far yielded promising results. This crossdisciplinary approach to complement research highlights the importance of integrating the core structure and dynamics of a biochemical reaction in the context of its pathophysiologic consequences. Overall, the “systems-wide” impact of complement is supported by evidence that complement-mediated pathways engage in functional “crosstalk” with other biological systems. Complement proteins appear to modulate key developmental and homeostatic processes, both in the course of inflammation and in noninflammatory settings. Here we outline this novel conceptual framework for the study of complement structure and function and integrate it into a wider pathophysiological perspective with examples from health and disease. We present a comprehensive account of how an integrated “systems” approach has contributed to elucidation of the structural–functional aspects of C3–ligand interactions and the rational design of small-size complement inhibitors. We outline the enormous capabilities offered by the integrated study of thermodynamics in protein binding and the bioenergetics of complement protein–protein interactions and consider new conceptual “avenues” that can be explored in elucidating key structural elements of complement function. We also present critical aspects of our studies on viral molecular mimicry and immune evasion and highlight the main mechanistic attributes of the “crosstalk” between complement and various biologic processes. It is our conviction that complement research will be spearheaded in the next decade by such combinatorial and crossdisciplinary approaches that will address basic biological networks modulated by complement in a global and integrated manner. Furthermore, the mining of biomolecular and textual databases will essentially complement these experimental strategies and enable scientists to form the integrative context for hypothesis-driven scientific discovery.

2. BIOPHYSICAL APPROACHES IN ELUCIDATING COMPLEMENT STRUCTURE AND BINDING ENERGETICS Cell regulatory networks are the key components of a unified biological system and are defined at the molecular level by the numerous biomolecular interactions that tilt the binding equilibriums and decide the fate of a cellular response 6 or the elicited phenotype upon specific stimulation . Defining the structural elements that underlie the various stages of a binding reaction between interacting

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proteins is integral to understanding a cellular response and also for devising means of intercepting, silencing, or enhancing its effect to the benefit of the host. The resolution of the fine structure of proteins by means of x-ray crystallography has assisted scientists to a great extent in defining such structural modules that regulate binding reactions. However, crystallographic data only refer to a static “snapshot” of a given interaction or conformation and fail to consider the complex and dynamic behaviour of the interacting partners in a protein–protein association. In an effort to circumvent this inherent drawback and shed light onto the dynamic nature of complement-mediated interactions, novel biophysical approaches are being adopted that allow the monitoring of the binding dy7 namics between various complement proteins and receptors . Such approaches also take into account a wide array of interactions that contribute to the formation of binding interfaces, including hydrophobic interactions among non-polar side chains, hydrogen bonding interactions, electrostatic interactions, and van der Waals interactions. Furthermore, these approaches also consider the electrostatic nature and shape constraints of the interacting partners within a complex, two parameters that dictate to a great extent the mechanism by which the optimum and more stable configuration is selected for recognition 7 and binding . In this respect, recent studies have yielded important information regarding the dynamics that govern complex interactions between various complement components, using a crossdisciplinary platform that integrates biochemical, physicochemical, and computational methods. Defining the binding interface and interacting structural elements of C3d and its receptor CR2 has been a major challenge in this direction. The application of electrostatic potential calculations 8–10 has essentially complemented the available crystallographic and site-directed 11 mutagenesis data and has indicated that the dynamics of the C3d–CR2 interaction is strongly dependent on the force of electrostatic fields applied between the two interacting molecules. Indeed, the analysis of the electrostatic potential of each protein in free form and in complex with each other has revealed that this interaction follows a two-step association model comprising distinct stages of 5 recognition and binding . The design of theoretical site-specific mutations within 5 the C3d moiety further supports this two-step association model . It is anticipated that such integrative approaches combining available crystallographic data, biochemical approaches, and biophysical calculations will shed more light on the complex C3d–CR2 association and provide a comprehensive platform for the development of effective complement therapeutics.

3. THERMODYNAMICS OF COMPLEMENT PROTEIN BINDING Distinct thermodynamic changes occur during a binding reaction, and the monitoring of such changes allows for a dynamic study of protein–protein interactions. Isothermal Titration Calorimetry (ITC) is a method that allows the longi-

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tudinal study of the thermodynamic changes that occur during protein complex 12 formation . It is essentially used for calculating the heat that is released in a biochemical reaction as a function of time and yields information on the stoichiometry, enthalpy, association constant, and free energy of binding. A distinct feature of ITC is that it can discriminate between entopy and enthalpy changes, thereby providing information on distinct chemical and structural (con13 formational) changes that contribute to protein binding. ITC has recently been applied for the study of energetics of the interaction of C3 with its inhibitor, 14 compstatin . Thermodynamic measurements have indicated that the binding of compstatin to C3 is 1:1 and occurs through hydrophobic interactions with possible conformational changes in C3 or compstatin. Some protonation changes, 14 occurring at the binding interface, have also been observed by ITC analysis . analysis will be extended to the energetics of various protein–protein interactions, with a goal to obtain the energetic parameters of complement activation and regulation pathways.

4. PROBING CONFORMATIONAL CHANGES OF COMPLEMENT PROTEINS WITH HYDROGEN/DEUTERIUM EXCHANGE AND MASS SPECTROMETRY Hydrogen/deuterium exchange has traditionally been used to understand the formation of protein core or stable intermediate or transient states in pathways of protein folding, because it provides a noninvasive method for identifying protected (or de-protected) exchanging amides. The same principles can be applied to studies of protein–protein association, where the loss in solvent-accessible surface area upon association can be correlated with amide protection from exchange for the amides that lose their contact with solvent. Recent advances in the use of mass spectrometry allow for rapid collection of data of free and com15–17 plexed proteins . Comparison of mass spectra of free and complexed proteins provides the sites of interaction without the need of previously available structural data. Hydrogen/deuterium exchange coupled to mass spectrometry has recently been used to probe the conformational changes of the C3 molecule in 18 its transition from a native to a hydrolyzed state , and it is becoming clear that such a methodology could provide valuable insight into the structural determinants that govern the interaction of C3 with various ligands and receptors (e.g., C3d–CR2).

5. COMBINATORIAL AND IN SILICO PROTEIN DESIGN: IN SEARCH FOR MORE POTENT C3 INHIBITORS Deregulated activation of complement on the surface of host cells and consumption of complement proteins in the fluid phase have been associated with detrimental proinflammatory effects leading to local tissue damage, perturbed ho-

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meostasis and remote organ failure in several pathological states . Over the years considerable effort has been devoted to the discovery of selective complement inhibitors that can intercept the complement cascade at distinct steps, 19 thus neutralizing its deleterious effects in the progression of disease pathology . Several complement inhibitors are currently under development, including small-size organic compounds, synthetic peptides, and also large monoclonal 20 antibodies . Compstatin, a potent small-size complement inhibitor that acts at the level of C3 by blocking all three pathways of complement activation, was discovered by screening a phage-displayed random peptide library for C321 binding peptides . This molecule stands out as a promising complement inhibitor that might be amenable to therapeutic applications in the clinic due to its small size, cost-effective and large-scale synthesis, and its ability to shut down all three pathways of complement activation by blocking the proteolytic cleavage of native C3 by the C3 convertases. The complement inhibitory activity of compstatin has been ascertained in 22-30 various in vitro, in vivo, ex vivo, and in vivo/ex vivo interface models . In a systematic effort to characterize the structural basis of the inhibitory activity of compstatin and design more potent analogs, a wide array of combinato31,40 rial, biophysical and in silico approaches have been used . Determination of the solution structure of compstatin by NMR-based strate31 gies paved the way for the rational design of more potent analogs through successive rounds of sequence and structure optimization. Instrumental to the success of these optimization approaches has been the integrated use of biophysical 33-40 methods and computational modeling . In conjunction with the high-throughput screening approaches, compstatin was also subjected to in silico combinatorial design, using a novel two-step computational optimization methodology. Interestingly, this round of theoretical design yielded a sixfold more active analog than the parent peptide with se41-44 quence Ac–I[CVYQDWGAHRC]T–NH2 . In addition to these rounds of experimental and combinatorial peptide design, a recent rational design effort was undertaken to generate analogs of compstatin with higher inhibitory activities, 45 incorporating in its structure non-natural and D-aminoacids . This approach was largely based on the hypothesis that the aromatic rings of y and w may contribute to the function of compstatin. This approach has led to identification of a more potent compstatin analog that exhibits 99-fold greater inhibitory activity 45 and contains a non-natural aminoacid in its sequence . The peptides derived from such computational and rational design approaches are now in the process of being tested experimentally, and a new generation of compstatin analogs (approx. 270-fold more active than the parent peptide with incorporation of nonnatural aminoacids in the sequence) are being produced in heterologous expression systems (Katragadda et al, unpublished observations). In conclusion, the integrated use of rational experimental and computational (in silico) peptide design approaches has provided a unique and crossdisciplinary platform for the discovery of more effective complement therapeu-

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tics targeting the C3 activation step in the complement cascade. Such integrated approaches should be integral to any drug design effort that involves peptide screening, synthesis, and structure manipulation.

6. DEFINING THE STRUCTURAL DETERMINANTS OF VIRAL IMMUNE EVASION: THE C3B/SPICE/VCP INTERACTION Considerable effort has been placed in the field of antiviral vaccine design toward elucidating the mechanism by which certain herpes and orthopox viruses escape the host immune response, through structural and functional mimicry of 46 complement regulatory proteins . SPICE and VCP are two secreted viral homologs of complement regulatory proteins that bear CCP modules and mediate immune evasion in the host by interacting with C3b and preventing comple47,48 ment-mediated neutralization of virus . Strikingly, despite the fact that it is 1000-fold more potent than VCP in deactivating human C3b, SPICE differs 48,49 from VCP in only 11 aminoacid residues . The generation of VCP–SPICE chimeras consisting of VCP and SPICE CCP modules has recently led to identification of the critical aminoacids that render SPICE a more potent inhibitor of 49 complement . Furthermore, electrostatic potential calculations using these chimeric proteins in interaction with human C3b have revealed an essential role of electrostatic forces in driving the VCP/C3b interaction. Electrostatic modeling has suggested a two-step association model for VCP/C3b that involves electrostatically driven recognition and enhanced binding. These studies revealed that a predominantly negative C3b and a predominantly positive VCP variant favor their electrostatically driven recognition and enhance their association. An increase in the positive charge of VCP variants occurs by mutations of acidic amino acids, which reduce the negative character of the electrostatic potential at the vicinity of SCR-2 and SCR-3 and enhance the positive character of the electrostatic po49 tential at SCR-1 . Electrostatic modeling of the VCP/C3b interaction, in conjunction with site-directed mutagenesis studies testing the ability of different VCP/SPICE variants to inhibit complement activation, have provided an integrated framework for better understanding the structural basis and dynamics of the VCP/SPICE–C3b interaction and the molecular mechanism by which viral RCA homologs mediate immune evasion. The important contribution of electrostatic forces to the formation of protein complexes is also highlighted in a recent study discussing the crystal structure of 66 human C3 . The findings presented in this study suggest that C3 takes up in solution a tertiary conformation that presents a “dipole” surface. Such a conformation strongly supports the electrostatic nature of C3 interactions, providing invaluable insight into the biophysical parameters (such as electrostatics) that drive the interaction of C3 with its multiple physiological ligands and receptors.

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It is our strong conviction that the reliable prediction and monitoring of the dynamic behavior of interacting proteins will essentially rely on an integrative platform combining both experimental and theoretical/biophysical approaches such as a survey of electrostatic forces.

7. A “SYSTEMS BIOLOGY” PERSPECTIVE OF INNATE IMMUNITY: NEWLY IDENTIFIED “CROSSTALKS” BETWEEN COMPLEMENT AND DIVERGENT BIOLOGICAL NETWORKS Biomolecular (structural and sequence) databases have been populated with an enormous amount of data generated by means of high-throughput screening and genome-wide profiling techniques. These databases essentially contain the core information on how complex biological networks are regulated at the transcriptome and proteome levels. The challenge facing contemporary bioscience is finding the means of managing these databases in such a way as to extract gene/protein associations that can model or predict the molecular circuits by 6 which individual cells and organisms elicit their responses to various stimuli . Systems biology is the field that integrates such approaches and helps create a comprehensive context for interpreting and predicting gene and pathway associations and also generates new knowledge in a systematic, hypothesis-driven 50 way . Integral to the success of such a systems-wide approach is the use of new text-mining algorithms that are being developed in an effort to enable scientists 51 to efficiently extract biological information from scientific literature databases . Text mining platforms enable researchers to manage complex ontogenies and cluster biologic entities in a meaningful manner that can shed light on novel 52 systems associations . Accumulating evidence suggests that inflammatory circuits interact with divergent pathways in modulating basic biological responses that do not 3 necessarily pertain to inflammation and the immune response per se . In this respect, complement components have been linked to regulatory networks that not only modulate innate immunity but also affect developmental, metabolic, 53 and homeostatic responses . An integrated survey of the scientifc literature using a high-throughput bioinformatics approach called “systems literature analysis” has revealed novel associations of complement with a wide array of biological processes that 53 extend well beyond the innate and adaptive immune response . Distinct associations of complement with such noninflammatory processes have also been verified experimentally. Indeed, recent studies using complement gene knockout models and highly selective complement receptor antagonists have demonstrated the involvement of complement in developmental processes, such 54,55 as limb and liver regeneration , stem cell engraftment/mobilzation, and 56 trafficking of hematopietic precursors to the bone marrow . The main attributes

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Figure 1. An illustration of a systems-wide overview of complement, as a complex network of protein–protein interactions that regulate the activation state of the cascade and also extend links to divergent biological processes; the systems associations of representative complement components have been retrieved through mining of the entire MEDLINE database.

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and mechanistic aspects of these newly identified crosstalks are discussed below. These processes have been selected as examples illustrating the multifaceted nature of the system, and the crossdisciplinary approaches that should be adopted in trying to elucidate its functions in diverse pathophysiological settings. Limb and lens regeneration in urodele amphibians represent the most challenging models for addressing key developmental questions that pertain to cell dedifferentiation, morphogenesis, and pattern formation. The role of complement components in this complex network of interactions that regulate cell fate decisions, tissue remodeling, and regeneration in lower vertebrates is discussed in greater detail in Chapter 5 by Tsonis et al. 7.1. Complement Intercepts Cytokine-Driven Regenerative Networks in the Liver Acute toxic and viral injury or surgical liver resection triggers a robust proliferative response in the liver that culminates in full restoration of hepatic structure 57 and function within days after the insult . Essential priming signals that drive the cell cycle re-entry of quiescent liver cells are provided by hormones, cytokines, and hepatic growth factor-mediated signaling pathways. Recent studies have underscored a previously elusive role of innate immunity in the regulation of the regenerative response of the liver. With the use of complement-deficient mouse strains it was demonstrated that complement components C3 and C5 and their downstream anaphylatoxin-mediated pathways provide essential signals that lead to activation of latent hepatic transcription factors and subsequent re58,55 lease of cytokines that mediate the early priming phase of liver regeneration . Similarly, in acute hepatotoxicity models it was shown that complement is required as a hepatic survival factor that contributes to the restoration of the liver parenchyma by promoting cell-cycle re-entry and proliferation of hepato59,58 cytes . These studies, collectively, provided evidence for crosstalk between complement receptor-mediated pathways and cytokine-driven signaling networks in modulating the early regenerative response in the liver. The global impact of inflammation on the regenerative response of the liver and the main mechanistic aspects of the involvement of complement in the early stages of hepatocyte regeneration are discussed in Chapter 2 by DeAngelis et al. Further delineating the mechanisms by which complement proteins and receptors interact with other signaling networks in the regenerating liver will provide insight into the molecular pathways that drive the early growth response of the liver and “prime” quiescent hepatocytes to re-enter the cell cycle.

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7.2. A Complement–Chemokine “Crosstalk” Regulates Hematopoietic Stem Cell Engraftment Complement regulatory molecules and receptors have been implicated in protecting blood cells from autologous complement-mediated lysis and promoting 60 their inflammatory recruitment during the course of infection . However, the distribution/expression of complement components in pluripotent hematopoietic precursors (HSCs) and early-committed myeloid/lymphoid progenitors at different stages of human hematopoiesis still remains ill-defined. Furthermore, the potential interaction of complement with chemokine and growth factordependent signaling networks that affect stem cell differentiation and regulate various homing activities of these cells in settings that elicit “danger” signals for the innate immune system (such as myeloablative injury, chemotherapy) has remained under intense scrutiny. + Recently, however, it was demonstrated that human CD34 hematopoietic progenitor cells express the C3a anaphylatoxin receptor, and that C3a receptor signaling synergistically promotes a-chemokine stromal-derived factor-1 (SDF1/CXCL12)-dependent responses of HSCs that are associated with trans56 endothelial migration, MMP, secretion and chemotaxis . These findings were further supported by the enhanced hematopoietic recovery observed in irradiated + mice that had been pretreated with C3a-primed Sca-1 hematopoietic progeni56 tors . Further studies have shown that C3a and its receptor, C3aR, promote retention of hematopoietic progenitor/stem cells in the bone marrow during stem cell 61 mobilization in mice . Taken together these studies support a crosstalk between complement and the SDF-1/CXCR4 signaling axis that appears to regulate the homing of hematopoietic stem cells into the bone marrow and, in synergy with chemokines, modulates the anchorage of progenitor cells in the bone marrow microenvironment, preventing their uncontrollable release to the peripheral circulation. Recent evidence that provides further insight into the role of complement in hematopoietic stem cell engraftment and supports its potential clinical application in stem cell transplantation/mobilization will be discussed in more detail in Chapter 3 by Reca et al. 7.3. Complement Modulates Coagulation Processes The inflammatory and coagulation cascades converge at several molecular targets, and both are considered amongst the earlier homeostatic responses to infec62 tion . Perturbations in the interactions between these two systems have been associated with morbidity and mortality in several infectious diseases that pro63,62 gress to disseminated thrombosis and multi-organ failure . Earlier studies have indicated a role for complement in procoagulant pathways by showing that interception of C5aR results in distinct changes of pro/anti-fibrinolytic protein 63,64 cascades , and also in the induction of Tissue Factor in endothelial cells and

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monocytes . Neutrophils together with complement are considered among the 64 earliest “innate sensors” for incoming inflammatory signals . They are rapidly chemoattracted by C5a and migrate through vascular endothelium barriers into tissues that have been exposed to inflammatory insults. To date, the potential interaction of complement and neutrophils in triggering the procoagulant response has remained elusive. Surprisingly, it was recently shown that antiphospholipid antibody-induced complement activation and downstream signalling via C5a receptors in neutrophils leads to the induction of tissue factor (TF), a key initiating component of the blood coagulation cascade (Ritis K. et al. [submitted]). Inhibition studies using the C3 inhibitor compstatin revealed that APS-autoantibodies trigger complement activation, which in turn leads to generation of C5a and induction of a TF-dependent coagulant activity in C5aR-bearing neutrophils. These findings provide further support to the concept that innate immunity and coagulation share common targets and identify a novel “crosstalk” between the complement and coagulation cascades that might be exploited therapeutically for the treatment of complement-associated thrombogenic diseases.

8. FUTURE PERSPECTIVES The complement system constitutes an attractive paradigm of how a complex network of protein–protein interactions can mediate divergent biologic activities, ranging from host defense and immune surveillance to modulation of adaptive immunity, stem cell trafficking, organ regeneration, and triggering of coagulation cascades. In the age of the “omics” revolution, new analytical tools and high-throughput profiling platforms are made available to complement researchers for elucidating key structure–function relationships and defining the distinct and dynamic conformational changes that underlie the wide array of complement protein interactions. Indeed, the recent resolution of the threedimensional structure of the central component of the system, C3, and its proteolytic fragment, C3c, provides new insights into the conformational dynamics of complement activation and lays the groundwork for future structural and guided66 mutagenesis studies that will unravel the complex regulation of the system . Furthermore, the integrated consideration of available 3D structures, genomewide profiling data, and the application of novel biophysical approaches to monitor the dynamics and energetics of complement protein interactions are anticipated to open up new avenues of opportunity for rational design of more effective complement therapeutics.

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9. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants AI 30040, GM56698, GM-62134, DK-059422, CA112162, and EB003968, and NSF Grant 0426691 to JDL.

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16. J.G. Mandell, A.M. Falick and E.A. Komives, Identification of protein-protein interfaces by decreased amide proton solvent accessibility, Proc Natl Acad Sci USA 95(25), 14705–14710 (1998). 17. J.G. Mandell, A.M. Falick and E.A. Komives, Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry, Anal Chem 70(19), 3987–3995 (1998). 18. M.S. Winters, D.S. Spellman and J.D. Lambris, Solvent accessibility of native and hydrolyzed human complement protein 3 analyzed by hydrogen/deuterium exchange and mass spectrometry, J Immunol 174(6), 3469–3474 (2005). 19. Sahu1 A and J.D. Lambris, Complement inhibitors: a resurgent concept in antiinflammatory therapeutics, Immunopharmacology 49(1–2), 133–148 (2000). 20. C.L. Harris, D.A. Fraser and B.P. Morgan, Tailoring anti-complement therapeutics, Biochem Soc Trans 30(Pt 6), 1019–1026 (2002). 21. A. Sahu, B.K. Kay and J.D. Lambris, Inhibition of human complement by a C3binding peptide isolated from a phage-displayed random peptide library, J Immunol 157(2), 884–891 (1996). 22. A.M. Soulika, M.M. Khan, T. Hattori, F.W. Bowen, B.A. Richardson, C.E. Hack, A. Sahu, L.H. Edmunds, Jr. and J.D. Lambris, Inhibition of heparin/protamine complex-induced complement activation by Compstatin in baboons, Clin Immunol 96(3), 212–221 (2000). 23. B. Nilsson, R. Larsson, J. Hong, G. Elgue, K.N. Ekdahl, A. Sahu and J.D. Lambris, Compstatin inhibits complement and cellular activation in whole blood in two models of extracorporeal circulation, Blood 92(5), 1661–1667 (1998). 24. A.E. Fiane, T.E. Mollnes, V. Videm, T. Hovig, K. Hogasen, O.J. Mellbye, L. Spruce, W.T. Moore, A. Sahu and J.D. Lambris, Prolongation of ex vivo-perfused pig xenograft survival by the complement inhibitor compstatin, Transplant Proc 31(1-2), 934–935 (1999). 25. A.E. Fiane, T.E. Mollnes, V. Videm, T. Hovig, K. Hogasen, O.J. Mellbye, L. Spruce, W.T. Moore, A. Sahu and J.D. Lambris, Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused pig xenografts, Xenotransplantation 6(1), 52–65 (1999). 26. A.E. Fiane, V. Videm, J.D. Lambris, O.R. Geiran, J.L. Svennevig and T.E. Mollnes, Modulation of fluid-phase complement activation inhibits hyperacute rejection in a porcine-to-human xenograft model, Transplant Proc 32(5), 899–900 (2000). 27. T.E. Mollnes, O.L. Brekke, M. Fung, H. Fure, D. Christiansen, G. Bergseth, V. Videm, K.T. Lappegard, J. Kohl and J.D. Lambris, Essential role of the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by a novel lepirudinbased human whole blood model of inflammation, Blood 100(5), 1869–1877 (2002). 28. A. Klegeris, E.A. Singh and P.L. McGeer, Effects of C-reactive protein and pentosan polysulphate on human complement activation, Immunology 106(3), 381–388 (2002). 29. S.T. Furlong, A.S. Dutta, M.M. Coath, J.J. Gormley, S.J. Hubbs, D. Lloyd, R.C. Mauger, A.M. Strimpler, M.A. Sylvester, C.W. Scott and P.D. Edwards, C3 activation is inhibited by analogs of compstatin but not by serine protease inhibitors or peptidyl alpha-ketoheterocycles, Immunopharmacology 48(2), 199–212 (2000). 30. A. Sahu, D. Morikis and J.D. Lambris, Compstatin, a peptide inhibitor of complement, exhibits species-specific binding to complement component C3, Mol Immunol 39(10), 557–566 (2003).

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31. D. Morikis, N. Assa-Munt, A. Sahu and J.D. Lambris, Solution structure of compstatin, a potent complement inhibitor, Protein Sci 7, 619–627 (1998). 32. D. Morikis, A. Sahu, W.T. Moore and J.D. Lambris, Design, structure, function and application of compstatin in Bioactive peptides in drug discovery and design: medical aspects, 235–246 (1999). 33. A. Sahu, A.M. Soulika, D. Morikis, L. Spruce, W.T. Moore and J.D. Lambris, Binding kinetics, structure-activity relationship, and biotransformation of the complement inhibitor compstatin, J Immunol 165(5), 2491–2499 (2000). 34. S.T. Furlong, A.S. Dutta, M.M. Coath, J.J. Gormley, S.J. Hubbs, D. Lloyd, R.C. Mauger, A.M. Strimpler, M.A. Sylvester, C.W. Scott and P.D. Edwards, C3 activation is inhibited by analogs of compstatin but not by serine protease inhibitors or peptidyl alpha-ketoheterocycles, Immunopharmacology 48(2), 199–212 (2000). 35. D. Morikis, M. Roy, A. Sahu, A. Troganis, P.A. Jennings, G.C. Tsokos and J.D. Lambris, The structural basis of compstatin activity examined by structure–functionbased design of peptide analogs and NMR, J Biol Chem 277(17), 14942–14953 (2002). 36. A.M. Soulika, D. Morikis, M.R. Sarrias, M. Roy, L.A. Spruce, A. Sahu and J.D. Lambris, Studies of structure–activity relations of complement inhibitor compstatin, J Immunol 171(4), 1881–1890 (2003). 37. A. Sahu, B.K. Kay and J.D. Lambris, Inhibition of human complement by a C3binding peptide isolated from a phage displayed random peptide library, J Immunol 157, 884–891 (1996). 38. A. Sahu, A.M. Soulika, D. Morikis, L. Spruce, W.T. Moore and J.D. Lambris, Binding kinetics, structure-activity relationship, and biotransformation of the complement inhibitor compstatin, J Immunol 165(5), 2491–2499 (2000). 39. D. Morikis, M. Roy, A. Sahu, A. Troganis, P.A. Jennings, G.C. Tsokos and J.D. Lambris, The structural basis of compstatin activity examined by structure–functionbased design of peptide analogs and NMR, J Biol Chem 277(17), 14942–14953 (2002). 40. A.M. Soulika, D. Morikis, M.R. Sarrias, M. Roy, L.A. Spruce, A. Sahu and J.D. Lambris, Studies of structure–activity relations of complement inhibitor compstatin, J Immunol 171(4), 1881–1890 (2003). 41. J.L. Klepeis, C.A. Floudas, D. Morikis, C.G. Tsokos, E. Argyropoulos, L. Spruce and J.D. Lambris, Integrated computational and experimental approach for lead optimization and design of compstatin variants with improved activity, J Am Chem Soc 125(28), 8422–8423 (2003). 42. J.L. Klepeis, C.A. Floudas, D. Morikis and J.D. Lambris, Predicting peptide structures using NMR data and deterministic global optimization, J Comput Chem 20, 1354–1370 (1999). 43. Klepeis JL and C.A. Floudas, Ab initio tertiary structure prediction of proteins, J Global Optim 25(1), 113–140 (2003). 44. J.L. Klepeis, Schafroth H.D., Westerberg K.M. and C.A. Floudas, Deterministic global optimization and ab initio approaches for the structure prediction of polypeptides, dynamics of protein folding, and protein–protein interactions, Comput Methods Protein Folding Adv Chem Phys 120, 265–457 (2002). 45. B. Mallik, M. Katragadda, L.A. Spruce, C. Carafides, C.G. Tsokos, D. Morikis and J.D. Lambris, Design and NMR characterization of active analogues of compstatin containing non-natural aminoacids, J Med Chem 48(1), 274–286 (2005).

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46. J. Mullick, A. Kadam and A. Sahu, Herpes and pox viral complement control proteins: “the mask of self”, Trends Immunol 24(9), 500–507 (2003). 47. A. Sahu, S.N. Isaacs, A.M. Soulika and J.D. Lambris, Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway, J Immunol 160(11), 5596–5604 (1998). 48. A.M. Rosengard, Y. Liu, Z. Nie and R. Jimenez, Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement, Proc Natl Acad Sci USA 99(13), 8808–8813 (2002). 49. G. Sfyroera, M. Katragadda, D. Morikis, S.N. Isaacs and J.D. Lambris, Electrostatic modeling predicts the activities of orthopoxvirus complement control proteins, J Immunol 174(4), 2143–2151 (2005). 50. T. Ideker, T. Galitski and L. Hood, A new approach to decoding life: systems biology, Annu Rev Genomics Hum Genet 2, 343–372 (2001). 51. R. Mack and M. Hehenberger, Text-based knowledge discovery: search and mining of life-sciences documents, Drug Discov Today 7(11 Suppl), S89–S98 (2002). 52. A. Persidis, S. Deftereos and A. Persidis, Systems literature analysis, Pharmacogenomics 5(7), 943–947 (2004). 53. D. Mastellos, C. Andronis, A. Persidis and J.D. Lambris, Novel biological networks modulated by complement, Clin Immunol 115(3), 225–235 (2005). 54. Y. Kimura, M. Madhavan, M.K. Call, W. Santiago, P.A. Tsonis, J.D. Lambris and K. Rio-Tsonis, Expression of complement 3 and complement 5 in newt limb and lens regeneration, J Immunol 170(5), 2331–2339 (2003). 55. C.W. Strey, M. Markiewski, D. Mastellos, R. Tudoran, L.A. Spruce, L.E. Greenbaum and J.D. Lambris, The proinflammatory mediators C3a and C5a are essential for liver regeneration, J Exp Med 198(6), 913–923 (2003). 56. R. Reca, D. Mastellos, M. Majka, L. Marquez, J. Ratajczak, S. Franchini, A. Glodek, M. Honczarenko, L.A. Spruce, A. Janowska-Wieczorek, J.D. Lambris and M.Z. Ratajczak, Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1, Blood 101(10), 3784–3793 (2003). 57. R. Taub, Liver regeneration: from myth to mechanism, Nat Rev Mol Cell Biol 5(10), 836–847 (2004). 58. D. Mastellos, J.C. Papadimitriou, S. Franchini, P.A. Tsonis and J.D. Lambris, A novel role of complement: mice deficient in the fifth component of complement (C5) exhibit impaired liver regeneration, J Immunol 166(4), 2479–2486 (2001). 59. M.M. Markiewski, D. Mastellos, R. Tudoran, R.A. Deangelis, C.W. Strey, S. Franchini, R.A. Wetsel, A. Erdei and J.D. Lambris, C3a and C3b activation products of the third component of complement (C3) are critical for normal liver recovery after toxic injury, J Immunol 173(2), 747–754 (2004). 60. X. Sun, C.D. Funk, C. Deng, A. Sahu, J.D. Lambris and W.C. Song, Role of decayaccelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting, Proc Natl Acad Sci USA 96(2), 628–633 (1999). 61. J. Ratajczak, R. Reca, M. Kucia, M. Majka, D.J. Allendorf, J.T. Baran, A. Janowska-Wieczorek, R.A. Wetsel, G.D. Ross and M.Z. Ratajczak, Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow, Blood 103(6), 2071–2078 (2004).

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62. C.T. Esmon, Interactions between the innate immune and blood coagulation systems, Trends Immunol 25(10), 536–542 (2004). 63. I.J. Laudes, J.C. Chu, S. Sikranth, M. Huber-Lang, R.F. Guo, N. Riedemann, J.V. Sarma, A.H. Schmaier and P.A. Ward, Anti-c5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis, Am J Pathol 160(5), 1867–1875 (2002). 64. R.F. Guo and P.A. Ward, Role of C5a in inflammatory responses, Annu Rev Immunol 23821–852 (2005). 65. K. Ikeda, K. Nagasawa, T. Horiuchi, T. Tsuru, H. Nishizaka and Y. Niho, C5a induces tissue factor activity on endothelial cells, Thromb Haemost 77(2), 394–398 (1997). 66. B.J.C. Janssen, E.G. Huizinga, H.C.A. Raaijmakers, A. Roos, M.R. Daha, K. Nilsson-Ekdahl, B. Nilsson and P. Gros, Structures of complement component C3 provide insights into the function and evolution of immunity, Nature 437, 505–511 (2005).

2 LIVER REGENERATION: A LINK TO INFLAMMATION THROUGH COMPLEMENT Robert A. DeAngelis, Maciej M. Markiewski, and John D. Lambris

1. INTRODUCTION The liver is one of the largest organs in the body, involved in various tasks such as the processing of dietary amino acids, carbohydrates, lipids, and vitamins, phagocytosis of particulate material in the portal circulation, synthesis of serum proteins, biotransformation of circulating metabolites, and detoxification and 1 excretion of endogenous waste products and pollutant xenobiotics into the bile . It is strategically located between the gastrointestinal tract and the rest of the body, with a unique dual blood supply including the portal venous system. This makes the liver an intermediate filter for most of the venous drainage of the ab2 dominal viscera, and a vital organ for maintaining metabolic homeostasis . These anatomical properties support the physiological functions of the liver but also make it vulnerable to a wide variety of metabolic, toxic, microbial, circulatory, and neoplastic insults. The large functional reserve of the liver usually prevents the appearance of clinical symptoms of liver failure even if a significant portion of the parenchyma is destroyed. However, the progression of diffuse liver disease or the strategic 3 disruption of bile flow may lead to life-threatening consequences , demanding regenerative capabilities from the liver to assure the restoration of structural and 4 functional integrity even after severe damage . Various insults, such as surgery or viral or toxic injury, can signal the mechanisms responsible for liver regen4 eration . Barring extensive severe injury or other circumstances preventing the

Robert A. DeAngelis*, Maciej M. Markiewski*, and John D. Lambris, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia, PA 19104 *These authors contributed equally to this work.

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normal division of hepatocytes, signaling for regeneration results in the reentry of mature quiescent liver cells into the cell cycle. The process of regeneration continues until the liver regains its original size, with complete recovery of tis4 sue architecture . Partial hepatectomy (PHx), in which two-thirds of the liver 5 mass is surgically removed , is one of the most common models used to study the regenerative response. PHx is considered a non-injurious procedure for the remaining liver tissue, which undergoes a compensatory hyperplasia. Conversely, another model involving toxic injury induced by the injection of carbon tetrachloride (CCl4) results in severe damage to liver tissue. Thus, after CCl4 injection the liver must both restore lost tissue mass and repair and remove injured parenchyma. Despite obvious differences between the surgical and injury models, the molecular mechanisms that govern the regenerative response seem to be quite similar. Furthermore, the factors involved in this phenomenon largely overlap with those responsible for the induction of innate immunity, suggesting that liver 6 regeneration may utilize the mechanisms of an inflammatory response . In addition, complement, an important player in innate immune reactions, has been implicated as a crucial factor for liver regeneration.

2. LIVER REGENERATION AND INFLAMMATORY MEDIATORS Liver regeneration requires the activity of multiple signaling pathways, assuring the synchronized proliferation of liver cells, protection from apoptotic signals, remodeling of extracellular matrix (ECM), and restoration of lobular architec7 ture . The initiation of regeneration through PHx is associated with minimal injury; therefore, an obvious inflammatory reaction that includes a significant inflammatory infiltrate is not seen in the liver parenchyma under these circumstances. However, elevated levels of acute-phase proteins in the blood, activation of liver macrophages, and release of cytokines that are involved in regulation of inflammatory responses to various pathogens suggest that PHx 4 does initiate an inflammatory reaction . Unlike PHx, injection of CCl4 results in an inflammatory infiltrate in the liver in response to necrosis. In this model, regeneration is associated with significant tissue injury and an inflammatory response not seen after PHx. Though cell death and the inflammatory reaction may interfere with attempts to clearly elucidate the molecular background of regeneration in the CCl4 model, it can be seen as a better reflection of liver diseases that trigger the regenerative response, such as viral hepatitis and toxic- or drug-induced injury, and of the regeneration of liver parenchyma that occurs after surgical resection carried out in response to various pathologies, including 3 primary or metastatic tumors .

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2.1. Cytokines and Transcription Factors Lipopolysaccharde (LPS), a strong activator of innate immunity, may be present 8,9 in increased concentration in the portal blood flow after PHx . This factor is known to be necessary for proper liver regeneration, as both germ-free athymic 10 and LPS-resistant mice show impaired regeneration after PHx . LPS is thought to be one of the earliest signals that starts the regenerative process, likely arriving from the gut to engage receptors on Kupffer cells, the resident macrophages 4 of the liver . Activation of Kupffer cells results in production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. TNF-α activates the nuclear factor-kappa B (NF-κB) transcription factor 11 in macrophages and hepatocytes. This response leads to secretion of IL-6, mainly from Kupffer cells, which in turn activates the transcription factor signal 12-14 transducer and activator of transcription (STAT) 3 in hepatocytes . Studies 14-16 13,17 using mice deficient in TNF receptor 1 (TNFR1) or IL-6 have shown that these cytokines are necessary for liver regeneration. The ability of IL-6 administration to correct the defect in hepatocyte DNA synthesis seen in TNFR1-/deficient mice (TNFR1 ) after PHx suggests that the role of TNF-α in liver re14 generation is mediated by IL-6 . NF-κB and STAT3 participate in the induction of immediate-early genes 13,18 important for liver cell growth and hepatoprotection . NF-κB regulates the cell 19,20 cycle regulator cyclin D1 . Stimulation of the IL-6 receptor (IL-6R/gp130) by 21 IL-6 promotes cell growth not only through STAT3 activation , but also through 22 activation of the mitogen-activated protein kinase (MAPK) signaling cascade . There is some debate that IL-6 may be more important as a hepatoprotective 17,23,24 factor rather than as a mitogen . IL-6 activates the pro-survival proteins phosphoinositol 3 kinase (PI3K) and Akt in addition to STAT3, which is also 25,26 involved in hepatoprotection . NF-κB has also been shown to be an antiapoptotic factor during liver regeneration. When NF-κB activation is inhibited, such as through the action of a superrepressor transgene of the NF-κB inhibitor, IκBα, or by treatment with gliotoxin, liver regeneration after PHx is impaired 27,28 and apoptosis of hepatocytes occurs instead of proliferation . NF-κB regulates 29-35 genes for anti-apoptotic proteins and prevents TNF-α-induced hepatocyte 36-41 death . The cytokines and transcription factors mentioned here, along with some of their targets, are also involved in inflammation. Most notable of these is TNF-α, which is released by mast cells and macrophages in the initial phase of the in42 flammatory response . In endothelium, TNF-α (along with IL-1, which is in7 volved in the acute-phase response in the liver ) induces a spectrum of changes, mostly regulated at the transcriptional level, referred to as endothelial activa43 tion . In particular, TNF-α induces the synthesis of endothelial adhesion molecules, other cytokines, chemokines, growth factors, eicosanoids, nitritic oxide, 43 and enzymes associated with matrix remodeling. Additionally, it increases the 44 surface thrombogenicty of the endothelium . TNF also causes aggregation and

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priming of neutrophils, leading to augmented responses of these cells to other mediators and release of proteolytic enzymes from mesenchymal cells, thus con45,46 tributing to tissue damage . LPS, thought to be an initial stimulator of liver regeneration, also activates 47 the complement system through the alternative pathway . In fact, there are many potential connections between complement and liver regeneration based on what is known about interactions of the factors discussed above with the complement pathways. The anaphylatoxin C5a, the effector molecule resulting from cleavage of complement protein C5, acts on macrophages to induce cyto48-54 kine release, including TNF-α . The role of the C3a anaphylatoxin (originating from cleavage of C3) in modulation of TNF-α and IL-1β production and release in macrophages is not as well characterized as that for C5a, but some published data indicate that C3a and C3a desArg may stimulate production of 55 these cytokines . C3a signaling appears to be costimulatory to LPS signaling and, depending on the pathophysiological background and target cell population, 55 may have stimulatory as well as inhibitory characteristics . C3a and C5a may therefore contribute to the induction of transcription factors indirectly through their effects on cytokines. Indeed, both C3a and C5a are known to enhance the release of IL-6 in response to LPS in peripheral blood mononuclear cells and 56-58 Kupffer cells in the liver . C5a is also important for NF-κB- and MAPK59 dependent release of IL-6 by neutrophils during sepsis . Additionally, C5a activates the lipooxygenase pathway of arachidonic acid (AA) metabolism in neutrophils and monocytes, leading to acceleration of eicosanoid production by 60 these cells . Previous reports have emphasized the importance of eicosanoids, specifically prostaglandins and their potential effect on CREB transcription fac61,62 tor signaling, in liver regeneration after PHx . 2.2. Growth Factors, Metalloproteases, Adhesion Molecules, and Acute Phase Proteins Growth factors, including hepatocyte growth factor (HGF), also contribute to hepatocyte proliferation during regeneration. Active HGF is produced by cleavage of pro-HGF by urokinase plasminogen activator (uPA) and plasminogen 63,64 proteases, a part of the fibrinolysis system . The involvement of this system in inflammation can be illustrated by the role of plasminogen activator (released from enodothelial cells and leukocytes activated during the inflammatory response) in cleaving plasminogen to generate the matrix protease plasmin, a multifunctional enzyme, which in turn can cleave complement C3 to produce C3 fragments. Also, plasminogen can degrade fibrin to form fibrin split products, 65 which may have permeability-inducing properties , and plasmin can activate Hageman factor, which can trigger multiple cascades to amplify the inflamma65 tory response . When uPA is blocked, production of HGF is delayed, as is liver regenera4,66 tion. HGF can activate pathways for PI3K, ERK (an MAPK protein), and Akt,

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which are involved in growth and hepatoprotection, as mentioned above . Conditional knockout mice for the HGF receptor Met have increased sensitivity to hepatocyte apoptosis and impaired recovery from damage when the liver is injured, further indicating that the HGF–Met pathway is involved in hepatocyte 68 survival . Interestingly, delayed recovery in these mice is associated with a persistent inflammatory reaction. C3 may promote the release of HGF from adhe69 sive granulocytes and monocytes , while in alveolar macrophages uPA and C5a act synergistically to upregulate TNF-α production, suggesting another potential 70 link between complement and cytokine production during regeneration . Proteases, especially metalloproteases (MMP), are intricately involved in liver regeneration by regulating activation of certain signaling factors, including HGF, and contributing to angiogenesis for restoration of liver architecture. Aside from being involved in HGF processing, plasminogen may also contribute to angiogenesis in the liver, as plasminogen-deficient mice have an impaired 71 increase in microvessel density during regeneration . Lack of tissue inhibitor of metalloprotease (TIMP)-1 leads to increased MMP activity in the regenerating liver and may result in greater release of HGF from the ECM. Indeed, an in-/72 crease in activated HGF is observed in TIMP-1-deficient (TIMP-1 ) mice . TNF-α can be shed from the cell surface by TNF-α-converting enzyme (TACE), 73 which is inhibited by TIMP-3 . TIMP-3 deficiency results in overproduction of 74 hepatic TNF-α, leading to hepatocyte apoptosis and liver failure . VEGF is another important angiogenic factor for liver regeneration, involved in the reconstruction of liver sinusoids through proliferation of sinusoidal endothelial cells, 75 which promotes hepatocyte proliferation . It can be released from the ECM to initiate signaling, perhaps by MMP-9, which is induced during liver regenera72,76,77 tion . Intriguingly, C5a can also be involved in angiogenesis. C5a-stimulated HUVECs reveal increased expression of genes involved in endothelial adhesion, 78 migration, and angiogenesis . In some cases, MMPs may be detrimental to the -/liver. After injection of CCl4, MMP-2 expression is increased in IL-6 livers, 79 which show greater damage and liver failure compared to wild-type livers . -/Additionally, injury and apoptosis in IL-6 livers is reduced when MMP-2 is inhibited. Providing yet another link between the complement system and liver regeneration, complement proteins have been shown to interact with MMPs as 80-84 targets and regulators of these enzymes . Adhesion molecule expression is necessary for interactions between endothelial cells and leukocytes during extravasation and migration, one of the most important events that occurs soon after induction of the innate immune re85 sponse . This process results in leukocytes leaving the bloodstream and entering the interstitial space to travel to the site of inflammation. Anaphylatoxins, mainly C5a, participate in endothelial activation during extravasation and migraton. C5a-stimulated HUVECs upregulate genes for E-selectin, ICAM-1, 78 VCAM-1, and IL-6 . Anaphylatoxins also influence the expression of adhesion molecules on leukocytes. It has been postulated that C5a is involved in eosinophil adhesion to bronchial epithelial cells during allergic inflammation in the

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airways . Another study showed that C5a is an activator of integrin-dependent 87 adhesion and transmigration in eosinophils and neutrophils . These examples indicate that anaphylatoxins can play a role in the process of extravasation, directly influencing the expression of adhesion molecules on endothelial cells as well as leukocytes. However, anaphylatoxins are also indirectly involved in this process through regulation of TNF-α and IL-1 expression. These two cytokines appear to be major regulators of adhesion molecule expression on both leukocytes and endothelium. Adhesion molecules are also necessary for liver regeneration. Mice deficient in ICAM-1 show impaired regeneration, with a decrease 88 in the recruitment of leukocytes and levels of TNF-α and IL-6 . Complement, which regulates ICAM-1 expression during inflammation, may potentially regulate ICAM-1 expression during regeneration, as well, perhaps to increase recruitment of lymphocytes to the liver through adhesion to sinusoidal endothelial 89 cells . Activation of transcription factors after PHx leads to upregulation of acute phase proteins, such as serum amyloid protein, hemopexin, and complement 13,90,91 (C)-reactive protein . Upregulation of serum amyloid A, serum amyloid A2, and haptoglobin precursor after PHx has also been noted in our recent study on 92 the liver proteome . Increased synthesis of acute-phase proteins is a hallmark of 85 acute inflammatory reactions mediated by the innate immune system . Complement is also involved in the acute-phase response. C5a contributes to produc93 tion of several acute-phase proteins in liver cell lines . C5a has also been shown to act synergistically with LPS to enhance production of the acute-phase protein 94 α2-macroglobulin in hepatocytes . It is possible that C5a acts during liver regeneration, either directly or indirectly through other signaling factors, to upregulate production of acute-phase proteins required for liver mass restoration. 2.3. Natural Killer T (NKT) Cells NKT cells display characteristics common to both classical T and natural killer 95 (NK) cells . Most NKT cells are reactive against the glycolipid-binding non96 polymorphic major histocompatibility complex class 1-like glycoprotein CD1d . Interestingly, besides being expressed on antigen-presenting cells of the immune 97,98 system, CD1d is constitutively expressed on parenchymal liver cells . NKT cells can contribute to both the Th1 and Th2 adaptive immune responses through their production of large amounts of, respectively, interferon-gamma (IFN-γ) 99,100 and IL-4 upon activation . IL-4 production by a liver population of NKT cells can also lead to production of antibody that activates complement to promote 101 recruitment of T cells during contact sensitivity . Recently, NKT cells have been suggested to play a role in liver regeneration. The number of NKT cells in the liver in mice increases very quickly after 102 PHx, dependent upon signaling through adrenergic receptors . Blocking of adrenergic receptors inhibits accumulation of NKT cells in the regenerating 95 liver . It is possible that expansion of the NKT cell population can impair re-

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generation of the liver. In one study, an increase in NKT cell numbers due to IL12 stimulation was shown to exacerbate injury during the early phases of liver 103 regeneration . Increased production of IFN-γ by the expanded population of NKT cells could partly explain this detrimental effect, as IFN-γ is known to play 104 a role in hepatitis-induced acute liver failure . Increased NKT cells could potentially lead to increased C3 production by liver epithelial cells as well. A mitogen for T-lymphocytes, which also contributed to NKT cell expansion in one 105 106 study , stimulated C3 release from rat epithelial cells in the liver . In this case, increased C3 may further contribute to liver damage caused by NKT cells.

3. THE ROLE OF COMPLEMENT IN LIVER REGENERATION The complement system plays a crucial role in the early innate immune response, and we have thus far postulated several potential links between this system and regeneration of the liver based on factors common to both. However, recent studies have provided evidence for a definitive role for complement in the regenerative response. C3 appears to be activated early after the initiation of liver regeneration, as cleavage products are observed in the serum 2–3 hours 107 after CCl4 injection and PHx (personal observation). Exemplifying the reciprocal nature of complement proteins and cytokines, C3 and C5 have been shown to be involved in cytokine production during early liver regeneration. Following 108 PHx, increases in TNF-α and IL-6 mRNA are observed . In mice lacking C3 -/(C3 ) and in mice treated with an inhibitory antagonist for the C5a receptor (C5aR), there is a reduction in TNF-α and IL-6 mRNA levels. Further, both of 108 these cohorts also show impaired activation of NF-κB and STAT3 . Thus, both C3 and C5 (most likely through the activities of C5a) are necessary for the initial priming events of liver regeneration. Due to this defect in priming, hepatocytes -/-/in both C3 and C5 mice do not enter the cell cycle and overall proliferation is 107-109 greatly reduced during liver regeneration . This lack of cell proliferation results in an inability of complement-deficient mice to completely restore their liver mass after insult. Complement is also involved in protection of the liver from damage during regeneration, perhaps again through its role in liver cell priming. After PHx, -/-/both C3 and C5 livers display severe damage and, in some animals, liver fail108 -/ure and mortality . A similar defect in liver regeneration is observed in C5 -/mice after CCl4 injection. Though CCl4 normally induces damage in livers, C5 mice have a much more diffuse and extensive pattern of liver necrosis and apoptosis after injection of CCl4 compared to wild-type mice, along with an increase 109, 110 in lipid content, known to be detrimental to liver regeneration and function . Thus, both C3 and C5 are necessary to prevent injury during the restoration of liver mass. C3 and C5 are not only separately required for cell division and hepatoprotection but also function in a cooperative manner. When both complement pro-

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teins are absent, in double-deficient C3/C5 animals, an even greater reduction in hepatocyte proliferation is observed after PHx than that seen in singly defi-/cient livers. In addition, the injury observed in C3/C5 livers is more severe than -/-/108 damage occurring in C3 or C5 livers. This additive effect suggests that C3 and C5 each have specific functions during regeneration of the liver, and do not just control redundant mechanisms. C3 and C5 are cleaved into their effector fragments following activation of the complement system. Further analysis of liver regeneration after PHx and CCl4 injection in complement-deficient mice has demonstrated the importance of these effector molecules, especially the anaphylatoxins C3a and C5a. Defects in cytokine signaling and transcription factor activation have already been noted when mice were treated with a C5aR antagonist. These mice also display im108 paired liver regeneration and increased liver injury and mortality after PHx . -/Reconstitution of C5 mice with murine C5 restores cell division and greatly 109 diminishes injury to near-wild-type levels following injection of CCl4 . C5a has been shown to be the main effector for this improvement in recovery, as it can be accomplished through reconstitution with only the C5a component of C5, while blockage of the C5aR with antagonist impairs regeneration in a manner -/similar to that seen in C5 mice. C3a has also been shown to be involved in -/proper liver regeneration. Reconstitution of C3 mice with murine C3a restores hepatocyte proliferation to wild-type levels and reverses post-PHx liver damage -/107,108 seen in C3 mice . The importance of both anaphylatoxins has been demon-/strated by single or double reconstitution of C3/C5 mice following PHx. When these mice are treated with only C3a or C5a, defects in regeneration are only partially corrected. However, double reconstitution with both anaphylatoxins restores regenerative parameters to levels similar to those seen in wild-type 108 mice, further confirming the cooperative effects of C3 and C5 . Finally, cell -/division in C3aR mice after CCl4 injection is impaired, again showing the im107 portance of C3a for regeneration . However, the defect is not as severe as in -/C3 mice, suggesting that C3 may have other functions aside from those mediated by C3a. In fact, C3 does appear to have an additional function. It is not only necessary for promoting liver cell growth and preventing injury through the actions of C3a, but also for clearing damaged tissue through deposition of C3b/iC3b. As mentioned, C3 cleavage products are present in serum soon after the initiation of liver regeneration. Twenty four to 36 hours after injection of CCl4, a second, even greater wave of C3 cleavage occurs. The timing of this second wave corre107 lates with local deposition of C3 within damaged liver parenchyma . Though -/wild-type and C3 mice show similar levels of initial liver injury early after -/CCl4 injection, there is delayed clearance of damaged parenchyma in C3 mice. Phagocytosis of necrotic and apoptotic tissue is facilitated by engagement of the 111, 112 CR3 receptor on macrophages with C3b/iC3b deposited in areas of injury . -/Thus, the delayed clearance of damaged tissue in C3 mice is caused by the absence of C3 deposition in these areas.

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Figure 1. Complement involvement in liver regeneration. Initiation of liver regeneration signals complement activation. C5a and possibly C3a bind to their receptors (C5aR, C3aR) on Kupffer cells, and, together with LPS signaling through Toll-like receptor (TLR) 4, stimulate release of TNF-α and IL-6. TNF-α acts in an autocrine manner on Kupffer cells and, with IL-6 and C5a, activates the NFκB and STAT3 transcription factors in hepatocytes. HGF binds to the Met receptor on hepatocytes after cleavage of pro-HGF by urokinase plasminogen activator (uPA) and plasminogen proteases. Immediate-early gene products and acute-phase proteins (APP) induced by NF-κB and STAT3, along with HGF, stimulate cell growth and hepatoprotective pathways. Complement also contributes to the clearance of damaged tissue, through C3b/iC3b deposition and its interaction with the CR3 receptor on macrophages. When complement components (C’) are absent or inhibited, regeneration can be impaired at any of these steps, resulting in liver failure and occasional mortality.

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A schematic representation of liver regeneration and the steps in which complement is involved is shown in Figure 1. In summary, complement seems to have multiple functions during liver regeneration: C3a and C5a are necessary for proper priming of liver cells and progression through the cell cycle, promoting growth and inhibiting cell death, while C3b/iC3b is needed to promote clearance of injured tissue and prevent development of a more severe inflammatory response that may lead to further injury. Without C3 and C5, the liver is unable to undergo proper tissue repair and cell proliferation. In many cases, this leads to liver injury, failure, and increased mortality. These studies demonstrate the important functions of complement during liver regeneration, and strengthen the connection between regeneration and the inflammatory response.

4. CONCLUSION We have emphasized here the similarities that exist between the inflammatory and regenerative responses in terms of mediators involved, providing evidence that liver regeneration includes an inflammatory reaction. Particular emphasis has been placed on the role of complement, an early and fundamental player in innate immunity, in regeneration of the liver. During the inflammatory response there are multiple interactions existing between several complement proteins and the network of pleiotropic mediators, the cytokines. Through cytokine functions, complement is involved in precise and balanced regulation of innate and adaptive immune responses and the control of cellular growth and apoptosis. Additionally, through cytokine signaling and other, more direct processes, complement is now known to be vital for proper liver regeneration. Thus, the interactions between various complement proteins and cytokines are essential for both immune responses and tissue regeneration, and provide a new link in the expanding chain connecting these two biological phenomena.

5. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants AI 30040, GM62134, DK-059422, and CA112162 to JDL.

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3 THE ROLE OF THIRD COMPLEMENT COMPONENT (C3) IN HOMING OF HEMATOPOIETIC STEM/PROGENITOR CELLS INTO BONE MARROW Ryan Reca, Marcin Wysoczynski, Jun Yan, John D. Lambris, and Mariusz Z. Ratajczak

1. INTRODUCTION Transplantation of hematopoietic stem/progenitor cells (HSPC) is routinely employed in the clinic to treat patients with hematological malignancies, inborn or acquired defects of hematopoiesis and immunodeficiencies. Prior to transplantation in order to destroy old pathological hematopoiesis, the patient receives a myeloablative dose of radiochemotherapy, and subsequently the patient’s own autologous- or harvested from histocompatible donor allogeneic-HSPC are infused intravenously in an attempt to establish new hematopoiesis. It is well established that the α-chemokine stromal derived factor-1 (SDF-1) and the Gprotein-coupled seven-span transmembrane receptor, CXCR4, axis plays a pivotal role in directing the homing of HSPC from the blood to the bone marrow 1,2 (BM) . Chemokines, small proinflammatory chemoattractant cytokines that bind to specific G-protein-coupled seven-span transmembrane receptors present on the plasma membranes of target cells, are the major regulators of cell trafficking. More than 50 different chemokines and 20 different chemokine receptors have 3 been cloned . Chemokines usually bind to multiple receptors, and the same receptor may bind to more than one chemokine. However, there is one exception to this rule: the α-chemokine SDF-1, which binds exclusively to CXCR4, and Ryan Reca, Marcin Wysoczynski, Jun Yan, Mariusz Z. Ratajczak, Stem Cell Biology Program, University of Louisville, Baxter II, STE 122, 580 S. Preston Street, Louisville, KY 40202, USA. John D. Lambris, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 401 Stellar-Chance Labs, 422 Curie Blvd., Philadelphia, PA 19104, USA. Web: www.complement.us.

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has CXCR4 as its only receptor. This fact alone suggests that the SDF-1– CXCR4 axis plays a uniquely important biological role. In fact from all the 1 chemokines tested so far, SDF-1 is the only chemoattractant for HSPC . 4,5 While SDF-1 is secreted by BM stroma ; CXCR4 is present on the surface 6 of HSPC . SDF-1 secreted by stromal cells in the developing BM is critical for 7-12 its colonization by fetal liver-derived HSPC during embryogenesis . In support of this, murine embryos with SDF-1 or CXCR4 knockout have a profound de9,13 crease in HSPC in their BM cavities . Furthermore, during adult life the SDF1-CXCR4 axis plays a pivotal role in retention of HSPC in the BM microenvi1-2,7-9,11-14 . Thus it is not surprising that proper functioning of the SDF-1– ronment CXCR4 axis is crucial in directing homing/engraftment of HSPC into BM after 1,15 transplantation . On the other hand, perturbation of the SDF-1–CXCR4 axis, for example, by mobilizing factors (e.g., G-CSF) is essential for egress of hema16-18 topoietic stem/progenitor cells from the BM into peripheral blood (PB) . Data from our laboratories demonstrate that complement (C) emerges as a new regulator of SDF-1-mediated homing of HSPC into BM as well as a regula19-21 tor of HSPC egress/mobilization from BM into peripheral blood . In this review we will present our data that the cleavage fragments of the third component (C3) of the complement system, C3a and C3adesArg, play an important role in modulating responsiveness of HSPC to an SDF-1 gradient. Thus a new concept emerges that trafficking of HSPC is tightly connected with activation of C and innate immunity. We will discuss these data in our review.

2. THE FUNCTION OF CXCR4 RECEPTOR DEPENDS ON LIPID RAFT FORMATION The function of CXCR4 on HSPC is regulated at multiple levels (Figure 1). First, expression of CXCR4 is regulated at the transcriptional level by several 22-30 factors (e.g., hypoxia) . Second, CXCR4 as well as its ligand SDF-1, are subject to proteolytic degradation by some proteases (e.g., metalloproteinases (MMPs), dipetidylpeptidase CD26 , and carboxypeptidase) that are expressed in 31,32 the hematopoietic microenvironment and serum . Third, CXCR4 after interaction with SDF-1 is internalized from the surface and subsequently may be recir15,33,34 . Fourth, culated from the endosomal compartment again to the cell surface CXCR4 is the subject of negative regulation by regulators of G-protein signaling 35 (RGS) proteins . Finally, it has been recently demonstrated that CXCR4 re36-39 quires association with membrane lipid rafts for optimal signaling (Figure 1) . Lipid rafts are membrane domains rich in sphingolipids and cholesterol, which form a lateral assembly in a saturated glycerophospholipid environment. The raft domains are known to serve as moving platforms on the cell surface, and are more ordered and resistant to nonionic detergents than other areas of a 40 membrane . These domains also act as good sites for crosstalk between various 39,41 cellular proteins . Lipid rafts have been shown to be important for T-cell

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Figure 1. Different levels of regulation of CXCR4 function on hematopoietic cells. First, expression of CXCR4 is regulated at the transcriptional level by several factors (e.g., hypoxia). Second, CXCR4 as well as its ligand SDF-1 are subject to proteolytic degradation by several proteases that are expressed in the hematopoietic microenvironment and serum. Third, CXCR4 after interaction with SDF-1 is internalized from the surface and is recirculated from the endosomal compartment at different rates. Fourth, CXCR4 is the subject of negative regulation by RGS proteins. Finally, functionality of the CXCR4 receptor depends of its incorporation into membrane lipid rafts, and several signals from other membrane receptors or integrins may increase the incorporation of CXCR4 into membrane lipid rafts, increasing its signaling.

39,42

polarization and chemotaxis . It has been recently reported that small GTPases such as Rac-1 and Rac-2, which are crucial for engraftment of hematopoietic 43,44 cells after transplantation, are present in lipid rafts of migrating HSPC . Thus, incorporation of CXCR4 into membrane lipid rafts results in better + responsiveness of CXCR4 cells to an SDF-1 chemotactic gradient. We noticed that several small inflammation-related molecules, such as fibrinogen and fibronectin cleavage fragments and hyaluronic acid, increase incorporation of CXCR4 into membrane lipid rafts and thus modulate the responsiveness of + 36 CXCR4 HSPC to an SDF-1 gradient .

3. COMPLEMENT IS ACTIVATED IN BM DURING MEYLOABLATIVE CONDTIONING FOR HEMATOPOIETIC TRANSPLANATION The protein components of complement (C) are activated through proteolysis in a cascade-like fashion (by pathways known as classical, alternative, or lectin),

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leading to generation of activated/cleaved proteins that bind to the C-activating surface and small liquid-phase activation peptides with potent proinflammatory properties that have been termed anaphylatoxins. Furthermore, all pathways of C activation merge at the level of C3, which is present at relatively high levels in serum (1 mg/ml). Cleavage of C3 is essential for activation of downstream C 45,46 components . Generally two groups of C3 cleavage fragments are distinguished — fluid phase (C3a, C3ades-Arg) and cell- or extracellular matrix-bound (C3b, iC3b, C3dg, C3d) fragments (Figure 2). C3a and C3b are the first cleavage products of C3, and each has a short half-life in plasma. C3a is processed by serum carboxypeptidase N to C3ades-Arg (long half-life cleavage product), and C3b is cleaved into iC3b (long half-life cleavage product) by factor I. The C3dg and C3d fragments (very long half-life) are generated from substrate-bound iC3b by proteolysis with various proteolytic enzymes including factor I, plasmin, and leukocyte elas45-47 tase .

Figure 2. Activation/cleavage of C3 and generation of bioactive peptides. Cleavage/activation of C3 secreted by BM stromal cells is initiated by a C3-convertase to generate fluid-phase C3a and stromal cell-bound C3b. Both C3a and C3b have a short half-life. Fluid C3a is rapidly degraded to C3ades-Arg and bound C3b is proteolyzed to iC3b. While C3a activates the C3aR and and another non-identified receptor (Receptor X?), C3ades-Arg binds to this yet-unidentified Receptor X only. iC3b tethers HSPC by interacting with CR3 (Mac-1).

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We noticed that during myeloablative conditioning for transplantation of HSPC (e.g., after lethal γ-irradiation or conditioning by cyclophosphamide) C3 21 is cleaved/activated in PB and BM . In support of this notion we have found that, during myeloablative conditioning for transplantation while C3 is cleaved and C3ades-Arg becomes detectable by ELISA, iC3b is deposited on BM-derived fibroblasts and endothelial cells as determined by FACS analysis of these cells 19 stained with anti-iC3b antibodies conjugated with Oregon green . Since BM tissue responds to myeloablative conditioning by activating C, we hypothesized that C3 activated in BM may play an important role in promoting regeneration 19 of BM after myeloablative treatment . C3 cleavage fragments bind to several specific C3 receptors (Figure 2). While C3a binds to the G-protein coupled, seven transmembrane-spanning C3areceptor (C3aR), the receptor for C3ades-Arg has not been identified yet (Receptor X). The C3aR is predominantly expressed on the surface of human mast cells, eosinophils, monocytes, and activated T-lymphocytes. Its major functions include chemotaxis of eosinophils and recruitment and degranulation of mast 48 cells . Recently, we showed that a functional C3aR is also expressed by normal human HSPC and lineage-expanded hematopoietic precursors from myeloid, 21 erythroid, and megakaryocytic lineages . The receptor for extracellular matrix-bound C3 cleavage fragment iC3b, CR3, is the αΜβ2-integrin also known as CD11b/CD18 or Mac-1. CR3 is expressed on granulocytes, monocytes/macrophages, and NK cells, and more importantly has been shown to be expressed on a significant proportion of HSPC. Thus, HSPC and their differentiated progeny express functional receptors for 49-51 both fluid-phase and surface-bound C3 cleavage fragments .

4. THE ROLE OF COMPLEMENT IN REGULATING THE BIOLOGY OF HSPC 21

49

Since HSPC expresses C3a and iC3b binding receptors, C3aR, and CR3, respectively, we became interested in whether C3 cleavage fragments play a role in regulating the biology of these cells. First, we tested if addition of C3 cleavage fragments to in-vitro growing hematopoietic clonogenic progenitor cells would increase their proliferation. However, neither C3a nor C3adesArg affected + + the clonogeneic growth of human CD34 or murine Sca-1 HSPC. Both the number and size of myeloid, erythroid, and megakaryocytic colonies for human and murine BM-derived cells growing in standard methylcellulose media were not affected regardless if C3a or C3adesArg were added (0–1 µg/ml) to the cultures. Thus C3 cleavage fragments do not directly affect the proliferation of human or 21 murine HSPC . 21 C3a and C3adesArg do not chemoattract HSPC alone , but both compounds, as + + mentioned above, sensitized the responses of human CD34 and murine Sca-1 cells to an SDF-1 gradient. We observed that C3a-C3aR axis signaling influ-

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enced the SDF-1-dependent homing of HSPC to BM by promoting their (i) chemotactic response to SDF-1, (ii) adhesion to VCAM-1, (iii) expression/secretion of metalloproteinase-9 (MMP-9), and (iv) migration across subendothelial basement membranes. Interestingly, a short exposure (priming) of Sca+ 1 cells to C3a before transplantation accelerated their engraftment in lethally irradiated mice. Hence, we concluded that the C3a–C3aR axis modulated responses dependent upon the SDF-1–CXCR4 axis, thereby regulating the homing of HSPC into BM. Based on these data, a new regulatory link between the C 21 system and the SDF-1–CXCR4 axis has been established . However, the question of whether C may also modulate in addition to the SDF-1–CXCR4 axis the chemokine–chemokine receptor axes as well requires further studies. On the other hand, as expected, we noticed that the receptor for extracellu+ lar matrix-bound iC3b, CR3, plays a role as reported in adhesion of CR3 HSPC in BM. However, while CR3 is mostly expressed on more differentiated hema49 topoietic progenitor cells (HPC) , recent data suggest that CR3 is also present on a substantial number of hematopoietic stem cells (HSC). In support of this, infusion of antibodies against CR3 into mice releases (mobilizes) both HSC and HPC from BM into PB. Based on this the CR3–iC3b interaction is crucial for 52 retention of HSPC in BM . To evaluate the effect of iC3b in tethering HSPC in BM we employed a different strategy. Since iC3b is deposited onto C-activating surfaces and our previous studies had shown that iC3b was deposited onto BM stroma damaged by 19 gamma radiation , to determine if such surface-bound iC3b deposited onto BM stroma also played a critical role in BM recovery, experiments were carried out to explore the role of CR3 in tethering HSPC to sites of BM damage marked by deposited iC3b. + -/In these experiments Sca-1 cells from wild-type (wt) and CR3 mice were tested for adhesion to iC3b deposited in vitro onto cultured BM stroma cells. -/BM stroma cells from C3 mice (to exclude the potential contribution of C3 produced by stromal cells) were γ−irradiated (to express a neoepitope recognized by a natural IgM antibody present in normal serum) and subsequently ex-/posed to serum (source of IgM and C3) from wt or C3 mice. The deposition of iC3b was confirmed on the surface of stroma cells exposed to wt serum only by 20,21 FACS using an affinity-purified goat anti-mouse C3 Ab-Oregon green . For + -/adhesion assays, Sca-1 cells were employed from wt or CR3 mice. After 1 and 6 hr of adhesion, non-adherent cells were discarded from the adhesion cultures and the cells in the wells were trypsinzed and subsequently plated in methylocel19 lulose cultures stimulated to grow CFU-GM colonies . We noticed that after 1 hr of adhesion CFU-GM was tethered to stroma cells in a CR3–iC3b-dependent manner. In prolonged adhesion (6 hr) this effect was not visible, suggesting that this CR3–iC3b-mediated interaction is important in early adhesion and may be compensated later on by other adhesion molecules. Thus, the CR3–iC3b axis may play an important role in the tethering, adhesion, and retention of HSPC in BM and thus also contribute to marrow reconstitution

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from myeloablative treatment (e.g., irradiation). We hypothesize that by increasing the adhesiveness of HSPC within the hematopoietic microenvironment the C system promotes their better interaction and exposure to BM stroma-expressed growth factors, adhesion molecules, and cytokines. These interactions are cru19 cial for self-renewal and expansion of HSPC .

5. HEMATOPOIESIS IN C3-DEFCIENT MICE UNDER NORMAL STEADY-STATE AND STRESS SITUTATIONS Since no obvious hematological deficiencies were described in mice deficient in -/complement C3 (C3 ), these mice are considered to be hematologically nor53 mal . To address this issue better we studied several hematological parameters +/+ in these mice and compared them to control C3 wt littermates. Table 1 shows -/that C3 mice display (i) normal peripheral blood cell counts as compared to +/+ control C3 wt littermates, (ii) similar numbers of clonogenic progenitors from the myeloid, erythroid, and megakaryocytic lineages in the bone marrow cavities as well as (iii) a similar total number of mononuclear cells present in their bone marrow cavities, and (iv) a number of transplantable colony forming unit-spleen (CFU-S) cells. Table I. Hematological Parameters in C3-/- and Normal Wild-Type C3

+/+

Littermates

However, normal hematopoiesis as observed in steady-state conditions does not preclude the possibility that hematological abnormalities may become visible under stressed situations when the demand for blood supply increases. Thus, -/+/+ we have sublethally irradiated C3 and control C3 animals (500 cGy) and evaluated the recovery of peripheral blood counts. Interestingly, we noticed that -/C3 mice had a delayed recovery of platelet and leukocyte counts by about 5–7 19 days .

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Similarly, C3 mice displayed a significant delay in hematopoietic recovery 19 + after transplantation of wt HSPC . Transplantation of histocompatible wt Sca-1 -/cells into C3 mice resulted in (i) a decrease in day 12 CFU-S, (ii) a 5–7 day delay in platelet and leukocyte recovery, and (iii) a reduced number of BM -/CFU-GM progenitors at day 16 after transplantation. Since HSPC from C3 mice engrafted normally into irradiated wt mice, the observed delay in recovery -/was likely related to a defect in the hematopoietic environment of C3 mice. Thus, our data strongly suggest that C3, even if dispensable for hematopoiesis under steady-state conditions, is needed for optimal regeneration of BM from 19 sublethal irradiation and for proper engraftment of HSPC after transplantation .

6. MOLECULAR EXPLANATION OF THE DEFECT IN HOMING/ ENGRAFTMENT OF HSPC IN C3-DEFICIENT MICE The early stages of BM seeding by HSPC, which precedes their proliferation/differentiation, are collectively termed “homing.” Homing can be divided + into several overlapping steps. CXCR4 HSPC infused into PB have to respond to a chemotactic SDF-1 gradient in BM, attach to BM endothelium, transmigrate through the basal membrane in a metalloproteinase-dependent manner, and finally home to a niche where they have to subsequently survive, expand, and 1,54 proliferate . Thus, homing of HSPC to BM niches is the first step in the engraftment process, during which HSPC self-renew and differentiate into precursor cells for all the hematopoietic lineages. The expansion of transplanted HSPC and proper colonization of BM tissue is directly responsible for final reconstitution of the BM tissue. -/Since C3 mice do not have C3, the C3 fragments C3a, C3ades-Arg, and iC3b were examined for a role in HSPC engraftment. Since C3a binds to the Gprotein-coupled seven transmembrane spanning C3aR and iC3b binds to the 48 integrin-type CR3 receptor , we assumed that the molecular action of C3 cleavage fragments will be different for fluid phase C3a/C3ades-Arg and for surface bound iC3b. As mentioned above, several small molecules (e.g., fibronectin and fibrinogen fragments, hylauronic acid) that are present in supernatants from leukopheresis products may modulate the responsiveness of HSPC to an SDF-1 gradient by enhancing, as we assumed, the incorporation of CXCR4 into membrane lipid rafts. Our recent research on human hematopoietic cells primed by fibrinogen and fibronectin confirmed that sensitization/priming of cell chemotaxis to an SDF-1 gradient is dependent on the cholesterol content in the cell membrane and incorporation of the SDF-1 binding receptor, CXCR4, and the small GTP-ase Rac-1 into membrane lipid rafts. This co-localization of CXCR4 36 and Rac-1 in lipid rafts facilitated GTP binding/activation of Rac-1 . Based on these observations, we asked if C3a and C3ades-Arg may also prime (increase) the responsiveness of HSPC to an SDF-1 gradient, similarly as fibronectin and fibrinogen fragments, by increasing incorporation of CXCR4 into

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membrane lipid rafts. Thus, the hypothesis was investigated that the priming 19 effect of C3a and C3ades-Arg is dependent on lipid raft formation . Accordingly, chemotaxis assays were performed with low doses of SDF-1, either alone or with a priming concentration of C3a or C3ades-Arg. To determine the contribution of lipid rafts, cells were preincubated for 30 min before chemotaxis in 2.5-mM hydroxypropyl-β-cyclodextrin to extract cholesterol from the membranes and to perturb lipid raft formation. These studies with hematopoietic cells suggested that the priming effect of C3a and C3ades-Arg on SDF-1 chemotaxis was blocked by this raft-disrupting agent. To provide more evidence that the priming effect of these small C3 fragments is dependent on lipid raft formation, confocal-like and Western blot analysis were carried out to assess CXCR4 incorporation into membrane lipid rafts in hematopoietic cells primed by C3a and C3ades-Arg. As expected, CXCR4 in hematopoietic cells stimulated by C3a and C3ades-Arg became 19 incorporated into cell membrane lipid rafts . Figure 3 depicts the role of C3 cleavage fragments in lipid raft formation. However, the type of C3 receptors involved in this process needs further clarification. Generally, it is widely accepted that, while the seven transmembranespanning G-protein coupled C3aR binds anaplylatoxin C3a, its degradation product, C3adesArg, binds to another unidentified receptor and the seven-transmembrane span C5L2 orphan receptor was postulated to be a possible candidate 55 for this latter interaction . However, based on our observations that blockade of C3aR by C3aR antagonist SB290157 only partially inhibited the priming effect, and not only C3a but also C3adesArg primes the responsiveness of HSPC to an SDF-1 gradient, we envisioned that another C3adesArg/C3a binding receptor may 19 exist that could be responsible for the observed priming effect . This was additionally supported by our data that cells from mice with C3aR knocked-out 19 showed a priming effect to both C3a and C3adesArg . To clarify this issue, another group of investigators recently took advantage of C5L2 knockout mice and demonstrated that in these animals, which do not have the C5L2 receptor, C3adesArg still is able to increase the responsiveness of 56 HSPC to an SDF-1 gradient . This strongly suggests that another C3a and C3adesArg binding receptor exists on HSPC. It is not clear at this point if this receptor would be another member of the seven-transmembrane span receptor 57 family, similar to C3aR and C5L2, or a different type of molecule . Next, we became interested in the molecular mechanisms of surface-bound C3 cleavage fragment, iC3b, in homing of HSPC to the BM. We noticed that fluid-phase iC3b may also prime the responsiveness of HSPC to an SDF-1 gradient and increase incorporation of CXCR4 into membrane lipid rafts (not published). More importantly, however, iC3b as discussed above seems to play a role as a tethering molecule in BM. The retention of HSPC in BM is controlled by an adhesive interaction between adhesion receptors on HSPC and stroma. These receptors include integrins (VLA-4, VLA-5, Mac-1), selectins (L-selectin), CD44, members of the immunoglobin superfamily, tyrosine kinase recep2,7,8,10,18,58-60 . Recent studies showing tor–c-kit, and chemokine receptor CXCR4

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Figure 3. Schematic diagram of the hypothetical role of C3 cleavage fragments (C3a and C3adesArg) in sensitizing the responsiveness of HSPC to an SDF-1 gradient. C3 is activated in BM in response to marrow damage (e.g., after conditioning for transplantation by TBI or chemotherapy). Cleavage/activation of C3 in the BM micorenvironment is initiated by a C3-convertase to generate C3a anaphylatoxin. Subsequently C3a is quickly cleaved by carboxypeptidases, which removes the terminal arginine residue from the 78-aminoacid C3a molecule. Although C3a has a short half-life, the C3a inactivation product, C3adesArg, has a long half-life. We postulate that C3a and C3adesArg fragments increase the responsiveness of HSPC to SDF-1 gradients by enhancing the incorporation of CXCR4 into membrane lipid rafts. A recent observation supports the concept that, while the seven transmembrane-span G-protein coupled C3aR binds C3a only, another non-identified receptor (Receptor X?) binds both C3a and C3adesArg. Activation of both of these receptors enhances incorporation of CXCR4 into membrane lipid rafts and thus its better association with downstream signaling proteins.

expression of CR3 on HPC and significant enhancement of G-CSF-induced mobilization by administration of blocking antibodies against β2-integrins indicated 61 the involvement of CR3 in the homing/retention of HPC in BM . Supporting -/this, our recent studies in CR3 mice revealed that these mice are more sensitive to mobilization by suboptimal doses of G-CSF as compared to their wt litter20 mates . Taken together, these data suggest that CR3 participates in retention of HSPC in the BM. Based on our investigations, the following model is proposed for the role of C3 cleavage fragments in hematopoiesis (see Figure 4). Cleavage/activation of C3 secreted by BM stromal cells and macrophages is initiated by a C3-

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Figure 4. Schematic diagram of the hypothetical role of C3 fragments in retention of HSPC in BM. C3 is activated in BM in response to marrow damage (e.g., after conditioning for transplantation by TBI). Cleavage/activation of C3 secreted by BM stromal cells and macrophages is initiated by a C3-convertase to generate fluid-phase C3a and stromal cell-bound C3b. Both C3a and C3b have a short half-life. Fluid C3a is rapidly degraded to C3ades-Arg and bound C3b is proteolyzed to iC3b. We postulate that while C3a and C3ades-Arg fragments increase the responsiveness of HSPC to SDF-1 gradients, iC3b tethers HSPC onto BM stroma. The HSPC tethered by the iC3b–CR3 axis are effectively targeted to the site of injury and may interact better with stroma cells because of both CR3 signaling resulting from ligation to iC3b and/or enhanced recognition of lower-affinity adhesion molecules.

convertase to generate fluid-phase C3a and stromal cell-bound C3b. Both C3a and C3b have a short half-life. Fluid C3a is rapidly degraded to C3ades-Arg and bound C3b is proteolyzed to iC3b. We postulate that while C3a and C3ades-Arg fragments increase the responsiveness of HSPC to SDF-1 gradients, iC3b tethers HSPC onto BM stroma. The HSPC tethered by the iC3b–CR3 axis are effectively targeted to the regenerating BM and may interact better with stroma cells because of both CR3 signaling resulting from ligation to iC3b or enhanced recognition of lower-affinity adhesion molecules. All of these investigations showing that C3 cleavage fragments play an important role in homing and retention -/of HSPC in BM explain at the molecular level why C3 mice display a defect in engraftment of HSPC after transplantation.

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7. CONCLUSIONS We presented evidence that a crosstalk between C and the SDF-1—CXCR4 axis plays an important and underappreciated role in engraftment of HSPC. Accordingly, a novel mechanism for HSPC engraftment was identified that involves (i) complement activation in BM after myeloablative conditioning for transplanta19-21 and (ii) release of specific CXCR4 incorporation into membrane lipid tion rafts (thus potentiating HSPC responses to SDF-1 gradients), whereas iC3b de+ posited onto irradiated BM cells functions to tether CR3(CD11b/CD18) HSPC 19 to damaged stroma . On the other hand, our data on BM regeneration strongly suggest that activation/cleavage of complement becomes important in stressed situations as seen in tissue/organ damage. In support of this notion, activation of C3 and the presence of biologically active cleavage fragments was reported in several other 62,63 models of tissue injury (e.g., liver damage) . These data support the concept that innate immunity plays an important role in regeneration of damaged organs. We postulate that it is likely that, similarly as in BM, activation of C that results in generation of C3 cleavage fragments plays an important role in regulating + chemoattraction and homing of CXCR4 non-hematopoietic tissue committed 64-66 stem cells (TCSC) for organ regeneration . We recently reported that these 65,66 TCSC reside in the BM and are released into circulation during organ injury . Trafficking of these cells, similarly as HSPC is directed by SDF-1, and C3 cleavage fragments may play an important role here as well. Thus, C emerges as an important co-regulator of trafficking not only of HSC but also of TCSC as well. Modulation of the C system along with the SDF-1–CXCR4 axis may result in the development of more efficient regeneration therapies.

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4 COMPLEMENT SYSTEM AND THE EYE Purushottam Jha, Puran S. Bora, Jeong-Hyeon Sohn, Henry J. Kaplan, and Nalini S. Bora. 1. INTRODUCTION The complement system is a key component of natural immunity and consists of a large group of plasma proteins that play a central role in the defense against 1-5 infection and in modulation of inflammatory responses . The complement system can be activated via three distinct pathways — namely, the classical, the alternative, and the lectin pathways — and complement activation triggers a 1-7 sequence of biological reactions . The classical pathway can be activated by immune complexes or by substances such as C-reactive protein, and the complement components involved 1-7 include C1, C2, C4, and C3 . The alternative pathway provides a rapid, antibody-independent route of C activation and amplification. The alternative pathway directly activates C3 when it interacts with certain activating surfaces (e.g., zymosan, lipopolysaccharides) and involves C3, Factor B, Factor D, and proper1-7 din . The activation of the lectin pathway is also independent of immune complex generation and can be achieved by interaction of certain serum lectins, such as mannose binding protein (MBL), with mannose and N-acetyl glucosamine 7,8 residues present in abundance in bacterial cell walls . Complement is a powerful defense system that has the potential to damage 1-7 host tissue . A critical step in the complement activation sequence is formation 6,9 of the C3 convertases . These proteases activate the third (C3) complement component, resulting in generation and deposition of the major opsonic fragments C3b and C4b on the cell membrane. In addition, the convertases promote assembly of the membrane attack complex (MAC). Thus, it is essential that the formation and function of C3 convertases be carefully regulated so that the opsonic activity of C3b and the cytolytic activity of MAC are directed against foreign cells and not to self-tissue. The host must be protected from inadvertent activation of complement on its own tissue.

Purushottam Jha, Puran S. Bora, and Nalini S, Bora, Department of Ophthalmology, Jones Eye Institute, 4301 West Markham, #523, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Tel: 501-686-8293; Fax: 501-686-8316; email: [email protected]. Jeong-Hyeon Sohn and Henry J. Kaplan, Department of Ophthalmology and Visual Science, Kentucky Lions Eye Center, 301 East Muhammad Ali Blvd., University of Louisville, Louisville, KY 40202, USA.

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Decay Accelerating Factor (DAF, CD55) and Membrane Cofactor Protein (MCP, CD46) are two important membrane-bound regulatory proteins that downregulate the complement cascade by regulating two critical enzymes — C3 10-14 and C5 convertases . Membrane Inhibitor of Reactive Lysis (MIRL, CD59) 15-16 has the capacity to regulate the assembly and activity of MAC . Crry is a widely distributed complement regulatory protein (CRP) in rodents and has both decay-accelerating and co-factor activities. It controls complement activation at 17-20 the critical step of C3 convertase formation . Tissue distribution studies have 21,22 shown that CD59 is a widely distributed protein in rodents also . Recently, MCP and DAF have also been identified in rodents.

2. COMPLEMENT AND OCULAR PROTECTION 23

Sohn et al. demonstrated that the functionally active complement system is present in the normal eye because anterior chamber injection of zymosan induced severe anterior uveitis. Immunohistologic staining of the normal rat eye revealed that, even when there was no inflammation, low levels of iC3b and MAC were present. These results suggested that the complement system is active at a low level in the normal eye and may provide immediate protection 23 against various pathogens. Sohn et al. also demonstrated that intraocular complement regulatory proteins tightly regulate this spontaneous complement activation. Control of complement activation at the level of C3 convertase was sufficient to prevent complement-mediated intraocular inflammation.

3. COMPLEMENT AND OCULAR DISEASES Inappropriate activation of complement has been reported to play an important role in the pathogenesis of various diseases, including ocular ones. 3.1. Complement and Corneal Disease Several investigators have shown that membrane-bound complement regulatory proteins (CRPs) — MCP, DAF, Crry, and CD59 — are differentially expressed 24-26 in the normal human and rodent eyes . All of these proteins are very strongly expressed in the corneal epithelium and the limbus, as well as on the central cornea. This overexpression is very crucial for the protection of cornea because the cornea is constantly being challenged by a variety of substances, including infectious organisms that produce phospholipase and other enzymes that can remove CRPs from ocular cell surfaces. The bacterially induced loss of CRPs on the cornea could lead to damage of ocular tissue by autologous complement activation in the course of complement attack on pathogens. Complement activation is believed to play an important role in ulceration of the human cornea 27 28 induced by Gram-negative bacteria . Cleveland and associates demonstrated

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that decomplemented mice had more severe corneal ulcers caused by Pseudomonas aeruginosa than mice with normal complement levels. 3.2. Complement and Autoimmune Uveitis Complement activation products such as C3b and C4b have been demonstrated 29,30 to be present in the eyes of patients with anterior uveitis . Recently, an experimental autoimmune anterior uveitis (EAAU) animal model was used to explore the role of complement in the pathogenesis of this ocular autoimmune dis31 ease . EAAU is an autoimmune disease of the eye that serves as an animal 32-37 model of idiopathic human anterior uveitis . In this report Jha et al. demonstrated that the induction and progression of autoimmune uveitis is complement restricted. Levels of iC3b, the cleavage product of C3, increased within the eye during the peak of EAAU. Activation of the complement system is necessary for 5,38 generation of C3 split products , and their increased levels within the eye of rats with EAAU provide indisputable evidence of local complement activation during autoimmune intraocular inflammation. The interaction between iC3b (generated as a result of local complement activation) and CR3 seems to play an important role in EAAU. The systemic injection of anti-CR3, which specifically blocks the interaction between iC3b and its receptor CR3, had a significant protective effect on EAAU, thus suggesting that the interaction of iC3b with CR3 was involved in the generation and progression of uveitis in EAAU. Furthermore, the expression of cytokines, chemokines, and adhesion molecules necessary for development of EAAU required complement activation. IFN-γ, IL-10, IP-10, ICAM-1, and LECAM-1 were significantly decreased following complement depletion, indicating that these molecules are regulated by complement. In addition, various ocular tissues upregulate the expression of complement regulatory proteins (CRPs) to avoid self-injury during autoimmune uveitis, and these CRPs played an active role in resolution of EAAU by downregulating complement activation in vivo. 3.3. Complement and Age-Related Macular Degeneration In recent years, studies have shown that the complement system is also involved 39,40 in immunopathogenesis of age-related macular degeneration (AMD) . AMD is the leading cause of visual loss in individuals over age 55. There are two clinical phenotypes of AMD — a nonexudative (dry) and an exudative (wet) type. One of the early signs of nonexudative AMD is accumulation of abnormal extracellular deposits, called drusen, adjacent to the basal surface of the retinal pigmented epithelium (RPE). Drusen are located between the basal lamina of RPE and the inner layer of Bruch’s membrane. Activation of the complement system has recently been implicated in formation of the drusen. Several proteins of the complement cascade have been 41-46 44 identified in the drusen . Johnson et al. reported that both MAC and IgG are

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co-distributed in drusen, indicating the involvement of the classical complement pathway in drusen formation. However, in 2001 Johnson and coworkers reported that antibody-dependent complement activation may not be responsible 45 for drusen formation and suggested that complement activation via the alternative pathway may be involved in this process. In the same study many complement regulators — including vitronectin, CR1, and MCP — were detected in drusen. Variation in factor H gene (HF1), which encodes a key regulatory protein of alternative pathway, has recently been associated with significantly in47 creased attributable risk (50%) of AMD in humans . Also, factor H protein has been shown to be present in the macular region in eyes of patients with early 47 AMD . Choroidal neovascularization (CNV) causes exudative (wet) type AMD. One of the ways to induce CNV in animals is to rupture Bruch’s membrane with 48,49 laser photocoagulation . Bora et al. have used a murine model of laser-induced 50 CNV to explore the role of complement activation in choroidal angiogenesis . In this report the requirement of complement system in the development of CNV -/was demonstrated by the inability to induce CNV in C3 mice. Furthermore, invivo complement depletion by CVF and inhibition of complement 6 (C6) with anti-C6 polyclonal antibody markedly reduced the incidence and development of CNV in C57BL/6 mice. There was an increased deposition of MAC in the CNV complex of complement sufficient animals. The absence of MAC coincided with low/basal levels of various growth factors — such as VEGF, TGFβ2, and β-FGF — in the laser spots. 50 Based on these observations Bora et al. proposed that laser photocoagulation causes complement activation in the posterior segment of the eye, which leads to increased deposition of MAC on RPE and/or choroid. The deposition of MAC results in increased membrane permeability, which in turn causes an increase in release of growth factors. These upregulated growth factors lead to abnormal proliferation of choroidal endothelial cells, resulting in the development of CNV (Figure 1) Taken together, the above-mentioned observations confirm the importance of complement activation and MAC deposition in the development of AMD, and suggest that complement inhibition may provide a novel approach for the treatment of AMD.

4. COMPLEMENT AND OCULAR TOLERANCE The eye is an immune-privileged site. One of the characteristic features of the immune-privileged sites is that if an antigen is introduced to these sites a systemic tolerance can be induced. Introduction of an antigen into the anterior chamber (AC) leads to a deviant systemic immune response, known as anterior 51-52 chamber-associated immune deviation (ACAID) . ACAID involves the gen-

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eration of antigen-specific regulatory T cells and suppression of delayed type hypersensitivity (DTH). Laser Photocoagulation

C3, C5 etc.

Release of growth factors-VEGF,TGFβ2, β-FGF MAC

Complement activation

Retinal Pigmented Epithelium layer Bruch’s membrane

formation Increased permeability

New blood vessels

MAC

Chorio-cappilaries

Figure 1. Role of complement activation and MAC deposition in development of CNV.

The complement system has been reported to be essential for the development of suppressed DTH after AC injection of antigen and hence plays an im53 portant role in the development of tolerance . In this report Sohn et al. proposed that binding of iC3b to CR3 present on APC induces production of TGF-β2, followed by upregulation of IL-10 and downregulation of IL-12. Under the in54 fluence of TGF-β2 there is increased production of IL-10 and downregulation of IL-12 production by APC, which directs the immune response away from the Th1 pathway and toward a Th2-like response, which in turn leads to suppression of DTH (Figure 2).

5. CONCLUSIONS The complement system is an important part of the innate immunity. In the normal eye, the complement system is continuously activated at low levels, and both membrane-bound and soluble intraocular complement regulatory proteins tightly regulate this spontaneous complement activation. This allows for destruction of the pathogens without induction of intraocular inflammation and vision loss. However, if control of complement activation is lost, intraocular inflammation occurs. Complement system and complement regulatory proteins control the intraocular inflammation in autoimmune anterior uveitis and play an important role in the development of corneal inflammation and age-related macular degeneration. Furthermore, the presence of a complement activation product, iC3b,

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OVA specific regulatory T-cells

DTH suppression

iC3b

DTH

OVA

iC3b

IL-10

iC3b

Inflammation

CR3 IL-10

OX42

IL-12 TGFβ2 IL-10 TGFβ2

IL-12

Antigen Presenting Cell (OVA – PEC)

Figure 2. Role of iC3b in ocular tolerance.

during the early phase of antigen and antigen-presenting cell (APC) contact is essential for induction of systemic tolerance to antigen injected into the anterior chamber of the eye and ocular immune privilege. Thus, in the eye complement functions as a double-edged sword —it provides innate immunity against pathogens while simultaneously instructing the adaptive immune response to suppress any harmful antigen specific DTH.

6. REFERENCES 1. 2. 3. 4.

J.P. Atkinson, M.M. Frank, Complement. In Clinical immunology, edited by C.W. Parker (WB Saunders, Philadelphia 1980), pp. 219–271. G.D. Ross, Immunobiology of the complement system (Academic Press Inc., Florida, 1986). H.J. Muller-Eberhard, Molecular organization and function of the complement system, Annu Rev Biochem 57, 321–347 (1988). M.M. Frank and L.F. Fries, The role of complement in inflammation and phagocytosis, Immunol Today 12, 322–326 (1991).

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22. Y.M. Qian, X. Qin, T. Miwa, X. Sun, J.A. Halperin, and W.C. Song, Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59, J Immunol 165, 2528–2534 (2000). 23. J.H. Sohn, H.J. Kaplan, H.J. Suk, P.S. Bora, and N.S. Bora, Chronic low level complement activation within the eye is controlled by intraocular complement regulatory proteins, Invest Ophthalmol Vis Sci 41(11), 3492–502 (2000). 24. J.H. Sohn, H.J. Kaplan, H.J. Suk, P.S. Bora, and N.S. Bora, Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59, Invest Ophthalmol Vis Sci 41(13), 4195–4202 (2000). 25. N.S. Bora, C.L. Gobleman, J.P. Atkinson, J.S. Pepose, and H.J. Kaplan, Differential expression of the complement regulatory proteins in the human eye, Invest Ophthalmol Vis Sci 34(13), 3579–3584 (1993). 26. D.S. Bardenstein, Y. Dietz, J.H. Lass, and M.E. Medof, Localization of the complement membrane attack complex inhibitor (CD59) in human conjunctiva and lacrimal gland, Curr Eye Res 13(12), 851–855 (1994). 27. B.J. Mondino, S.I. Brown, B.S. Rabin, and J. Bruno, Alternate pathway activation of complement in a Proteus mirabilis ulceration of the cornea, Arch Ophthalmol 96(9), 1659–1661 (1978). 28. R.P. Cleveland, L.D. Hazlett, M.A. Leon, R.S. Berk, Role of complement in murine corneal infection caused by Pseudomonas aeruginosa, Invest Ophthalmol Vis Sci 24(2), 237–242 (1983). 29. B.J. Mondino, M.M. Glovsky, and L. Chekiere. Activated complement in inflamed aqueous humor, Invest Ophthalmol Vis Sci 25, 871–873 (1984). 30. B.J. Mondino and H. Sumner, Anaphylatoxin levels in human aqueous humor, Invest Ophthalmol Vis Sci 27, 1288–1292 (1986). 31. P. Jha, J.H. Sohn, Q. Xu, H. Nishimori, Y. Wang, S. Nishihori, B. Manickam, H.J. Kaplan, P.S. Bora, and N.S. Bora, The complement system plays a critical role in the development of experimental autoimmune anterior uveitis, Invest Ophthalmol Vis Sci 47 (2006). 32. R.M. Broekhuyse, E.D. Kuhlman, H.J. Winkens, and A.H.M. Van Vugt, Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis, I: induction by a detergent-insoluble, intrinsic protein fraction of the retina pigment epithelium, Exp Eye Res 52, 465–474 (1991). 33. N.S. Bora, M.C. Kim, N.H. Kabeer, S.C. Simpson, M.T. Tandhasetti, T.P. Cirrito, A.D. Kaplan, and H.J. Kaplan, Experimental autoimmune anterior uveitis. Induction with melanin-associated antigen from the iris and ciliary body, Invest Ophthalmol Vis Sci 36(6), 1056–1066 (1995). 34. N.S. Bora, M. D. Woon, M. T. Tandhasetti, T. P. Cirrito, and H. J. Kaplan, Induction of experimental autoimmune anterior uveitis by a self-antigen: melanin complex without adjuvant, Invest Ophthalmol Vis Sci 38(10), 2171–2175 (1997). 35. M.C. Kim, N.H. Kabeer, M.T. Tandhasetti, H.J. Kaplan, and N.S. Bora, Immunohistochemical studies in melanin associated antigen (MAA) induced experimental autoimmune anterior uveitis (EAAU), Curr Eye Res 14(8), 703–710 (1995). 36. M.D. Woon, H.J. Kaplan, and N.S. Bora, Kinetics of cytokine production in experimental autoimmune anterior uveitis (EAAU), Curr Eye Res 17(10), 955–961 (1998). 37. N.S. Bora, J.H. Sohn, S.G. Kang, J.M. Cruz, H. Nishihori, H.J. Suk, Y. Wang, H.J. Kaplan, and P.S. Bora, Type I collagen is the autoantigen in experimental autoimmune anterior uveitis, J Immunol 172(11), 7086–7094 (2004).

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38. A.E. Davis 3d, R.A. Harrison, and P.J. Lachmann, Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d,g (alpha 2D), and C3g, J Immunol 132, 1960–1966 (1984). 39. F.L. Ferris, S.L. Fine, L. Hyman, Age-related macular degeneration and blindness due to neovascular maculopathy, Arch Ophthalmol 102, 1640–1642 (1984). 40. S.L. Fine, J.W. Berger, M.G. Maguire, A.C. Ho. Age-related macular degeneration. N Engl J Med 342, 483–492 (2000). 41. L.V. Johnson, M.K. Staples, M.A. de los Rios, M.J. Nealon, S. Ozaki, P.J. Kappel, M.B. Stenkilsson, and D.A. Anderson, Inflammation and the immune response in age-related macular degeneration, Exp Eye Res 67, p. S102 (1998). 42. L.V. Johnson, P.A. Erickson and D.A. Anderson, Age-related maculopathy: RPE, immune response molecules and drusen biogenesis, Invest Ophthalmol Vis Sci 40, S992 (1999). 43. R.F. Mullins, S.R. Russell, D.H. Anderson, and G.S. Hageman, Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease, FASEB J 14(7), 835–846 (2000). 44. L.V. Johnson, S. Ozaki, M.K. Staples, P.A. Erickson, and D.A. Anderson, A potential role for immune complex pathogenesis in drusen formation, Exp Eye Res 70, 441–449 (2000). 45. L.V. Johnson, W.P. Leitner, M.K. Staples, and D.H. Anderson, Complement activation and inflammatory processes in drusen formation and age related macular degeneration, Exp Eye Res 73, 887–896 (2001). 46. G.S. Hageman, P.J. Luthert, N.H. Victor Chong, L.V. Johnson, D.H. Anderson, and R.F. Mullins, An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and agerelated macular degeneration, Prog Retin Eye Res 20(6), 705–732 (2001). 47. G.S. Hageman, D.H. Anderson, L.V. Johnson, L.S. Hancox, A.J. Taiber, L.I. Hardisty, J.L. Hageman, H.A. Stockman, J.D. Borchardt, K.M. Gehrs, R.J. Smith, G. Silvestri, S.R. Russell, C.C. Klaver, I. Barbazetto, S. Chang, L.A. Yannuzzi, G.R. Barile, J.C. Merriam, R.T. Smith, A.K. Olsh, J. Bergeron, J. Zernant, J.E. Merriam, B. Gold, M. Dean, and R. Allikmets, A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration, Proc Natl Acad Sci USA 102(20), 7227–7232 (2005). 48. H. Miller, B. Miller, T. Ishibashi, and S.J. Ryan, Pathogenesis of laser-induced choroidal subretinal neovascularization, Invest Ophthalmol Vis Sci 31, 899–908 (1990). 49. H. Kimura, T. Sakamoto, D.R. Hinton, C. Spee, Y. Ogura, and S.J. Ryan, A new model of subretinal neovascularization in the rabbit, Invest Ophthalmol Vis Sci 36, 2110–2119 (1995). 50. P.S. Bora, J.H. Sohn, J.M. Cruz, P. Jha, H. Nishihori, Y. Wang, S. Kaliappan, H.J. Kaplan, and N.S. Bora, Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization, J Immunol 174(1) 491–497 (2005). 51. J.W. Streilein, B.R. Ksander, and A.W. Taylor, Immune deviation in relation to ocular immune privilege, J Immunol 158(8), 3557–3560 (1997). 52. J.W. Streilein, Immunologic privilege of the eye, Springer Semin Immunopathol 21(2), 95–111 (1999).

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53. J.H. Sohn, P.S. Bora, H.J. Suk, H. Molina, H.J. Kaplan, and N.S. Bora, Tolerance is dependent on complement C3 fragment iC3b binding to antigen-presenting cells, Nat Med 9(2), 206–212 (2003). 54. T.J. D'Orazio, and J.Y. Niederkorn, A novel role for TGF-beta and IL-10 in the induction of immune privilege, J Immunol 160(5), 2089–2098 (1998).

5 TO REGENERATION … WITH COMPLEMENT Panagiotis A. Tsonis, John D. Lambris, and Katia Del Rio-Tsonis

1. REGENERATIVE ABILITIES IN VERTEBRATES Apart from wound healing (or wound repair), which is mostly closure of a wound by scar tissue, the degree of tissue renewal or regeneration in vertebrates varies in different tissues. In fact, what is different is the complexity involved in the mechanisms and magnitude of regeneration. The simplest form of regeneration is the axonal outgrowth seen in a severed nervous system. Regeneration by simple proliferation seen in organs, such as intestines, liver, or adrenal gland, is somewhat more complex. It involves proliferation of cells that compose the particular organ. Regeneration of other organs and tissues, on the other hand, can be channeled through proliferation and differentiation of stem cells. More complex types of regeneration involve dedifferentiation. In these cases (mostly seen in amphibia), a particular cell type at the damaged site dedifferentiates and then redifferentiates into the same type. Regeneration of CNS (brain, spinal cord), intestine and heart can be achieved by this mechanism of dedifferentiation. An even more complex type of regeneration involves transdifferentiation from one cell type to another and can be seen during pancreas regeneration. The most complex type of regeneration, however, is seen in amphibian limb and lens regeneration, in which cells at the damaged or amputated site dedifferentiate and transdifferentiate, but they are also able to build back an exact replica of the lost part. This is indeed the most spectacular type of regeneration, and is restricted to some urodeles. The other types of regeneration are rather widespread among 1 vertebrates . In our laboratories we have been committed to work with the amphibian models because they can provide information on all the mechanisms that are Panagiotis A. Tsonis, Department of Biology, University of Dayton, Dayton, OH 45469-2320, USA. John D. Lambris, University of Pennsylvania, Philadelphia, PA 19104, USA. Katia Del Rio-Tsonis, Department of Zoology, Miami University, Oxford, OH 45056, USA.

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involved in the different types of regeneration. We strongly believe that knowledge from the regenerative abilities of amphibia could be applied to other animals as well. In the following sections, we will familiarize the reader with the process of limb and lens regeneration, the two systems we have selected in which to examine the role of the complement system.

2. LIMB REGENERATION Among amphibia, both anura and urodeles are capable of limb regeneration; however, only some urodeles are capable of regeneration as adults. Most of the anura (frogs) lose their regenerative properties after metamorphosis. This makes some newts and salamanders the most gifted ones. These animals can regenerate 2,3 their limbs throughout their lives and as many times as they are amputated . Upon amputation the events that lead to regeneration of the missing part are initiated immediately. The first apparent histological event to be noticed is coverage of the wound by the so-called wound epithelium, which starts soon and ends within a few hours after amputation. The presence of this specialized wound epithelium is of great importance since its removal would not allow the subsequent events of dedifferentiation and regeneration to take place. Once the cover has been established, the remaining tissues of the intact limb (called the stump) undergo a dramatic cellular event and lose the characteristics of their origin. It is believed that the wound epithelium provides the critical signal for 4 dedifferentiation . The event of dedifferentiation is unique in these animals and is a necessary prerequisite for regeneration to occur. Terminally differentiated cells, such as muscle, virtually melt down and become mononucleated cells. The remodeling of the extracellular matrix is paramount for the process of dedifferentiation. Many proteins and enzymes — such as intergins, collagens, and collagenases — 1,2 are expressed specifically during this period . Dedifferentiation of the normal terminally differentiated tissues is the key to the formation of the blastema, which is a mass of dedifferentiated cells that proliferate for about 2 weeks and then redifferentiate to give rise to the missing part. In other words, the blastema cells are “embryonic-like,” because they have the potential to form a normally 2,3 patterned limb similar to the one formed during embryonic development . The blastema is the product of dedifferentiation of many different tissues, such as muscle, cartilage, bone, and mesodermal cells. The blastema cells redifferentiate and produce the lost part of these tissues. A blastema cell needs not to differentiate to a cell it came from. In other words, blastema cells from muscle can transdifferentiate to form cartilage. This has been shown by clonal blastema 2,5 cells derived from muscle and transplanted back onto a host-regenerating limb . Several important factors have been found to be expressed during the above-mentioned phenomena. Expression of fibroblast growth factors and their receptors in the wound epithelium have been associated with the critical signal-

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ing for dedifferentiation . It has been found that expression of FGF-8, FGFR-1, and FGFR-2 is correlated with the ability of limb regeneration in premetamor8,9 phic frogs . On the other hand, retinoblastoma protein, Rb, has been implicated 10 in reentry of the cell cycle during muscle dedifferentiation . When multinucleated muscle cells are allowed to enter the cell cycle by serum stimulation (possible only in newt muscle), Rb becomes mostly hyperphosphorylated. This represents its inactive form. The active hypophosphorylated form inhibits entry into the S phase in myotubes. The serum factor that stimulates myotubes to dediffer11 entiate and enter the cell cycle seems to be thrombin . The cascade of mechanisms involved in these events is still elusive.

3. LENS REGENERATION When the lens of a newt is removed, the process of regeneration is always initiated from the dorsal iris. The pigment epithelial cells (PECs) from the dorsal iris proliferate, dedifferentiate, and then transdifferentiate into lens cells. The process of proliferation and dedifferentiation takes a few days. During this period PECs initiate DNA synthesis and eventually lose their characteristics of origin, such as pigmentation. At about 7–10 days post-lentectomy a small vesicle has been formed at the tip of the dorsal iris. Cells in this vesicle then transdifferentiate into lens cells and form the lens vesicle (10–15 days). Cells from the posterior part of the lens vesicle differentiate to form the lens fibers (15–20 days). 1,12 Lens regeneration is complete by 25 days post-lentectomy . FGF signaling seems paramount in lens regeneration as well. FGFR-1 protein has been found to be specifically expressed during dedifferentiation in the dorsal iris, and its inhibition by specific FGFR-1 inhibitors results in no lens 13,14 regeneration . Other factors that seem to be involved in lens regeneration are 15-17 the homeo-box-containing Pax-6 and Prox-1 genes as well as retinoid recep18 tors . These regulatory factors are also expressed in regeneration-competent tissues of the dorsal iris and might regulate the normal ability of eye tissues to transdifferentiate to lens. The reader should not fail to see that some of the mechanisms involved in limb and lens regeneration are similar. Especially FGF signaling could be com1 mon in both regenerative systems . Indeed, the eye, for example, is not a necessary physical environment for transdifferentiation and lens formation. If dorsal PECs are transplanted in the regenerating limb blastema they transdifferentiate 19 and form a perfect lens! The ventral PECs are not able to do this . This means that the factor(s) responsible for signaling must be common during limb and lens regeneration. However, when PECs from the dorsal or ventral iris from any animal, including old humans, are placed in vitro they all have the capacity for transdiffer20 entiation . It seems that dissociation and culturing of cells, which alters their extracellular environment considerably, can be an inductive mechanism. The

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study, therefore, of limb and lens regeneration in parallel could be very informative when it relates to mechanisms of regeneration. That is why in this proposal both systems will be studied in order to pinpoint common themes in regeneration. This could be more useful when it comes to more broad applications in the different fields of regeneration.

4. THE COMPLEMENT SYSTEM The complement system is comprised of several serum proteins, membranebound receptors, and regulatory proteins that constitute a phylogenetically an21 cient mechanism of innate immunity . The functions of the complement system in host defense and inflammation are mediated mainly through sequential activation and proteolytic cleavage of serum proteins. Complement activation occurs through three distinct pathways (classical, alternative, and lectin) — all of which converge at activation of C3, the third component of complement. C3 can interact with a wide spectrum of factors, and because of that it is able to mediate a 22,23 wide variety of functions . C3 interacts with several proteins that are involved 24,25 in differentiation, such as fibronectin and integrins . Other complement factors share homologies with domains of extracellular matrix proteins, such as collagen binding, which might indicate that complement factors could be involved in 26 such interactions in the extracellular matrix . Therefore, some complement functions might not be immunologic. For example, C3 is expressed in myoblasts 27 and is also associated with proliferation and growth of B-cells in vitro . C5, the fifth component of complement, has also been found to have novel noninflammatory functions in various tissues. Studies in a human neuroblastoma cell line have suggested that C5a (a fragment of C5) participates in apoptotic signal transduction pathways through its binding to the neuronal C5a recep28,29 tor . It has also been shown that the terminal complex system C5b-9 (MAC) in sublytic doses can induce DNA synthesis and cell proliferation in cultured 30 31 32 mouse fibroblasts , human aortic smooth muscle cells , oligodendrocytes , and 33 glomerular epithelial cells, in the absence of other growth factors . Also, sublytic concentrations can activate monocytes and induce cytokine release through activation of NF-kappaB signaling pathways, which are critical for cell 34 cycle transition into DNA synthesis . The possible role of complement in differentiation of muscle and its interaction with molecules that are involved in cell-to-cell communication and signaling prompted us to examine expression of complement factor C3 in limb regeneration. We have found expression of C3 during blastema formation and limb 35 regeneration . The fact that C3 is not expressed in the intact or developing limb indicated to us specificity for the regeneration process. Furthermore, we were able to show expression of C3 in dedifferentiated muscle cells in vitro.

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Figure 1 (see color insert, Fig. 5.1). Expression of C3 (A,B) and C5 (C,D) during limb regeneration, 3 weeks after amputation. Note expression of C3 in the blastema cells (bl; arrow) and of C5 in the wound epithelium (we; arrow). B and D have been counter-stained with DAPI.

Encouraged by these interesting results, we asked the question of whether complement factors are expressed in other regenerative tissues. C3 was expressed during lens regeneration as well, strengthening our conviction for a role 36 in the control of regeneration processes in amphibia . Experiments from our team dealing with liver regeneration in mice, which can be regenerated by proliferation of the remaining hepatic cells, showed that C5 is imperative for the process. Mice lacking C5 were not able to repair the liver, and they were able to 37 do so only when they were reconstituted with C5 . All this information gives credit to our idea that the complement system has a new role — namely, control of regenerative processes. The long-term goal of this research is to establish the relationship between complement and regeneration and identify the mechanisms by which complement controls regeneration. Biochemistry of the complement factors is well understood, and this will enable us to dissect the mechanisms whereby the complement factors control regenerative processes. This knowledge can be applied in other systems and eventually might allow us to devise strategies that can be used to induce regeneration.

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Figure 2 (see color insert, Fig. 5.2). Expression of C3 (A–C) and C5 (D–F) during lens regeneration 20 days post-lentectomy. Note expression of C3 on the iris (B,C; arrow) and of C5 in the regenerating lens (E,F; arrow). B and E have been counter-stained with DAPI.

5. REFERENCES 1. 2. 3. 4. 5. 6.

P.A. Tsonis. Regeneration in vertebrates. Develop Biol 221, 73–284 (2000). P.A. Tsonis. Limb Regeneration (Cambridge University Press, New York, 1996). D.L. Stocum. Wound repair, regeneration and artificial tissues. (Springer-Verlag, Heidelberg, Germany, 1995). C.S. Thornton. The effetcs of apical cap removal on limb regeneration in Amblystoma larvae. J Exp Zool 134, 357–382 (1957). D.C. Lo, F. Allen, and J.P. Brockes. Reversal of muscle differentiation during urodele limb regeneration. Proc Natl Acad Sci USA 90(15), 7230–7234 (1993). L.M. Mullen, S.V. Bryant, M.A. Torok, B. Blumberg, and D.M. Gardiner. Nerve dependency of regeneration: the role of distal-less and FGF signaling in amphibia limb regeneration. Development 122, 3487–3497 (1996).

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25. I. Leivo, and E. Engvall. C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast. J Cell Biol 103(3), 1091–1100 (1986). 26. I. Kiss, F. Deak, R.G. Holloway Jr., H. Delius, K.A. Mebust, E. Frimberger, W.S. Argraves, P.A. Tsonis, N. Winterbottom, and P.F. Goetinck. Structure of the gene for cartilage matrix protein, a modular protein of the extracellular matrix: exon/ intron organization, unusual splice sites, and relation to alpha chains of beta 2 integrins, von Willebrand factor, complement factors B and C2, and epidermal growth factor. J Biol Chem 264(14), 8126–8134 (1989). 27. C. Servis, and J.D. Lambris. C3 synthetic peptides support growth of human CR2positive lymphoblastoid B cells. J Immunol 142(7), 2207–2212 (1989). 28. I. Farkas, L. Baranyi, Z. S. Liposits, T. Yamamoto, and H. Okada. Complement C5a anaphylatoxin fragment causes apoptosis in TGW neuroblastoma cells. Neuroscience 86, 903–911 (1998). 29. I. Farkas, L. Baranyi, M. Takahashi, A. Fukuda, Z. Liposits, T. Yamamoto, and H. Okada. A neuronal C5a receptor and an associated apoptotic signal transduction pathway. J.Physiol. (Lond) 507 (Pt 3), 679–687 (1998). 30. J.A. Halperin, A. Taratuska, and A. Nicholsonweller. Terminal complement complex C5b-9 stimulates mitogenesis in 3T3 cells. J Clin Invest 91, 1974–1978 (1993). 31. F. Niculescu, T. Badea, and H. Rus. Sublytic C5b-9 induces proliferation of human aortic smooth muscle cells: role of mitogen activated protein kinase and phosphatidylinositol 3-kinase. Atherosclerosis 142, 47–56 (1999). 32. H. Rus, F. Niculescu, T. Badea, and M.L. Shin. Terminal complement complexes induce cell cycle entry in oligodendrocytes through mitogen activated protein kinase pathway. Immunopharmacology 38, 177–187 (1997). 33. S.J. Shankland, J.W. Pippin, and W.G. Couser. Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro. Kidney Int 56, 538–548 (1999). 34. K.S. Kilgore, E. Schmid, T.P. Shanley, C.M. Flory, V. Maheswari, N.L. Tramontini, H. Cohen, P.A. Ward, H.P Friedl, and J.S. Warren, Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation. Am J Pathol 150, 2019–2031 (1997). 35. K. Del Rio-Tsonis, P.A. Tsonis, I.K. Zarkadis, A.G. Tsagas, and J.D. Lambris. Complement factor C3 expression during limb regeneration. J Immunol 161, 6819– 6824 (1998). 36. Y. Kimura, M. Madhavan, M.K. Call, W. Santiago, P.A. Tsonis, J.D. Lambris, and K. Del Rio-Tsonis. Expression of complement 3 and complement 5 in newt limb and lens regeneration. J Immunol 170(5), 2331–2339 (2003). 37. D.J. Mastellos, C. Papadimitriou, S. Franchini, P.A. Tsonis, and Lambris, J.D. A novel role of complement: Mice deficient in the fifth component of complement (C5) exhibit impaired liver regeneration. J Immunol 166, 2479–2486 (2001).

6 SELF, NON-SELF, AND DANGER: A COMPLEMENTARY VIEW Jörg Köhl 1. INTRODUCTION The main purpose of the immune system is to guarantee the functional integrity of the body. As such it needs to recognize potential threats that could jeopardize functional body integrity and to eliminate such threats. This view demands sensors able to identify all kinds of potential danger. Initially, immunologists considered everything that is non-self as a threat, in particular pathogenic microorganisms. Vice versa, this model implied that self-derived molecules are innocuous and that the immune system is designed to discriminate self from 1,2 non-self . Importantly, immunity was mainly considered as cellular immunity, neglecting “unspecific” immune responses of innate immunity, including complement. As we know today, this view is certainly oversimplified as the immune system can recognize self and promote strong inflammatory responses against self-derived molecules, eventually leading to autoimmunity. Clearly, antigenpresenting cells (APCs) randomly sample molecules in their surroundings, present those antigens to T cells, and activate T cells through costimulatory signals, suggesting that cellular immunity per se cannot discriminate between self and non-self. A loophole for this dilemma was provided by the suggestion that germlineencoded structures may exist on APCs that recognize conserved pathogenassociated molecular patterns and that only this recognition licenses APCs to 3 induce T cell activation . This infectious non-self (INS) model fueled new interest in innate immunity research, resulting in the discovery of Toll-like receptors (TLR) that recognize conserved structures on all sorts of microorganisms. Data showing that TLR activation on DCs induces upregulation of costimulatory molecules and secretion of T cell-activating cytokines supported the model of 4 non-infectious self versus infectious non-self discrimination . However, sev-

Jörg Köhl, Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, MLC 7021, Cincinnati, OH 45229, USA. Phone: 513-636-1219; Fax: 513-636-5355, email: [email protected].

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eral obstacles remained, as the model does not provide a satisfactory explanation of the mechanisms underlying transplant rejection, development of autoimmunity, adjuvant activity of non-bacterial origin, or spontaneous regression of 5 occasional tumors . In order to reconcile these pitfalls, Matzinger proposed a 5,6 “danger model” of immunity . In this model, immunity is not designed to discriminate self from non-self but to recognize danger signals irrespective of their nature. In other words, immunity evolved as a system to recognize tissue damage rather than foreignness. In support of this view, endogenous ligands of pattern-recognition receptors (such as TLR) have been found that have the ability to 7 license APCs for T-cell activation independent of infection . The functional integrity of tissues is not only jeopardized by infectious or injury-related noninfectious threats but by ongoing cell death and the need to adequately clear and replace these cells. Obviously, immune responses are necessary to adequately address such physiological threats; however, the quality of such physiologic immunity is different. Inflammatory responses that are beneficial under pathological conditions — i.e., infection, toxic or traumatic tissue injury — are undesirable in response to physiologic threats. If so, how does the immune system discriminate between physiological and pathological threats? Further, certain humoral and cellular effectors such as complement factor 3 (C3) and natural killer (NK) cells are powerful weapons of innate immunity that can induce cell killing independent of any danger motifs. Tissue cells are protected from killing through expression of self-derived inhibitory molecules, and only the lack of these regulators instructs complement or NK-mediated suicide. These examples suggest that the INS or the danger model should not be considered as apodictic dogmas but as helpful frameworks that allow for a better understanding of how innate and adaptive immunity responses face the challenge of tissue integrity. INS and danger model-driven research mainly focused on the search for novel pattern-recognition receptors and ligands for such receptors. This focus neglected contributions of other arms of innate immunity, such as the complement system, which have the potential to recognize exogenous and endogenous threats and to shape innate as well as adaptive immune response. Complement has been primarily regarded as a host-defense system against infection that attracts leukocytes, flags microorganisms for facilitated phagocytosis, and kills bacteria through cell lysis. The purpose of this article is to discuss recent findings suggesting that complement is critically involved in tissue homeostasis, danger sensing, translation of danger recognition to cellular innate and adaptive responses and missing self-responses.

2. COMPLEMENT AS A “MASTER ALARM SYSTEM” OF INNATE IMMUNITY The complement system is a central arm of innate immunity comprising a network of more than 30 serum and cell-surface proteins, whose primary functions

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are recognition and elimination of microorganisms , clearing of immune com9 10,11 plexes and apoptotic cells , and mediation of inflammation . Recent findings provide evidence that complement also regulates innate as well as adaptive im12,13 mune responses . In evolutionary terms, complement is a phylogenetically ancient arm of innate immunity that evolved more than 700 million years ago. Currently available data suggest alternative and lectin pathway functions even in 14 such invertebrates as Ciona intestinales . During evolution the complement system was built around C3 (e.g., by gene duplication in the case of C4 and C5), stressing the critical importance of C3 and complement in general for survival of complex organisms. C3 can be cleaved by different mechanisms, i.e., in response to lectin, alternative, and classical pathway activation. As can be found in textbooks, the lectin pathway becomes activated when Mannan-binding lectin (MBL), a C-type lectin, recognizes conserved carbohydrate patterns and binds to MBL-associated 15 serine proteases MASP-1 and MASP-2 . The thioester within C3 preferentially 16 reacts with hydroxyl groups in polysaccharides as a signal to cleave C3 , nucleating the formation of a protease complex as the initial step of alternative complement pathway activation. Finally, natural or induced antibodies recognize conserved or variable molecule structures to form immune complexes that bind C1q and activate the classical complement pathway. As I will outline below, complement activation is much more complex than that. 5 Considering that the immune system has evolved to prevent tissue damage , complement may be regarded as a “master alarm system,” providing: (a) critical “danger sensors” that recognize harmful events of either infectious or noninfectious origin; and (b) “danger transmitters” that translate the danger signals into adequate immune responses. A prerequisite for the role of complement as a critical alarm system is that the initial “sensors” of the distinct complement pathways — i.e., C1q, MBL and C3 — recognize exogenous as well as endogenous danger motifs. Indeed, in addition to the well-appreciated binding to immune complexes, C1q has been found to bind cell-wall components and mem17 brane proteins of all kinds of microorganisms — the prion infectious agent as well as fragments of cellular and subcellular membranes (e.g., mitochondrial 18-21 membranes) and other modified host proteins and phospholipids . Further, it can bind C-reactive protein (CRP) and serum amyloid P (SAP) protein of the 22 pentraxin family . MBL recognizes polysaccharide structures on bacteria, fungi, 23 24 viruses, and parasites as well as on apoptotic host cells . C3 has a complex role as it functions as a sensor of the alternative pathway, serves as a substrate for proteolytic cleavage by the C3 convertases formed in response to classic and lectin pathway activation, and nucleates formation of the C3 amplification loop. The thioester in nascent C3, which allows covalent attachment to surfaces, is highly protected. Data from the recently uncovered C3 crystal structure provide a rationale for the protection of the thioester from rapid hydrolysis or reaction with nucleophils. C3 (as well as C4 and C5) belongs to the α2-macroglobulin family of proteins, comprising a core of eight homologous macroglublin

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domains (MGs; already found in metazoans) that evolved from a common an25 cestor . The inherent structural properties of the MG domains within the C3 25 molecule restrict the accessibility of the thioester . However, the protection is not absolute as C3 spontaneously hydrolyzes to form C3(H2O) at a very low rate (in which C3a is retained) and, in addition, reacts with nucleophils at low pace eventually leading to C3 cleavage. In fact, C3a can be found in plasma in the -8 26 range of 10 M in healthy individuals , supporting the view of permanent low27 level C3 cleavage, something that has also been observed in vitro . Numerous examples exist for spontaneous C3b deposition on microorganisms as well as on tissues (e.g., tumors) in response to alternative pathway activation. When the cells express complement regulators such as membrane cofactor protein (CD46) or complement receptor 1 (CR1;CD35), C3b is converted to inactive products through cleavage by factor I. If the integrity of the cell is distracted, either by apoptosis, injury, infection, or tumor transformation, cells can lose these regulators and become a target of complement-mediated immune responses. Collectively, these data suggest that the complement system provides a sophisticated system of “alarm sensors” that are able to detect changes in the physiologic environment of tissues.

3. COMPLEMENT-DERIVED DANGER-TRANSMITTERS SHAPE INNATE AND ADAPTIVE IMMUNE RESPONSES FOLLOWING PHYSIOLOGICAL AND PATHOLOGICAL THREATS The immune system needs to distinguish between physiologic changes in the environment, e.g., physiologic cell death and the necessity to clear these cells without substantial inflammation and pathological changes that demand inflammation. In short, danger-sensing needs to translate into adequate dangertransmission that tailors the immune response. The complement system is well suited for this task as it releases a panel of bioactive cleavage fragments, the function of which is regulated by interaction with soluble and cell-bound proteins. Cleavage fragments of C3 (e.g., C3a, C3b, iC3b, C3dg) have the ability to act as “danger-transmitters” that instruct cells of the innate and adaptive immune systems through interaction with specific receptors (C3aR, CR1 [CD35], CR2 [CD21], CR3 [CD11b/CD18], and CR4 [CD11c/CD18], Likewise, cleavage products of C5 — i.e., C5a as well as the membrane attack complex (C5b–9; MAC) — can transmit humoral inflammation and translate humoral into cellular inflammatory responses through activation of G-protein-coupled receptors (GPCRs). Differential expression of the distinct complement receptors on distinct cell populations as well as regulatory feedback loops that regulate their expression provide an important platform to tailor innate and adaptive immune response. In addition to the cleavage fragments of C3 and C5, C1q and MBL can function as danger-transmitters, besides their roles as danger-sensors (see above). As I will outline below, complement-derived signals shape innate and

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adaptive immune responses at many levels necessary to adequately address physiological and pathological challenges, including clearance of apoptotic and necrotic cells, removal of self-reacting B cells, building the natural antibody repertoire, keeping tolerance at mucosal surfaces, and providing powerful defense mechanisms against pathogens. 3.1. Danger Transmission through C1q Receptors C1q and MBL bind to several receptors, including cC1q-R/CR, or calreticulin (CR), which is also known as collectin receptor, gC1q-R, C1q-Rp (CD93), and 28 CR1 (CD35), the receptor for C3b . On macrophages and dendritic cells (DC), cC1q-R/CR and gC1q-R promote the uptake of apoptotic cells as well as immune complexes. Further, they mediate phagocytosis and killing of bacteria, viruses, fungi, and parasites. In addition to cC1q-R/CR and gC1q-R, C1q-RP 29 was found to enhance ingestion of C1q-targeted cells . The view of C1q-coating as an important physiological clearance mechanism for apoptotic cells has been supported by experiments with C1q-deficient mice that show profound impair30 ment in such clearance, resulting in severe glomerulonephritis . Further, the 31 lack of C1 in humans promotes a form of cutaneous lupus . The activation of cC1q-R/CR and gC1q-R on platelets and endothelial cells has been associated with increased cell adhesion and thrombosis, suggesting a link between complement and coagulation. This view is further supported by data showing that high-molecular-weight kininogens bind to a complex composed of gC1q-R, cytokeratin1, and urokinase plasminogen activator receptor on endothelial cells that is crucial for the assembly and activation of the intrinsic coagulation/kinin32 forming cascade . On B and T cells, cC1q-R/CR and gC1q-R regulate cell activation and proliferation. As may have been expected in terms of evolutionary pressure, pathogens have found ways to exploit many of the physiological functions of complement receptors for their survival. As an example, Hepatitis C virus can bind through its core protein to gC1q-R, leading to suppression of T33 cell proliferation . 3.2. Danger Transmission through C3 Cleavage Fragments 3.2.1. C3b Binding to Complement Receptor 1 C3 cleavage products provide a remarkably complex pattern of signals to direct immune responses. The primary cleavage product of C3 — C3b — binds to CR1, which is a single-chain type 1 transmembrane glycoprotein. In the circulation it is predominantly expressed on red blood cells. Here it serves as an immune adherence receptor for C3b-loaded immune complexes (IC) that are shuttled to liver and spleen, where they are transferred to and processed by tissue 34,35 macrophages through an Fc receptor-mediated process . This is an important physiological mechanism for safe removal of IC. When defective, it results in deposition of IC in tissues and promotes severe inflammation, as observed in

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SLE. In B cells, it is supposed to play important roles in maintaining tolerance against tissue-derived antigens in conjunction with CR2 (see below). In neutro36 phils and monocytes, CR1 can mediate phagocytosis , although other complement receptors appear more important in this respect (i.e., C1qR and CR3). Importantly, CR1 is the only cofactor protein for factor I that is able to promote degradation of C3b to C3dg, which shifts the specificity from CR1 to CR2 (discussed below). In addition to this physiologic properties, pathogens such as Leishmania, Mycobacteria, and HIV use C3b deposition on their surface to enter 37 host cells through CR1 . Further, several publications suggest an important role 38 for CR1 in the pathogenesis of severe malaria related to rosette formation . 3.2.2. C3dg Binding to Complement Receptor 2 The terminal cleavage fragments of C3, C3dg, or C3d bind to complement receptor 2 (CR2; CD21), which is primarily expressed on B cells and follicular dendritic cells. Further, CR2 expression has been found on subsets of CD4 and 39,40 41 CD8 thymic and peripheral T cells , on activated T cells , basophils, mast cells, keratinocytes, and epithelial cells. On B cells, CR2 is part of the B-cell receptor complex. C3d binding to CR2 results in a dramatic (1000-fold) reduc42 tion of the B cell activation threshold . Further, B-cell responses are substantially impaired in mice when the Cr2 locus is disrupted, resulting in a deficiency of both CR1 and CR2. These mice suffer from a defect in T cell-dependent anti43 gen responses and are prone to develop autoimmunity as a result of defective 44 elimination of self-reactive B-cells . In addition to the regulatory effects on T cell-dependent antigens, an increasing body of data suggests a critical role for CR2 in shaping the natural antibody repertoire (reviewed in [45]). Natural antibodies are mainly produced by a subset of long-lived, self-replenishing B cells, i.e., B-1 cells. As germline-encoded molecules, they serve as low-affinity pattern recognition receptors that recognize pathogen as well as tissue-derived -/patterns. Importantly, Cr2 mice lack a subset of the natural antibody repertoire that is critical to recognize hypoxia-related damage on endothelial cells. On the -/one hand, this can be beneficial, as Cr2 mice are protected from intestinal 46,47 ischemia/reperfusion injury . However, the impaired ability to recognize damaged cells may also lead to defective clearance and promote autoimmune responses. Collectively, these data demonstrate that the complement system is an integral part of the B-cell compartment that shapes and regulates both the innate and the adaptive part of the B-cell response (reviewed in [48]). 3.2.3. iC3b Binding to Complement Receptors 3 and 4 Once C3b is covalently attached to a cell membrane, it can become part of the amplification loop of the alternative pathway, which results in accelerated C3b deposition, or it can be further degraded to iC3b, C3c, and C3dg, depending on the soluble (factors B, H) and membrane-bound (CR1, CD46) molecules that serve as binding partners for C3b. The decision on the fate of C3b is primarily

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dependent on the carbohydrate environment . Provided that a sialic acid-rich environment favors factor H binding to C3b, it acts as a cofactor for factor I to cleave C3b into iC3b and C3f. iC3b can bind to CR3 and CR4, both of which are β2 integrins, functioning as important phagocytic receptors. CR3/4 are promiscuous receptors that bind a panel of different endogenous and exogenous 50 ligands such as fibronectin, ICAM-1/2, β-glucan, or LPS . Importantly, CR3/4mediated phagocytosis does not per se elicit proinflammatory signals in phagocytes, nor does it provoke respiratory burst. Considering that CR3 has primarily evolved to serve physiologic functions necessary to balance tissue homeostasis, i.e., by clearing of apoptotic cells, the lack of inflammation is not surprising. However, CR3 is also considered an important receptor in innate immune defense against pathogens, promoting leukocyte adhesion and transmigration through the endothelium as well inflammatory response. Importantly, CR3 needs to be pre-activated to adopt a high-affinity state before ligation results in an inflammatory response. Signaling pathways that induce the high-affinity status include those downstream of activating immunoglobulin G receptors 51 52 (FcγRs) or chemoattractant GPCR . Of note, oleic acid and the 14-kDA mye53 loid related protein (MRP-14) , which are released after tissue or cell injury, are also able to induce the high-affinity state of CR3, suggesting that danger signals per se, but not the discrimination between infectious- or noninfectious danger signals, are critical to CR3-mediated inflammatory effector functions. As described for other complement receptors, several pathogens exploit CR3 to invade phagocytes and to escape the immune response. Examples are Histoplasma capsulatum, Leishmania major, and several Mycobacteria. Cellmediated immunity is a critical defense mechanism to kill these microorganisms and/or to limit their systemic spreading. Interferon gamma (IFN-γ) released from + Th1-polarized CD4 lymphocytes is crucial to infection control. Importantly, ligation of CR3 (by iC3b and other ligands) has been found to suppress produc54 tion of IL-12 from monocytes in response to bacterial stimulation . IL-12 is a heterodimeric cytokine that plays a key role in bridging innate and adaptive im+ mune responses, as it activates and skews CD4 T cells toward a Th1 pheno55 type . Thus, suppression of IL-12 provides an important mechanism for these pathogens to escape elimination by adaptive immune responses. Obviously, it is harmful for the host to shut down adaptive immunity following infection with these intracellular pathogens. In light of the physiologic functions of CR3, i.e., apoptotic cell clearance (see above), IL-12 suppression might be beneficial as it prevents unwanted activation of cell-mediated immunity. It will be interesting to see whether the status of CR3 (high or low affinity) impacts the suppressive effect on IL-12 production. 3.2.4. C3b Binding to Membrane Cofactor Protein (CD46) Another example of the regulatory impact of the danger-transmitter C3b comes from its interaction with the regulator protein CD46, which is ubiquitously expressed on all nucleated cells. CD46 acts as a cofactor for factor I and promotes

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cleavage of C3b into iC3b. Its main control function is to protect tissues from complement-mediated attack by preventing deposition of C3b in response to alternative pathway activation and formation of the amplification loop. Impor56 tantly, CD46 expression is reduced in dying cells , suggesting that the decrease of this self-derived molecule is important in the recognition and clearing of apoptotic cells. In support of this view, the Gasque lab has recently shown that CD46 is rapidly translocated from cytoplasmic, nuclear, and membrane stores 57 into membrane-bound apoptotic blebs, budding from the cell surface . They further demonstrated early exposure of nucleic acids on the cell membrane, functioning as a pattern recognition motif for C1q, activating the classical pathway. In consequence of the CD46 deprivation, associated with preserved CD55 and CD59 expression, preventing MAC formation, the cells become heavily loaded with C3b, making them an excellent target for CR3-mediated phagocytosis. Collectively, these data suggest that CD46 may play a similar role as major histocompatibility complex classs I, the lack of which allows innate immunity to recognize altered self and to eliminate the altered cells through NK cellmediated cytotoxicity. In addition to its regulatory role on apoptotic cell elimination, CD46 impacts the development and maintenance of adaptive immune responses. Similar to the effect of iC3b binding to CR3, ligation of CD46 on monocytes by C3b 58 dimers suppresses production of IL-12 . This pathway has been demonstrated to be exploited by measles virus and may explain the immunosuppression follow58 ing measles virus infection . CD46 is also a ligand for several other pathogens such as Neisseria, Herpesvirus 6, Streptococcus pyogenes, and Adenoviruses + (for review see [59]). On CD4 T cells, C3b-mediated activation of CD46 in the presence of TCR stimulation results in proliferation of a T cell population with a 60 regulatory phenotype .

4. DANGER TRANSMISSION MEDIATED THROUGH THE ANAPHYLATOXIC PEPTIDES C3A AND C5A Serine proteases generated in response to activation of the three pathways of the complement system, as well as allergen- and tissue-derived proteases, can cleave the low-molecular-weight anaphylatoxic peptides (AT) C3a and C5a from C3 and C5, respectively. The ATs act as potent mediators of inflammatory effector functions, such as phagocyte and mast cell recruitment and activation, including the release of granule enzymes, reactive oxygen species, cytokines, and 11 chemokines, as well as upregulation of adhesion molecules . Most of these properties are mediated through ligation of specific and distinct C3a and C5a receptors (see below). Consonant with the proinflammatory properties, ATs have been implicated in the pathogenesis of several infectious, autoimmune, and 61 62 63,64 allergic diseases such as pneumonia , sepsis , rheumatoid arthritis , systemic 9 65 66-68 lupus erythematosus , multiple sclerosis , and allergic asthma . In short, the

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release of the AT is considered to contribute significantly to the benefit and burden of inflammation. In line with the view of complement as a crucial alarm system, several reports provide evidence for an important role of the ATs (and the membrane attack complex) in the development of the inflammatory response following ischemia/reperfusion injury (reviewed in [11]). Here, exposure of hypoxia-induced expression of neoepitopes on endothelial cells can activate the three different pathways of the complement system to release C3a and C5a. These well-appreciated proinflammatory properties of the AT are critical to shape the innate inflammatory response in response to tissue damage/destruction and/or attack by pathogens. As I will outline below, ATs exert regulatory functions far beyond their role as proinflammatory effector molecules of innate immunity. I will provide examples demonstrating that ATs regulate tissue repair, tissue remodeling, and shape adaptive immune responses at the mucosal surface, and impact TLR-induced danger responses. 4.1. Anaphylatoxin Receptor-Dependent and Independent Effects The ATs unfold their functions through distinct G-protein-coupled receptors 69-72 (GPCR) expressed preferentially on myeloid lineage-derived leukocytes . In leukocytes, C3aR and C5aR (CD88) mediate their effects via coupling to the 73-76 pertussis-sensitive and -insensitive G-proteins Gαi and Gα16, respectively . In 77 endothelial cells the C3aR also couples to Gα12 and Gα13 . The downstream pathways have been intensively studied in neutrophils, monocytes, and macrophages. In neutrophils C5aR signals through Gαi and activates phosphoinositide3-kinase γ (PI3Kγ). PI3Kγ belongs to a family of lipid kinases involved in generating distinct phosphoinositides that are important second messengers for intracellular signaling. C5a activates PI3Kγ through coupling to distinct Gproteins: (1) Gαi, which activates the small GTPase Ras, and (2) Gβγ. In turn, Ras or PI3Kγ can activate the Raf/mitogen-activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) kinase (MEK)/ERK cascade, which links C5aR ligation to gene expression through effects on transcription factor activation and translocation to the nucleus. In monocytes and macrophages coupling of the C5aR to Gαi and Gα16 has been described. The Gα16 subunit and the βγ-subunit can activate phosphoinositide-specific phospholipase C (PLC) subtype β, which is a multidomain phosphodiesterase that generates the second messenger inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These 2+ latter molecules mediate Caҙ mobilization and activation of protein kinase C (PKC). Downstream of PKC, signal transducer and activator of transcription 3 (STAT3) can be activated by C5a (through Janus kinase (Jak) phosphoryla78,78 tion) , suggesting a role for C5a in cell proliferation. In fact, C5a (and C3a) 79 were found to be essential for liver regeneration . In further support of this view, C5aR signaling induces epidermal growth factor (EGF) receptor phos77 phorylation in endothelial cells . EGF is a strong mitogen for human airway 80 smooth muscle cells via activation of the ERK MAPK pathway .

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In addition to CD88, the orphan receptor C5L2 has been described as a sec81-83 ond receptor for C5a . Interestingly, C5L2 binds C5adesArg with a tenfold 82,83 higher affinity than CD88 . In contrast to CD88, C5L2 is uncoupled from Gproteins. C5L2 ligation does not result in degranulation, an increase in intracel2+ lular Ca , or receptor internalization, suggesting that C5L2 signaling does not follow the classical pathways of chemoattractant receptors. The overall mRNA 84 expression levels of C5L2 in murine tissues is much lower than that of CD88 . However, the situation can change dramatically in acute inflammation, which may impact the inflammatory response. During CLP-mediated septic peritonitis, C5L2 surface expression markedly increased on neutrophils, whereas CD88 expression decreased. In contrast, expression of both receptors increased in tissues (lung, liver, kidney, and heart). Blockade of either receptor had an opposite effect on production of IL-6, i.e., C5aR blockade resulted in reduction whereas 85 C5L2 blockade resulted in an increase of IL-6 . Similarly, inflammation was -/enhanced in C5L2 mice in a model of immune complex alveolitis, as evidenced 84 by increased cellularity and proinflammatory cytokines in BAL . Collectively, these data suggest that C5a/C5adesArg binding of C5L2 limits the proinflammatory properties of these ATs. The opposing roles of the CD88/C5L2 tandem provide a sophisticated feedback mechanism that allows the complement system to regulate C5a-mediated danger transmission. Several reports in the literature suggest AT effects that are independent of AT receptor ligation. This is particularly true for C3a and its degradation product C3adesArg. The latter is devoid of binding to C3aR; however, similar to C3a, it can suppress LPS-induced cytokine production from tonsil-derived B 86-88 cells and monocytes . Further, C3adesArg stimulates pituitary cells to release 89 prolactin, growth hormone, and adrenocorticotropin . C3adesArg has also been termed acylation-stimulating protein (ASP) by one group referring to its role in 90 triacylglycerol synthesis in human adipocytes . All of the C3adesArg biology has been enigmatic, as it does not fit in our mechanistic understanding of biological functions as receptor mediated entities. Although a recent publication suggests that C5L2 may be the missing link, C3adesArg binding to this receptor 91,92 and signaling is controversial . Data from the crystal structure of C3 may provide a loophole to this di25 lemma . The paper shows that important domains with specialized functions have been added to the core region of C3 through a series of gene insertion events, one of which is the insertion of C3a into a loop within the MG6 domain. Thus the “original” function of C3a prior to this insertion may have been distinct from its function related to C3aR signaling found later in evolution. C3a is a highly cationic peptide containing four α-helical regions, structural features that resemble those of antimicrobial peptides. Indeed, C3a and C3adesArg exert antimicrobial activity toward Gram-negative and -positive bacteria in the low-µM 93 range . As most of the C3a generated in the circulation is rapidly converted into C3adesArg by means of serum carboxypepidase N, the original function of C3a (direct microbial killing) may be preserved in situations of massive systemic

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complement activation such as in sepsis. Interestingly, antimicrobial peptides 94 have been found to exert immunoregulatory functions as described for C3a . These functions may originate from cell activation by peptide insertion into cell membranes or through binding to promiscuous GPCRs, as has been shown for cathelicidins, which can activate FPRL1 or β-defensins, which can signal through CCR6.

5. ANAPHYLATOXIN-MEDIATED DANGER TRANSMISSION IN NON-MYELOID CELLS In addition to expression on professional inflammatory cells, AT receptor expression has been demonstrated on non-myeloid cells such as endothelial 77,95-97 96,98 98 99,100 cells , epithelial cells , smooth muscle cells , and fibroblasts , as well as 101,102 98,103 100,103 104,105 on tissue cells of kidney , lung , liver , and brain . The presence of AT receptors on tissue cells suggests AT-mediated immune functions beyond inflammation. Complement activation in response to exogenous and endogenous danger signals is often associated with cell and/or tissue injury. In addition to the safe removal of cells (discussed above), cells need to be replaced to maintain or reestablish normal tissue function. The mechanisms underlying tissue repair and remodeling are complex. Out of all organs, the liver has the greatest potential to regenerate damaged or destroyed cell mass. Liver regeneration proceeds through distinct stages, including priming of hepatocytes, cell cycle progression, proliferation, and cessation of regeneration. In models of acute toxic liver injury and partial hepatectomy, the Lambris lab has recently shown that liver regeneration 79,106 is severely impaired in AT-receptor-deficient mice . Mechanistically, they found direct effects of AT-receptor signaling on hepatocytes and on Kupffer cells, which increased IL-6 and TNF-α production, which in concert activated transcription factors (NFk-B, STAT-3) crucial for liver cell regeneration. Chronic liver tissue injury of infectious or noninfectious origin often leads to fibrotic remodeling of the tissue architecture. Recently, a gene locus in the mouse genome was identified by quantitative trait locus analysis that confers susceptibility to liver fibrosis. Importantly, the gene locus included the C5 gene. In a murine model of chronic liver injury, pharmacological targeting of the C5aR had antifibrotic effects in vivo. Further, some common polymorphisms of the human gene C5 were found to be associated with advanced fibrosis in chronic hepatitis C virus infection. These data support the idea that C5 has a 100 causal role in fibrogenesis across species . Similarly, C5 had a detrimental effect during chronic stages of bleomycin-induced pulmonary injury, indicating a profibrotic role for C5. This deleterious activity of C5 was associated with expression of the fibrogenic cytokine TGF-β1 and matrix metalloproteinase-3, an 99 important mediator in fibroblast contraction . Collectively, these data suggest that, in case of acute cell injury, ATreceptor activation on tissue cells and on tissue macrophages is an important

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signal for proper tissue repair. In contrast, triggering of C5aRs on fibroblasts through ongoing activation of complement-derived danger sensors and subsequent release of the danger transmitter C5a may result in defective repair, i.e., fibrotic remodeling of chronically injured tissue.

6. C5A RECEPTOR SIGNALING IN PULMONARY DENDRITIC CELLS REGULATES INHALATION TOLERANCE As outlined above, ATs instruct innate immune responses in many ways. The presence of AT receptors on APCs suggests that they may also be involved in the development of adaptive immune responses. AT receptor expression has been found on distinct DC populations and was shown to induce several effector functions: (a) DCs isolated from rat respiratory tract tissue, or generated in vitro 107 from human monocytes, migrate in response to C5a ; (b) human Langerhans cells (a type of dermal immature DC) express C5aR and respond chemotacti108 cally to C5a ; (c) Langerhans cell C5aR expression increases during maturation 109 and tissue trafficking ; (d) human dermal DCs and monocyte-derived immature 110 DCs express C5aR and C3aR ; (e) C5a induces calcium fluxes in such dermal DCs, whereas C3a does not; (f) expression of both AT receptors is downregu111 lated on such dermal DC by TNF-α ; and (g) both human and murine monocyte-derived immature DCs and mature DCs respond to C5a as determined by 2+ 111 chemotaxis and Ca mobilization . Thus, interactions involving C5a have potent functional effects on both immature and mature DCs. Mucosal DCs play important regulatory roles in the respiratory and intestinal tracts, where they control the immune response to inhaled or ingested antigens. As immature DCs, they continuously sample proteins, process these proteins, and migrate to the regional lymph nodes. During this process they change their phenotype and express high levels of MHC II and a panel of costimulatory molecules. Upon contact with naive T cells, they stimulate T-cell proliferation. Importantly, this DC/T cell interaction does not induce immunity, but toler112 ance . Under steady-state conditions, immature DCs take up harmless exogeneous as well as endogeneous antigens such as airborne particles or apoptotic cells. According to the INS model, endogenous antigens or harmless exogenous antigens are unable to promote full T-cell activation, as the mucosal DC has not received an infectious (non-self) danger signal that would then induce/upregulate all of the pathways (MHC II, costimulatory molecules, and cytokines) necessary to promote adaptive immunity. As pointed out above, apoptotic cells are recognized by complement-derived danger sensors, suggesting low-level complement activation at mucosal surfaces. Indeed, trace amounts of C3a and C5a have been found in bronchoalveolar lavage samples from healthy volun113,114 teers . Further, C5aR are expressed on distinct subsets of airway DCs. Together these data suggest a possible role for C5a in mucosal tolerance, particularly in the lung. In support of this view, we recently found that ablation of

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C5aR signaling in mice results in spontaneous development of a Th2-polarized immune response. Moreover, repeated pulmonary exposure to ovalbumin, resulting in inhalation tolerance, induced a strong Th2 adaptive immune response, associated with eosinophilic and lymphocytic airway inflammation and in115 creased airway responsiveness in the absence of C5aR signaling . These data suggest a dynamic model of inhalation tolerance in which C5aR signaling in pulmonary DCs is necessary to prevent their full activation and subsequent induction of adaptive immunity. Thus, adaptive immune responses not only develop when infectious (non-self) or noninfectious danger signals trigger TLRs, but also in the absence of complement-derived danger transmitters. As is evident from these data, the term “danger transmitter” does not adequately describe the effect of C5a on DCs, which is suppressive but not activating. Rather, C5a needs to be considered an endogenous, soluble self-recognition signal, the lack of which indicates danger to the DC and initiates T-cell activation. Thus, C5a may be considered as a “homeostasis transmitter” that prevents inadequate and potentially harmful immune responses. The decision as to which of the functions dominates (danger vs. homeostasis) is likely to depend on the cell type and/or additional signals, such as TLR-signaling (see below). Collectively, these data complement the missing-self concept, in which the lack of cell-bound receptors instructs the innate immune system (NK cells, complement system) to eliminate cells with altered self motifs. Here, the lack of C5a or, more precisely, that of C5aR signaling, instructs innate immunity (DCs) to activate adaptive immune responses. Accordingly, “homeostasis transmitters” are likely to play important roles in preventing autoimmune responses, e.g., following apoptotic cell uptake by APCs. Recent studies provide evidence that distinct properties of two phenotypic and functional distinct pulmonary DC subsets, i.e., myeloid DC (mDC) (CD11c+, CD11b+, Gr1–) and plasmacytoid DC (pDC) (CD11c+, CD11b–, Gr1+) are crucial to the development of adaptive immune responses in the 116 lung . In a murine model of experimental allergic asthma, adoptive transfer of 117 mDCs confers immunogenicity , whereas adoptive transfer of pDCs results in 118 inhalation tolerance . In agreement with these data, we recently found that only + mDCs but not pDCs stimulate sensitized CD4 lymphocytes to produce high amounts of IL-5, IL-13, and IL-10 ex vivo. In addition, our data demonstrate that pulmonary pDCs have the ability to actively suppress the mDC-induced + 115 activation of sensitized CD4 lymphocytes ex vivo . In support of the view that pDCs play a critical role in inhalation tolerance, Oriss et al. reported increased accumulation of pDCs in lymph nodes of tolerized mice, whereas under conditions that induce airway inflammation mDCs became the predominant cell 119 type . We found a shift in the relative proportion of mDCs to pDCs in response to: (a) allergen exposure; and, more importantly, (b) C5aR ablation. In fact, in the absence of C5aR signaling, the mDC/pDC ratio was twice or three times as high as in the presence of C5aR signaling. These data not only support the concept that airway tolerance vs. allergy is determined by the relative proportion of

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distinct DC subsets, but that C5aR signaling during allergen sensitization regulates this balance. At this point, the mechanisms underlying this regulation remain elusive. C5a may affect the mobilization of mDC precursors from bone marrow, increase mDC recruitment, and/or increase mDC survival. The importance of this finding is supported by recent data demonstrating that asthmatic 120 children have lower numbers of circulating pDCs than healthy controls . Collectively, these data suggest a critical role of C5aR signaling at the DC/T cell interface that prevents the development of maladaptive immune responses at the mucosal surface.

7. C5A RECEPTOR SIGNALING ON APCS IMPACTS DANGER TRANSMISSION THROUGH TLRS Many exogenous or endogenous molecular patterns, either derived from pathogens such as LPS, glycolipds, and teichoic acid, or from tissues such as modified LDL or necrotic cells, can be recognized by complement-derived danger sensors and TLRs, suggesting that complement receptor pathways may intersect with TLR pathways. The first evidence of a regulatory link between the complement system and TLR-mediated immune responses was provided by reports demon54 58 strating that activation of CR3 and the complement regulator molecule CD46 by C3 cleavage products promote efficient control of TLR-induced synthesis of IL-12 from human monocytes. Further, the C5 cleavage fragment C5a was found to downregulate LPS or Staphylococcus Cowan strain 1 (SAC)-induced 121,122 synthesis of IL-12 in human monocytes but not in monocyte-derived DCs . In contrast, ablation of C5 in murine macrophages led to reduced IL-12 production in response to IFN-γ + SAC stimulation, suggesting that C5a enhances IL123 12 production . These conflicting data suggest a complex, cell-dependent role of C5a in IL-12 regulation. The underlying molecular mechanisms and the consequences of such regulation on adaptive immunity and infection are poorly understood. First mechanistic insights into the crosstalk between TLR4 and C5aR signaling pathways have recently been provided in a report, demonstrating that C5a negatively impacts TLR4-induced synthesis of IL-12 family cytokines IL-12, IL-23, and IL-27 from murine macrophages through activation of signaling pathways that involve extracellular signal-regulated kinase (ERK) and phospho124 inositide 3-kinase (PI3K) . In addition to its role in dampening TLR4-driven synthesis, C5a also suppressed CD40-induced IL-12 family cytokine production through the signaling intermediate ERK1/2. The CD40–CD154 (CD40 ligand) + interaction provides an important feedback loop by which activated CD4 T cells amplify production of IL-12. Further, IFN regulatory factor 1 (IRF-1) and IFN consensus sequence binding protein (ICSBP; IRF-8) were identified as crucial transcription factors downstream of the ERK- and PI3K pathways, respectively. These data indicate that C5a modulates both innate (TLR4) and adaptive (CD40)

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immune responses that drive the production of IL-12 family cytokines. Such broad inhibition is likely to affect the host response to infection and to modulate autoimmunity. In support of this view, a central role of C5/C5a was suggested in 64 125 126 the pathogenesis of autoimmune arthritis , systemic lupus , DTH responses , 123 127 and allergy , as well as in resistance to Listeria and to blood stage-malaria 128 infection . Common to all of these models is their dependency on or exacerba124 tion by IL-12 family cytokines. In the same report , a suppressive effect of C5a on Th1 polarization was found the in vivo relevance of which was documented by the acquisition of resistance to Leishmania major infection by the genetic deficiency of the C5a receptor in normally susceptible BALB/c mice. In this model, L. major takes advantage of the activation of one important defense mechanism of innate immunity (the complement system) to suppress cellmediated immunity induced by another crucial arm of innate immunity, i.e., the TLR system. This immune evasion strategy may be used by other intracellular pathogens as well, such as Mycobacteria, Listeria, Histoplasma, or even HIV. The crosstalk between TLR4 and the C5aR is not restricted to release of IL12 family cytokines. The febrile response following TLR4 ligation appears to depend on complement activation and the interaction of C5a with its receptor as pharmacological targeting of the C5aR prevents LPS-induced fever, at least in mice. Several lines of evidence suggest that C5a stimulates Kupffer cells to release prostaglandin E2, which binds to vagal afferents in the liver transmitting 129 the pyrogenic information to the brain .

8. SUMMARY Complement is a sophisticated system of molecules that is critical to the functional integrity of the body. Initially considered as a defense system to ward off infections, it becomes increasingly clear that the complement system is one of the most important humoral systems to sense danger, i.e., to recognize conserved patterns on pathogens and on altered/damaged self. In addition to this important role in danger recognition, the complement system has the ability to translate the danger information into an adequate cellular innate or adaptive immune response. This is accomplished by two distinct mechanisms: (a) danger sensors that have recognized altered cells or pathogens can directly activate cellbound receptors (e.g., C1q / C1q receptor interaction), and/or (b) danger sensors initiate cleavage of complement factors C3 and C5, the fragments of which acquire the ability to bind to complement receptors and/or regulators. It is the specific interaction of the danger sensors and of the cleavage fragments with distinct cell-bound receptors/regulators that directs the immune response toward an innate or an adaptive phenotype. Further, the expression pattern of the complement receptors critically impacts the shape of the immune response. Complement has the ability to discriminate between physiological and pathological danger, i.e., physiological cell death and death in response to injury. In the former

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case, cells are merely flagged for enhanced phagocytosis (by C3 fragments) without accompanying inflammation (through CR3), whereas in the latter case inflammatory signals are accessorily triggered (e.g., by the release of ATs, which recruit and activate neutrophils, eosinophils, etc.). This function is of major importance for apoptotic cell clearance and tissue repair but plays also important roles in fibrotic tissue remodeling in response to chronic tissue injury. Further, complement cleavage fragments may prevent the development of maldaptive immune responses at the mucosal surface. Here, complement fragment C5a does not act as a danger transmitter but as a “homeostasis transmitter,” as its interaction with the C5a receptor on DCs provides a signal that prevents DCs + from activating CD4 T cells. The generation of regulatory T cells in response to CD46 ligation may have a similar function, as injured cells lose CD46 expresssion, which may lead to decreased proliferation of Tregs and, consecutively, increased production of T effector cells. Although we are still at the beginning of understanding the complex interaction patterns within the complement system, recent data suggest substantial crosstalk between the signaling pathways downstream of complement receptors and other receptors of the innate immune system that function as immune sen130,131 sors and/or transmitters (i.e., TLRs, FcγRs ). Given the importance of complement as a sensor and effector system of innate and adaptive immune responses, a complement-related view of the immune system might help to unravel some enigmas of autoimmunity, allergy, and transplantation.

9. ACKNOWLEDGMENTS: This work is funded by National Institute of Health grants R01-AI057839 and R01-AI059305 to JK.

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7 GC1QR/P33 SERVES AS A MOLECULAR BRIDGE BETWEEN THE COMPLEMENT AND CONTACT ACTIVATION SYSTEMS AND IS AN IMPORTANT CATALYST IN INFLAMMATION Berhane Ghebrehiwet, Claudia CebadaMora, Lee Tantral, Jolyon Jesty, and Ellinor I. B. Peerschke

1. ABSTRACT The receptor for the globular heads of C1q, gC1qR/p33, is a ubiquitously expressed protein, which is distributed both intracellularly and on the cell-surface protein. In addition to C1q, this molecule also is able to bind several other biologically important plasma ligands, including high-molecular-weight kininogen (HK), factor XII (FXII), and multimeric vitronectin. Previous studies have shown that incubation of FXII, prekallikrein, and HK with gC1qR leads to a zinc-dependent and FXII-dependent conversion of prekallikrein to kallikrein, a requisite for kinin generation. In addition, these studies showed that normal plasma, but not plasma deficient in FXII, PK, or HK, activate upon binding to endothelial cells (EC), and that this activation could be inhibited by antibody to gC1qR. In these studies, we show that incubation of serum with microtiter plate bound gC1qR results in complement activation, as evidenced by the binding and activation of C1 and generation of C4d. However, neither C1q-deficient serum nor a truncated form of gC1qR (gC1qR∆74-96), supported complement activation. Taken together, the data strongly suggest that at sites of inflammation, such as vasculitis and atherosclerosis, where gC1qR as well as its two important plasma ligands, C1q and HK, have been shown to be simultaneously present, soluble or cell-surface-expressed gC1qR may contribute to the inflam-

Berhane Ghebrehiwet, Claudia CebadaMora, Lee Tantral, Jolyon Jesty, Department of Medicine, SUNY at Stony Brook, Stony Brook, NY 11794, USA. Ellinor I.B. Peerschke, Department of Pathology, Weill Medical College of Cornell University, NY, 10021, USA.

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matory process by modulating complement activation, kinin generation, and perhaps even initiation of clotting via the contact system. Based on these and other published data, we propose a model of inflammation in which atherogenic factors (e.g., immune complexes, virus, or bacteria) are perceived not only to convert the endothelium into a procoagulant and proinflammatory surface, but also to induce enhanced expression of cell surface molecules such as gC1qR. Enhanced expression of gC1qR in turn leads to: (i) high-affinity C1q binding and cell production of proinflammatory factors, and (ii) high-affinity HK binding and facilitation of the assembly of contact activation proteins leading to generation of bradykinin and possibly coagulation through activation of FXI.

2. INTRODUCTION The receptor for the globular heads of human C1q, gC1qR/p33 ⎯ henceforth referred to as gC1qR ⎯ consists of three identical 33-kDa monomers that form a stable homotrimeric protein, capable of binding C1q with high affinity and 1-2 specificity . This conclusion is derived not only from direct radioligand binding studies, which showed specific binding with an estimated binding affinity of approximately 15–35 nM, but also from studies that demonstrated selective capture of C1q when normal human serum was passed over a gC1qR affinity col1 umn . Previous experiments have also shown that native gC1qR purified from Raji cell membranes, or highly purified, and enzyme-free recombinant gC1qR, inhibits serum hemolytic activity by apparently competing with immune com1 plexes for the same binding site(s) on C1q . On the basis of these results, we had hypothesized that the binding site(s) for gC1qR and IgG on the C1q molecule 1 may be identical or perhaps overlap each other . In fact, subsequent ligand blot analysis has shown that not only does gC1qR bind preferentially to the A chain of C1q but also to a synthetic peptide corresponding to residues 155–164 of the 3 A chain, postulated to contain a putative site for IgG . Furthermore, residues 155–164, which contain the two adjacent arginine residues at positions 162 and 3 163, which had been shown to be important for IgG binding , also supports bind2 ing of gC1q-R . The specificity of this interaction was validated when substitution of the two arginine residues (R) by glutamine (Q) failed to support gC1qR 2 binding . Functional peptide mapping analysis revealed that the N-terminus of gC1qR corresponding to residues 76–93 contains a putative binding site for C1q and that this binding is inhibited by monoclonal antibody (mAb 60.11), which rec4 ognizes an epitope within this domain . Analysis of the geometry and topogra5 phy of the three-dimensional structure of gC1qR together with localization of 6 the mAb 60.11 sites indicate that gC1qR could potentially engage C1q via at least two, if not three, of its globular heads without much spatial constraint. This in turn can potentially induce the requisite conformational change that is neces7 sary to produce the subtle signal for C1 activation .

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In addition to serving as a cell-surface receptor for C1q, gC1qR is also released as a soluble protein at sites of tissue injury and inflammation probably from infected or damaged cells, or cells undergoing apoptosis. Therefore, because the potential for this molecule to contribute to the complex pathways of the inflammatory process by activation of complement is high, the present studies were undertaken to examine more closely the interaction between gC1qR and C1q within the context of complement activation using a highly sensitive microplate assay for detection of the activation product C4d.

3. MATERIALS AND METHODS 3.1. Chemicals and Reagents The following chemicals and reagents were purchased from the sources indicated: alkaline phosphatase conjugated goat anti-mouse or goat anti-rabbit IgG, alkaline phosphatase conjugated extravidin or streptavidin, and NHS-LC-biotin (556.58 kDa) (Pierce, Rockford, IL); Phe–Pro–Arg–chloromethylketone (PPACK) (Haematologic Technologies, Inc., Essex Junction, VT); biotinlabeled monoclonal anti-human C4d, C1q, and C1q depleted sera (C1qDS) (Quidel Corp., Santa Clara, CA); human thrombin, bovine serum albumin, IgG anti-sheep red blood cell stroma (hemolysin), isopropyl β-D-1 thiogalacto pyranoside (IPTG) (Sigma-Aldrich, St Louis, MO); and sheep red blood cells (Colorado Serum Co, Denver, CO). 3.2. Proteins and Antibodies The human C1q that was used to generate antibodies was prepared by the 8 method of Reid as described earlier . Polyclonal anti-C1q antibodies were generated by immunization of either goats or rabbits with highly purified C1q that was free of IgG, IgM, or C1q inhibitor (chondrotin 4-sulfate proteoglycan) that 9 often contaminates C1q preparations . These antibodies are therefore part of our antibody bank. 3.3. Biotinylation of Proteins Biotinylation of proteins was accomplished using NHS-LC-biotin (556.58 kDa) 10 according to well-established procedure . Proteins to be labeled (0.5–1 mg/ml) were first dialyzed against 2 X 1L of 0.2-M NaHCO3 buffer, pH 8.3, and labeling initiated by addition of 50 µg/ml biotin freshly dissolved in double-distilled water or DMSO. The reaction was allowed to proceed for 4–5 h at room temperature with a constant but gentle tumble mixing, after which the reaction was stopped and the labeled protein separated from free biotin by filtration on a PD10 column. The pooled protein sample was further dialyzed against 2 x 1 L PBS, pH 7.5, and the degree of biotinylation verified by a standard ELISA using wells

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coated with serial dilutions of the labeled protein and probing with either alkaline phosphatase conjugated streptavidin or ExtrAvidin. 3.4. Expression of Recombinant gC1qR The construction and expression of a plasmid pGex-2T containing an insert encoding the mature form (residues 74–282) as well as truncated forms ∆74–96 and (∆276–282) of gC1qR, as well as expression, purification, and characteriza1,11 tion of the recombinant gC1qR have been described in detail previously . Briefly, the cDNAs were subcloned downstream of the glutathione-S-transferase (GST) gene in the expression plasmid, pGex-2T (Pharmacia Biotech Inc.), transformed into E. coli BL-21 (DE3) and protein expression induced by 0.5-mM IPTG (isopropyl β-D-1 thiogalacto pyranoside). The proteins were expressed as GST fusion products with the GST at the N terminus of the gC1qR. The fusion products were purified on a glutathione-Sepharose 4B column. The gC1qR fusion products were then cleaved by thrombin and the GST-free gC1qR purified on fast protein liquid chromatography (FPLC, Pharmacia) using a Mono-Q ionexchange column. After verification by ELISA and Western blotting using monoclonal antibody to gC1qR, the single peak containing the gC1qR was pooled, concentrated to 1–2 mg/ml, and stored at –80°C. To eliminate the possibility of interference with a trace amount of thrombin, 5-mM Phe–Pro–Arg– chloromethylketone (PPACK), which is a specific and rapid inhibitor of thrombin, was added before each experiment. 3.5. Collection of Normal Human Serum Blood was individually collected from 10 apparently healthy volunteers, and the serum collected after clotting for 30 min at 37°C followed by another 30 min on ice. Each serum was then individually tested for complement activity by a hemolytic assay to exclude those with unusually low complement profile. Then the sera with similar complement activity were pooled, aliquoted into 0.5-ml portions, and kept frozen at –80°C until needed. Once the serum was thawed and used, the remainder was never refrozen and/or used. 3.6. Hemolytic Assay To test the effect of gC1qR on normal human serum (NHS), a standard hemo8 2+ lytic assay was used. Briefly, 5x10 /ml sheep erythrocytes (E), in GVB (isotonic Veronal-buffered saline, pH 7.4, containing 0.15-mM CaCl2, 0.5-mM MgCl2, and 0.1% gelatin) were sensitized (37°C, 30 min) with a predetermined dilution of IgG anti-sheep red cell stroma (A). After incubation, the sensitized 2+ EAs were pelleted, washed 2X in GVB and resuspended in the same buffer. The EAs were then tested by incubating (37°C, 30 min) 50 µl of the EA with 0.5 2+ ml of a 1:100 final dilution of NHS in GVB .The effect of purified recombinant

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gC1qR on NHS was then tested by incubating (60 min, 37°C) concentrations of gC1qR (containing 5-mM PPACK, final concentration) ranging from 0 to 50 µg/ml with 100 µl of a 1:5 dilution of NHS. The volume was brought up to 450 µl, and after addition of 50 µl of EAs incubated for an additional 60 min at 37°C. After incubation, the reaction mixtures were centrifuged and the amount of hemoglobin released into the supernatants measured spectrophotometrically at an optical density (OD) of 512 nm. NHS with buffer alone served as a negative control and NHS incubated with 10 µl of a 10-mg/ml aggregated IgG (45 min, 56°C), served as a positive control. EAs lysed with 0.5 ml of H2O served to determine a 100% , or maximal, hemoglobin release. 3.7. Microplate Assay for Complement Activation Solid-phase complement activation was performed by incubation of NHS with microtiter well-coated gC1qR using the C4d enzyme microassay (Quidel). Briefly, protein dilutions (in CO3 buffer, pH 9.3) were first prepared as follows: gC1qR (10 µg/ml), BSA (10 µg/ml), or aggregated IgG (10 mg/ml); then duplicate wells of a microtiter plate (MaxiSorb, Nunc, Kamstrup, Denmark) were coated with 50 µl of these dilutions and incubated for1 hr at 37°C. After washing with Tris buffer (20 mM Tris-HCl, pH 7.5), the unreacted sites were blocked with 1% BSA that had been heat inactivated (1 h, 56°C) to destroy trace amounts of bovine C1q. Complement activation was initiated by applying 50 µl 2+ of a 1:10 dilution (in GVB ) of NHS to each of the wells. After 1 hr at 37°C, the 2+ serum was removed, the wells washed (3X with GVB ), and the bound C1q and C4d probed with biotinylated anti-C1q or anti-C4d. The reaction was developed with alkaline phosphatase conjugated ExtrAvidin and pNPP following a standard ELISA.

4. RESULTS 4.1. Inhibition of Hemolytic Activity by gC1qR Normal human serum was first incubated with either buffer, BSA, or various concentrations of recombinant gC1q-R (rgC1qR) or soluble gC1qR (sgC1q-R) 12 isolated from culture supernatants of Raji cells . The residual hemolytic activity was measured by standard hemolytic assay. As shown in Figure 1, incubation of either soluble gC1qR or the recombinant form of gC1qR with NHS is able to inhibit complement hemolytic activity in a dose-dependent manner and under 12 physiologic ionic strength as has been shown previously . The inhibition is even stronger, when EDTA-serum is first incubated with gC1qR, then recalcified to 2,12 reassemble the C1 complex, and then tested with EA .

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Figure 1. Inhibition of hemolytic activity. Normal human serum was first incubated with either buffer or concentrations of recombinant gC1q-R (rgC1q-R) or membrane-solubilized gC1q-R (sgC1q-R). Residual hemolytic activity was measured by standard assay.

4.2. Soluble gC1qR but not ∆74-96 gC1qR Can Activate the Classical Pathway In order to examine whether the inhibition seen in Figure 1 is due to complement activation or selective and site-specific interference with the C1q–IgG interaction site, an enzyme assay of complement activation was performed in which microplate wells coated with activating or control proteins are reacted with NHS and the state of activation measured by quantifying the amount of C4d generated. As shown in Figure 2, gC1qR is a very efficient activator of the classical pathway, as evidenced by the substantial amount of C1q bound and C4d generated. This was comparable to that generated by aggregated IgG. However, when the truncated form of gC1qR, (∆74–96 gC1qR) was used, the amount of C1q bound or C4d generated was at least 50% less than that of the mature form, indicating that the N-terminal domain is important for activation (not shown). Furthermore, the ability of gC1qR to activate complement was strongly diminished when ∆276–282gC1qR was used instead of gC1qR (not shown).

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1.8 1.6

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Figure. 2. Activation of the classical pathway by soluble gC1q-R/p33. Microtiter plate wells were first coated in with eitherBSA [10 µg/ml of heat-inactivated BSA (56°C, 1 hr) in carbonate, pH 9.6] or gC1q-R (10 µg/ml, carbonate pH 9.6). After 1 hr incubation at 37°C, the wells were blocked with 1% heat-inactivated BSA following standard ELISA procedures. The wells were then washed with GVB2+ (isotonic Veronal-buffered saline containing 0.15 mM CaCl2, 0.5 mM MgCl2 and 0.1% gelatin) and then incubated with a dilution of normal human serum (1:50 in GVB2+). After 1 hr, the wells were washed and probed with either biotinylated anti-C1q or anti-C4d followed by reaction with alkaline phosphatase conjugated ExtrAvidin and pNPP. Each data bar represents a mean of experiments (n = 5) run in quadruplicates.

5. DISCUSSION The data presented in this paper demonstrate and support the long-held, but heretofore unproven, hypothesis that soluble gC1qR can activate complement via the classical pathway. Activation seems to be dependent on the interaction of C1q with the N-terminal domains (residues 74–96) of the intact homotrimeric protein, since deletion of these domains adversely affects its ability to activate complement. Trimer formation is critical for the function of gC1qR, and the Cterminal residues have been shown to play a role in trimerization of the molecule (Kumar and Ghebrehiwet, unpublished data). Deletion of the C-terminal residues (∆276–282), which seems to be critical in the trimerization of gC1qR, also reduces its activity. This implies that at sites of inflammation, where it is abundantly expressed both in soluble form and on the cell surface, gC1qR has the potential to exacerbate the inflammatory process by generating vasoactive peptides from the complement system, and bradykinin from the contact system.

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The inflammatory process is the result of multiple interactions, including important ones with the complement and contact systems. Since gC1qR can activate both systems, it is plausible to assume that it can serve as a bifunctional molecule that not only is expressed on cell surfaces — in particular, endothelial cells (DCs) — but also recognizes and binds diverse ligands that are involved in various ways in the inflammatory response. Among them are microbial and viral antigens and plasma proteins, of which the two most important ones are C1q and 3 HK . ECs play a major role not only in maintaining vessel-wall integrity but also in the pathophysiology of thrombosis, inflammation, and atherosclerosis. This is largely due to their ability to respond to a plethora of environmental stimuli that include bacterial endotoxin, virus, cytokines, chemokines, growth factors, and 13-15 complement proteins . The response of blood vessels to injury is comprised of a series of interrelated actions involving ECs, platelets, and proteins of the coagulation, comple13-15 ment, kinin, and fibrinolytic systems . Inflammation may contribute to thrombosis and atherosclerosis through the complement system, or via inflammatory cytokines secreted by activated macrophages and lymphocytes, all of which have been shown to be present at inflammatory sites including atherosclerotic 16 plaques . It is interesting in this context that complement proteins such as C1q 15 and C3, but not their inhibitors, are seen in atherosclerotic plaques . In addition, 17 C1q, which is synthesized by activated macrophages and immature dendritic 18,19 cells , may by itself contribute to local inflammation — not only by chemotac20 tic recruitment of neutrophils but also by inducing the expression of several 21 CAMs such as ICAM-1, E-selectin, and VCAM-1 , and triggering the produc22 tion of cytokines such as MCP-1, IL-6, and IL-8 from endothelial , and possibly other cells present at the inflammatory site. There is also evidence that the inflammatory process entails the interaction of key blood proteins involved in thrombosis with gC1qR on many cell types, including macrophages, lympho23,24 cytes, ECs, and platelets . The binding of HK and FXII to endothelial cell 24 gC1qR strongly suggests a role in the generation of bradykinin (BK) . This in turn can contribute to the inflammatory process by binding to BK receptors (BK2) and causing vascular permeability and infiltration of vascular tissue by 23 proinflammatory cells . In fact, this mechanism is postulated to play a major role in angioedema, either due to C1-INH deficiency or related to ACE (angio25-27 tensin converting enzyme) inhibitor . In addition, BK can directly stimulate cancer growth, enhance angiogenesis by stimulating release of fibroblast growth factor, and vascular endothelial growth factor (VEGF), and facilitate tumor in28-29 vasion by stimulating activity of membrane metalloproteinases . Therefore, at sites of atherosclerotic, inflammatory, and vascular lesions, where gC1qR is heavily expressed and both C1q and HK are present, we see both molecules to participate in the inflammatory process: C1q via activation of complement, by binding to gC1qR and inducing the release of proinflammatory cytokines from monocytes and macrophages; and HK by FXII-dependent generation of bradykinin (Figure 3). In addition, enzymes generated during contact activation,

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atherogenic factors (ICs, viruses, bacteria, etc)

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Figure 3. Schematic proposal of the contribution of the complement and kinin-generating systems on the endothelium. In this model, atherogenic factors are perceived (I) to convert the endothelial cell surface into a procoagulant and proinflammatory surface, and (II) to trigger complement activation and generation of chemotactic peptides, causing enhanced expression of gC1q-R. Expression leads to (III) high-affinity C1q binding and cell production of IL-6, IL-8, and MCP-1, and (IV) facilitation of the assembly of contact activation proteins leading to the generation of bradykinin and perhaps coagulation. Chemotactic factors may also (V) recruit gC1q-R-expressing monocytes and macrophages and enhance the local inflammatory response by generating proinflammatory molecules.

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such as FXIIa and FXIIf can directly activate C1 and can thus enhance the crosstalk between the two proinflammatory pathways, with gC1qR playing as the molecular orchestrator. Taking this information into account, we propose the following model (see Figure 3). At inflammatory sites we see the two pathways — (i) contact activation leading to kinin generation, and (ii) complement activation leading to generation of vasoactive peptides — as complementary. We propose that gC1q-R (shown by the doughnuts on the activated EC) is of major significance in the initiation and regulation of both processes.

6. ACKNOWLEDGMENTS This work was supported in part by grants from the National Institutes of Health (NHLBI 67211,EIBP) and a generous gift from Larry and Sheila Dalzell (B.G).

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16. E.I.B. Peerschke, J.O. Minta, S.Z. Zhou, A. Bini, A. Gotlieb, R.W. Colman and B. Ghebrehiwet, gC1q-R/p33 expression on human coronary artery atherosclerotic lesions. Mol Immunol 41,759–766 (2004). 17. M. Kaul, and M. Loos, The Fc-recognizing collagen-like C1q molecule is a putative type II membrane protein of macrophages. Behring Mitt 93, 171–179 (1993). 18. Z. Vegh-Goyarts, E. Goyarts, K. Rozengarten, A. Mazumder and B. Ghebrehiwet, Maturation-dpendent expression of cC1q-R and gC1q-R on human monocyte derived dendritic cells. Int Immunopharmacol 3, 345–357 (2003). 19. G. Castellano, A.M. Woltman, A.J. Nauta, A. Ross, L.A. Trouw, M.A. Seelen, F.P. Schena, M. Daha, and C. van Kooten. 2004. Maturation of dendritic cells abrogates C1q production in vivo and in vitro. Blood 103, 3813–3820. 20. L.E.A. Leigh, B. Ghebrehiwet, T.P.S. Perera, I.N. Bird, P. Strong, U. Kishore, K.B.M. Reid and P.Eggleton, C1q-mediated chemotaxis by human neutrophils: involvement of gC1q-R and G-protein signaling mechanisms. Biochem J 330, 247– 254(1998). 21. C. Lozada, R. I. Levin, M. Huie, R. Hirschhorn, D. Naime, M. Whittlow, P. A. Recht, B. Golden, and B. Cronstein, Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intracellular and vascular cell adhesion molecules 1. Proc Natl Acad Sci USA 92, 8378–8382(1995). 22. R.H. van den Berg, M.C. Faber-Krol, R.B. Sim, and M.R. Daha The first subcomponent of complement C1q triggers the production of IL-6,Il-8 and chemoattractant peptide 1 by human umbilical vein endothelial cells. J Immunol 161, 6924–6934 (1998). 23. A.P. Kaplan, K. Joseph, Y. Shibayama, S. Reddigari, B. Ghebrehiwet and M. Silverberg, The intrinsic coagulation-kinin forming cascade: assembly in plasma and cell surface in inflammation. Adv Immunol 66, 225–272 (1997). 24. K. Joseph, B. Ghebrehiwet, and A.P. Kaplan, Activation of the kinin-forming cascade on the surface of endothelial cells. Biochem J 382, 71–75 (2001). 25. D. Regoli and J. Barabe, Pharmacology of bradykinin and related kinins, Pharmacol Rev 32, 1–46 (1980). 26. M. Cugno, J. Nussberger, M. Cicardi, and A. Agostoni, Bradykinin and the pathophysiology of angioedema. Int Immunopharmacol 3, 311–317 (2003). 27. T.R. Fields, A.P. Kaplan, and B. Ghebrehiwet, Kinin formation in hereditary angioedema plasma: Evidence against kinin derivation from C2 and in support of "spontaneous" formation of bradykinin. J Allergy Clin Immunol 72, 54–60 (1983). 28. J. Wu, T. Akaike, T. Havashida, T. Okamoto, A. Okuvama, and H. Maeda, Enhanced vascular permeability in solid tumor involving peroxinitrite and matrix metalloproteinases. Jpn J Cancer Res 92, 439–451 (2001). 29. K. Ishihara, M. Kamata, I. Hayashi, S. Yamashina, M. Majima, Role of bradykinin in vascular permeability and angiogenesis in solid tumor. Int Immunopharmacol 2, 499–509 (2002). 30. B. Ghebrehiwet, M. Silverberg, and A.P. Kaplan, Activation of the classical pathway of complement by Hageman factor fragment. J Exp Med 153, 665–676 (1981).

8 POSSIBLE IMMUNOPROTECTIVE AND ANGIOGENESIS-PROMOTING ROLES FOR MALIGNANT CELL-DERIVED PROSTASOMES: A NEW PARADIGM FOR PROSTATIC CANCER? Kristina Nilsson Ekdahl, Gunnar Ronquist, Bo Nilsson, and Adil A. Babiker

1. INTRODUCTION Prostate cancer is the most commonly diagnosed cancer in men in the Western 1 world, as well as the second leading cause of cancer death . During the past 10 years, the increase in incidence was approximately 3 per 100,000 in Western Europe. During his lifetime a man of age 80 has a 70% chance of harboring 1 latent cancer in the prostate gland . This information is derived from autopsy reports and reveals that the prevalence of histological prostate cancer is much higher than the number diagnosed with clinical disease while alive, which means that most prostate cancers will never be clinically evident or life-threatening. A number of biomarkers (including prostate-specific antigen [PSA], despite its low specificity and sensitivity, are being analyzed as tools to diagnose primary prostate cancer, but at present there are still no clinical markers available with the potential to identify prostate cancer patients at risk of developing metastases (see [2] and the references therein). 3,4 Prostasomes, which were first described during the late 1970s , are membrane-surrounded organelles with a diameter of 40–500 nm (Figure 1). They are produced by the epithelial cells of the human prostate gland and are present in human semen at a protein concentration of approximately 2 mg/mL.

Kristina Nilsson Ekdahl, Department of Chemistry and Biomedical Sciences, University of Kalmar, Sweden. Gunnar Ronquist, Bo Nilsson, Adil A Babiker, Department of Oncology, Radiology, and Clinical Immunology, Department of Medical Sciences, University Hospital, Uppsala Sweden.

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Figure 1. Scanning electron microscope picture of purified seminal prostasomes 40,000x magnification. The length of the bar is equal to 500 nm.

Prostasomes are surrounded by a lipid bilayer membrane and are found encased in bigger storage vesicles together with electron-dense material in prostate secretory cells. The composition of the membrane is unusual in that the ratio of cholesterol to phospholipid is approximately 2:1 as compared to about 0.8:1 for 5 most other mammalian cell membranes . Prostasomes are released by exocytosis into the prostatic fluid (and semen) in the form of small intact organelles. Using a proteomic approach, Utleg and coworkers have identified a total number of 6 139 proteins in human seminal prostasomes . Approximately 1/3 of the 4,7 identified proteins are enzymes, including ATPase , arachidonic acid 158 9 10 lipoxygenase , aminopeptidase N (CD13) , dipeptidyl peptidase IV (CD26) , 11 12 enkephalinase (CD10) and Ser/Thr protein kinase(s) . Prostasomes have been shown to possess complement-regulatory properties because of their high expression of CD59 and, to a lesser degree, of their much 13 lower concentrations of CD55 and CD46 . (The function of these proteins is discussed below). Both benign and malignant human prostatic cells express CD59 that is glycosyl phosphatidyl inositol-(GPI)-linked to the cell surface, and this CD59 may protect the cells from the cytolytic activity of the complement 14 system . In two recent studies (summarized below in greater detail) we have demonstrated that CD59 from seminal and malignant cell-derived prostasomes can be transferred to both xenogeneic and allogeneic CD59-deficient erythrocytes as well as to autologous cancer cells, resulting in protection against 15,16 complement-mediated lysis .

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Prostasomes have been shown to interact with spermatozoa, enhancing 17,18 19 and neutralizing formation of free radicals . These two sperm motility functions produce an improved microenvironment for the spermatozoa and are of importance for fertilization. Recently it has been demonstrated that seminal 20 prostasomes are able to inhibit angiogenesis in vitro , and it is postulated that 21 prostasomes may play a role in prostatic disease . In addition, prostasomes have been shown to be produced by metastatic prostate cancer cells, both from bone 16,22 metastases and from metastasis-derived cell lines , but thus far the possible differences between prostasomes of malignant and nonmalignant origin have attracted little attention and the pathophysiological roles of prostasomes remain to be clarified.

2. HYPOTHESIS: MALIGNANT CELL-DERIVED PROSTASOMES PROVIDE CANCER CELLS WITH A ZONE OF INNATE IMMUNE PRIVILEGE The properties of prostate cancer alter dramatically when the cells become 23 androgen independent and metastasis occurs. Research in our laboratory and 24 that of others has revealed that dedifferentiated metastatic prostate cancer cells are able to produce prostasomes and export them to the extracellular space around the metastatic cells. In a series of papers we have mapped the phenotype of metastatic cell-derived prostasomes as compared to normal seminal prostasomes. Three main traits were found to be associated with the malignant phenotype: (a) Metastatic cell-derived prostasomes exhibit a high level of expression of the complement-modulatory protein CD59, which can be trans16 ferred to CD59-deficient cells and malignant cells . (b) We detected high levels of protein kinases (PKs) on prostasomes of malignant cell origin. These PKs are able to phosphorylate key molecules of the innate immune system, including complement component C3. (c) The high level of ATP-splitting activity found on seminal prostasomes is almost nonexistent on malignant cell-derived prostasomes, resulting in high levels of ATP being available for phosphorylation 25 reactions . (d) We have confirmed earlier observations that prostasomes express 26 high levels of tissue factor (Babiker et al., manuscript in preparation). This last observation, however, is beyond the scope of this review article and will not be discussed further. Based on these collective results, we hypothesize that prostasomes produced by the prostate cancer cells are themselves major players in the malignant process and that resistance against the (innate) immune system is a central driving force in the selection process. Unlike prostasomes in seminal plasma, prostasomes produced by malignant cells are not cleared when released into the extracellular space but remain trapped within or in the close proximity to the tumor. This situation can create a microenvironment of innate immunity privi-

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lege in which extracellular prostasomes protect the prostatic malignant cells against immune attack and promote angiogenesis, cell migration, and tumor cell invasion, thereby promoting their metastasis. One question that needs to be addressed in this context is whether malignant cell-derived prostasomes are as potent in transferring CD59 with concomitant resistance to complement mediated lysis, as are their seminal counterparts (see below). Furthermore, it remains to be established whether prostasomes of either origin possess other means by which they can attenuate complement activation and other functions of innate immunity.

3. COMPLEMENT ACTIVATION AND EXPRESSION OF COMPLEMENT REGULATORY PROTEINS BY MALIGNANT CELLS The complement system consists of approximately 30 plasma and cellular proteins (receptors and regulators) that play a primary role in host defense differentiating between self and non-self and acting as a purging system for the body. The main event in the activation of complement is proteolytic cleavage of C3, producing C3b and C3a. This cleavage event is accomplished by two enzyme complexes called convertases, which are assembled by three different activation pathways. These converge in a common pathway, forming the membrane attack complex (MAC, C5b-9), which elicits cell lysis by insertion into the lipid bilayer of plasma membranes. The anaphylatoxins (C3a and C5a) activate and recruit polymorphnuclear leukocytes and monocytes to the target, while target-bound C3 fragments facilitate binding and activation of the recruited cells. Complement activation on autologous cells is controlled by several soluble and membrane-bound regulators. Most of the regulators are homologous proteins that are members of the regulator of complement activation (RCA) superfamily and mainly regulate the two types of convertases: factor H, C4BP, complement receptor 1 (CR1; CD35), DAF (CD55), and MCP (CD46) belong to this family. In addition, C1 inhibitor (C1INH) regulates the C1 complex C1qC1r2C1s2. CD59 is a membrane protein that is a regulator of the MAC, while vitronectin (S-protein) and protectin are soluble MAC inhibitors. The role of complement activation as a mechanism for destruction of malignant cells is not yet well defined. It is, however, highly probable that the complement system is involved in control of malignant tumors since this system is extremely sensitive to altered self and non-self structures on cell surfaces. It is therefore expected that cells that are unable to protect themselves against complement attack will be eliminated early in the process of cancer development. One consequence of this situation would be that the surviving clones would have an increased expression of complement regulatory proteins. Consistent with this hypothesis are several reports that various malignant cell lines and tumors, including thyroid, colon and lung cancers, can activate and

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deposit complement fragments on the cell surface . Numerous investigations have shown that various mechanisms for complement protection are upregulated in malignant cells and cell lines. These include, for example, an increased expression of the complement-regulatory proteins CD35, CD46, CD55, or CD59 14,30-32 . It has been observed that loss of one complementin prostate cancer inhibitory protein may be compensated for by increased expression of another. We and others have demonstrated that some malignant cells express ectokinases (cell bound extracellular protein kinases, PKs) with specificity for complement proteins, and we postulate that extracellular phosphorylation may be one mechanism by which malignant cells can attenuate or disarm the complement system. We have shown that the myeloid leukaemia cell line U937 releases a CK2-like PK that phosphorylates C3 and increases its cleavage by elastase into 33 34 C3 fragments . Similar results have been reported by Paas and coworkers , who observed release of ectokinases that have complement-regulatory activity from K562 cells (a human leukemic cell line). Complement inhibitors are not uniformly expressed in all cancers. In some studies the expression of complement-regulatory proteins has been found to 14,35 increase with tumor grade , whereas other investigators have reported that a loss of complement-regulatory proteins on tumor cells correlates with poor 36,37 clinical outcome . However, the link to complement in the last two studies can be questioned since the missing complement regulators belong to the large phosphatidylinositol-anchored protein family and may therefore reflect loss of other important proteins.

4. CD59 TRANSFER BY PROSTASOMES RESULTS IN PROTECTION AGAINST COMPLEMENT MEDIATED LYSIS Prostasomes are isolated from seminal plasma and from human prostatic carcinoma cell lines by differential centrifugations and subsequent gel chroma5,22,38 . Prostasomes have been prepared according to this protocol from tography seminal plasma and from the cell lines PC-3, DU145, and LNCaP, which 39 40 41 originate from metastases of bone , brain , and lymph node , respectively. The purified prostasomes were analyzed using various assays, with the overall aim of elucidating whether differences in the function or expression of potential clinical markers can be found between prostasomes of malignant and nonmalignant origin. 15 Initial experiments in our laboratory have addressed the issue of whether CD59 can be transferred from seminal prostasomes to CD59 deficient cells (rabbit erythrocytes [RE] and erythrocytes from patients with paroxysmal nocturnal hemoglobinuria [PNH]). Incubation of the erythrocytes with prostasomes led to a transfer of CD59 that could be detected by flow cytometry. In both cases, the erythrocytes acquired resistance to complement-mediated lysis during subsequent incubation with normal human serum, while erythrocytes not

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incubated with prostasomes remained susceptible to lysis. In the case of RE, saturation was already seen after incubation with prostasomes at the very low concentration of 40 µg/mL, which corresponds to 1/50 of the concentration found in seminal plasma. The mechanism of transfer of CD59 from prostasomes to erythrocytes remains to be elucidated. Differences were found in the behavior of erythrocytes from different species. After removal of GPI-anchored proteins from prostasome-incubated erythrocytes by digestion with PIPLC (phosphoinositol-specific phospholipase C), RE lost most of the prostasome-induced resistance against lysis while the human PNH cells remained protected, suggesting that the CD59 bound to RE mainly through the GPI-anchor and that other mechanisms are operative during transfer to the human cells. These results support the idea that the transfer of CD59 to allogeneic cells and, perhaps also to 15 autologous cells, can protect them from lysis elicited by C5b-9 complexes . The aim of subsequent experiments was to elucidate whether malignant cell-derived prostasomes expressed CD59 to a degree similar to that of their seminal counterparts and whether they are as potent in transmitting resistance to complement-mediated lysis. A further aim was to investigate whether prostasomes are able to interact with autologous cells, i.e., the malignant cells from which they originate. Variable amounts of CD59 were detected by flow cytometry in all types of prostasomes. Low expression of CD59 was seen in seminal and nonmalignant prostasomes; prostasomes isolated from cell lines DU145 (brain metastasis) and PC-3 (bone metastasis) contained high amounts of CD59, while the levels found on prostasomes isolated from LNCaP (lymph node 16 metastasis) cells contained intermediate amounts (Table 1). All malignant cellderived prostasomes were able to transfer CD59 to RE to a similar degree. In contrast, seminal prostasomes that had the lowest expression of CD59 were able to transfer higher levels of CD59 to RE than could any of the malignant cellderived prostasomes (Table 1). PC-3- and DU145-derived prostasomes were able to transmit a very high resistance to complement-mediated lysis; the protective effect transferred by LNCaP-derived or seminal prostasomes was negligible by comparison (Table 1). Taken together, the lack of correlation between CD59 transfer and induced resistance to complement suggests either that malignant cell-derived prostasomes deliver CD59 in a different conformation or that they transfer other complement-attenuating factors in addition to CD59. The latter possibility is supported by the observation that the complement-protective effect of prostasomes can be counteracted in part, but 16 not in total, by monoclonal anti-human-CD59 antibodies . A further intriguing observation is that CD59 from prostasomes can be transferred not only to CD59-deficient, xenogeneic RE, but also to autologous cells, i.e., the cancer cell lines from which they were derived. In the case of DU145 and PC-3 cells, transfer of CD59 was seen only after the GPI-anchored proteins had been removed by PIPLC. In contrast, LNCaP cells, which initially had lower expression of CD59 than did the other two cell lines, were saturated by CD59 after incubation with either LNCaP-derived (“autologous”) or seminal

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(“allogeneic”) prostasomes. Thus, transfer of CD59 (and other immunomodulatory proteins) might be a mechanism by which prostasomes interact with malignant or other cells, with suboptimal expression of CD59, in a secluded 16 extracellular compartment .

Table 1. Comparison of Phenotypes of Malignant Cell-Derived and Seminal Prostasomes Source of prostasomes DU145 cells PC-3 cells LNCaP cells Seminal

CD59 expr +++

CD59 transfer ++

AHE ++

PKA ++

PKC (+)

CK2 +++

ATP ase (+)

++

++

+++

++

(+)

++

-

+

++

+

+++

(+)

+

-

(+)

+++

+

+

(+)

+

+++

CD59 expr = amount of CD59 on prostasomes detected by flow cytometry CD59 transfer = ability to transfer CD59 to rabbit erythrocytes AHE = antihemolytic effect after transfer of CD59 to rabbit erythrocytes PKA = activity of protein kinase A on prostasomes PKC = activity of protein kinase C on prostasomes CK2 = activity of casein kinase 2 on prostasomes ATPase = ATP splitting activity on prostasomes

5. EXTRACELLULAR PHOSPHORYLATION OF PLASMA PROTEINS Phosphorylation is a rapid mode of altering the properties of a protein; for example, its enzymatic or cofactor activity, its affinity for ligands, or its susceptibility to proteolytic degradation. PKs are enzymes that catalyze the transfer of the γ-phosphoryl group of ATP (which is thus a co-substrate in the phosphorylation reaction) to an acceptor amino acid side chain of a protein substrate in the presence of divalent cations. Unlike many other regulatory mechanisms, protein phosphorylation is a reversible process since the phosphate group(s) may be removed by phosphoprotein phosphatases, thereby restoring the original properties of the protein. It has been shown that C3 can be phosphorylated in vitro by at least five different PKs, in different domains of the molecule, and with different functional effects. These PKs include Ser/Thr protein kinases, which require cationic amino acids in the vicinity of the

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phosphorylation site (e.g., PKA and PKC) and casein kinases 1 and 2 (CK1 and CK2) that are specific for anionic protein sequences (reviewed in [42]). 43 The C3a domain of C3 is phosphorylated by PKA and PKC and by an 44 ectoprotein kinase from the human parasite Leishmania major . PKA and PKC 43 phosphorylate native C3 but not iC3 or C3a . In contrast to the case of CK2, phosphorylation by PKA, PKC, or leishmanial PK protects the C3 molecule from activation, either by trypsin (all three PKs) or by both the classical and alternative pathway convertases (PKA and PKC). Phosphorylation of C3 by PKA or PKC may thus be regarded as a complement regulatory mechanism that operates on the convertase level, i.e., is related to the mode of action of the regulators of complement activation (RCAs) DAF, MCP, CR1, and factor H. Like C3, human fibrinogen is phosphorylated by PKA, PKC, CK1, and CK2; this modification affects the thickness of the generated fibrin fibers as well 45,46 as their degradation by plasmin . C3 and fibrinogen are phosphorylated by a CK1-like PK that is released from activated human platelets and that has a 47,48 possible association with thrombosis . Finally, CK2 phosphorylates both polypeptide chains of C3 and increases the susceptibility of C3 to cleavage by 33 elastase .

6. MAPPING OF PROTEIN KINASES ON PROSTASOMES Using combinations of model phosphorylation substrates and well-defined PK inhibitors, we have characterized the activity and specificity of ectoprotein kinases with regard to prostasomes of different origin. The identities of the 4,7 prostasomal PKs were confirmed by flow cytometry. In earlier studies , seminal prostasomes were reported to be associated with a high ATP-splitting activity. ATP is a co-substrate, and its stability must thus be taken into consideration in evaluating the apparent efficiency of a phosphorylation reaction. For our quantification we therefore utilized prostasomes that had been solubilized with octylglucoside, which substantially reduces the ATPase activity, and also lowers the cholesterol content that may interfere with proteins and other compounds, such as the PK inhibitors. PKA, PKC, and CK2, but not CK1, were detected in all the prostasome preparations examined. Significantly higher PKA activity was found in prostasomes of malignant cell origin. LNCaP had a fivefold higher activity than did seminal prostasomes. Expression of CK2 activity was moderately increased (up to twofold) when cell line-derived prostasomes were examined. In contrast, similar (low) PKC activity was seen for all preparations. The profile of PK expression differed between the preparations: DU145 was highest in CK2 activity and lowest in PKA of all the prostasomal preparations from malignant cells, while LNCaP was highest in PKA and lowest in CK activity. In additon, in accordance with previous reports, ATPase activity was found in high amounts

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on seminal prostasomes but was almost undetectable on all other prostasomal 25 preparations (Table 1) . Thus far, all prostasomes of malignant cell origin have been found to have significantly higher PK activity than do seminal prostasomes. It is particularly intriguing that LNCaP prostasomes associated with the highest PKA activity are devoid of ATPase activity, suggesting that prostasomes with these properties would be capable of substantial phosphorylation of available biomolecules if trapped in a secluded milieu.

7. C3 AND OTHER SUBSTRATES FOR PROSTASOMAL PKS In order to establish that the presence of PKs on prostasomes has any physiological relevance, it is necessary to identify physiological substrates for the prostasomal PKs. Using prostasomes solubilized with octylglucoside as a source of PKs, we have identified three highly interesting substrates: complement component C3, fibrinogen, and vitronectin. C3, the key protein in the complement cascade, was phosphorylated in the α-chain by PKA from all preparations, but to at least a tenfold higher degree by malignant cell-derived 25 prostasomes than by seminal prostasomes . Solubilization with octylglucoside substantially reduces ATPase activity. Therefore, the differences between malignant cell-derived (low in ATPase) and seminal (high in ATPase) prostasomes are expected to be even more pronounced under physiological conditions. The observation that C3 is phosphorylated by malignant prostasomal PKA provides strong evidence that prostasomes may indeed have the capability to disarm complement activation by regulatory phosphorylation, since earlier studies have shown that C3 that has been phosphorylated by PKA (or PKC) in vitro is rendered inaccessible to physiological activation by both of the 43 convertases . Fibrinogen, which plays a role in tumor angiogenesis and metastasis, has also been found to be phosphorylated in the α-chain by PKA, PKC, and CK2 25 from all the prostasome preparations tested . This observation is intriguing since fibrinogen has been implicated in playing a role in tumor angiogenesis and 49 metastasis , and it has been reported that several of its functions are affected by 46 phosphorylation with various PKs . In addition, vitronectin, which is involved in cell adhesion and cancer proliferation, is phosphorylated to a high degree by PKA and CK2 (Babiker et al., manuscript in preparation). As in the case when C3 is used as substrate, the phosphorylation of vitronectin was at least 10-fold greater with prostasomes derived from malignant cells than with seminal prostasomes. Suppression of fibrinolysis is important for metastatic prostate cancer, and phosphorylation of vitronectin has previously been reported to 50 suppress fibrinolysis . Taken together, these data indicate that malignant cell-derived prostasomes have the potential to phosphorylate and thereby modulate the function of a

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number of proteins critically involved in innate immune response and cell adhesion.

8. CONCLUSIONS Understanding the protective mechanisms utilized by metastatic prostate cancer cells in order to avoid attack by complement or other parts of the innate immune system and to affect tumor angiogenesis and metastasis will help us to identify suitable targets for pharmaceutical intervention. We have shown that at least two different complement-attenuating mechanisms are at work in close proximity to prostasomes: transfer of CD59, which inhibits complement at the level of MAC insertion, and phosphorylation of C3 so as to make it resistant to (physiological) activation and thereby regulate complement at the convertase level. In addition, we have shown that both fibrinogen and vitronectin, which play critical roles in cell adhesion, are targets for prostasome-mediated phosphorylation. Given the broad specificity of the various PKs, it is most likely that other relevant substrates such as proteins involved in angiogenesis or different matrix proteins may be found. Finally, we have demonstrated that expression and function of different proteins capable of mediating these effects (CD59 and PKs, particularly PKA) are highly upregulated on prostasomes derived from malignant cell lines as compared to seminal prostasomes, suggesting that the malignant cell-associated prostasomes have a higher potential to interact with neighboring cells. The fact that substantial differences are found in protein expression profiles between physiological and pathological prostasomes may be relevant in the search for suitable clinical markers to identify patients with primary prostate cancer who are at risk for developing metastases. In addition, possible targets for therapeutic intervention may include GPI-anchored proteins and specific PKs present at high concentrations in close proximity to metastases. If the overexpression of RCAs and PKs on metastatic prostate cancer cells can be controlled or counteracted, these modifications could possibly also be used to potientate other types of immunotherapy.

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21. A. B. Stewart, W. Anderson, G. Delves G, B. A. Lwaleed, B. Birch and A. Cooper, Prostasomes: a role in prostatic disease? BJU Int 94(7), 985–989 (2004). 22. L. Carlsson, O. Nilsson, A. Larsson, M. Stridsberg, G. Sahlén and G. Ronquist, Characteristics of human prostasomes isolated from three different sources, Prostate 54(4), 322–330 (2003). 23. G. Sahlén, A. Ahlander, A. Frost, G. Ronquist, B. J. Norlen and B. O. Nilsson, Prostasomes are secreted from poorly differentiated cells of prostate cancer metastases, Prostate 61(3), 291–297 (2004). 24. D. Floryk, S. L. Tollaksen, C. S. Giometti and E. Huberman, Differentiation of human prostate cancer PC-3 cells induced by inhibitors of inosine 5′monophosphate dehydrogenase, Cancer Res 64(24), 9049–9056 (2004). 25. A. A. Babiker, B. Nilsson, G. Ronquist G and K. N. Ekdahl, Overexpression of ectoprotein kinases in prostasomes of metastatic cell origin, Prostate, in press (2006). 26. J. A. Fernández, M. J. Heeb, K. P. Radtke, and J. H. Griffin, Potent blood coagulant activity of human semen due to prostasome-bound tissue factor, Biol Reprod 56, 757–763 (1997). 27. M. F. Bernet-Camard, M. H. Coconnier, S. Hudault and A. L. Servin, Differential expression of complement proteins and regulatory decay accelerating factor in relation to differentiation of cultured human colon adenocarcinoma cell lines, Gut 38(2), 248–253 (1996). 28. S. D. Lucas, A. Karlsson-Parra, B. Nilsson, L. Grimelius, G. Akerstrom, J. Rastad and C. Juhlin, Tumor-specific deposition of immunoglobulin G and complement in papillary thyroid carcinoma, Hum Pathol 27(12), 1329–1335 (1996). 29. F. Niculescu, H. G. Rus, M. Retegan and R. Vlaicu, Persistent complement activation on tumor cells in breast cancer, Am J Pathol 140, 1039–1043 (1992). 30. G. A. Niehans, D. L. Cherwitz, N. A. Staley, D. J. Knapp and A. P. Dalmasso. Human carcinomas variably express the complement inhibitory proteins CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin), Am J Pathol 149(1), 129–142 (1996). 31. L. Bjorge, C. A. Vedeler, E. Ulvestad and R. Matre, Expression and function of CD59 on colonic adenocarcinoma cells, Eur J Immunol 24(7), 1597–1603 (1994). 32. M. Yamakawa, K. Yamada, T. Tsuge, H. Ohrui, T. Ogata, M. Dobashi and Y. Imai, Protection of thyroid cancer cells by complement-regulatory factors, Cancer 73(11), 2808–2817 (1994). 33. K.N. Ekdahl and B. Nilsson, Phosphorylation of complement component C3 after synthesis in U937 cells by a putative protein kinase, casein kinase 2, which is regulated by CD11b: evidence that membrane-bound proteases preferentially cleave phosphorylated C3, Biochem J 328(2), 625–633 (1997). 34. Y. Paas, O. Bohana-Kashtan and Z. Fishelson, Phosphorylation of the complement component, C9, by an ecto-protein kinase of human leukemic cells, Immunopharmacology 42(1–3), 175–185 (1999). 35. P. Hofman, B. Hsi, S. Maine, P. Fenichel, A. Thyss and B. Rossi B, High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: A preventive mechanism of malignant tumor cells against complement attack? Breast Cancer Res Treat 34, 213–219 (1994). 36. Z. Madjd, S. Pinder, C. Paish, I. Ellis, J. Carmichael, and L. Durrand, Loss of CD59 expression in breast tumours correlates with poor survival. J Pathol 200, 633–639 (2003).

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37. Z. Madjd, L. Durrand, R. Bradley, I. Spendlove, I. Ellis and S. Pinder, Loss of CD55 is associated with aggressive breast tumors, Clin Cancer Res 10, 2797–2803 (2004). 38. G. Ronquist, G. Frithz and A. Jansson, Prostasome membrane associated enzyme activities and semen parameters in men attending an infertility clinic, Urol Int 43(3), 133–138 (1988). 39. M. E. Kaighn, K. S. Narayan, Y. Ohnuki, J. F. Lechner and L. W. Jones, Establishment and characterization of a human prostatic carcinoma cell line (PC-3), Invest Urol 17(1), 16–23 (1979). 40. K. R. Stone, D. D. Mickey, H. Wunderli, G. H. Mickey and D. F. Paulson, Isolation of a human prostate carcinoma cell line (DU 145), Int J Cancer 21(3), 274–281 (1978). 41. J. S. Horoszewicz, S. S. Leong, T. M. Chu, Z. L. Wajsman, M. Friedman, L. Papsidero, U. Kim, L. S. Chai, S. Kakati, S. K. Arya and A. A. Sandberg, The LNCaP cell line - a new model for studies on human prostatic carcinoma, Prog Clin Biol Res 37, 115–132 (1980). 42. K. N. Ekdahl and B. Nilsson, Phosphorylation of plasma proteins with emphasis on complement component C3, Mol Immunol 36(4–5), 233–239 (1999). 43. P. O. Forsberg, S. C. Martin, B. Nilsson, P. Ekman, U. R. Nilsson and L. Engstrom, In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways, J Biol Chem 265(5), 2941–2946 (1990). 44. T. Hermoso, Z. Fishelson, S. I. Becker, K. Hirschberg and C. L. Jaffe, Leishmanial protein kinases phosphorylate components of the complement system, Embo J 10(13), 4061–4067 (1991). 45. P. O. Forsberg and S. C. Martin, Plasmin digestion of human fibrinogen previously phosphorylated by protein kinase C or dephosphorylated by alkaline phosphatase in vitro, Thromb Res 58(2), 119–127 (1990). 46. S. C. Martin, P. O. Forsberg and S. D. Eriksson, The effects of in vitro phosphorylation and dephosphorylation on the thrombin-induced gelation and plasmin degradation of fibrinogen, Thromb Res 61, 243–252 (1991). 47. K. N. Ekdahl, L. Rönnblom, G. Sturfelt and B. Nilsson, Increased phosphate content in complement component C3, fibrinogen, vitronectin, and other plasma proteins in systemic lupus erythematosus: covariation with platelet activation and possible association with thrombosis, Arthritis Rheum 40(12), 2178–2186 (1997). 48. K. N. Ekdahl, A. A. Bengtsson, J. Andersson, G. Elgue, L. Rönnblom, G. Sturfelt and B. Nilsson, Thrombotic disease in systemic lupus erythematosus is associated with a maintained systemic platelet activation, Br J Haematol 125(1), 74–78 (2004). 49. Y. Roche, D. Pasquier, J. Rambeaud, D. Seigneurin and A. Duperray, Fibrinogen mediates bladder cancer migration in an ICAM-1-dependent pathway, Thromb Haemost 89(6), 1089–1097 (2003). 50. I. Schvartz, T. Kreizman, V. Brumfeld, Z. Gechtman, D. Seger and S. Shaltiel, The PKA phosphorylation of vitronectin: effect on conformation and function, Arch Biochem Biophys 397(2), 246–252 (2002).

9 DIVERSIFIED COMPONENTS OF THE BONY FISH COMPLEMENT SYSTEM: MORE GENES FOR ROBUSTER INNATE DEFENSE? Miki Nakao, Yoko Kato-Unoki, Makiko Nakahara, Junichi Mutsuro, and Tomonori Somamoto

1. INTRODUCTION 1

The complement system is a major humoral factor of the immune system . From the phylogenetic point of view, a prototypic complement system has been found in a variety of deuterostomic invertebrates such as the sea urchin (echinoderm) 2 and ascidians (urochordates) . Moreover, recent discoveries of C3 and/or factor 3 4 B in coral (cnidarian) and horseshoe crab (arthropod) suggest that emergence of the complement system predates the separation of protostomes and deuterostomes. Molecular and functional information currently available from the invertebrates suggests that the complement prototype could have been composed of reaction cascades similar to mammalian lectin and alternative activation 4,5 pathways . Namely, the central component (C3), a collectin-like lectin for recognition of pathogen-associated molecular patterns on bacterial cell walls, a serine protease associated with the lectin for specific cleavage of C3, and C3b/iC3b-receptor on phagocytes would have functioned as an opsonic system in the prototype. In bony fish, one of the most primitive groups of vertebrate, the complement system is equipped with all three activation pathways (the lectin, alternative, and classical pathways), and the lytic pathway as found in mam6-8 mals (Figure 1). Recent molecular analyses have uncovered a striking feature of bony fish complement that several complement components are encoded by 9 multiple genes . In particular, presence of multiple isoforms of the components

Miki Nakao, Yoko Kato-Unoki, Makiko Nakahara, Junichi Mutsuro, and Tomonori Somamoto, Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan.

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Ag-Ab complex

Mannose-rich glycans

LPS β-glucans

Classical pathway Lectin pathway C1 MBL-MASPs (C1qC1r2C1s2 ) C4, C2

Alternative pathway C3(H2O)

*

C3(H2O)B

C3

C4bC2a

Bf Df C3(H2O)Bb

C3a C3b

Bf Df

C3bBb

C3bB C4bC2aC3b

C3bBbC3b

C5

C5b C5a

C6 C8

Lytic pathway C7 C9

C5bC6C7C8C9n (Membrane-attack complex) Figure 1. Simplified scheme of the mammalian complement system. Only the components directly involved in the activation cascades are shown. Arrows with a solid line show proteolytic actions, and those with a broken line indicate transition or conversion of the components. An asterisk shows spontaneous hydrolysis of the thioester (C3-tick over). Abbreviations: Ag, antigen; Ab, antibody; LPS, lipopolysaccharides.

have been found in all the complement pathways of carp, a pseudotetraploid species. In this review, we summarize the status of the current research on the structural diversity of bony fish complement components in each pathway and their possible functional differentiation. Although the biological significance of such complement diversity in bony fish is still unclear, possible approaches to elucidate the role of diversification of the complement components in bony fish immunity are discussed.

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2. CLASSICAL PATHWAY COMPONENTS The recognition molecule of the classical pathway is C1q, which binds to the Fc part of IgM and IgG. C1q is complexed with two molecules each of serine proteases, termed C1r and C1s, and binding of C1q to the immunoglobulins leads to activation of C1r and C1s. The activated C1s cleaves C4 into C4a and C4b and C2 into C2a and C2b, resulting in formation of a C3-convertase complex, C4bC2a, in which C4b covalently binds to the target surface through the internal thioester site, and C2a proteolytically activates C3 to generate C3a and C3b 1 fragments . C3a is a small (~9 kDa) molecule that shows anaphylatoxic activities. On the other hand, C3b can bind covalently, like C4b, to the target surface, serving as a ligand for complement receptors on phagocytes and also as a subunit of a C5–convertase complex. The classical pathway components are poorly characterized at the molecular level in bony fish, though hemolytic activity attributable to the classical pathway has been detected from bony fish serum based on its antibody dependency and 6 calcium ion requirement . Bony fish C1q has not appeared in the literature, whereas only a C1r-like serine protease, but not a C1s-like one, has been identi10,11 fied at the molecular level in carp and trout . A serine protease functionally equivalent to C1s has been purified from trout plasma solely based on its activ12 ity to cleave C4 . This activity, however, is also shared by MASP2, suspending the final assignment of the trout molecule to C1s or MASP2. In carp, two distinct C1r-like molecules have been cloned and designated 10 C1r/s-A and C1r/s-B . Deduced amino-acid sequences of these molecules, shar667 666 ing 83% identity, differ at the position (Gly in C1r/s-A and Ala in C1r/s-B) that corresponds to the entrance of the S1 specificity pocket of bovine trypsin. The Gly-to-Ala substitution of bovine trypsin by targeted mutagenesis has been reported to attenuate proteolytic activity and shift substrate specificity toward 13 Lys from Arg as an S1 site . Since a crystallographic study of the serine protease domain of human C1s has indicated that C1s has a trypsin-like S1 specificity 14 pocket , it seems likely that the Gly/Ala substitution would affect the substrate specificity and proteolytic efficiency of C1r/s-A and C1r/s-B, as in the case of trypsin. If C1r/s-A and C1r/s-B are C1r-equivalent, as predicted by phylogenetic analysis, C1r/s-B isotype with the predicted substrate specificity to Lys-X would be less active than C1r/s-A to cleave C1r/s-A or C1r/s-B itself, because both isotypes share an Arg–Val segment at the putative cleavage site into H- and L10 chains of their active forms . Even if the two isotypes were C1s-equivalent, C1r/s-B could again be poorly active as an enzyme to cleave carp C4, because two carp C4 isotypes share Arg-X at the cleavage site to yield C4a and C4b 15 fragments . Therefore, it seems likely that C1r/s-A and C1r/s-B are functionally differentiated. It is of particular interest to know a cleavage site sequence in carp C1s to generate its H- and L-chain. 11,12 16 15 C4 has been purified and cloned from trout , fugu , and carp . Trout C4 and fugu C4 have been reported to be encoded by a single gene in respective

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Figure 2. Phylogenetic tree of the thioester-containing protein family containing complement components C3, C4 and C5 and α-macroglobulins, drawn by the maximum likelihood method. Bootstrap percentages supporting the partitionings are shown. Abbreviations: Haro, the solitary Japanese ascidian; Ciin, Ciona intestinalis (an ascidian); Hosa, human; Mumu, mouse; Epbu, hagfish; Laja, lamprey; Onmy, rainbow trout; Anmi, spotted wolffish; Orla, medaka fish; Paol, Japanese flounder; Cyca, carp; Gaga, chicken; Nana, cobra; Capo, guinea pig; Trsc, banded hound shark; Xela, Xenopus laevis; Rano, rat; Stpu, sea urchin; Swex, coral; Brbe, amphioxus; Drme, fruit fly.

genomes, though two C4 isoforms with different glycosylations have been isolated from trout plasma. In contrast, two divergent C4 isotypes — C4-1 and C42 — sharing only 32% amino-acid identity, have been identified in carp. Phylogenetic analyses suggest that C4-1 and C4-2 represent two ancient lineages of

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C4 in vertebrates (Figure 2). Database mining implies the presence of C4-1 and 17 C4-2 in trout, salmon, and zebrafish , while the fugu genome seems to contain 16 only a single copy of the C4 gene . The most intriguing difference between carp C4-1 and C4-2 is an amino-acid substitution of the catalytic histidine, which primarily determines the substrate specificity of the cleavage and binding reaction of the internal thioester bond. Human C4B isotype possessing the catalytic 1106 histidine (His ) binds to hydroxy- and amino-groups, making ester- and amide1106 linkages, respectively. On the other hand, C4A isotype, in which His is re18 placed by Asp, binds predominantly to the amino-group . Similarly, carp C4-1 has a His, and C4-2 possesses an Asp at the equivalent position, suggesting their 15 difference in the binding specificity of the thioester . In the bony fish complement system, the presence of C2 has not been 8 proven either at the cDNA or protein level .

3. LECTIN PATHWAY COMPONENTS Activation of the lectin pathway is triggered by recognition of pathogenassociated molecular patterns such as high-density array of terminal mannose moiety presented by the bacterial cell wall. In mammals, two groups of collectins, mannose-binding lectin (MBL) and ficolins (FCN), are responsible for 19 recognition . These lectins in serum are complexed with three homologous serine proteases — termed MBL-associated serine proteases-1, -2, and -3 (MASP1, MASP2, and MASP3) — and a truncated form of MASP2 (sMAP or Map19). MASP2 proteolytically activates C4 and C2, and MASP1 is suggested to cleave C3. The roles of MASP3 and sMAP have not been well characterized. In bony fish, MBL-like molecules have been cloned from three cyprinid species (carp, goldfish, and zebrafish) and found to be produced mainly in 20 spleen rather than liver, which is the major source of mammalian MBL . Their deduced primary structures predict a specificity toward galactose, unlike mammalian MBL, which is specific for mannose, N-acetyl-D-glucosamine, and glucose. Recently we have cloned and purified another MBL-like molecule that show the same carbohydrate specificity as that of mammalian MBL, indicating 21 the presence of two distinct MBL lineages in bony fish . In-silico sequence analysis using the zebrafish genome database suggests that the MBL-like galactose-specific lectin and the authentic MBL are encoded by tandemly duplicated genes (Figure 3). 22 23 The mammalian MASP3-like molecule (caMASP) and its truncated form (MRP) have been cloned from carp. Interestingly, caMASP and MRP are generated by alternative polyadenylation and splicing, as are human MASP2 and 24 sMAP , and carp have duplicated genes of caMASP, both of which produce the two splicing variants. However, the role of caMASP and MRP in the carp complement system is totally unknown. We have recently identified a human MASP2-like molecule in carp serum. Carp MASP2 was purified from serum as

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M. NAKAO ET AL. Amino acid identity with zebrafish GalBL (AF227738) 67%

63%

99%

45%

Gene 1

Gene2

Gene 3

Gene 4

47%

46%

420,000

430,000

440,000 49%

81%

Amino acid identity with carp MBL (AB110825 )

Figure 3. Gene organization of the mannose-binding lectin (MBL)-like lectins identified in a zebrafish genomic contig (AL954692). Boxes show putative exons. Nucleotide numbers within the contig are also shown below the scale. Sequence similarity at the amino-acid level with zebrafish MBLlike galactose-binding lectin (designated GalBL) and with carp MBL suggests that Gene 3 corresponds to the reported GalBL and Gene 4 encodes zebrafish MBL. Note that Gene 1 and Gene 2 also specify GalBL-like amino-acid sequences, suggesting that GalBL-like lectin is further diversified.

21

a complex with MBL, and its cDNA was also cloned from the hepatopancreas . The carp MBL–MASP2 complex cleaves human intact C4 into C4a and C4b but not C4i, a C4 with thioester hydrolyzed, suggesting that complex functions in the lectin pathway activation. It is to be analyzed whether the MASP2 gene is duplicated in the carp genome.

4. ALTERNATIVE PATHWAY COMPONENTS The alternative pathway does not contain any obvious recognition molecule to launch its activation. Intrinsically, this pathway is rendered to low-level activation triggered by spontaneous hydrolysis of the thioester in C3, and this activation is continuously suppressed in normal conditions by several regulatory components described below. Upon contacts with so-called activators such as bacterial lipopolysaccharides and β-glucans, the spontaneous activation is released from the regulation, allowing amplified activation of C3 in a positive1 feedback manner . The central component in the alternative pathway is C3, a 200-kDa glycoprotein composed of two polypeptide chains (α-chain, ~120 kDa; β-chain, ~70 kDa) linked by a disulfide bond. Upon activation, C3 is cleaved by a C3– convertase complex into C3a and C3b, which is then degraded into iC3b, C3dg, and C3d by factor I and its cofactors, and by other endogenous proteases. C3a is a potent anaphylatoxin recognized by C3aR on leukocytes, eliciting inflammatory responses. C3b and iC3b act as opsonic ligands for CR1 and CR3 on phagocytes, respectively, whereas C3d stimulates B cells by binding to CR2. Thus, C3 plays biologically key roles in host defense.

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C3 has been purified and/or cloned from a number of bony fish species, in8 cluding trout, carp, medaka fish, zebrafish, and others . All the bony fish C3 has a well conserved molecular architecture that is specified by the disulfide-linked two-chain structure, the specific cleavage site for the C3-convertase, the internal 25 thioester bond, and the C-terminal extended domain termed the C345C domain . The most intriguing finding on bony fish C3 is that all the bony fish species 9 studied so far have multiple C3 isoforms, in contrast to mammals . Although some bony fish species are tetraploid or pseudotetraploid like trout (2n = 58–62) and carp (2n = 100), the multiple C3 isoforms have been found not only in the 26 tetraploid species but also in diploid species such as sea bream (2n = 48) , 27 28 medaka fish (2n = 48) , and zebrafish (2n = 50) . The primary structures of the 29 27 multiple C3 isoforms have been extensively analyzed in carp , medaka fish , 30 and trout . At the protein level, various functions including hemolytic activity, covalent binding to a variety of targets, and fragmentation after activation have 31 26 29 been analyzed for the C3 isoforms of trout , sea bream , and carp . The most interesting finding about C3 divergence is an amino-acid substitu1106 tion of the thioester-catalytic histidine, which corresponds to His of human C4A/C4B isotypes. As summarized in Figure 4, the catalytic His is replaced by Thr in trout C3-4, Ala in medaka fish C3-2, Ser in carp C3-S, and Gln in carp

β-chain S

S α-chain

Thioester site Trout C3-1 Trout C3-3 Trout C3-4 Carp C3-H1 Carp C3-H2 Carp C3-S Carp C3-Q1 Medaka C3-1 Medaka C3-2 Human C3

The catalytic site

S——C=O | | GCGEQ........... PVIHAEM GCGEQ........... KLLHPTM GCGEQ........... KILTPSM GCGEQ........... AVIHGEM GCGEQ........... AVIHGEM GCGEQ........... AVISSGM GCGEQ........... AVRQSEM GCGEQ........... KMYHGEM GCGEQ........... RVSAGYM GCGEQ........... PVIHQEM H1126 E1128

Figure 4. Conservation of the thioester site and diversity of the thioester-catalytic site in bony fish C3 isoforms. A schematic polypeptide structure of C3 shows its two-chain structure. The thioester site (the thioester linkage is shown above the sequence) is perfectly conserved in all the species. The catalytic histidine (H1126 in human C3) is replaced in at least one of the isoforms in each species. Note that the residue corresponding to human E1128, which is predicted to affect the catalytic activity of H1126, is also substituted in the bony fish C3 isoforms.

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C3-Q1 and C3-Q2. A study on human C3 using a panel of site-directed mutants has shown substitution of the catalytic His with a less nucleophilic residue such as Ala and Asp alters the substrate specificity of the covalent binding reaction of 32 the thioester from hydroxyl- to amino-group . Therefore, it is most likely that those carp C3 isotypes without the catalytic His do not bind to hydroxyl-groups and, in turn, prefer amino-groups as a substrate. This was confirmed at the protein level using the carp C3 isotypes; C3-S with Ser at the catalytic site did bind to glycine, a model molecule containing an amino-group, whereas C3-H1 and 29 C3-H2 with the catalytic His did not . The hemolytic activity of carp C3 isoforms also shows considerable difference from among others (Table 1). Especially, the hemolytic activity of C3-S without the catalytic His is much higher than that of C3-H1, suggesting that both C3-H1 and C3-S play a significant role in carp complement. In contrast, C3-H2 carrying the catalytic His shows no hemolytic activity, indicating that undefined structural elements other than those related in the thioester-catalytic mechanism affect C3 activation. C3 isoforms purified from trout and gilthead sea bream have been analyzed for their efficiency in covalent binding to different target particles: sheep eryth26,31 rocytes, rabbit erythrocytes, E. coli, and zymosan (Table 1). The C3 isoforms in each species show considerably different degrees of binding efficiency. In particular, a dramatic difference in the binding to erythrocytes and zymosan was noted between C3-1 and C3-3/C3-4 in trout and between C3-1/C3-2 and C33/C3-4/C3-5 in sea bream. For trout C3 isoforms, structural elements that explain the functional diversity was implicated to be the catalytic His and the resi1128 30 due equivalent to Glu of human C3 . However, it is still difficult to identify amino-acid residues responsible for the diversity in the binding efficiency against the natural targets. The “net” binding efficiency could be affected not only by the substrate specificity of the covalent binding reaction of the thioester but also by the relative abundance and surface distribution of hydroxyl- and amino-groups on natural targets. C3a anaphylatoxins have been purified from trout and carp. Carp C3a released from C3-H1 isoform showed undetectable activity in the chemotaxis as33 say for neutrophils, as does mammalian C3a . In trout, C3a molecules were prepared from three C3 isoforms (C3-1, C3-3, and C3-4) and analyzed for their 34 functions . All the C3a isoforms induced a respiratory burst of the head kidney leukocytes at a similar level of the activity, with no indication of functional differentiation among the three C3a forms. As mentioned above, the activated form of C3, or C3b, is degraded into smaller fragments, shifting their specific receptors and thereby biological activities. Carp C3-H1 and C3-S, the two major isoforms in the serum, have been 35 demonstrated to yield a mammalian C3d-like final fragment . The efficiency of C3d production seems different between C3-H1 and C3-S. More interestingly, recombinant C3d fragments of C3-H1 and C3-S, expressed as fusion proteins with N-terminal glutathione-S-transferase, were shown to attach to carp lymphocytes at different affinity. It is therefore suggested that C3 isoforms in bony

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Table 1. Functional Properties of Bony Fish C3 Isoforms Species/Properties

Isoforms

Rainbow trout a,b Binding to sheep E a,b Binding to rabbit E b Binding to zymosan Serum level (mg/ml)

C3-1 – ++++ ++++ 1.5-2.0

C3-3 + +++ – 0.3-0.4

C3-4 + +++ – 0.3-0.4

Gilthead sea bream a,b Binding to sheep E a,b Binding to rabbit E b Binding to zymosan

C3-1 ++++ ++++ +++

C3-2 ++ ++++ +++

C3-3 ++ ++ –

C3-4 ++ ++ –

C3-1 c (C3-S) + ++++ ~0.3

C3-2/3 (C3-H1) – ++ ~0.6

C3-4 (C3-Q1) a ND a ND a ND

C3-5 (C3-H2) – – a ND

wtC3 – + ++++ +++ +++

C3-H A + – a ND a ND a ND

Carp b

Binding to glycine b Hemolytic activity Serum level (mg/ml) d

Human Binding to glycine Binding to glycerol a,b Binding to sheep E a,b Binding to rabbit E b Binding to zymosan

9

1126

C3-5 +++ +++ –

1126

C3-H D + – a ND a ND a ND

a

Abbreviations: E, erythrocytes; ND, not determined. Approximate relative activity is shown: –, none; +, low; ++, moderate; +++, high; ++++, very high. c Corresponding name of the isoform identified at the cDNA level. d Data from the artificial mutants. b

fish show diverged function not only in the binding specificity of thioester, but also in their biological effector functions associated with leukocytes. Factor B (Bf) is a serine protease precursor that shares the same domain structure with C2. Bf binds to C3b, and the complexed Bf is cleaved into Ba and Bb, which stays bound to C3b and functions as a catalytic subunit of the C3/C536,37 38 convertases, C3bBb and C3bBbC3b. In contrast to mammals, carp , trout , 28 and zebrafish have been shown to possess multiple copies of Bf genes. In trout, two B/C2-like isotypes sharing 75% amino-acid sequence identity 38 have been cloned and named Bf-1 and Bf-2 . In a hemolytic assay using trout serum depleted of Bf-1 and Bf-2, Bf-2 alone was enough to restore the hemolytic activity, suggesting that the two isotypes play a similar role in trout com-

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plement. Two cDNA sequences, one complete and one partial, encoding B/C2 have also been isolated from zebrafish and designated BfA and BfB, respec28 tively. They share 82% identity when compared over the overlapping region . We have cloned three Bf-like molecules from carp and designated them 36,37 B/C2-A1, B/C2-A2, and B/C2-A3 , proving the presence of at least three Bflike genes. B/C2-A1 and B/C2-A2 share 78% amino-acid sequence identity, as in the case of the B/C2-isotypes of zebrafish and trout. B/C2-A3 is a novel isotype in that it shows less similarity (~60%) with the two other carp B/C2-A isotypes. A phylogenetic analysis suggests that B/C2-A3 emerged first in the bony fish lineage after separation of cyprinid and other families and that B/C2-A1 and B/C2-A2 then diverged perhaps by tetraploidization of the carp ancestor. It is of particular interest that B/C2-A3 is mainly expressed in lymphoid organs such as the kidney and spleen, in contrast to B/C2-A1 and B/C2-A2 expressed mainly in 37 hepatopancreas, the organ equivalent to liver (Table 2). Furthermore, expression of B/C2-A3 in carp kidney and spleen is upregulated by intraperitoneal stimulation of carp with sodium alginate (acidic polysaccharide from sea algae) or scleroglucan (fungus-derived β-1,3-glucan), known as immunopotentiators 39,40 for fish . Table 2. Three Factor B Isotypes of Carp Properties

Amino acid identity with B/C2-A1 B/C2-A2

37

Isotype name B/C2-A1

B/C2-A2

B/C2-A3

-

78% -

60% 62%

+ (+) ± (–) – (+) – (–)

– (±) + (++) – (++) ± (++)

a

Expression site Hepatopancreas Head kidney Renal kidney Spleen a b

++ (++) ± (–) – (–) – (–)

b

Determined by semi-quantitative RT-PCR. Relative expression levels in normal (unstimulated) fish: –, not detected; ±, faint; +, low; +, high. Data in parenthesis show that of fish which had been intraperitoneally injected with β-1,3-glucan (scleroglucan) two days before.

This extrahepatic and inducible expression suggests that carp B/C2-A3 may have evolved as a novel Bf molecule specialized as an acute-phase reactant to enhance complement activation at the early stage of infection. We have recently prepared recombinant proteins of the carp factor B isotypes using the baculovirus-mediated system. Further functional analyses at the protein level are in progress.

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5. LYTIC PATHWAY COMPONENTS The lytic pathway is composed of C5, C6, C7, C8, and C9. Like C3, C5 is proteolytically activated into C5a and C5b by the C5-convertase, a C4bC2aC3b complex in the classical and lectin pathways or a C3bBbC3b complex in the alternative pathway. C5a is a potent anaphylatoxin, and C5b is anchored on the target membrane mainly by a hydrophobic interaction. C5b on the target membrane attracts C6, C7, and C8 to form a membrane-bound complex, and then multiple molecules of C9 are integrated to form a tunnel-shaped cytotoxic complex, termed the “membrane-attack complex” (MAC). Earlier studies at the protein level have revealed that bony fish have a well-developed lytic pathway 41-43 composed of C5 through C9 . Later researches at the molecular level have shown that all the lytic components have well-conserved domain architectures similar to those of mammalian counterparts. To date, a number of cDNAs en44 coding the lytic pathway components have been cloned as follows: trout C5 , 45 46 47 48 49 50 51 carp C5 , trout C6 , C7 , C8α-chain , C8β-chain , and C9 , fugu C9 , and 52 52 flounder C8β and C9 . 44 Whereas trout C5 has been suggested to be encoded by a single-copy gene , 45 two distinct C5 isotypes, C5-I and C5-II, have been identified from carp . Carp C5-I and C5-II share a high degree of amino-acid sequence similarity (83%), but only the C5-I gene generates a novel variant, in which the C5-convertase cleavage site (LSR) corresponding to the C terminus of C5a is replaced by LSH. The Arg-to-His substitution probably makes the C5 variant impossible to be activated by the C5-convertase, because a catalytic subunit of the C5 convertase, Bb, is predicted to show a typical trypsin-like substrate specificity, favoring Arg 37 and Lys as the S1 site . If it is the actual case, the C5 variant could act as a competitive inhibitor for normal C5 activation, since the variant would interact with the C5-convertase but fail to be cleaved. It is intriguing that only C5-II isotypes behave as an acute-phase reactant, as assayed by northern hybridization, after stimulation of fish with LPS and β-glucan (Table 3). Trout C5a, generated as a recombinant protein, showed potent chemoattrac53 tive and respiratory burst-inducing activities, as does mammalian C5a . In carp, two C5a forms that probably represent allotypic variants of C5-I showed differ33 ent levels of the chemoattractive activity against the head kidney leukocytes . It would be intriguing to test whether such a functional difference could be detected between C5a forms from C5-I and C5-II.

6. COMPLEMENT RECEPTORS The following complement receptors have been identified in mammals: CR1, 54-56 57 58-60 CR2, CR3, C3aR, C5aR, and C1qR. Of these, CR3 , C3aR , and C5aR have been identified in bony fish.

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Table 3. Expression of C5 Isotypes of Carp

45

Stimulant and isotype ab

Expression site

Hepatopancreas Head kidney Renal kidney a b

None (saline) C5-I C5-II + – –

+ ± ±

LPS C5-I

C5-II

+ – –

+++ ± ±

β-1,3-glucan C5-I C5-II ++ – –

++++ + +

Relative expression levels shown in the number of ‘+’. (–, not detected; ±, faint) Spleen and intestine were also analyzed and gave no signal either in normal or stimulated fish.

CR3 is a member of the leukocyte integrins, consisting of CD11b (αM61 chain) and CD18 (β2-chain) . A CD18 ortholog has been identified in the chan56 nel catfish . Catfish CD18 expression in the leukocytes has been shown to increase in response to fMLP stimulation. In addition, a 95-kDa polypeptide was coprecipitated with the CD18 by anti-recombinant CD18 from catfish leukocytes, indicating that catfish CD18 forms a mammalian CR3-like heterodimer. In carp two CD11-like and two CD18-like cDNA sequences, each sharing 90% amino-acid sequence identity, have been cloned (unpublished observation). Interestingly, carp CD11-like sequences show almost equal similarity to mammalian CD11a (αL-chain of LFA), CD11b (αM-chain of CR3), and CD11c (αX-chain of CR4). Therefore, the carp CD11 may represent an ancestral subunit of the leukocyte integrins that later diverged into LFA, CR3, and CR4. If this is the actual case, it is very interesting to analyze the possible multifunctions of the carp CD11–CD18 complex in cell-adherence and opsonization. A difference in expression level was observed between the isotypes of carp CD11 and CD18 (data not shown). C3aR and C5aR are members of the G protein-coupled receptors containing 57 58-60 seven transmembrane domains. Recently, C3aR and C5aR orthologs have been identified in trout. Trout C5aR are suggested to be duplicated genes, but analysis of the transcript implies that only one of them is expressed.

7. REGULATORY FACTORS To control excessive activation and activation on host cells, the complement system contains various regulatory factors. C1Inh, a member of the serpin family of protease inhibitors, is an inactivator for the activated forms of C1r/C1s/ MASPs. Regulation of C3/C5-convertase formation is accomplished in two distinct manners: decay-acceleration and C3b/C4b-degradation. The decay-accelerating factor (DAF), factor H, and complement receptor type 1 (CR1) enhance

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dissociation of C3bBb and C4bC2a to limit C3-convertase. On the other hand, degradation of C3b and C4b is catalyzed by a specific serine protease, factor I, which needs appropriate cofactors such as C4bp, factor H, CR1, and MCP. In bony fish, a factor H-like regulatory cofactor from the striped sand bass 62,63 has been well characterized . The sand bass cofactor, designated SBP1, has been reported to function as both C4bp and factor H, supporting degradations of 62 C4b and C3b . Thus, bony fish may have a prototypic cofactor for factor Imediated regulation of C3b and C4b. Two factor I isotypes have been identified in carp at the cDNA level and 64 designated FI-A and FI-B . These isotypes have a similar domain organization, sharing 75% amino-acid identity, except that FI-A has a long (~100 amino acid) N-terminal extension that shows no obvious homology to any other sequences in databases (Figure 5). The functional significance of the N-terminal extension of FI-B is still to be assessed. FI-A mRNA is detected only in the hepatopancreas and ovary, whereas FI-B mRNA is found in the liver, kidney, and spleen. Although no functional data at the protein level has been obtained for carp FI-A and FI-B, the different expression profiles imply that they are functionally differentiated to some extent, though biological meaning of the FI-A expression in ovary is unknown.

FI-A FIMAC

SRCR

LDLR-1 LDLR-2

SP

FI-B Figure 5. Domain structures of two factor I isotypes (FI-A and FI-B) of carp. The N-terminal extension of FI-B is shared. Abbreviations of the domain names: FIMAC, factor I/membrane-attack complex; SRCR, scavenger receptor cysteine-rich; LDLR, low-density lipoprotein receptor type A; SP, serine protease.

8. CONCLUDING REMARKS AND FUTURE DIRECTIONS It has become increasingly evident that bony fish possess an expanded repertoire of complement component genes. In many cases, additional copies of a complement gene have not been abandoned as pseudogenes in the fish species but seem to remain functional. It is interesting that such diversity is prominent in carp, a pseudotetraploid species, but not so much in trout, which also has a tetraploid past. How does carp utilize the multiple isoforms of its complement components? The present data imply that the multiplied isoforms of the carp complement

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components have not merely been maintained as redundant copies with an identical function. Rather, they have likely been differentiated so as to show preferential interactions with foreign targets and/or other complement components/receptors or expression profiles. Although the findings described in this chapter give us only a glimpse into the expansion of complement-related genes in bony fish, further analysis of their functional differentiations will uncover the biological significance of this diversity. A plausible hypothesis for the diversity of the complement component has been postulated as a unique evolutionary strategy to enhance innate immune defense in bony fish, of which adaptive immunity seems not to have been developed well compared with that of mammals. 65 66 In this context, it is interesting to note that multiple copies of C3 and factor B genes have been identified in the genome of Ciona intestinalis, which lacks adaptive immunity. To examine the hypothesis, the roles of each isoform in the host defense of bony fish have to be evaluated in vivo. Application of gene knock-down and gene knock-out techniques will be desirable in solving these questions.

9. ACKNOWLEDGMENTS The authors thank Ms. Ikuko Takahashi for editorial assistance and Dr. John D. Lambris for helpful discussion. This work was supported in part by a Grant-inAid for Scientific Research from the Japanese Society for Promotion of Science (16380135 to Nakao).

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10 C5b-9 COMPLEMENT COMPLEX IN AUTOIMMUNE DEMYELINATION: DUAL ROLE IN NEUROINFLAMMATION AND NEUROPROTECTION Horea Rus, Cornelia Cudrici, and Florin Niculescu 1. INTRODUCTION Multiple sclerosis (MS) is the prototype for central nervous system (CNS) demyelinating diseases in humans. MS is considered an autoimmune disease in which myelin and the myelin-producing oligodendrocytes (OLGs) are the targets of immune attack. The essential requirements for initiating CNS inflammation characteristic of MS are the expression of encephalitogenic antigens, generation of chemotactic signals in the CNS, expression and upregulation of + adhesion molecules on endothelial cells, and activation of antigen-specific CD4 1 T cells . CD4 T cells are primed in the periphery and then enter the CNS. In the perivascular space they encounter myelin antigen expressed by local antigen2,3 presenting cells, microglia, and possibly dendritic cells . The reactivated CD4 T cells then invade the parenchyma of the CNS and release proinflammatory cyto4 kines and activate microglia . Subsequent demyelination may occur through 5 activation of macrophages and cytotoxic T cells . Activation of complement by 6,7 antibodies against myelin proteins found in MS patients or by myelin and 8,9 OLGs themselves can also contribute to myelin damage. Recent observations have indicated that inflammatory cells and mediators can also have protective effects, playing an important role in recovery and repair. In the context of inflammatory demyelination, these protective effects lead to axon preservation/repair, limitation of glial scarring, remyelination, and protection from cell death within the CNS. For example, a neuroprotective role for T cells after CNS injury has been demonstrated in a lysolecithin-induced demyelination model, in which T cell-deficient mice show significantly reduced

Horea Rus, Department of Neurology, University of Maryland, School of Medicine, Baltimore, MD 21201, USA. Veterans Administration Maryland Health Care System, Multiple Sclerosis Center of Excellence, Baltimore, MD 21201, USA. Cornelia Cudrici, Department of Neurology, University of Maryland, School of Medicine, Baltimore, MD 21201, USA. Florin Niculescu, Department of Medicine, Division of Rheumatology and Clinical Immunology, University of Maryland, School of Medicine, Baltimore, MD 21201, USA.

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spontaneous remyelination when compared to control animals .This repair may be mediated at least in part through the antigen-stimulated production of growth factors, particularly brain-derived (BDNF) and glial cell line-derived neurotrophic factors, by T cells and macrophages. BDNF-positive mononuclear cells are 11 localized throughout demyelinating MS lesions . BDNF is involved in regulating survival and differentiation of neurons, which express BDNF receptor gp145trkb. These data lead to the introduction of new paradigm, that of neuroprotective autoimmunity. A similar protective role has been shown for macrophages, which also secrete growth factors promoting differentiation of oligoden12 drocyte progenitors , and for several cytokines, such as TNFα, IL-1β, and 13 IFNγ . An enhanced myelinogenesis is observed in macrophage-supplemented CNS aggregate cell cultures, and TGF-β1 produced by macrophages may have the potential to promote remyelination by modulating the synthesis of growth 14 factors . The neuroprotective effects of complement activation have recently been documented, suggesting that the C5b-9 complement complex also plays an im15 portant role in the repair process . In this review we present experimental and clinical evidence that point to the involvement of complement activation and the subsequent assembly of the C5b-9 complement complex in both neuroinflammation and neuroprotection in MS and in experimental autoimmune encephalomyelitis (EAE).

2. ROLE OF C5b-9 IN NEUROINFLAMMATION 2.1. In Vitro Demyelination by C5b-9 The requirement for anti-myelin antibodies and serum complement in demyeli16 nation was studied in vitro by using myelinated CNS explants cultures . Treatment of myelinated explants with IgG or IgM anti-myelin antibodies and fresh serum induced extensive demyelination. To study the possible role of C5b-9, serum complement was activated using the IgM fraction of anti-guinea pig spinal cord antiserum and human serum depleted in late component C8 (C8D) with 17 and without C8 to assemble C5b-7 and C5b-9 , respectively. Exposure to antibody and C8D failed to induce demyelination; in contrast, C8 addition to C8D to complete C5b-9 assembly induced extensive demyelination and eventual myelin loss after 20 h (Figure 1). Removal of antibody and complement after 2 h failed 17 to inhibit myelin vesiculation , suggesting that molecular changes of myelin damage were initiated by C5b-9 within 2 h, and continuous activation of complement may not be required. These findings unequivocally demonstrated the C5b-9 complex as an effector in myelin damage and demyelination. Although 18-20 9, 21 and OLGs can activate C1 and generate C5b-9 in vitro, isolated myelin anti- myelin antibodies, including anti-galactocerebroside, are required for complement to produce significant demyelination in explants by increasing the level of C5b-9. Demyelination induced in explants, as shown by massive myelin

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Figure 1. In vitro demyelination by C5b-9. To study the role of C5b-9 in antibody-mediated demyelination in vitro, myelinated rat cellebelar explants were incubated with anti-guinea pig spinal cord antibody in the presence of C8D serum (CTR) and C8D serum reconstituted with C8. Activation of complement up to C7 failed to cause significant myelin vesiculation, whereas addition of C8 to form C5b-9 (Ab+C8D (10%) +C8) induced extensive demyelination. In the presence of 2.5% C8D (Ab+C8D (2.5%)) and C8, only part of the axons were demyelinated.

vesiculation, may differ from that of in-vivo demyelination since damaged myelin is opsonized by C4b/C3b and iC3b and will be actively removed by macrophages derived from circulating monocytes. 2.2. Role of C5b-9 in Demyelination during EAE The involvement of complement in demyelination in vivo has been investigated in EAE by inhibiting complement activation and by using rodents deficient in particular complement components. In rats, depletion or inhibition of complement using cobra venom factor or soluble CR1 has been shown to ameliorate 22,23 EAE . In an antibody-mediated model of EAE induced with Myelin Oligodendrocytes Glycoprotein (MOG), complement-fixing anti-MOG antibody 24,25 was found to be essential in inducing demyelination . In C3 knockout mice, MOG-induced EAE has produced conflicting results: one group has reported lower clinical scores with less inflammation, indicating 26 protection from demyelination in the absence of C3 , while another group, using a higher dose of MOG, has seen similar clinical signs in EAE knockout and con27 trol mice . On the other hand, deletion of C3aR has been found to be protective 28 in MOG-induced EAE . Factor B knockout mice developed less severe EAE than did control mice, indicating a role for the alternative pathway in comple26 ment activation . When EAE was induced by MOG in C5a receptor (C5aR)deficient mice, no difference was observed in disease onset or severity in defi29 cient and control mice . Blockade of C5a receptors using small peptides failed 30 to protect against EAE . Thus, C5aR might not play an important role in mediation of inflammation in EAE.

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The importance of C5b-9 in demyelination and axonal damage has been re31,32 ported in an EAE model in C6-deficient rats . In these rats, which are unable to form C5b-9, disease activity and demyelination, as well as T-cell and macro31,32 phage infiltrates, were significantly reduced . Axonal fragmentation and swelling was seen in the demyelinated area of C6-sufficient rats, compared with the normal axonal structure in the C6-deficient rats. Strong staining for C9, indicative of C5b-9 deposition, correlates well with the area of axonal degeneration and demyelination in the C6 sufficient rats. The expression of P-selectin is also lower on endothelial cells in C6-deficient rats than in normal animals, but no 32 differences are seen in the profile of Th1 or Th2 cytokines . These data indicate that the terminal complement complex C5b-9 plays a proinflammatory role in the acute phase of EAE and is required for demyelination, mononuclear infiltration, and axonal damage.

3. ROLE OF C5b-9 IN NEUROPROTECTION 3.1. Inhibition of Oligodendrocyte Apoptosis by Sublytic C5b-9 Differentiation of OLGs in serum-free medium is associated with apoptotic cell death. Serum withdrawal-induced apoptosis in OLGs is associated with caspase 33-35 activation, that includes caspase-8, caspase 9, and caspase-3 . In addition, BID cleavage, cytochrome c release, and a markedly reduced expression of c-FLIPL are seen. Apoptosis of OLGs is inhibited by a specific caspase-3 inhibitor, 33 DEVD-CHO , and the caspase-8 inhibitor Z-IETD-FMK in a dose-dependent 35 manner . We have found that both phosphatidylinositol 3-kinase (PI3K) and Akt are downregulated during OLG differentiation and are induced in response to sublytic C5b-9 (Figure 2). Furthermore, we observed that sublytic C5b-9 was effective in inhibiting cytochrome c release (Figure 3) and activation of caspase 9 and caspase-3, thus rescuing OLGs from serum deprivation-induced apoptotic cell death. C5b-9-mediated activation of the PI3K/Akt signaling pathway also 34 led to phosphorylation of BAD . Association of BAD with BCL-XL is thought to produce mitochondrial damage by allowing oligomerization of proapoptotic BAX and BAK. On the other hand, dissociation of BAD from BCL-XL and binding to cytoplasmic 14-33 proteins increase cell survival and require phosphorylation of BAD at Ser112, 36 Ser136, and possibly Ser156 . C5b-9 has been shown to stimulate phosphorylation of BAD at Ser112 and Ser136 and cause dissociation of the BAD/BCL-XL 34 complex . Both processes can be reversed by pertussis toxin (PTX) and the PI3K inhibitor LY 240092. Therefore, sublytic complement attack appears to increase OLG survival in part by activating signaling pathways that phosphorylate BAD and reduce its association with BCL-XL. These results suggest that C5b-9 inhibits the mitochondrial pathway of apoptosis in OLGs.

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Figure 2. Activation of PI3K and Akt by C5b-9. (A) OLGs were treated with C5b-9 or C5b6 for the indicated time periods. Anti-PI3K immunoprecipitates were examined for PI3K activity by assessing phosphorylated phosphatidylinositol by TLC. Radioactive spots corresponding to PI3K activity were excised and counted. Results from triplicate samples are shown as cpm ± SEM. (B) OLGs treated with C5b-9 or C5b6 for the indicated time period were examined for Akt kinase activity. Anti-Akt1 immunoprecipitates were examined by in vitro kinase assay using an Akt-specific peptide substrate. From L Soane, H-J Cho, F Niculescu, H Rus, ML Shin. J Immunol 167, 2305 (2001). Copyright 2001, The American Association of Immunologists Inc.

Figure 3. Effect of C5b-9 on cyto-c release in OLGs. OLGs differentiated from OLG progenitors cells for 56 h were exposed to sublytic C5b-9. After 18h cells were permeabilized with digitonin, and cytochrome c (cyto-c) release was assessed by immunoblotting. Controls (CTR) included cells treated with C5b6 or medium. Results are also shown as density ratios to actin and to cox IV, for supernatant and platelet, respectively. From L Soane, H-J Cho, F Niculescu, H Rus, ML Shin. J Immunol 167, 2305 (2001). Copyright 2001, The American Association of Immunologists Inc.

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To identify initiators of apoptosis in OLGs, we evaluated the ability of TNFα and Fas L to induce apoptosis and examined the effect of C5b-9 on these pathways. Both TNFα and Fas L were able to induce apoptosis of OLGs, and 33,37 C5b-9 inhibited FasL and TNF-α-induced cell death . This effect is most 35 probably mediated through inhibition of caspase-8 activation by C5b-9 . Since cleavage of BID by caspase-8 has been shown to directly trigger the release of 38 cytochrome c from mitochondria (Figure 4), we monitored BID cleavage as 39 well as the levels of c-FLIP, an endogenous inhibitor of caspase-8 . Exposure to C5b-9 inhibited BID cleavage (Figure 5) and caused a significant increase in c35,37 FLIPL expression . These results suggest that C5b-9 prevents further caspase-8 processing, possibly through a c-FLIPL-dependent mechanism. All these C5b-9 effects on OLG apoptosis are mediated through activation of PI3K. Thus, our data indicate that C5b-9, acting through PI3K signaling, is able to rescue OLG apoptosis by regulating caspase-8 activation and preventing mitochondrial insertion of proapototic proteins BAD and BID (Figure 4).

Figure 4. Protection of oligodendrocytes from apoptosis by sublytic C5b-9. Assembly of sublytic C5b-9 leads to G-protein activation, followed by activation of PI3K and Akt. Activation of PI3K induces upregulation of FLIP and inhibition of caspase-8 processing, thereby inhibiting Fas-induced apoptosis in oligodendrocytes. PI3K is also responsible for inhibition of Bid cleavage. On the other hand, Akt is able to induce phosphorylation of BAD, which is sequestered in the cytoplasm by the 14-3-3 protein. All these C5b-9-induced effects result in inhibition of cytochrome c release and of mitochondrial apoptosis.

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Figure 5. Effect of C5b-9 of Bid cleavage. OLGs were exposed to either C5b-9 or C5b6 and examined by Western blotting for expression of tBid. The same blot was stripped and then probed with βactin (A). Results are shown as density ratios to β-actin in the lower panel (B). tBid levels were significantly reduced in the presence of C5b-9 when compared with cells treated with C5b6.

3.2. Contribution of Complement C5 to Neuroprotection in EAE We have recently analyzed the influence of C5 on inflammatory demyelination 15 during the course of EAE in C5-deficient (C5-d) and C5-sufficient (C5-s) mice . Both groups of mice displayed early-onset EAE, with a short recovery phase followed by a stable chronic course. During acute EAE, the lesions were more diffuse and extensive in C5-s than in C5-d mice. More striking differences attributed to the presence of C5 were found in the chronic lesions. In the absence of C5, EAE was associated with greater inflammatory demyelination, axonal loss, and progression to gliosis. On the other hand, C5-s mice showed prominent myelin repair and axon preservation during the chronic phase of EAE. These results in the EAE model indicate that the absence of C5 leads to fiber loss and extensive scarring, whereas the presence of C5 favors axonal sur15 vival and more efficient remyelination . These data also point to a dual role for C5: enhancing inflammatory demyelination in acute EAE and promoting remyelination and repair during recovery. We next analyzed the effect of complement 40 C5 on the apoptosis of OLGs during EAE . In acute EAE, C5-d and C5-s mice

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had similar numbers of total apoptotic cells; during recovery, however, C5-s mice showed significantly fewer apoptotic cells than did C5-d mice. In addition, while both groups of mice displayed TUNEL-positive OLGs, there were significantly fewer positive OLGs in C5-s than in C5-d mice during both acute EAE and recovery (Figure 6). We also investigated the effect of complement C5 on Fas-mediated apoptosis during EAE. Our results indicated that the expression of Fas and FasL is high in acute EAE but decreases during recovery in C5-s mice, whereas the number of Fas- and FasL-positive cells re40 mains high in C5-d mice during recovery . Fas expression was upregulated on the OLG surface (Figure 7) and FasL on inflammatory cells. These findings are consistent with a role for complement C5 in protecting OLGs from apoptosis in EAE, possibly by forming C5b-9 and thus promoting remyelination and preserving axon fibers during recovery. Recent data have suggested that neuronal protection is mediated by an increase in surface expression of CD55 that is induced by complement activation during chronic autoimmune inflammation of the 41 CNS .

Figure 6. Apoptotic OLGs during acute EAE and remission phase. Quantitative evaluation of apoptotic OLGs was performed on C5-s and C5-d mice. Data are expressed as percentage of TUNEL+ OLGs (mean ± SEM) relative to total TUNEL+ cells counted. In C5-d mice, the number of apoptotic OLG was significantly higher, both during acute EAE and in remission.

4. DOES C5b-9 PROTECT OLIGODENDROCYTES FROM APOPTOSIS IN MULTIPLE SCLEROSIS? In MS white matter lesion deposition of C1q, C3d, and C5b-9 has been detected on and within macrophages/microglia and astrocytes, and in blood vessel

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Figure 7. Fas expression by oligodendrocytes in acute EAE. Spinal cords were examined by double staining for expression of Fas and the myelin/OLG marker MAB328 (Chemicon, Temecula, CA). Many Fas+ OLG (arrowheads) are present in an area of actively demyelinating lesion, during acute EAE in C5-s mice (X400).

5,42-44,45

. C3d and C4d were detected along myelin sheaths at the edge of the walls lesions. The pathological lesions seen in MS have been confined to four patterns. In pattern II, the most common (39 of 73 cases), high levels of immunoglobulins and C5b-9 deposition at sites of active myelin destruction were found. Moreover, the C5b-9 deposits were present only in Pattern II demyelinat5,46 ing lesions . Although apoptosis of OLGs may occur in all four types of lesions, only pattern III generally shows a significant number of apoptotic OLGs. It is tempting to speculate that apoptotic OLGs are less frequently seen in pattern II as a result of activation of complement and subsequent C5b-9 assembly, leading to rescue of OLGs from apoptosis. While incidence of pattern II and III lesions were similar in acute MS, the latter become rare in chronic MS, indicating that OLG apoptosis is an early event in MS lesion evolution. When acute MS lesions were examined in a different study, extensive OLG apoptosis and microglial activation was observed in tissues containing few or no lymphocytes 44 or myelin phagocytes . In acute lesions, within hours, OLGs throughout the affected tissue appear apoptotic, and myelin sheaths stain positively for activated complement components C3d and C5b-9, while immunoreactivity for the 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin-associated glycoprotein is diminished, and ramified microglia with thickened processes appear in in44 creased numbers . Engulfed myelin positive for complement activation products is present in macrophages. These data suggest that complement activation 44 plays a role in the clearance of vacuolated myelin by macrophages . In purely cortical lesions, the extent of complement deposition was generally low and suggests that the role of complement in the pathogenesis of MS lesions is also 45 location-dependent .

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Increased levels of SC5b-9 have been detected in the spinal fluid of MS pa47-49 tients during relapses , and these levels have been shown to correlate with 49 neurological disability as measured by the Expanded Disability Status Scale . These findings suggest that complement activation and C5b-9 assembly also take place in MS spinal fluid when a breakdown has occurred in the blood–brain barrier. In conclusion, complement activation and C5b-9 assembly may contribute to neuroinflammation and demyelination in the acute phase of MS, while reducing the level of OLG apoptosis in the chronic phase. Taken together, these data indicate that complement activation can play both proinflammatory and neuroprotective roles in MS. Because activation of complement has many beneficial effects, long-term inhibition of complement activation may have undesirable consequences. Therefore, designing drugs that modulate rather than inhibit activation of the complement system may represent a new direction in therapy for demyelinating disorders.

5. ACKNOWLEDGMENTS We thank Dr. Deborah McClellan for editing this manuscript. This work was supported in part by US Public Health Grant RO-1 NS42011 (to H.R.) and by the Veterans Administration Maryland Health Care System Multiple Sclerosis Center of Excellence in Baltimore (H.R.).

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11 THE DOUBLE-EDGED FLOWER: ROLES OF COMPLEMENT PROTEIN C1q IN NEURODEGENERATIVE DISEASES Andrea J. Tenner and Maria I. Fonseca

1. INTRODUCTION C1q, the recognition component of the classical complement activation pathway, is a multifunctional protein of the immune system that has been shown to be expressed in the brain of Alzheimer’s disease (AD) patients. To experimentally address the role of C1q and the classical complement pathway in AD, a mouse model lacking C1q (APPQ-/-) was generated by crossing a transgenic mouse overexpressing the human mutant amyloid precursor protein (APP) with a C1qdeficient mouse. While displaying the characteristic “plaque” structures seen in Alzheimer’s disease, those APPQ-/- mice lacking the complement pathway had significantly less inflammation in the brain surrounding these plaques and significantly less loss of neuronal integrity than the transgenic mice with an intact complement system. Thus, at ages when the fibrillar plaque pathology is present, C1q exerts a detrimental effect on neuronal integrity, most likely through activation of the classical complement cascade and enhancement of inflammation. C1q synthesis is induced in several injury models, as well as in AD. However, since C1q has been shown to facilitate the rapid removal of apoptotic cells and blebs, and recently to modulate cytokine expression, C1q could play a critical role in avoiding the release of potentially damaging intracellular components in the initial stages of CNS injury by enhancing the phagocytic activity of resident microglia and macrophages and modulating inflammation. Thus, C1q, due to its diverse functional capabilities, could play a dual role in CNS disease, one being antiinflammatory and protective by rapidly clearing cell debris/neuronal blebs and modulating cytokine expression, and one enhancing neurotoxic inflammation in the presence of other complement components via

Andrea J. Tenner and Maria I. Fonseca, Departments of Molecular Biology and Biochemistry, and Pathology Institute for Brain Aging and Dementia, Center for Immunology, University of California, Irvine, CA 92697.

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activation of the classical complement cascade. This new perspective provides a basis for identifying new candidate drug targets to slow the progression of AD in humans. Alzheimer’s disease (AD) is a neurodegenerative disorder associated with the loss of cognitive function and the presence of characteristic neuropathological changes that include synaptic and neuronal loss, neurofibrillary tangles (NFTs), extracellular senile plaques composed of β-amyloid (Aβ) protein deposits, and evidence of glial inflammatory events. While compelling arguments can be made for the correlation between these pathologic structures and neurodegeneration, controversy remains as to the contributions of this pathology to cognitive dysfunction in AD, a critical issue for identification and development of therapeutic strategies for this disease, which afflicts more than 4.5 million peo1 ple in the United States . One hypothesis currently being tested is that Aß plays a major role in the progressive pathology and cognitive dysfunction seen in AD. The presence of mutations in various genes that lead to accumulation of amyloid in cases of fa2,3 milial AD provides genetic evidence for this “amyloid hypothesis” . However, there are distinct pools of amyloid peptide, including intraneuronal accumulations, soluble oligomeric structures, as well as prominent fibrillar extracellular plaques, and the precise relationships between different pools of amyloid and the major learning and memory deficits are still being investigated. Intraneuronal amyloid peptide is found early both in human Alzheimer’s disease and in some murine models of the disease. Several transgenic models have shown synaptic and behavioral deficits prior to plaque detection, suggesting that soluble Aβ 4-7 intermediates and/or intraneuronal Aβ are responsible for early synaptic loss , and supporting multiple levels of amyloid-induced injury. For example, in a novel triple transgenic mouse model (3xTg) of Alzheimer’s disease, LTP dysfunction correlates with the appearance of intraneuronal Aβ, much earlier than 7 detectable extracellular amyloid and tangle pathology . Ashe and colleagues have concluded from an analysis of behavior (the reference memory version of the Morris water maze test) that while the appearance of senile plaques correlates with onset of impairment of memory loss in their APP transgenic model, the absolute amount of insoluble Aβ (a reflection of fibrillar plaque load) does 8 not correlate with severity of performance loss . These authors suggest that plaques are a surrogate marker for another pool of “pathogenic Aβ assemblies,” which may include soluble oligomers that alter LTP in rodents and can be toxic 4,9,10 to neuronal cell cultures . Nevertheless, discrete neuronal loss is seen localized only to thioflavine-positive plaques when a cross-correlation density map method is applied to analyze Aβ, thio-S, and NeuN from both AD brains and 11 that of APPPS1 mice . In human studies, clinically significant cognitive decline occurs at the stage of the disease in which fibrillar amyloid and NFT deposits accumulate. It is well established that complement factors (including C1q, Figure 1) and reactive microglia and astrocytes are found associated with Aβ plaques containing the beta

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sheet fibrillar amyloid peptide rather than diffuse amyloid plaques. Thus, it is suggested that activation of complement by fibrillar Aß contributes to local activation of glia and proinflammatory cascades, ultimately accelerating the pathol14,15 ogy, neuronal dysfunction, and cognitive decline seen in the human disease. However, several reports suggest a protective role of complement in injury models as well. In this mini-review, we present an overview of our work and that of others in this area suggesting distinct pathways for C1q in AD progression, and discuss future approaches to understanding the basis of the loss of cognition as this disease progresses and applying that information to design and test new therapeutic strategies.

Figure 1 (see color insert, Fig. 11.1). C1q (red) colocalizes with thioflavine (green) in cortex of Alzheimer’s disease brain. Scale bar: 100 µm.

2. COMPLEMENT IN THE BRAIN The complement (C′) system is a powerful effector mechanism of the immune system that, upon activation, generates activation fragments (C3a and C5a) responsible for initiation of a local inflammatory response by recruitment of leukocytes to the area of infection or injury, and results in assembly of the membranolytic membrane attack complex (MAC or C5b-9) (Figure 2). In addition, C3b, C4b, and C1q can opsonize (target to professional phagocytes for inges16-18 tion) or facilitate rapid clearance of pathogens or cellular debris . While evolutionarily highly protective to the host, particularly in killing pathogens and clearing cellular debris, tissue damage can result from chronic or unregulated activation of the complement system by antibody–antigen complexes, specific pathogens, cellular debris, or misfolded proteins that are recurring or not de19,20 graded and cleared . As mentioned above, in vivo C1q (Figure 1) and other complement components have been shown to be associated with fibrillar Aß plaques and activated

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Figure 2. C1q is the recognition component of the classical complement pathway (CCP). The result of CCP activation is the recruitment of myeloid/glial cells to the area of activation and generation of the membranolytic MAC (membrane attack complex).

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glia , but not with diffuse plaques that can be present in brain tissue from nondemented elderly individuals and that are not surrounded by glial cells. In addition, McGeer and colleagues originally demonstrated the colocalization of com25 plement components with neurofibrillary tangles . We and others verified C1q 21,26 association with neurofibrillary tangles (PHF-1 immunoreactivity) . Synthesis 27,28 of most complement factors has been shown to occur within the AD brain , receptors for complement activation products are on neurons as well as on microglia and astrocytes (reviewed in [29]), and the complement membranolytic complex, C5b-9, has been detected in areas containing the fibrillar plaques in 30 AD . Thus, there is a source of complement components in the CNS and signature markers that indicate that complement activation does occur in the AD brain. Brain tissue from Down’s syndrome patients who develop age-related dementia was also examined for these pathological markers and were found to mimic AD cases in the correlation between C1q colocalization with plaques and 31-33 presentation of cognitive loss . Extensive in vitro studies by this laboratory and others have shown that C1q, the first component of the classical complement pathway (CCP), binds to

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fibrillar Aß and activates the classical complement pathway , while activation of the alternative complement pathway by interaction of Aß with C3 has also 37,38 been documented . This activating ability was shown to correlate with the assembly state of the peptides, i.e., with β-sheet fibril formation, as documented 35 39 by sedimentation properties , circular dichroism and electron microscopy , and 40 later by thioflavine staining by Webster and colleagues . In vitro phosphorylated tau, a component of the neurofibillary tangles, also activates the classical 26 complement cascade . These data provided the in-vitro correlates to the immunohistochemical findings in AD brain in which C1q was associated only with 21 thioflavine-staining (fibrillar) Aß and extracellular PHF-1-positive tangles . It was subsequently determined that the acidic amino-acid residues 7 and 11 in the N-terminal portion of the amyloid peptide were critical for the interaction with 39,41 C1q and initiation of the classical complement pathway . These data, con36,42,43 firmed by others , allow construction of a model of this protein:protein interaction in which properly spaced negative aspartic acid and glutamic acid charges are able to interact with an arginine ladder of positive charges on C1q (Figure 39 43 3) . Additional interaction sites within the C1q molecule were proposed , and the recent crystal structure of the C1q globular domain reveals the presence of 44 the properly spaced arginine residues to provide additional interaction sites for Asp7/Glu 11 on fAß. Activation of the complement cascade results in the production of chemotactic factors (C3a and C5a) that can recruit microglia and astrocytes to the 45 plaque area (Figure 2). Activated microglia can be phagocytic, but can also secrete several proinflammatory cytokines, as well as reactive oxygen species and nitric oxide, essentially an inflammatory reaction in the area of the plaque 46 that could lead to enhanced neurodegeneration . In addition, the cytolytic C5b-9 30 complex has been detected associated with membranous material in AD brain . Thus, complement activation in AD induced by the fibrillar form of the Aß peptide (and perhaps hyperphosphorylated tau) can lead to detrimental conse47,48 quences, particularly if regulation of the activated cascade is deficient . If complement activation and the subsequent inflammation induced does contribute significantly to the neuropathology and cognitive function, then a specific inhibitor of the activation interaction (for example, to the Aß peptide C1q interaction domain and/or other molecularly defined complement activators) would be invaluable as it would block the pathogenic activation but should not affect the systemic protective functions of the complement system.

3. MURINE MODELS OF ALZHEIMER’S DISEASE Several mouse models that mimic some of the neuropathological features of AD have been developed to investigate mechanisms of pathogenesis and permit sub49-51 52 sequent testing of therapeutic interventions , some of which are reviewed in . Tg2576 mice that overexpress the 695aa form of human APP (also containing

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Figure 3. Model of triple helix portion of C1q interacting with Aβ constrained to the β-sheet.

the Swedish mutations), display age-dependent increases in Aß plaques with the 51 first deposits appearing at 9–10 months . Microglial and astrocytic activation and dystrophic neurites are also present in the plaque area, increasing with 51,53 age . Double transgenic mice generated from the cross of Tg2576 and mutant PS1 (M146L) develop larger numbers of fibrillar Aß deposits with age, and 54 these deposits begin to appear earlier than in Tg2576 (3–4 months) . Activated glia and complement factors associated with the plaque, as well as proinflamma55 56 tory cytokines , are also prominent in the brain tissue of these mice . Although 57,58 neuronal loss is minimal in these models , behavioral deficits have been re51,59 ported . To test the in vivo role of C1q in AD neuropathology we generated 2 C1qdeficient mouse models by completely eliminating the C1q protein and thus 51 CCP activation. Tg2576 APP and the APP (Tg2576) transgenic with PS1 muta54 60,61 tion (APPPS1) were crossed to a C1q knockout mouse (Q-/-) , which has been characterized as a model for studying effects of C1q-mediated activation of complement and C1q-mediated enhancement of apoptotic cell clearance. The resulting mice (APPQ-/- and APPPS1Q-/-) have the age-specific characteristic amyloid pathology of the corresponding APP Q+/+ lines but lack classical

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Figure 4 (see color insert, Fig. 11.4). Representative images from APP (left) and APPQ-/- (right) mice at 12 (middle) or 16 (top, bottom) months of age. Comparable levels of thioflavine staining are seen in APP and APPQ-/- mice (top panel). Scale bar: 100 µm. Decreased astrocytic reactivity (GFAP,red) around fibrillar Aß (thioflavine, green) plaques in APPQ-/- mice relative to APP C1qsufficient mice is evident (middle panel). Higher density of synaptophysin staining is seen in CA3 area of hippocampus of APPQ-/- mice relative to APP mice (bottom panel). Scale bar: 20 µm (middle and bottom panels).

pathway complement activation. Plaque area (total Aß and separately fibrillar Aß as detected with thioflavine) in cortex and hippocampus were identical in the C1q-sufficient and -deficient APP and APP/PS1 transgenic mice (Figure 4). As in the AD brain, C1q immunoreactivity was seen associated with thioflavine62 56 positive Aß plaques in the Tg2576 animals and APPS mice models , with C1q colocalized almost exclusively with thioflavine-positive plaques. No C1q stain62 ing was seen in APP Q-/- animals at any age , as expected. However, a prominent difference in the C1q-negative animals was a reduction in activated glial cell markers, GFAP and MAC-1, surrounding the fibrillar plaques at 12 and 16 months of age, the ages at which thioflavine plaques and C1q increase significantly. Image analysis showed that there was 50–60% less astrocytic reactivity

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at 12 (Figure 4, middle panels) and 16 months of age, and 50% lower microglial immunoreactivity at 16 months in the APPQ-/- mice relative to the APPQ+/+ group. At 3 and 6 months of age, at which time no Aß deposition was detected, no significant differences in these glial cell markers were seen. Similar immu62 nohistochemical results were found with the APPPS1 double transgenic . The significant decrease in activated microglia and astrocytes seen in the C1qdeficient animals support the hypothesis that complement activation, initiated by the binding of C1q to fibrillar Aß, is a trigger for recruitment of activated glia to 45 the plaques, likely due to C3a and/or C5a . Synaptophysin (SP), a protein localized in the neuronal synaptic vesicles and widely used as a marker for presynaptic terminals, has been shown to be decreased in AD brains and correlates well with severity of the cognitive 63,64 state . A significant decrease in SP at 12 and 16 months was observed in the APP (Tg2576) in the stratum lucidum of the CA3 area, where the strongest SP staining is usually detected. However, significantly less loss of synaptophysin (and MAP-2, a marker for cell body and dendritic integrity) was reproducibly 62 seen in the APPQ-/- mice (Figure 4, bottom panels) . Thus, decreases in activated glia in the APPQ-/- mice were accompanied by improved neuronal integrity. Interestingly, alterations in the distribution of mossy fibers in the stratum 65 66 lucidum is related with long-term plasticity and long-term memory . Levels of SP and MAP-2 in the APPQ-/-, however, were lower than in the age-matched control B6SJL mice, indicating that classical pathway complement activation and its resultant inflammation are not the only cause of neuronal injury. However, collectively, the effect of C1q on the inflammatory capacity of fibrillar plaques, and the subsequent neuronal damage at later stages of the disease, are compatible with the hypothesis that therapies inhibiting plaque-induced classical complement pathway activation in later stages of the disease may avoid the rapid/steep decline of cognitive ability seen in later stages of human AD. Animal models are an essential component of assessing the relevance of molecular and biochemical pathways in disease processes. However, the animal models (Tg2576 and Tg2576xPS1) used in our study, and those used by many other investigators in the field, while displaying age-dependent accumulation of amyloid deposits in the brain, are not a perfect mimic of the human disease, as 67 mentioned above and as discussed in reviews by Loeffler and McGeer and col68 leagues . Specifically, neurofibrillary tangles and neuronal loss were not seen or not nearly as prominent as in AD. However, a more recently generated triple transgenic mouse (3xTg) contains a human mutant APP transgene and mutated PS1 knock-in along with a form of human tau associated with frontotemporal 7 dementia . Thus, in addition to extracellular plaque pathology (of multiple morphological types), this transgenic animal also displays tau pathology in an agedependent manner. As alluded to above, it is important to point out that results of many studies are bringing into question the precise relationships between different pools of amyloid and the major learning and memory deficits that are the first and argua-

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bly the most debilitating human clinical presentation of the disease. Indeed, complement activation that is apparent at the time of fAß deposits may be later than the initial neural dysfunction and cognitive deficits in mouse models. It remains unknown whether complement activation influences the appearance and kinetics of accumulation of tau and the effect on cognitive functions, a question that can now be addressed utilizing manipulated the 3xTG murine model. In the end, however, it will be important to determine if at the stage of human disease in which fibrillar amyloid and NFT deposits accumulate (which correlates with the stage at which cognitive decline is evident in the human disease), the activation of complement contributes to and/or accelerates neuronal dystrophy and significantly impinges on cognitive ability in humans. Another difference exists between the mouse models previously used to study AD and human systems that may influence the observable extent of the 47,67 contribution of complement to pathology and dysfunction . Some laboratory strains of mice including those commonly used for AD transgenics have lower 69,70 complement lytic activity than in human as measured by in-vitro complement lysis assays. The basis for this apparent deficiency has not been established, and thus basic research on the murine complement system is required to verify that these in-vitro results are indicative of the in-vivo situation. However, if there are differences, classical complement pathway activation in human Alzheimer’s disease may have a greater contribution to human neuropathology and neuronal cell injury than that seen in the transgenic mouse model Tg2576 reported above, and therapeutic intervention in complement mediated events may result in even greater benefit in humans than predicted from the murine data via slowing the progression of pathology and cognitive dysfunction. Thus, mouse models that more closely mimic the human condition are needed to validate the contribution of complement applicable to human Alzheimer’s disease. If there is a detrimental effect of CCP, specific targeted inhibitors of complement activation by Aß should have a beneficial effect for individuals in which deposits have already begun to accumulate, by slowing the progression of this stage of the disease (with or without other therapeutic interventions). While there are multiple potential targets for therapeutic intervention in AD including immunization to prevent 71-73 amyloid accumulation , different approaches will likely be beneficial at different stages of the disease, and thus a cocktail of therapeutic reagents may be more successful in delaying CNS degeneration. In addition to its possible contribution to the pathogenesis of Alzheimer’s disease, complement has been implicated in a number of other neurodegenera74,75 76-78 tive diseases, including multiple sclerosis , stroke and ischemia/reperfusion , 79 80 81 as well as other dementias , age-related macular degeneration , prion disease 82,83 and spinal cord injury . In addition, a polymorphism in factor H that may affect the ability to limit the activated complement cascade has recently been identified by four independent groups to contribute to the risk of aged-related macu84-87 lar degeneration , supporting a role for subtle alteration of the regulation of activated complement in the pathogenesis of degenerative disorders. Thus, an

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enhanced understanding of the murine complement system, and development of reagents to analyze murine complement proteins and animal models with manipulated complement capabilities may be relevant to these other diseases as well.

4. POTENTIAL PROTECTIVE ROLES OF COMPLEMENT IN THE CNS It is becoming increasingly evident that some complement components also provide protective functions in areas of injury (reviewed in [47]). Thus, the potential protective effects of specific complement components in this disorder could also be targets for intervention. Recent work suggests that at least some of the complement factors (C3 and C5) can decrease neuropathology in mouse models 88 of AD or limit the detrimental responses to neurodegenerative stimuli in other 89,90 injury models . For example, when the cleavage of complement component C3 was inhibited in a transgenic APP model by overexpressing the soluble Crry protein (which inhibits formation of the enzyme that amplifies cleavage of C3 to C3b), Aß deposition was higher than in comparable mice that did not overexpress Crry and was accompanied by less microgliosis but elevated loss of NeuN 88 relative to the APP wild type . One explanation provided by the investigators was that this lack of microglia (and/or lack of C3b as an opsonin to enhance phagocytosis) prevented clearance of Aß and ultimately could lead to increased degenerating neurons. At this point the mechanisms resulting in the difference in outcome between the APP Q-/- mouse described by us and that of the Crry transgene is unknown. However, the outcome of greater plaque density in the APP/Crry transgenic and greater NeuN loss clearly indicates other factors are involved (perhaps including alternative pathway complement activation in the Q-/- mouse, which is not as effective in the Crry overexpressing mouse). Thus, in the APP/Q-/- mouse alternative pathway deposition of C3 on the plaques may take place providing an opsonic effect, and thus may provide an explanation for this apparent discrepancy. While the extent of C3 deposition due to the alternative pathway is not yet known, it is clear from other systems that very low levels of C3b can trigger significant enhancement of phagocytosis, and thus it may be that limited, but some, complement activation is beneficial. (Alternatively, the constitutive overexpression of Crry may have as of yet undiscovered effects.) Seemingly contradictory results were also observed in studies of the influence of the complement component C5 on inflammation. Amyloid-induced in91 flammation was decreased in one model of C5-deficient mice relative to C5 sufficient mice, while Pasinetti and colleagues reported that C5-deficient mice 89,92 showed enhanced neurodegeneration in response to some excitotoxic stimuli , and Cooper and O’Barr reported that the activation peptide C5a was neuroprotective in an in-vitro assay of toxicity using SH-SY5Y cells (90), again suggesting complex mechanisms are involved, and a protective role for complement

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exists under some conditions. Recent discovery of a second C5a receptor (C5L2), which has been proposed as a “decoy” receptor that binds C5a but does 93 not signal activation, may provide an explanation for these differences . Importantly, neuronal synthesis of C1q has been seen in several injury 94-98 99 models , including oxidative stress during aging , in the absence of induction of other C1 subcomponents C1r and Cls. Hippocampal organotypic slice cultures have been established as a useful model of amyloid interactions with neu100 rons and glia . After addition of amyloid to these cultures, slices were immunostained with antibody against rat C1q. A distinct reactivity for C1q in the CA1/CA2 pyramidal neurons of the hippocampus was observed in the Aß101 treated slices (Figure 5) . C1q immunoreactivity was largely overlapping with the neurons that had internalized Aß (verified by confocal microscopy). No such immunoreactivity was detected in untreated cultures or upon addition of control/scrambled Aß peptides. ELISA assays documented the increase in C1q in tissue extracts from slices of the treated group. In addition, C1q mRNA levels 101,102 were increased in these slices . These data demonstrate for the first time that, upon acute exposure to Aß, C1q expression is induced in neurons in this organotypic system. These findings are similar to the induction of C1q synthesis in brain by numerous other acute injuries, such as viral infection, kainic acid treat94,95,103-106 ment, and ischemia/ reperfusion .

Figure 5 (see color insert, Fig. 11.5). Neurons in rat hippocampal organotypic slice cultures that have ingested exogenously added Aß are immunoreactive for C1q.

21,28,107

Synthesis of C1q has been detected in Alzheimer’s disease , other human neurodegenerative diseases, such as Huntington’s disease (94), or mouse 108 models of the diseases such as Sanfilippo syndrome , and in response to ag99 ing , conditions not generally thought of as acute. Importantly, C1q synthesis is known to be regulated independent of other complement components (including C1r and C1s, the enzymatic components necessary for classical complement 109 pathway activation) in peripheral systems where it has been investigated . Indeed, C1q is a member of the family of defense collagens that have been shown

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Figure 6. C1q enhances phagocytosis and modulates inflammatory cytokines.

to enhance phagocytic activity of monocytes, macrophages and microglia in 16,110,111 61,112 vitro , and clearance of apoptotic cells both in vivo and in vitro (Figure 6). Therefore, neuronal C1q may flag either damaged neurons or neuronal 112-114 blebs to be rapidly removed to prevent release of neurotoxic levels of intra46 cellular components (such as glutamate) in the initial stages of CNS injury . Recent work in our laboratory and others have shown that under some circum115 stances C1q downregulates production of proinflammatory molecules (and Fraser et al., submitted for publication). Indeed, several reports now implicate a deficiency of C1q as a risk factor for development of autoimmunity in both hu116-118 man and mouse . As a result, C1q could play a protective role in the early stages of neuronal injury by suppressing the progression of an inflammatory state. It should be noted however, that no differences were seen between APP and APPQ-/- mice in glial or neuronal markers at younger ages when there is no detectable plaque pathology. Thus, in the early stages damage may be below the

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threshold for detection or the function of C1q is compensated by other unknown mechanisms in the APPQ-/- mouse. In addition, we have described an unusually robust display of neuronal C1q in the cortex and hippocampus of a patient with a 32 genetic relation to a late-onset AD patient . This patient had abundant diffuse amyloid in hippocampus and cortex but no detectable thioflavine staining (and no known cognitive impairment). These observations are consistent with, though does not prove, the hypothesis that C1q synthesis at this early stage of the disease may be a protective response to injury.

5. POTENTIAL COMPLEMENT-BASED THERAPEUTICS If complement does contribute significantly to the progression of pathology in these and improved mouse models, many mechanistic questions would remain to be addressed. However, if correlations to changes in neuronal activity or behavior are determined, investigations of potential therapies directed at eliminating specific activation of CCP by fibrillar Aß may be considered without complete knowledge of the downstream mechanisms. The development of complement inhibitors that can be used in vivo, while 119 still in the developmental stages is rapidly accelerating . Acute injury/inflammation is much more amenable to complement inhibition, since the drug 120,121 need only be administered transiently , whereas inhibition of complement for chronic disorders for longer periods of time may generally lead to susceptibility to infection and/or autoimmunity. However, Bergamaschini et al. administered Enoxaparin, a low-molecular-weight heparin, to the APP23 transgenic mouse three times a week for 6 months. This compound, which had no noted toxicity and was demonstrated to inhibit Aß-induced complement and contact system activation and amyloid-induced toxicity of differentiated PC-12 cells, demonstrated a greater than 2-fold decrease in amyloid deposits, total amyloid load, 122 and decreased astrogliosis , similar to the reduction reported by Fonseca et al. in C1q-deficient animals. Compstatin is perhaps the most promising specific 123,124 inhibitor, as it has been shown to be an effective inhibitor of C3 in baboons . While effective and nontoxic, it specifically binds to primate C3 and thus is not applicable for preclinical testing in rodents. However, several antagonists of the 125 C5a receptor have been described, three of which have shown activity in vivo . One of these, AcF-[OPdChaWR], was developed by Taylor's group at the University of Queensland, Australia, and has been shown to be active against a 126 model of inflammatory bowel disease when given orally to rats . Other strategic designs for targeting inhibitors to specific tissues or sites are emerging, in127 cluding tissue-specific expression of complement inhibitors , and generation of soluble chimeric molecules consisting of single-chain antibody targeting a specific cell type fused to the amino terminus of inhibitor, such as Crry (C3 inhibi128 tor) . Such a design would eliminate systemic and lifetime inhibition of the 88 transgenic overexpression of Crry . Another candidate designer therapeutic

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would be a small molecule that bound specifically to beta sheet amyloid, blocking the Asp7 and Glu11 sites that interact with C1q with an anchor binding site to some other residues nearby on Aß, enhancing its affinity and specificity for beta sheet Aß. One example of a candidate small molecule has already been identified and shown to inhibit binding of C1q to Aß selectively, and protect 129 neurons in vitro from Aß-induced complement-mediated cell death . Such therapeutic interventions should be particularly beneficial for individuals entering what has been called the “catastrophic” phase of AD after cognitive dysfunction becomes clinically apparent, where the rate of decline appears to accelerate 130 and neuritic plaques are seen upon autopsy .

6. SUMMARY A role for the complement cascade in AD neuropathology was hypothesized 24 over a decade ago , and the results of a significant number of in vitro studies are consistent with the involvement of this pathway in AD pathogenesis (reviewed in [47,131]). Since C1q is colocalized with thioflavine-positive plaques and the C5b-9 complement membrane attack complex is detected in AD brain at autopsy, it is reasonable to hypothesize that complement activation has a role in 30 the manifestation of AD either by its lytic capacity or as a trigger of glial infil132 tration and initiation of potentially damaging inflammation . The observed diminished glial activation and reduced loss of neuronal integrity in a murine model overexpressing mutant human APP but lacking the ability to activate the classical complement cascade provide the first direct evidence for a detrimental role of C1q, and presumably activation of the classical complement pathway in an animal model of AD. Research is now focused on generating mouse models that more closely mimic the human disease, so that the role of complement activation and inflammation on the behavioral/learning and memory dysfunction that occurs in this disease can be assessed. In addition, candidate therapies such as targeted inhibition of complement activation will need to be tested in these animal models as a step toward treatment of humans with the disease. However, it is important that the potential for a protective effect of C1q early on in disease progression should not be overlooked. Rather, strategies that enhance or mimic the protective effects of C1q as well as strategies that inhibit the detrimental processes should be fully investigated.

7. ACKNOWLEDGMENTS The studies from the authors’ laboratory mentioned here have been supported by grants from the National Institutes of Health (NS35144 and AG 00538). The authors thank Rong Fan for Figure 5 and Cheryl Cotman for help with the other illustrations.

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12 THE ROLE OF THE COMPLEMENT SYSTEM IN THE PATHOGENESIS OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS AND MULTIPLE SCLEROSIS Nóra Terényi, József Prechl, and Anna Erdei

1. INTRODUCTION The central nervous system (CNS) has long been known as an immunologically privileged site, isolated from systemic circulation by the so-called blood–brain barrier (BBB): a special anatomical structure formed by endothelial cells and astrocytes. However, increasing evidence is available now that there is immune surveillance in the healthy brain, as a wide variety of leukocytes have the ability 1 to enter the CNS . Activated immune cells gain further capabilities to cross the BBB, while inflammatory mediators can adversely affect the barrier function of endothelial cells. Hence, under conditions of CNS-directed immune activity, the stage is set for initiation of the complement cascade, utilizing systemic or locally produced complement components and regulated by both the infiltrating leukocytes and proper cells of the CNS. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the CNS, affecting approximately 1 in 700 people in the United

Nóra Terényi, Department of Immunology, Eötvös Loránd University, Pázmány P.s.1/C, 1117 Budapest, Hungary. József Prechl, Research Group of the Hungarian Academy of Sciences at the Department of Immunology, Eötvös Loránd University, Budapest, Hungary. Anna Erdei, Department of Immunology, Eötvös Loránd University, Pázmány P.s.1/C, 1117 Budapest, Hungary. Research Group of the Hungarian Academy of Sciences at the Department of Immunology, Eötvös Loránd University, Budapest, Hungary.

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States. Demyelination in MS is caused by activated immune effectors, like Th1 and Tc cells, macrophages, and the complement system. The clinical course of MS is highly variable both within and between patients. Differences in the pathological character are also so wide that it raised the assumption about diverse causes of demyelination. Investigating biopsies and autopsies, Lucchinetti et al. have described four main types of pattern based on the structure of le2 sions . In patterns I and II active demyelination was associated with a Tlymphocyte- and macrophage-dominated inflammation. Prominent deposition of Igs and C9neo antigen at lesions was found exclusively in pattern II. Sharply demarcated plaques were typically centered on small veins or venules in both types, with a high incidence of remyelinated shadow plaques. Pattern III inflammatory infiltrates contain T lymphocytes, macrophages and microglia, while Ig and complement deposition is absent. The striking feature in these cases was a preferential loss of Myelin Associated Glycoprotein (MAG), while other myelin proteins, including Proteolipid Protein (PLP), Myelin Basic Protein (MBP) and Myelin Oligodendrocyte Glycoprotein (MOG) were still present. In pattern IV, inflammatory infiltrates were also dominated by T lymphocytes and macrophages, but neither immune complex deposition nor a difference in staining patterns of myelin-specific proteins (such as MAG, MBP, PLP, MOG) was observed. Overall, the most frequently observed pattern was type II, followed by patterns III, IV and I. Thus, pathological evidence supports that the complement system is activated at the most frequently found type II lesions. Experimental Autoimmune Encephalomyelitis (EAE) is a widely used animal model of MS. EAE, too, has many types depending on the species, strains, quality of antigen and adjuvants, and method of antigen administration used. Basically, EAE can be induced by active immunization with proteins or peptides derived from the myelin sheath of neurons, or by passive transfer of myelin antigen-stimulated lymphocytes. Both treatments result in an immune-mediated attack on the myelin sheath of neurons, demyelination, and MS-like symptoms. Pathological assessment of disease severity relies on quantification and qualification of infiltrating cells in the CNS and measurement of the extent of demyelinization. Clinical assessment relies on motoneuron involvement in CNS damage and is carried out by scoring the degree of paralysis, ranging from tail weakness to tetraplegia. The most susceptible and very extensively studied mouse strains are SJL/J, and B10.PL. EAE in B10.PL mice is a chronic disease characterized by MBP epitope specificity, while in SJL/J mice it manifests as a relapsing-remitting disease with PLP as the predominant antigen. Although less frequently used, EAE has been worked out for many other inbred, less susceptible mouse strains (A.SW, B10.S, PL/J, B10RIII, SWR, 129/J, C3H, Balb/c, Biozzi AB/H, NOD/Lt) as well. While C57BL/6 is presumed to be a less susceptible strain, these mice are also widely used in EAE experiments because of the availability of numerous genetically modified mice on this background. MOG 3 35-55 peptide has been successfully used in C57BL/6 to induce active EAE .

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Rats (Lewis), guinea pigs, marmosets and rhesus monkeys have also been used with various antigens and experimental regimen. The complement system consists of some 30 soluble or membrane-bound proteins, which play an important role in immune homeostasis and defense against pathogens. Its major effects are the direct lysis of target cells by forming the membrane attack complex (MAC), enhancement of phagocytosis via opsonization by C1q and C3 fragments, and chemoattraction of immune cells like macrophages and T cells. The complement cascade can be activated by one of three pathways: the C1q-dependent classical, the lectin-dependent, and the alternative pathways. Initiation of any of these cascades leads to formation of C3 and C5 convertase complexes, followed by organization of the MAC. Dissecting the complex roles of complement in EAE, it seems important to point out that complement may act during the induction phase, enabling the development of a CNS-targeted immune response, or during the effector phase of the autoimmune response itself, sustaining or mediating myelin damage. Depending on the experimental system, investigation of one or both mechanisms is possible. A summary of the possible points of interaction between complement activation and the development of demyelinating disease is presented in Figure 1.

Figure 1. Possible roles of complement proteins in demyelinating encephalomyelitis. The diagram is based mainly on receptor expression data in [5].

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2. LOCAL PRODUCTION AS A COMPLEMENT SOURCE IN THE CNS Soluble complement proteins cannot freely enter the CNS: concentrations of C3 and factor B in the cerebrospinal fluid are more than two orders of magnitude 4 lower than in blood, about 2.5 and 0.29 µg/ml , respectively. In the CNS there are four main types of cells: microglia, astrocytes, oligodendrocyte and neurons; all of them have the ability to express various elements of the complement system. The main source of soluble complement components in the CNS is the as5 trocyte, the other cell types mainly generating receptors and regulators . Accumulating evidence suggests, though, that extrahepatic synthesis of complement has an important role in local inflammation. In an early study Andrews et al. reported that in kidney transplanted donor–recipient pairs, mismatched at the C3 S/F locus, donor-specific C3 mRNA was found in the recipi6 -/ents, at up to 61 days post-transplantation . Survival of renal allografts from C3 +/+ +/+ and C3 mice was investigated by Pratt et al., who found that C3 grafts were -/7 shortly rejected, while C3 grafts had long-time function . Macrophages are thought to be the main producers of C components after hepatocytes; these mobile cells migrating to the CNS are well suited for local, targeted production of complement components at inflammatory lesions. Early studies reported that MS patients had higher levels of terminal complement components in the CNS 8 than those who had non-inflammatory CNS disease . Underlining the susceptibility of the CNS to complement-mediated attack, it is known that central nerve myelin directly activates the classical pathway of complement even in the ab9,10 sence of myelin-specific antibody , and neurons are susceptible to comple11,12 ment-mediated lysis .

3. THE ROLE OF COMPLEMENT DEPOSITION IN MYELIN DAMAGE The focus of most investigations was the role of the classical and alternative pathways of complement in EAE or MS, and almost all proteins have been studied one by one from C1 to C9. In the following section we summarize data obtained from mice with targeted or total depletion of complement, and studies on particular soluble C components. 3.1. Decomplementation by CVF Cobra venom factor (CVF) is a protein isolated from cobra venom (Naja naja or Naja haje), which forms a stable C3 convertase with factor B and depletes C3 from the serum. Its effect is reversible — normal complement activity is restored after about 2 weeks. The earliest studies attempting to address the role of complement in EAE used CVF with the common conclusion that complement deficiency ameliorates disease symptoms. In Lewis rats CVF suppressed EAE

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induced by either active immunization or by passive transfer of 10 splenocytes 7 13 but had no effect when 5 x 10 cells were used . Our aim was to investigate in mice how the onset of the disease is influenced by the lack of complement. To this end, C57BL/6 mice were decomplemented by CVF injection 5 days before induction of EAE. As shown in Figure 2, mice without an active complement system did not develop the disease. We also found that mice with severe symptoms had higher MOG specific antibody levels than controls. Therefore, we next generated a group of animals with higher antibody levels by injecting MOG peptide into their footpad. As opposed to our expectations, however, mice with high specific antibody levels stayed healthy, even if these animals also generated MOG-specific T cells. Deposition of C3 fragments after in-vitro serum treatment of the antigen-presenting cells increased proliferation of the T cells isolated from the lymph node of MOG14 injected animals (data not shown), indicating a role of the complement system . In summary, our results suggest that complement plays a role in the development of EAE, but most probably not by the involvement of MOG-specific antibodies.

Figure 2. EAE was induced in C57/B6 mice by injecting 100 µg MOG-peptide, representing aminoacid residues 35–55, emulsified in complete Freund’s adjuvant. To study the effect of the absence of complement activity, one group was treated by high-dose CVF 5 days prior to EAE induction. Parallel to this pertusiss toxin was also injected i.p. on days 1 and 3, as well. Clinical signs of EAE were rated daily using a standard scale of 0 to 5. Values represent mean clinical score (n = 4).

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3.2. C1 The level of C1q mRNA, which is not detected in normal brain, becomes clearly detectable using RT-PCR analysis when the clinical symptoms of EAE become evident in Lewis rats transferred with MBP specific T cells. Mostly microglia 15 were positive . It is interesting to note that myelin oligodendrocyte protein has a specific C1q binding motif. MOG is expressed predominantly on the outer sur16 face of the myelin sheath; it is available to C1q . This may account for the above-mentioned ability of the myelin sheath to activate the classical pathway. 3.3. C3 C3 activation is the converging point of all three pathways of complement activation; C3 cleavage is therefore a highly important step in the process. Two groups investigated the role of C3 using genetically C3-deficient mice with con-/tradictory results. According to Calida et al., C3 animals were just as susceptible to EAE as wild-type mice, while Nataf et al. showed reduced severity of -/EAE accompanied by moderate demyelination and cellular infiltration of C3 -/animals. This latter study revealed an essentially identical response in C3 and -/factor B mice, implying a role of the alternative pathway. Although the animals used in these studies were of the same genetic background, immunization protocols were different, possibly accounting for the observed discrepancy. Interest-/ingly, Calida found that C3 animals raised in a germ-free environment were even more susceptible to EAE: the mortality of these animals was 50%, as op17,18 posed to the 0% in wild-type mice . In-vitro data suggests that C3 deposition on myelin enhances phagocytosis, an effect important in mediating direct damage to oligodendrocytes and sustaining inflammation by the presentation of myelin-derived epitopes to T cells. Serum-treated myelin vesicles, opsonized by C3 fragments, were taken up by 19,20 macrophages and microglia in a CR3-dependent fashion . 3.4. C4 -/-

EAE has also been induced in C4 mice (B6), and no significant difference com+/+ pared to the wild-type animals was observed. EAE incidence was 100% in C4 -/and 91% in C4 animals, and the time of onset and severity, cellular infiltrate of T cells and macrophages, and extent of demyelination by toluidine blue staining were practically identical. However, they found elevated levels of IL-1β, TGFβ, TNFα, IP-10 (CXCL10), and RANTES (CCL5) mRNA expression in C421 deficient animals .

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3.5. C5 -/-

+/+

Wreeth et al. immunized C5 and C5 mice with purified guinea pig myelin. According to the symptoms, disease course was divided into an early acute, a short recovery, and a stable chronic phase. Incidence of EAE was 100% in both -/groups, but in general C5 mice developed less progressive disease. In the early -/acute phase, C5 mice showed less inflammation and demyelination, but during the recovery phase inflammation and demyelination were more extensive in -/22 lesions of C5 mice . The number of apoptotic oligodendrocytes was signifi-/cantly higher during both the acute and recovery phase in C5 mice. 3.6. C6-C9, MAC The role of C6 in EAE was studied in C6-deficient rats. Tran et al. found that -/EAE is less severe in C6 animals: incidence was 90% in the control group and 70% in C6-deficient animals. After exclusion of animals that did not develop -/clinical disease, C6 rats still demonstrated milder signs, delayed onset, and reduced weight loss. C3 deposition was similar in both groups, but no C9 depos-/23 its were found in C6 animals . Mead et al. found no difference in EAE development between C6-sufficient and -deficient animals with the same genetic background as Tran used. On the other hand, the severity of ADEAE (antibodymediated demyelinating EAE) was found to depend on C6: wild-type animals -/developed far more severe disease than C6 when antibody was administered at 24 onset of clinical signs . These observations suggest that C6-dependent MAC formation has a role in myelin damage especially when pathological antibodies are present. Of note, the C5b-9 complex may have a different function than direct lysis of the attacked cells. At a sublytic concentration the complex enhances oligodendrocyte survival, supposedly by increasing the level of BCL-2 protein, and 25 inhibition of caspase-9 and caspase-3 .

4. ANAPHYLATOXIN EFFECTS IN DEMYELINIZATION Anaphylatoxins are generated upon activation of the complement cascade. These small molecules (~9–11 kDa) have several effects, from being powerful chemoattractants of all cell types from the myeloid lineage, increasing vascular permeability, inducing degranulation of mast cells and basophils, to inducing 26 expression of pro-inflammatory cytokines . Studies on primary cultures of fetal and adult brain have shown that C3a receptor was expressed on microglia and astrocytes, and not on oligodendrocytes and neurons. Stimulation by IFNγ, IL1β, TNFα, or PMA had no effect on the expression pattern of C3aR. In MS brain sections there was a consistent and strong staining of astrocytes, microglia, 27 and vascular smooth muscle cells for C3aR .

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Table 1. Effects of Complement Deficiencies on Development of EAE* Incidence CVF rat

Score, severity

Demyelination

Infiltrating cells

Ref.

reduced

reduced

no difference

[13]

C3 -/mouse

100% [17] or 84% [18]

reduced [18] no diff. [17]

reduced [18]

reduced [18]

[17,18]

fB -/mouse

75%

reduced

reduced

reduced

[18]

C4 -/mouse

91%

no difference

no difference

no difference

[21]

C5 -/mouse

100%

less progressive

later onset reduced

ADEAE: 100% [23] 91% [23] 74% [24]

reduced [23]

C6-/rat

no difference [23]

absent [23] absent [23]

[22] no difference [23] reduced [24]

[23,24]

reduced [24]

* as compared to complement sufficient animals.

Deletion of C3aR attenuated MOG-induced EAE: disease severity was more intense in control mice, yet the incidence was 100% in both groups. To investigate the role of C3a expression in the CNS, EAE was induced in C3a/GFAP transgenic mice, which express C3a under the astrocyte-specific promoter glial fibrillary acidic protein (GFAP). In these animals disease severity increased significantly, and mortality was 60% as opposed to 16% in the control -/28 group. In C3a/GFAPxC3aR mice disease severity was reduced , however, verifying the presumed role of C3a in CNS inflammation. -/In order to evaluate the function of C5a and its receptor in EAE, C5aR mice on C57BL/6 background were generated and EAE was induced by MOG 35–55 peptide. No difference was found in any of the parameters of the dis29 ease . A concordant study from Reiman et al. analyzed C5a expression in the CNS. They reported that C5a/GFAP mice with EAE showed no significant dif30 ference from control animals, in sharp contrast to C3a/GFAP animals . On the other hand, C5a may have direct effects on the development of EAE by inducing apoptosis of neurons. Farkas et al. described that a C5a-derived peptide induces 31 rapid apoptosis in TGW neuroblastoma cell line . T cells, the presumable initia-

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tor cells in EAE and MS, express C5aR, and receptor levels increase after acti32 vation and T cells are positively chemoattracted by C5a .

5. COMPLEMENT REGULATION IN THE CNS Complement regulators are able to block cascade activation at various stages. Expression pattern of complement regulatory proteins on neurons, oligodendrocytes, microglia, and astrocytes is different; this may contribute to their distinct reactions to complement effects. Using an outbred primate model of EAE, strong C1q and C3 staining was observed next to inflammatory infiltrates in marmosets on neurons, axons, and in cell bodies. While neurons in the control brain were negative for DAF, its expression was found surrounding the inflammatory infiltrates. Neuronal sites that stained for C3 displayed strong DAF staining; distant from the lesions DAF was not observed. While marmosets developed a relapsing-remitting or primary progressive form of EAE, rhesus monkeys represented the acute type of the dis33 ease. No upregulation of DAF expression was observed in the latter group . Human neuronal cell lines have also been examined in this respect, and all of them failed to express CR1, but DAF, MCP, and CD59 were detected on the cells. Concerning susceptibility to complement-mediated lysis, a cell line (IMR32) expressing very low levels of DAF was found to be most sensitive. Transgenic overexpression of DAF significantly protected IMR32 cells against 33 complement-mediated lysis . Scolding et al. studied the expression of complement regulatory proteins on cultured adult human oligodendrocytes. Their studies revealed that there was no CR1 and MCP on oligodendrocytes; only DAF 34 and CD59 expression were detectable . Following incubation of primary human fetal brain cultures with normal human serum, C1q, C4, C3b, iC3b, C3c, and C9 were detected on the cells, mostly on neurons. Neurons were susceptible to complement-mediated lysis. Investigating the presence of complementregulatory proteins by different techniques, they found that CD59 but not CR1 was expressed on both cell types, while DAF was expressed on astrocytes, but 12 not on neurons .

6. COMPLEMENT AND THERAPY OF EAE Soluble forms of complement receptors can be used for complement inhibition by virtue of their ability to neutralize active fragments and to act as factor I cofactors. Piddlesden et al. reported that ADEAE rats treated by human recombinant soluble CR1 (sCR1) showed significantly reduced clinical signs and abolished hemolytic complement activity for at least 6 days. Administration of sCR1 successfully inhibited inflammation, complement deposition, and demyelina35 tion . A functional homolog of human CR1 and MCP in rodents is Crry. CNSspecific expression of its soluble form completely inhibited EAE in SJL/J and

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partially in C57BL/6 or C57BL/6xSLJ/J mice . These investigations suggest that targeted modulation of complement activation can interfere with disease progression. Thus, even if the complement system is not the central player in the development of demyelinating encephalitis, the complement-inhibitory activity of therapeutic interventions may be beneficial.

7. ACKNOWLEDGMENTS We wish to thank József Kármán and Árpád Mikessy for their expert help with mice, and Gábor Tóth for peptide synthesis. The financial support of the Hungarian National Science Fund (OTKA) (TS044711, T047151) and that of NKFP (1A/040/04) are gratefully acknowledged.

8. REFERENCES 1.

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13 THE COMPLEMENT SYSTEM: A POTENTIAL TARGET FOR STROKE THERAPY J Mocco, Michael E. Sughrue, Andrew F. Ducruet, Ricardo J. Komotar, Sergei A. Sosunov, and E. Sander Connolly, Jr.

1. INTRODUCTION Ischemic stroke remains the leading cause of disability and third leading cause 1 of death in the United States and other industrialized nations . The loss in terms 1-3 of quality years of life and health-care resources is staggering . Compounding this problem is the lack of any universally administered therapy: the existing modalities are not an option for approximately 97–98% of patients presenting with acute ischemic stroke, as these treatments carry a significant risk of devastating intracerebral hemorrhage when administered outside a narrow time win4 dow . Therefore, efforts have been made to investigate therapeutic avenues that reduce ischemic brain injury and can be administered safely at delayed time points. The conceptual basis for these efforts stems from the observation that brain infarction following occlusion of a major artery by thrombus is neither a homogenous, nor an instantaneous process, but rather has distinct spatial and temporal variation that can be exploited for therapeutic benefit. More specifically, it has been demonstrated that while the area most central to, and dependent on, the vascular supply of the occluded artery (termed the infarct core), likely suffers irreversible damage early on in the course of ischemia, the more peripheral portions (termed the penumbra) receive some collateral blood supply sufficient to delay the occurrence of irreversible damage, and thus can potentially be salvaged.

J Mocco, Michael E. Sughrue, Andrew F. Ducruet, Ricardo J. Komotar, Sergei A. Sosunov, E. Sander Connolly Jr., Department of Neurological Surgery, Columbia University, College of Physicians & Surgeons, New York, New York 10032.

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While the success of reperfusion-based therapies clearly stresses the importance of prompt return of blood flow in salvaging brain tissue following stroke, it is becoming increasingly clear that simply reperfusing the brain does not represent adequate therapy, as other pathophysiologic mechanisms appear capable of spreading the infarction process into the tenuous penumbral regions, even after blood flow is restored. These mechanisms have been loosely grouped under the rubric “reperfusion injury,” and include such events as programmed cell death, generation of oxygen free radicals, and inflammation. The exciting recent announcement of the encouraging results of the SAINT 1 trial of the free-radical agent NXY-059 provides further support for the potential benefit of penumbra 5 salvaging therapy, also known as neuroprotection . Therapeutic strategies targeting inflammatory cascades have been of particular interest to stroke investigators for a number of reasons, most notably the large number of potential targets, the rapidly expanding arsenal of immunomodulatory agents, and the relatively delayed nature of neuroinflammatory pathophysiology in ischemic cerebral infarction. Recently, the complement cascade, a potent inflammatory mediator, has received attention for all of the above reasons. It is within this context that we perform the current review.

2. RATIONALE FOR BLOCKING COMPLEMENT ACTIVATION TO TREAT STROKE 2.1. Inflammation Is Deleterious in Stroke, and Complement Is Activated in Stroke A variety of proinflammatory effectors are activated following stroke, particularly during reperfusion. There is mounting evidence that this is detrimental, and that inflammation exacerbates injury following cerebral ischemia/reperfusion 1,6-11 (I/R) in both animals and humans . The mechanisms by which inflammation causes injury are many; however, release of proteases and free radicals by infil12,13 and increased leukocyte adhesion leading to capillary trating neutrophils 14 plugging, referred to as microvascular failure , are two well-studied examples. Complement components are deposited on neurons in the brain following 15-17 cerebral ischemia . Currently, the origin of this deposition is not known. While the liver is the body’s principal producer of complement proteins, it has recently been discovered that numerous extrahepatic cells are capable of manu18,19 facturing complement proteins . Particularly, various brain cells have been 20-22 demonstrated to produce mRNA for the entire complement cascade in vitro . As well, cerebral ischemia appears to upregulate the expression of at least some 15 complement proteins, notably C1q . However, given the large pool of complement protein in the blood, it is likely that some portion of the complement protein deposited in the brain following stroke enters with the loss of blood–brain 23 barrier integrity that begins between 3 and 6 hours following ischemic onset .

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Whatever the complement proteins’ source, given the diffusible nature of many complement cascade byproducts (notably C3a and C5a), it is reasonable to hypothesize that injured and dying cells likely injure neighboring, otherwise viable cells by promoting inflammation in adjacent tissues and vascular beds via 24 complement activation . 2.2. Complement Activation Exacerbates Ischemic Injury in Other Organs Beginning in 1970, with Hill and Ward’s observation that systemic complement depletion using cobra venom factor (CVF) prevented neutrophil influx into ischemic rat myocardium, a mounting body of evidence has implicated the complement cascade in the pathogenesis of I/R throughout the body. The majority of this work has focused on myocardial ischemia, where complement deposition occurs by 3 hours following the onset of ischemia, which may be related to focal 25 loss of regulators of complement activation (RCA) in the infarct region . As well, more complement-specific experiments (as CVF also affects other, non26 complement, proteins), such as animals genetically deficient in complement , soluble complement inhibitor-1 (sCR1) (which binds C3b, C4b, and probably 27,28 29 25 C1q) , C1-esterase inhibitor , and C5 activation blocking antibodies , have demonstrated an ability to limit the extent of post-ischemic myocardial injury. 30 31 32 Similar benefits observed in renal , intestinal , and limb ischemia has further strengthened the concept of complement-mediated injury following I/R as a biological principle. 2.3. Complement Activation Causes Injury in Other Nervous System Diseases 20-22,33-39

. The complement cascade is activated in a host of neurological diseases Evidence indicates that, while complement activation is not the primary inciting event in most neurological diseases, in the acute stages it probably exacerbates the injury. For example, complement has been demonstrated to have pro40-43 inflammatory and deleterious effects in Alzheimer’s disease , and in Pick’s 34,44,45 . Perhaps more relevant to stroke, transgenic mice with astrocytes disease that produce a soluble form of the murine complement inhibitor Crry (sCrry) were shown to develop less profound brain injury secondary to head trauma than 46 the complement replete wild-types . 2.4. Neurons Seem to Be Unusually Susceptible to Complement Activation Neurons, in vitro, spontaneously activate the complement cascade without an 47,48 inciting stimulus . Singhrao et al. reported that human primary neuronal cell cultures cultured in the presence of complement are capable of activating the

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entire complement cascade in the absence of any obvious stimulus, eventually 49 leading to neuronal lysis by MAC . They hypothesized that this was the result of a relative deficiency in decay-activating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), which rendered neurons unable to prevent spon49 taneous complement activation . While these interesting data suggest that neurons may, in fact, be more susceptible to lysis by complement, caution should be utilized in drawing such a conclusion, as many soluble complement inhibitors, such as C4-binding protein and factor H, largely produced by astrocytes, are 50 likely lacking in a pure culture of neurons . 2.5. In Vivo Evidence Suggests that Complement Is Involved in Cerebral I/R Pathogenesis In 2001, the results of the failed Enlimomab Acute Stroke Trial, a clinical trial 51 of an anti-ICAM-1 antibody, were published . The study, which was halted following administration of the antibody to 625 patients, concluded that Enlimomab-treated patients demonstrated worse outcomes than placebo-treated patients. Subsequent investigation revealed that the mouse antibody utilized in this study activated the complement cascade when incubated with blood from 52 healthy human volunteers . This was hypothesized to be a possible explanation for the adverse effects of the antibody. Early work involving nonspecific pharmacologic complement inhibition us16,53,54 55,56 , and C1-esterase inhibitor , suping agents such as cobra venom factor ports the hypothesis that complement activation contributes to cerebral I/R injury (Table 1). However, studies using CVF in both a thromboembolic model of stroke in a rabbit and permanent focal ischemia in rats failed to show a protec57,58 tive benefit of cobra venom factor administration . Drawing conclusions from experiments relying on these techniques of complement inhibition is problematic due in part to their lack of specificity. For instance, recent work utilizing C1-esterase inhibitor has demonstrated that the protection mediated by this inhibitor does not involve C1q but instead results from inhibition of cell recruit59,60 ment and inflammation . A more specific complement inhibitor, soluble complement receptor-1 (sCR1), led to significant reductions in neutrophil and platelet aggregation and significantly improved neurological function following experimental stroke, as well as a modest, but non-statistically significant reduction in cerebral infarct 17 volume . These results were more marked when a sialyated form of the sCR1 molecule, which both inhibits complement activation and blocks neutrophil ad17 hesion via P/E-selectin, was administered . Taken together, these data strongly suggest that blocking the complement cascade during the early phases of infarct evolution may be able to decrease recruitment of penumbral tissue into the infarct and thereby limit the extent of brain injury following stroke.

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Table 1. Complement Manipulation in Models of Cerebral Ischemia Study US Vasthare et al., 199853 SM Lew et al., 199957 J Huang et al., 199917 NH Akita et al., 200155 MD Imm et al., 200261 RM Cowell et al., 200316 MG De Simoni et al., 200356 R Gill et al., 200458 MG De Simoni et al., 200460 C Storini et al., 200559 E Figueroa et al., 200554

Model system Rat bilateral CCA occlusion Rabbit ICA autologous clot Mouse MCAO (trans) Rat MCAO (trans) Rat neonatal HIE Rat neonatal HIE Mouse MCAO (trans) Rat MCAO (perm) Mouse MCAO (trans) Mouse MCAO (trans) Rat MCAO (trans), neonatal HIE

Agent

Summary of findings

CVF

Improved outcomes and cerebral blood flow in complement-depleted animals

CVF

No difference in infarct size between controls and complement depleted animals

sCR1/sCR1sLex

Inhibition of platelet and neutrophil accumulation, reduced infarct volume in treated animals

C1-inhibitor

Reduced infarct volume and myeloperoxidase activity in treated animals Larger infarcts in treated animals; no effect using CVF, sCR1, or sCR1-sLex Reduction in infarct size and depletion of brain and systemic C3 in treated animals Reduction in infarct volume and improved neurological outcome in treated animals

Human C9 CVF C1-inhibitor

CVF C1inhibitor, C1q -/C1-inhibitor

CVF

Reduced circulating C3, no improvement in infarct volume in treated animals No significant reduction of infarct in C1-inh treated C1q knockout mice Reduction of P-selectin/ICAM-1/pro-caspase 3; increase in IL-6/IL-10/NFH; inhibition of macrophages Reduction of infarct volumes in MCAO, reduction of cerebral atrophy in HIE

3. POTENTIAL NEGATIVES OF COMPLEMENT BLOCKADE FOLLOWING STROKE While the above data present a compelling case for wholesale inhibition of complement in stroke patients, more recent advances in complement research indicate that complement components are multifunctional, and that many are critical in the proper functioning of mammalian cells and tissues. Thus, prior to clinical translation a greater understanding of the proper targeting, timing, duration, and potency of anti-complement agents is needed in order to avoid interfering with critical processes related to post-I/R brain repair, healing, and functioning.

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3.1. Complement May be Needed to Opsonize Cellular Debris after Stroke It has long been accepted that complement deposition on the surface of pathogens greatly increases the rate at which these opsonized pathogens are phagocy62 tosed and destroyed . More recently, a number of studies have demonstrated that complement, particularly early components such as C1q, C3, and C4, plays 62-66 a critical role in facilitating clearance of apoptotic cells . Additionally, the presence of genetic complement deficiencies strongly predispose to the development of autoimmune diseases, such as lupus, which are generally considered 62,63 to result in part from the failure to clear apoptotic cells . It is hypothesized that apoptotic cells that are not rapidly cleared progress to lysis with release of potentially damaging intracellular substances into the surrounding extracellular 67-78 tissue space . Most relevantly, in cases of acute tissue injury it is likely that necrotic cell debris from uncleared apoptotic cells exacerbate the proinflammatory response; however, this proinflammatory pathway remains in79-83 completely elucidated . Beyond preventing leakage of hazardous cell components, it has recently been discovered that phagocytosis of apoptotic cells is not a biologically neutral 63,71,84,85 . For example, event but rather has profound effects on cytokine expression macrophages that ingest apoptotic cells reduce expression of proinflammatory cytokines such as TNF-α and IL-1, and increase expression of antiinflammatory 67,73,77,82 . This phenomenon has also been demonstrated to cytokines such as TGF-β occur in human microglia, with the interesting caveat that growth factors such as 86 NGF are also released . In either case, complement inhibition appears to exacerbate certain disease processes as a result of decreased clearance of cellular debris. For example, in one study mice doubly transgenic for human amyloid precursor peptide (hAPP) and sCrry experienced significantly more extensive Alzheimer’s-type neuro61 degeneration compared to mice only transgenic for hAPP . While the exact mechanism for this observation is currently unclear, it is hypothesized that this increase in neuronal loss is secondary to failed amyloid plaque clearance in the 61 complement-inhibited mice . 3.2. Complement Aids in Tissue Recovery/Repair in Other Organs While the anaphylotoxins C3a and C5a are best known for their profound proinflammatory effects, more recently it has been appreciated that these molecules play critical roles in promoting cell survival, differentiation, and regeneration 87-89 following injury . For example, it has been shown that C3-/- knockout mice subjected to either toxic injury or partial hepatectomy demonstrate marked impairment of liver regeneration secondary to their failure to produce C3a and 88,89 C5a . C3a and C5a are thought to prime cells for rapid tissue regeneration/re88,89 pair by increasing their responsiveness to specific growth factors .

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Similarly, work by Ratajczak and colleagues has implicated C3a in the en87 graftment of hematopoetic stem cells into bone marrow stroma . They demonstrated that C3-/- knockout mice irradiated and then administered bone marrow mononuclear cells demonstrate a profound impairment in the ability of these stem cells to form functional colony-forming units when compared to wild-type controls. This effect was hypothesized to result from a combination of two C3mediated mechanisms. First, C3 activation releases C3a, which, acting in a large part through its catabolite C3ades-Arg, binds the C5L2 receptor, potentiating the stem cell chemotactic response to the growth factor stromal-derived factor-1. Second, C3b deposited in the stroma is broken down to iC3b, mediating stem cell stroma attachment via the iC3b–CR3 interaction. 3.3. Complement Activation Products May Be Neuroprotective While the effects of C3a and C5a on neurorecovery remain unclear, there is evidence that these molecules have noninflammatory effects in the CNS, and that they may affect neuronal survival following stress. Mice genetically deficient in C5 demonstrate markedly increased susceptibility to glutamate excitotoxic neu90 ronal death following an intrahippocampal injection of kainate . Additionally, more recent in-vitro work has demonstrated that pretreatment of neuronal cultures with C5a reduces induced neuronal cell death, in part due to MAP kinase91-93 dependent inhibition of Caspase activation .

4. CONCLUSION Anti-complement strategies appear to hold great promise for the development of stroke therapeutics. Yet caution should be exercised. It is clear that the complement cascade is a complex and intricate system with widely varied effects, and if any knowledge has been gained from the many failed attempts at translating stroke therapies to the bedside, it is that cavalier application of under-elucidated therapies that leads to wasted resources and the potential for poor patient outcomes.

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14 OBSERVATIONS ON COMPLEMENT ACTIVITY IN THE TWO-STAGE INFLAMMATORY/HEMOSTATIC RESPONSE IN THE BABOON AND HUMAN MODELS OF E. COLI SEPSIS AND ENDOTOXEMIA Fletcher B. Taylor Jr., Eric Hack, and Florea Lupu 1. INTRODUCTION The observation that baboons infused with sublethal concentrations of Escherichia coli (E. coli) exhibited a two-phase activation of complement suggested that the response to E. coli could consist of two distinct stages. This observation raised the question of whether each stage could be driven by different pathophysiologic processes involving complement. Should this be true, it could form a basis for understanding the mechanisms and conditions that give rise to the clinical counterparts seen in severe sepsis ranging from sudden capillary leak (toxic shock) to gradual progressive organ failure. This question resurfaced when we observed that those baboons infused with LD30 E. coli that did die exhibited a delayed clinical course suggestive of progressive organ failure rather than the cardiovascular collapse, disseminated intravascular coagulation (DIC), and shock that followed infusions of LD100 concentrations of E. coli. We therefore have reexamined previous studies of the baboon responses to lethal and 1 2 sublethal E. coli and of the human response to endotoxin . We wished to (i) establish whether there were two stages in the sublethal response and characterize the behavior of the complement system in each; (ii) determine how these two stages differ from each other, and (iii) show whether the lethal variants of the two stages seen in the compensated baboon response to sublethal of E. coli are models of the cardiovascular collapse and shock, and the progressive organ failure variants seen in severe sepsis. Fletcher B. Taylor Jr., Florea Lupu, Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.. Eric Hack, Department of Immunopathology, Sanquin Research, P.O. Box 9190, 1006 AD Amsterdam, The Netherlands.

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2. DESCRIPTION OF THE BABOON AND HUMAN MODELS OF E. COLI SEPSIS AND ENDOTOXEMIA 2.1. Baboon E. Coli Sepsis Model The baboon model for sepsis that was used in this study has been described in 3,4,5,6 detail elsewhere . Briefly, a mixed breed of Papio C. cynocephalus and Papio C. anubis baboons were purchased from Charles River Breeding Laboratories Inc., Wilmington, Delaware. The baboons were sedated with ketamine hydrochloride (Aveco Company Inc., Fort Dodge, Iowa; 14 mg/kg of body weight intramuscularly) on the morning of the study and anesthetized with sodium pentobarbital (2 mg/kg). The animals were intubated orally and allowed to breathe spontaneously. The femoral artery and both femoral veins were cannulated aseptically and used for measuring aortic pressure, obtaining blood samples, and the infusion of live organisms, fluids, and anesthetic. In these studies, either a lethal 10 dose (4 baboons [lethal group]), i.e., 0.4 x 10 CFU/kg, or a sublethal dose (10 8 baboons [sublethal group]), i.e., 0.4 x 10 CFU/kg of E. coli type B isolated as 4 described previously was infused. The animals receiving the lethal doses died within 6 to 10 hours. The clinical and laboratory data of these 14 animals have 3,7 been described elsewhere . In a subsequent experiment, three additional baboons were challenged with a sublethal dose of E. coli, and one baboon was challenged with a lethal dose of E. coli. The latter animal died 28 hours after the challenge. Mean systemic and arterial pressure and heart rate were measured in each animal every hour during the 6-hour observation period. Hematological parameters were assessed in blood samples collected at T+0, T+30, T+60, T+120, T+180, T+240, T+300, T+360, and T+1440 minutes, as described previously, 5 where T is the time in minutes after the start of the challenge . In addition, at each of these time points, 5-ml blood samples were also collected in 10-mM EDTA and 0.05% (wt/vol) Polybrene (final concentrations) as described previ7 ously . The plasma samples were stored in aliquots at –70°C. The study protocol used received prior approval by the Institutional Animal Care and Use Committee of the Oklahoma Medical Research Foundation and the Oklahoma Health Sciences Center. Assays and reagents used for assay of C3b/c, C4b/c, C5b-9, and C-reactive protein (CRP) and the statistical analysis of data shown in Figures 1 and 2 were 1 described previously . 2.2. Human Endotoxin Model The subjects reported to the Oklahoma City Veterans Administration Medical Center, where the studies were carried out, at 7:30 a.m. without having eaten since 10:00 p.m. the previous evening. After the electrocardiographic monitors were connected, intravenous catheters were established and subjects were allowed to equilibrate for 30 min, during which time baseline vital signs and blood

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Figure 1. Levels of complement activation products (mean and SD, expressed as changes relative to baseline values) C3b/c (A), C4b/c (B), and C5b-9 (C) in baboons who received a lethal (black circles, n = 4) or sublethal (open triangle, n = 10) dose of E. coli intravenously. Significant differences between both groups at each time point are indicated with an asterisk for p < 0.05 or ** for p < 0.005. From J Infect Immun 61(10), 4293–4301 (1993).

samples were taken. After this period, 4 ng/kg of Lot F, Difco Escherichia coli endotoxin (lipopolysaccharide, LPS) was infused over a 1-min period. The subjects generally experienced chills, headache, and rigors after 1 hour, followed by fever, myalgia, and nausea between 2 and 7 hours. Recovery to normal temperature and appetite occurred between 8 to 12 hours, at which time the subjects were discharged. No medications for relief of myalgia or fever were required. Nursing notes were used to grade clinical symptoms. Blood samples were taken

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Figure 2. Levels of C5b-9 complexes (solid lines), expressed as changes relative to the baseline value, and CRP (broken lines), expressed as arbitrary units per milliliter, in four baboons who received either a sublethal (A; n = 3, data represent mean and SD or lethal (B; n = 1) dose of E. coli, and were sampled for more than 6 hours after the challenge. From J Infect Immun 61(10), 4293– 4301, (1993).

at T–0 and 0.5, 2, 4, 8, 12, 24, and 48 hours post-LPS injection. T–0 is defined as the time at which the first blood sample was drawn just before the bolus infusions of endotoxin. These samples were taken via a short 6-cm catheter from the cephalic vein during the first 8 hours and by venipuncture at 12, 24, and 48 hours. Assays and reagents for assay of cytokines, hemostatic factors, elastase/alpha 1 AT complex, C-reactive protein/complement product complexes, endotoxin, neutrophil oxidase, and myloperoxidase activity were as described 2 previously .

3. RESULTS 3.1. Activation Parameters of the Complement System in Baboons after Lethal and Sublethal E. Coli Challenge Administration of a lethal dose of E. coli resulted in pronounced activation of the complement system. As shown in Figure 1a, almost immediately after the start of E. coli infusion, levels of C3b/c steeply increased to eight times the baseline value at T+60 minutes. After a slight decrease, a peak value of 13 times the baseline value occurred at T+180 minutes. Circulating C4b/c also sharply increased to five times the baseline value T+180 minutes (Figure 1b), indicating that at least part of the C3 was activated

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via the classical pathway. Plasma levels of the membrane attack complex, i.e., C5b-9 also rose to 12 times the baseline value at T+60 minutes (Figure 1c). These high levels of the C5b-9 complexes remained constant until T+240 minutes, after which a slight decrease to eight times the baseline value at T+360 minutes was seen. In contrast with the marked and rapid increase in circulating levels of complement activation products observed in the animals that received the lethal dose of E. coli, only a modest increase in these parameters was observed in baboons challenged with the sublethal dose. During the first 6 hours a small increase with peak values of three times the baseline value at T+120 in C3b/c levels occurred (Figure 1a), whereas during this period no significant change in C4b/c levels were noted (Figure 1b). Finally, a moderate increase of two times the baseline value was observed for plasma levels of C5b-9 complexes during the first 6 hours (Figure 1c). No significant changes were observed in total C3 and C4 levels in both groups during the observation period (data not shown). The rapid rise in circulating complement activation products upon infusion with the lethal dose of E. coli suggested direct activation of the complement system by E. coli. Samples obtained from the animals challenged with the sublethal dose of E. coli at 24 hours after the E. coli administration were also analyzed. Surprisingly, levels of all complement activation parameters measured were increased at this 24-hour time point in all animals tested (n = 7) and exceeded the maximal levels observed during the first 6 hours (Table 1).

Table 1. Complement Activation at 3 (T + 180) and 24 (T + 1440) Hours After Administration of Live E. coli Baboon Complement activation atb: a Complement group T+180 T+1440 SLEC 1.76±0.85* 5.95±3.64* C3b/c LDEC 12.89±3.76 NDc C4b/c SLEC 1.33±0.36* 7.65±6.62* LDEC 5.48±3.68 ND C5b-9 SLEC 1.71±0.43** 4.59±1.93** LDEC 11.37±1.02 ND Elastase/•1AT 1AT SLEC 12.1±5.2 20.4±6.91 LDEC 44.3±12.1 ND a: SLEC, baboons that received a sublethal dose of E. coli (N=10). LDEC, baboons that received a lethal dose of E. coli (N=4). b: expressed as increases relative to baseline values (mean ± SD). Statistical significance values for the difference between values at T+180 and T+1440 are shown as follows: asterisk for p<0.05; double asterisk for p <0.005. c: ND = not determined.

This suggested the presence of an activation mechanism other than that triggered directly by the bacteria or their constituents. However, samples taken from

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from the animals during the period from 6 to 24 hours after the challenge were not available. Therefore, we decided to challenge three other baboons with a sublethal dose and one additional baboon with a lethal dose of E. coli to further substantiate this presumed biphasic activation pattern of complement activity. In the three baboons challenged with a sublethal dose, an initial rise in C5b-9 complexes was observed, with peak levels at T+120 minutes (Figure 2). A second rise in C5b-9 complexes, which started at T+360 minutes and exceeded the activation seen during the first few hours, was then observed (Figure 2). In the baboon challenged with a lethal dose, the second rise in C5b-9 complexes was less pronounced than the initial activation observed during and immediately after E. coli infusion. Nevertheless, levels at T+1440 minutes (24 hours) represented a fivefold increase compared with the baseline value (the four previous animals that received the lethal dose of E. coli had died at this time point). The second increase in C5b-9 complexes in the three baboons challenged with a sublethal dose was accompanied by a steep rise in plasma levels of CRP (Figure 2a), which suggested the involvement of this acute-phase reactant in the second phase of complement activation. Spearman rank analysis of the C5b-9 and CRP levels from T+240 to T+1440 minutes (24 hours) yielded a correlation coefficient of 0.64 (p = 0.001). In the baboon challenged with a lethal dose, a marked increase in CRP was also observed, but probably because of the excessive complement activation in the initial phase and the low number of samples, no clear correlation was seen between the rise in CRP and elevated C5b-9 levels during this period (Figure 2b). Finally, the neutrophil response to activation of complement following activation of complement by E. coli is shown in Table 1 (elastase/alpha 1 AT). The pattern of elastase/alpha 1 AT complex generation follows the pattern of maximum complement activation after challenge with lethal and sublethal E. coli, respectively (Figures 1 and 2). 3.2. Activation Parameters of Cytokine Complement and Hemostatic Systems in Humans after Endotoxin Challenge: Evidence Establishing Two Distinct Sequential Pathophysiologic Events Figure 3 shows the two stages of the inflammatory (left column) and hemostatic (right column) responses of human subjects to endotoxin. The inflammatory mediators and regulatory cytokines (TNF, Il-6, Il-10) peak during the first stage (to T+8 hours), while the complement activation products bound to C-reactive protein peak during the second stage following a low initial sign of activity during the first stage. Note that the cytokines had returned toward baseline at the time the second stage began and that the clinical symptoms also had subsided. The hemostatic mediators thrombin-antithrombin (TAT) and plasminantiplasmin (PAP) and the regulator protein C hemostatic factor also exhibited their maximum responses during the first stage before returning to baseline. At this point the hemostatic response going into the second stage was dominated by

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Figure 3. Two-stage human inflammatory and hemostatic responses to endotoxin: (A) Oxidase (o— o) and COR/MOR (x—x) responses to endotoxin. (B) Left-hand column: TNF, IL-6 and IL-10 cytokines (o—o, x—x, U—U) responses to endotoxin. Right-hand column: protein C, thrombin/antithrombin (TAT) and plasmin/antiplamsin complexes (PAP), (o—o, Ɣ—Ɣ, x—x) responses to endotoxin. (C) Left-hand column: soluble thrombomodulin (sTM), C-reactive protein (CRP), and CRP/C3b complex (x—x, Ɣ—Ɣ, o—o) responses to endotoxin. Right-hand column: tissue factor antigen (TF), factor VIIa, and soluble fibrin monomer (sFM) (o—o, x—x, Ɣ—Ɣ) responses to endotoxin.

the appearance of circulating tissue factor antigen, soluble fibrin monomer (sFM), and soluble thrombomodulin (sTM). This was accompanied by a second, much more significant drop in circulating FVIIa and protein C at T+12 hours in

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the second stage. Note that there were two distinct peaks of phagocytic oxidase activity (top panels) that corresponded with these two stages.

3.3. Evidence Suggesting that There Is a Unique Counterpart to the Second Stage of the Compensated Response to E. Coli that Is Distinct from the Lethal Counterpart to the First Stage Figure 4 compares the response of biomarkers of neutrophil count, elastase/alpha-1 AT complexes, soluble thrombomodulin, and complement activation of animals infused with LD100 concentrations of E. coli (left column) and those infused with LD30 concentrations of E. coli (right column) in which the responses of survivors versus non-survivors also are compared. Soluble thrombomodulin is included because it reflects the degree and timing of endothelial injury arising from the aberrant complement/neutrophil interaction. The response to LD100 concentrations of E. coli (left column) was rapid and intense. It included all the inflammatory cytokine complement and hemostatic mediators. Therefore, it is considered to be the lethal counterpart to the first stage of the compensated response to sublethal E. coli, leading to death from capillary leak, consumptive coagulopathy, and shock in hours. The response to LD30 concentrations of E. coli (right column) was delayed, and it shows that the biomarkers of phagocyte (neutrophil and macrophage) activity and endothelial injury occur after surviving a relatively moderate first-stage response. It also shows that the biomarker responses of those animals that survived the LD30 concentrations of E. coli were five- to tenfold less than those of the non-survivors. Figure 5a–c compares the pulmonary histopathology of the animal that received the LD100 concentration of E. coli with two animals that received LD30 concentration of E. coli, one of which was dying at 24 hours while the other was in full recovery at 24 hours. The animal that received the LD30 concentration of E. coli and that was dying exhibited a remarkably early proliferation of fibroblasts with secretion of collagen and influx of macrophages (Figure 5a, inset). The LD100 animal exhibited an intense influx of neutrophils into the alveolar capillary bed with effusion of fluid into the alveolar spaces (Figure 5b, inset). The animal that received the LD30 concentration of E. coli and that was recovering exhibited a normal pulmonary histology (Figure 5c).

4. CONCLUSIONS The observation in earlier studies that the complement system exhibited a biphasic response to sublethal E. coli raised the question of whether there were two stages in the compensated response to E. coli. It also raised the question of

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Figure 4. Comparison of baboon responses to LD100 E. coli to survivor and non-survivor responses to LD30 E. coli. The left-hand column shows the white blood cells (WBC), elastase, thrombomodulin, and C5b-9 response to an LD100 concentration of E. coli. The right-hand column shows the response of these same parameters to LD30 E. coli and compares that of the survivors (Ɣ—Ɣ) with that of the non-survivors (U—U).

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Figure 5. Hematoxylin-eosin staining of lung tissue sections from an animal challenged with LD30 non-survivor (a), LD100 E. coli (b), and LD30 survivor (c). High-magnification insets are presented for panels a and b. White arrows indicate macrophages in (a) and neutrophils in (b). av = alveolar space. Magnification bars are provided for each micrograph.

what the implications might be concerning the tissue pathophysiology of E. coli sepsis and possibly, by extension, of severe sepsis in humans. The first set of studies of the baboon model of E. coli sepsis demonstrated that challenge with a lethal concentration of E. coli produced a pronounced activation of the complement system within the first 6 hours. While this occurred 8 via the alternative pathway as shown by in-vitro studies , this activation also occurred via the classical pathway since C4b/c levels also increased upon challenge with E. coli. This same study showed that a challenge with sublethal concentrations of E. coli also produced a moderate activation of complement within the first 6 hours. We again observed that there was an even greater degree of activity in samples collected at T+24 hours. This confirmed our conclusion that the compensated response to E. coli exhibited two stages. Additional experiments in three animals that received a sublethal concentration of E. coli revealed that this second stage started at 6 hours after the start of the E. coli challenge (Figure 2). This was not due to direct activation of the system by intact E. coli or

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endotoxin, since neither was detected in the circulation after 6 hours . We concluded that this was more likely due to endogenous activation. Our second set of studies further characterized these two stages both in the baboon model of E. coli sepsis and in the human model of endotoxemia. The human studies demonstrated that the first stage was symptomatic and that it was dominated by the appearance of peak levels of mediator and regulator cytokines and of thrombin/plasmin inhibitor complexes. This appearance was coincident with the presence of endotoxin or, in the case of the baboon studies, E. coli. After these agents were cleared and these biomarkers returned to baseline (T+6 to 8 hours), a second asymptomatic stage followed. This consisted of signs of a second round of inflammatory and hemostatic activity dominated by peak levels of soluble thrombomodulin, tissue factor antigen that was coupled with a second, more profound drop in factor VIIa, and the appearance of soluble fibrin monomer. This occurred in the absence of thrombin/plasmin inhibitor complexes and was coincident with the appearance of a second, much higher peak of C-reactive protein/complement product complexes that was sustained over 48 hours. We concluded that this was a compensated innate response to endotoxin and that it occurred in two stages. The first stage was driven by the immediate toxic effects of exogenous, foreign organisms, plus the host’s initial responses to dispose of them and counteract their effects. We concluded that this first stage occurred largely within the vasculature. The second stage was driven by endogenous effects arising from the host’s initial “overcorrection.” We concluded that this was the host’s response to its initial response and that having cleared the organisms was experiencing the “aftereffects” of the intravascular events that now had extended to involve the perivascular tissues. This conclusion is supported by the appearance of soluble thrombomodulin, which is a marker of endothelial injury, and of circulating tissue factor antigen, which is an indicator of exposure of subendothelial structure and disruption of cellular membranes with the release into the circulation of membrane fragments bearing tissue factor. Figure 6 illustrates the two-stage concept of the compensated human response to endotoxin. It also presents the hypothesis that the endogenous host response that initiates the second stage is an ischemic/reperfusion event that produces oxidative stress involving the tissues leading in turn to activation of 10 complement by the mannose-binding ligand, . In a final set of studies we returned to the baboon model of E. coli sepsis to determine if there was a lethal counterpart to the second stage. We already had determined that the response to LD100 E. coli constituted the lethal counterpart to first stages of the compensated response and that it was characterized by increased capillary permeability, DIC, and shock. We then asked whether there was a lethal counterpart to the second stage of the compensated response in which the subjects survived the first stage, only to die later. We hypothesized that those animals that died upon receiving LD30 concentrations of E. coli might constitute such a group, and that the complement system might play a similar

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role in an uncompensated version as it did in the compensated version of the second-stage response.

Figure 6. Diagram illustrating the two-stage concept of compensated human response to endotoxin.

Of those animals that were infused with LD30 concentrations of E. coli that died, all of them survived the initial first-stage cytokine, hemostatic coagulopathic events only to die 2–5 days later of progressive organ (pulmonary, renal) failure. We concluded that this was the lethal counterpart of the second stage of the compensated response. Figure 5a,b clearly shows the difference in the pulmonary histopathology between the two animals, one of which was dying of capillary leak and shock while the other was dying of organ failure after receiving LD30 concentrations of E. coli. Both correspond to their rapid and delayed clinical courses as reflected by the responses of the biomarkers shown in Figure 4. Of equal interest is the observation that these two cases (LD100 and LD30) exhibit two distinctly different lethal pathophysiologic processes. We concluded that the LD100 animal was dying of the toxic effects and aberrant host response secondary to the infusion of E. coli organisms consisting of an intense complement, cytokine response accompanied by an intense influx of activated neutrophils into the pulmonary vascular bed and capillary leak. We concluded that the LD30 animal died of an entirely different pathophysiologic process involving a surprisingly rapid aberrant remodeling/repair process secondary to ischemic/re-

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perfusion with oxidative stress consisting of an unusually intense complement response in the absence of circulating organisms or cytokines. This response included a massive influx of macrophages, proliferation of fibroblasts with secretion of collagen leading to acute respiratory distress syndrome (ARDS), and finally to pulmonary failure. These observations are of clinical importance because if the pathophysiologic processes described above can be distinguished from each other at the time of admission to the hospital and identified as the primary driver of a given variant of severe sepsis, they can offer a rational for a more specific effective treatment. The fact that complement is operative in both the stage-one and stagetwo pathophysiologic variants is of special interest in this regard. It suggests that, depending on the conditions, its role in defending the host might be critical during the first-stage toxic/initial host response, whereas a role in harming the host might be more likely during the second-stage ischemic reperfusion events.

5. REFERENCES 1.

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F.B. Taylor Jr, P.A. Haddad, E. Hack, A.C. Chang, G.T. Peer, J.H. Morrissey, A. Li, R.C. Allen, H. Wada and G.T. Kinasewitz. Two-stage response to endotoxin infusion into normal human subjects: correlation of blood phagocyte luminescence with clinical and laboratory markers of the inflammatory, hemostatic response. Crit Care Med 29(2), 326–334 (2001) J.P. deBoer, A.A. Creasey, A. Chang, D. Roem, A.J.M. Eerenberg, C.E. Hack and F.B. Taylor Jr. Activation of the complement system in baboons challenged with live Escherichia coli: correlation with mortality and evidence for a biphasic activation pattern. J Infection Immun 61(10), 4293–4301 (1993) A.A. Creasey, P. Stevens, J. Kenney, A.C. Allison, K. Warren, R. Catlett, L. Hinshaw and F.B. Taylor Jr. Endotoxin and cytokine profile in plasma of baboons challenged with lethal and sublethal Escherichia coli. Circ Shock 33, 84–91 (1991) L.B. Hinshaw, D.J. Brackett, L.T. Archer, B.K. Beller and M.F. Wilson, Detection of the “hyperdynamic state” of sepsis in the baboon during lethal E. coli infusion. J Trauma 23, 361–368 (1983) F.B. Taylor Jr, A. Chang, C.T. Esmon, A. D’Angelo, S. Vigano-D’Angelo and K.E. Blick, Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest, 79, 918–925 (1987) F.B. Taylor Jr, T.E. Emerson, R. Jordan, A.K. Chang and K. E. Blick, AntithrombinIII prevents the lethal effects of Escherichia coli infusion in baboons. Circ Shock 26, 227–235 (1988) J.P. deBoer, A.A. Creasey, A. Chang, D. Roem, M.C. Brouwer, A.J.M. Eerenberg, C.E. Hack and F.B. Taylor Jr. Activation patterns of coagulation and fibrinolysis in baboons following infusion with lethal or sublethal doses of E. coli. Circ Shock 39, 59–67 (1993). H.J. Muller-Eberhard and R.D. Schreiber, Molecular biology and chemistry of the alternative pathway of complement. Adv Immunol 29, 1–53 (1980)

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F.B. Taylor Jr. Studies of the natural history and mechanism of the primate (baboon) response to E. coli. In D.C. Morrisson and J.L Ryan (ed.), Bacterial endotoxic lipopolysaccharides, Vol. 2, CRC Press, Inc., Boca Raton, FL (1992). 10. C.D. Collard, A. Vähevä, M.A. Morrissey, et al. Complement activation with oxidative stress: role of the lectin complement pathway, Am J Pathol 156, 1549–1556 (2000).

15 COMPLEMENT ACTIVATION DURING SEPSIS IN HUMANS Heike Schreiber, Daniel Rittirsch, Michael Flierl, Uwe Brueckner, Marion Schneider, Manfred Weiss, Florian Gebhard, and Markus Huber-Lang

1. INTRODUCTION In critically ill patients, sepsis is currently one of the leading causes of death in 1,2 both the United States and Europe . Despite intensive scientific and clinical efforts, the deleterious pathophysiological consequences of sepsis remain enigmatic. During experimental sepsis in rodents, uncontrolled activation of the 3,4 complement system results in excessive generation of the anaphylatoxin, C5a , 5,6 and reduction of the C5a receptor (C5aR) on neutrophils , both of which have 7 been associated with impairment of the innate cellular immune system and a 5,6 subsequent poor clinical outcome . The development and manifestation of sepsis has been successfully averted in animals by blockade of C5a interaction with C5aR in rodents in which sepsis has been induced by cecal ligation and puncture 8 (CLP) . Furthermore, sepsis-induced multiple-organ dysfunction was dramati5 cally attenuated by C5a-blockade . In-vitro exposure of neutrophils with concentrations of C5a found in humans with sepsis have revealed at a molecular level 7 several cellular signaling defects , mainly involving mitogen-activated protein phox kinase (MAPK), protein kinase C, p47 , and phosphoinositol-3-kinase (PI37,9 K) . These alterations have resulted in a functional disturbance of the MAPK

Markus Huber-Lang, Dept. of Traumatology, Hand-, Plastic-, and Reconstructive Surgery, Steinhoevelstr.9, 89075 Ulm, Germany. Heike Schreiber, Daniel Rittirsch, Michael Flierl, Uwe Brueckner, Florian Gebhard, Dept. of Traumatology, Hand-, Plastic-, and Reconstructive Surgery, 89075 Ulm, Germany. Marion Schneider, Manfred Weiss, Dept. of Anesthesiology, University of Ulm Medical School, 89075 Ulm, Germany.

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system, resulting in defects in chemotactic and phagocytotic activities and as7 sembly of the NADPH oxidase-dependent “oxidative burst,” finally leading to an impairment in bacterial clearance. Whereas many studies have elucidated the role and crosstalk of complement and neutrophil activation during experimen10,11,12 7 tal and in-vitro simulated sepsis , complement function and C5aR status during progressive sepsis in humans are still largely unknown. To transfer the animal results to the condition of human sepsis, the present prospective study was designed to determine changes of complement in serum, expression of C5aR on neutrophils, and the outcome of sepsis in humans. After permission from the Ethics Committee of the University of Ulm (no. 82/2002), 40 patients with severe sepsis or septic shock (in accordance with the consensus criteria) and 20 healthy volunteers were enrolled for measurement of C5aR content on blood neutrophils by flow cytometry. In parallel, complement hemolytic activity, C3a, C4a, and membrane attack complex (MAC) were determined in the preserved sera. During experimental sepsis, excessive generation of the complement activation product C5a and downregulation of the C5a receptor (C5aR) on neutrophils have been described to be associated with both an impaired innate immune response and a bad outcome. Therefore, in the present study complement monitoring was performed in humans with sepsis and C5aR expression on neutrophils was evaluated. In comparison to healthy volunteers, serum levels of C3a, C5a, and MAC were all significantly increased in patients with sepsis (n = 40). In contrast, the complement hemolytic activity of patients with sepsis was significantly reduced. Patients with sepsis who died (within 30 days after measurement) exhibited a significantly lower hemolytic activity in comparison to those who survived the lethal consequences of sepsis. Blood neutrophils isolated from patients with sepsis demonstrated a significant decrease in C5aR expression in comparison to that found in healthy volunteers. We found evidence that in humans with severe sepsis or septic shock there is excessive generation of complement activation products, impaired complement function, and and a reduction of C5aR on neutrophils, all of which are associated with a lethal outcome. 2. MATERIAL AND METHODS 2.1. Reagents Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich. 2.2. Patient Selection The study was approved by the Local Ethics Committee of the University of Ulm. Written informed consent was obtained or consent was presumed if the

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patient was not capable of making decisions because of sedation or altered mental function. Patients who fulfilled the clinical criteria for severe sepsis or septic 13 shock according to Bone’s definition were prospectively enrolled in the study (n = 40). The exclusion criteria were: age <18 years, pregnancy, HIV/AIDS, cardiogenic shock as the primary underlying disease, underlying hematologic disease, and cytotoxic therapy given within the previous 6 months. Twenty sexand-age-matched healthy volunteers served as the control group. All patients were followed throughout the 30-day study period or until death occurred. Patient enrollment in the study was related to the clinical designation of sepsis and the need for pressor support in order to maintain blood pressure and cardiac output. Samples of blood were obtained at the time patients required continuous infusion of vasopressors or inotropic agents to maintain blood pressure despite adequate fluid resuscitation. 2.3. Measurement of Serum Concentrations of C3a, C5a, and MAC The serum concentrations of complement activation products C3a and C5a and membrane attack complex (MAC) were measured by a C3a and MAC enzymelinked immunoassay (ELISA) (Quidel, San Diego, CA) and by a C5a ELISA (IBL Immuno-Biological Laboratories, Hamburg, Germany) according to the manufacturer’s protocol. 2.4. Hemolytic Complement Assay Serum was serially diluted in TBS (75 g/l NaCl, 177 ml/l 1-N HCl, 28 ml/l triethanolamine, 1.2 ml/l 1.25-M CaCl2, 1.2 ml/l 4.16-M MgCl2), and the dilution causing 50% lysis of sensitized sheep red blood cells (Colorado Serum Company, Denver, CO) after incubation at 37°C for 1 h determined (CH50). The complement reaction was stopped by addition of ice-cold TBS (with 0.05% gelatin). After centrifugation (2500 rpm, 5 min), absorbance of the supernatant fluids was measured (at 541 nm). 2.5. Neutrophil Isolation Neutrophils were isolated from whole blood by Ficoll-Hypaque gradient centrifugation (Pharmacia Biotech AB, Uppsala, Sweden) and dextran sedimentation followed by hypotonic lysis of residual red blood cells. 2.6. Analysis of C5aR Content on Neutrophils Flow cytometric analysis was conducted immediately after whole blood collection in an EDTA-containing syringe. A total of 10 µl of FITC-labeled anti-CD 88 (Serotec GmBH, Duesseldorf, Germany) was incubated with 100 µl human whole blood for 20 min at RT. For control purposes, non-labeled Isotype IgG

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(Serotec) or FITC-labeled Isotype IgG (Serotec), respectively, was added to control samples in equal amounts. After washing, the cells were suspended in phycoerythrin-labeled anti-rabbit IgG (Serotec) diluted 1/50 in staining buffer (PBS with 0.1% sodium azide and 1% FBS) for further 20 min. Erythrocytes were lysed for 10 min by addition of 2 ml of 1 x FACS lysing solution (BD PharMingen, San Diego, CA). After 2x washing with PBS, the leukocytes were reconstituted in a fixing solution (1% paraformaldehyde prepared in PBS plus 0.1% sodium azide) and analyzed on a flow cytometer (BD PharMingen, San Diego, CA). 2.7. Statistical Analysis All values were expressed as mean + SEM. Data sets were analyzed by KruskalWallis one-way ANOVA on ranks; differences in the mean values among experimental groups were then compared using a multiple-comparison procedure (Dunn’s method). Results were considered statistically significant where p < 0.05.

3. RESULTS 3.1. Epidemiological Assessments All patients studied were admitted to the Surgical Intensive Care Unit. The mean age of the patients with sepsis was 65 ± 2 years (range: 36–82 years). The age distribution was similar to the healthy control group, with a mean age of 61 ± 4 years (range: 40–75 years). The site of infection in more than 2/3 of cases was localized either to the abdominal cavity or to the retroperitoneum. In 15% (6/40) the lungs were the primary locus of the infection while peritonitis was found in 53% (21/40) of septic patients and in other sites as indicated (Figure 1). Regarding the lethality rate, 45% (18/40) of the patients failed to survive the lethal complications of septic shock during an observation period of 30 days commencing on the day of admission. 3.2. Sepsis-Induced Complement Activation during Septic Shock in Humans In the past, an increased C3a/C3 ratio in serum from patients who developed 14 sepsis has been reported . However, little is known about changes of complement activation products and the membrane attack complex (C5b-9, MAC) in 15,16 serum during the shock phase of human sepsis . Accordingly, complement activation products were determined in the present study as measured by ELISA. As expected, in comparison to healthy volunteers enhanced levels of the anaphylatoxin C3a were found in sera obtained from patients with septic shock. The increase was more than threefold when compared to normal controls (probands)

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Figure 1. Site of infection in patients with septic shock.

(p < 0.01, Figure 2). A similar pattern was found for C5a, with a more than fivefold increase (p < 0.01, Figure 3). Furthermore, there was a modest but significant increase (p < 0.05) in the presence of the membrane attack complex (Figure 4). 3.3. Sepsis-Induced Impairment of Complement Function during Septic Shock in Humans Sepsis-induced complement activation was reflected by a significantly reduced hemolytic activity in sera from patients with septic shock versus (age-matched) healthy volunteers (Table 1). In comparison to patients who survived the septic shock period, non-survivors exhibited a further significant decrease in serum hemolytic activity. The data indicate complement activation and consumption during septic shock. 3.4. Loss of C5aR on Neutrophils Is Associated with a Lethal Outcome during Sepsis in Humans 5,6

Based on recent findings during experimental sepsis in rats , we next determined whether the C5aR status on neutrophils was altered during septic shock in humans, using flow cytometric analyses. As expected, virtually all neutrophils obtained from healthy volunteers (probands) expressed the C5aR on their surface with a mean fluorescence intensity (MFI) of 1019 ± 77 (= 100%) and a background MFI of 108 ± 44 (isotype-matched IgG negative control). In contrast, C5aR expression was significantly reduced on neutrophils from patients during septic shock (Table 1). There was a further significant decrease to nearly background levels in neutrophils from patients who did not survive septic shock, indicating nearly total loss of C5aR from surfaces of neutrophils.

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Figure 2. Levels of C3a in serum obtained from healthy volunteers (probands, n = 20) or patients with severe sepsis or septic shock (patients with sepsis, n = 40).

Figure 3. Levels of C5a in serum from patients with severe sepsis or septic shock (n = 40).

4. DISCUSSION In experimental sepsis, there is compelling evidence that complement activation and consumption have occurred. In the cecal ligation and puncture (CLP) model, 17 which closely mimics the pathophysiology of sepsis in humans , complement hemolytic activity (CH50) levels has been reported to fall by 30–50% during the first 24 hr of sepsis and then gradually returning towards normal levels in 18 survivors . The complement activation products, including the anaphylatoxins

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Figure 4. Detection of C5b-9 membrane attack complex (MAC) in serum from patients with severe sepsis or septic shock (n = 40).

Table 1. Changes in Serum CH50 and PMN C5aR Content during Septic Shock* Parameter

Controls

Sepsis survivors

Sepsis nonsurvivors

Serum Ch50 C5aR (MFI)**

100 100

82.1 + 10.0 49.6 + 2.1

49.6 + 4.4 31.6 + 1.1

* Data are presented as percentages, with controls set at 100% values. N = 40 for each group. ** MFI = mean fluorescence intensity

(C3a, C4a, C5a) and the membrane attack complex (C5b-9), have been detected 19,20 in the plasma of primates and humans with sepsis . However, confusing and contradictory claims concerning the concentration of C5a in serum obtained 16,21 from patients with systemic inflammatory diseases have been made . The ability to detect the anaphylatoxins, especially C5a, is technically difficult because of the presence of high-affinity C5a receptors (C5aR) on circulating neutrophils (PMN). These receptors, which number approximately 50,000/neutrophil (which 15 translates to ca. 2.5 x 10 C5aR on all neutrophils within the circulation), would 15 absorb C5a (1 nM of C5a equals ca. 0.6 x 10 C5a molecules), implying theoretically that C5a can only be detected in serum after saturation of C5aR with C5a has occurred on blood neutrophils. However, in the present study we found in a representative cohort of patients with septic shock (n = 40) an increase in both C3a and C5a levels (Figures 2 and 3) and a corresponding significant de-

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crease in complement hemolytic activity of more than 50% (Table 1). While it might be argued that C5a detection on blood PMN could be an effective alternative to measuring plasma C5a, it is known that once C5a is bound to C5aR the receptor-ligand is rapidly internalized, with subsequent partial C5aR recycling 22,23 via the Golgi apparatus, with return of C5aR to the cell surface . Therefore, the measurement of leukocyte-bound C5a seems technically not a reliable strategy for estimation of C5a generation in vivo. There are data that indirectly suggest excessive C5a generation in humans with sepsis. In rats with CLP there was a substantial reduction of C5a binding on neutrophils with the progression of 5 sepsis and similar reductions on C5aR content on blood neutrophils in the first 6 36 hr after CLP , in a manner that was predictive of survival or lethality. How24 ever, more extensive accumulation of data in humans is required , especially during the more advanced phases of sepsis. Here, we present data on blood neutrophils, indicating a loss of C5aR during septic shock, especially in those patients who failed to survive the vicious spiral of sepsis and its complications (Table 1). These data are in accordance with the experimental findings indicative of an early reduction in C5aR content on surfaces of blood neutrophils in the first 24–36 hr of sepsis and gradual restoration during subsequent days in the 5,6 surviving groups . Further experimental studies with immune modulation by 3,5,6,8,25 inhibition of the C5a–C5aR interaction has shown it to be highly protective . Although the mechanisms of these protective effects is not known, it is presumed that infusion of antibody (anti-C5a or anti-C5aR) prevents interaction of C5a with C5aR, protecting innate immune functions of phagocytic cells that otherwise would deteriorate during sepsis. These data based on evaluation of sera and neutrophils from patients in septic shock suggest that activation and the corresponding consumption of complement, with loss of cellular C5aR, may play a crucial role in the pathophysiology of sepsis and its outcome. The status of C5aR on neutrophils, as detected by flow-based methods, may be used as a marker defining the immune status of the patient and might be helpful for effective patient monitoring and patient-tailored treatment.

5. ACKNOWLEDGMENTS This work was supported by research grants from the Deutsche Forschungsgemeinschaft (DFG HU 823/2-2) and the National Institutes of Health (GM61656). We thank Sonja Albers and Barbara Acker for outstanding laboratory assistance and Beverly Schumann for secretarial assistance in preparation of the manuscript.

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16. H. Nakae, S. Endo, K. Inada, T. Takakuwa, and M. Yoshida, Chronological changes in the complement system in sepsis, Surg Today 26, 225–229 (1996). 17. K.A. Wichterman, A. E. Baue, and I. H. Chaudry, Sepsis and septic shock — a review of laboratory models and a proposal. J Surg Res 29, 189–201 (1980). 18. B. J. Czermak, M. Breckwoldt, Z. B. Ravage, M. Huber-Lang, H. Schmal, N. M. Bless, H. P. Friedl, and P. A. Ward, Mechanism of enhanced lung injury during sepsis. Am J Pathol 154, 1057–1065 (1999). 19. D. H. Hangen, J. H. Stevens, P. S. Satoh, E. W. Hall, P. T. O’Hanley, and T. A. Raffin, Complement levels in septic primates treated with anti-C5a antibodies. J Surg Res 46, 195–199 (1989). 20. A. Bengtson and M. Heideman, Anaphylatoxin formation of sepsis. Arch Surg 123, 645–649 (1988). 21. C. Leon, M. J. Rodrigo, A. Tomasa, M. T. Gallart, F. J. Latorre, J. Rius, and J. Brugues, Complement activation in septic shock due to gram-negative and grampositive bacteria. Crit Care Med 10, 308–310 (1982). 22. N. P. Gerard and C. Gerard, The chemotactic receptor for human C5a anaphylatoxin. Nature 349, 614–617 (1991). 23. N. Naik, E. Giannini, L. Brouchon, and F. Boulay, Internalization and recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated internalization is modulated by phosphorylation of the C-terminal domain. J Cell Sci 110, 2381–2390 (1997). 24. M. Furebring, L. D. Hakannsson, P. Venge, B. Nilsson, and J. Sjölin, Expression of the C5a receptor (CD88) on granulocytes and monocytes in patients with severe sepsis. Crit Care 6, 363–370 (2002). 25. I. J. Laudes, J. C. Chur, S. Sikranth, M. Huber-Lang, R. F. Guo, N. Riedemann, J. V. Sarma, A. H. Schmair, and P. A. Ward, Anti-C5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis. Am J Pathol 160, 1867– 1875 (2002).

16 THREE DISTINCT PROFILES OF SERUM COMPLEMENT C4 PROTEINS IN PEDIATRIC SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) PATIENTS: TIGHT ASSOCIATIONS OF COMPLEMENT C4 AND C3 PROTEIN LEVELS IN SLE BUT NOT IN HEALTHY SUBJECTS Yee-Ling Wu, Gloria C. Higgins, Robert M. Rennebohm, Erwin K. Chung, Yan Yang, Bi Zhou, Haikady N. Nagaraja, Dan J. Birmingham, Brad H. Rovin, Lee A. Hebert, and C. Yung Yu

1. ABSTRACT The serial changes of serum complement proteins C4 and C3 in SLE were characterized in 33 pediatric SLE patients with defined C4 genotypes. Three distinct groups of C4 protein profiles were observed. The first group was characterized by persistently low C4 levels (<10 mg/dL) throughout the course of the study. Patients with this profile had mild disease manifestations and low to medium copy numbers of C4 genes. The second group featured periodic fluctuations of serum C4 protein concentrations above and below 10 mg/dL, paralleled with ups and downs of SLE disease activities. Most patients with the second profile had unequal copy numbers of C4A and C4B genes and relatively severe disease. The third group had normal serum C4 levels (>15 mg/dL) most of the time and occasionally low C4 and C3 levels that were mostly coincident with disease flares prior to effective medical treatment. Most patients in this group

Yee-Ling Wu,1,2,3 Gloria C. Higgins,1,3 Robert M. Rennebohm,1,3 Erwin K. Chung,1,3 Yan Yang,1,3 Bi Zhou,1 Haikady N. Nagaraja,4 Dan J. Birmingham,2,5 Brad H. Rovin,2,5 Lee A. Hebert 5 and C. Yung Yu1,2,3. 1Columbus Children's Research Institute, 2Integrated Biomedical Graduate Program, 3 Department of Pediatrics, 4Department of Statistics, 5Department of Internal Medicine, The Ohio State University, Columbus, Ohio 43205.

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had medium to high C4 gene dosages. In SLE patients but not healthy subjects the serum C3 protein levels were tightly associated with serum C4 protein levels. It was found that a subject's body mass index (BMI) is an important parameter in determining the serum C3 and C4 levels, both in SLE patients and healthy subjects. However, the BMI is unlikely to be involved in the parallel changes of C3 and C4 in SLE. As expected, the maximum serum C4 concentration present in a patient, which probably reflects the patient's inherent capacity to produce a physiologic level of C4, was a function of her/his C4 gene dosage. Therefore, classifying C4 and C3 protein profiles and elucidating the genotypic and phenotypic variations of C4 may improve prediction of SLE disease flares and severity, and facilitate more effective management of the disease.

2. INTRODUCTION Low complement activity or hypocomplementemia of serum C4 and C3 proteins 1,2 are features of human systemic lupus erythematosus (SLE) . The cause-andeffect relationship of complement in SLE has been perplexing. Deficiencies of complement C4 and other early components of the complement classical activa3-10 tion pathway including C1q and C2 are strong genetic risk factors for SLE . A marked fall in complement C3 and C4 serum protein levels preceding disease 11-14 flare was observed in many SLE patients . However, a wide range of serum C4 protein levels in healthy subjects that overlap with those in SLE patients hinders a clear understanding of complement C4 in SLE disease pathogenesis and disease progression. The complex genetics of complement C4A and 15-18 plus undefined epigenetic C4B with polygene and gene size variations and environmental factors affecting C4 protein expression levels probably contribute to what resembles a continuous, quantitative genetic trait for human serum C4A and C4B proteins. Our long-term goal is to identify and decipher how those genetic and environmental cues for the qualitative and quantitative variations of C4A and C4B proteins interact and contribute to the health and disease processes. In this study we focus on investigating the patterns of serial C4 and C3 serum protein profiles in pediatric SLE patients with defined C4 phenotypes and genotypes. Three distinct C4 protein profiles with persistently low, fluctuating, and relatively high levels of serum C4 were identified. The patient group with serum C4 proteins fluctuating above and below 10 mg/dL was also characterized by severe and frequent disease flares, and an unequal number of C4A and C4B genes. Further analysis revealed a tight association of serum C4 and C3 protein levels in SLE but not in healthy subjects, and provided evidence of the influence of body mass index (BMI) in determining serum C3 and C4 protein levels in both patients and healthy subjects.

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3. MATERIALS AND METHODS 3.1. Study Populations The study populations consist of 33 consecutive pediatric SLE patients being treated at the Columbus Children's Hospital Rheumatology Clinics over the past ten years. All but two of the SLE patients were female. The mean age of disease onset was 13.2 ± 3.6 years. The mean age of the patients at the time of enrollment was 15.5 ± 3.8 years. Included in the study were 35 first-degree relatives and 102 healthy Caucasian females. Informed consents (and assents for children between aged 8 and 17 years) were obtained in accordance with approved protocols on human subject research by the Columbus Children’s Hospital Institutional Review Broad. 3.2. Preparation of EDTA-Plasma and Genomic DNAs Peripheral blood was collected by venipuncture. Plasma samples were harvested by centrifugation and immediately frozen at –80°C for C4 phenotyping experiments. Genomic DNA samples for TaqI, or PshAI-PvuII restriction fragment length polymorphism (RFLP) were prepared from EDTA-blood by the Puregene DNA isolation kit (Gentra systems) following the manufacturer’s instructions. Genomic DNA for long-range mapping were prepared from Ficoll gradient purified white blood cells, which were embedded in low gelling temperature agarose and subjected to proteinase K digestion. 3.3. C4 Phenotyping and Genotyping The techniques for phenotyping and genotyping analyses of human C4 proteins and genes are essentially as described as in [19–21]. Briefly, plasma C4 (and C3) concentrations were determined by single radial immunodiffusion using EDTA-plasma and commercial kits (The Binding Site, England). Complement C4A and C4B protein allotypes were determined by immunofixation of EDTAplasma resolved by high-voltage agarose gel electrophoresis, using goat antiserum against human C4 (DiaSorin). The copy number of long and short C4 genes and RCCX modular variations were determined by genomic TaqI RFLP, and confirmed by Southern blot analysis of PmeI digested genomic DNA in agarose plugs, resolved by pulsed field gel electrophoresis using a Biorad CHEF Mapper. C4A and C4B gene dosages were elucidated by genomic PshAI-PvuII RFLP, employing a C4d probe for hybridization.

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3.4. Mutations of Complement C4 and C2 Genes Sequence-specific primer (SSP)-PCRs were employed to screen for the 2-bp (TC) insertion in codon 1213 and the 2-bp (GT) deletion in codon 497 of the C4 5,22 23 genes , and the 28-bp deletion in exon 6 of the C2 genes . 3.5. Clinical Information Clinical data including serum complement C3 and C4 concentrations, titers for autoantibodies including anticardiolipin, anti-ds DNA, and anti-Smith were determined by Clinical Laboratories of the Columbus Children's Hospital. 3.6. Statistics Correlation and multiple regression analyses were performed by JMP version 5 (SAS Institute, Cary, NC) and SPSS version 13.01 (SPSS Inc., Chicago, IL). Models that account for repeated measures per subject were used. Scatterplots were produced by SPSS. Least-squares plot for mean C3 and C4 level groups was produced by Excel (Microsoft).

4. RESULTS 4.1. Demographics and Clinical Features of the Pediatric SLE Study Population Thirty-three pediatric SLE patients from the Columbus Children's Hospital were studied (Table 1). The mean age of disease onset was 13.2 ± 3.6 years. The majority of patients had their disease onset during or after their puberty (i.e., 10–18 years old), but four patients had disease onset before 8 years old. Thirty-one patients were female and two patients were male (female to male ratio: 15.5 to 1). The ethnicities of the American patients were 20 Caucasians, 8 Africans, 1 Asian, and 4 with mixed racial backgrounds. All patients satisfied a minimum of 24 four diagnostic criteria proposed by the American College of Rheumatology , and twenty-three (69.7%) satisfied five or more ACR criteria. The duration of laboratory test records ranged from 9 months to 9.4 years (mean: 3.5 ± 2.2 years). All patients had antinuclear antibodies. Of the immunological parameters examined, 25 patients (75.8%) were positive for anti-dsDNA, 20 (60.6%) were positive for anticardiolipin or antiphospholipid antibodies, and 9 (27.3%) were positive for anti-Smith. Fifteen patients (45%) had renal involvement such as proteinuria, renal casts, and/or biopsy-proven diffused proliferative glomerulonephritis.

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Table 1. Demographics of the Pediatric SLE Patients Age of Age of Duration of Patient No. Race Sex Disease Onset Enrollment Record

BMI

ACR Criteria

1

C

F

16

16.5

3

41.1 ± 3.3

5, 9, 10b, 10c, 11

2

AA

F

16

17.7

3.08

35.7 ± 1.8

6, 7, 9, 10a, 10c, 11

3

C

F

13.5

14.1

3.08

31.6 ± 2.3

1, 3, 4, 8, 9, 11

4

A

F

12

12.7

4.75

18.0 ± 1.2

1, 5, 9, 10a, 10b, 10c, 11

5

C

F

16

17.8

2.5

27.6 ± 1.3

1, 5, 7, 8, 11

6

C

F

10.8

11

4.58

25.6 ± 2.1

1, 5, 10a, 10c, 11

7

C

F

12

12.5

0.75

30.5 ± 3.6

1, 4, 5, 9, 10a, 10c, 11

8

AA

F

17.5

17.7

3.75

23.5 ± 0.8

4, 5, 9, 10b, 11

9

C

F

14

14.3

2.92

22.7 ± 1.1

1, 7, 9, 10c, 11

10

C

F

13

16.6

6.5

23.1 ± 1.1

4, 5, 6, 7, 9, 10a, 10b, 10c, 11

11

C/A

F

14.5

16.7

1.33

23.2 ± 0.6

7, 9, 10a, 10b, 11

12

C

F

16

16.5

2.33

18.6 ± 1.8

5, 9, 10a, 11

13

C

F

15

18.3

5

33.8 ± 1.4

6, 7, 9, 10a, 11

14

AA

F

5

7.3

2.17

16.0 ± 0.6

5, 9, 10a, 10c, 11

15

C

F

12

18.2

5.08

25.1 ± 2.2

1, 9, 10a, 11

16

C

M

11

12

3.25

29.2 ± 1.1

5, 7, 9, 10a, 10c, 11

17

AA/A

F

16

18.1

1.92

33.3 ± 1.1

1, 5, 7, 10a, 10c, 11

18

AA

F

15

18.4

4

24.9 ± 0.4

2, 5, 6, 7, 9, 10a, 10b, 10c, 11

19

C

M

11

11.2

2.92

35.1 ± 3.9

4, 7, 9, 10b, 11

20

C

F

15

20.2

5.68

25.9 ± 2.4

1, 3, 7, 11

21

AA

F

17

24.6

7.92

28.6 ± 3.6

2, 7, 10a, 10c, 11

22

C

F

13.5

16.2

2.75

24.9 ± 0.7

1, 3, 4, 5, 7, 9, 10a, 11

23

C

F

6

7.6

2.17

16.5± 0.5

5, 8, 10a, 10c, 11

24

C

F

4

17.3

4.33

27.2 ± 3.2

1, 4, 5, 6, 10a, 11

25

AA/C

F

7.5

7.9

2.17

28.2 ± 4.2

1, 4, 9, 10a, 10b, 10c, 11

26

C

F

10

17.2

9.25

26.1 ± 0.6

1, 5, 10a, 10c, 11

27

C

F

13

13.7

2.08

19.1 ± 0.7

1, 7, 9, 10a, 10c, 11

28

AA

F

16

17

2

34.6 ± 1.8

4, 7, 9, 10a, 10b, 10c, 11

29

C

F

16

16.2

0.92

25.5 ± NA

1, 4, 5, 9, 10a, 10c, 11

30

AA

F

11

16.5

9.42

41.4 ± 6.1

1, 5, 9, 10a, 10c, 11

31

AA

F

18

18.8

1.5

30.7 ± 2.2

1, 4, 5, 7, 9, 10a, 11

32

C

F

18

18.5

1

19.1 ± 0.4

5, 6, 9, 10a, 11

33

AA/C

F

12

12.6

1.75

23.4 ± 3.3

1, 4, 6, 9, 10a, 10c, 11

Race: A, Asian; AA, African American; C, Caucasian. Sex: F, female; M, male; Age and duration of records are in years; ACR (American College of Rheumatology) SLE diagnostic criteria: 1, malar rash; 2, discoid rash; 3, photosensitive rash; 4, oral or nasal ulceration; 5, arthritis; 6, serositis; 7, renal disorder; 8, neurologic disorder; 9, hematologic disorder; 10a, anti-dsDNA, 10b, anti-Smith, 10c, antiphospholipid; 11, antinuclear antibody.

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A.

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

Serum C4 Conc. (mg/dL)

40 35 30 25 20 15

200 180 160 140 120 100 80 60 40 20 0

10 5 0 09/16/01

04/04/02

10/21/02

05/09/03

Serum C3 Conc. (mg/dL)

Patient No. 23

11/25/03

Date

B.

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

Serum C4 Conc. (mg/dL)

40 35 30 25

200 180 160 140 120 100 80 60 40 20 0

20 15 10 5 0 01/05/98

08/28/99

04/19/01

12/10/02

Serum C3 Conc. (mg/dL)

Patient No. 10

08/01/04

Date

C.

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

Serum C4 Conc. (mg/dL)

40 35 30 25 20 15

60 40 20 0

10 5 0 10/26/01

200 180 160 140 120 100 80

08/22/02

06/18/03

04/13/04

Serum C3 Conc. (m g/dL)

Patient No. 16

02/07/05

Date

Figure 1. Distinct patterns of serum complement C4 profiles in three pediatric SLE patients. In each panel serum C4 concentrations are shown in solid lines (blue) and serum C3 concentrations in dotted lines (purple). The X-axis shows the dates of blood tests. The RCCX structures, C4A and C4B protein allotypes, the mean C4 protein concentration and the mean C3 protein concentration of the patient are listed in each panel. Panel A shows the profile of patient 23, whose serum C4 concentrations were persistently low (mean C4 level: 8.3 ± 3.2 mg/dL). Panel B shows the profile of patient 10, whose serum C4 levels fluctuated above and below 10 mg/dL (mean C4 level: 13.5 ± 4.7 mg/dL). Panel C shows the profile of patient 16, whose serum C4 levels were mostly within the normal range (mean C4 level: 19.1 ± 7.5 mg/dL).

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233

4.2. Three Types of C3–C4 Protein Profiles in SLE Patients A serum C3–C4 protein profile was constructed by plotting serum C3 and C4 protein levels over time for each of the 33 patients. Altogether 899 sets of datapoints for C3 and C4 were available. Very low C3 levels in a patient (C3 level < 50 mg/dL) were often coincident with severe disease activity, including manifestations of lupus nephritis, severe skin lesions, serositis, and cytopenias. Overall three major patterns of C4/C3 protein profiles were observed (Figure 1). The first pattern consists of 8 patients whose C4 levels were persistently low. The mean serum C4 levels were lower than 10 mg/dL (i.e., Group I, low mean C4 levels). The maximum C4 protein levels in these patients were mostly between 13 and 15 mg/dL (mean 14.5 ± 4.1 mg/dL). The ratios of mean C3 concentrations to mean C4 concentrations in this group of patients ranged from 9.0 to 13.4. All of these patients had anti-dsDNA antibodies at some point during their disease but only two patients had experienced renal manifestation of lupus. The disease activities among these patients had been relatively mild and well "controlled," according to the assessment of the treating physicians. The second pattern consists of 12 patients who manifested periodic or frequent fluctuations of C4 concentrations above and below the 10-mg/dL threshold. The mean C4 concentrations among these patients generally ranged between 10 and 15 mg/dL (i.e., Group II, medium mean C4 levels). The mean maximum C4 protein concentration in these patients was 21.7 ± 4.0 mg/dL. The ratios of mean C3 concentrations to mean C4 concentrations varied from 5.1 to 11.4. Among these patients, 11 (92%) fulfilled five or more ACR criteria, 10 (77%) had anti-dsDNA antibodies, 10 (77%) had hematologic abnormality (leucopenia, hemolytic anemia, or thrombocytopenia), and 7 (54%) had renal disease. This group of patients appeared to have relatively frequent and disease exacerbations. The third pattern consisted of 13 patients whose C4 protein concentrations were generally at the normal range but had occasionally low C4 (and low C3) levels. The mean serum C4 protein levels were generally higher than 15 mg/dL (i.e., Group III, high mean C4 levels). The mean maximum C4 protein concentration was 32.2 ± 7.5 mg/dL. The ratios of mean C3 concentrations to mean C4 concentrations varied from 3.5 to 6.9 (Table 2). The low C4 and low C3 levels usually coincided with the initial time of disease diagnosis before effective medical treatment, or at the time of disease flares. A wide spectrum of disease activity from relatively mild to severe was observed in the Group III patients, by assessment of the treating physicians. 4.3. Tight Correlation between Serum C3 and C4 Concentrations in SLE Patients but not in Healthy Subjects A cross-sectional study of plasma complement C4 and C3 protein concentrations was performed on 102 healthy Caucasian subjects. The overall Pearson’s corre-

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Table 2. Quantitative Variations of Serum Complement C4 and C3 in Pediatric SLE Patient No. of No. M. Group I 6 41 7 7 11 8 12 18 14 18 23 16 27 23 29 21

C4 Mean

C4 max

C4 min

C3 Mean

C3 max

C3 min

[C3]/ [C4]

R

7.2 ± 3.5 5.7 ± 1.9 7.5 ± 3.5 6.6 ± 2.5 7.2 ± 2.8 8.3 ± 3.2 8.5 ± 2.8 7.0 ± 3.2

24 10 13 13 15 13 14 14

5 5 5 4 5 5 5 2

94.3 ± 15.4 76.7 ± 30.3 83.8 ± 4.9 65.3 ± 9.2 71.2 ± 18.6 74.7 ± 6.8 97.2 ± 26.4 77.1 ± 19.5

137 114 92 82 115 87 130 102

49 32 77 38 47 66 26 30

13.1 13.4 11.2 9.9 9.9 9.0 11.4 11.0

0.543 0.543 0.261 0.341 0.666 0.545 0.819 0.829

Group II 2 4 8 9 10 17 18 19 22 26 30 33

26 36 13 43 56 13 23 22 16 36 92 16

10.2 ± 3.0 12.0 ± 5.5 11.5 ± 4.6 10.6 ± 5.2 13.5 ± 4.7 12.2 ± 3.6 10.0 ± 5.0 11.6 ± 6.3 11.6 ± 4.5 14.2 ± 2.4 11.5 ± 5.1 14.1 ± 5.6

14 29 18 23 21 18 20 24 23 20 24 26

5 5 5 3 5 5 5 5 5 6 5 6

110.8 ± 17.8 81.2 ± 18.3 63.1 ± 16.9 76.1 ± 26.0 74.1 ± 17.2 90.2 ± 17.7 71.7 ± 26.5 113.6 ± 30.4 108.7 ± 17.2 72.8 ± 11.5 89.6 ± 15.1 105.4 ± 27.9

148 130 94 122 107 116 117 157 147 99 119 137

62 20 43 23 37 61 38 37 79 46 49 39

10.9 6.8 5.5 7.2 5.5 7.4 7.2 9.8 9.4 5.1 7.8 7.5

0.640 0.444 0.952 0.769 0.794 0.844 0.910 0.714 0.626 0.616 0.845 0.639

Group III 1 3 5 13 15 16 20 21 24 25 28 31 32

34 21 30 25 16 27 20 39 63 29 22 24 15

18.9 ± 4.0 18.5 ± 3.5 21.7 ± 5.2 16.8 ± 5.7 18.8 ± 4.0 19.1 ± 7.5 19.9 ± 5.5 35.6 ± 10.1 14.9 ± 4.2 22.0 ± 8.9 15.7 ± 7.3 18.4 ± 9.9 26.1 ± 4.0

30 26 32 27 22 29 33 51 22 38 33 33 33

12 11 5 5 5 5 11 14 6 5 5 3 19

104.5 ± 18.7 127.7 ± 12.9 102.2 ± 14.4 115.9 ± 26.0 106.2 ± 23.2 108.2 ± 23.2 120.9 ± 12.9 144.3 ± 36.7 90.1 ± 15.9 129 ± 28.8 69.5 ± 19.1 70.7 ± 34.5 92.3 ± 17.8

138 152 128 146 129 132 153 209 121 190 105 133 135

73 106 41 55 29 58 100 62 37 36 23 17 72

5.5 6.9 4.7 6.9 5.6 5.7 6.1 4.1 6.0 5.9 4.4 3.8 3.5

0.826 0.540 0.786 0.896 0.914 0.913 0.498 0.947 0.843 0.539 0.831 0.966 0.281

Key: No. of M., number of measurements for C3 and C4 serum protein concentrations; max, maximum; min, minimum; [C3]/[C4], ratio of mean C3 to mean C4 serum protein concentrations. R, coefficient of correlation between serum C3 and C4 protein concentrations.

lation coefficient (R) between plasma C3 and plasma C4 levels was 0.127 with a p-value of 0.202 (Figure 2). This implied that no significant correlation between the protein concentrations of C4 and C3 was present in healthy subjects.

COMPLEMENT C4 PROFILES IN SLE

235

R = 0.127, p = 0.202 N = 102

Figure 2. A scattered plot of complement C3 concentrations against complement C4 concentrations in 102 healthy Caucasian females. EDTA-plasma samples were used for radial immunodiffusion assays to determine plasma C3 and plasma C4 concentrations. In our experience C4 protein levels in a subject measured by radial immunodiffusion assays of EDTA-plasma are 1.44 ± 0.20-fold higher than that measured by nephelometry of serum.

When the C3 and C4 protein profiles in pediatric SLE patients were analyzed individually, however, a very tight correlation between C3 and C4 protein levels in all but two of the patients emerged (Table 2). When the three groups of C4 profiles were analyzed for the correlation between their C3 and C4 levels, Pearson’s correlation coefficients for Group I, Group II, and Group III were 0.530 (number of measurements or n = 150), 0.530 (n = 391) and 0.749 (n = 358), respectively, and they were all at significant levels, with p < 0.01 (Figure 3). However, the above correlation produced by bivariate fit model did not take into account the multiple measurements from the same subjects. Therefore, linear regression analysis that treated subjects as random effects was employed to delineate the effects of multiple measurements and the effect of serum C4 level on serum C3 level. In addition, in Group I and Group II many patients exhibited incidences of serum C4 levels below the detection threshold by nephelometry, by which an arbitrary C4 level of 5 mg/dL was assigned to assist the correlation study (panels A and B, Figure 3). Such arbitrary assignment could skew the outcome of the data analysis toward a negative result for an association between C3 and C4. To circumvent these artifacts, we grouped the C3–C4 data sets into five categories (referred as C4 level groups in Figure 4) with reference to their C4 levels: ≤5 mg/dL, between 5.1 and 9.9 mg/dL, between 10 and 14.9 mg/dL, be2 tween 15 and 19.9 mg/dL, and ≥20 mg/dL. The R for the mixed effect

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Y.L. WU ET AL.

A.

G roup I

R = 0.5 .011 0 .5 30 3 0,, p < 00.0 N = 115 5 0, tie nnts ts 0 , 8 P aatie

B.

G roup II

R = 0.5 .011 0 .5 30 3 0,, p < 00.0 N = 339 9 1, tie n ts 1 , 1 2 P aatien

C.

G roup III

R = 0.7 .011 0 .7 49 4 9,, p < 00.0 N = 335 5 8, tie n ts 8 , 1 3 P aatien

Figure 3. Correlation of serum C3 levels with serum C4 levels in pediatric SLE. SLE patients were categorized into three groups according to their C4 serum protein profiles as shown in Figure 1. Scatterplots of serum C3 levels versus serum C4 levels are shown. Panel A is from Group I patients who have persistently low serum C4 levels (mean serum C4 concentration <10 mg/dL). Panel B is from Group II patients who have serum C4 levels fluctuating above and below 10 mg/dL (mean serum C4 concentrations = 10–15 mg/dL). Panel C is from Group III patients who have serum C4 levels within the normal range most of the time. Serum C4 levels below the detection threshold by nephelometry were assigned 5 mg/dL.

COMPLEMENT C4 PROFILES IN SLE

237

Least Square Mean of Serum C3 Conc. (mg/dL)

regression model that took into account the association contributed by multiple measurements from the same subjects explained 73.0% of the variability in C3 (Figure 4). It showed that at the higher C4 level group, the mean of C3 was significantly higher (p < 0.0001). The Tukey Honestly-Significant Difference (HSD) test was further applied to determine how distinct the least-squares mean C3 levels were among different C4 level groups. At the stringent condition with alpha equal to 0.05, all pairwise comparisons taking the least-squares mean C3 from any two C4 level groups showed that all 10 differences were significant. In other words, all five groups were distinctly different, and this suggested that the correlation between C3 and C4 levels were tight enough such that no overlap was present between groups.

140 120 100 80 60 40

R2=0.730 p<0.0001

20 0 0

≤15

(5,9.9) 2

(10,314.9) (15, 4 20)

≥520

6

C4 Level Groups

Figure 4. Least-squares mean plot of the serum C3 concentrations with respect to different C4 level groups. 899 sets of serum C4–C3 concentration data were separated into five different groups with reference to their serum C4 protein levels (referred as C4 level groups): ”5 mg/dL, between 5.1 and 9.9 mg/dL, between 10 and 14.9 mg/dL, between 15 and 19.9 mg/dL, and •20 mg/dL. The leastsquares mean of C3 concentration along with its standard error were calculated for each C4 level group. Linear regression analysis gives an R2 of 0.730 for the relationship between C4 level groups and least-squares mean of C3 with C4 level group as a significant predictor of variability in C3 concentration with a p value less than 0.0001.

Further correlation analyses were performed between the mean concentrations of C3 and C4, and between the maximum concentrations of C3 and C4, of the thirty-three patients. A correlation coefficient of 0.629 (p < 0.0001) was obtained for the mean C3 and C4 concentrations, and of 0.729 (p < 0.0001) for the maximum C3 and C4 concentrations. Thus, serum complement C3 protein levels in pediatric SLE was tightly associated with serum complement C4 protein levels, irrespective of analysis

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methods in individual patients, in groups according to the serum C4 protein profiles, and in the entire pediatric SLE patient cohort. 4.4. Serum C3 and C4 Levels Both Correlated with BMI but BMI Alone Could Not Account for the Tight Association between Serum C3 and C4 Levels We previously reported a positive correlation between BMI and serum C3 protein concentrations, and between BMI and serum C4 protein concentrations in 25 healthy Hungarian subjects . In 102 healthy female Caucasian subjects from Ohio, we also observed a positive correlation between BMI and plasma C3 protein levels, and between BMI and plasma C4 protein levels. The correlation coefficient (R) between BMI and plasma C4 level was 0.270 (p = 0.0016); the R value between BMI and plasma C3 level was 0.375 (p < 0.0001). For the pediatric SLE cohort studied here, linear regression analyses taking into account the multiple measurements available for each patient were applied to assess the relationship between the two complement protein levels with BMI. In this analysis, both serum C3 and C4 levels were found correlated with BMI. Per unit of BMI changes, the ANOVA model predicted 0.437 unit change in serum C4 level, and 1.937 units change in serum C3 level. The model produced 2 2 an R of 0.648 for the analysis of C4 level with BMI, and an R of 0.632 for the analysis of C3 level with BMI, by which the p values were less than 0.0001 in both analyses (Table 3). Table 3: Correlations between BMI and C3, and between BMI and C4 Protein Levels in Healthy Subjects and Pediatric SLE

BMI vs C4

Healthy subjects (N = 102)

Pediatric SLE (N = 422)

R = 0.270

R2 = 0.632

p = 0.0016

Slope = 0.437 p < 0.0001

BMI vs C3

R = 0.375

R2 = 0.648

p < 0.0001

Slope = 1.938 p < 0.0001

N, number of data points.

To address whether the common association with BMI could explain the tight correlation between C3 and C4 levels in SLE, we added BMI as a factor in linear regression analyses on the relationship between C3 and C4 levels. If C4 protein levels were “correlated” with C3 protein levels only through their association with BMI, by including BMI in this analysis model, the contribution of C4 levels in the relationship would no longer be significant. Yet, the results of

COMPLEMENT C4 PROFILES IN SLE

239

regression analyses showed that both BMI and C4 levels were significant factors in predicting C3 levels in SLE, with a p value of 0.0018 for BMI and a p value 2 less than 0.0001 for C4 levels. The R in this ANOVA model is 0.765, meaning that the quality of this ANOVA model adjusting multiple measurements of BMI and C4 levels from all 33 subjects account for 76.5% of the variability in C3 levels. Therefore, there was a significant association between serum C3 and C4 levels in SLE, and this association was not through their common associations with BMI. 4.5. C4 Genotypic and Phenotypic Variations in Pediatric SLE The C4 genotypic and phenotypic variations among the 33 patients and 35 firstdegree relatives were determined. Common mutations in complement components C4 and C2 genes were also investigated. Figure 5 illustrates such analyses to determine the C4 genotypes and phenotypes in the 3 patients with low, medium, and high mean C4 protein levels, respectively, together with their firstdegree relatives. TaqI RFLP revealed that patient P23 had two C4 genes with monomodular-long (L) and monomodular-short (S) (i.e., L/S) RCCX modules (panel A). Results of the PshAI-PvuII RFLP revealed equal band intensities of C4A and C4B genes and therefore the presence of one C4A and one C4B (panel B). C4 allotyping by immunofixation revealed that these two C4 genes were coding for a C4A3 and a C4B1 protein (Panel C). Such interpretations were corroborated by results of the father. The father F23 had three C4 genes arranged in the LS/L RCCX configuration. The band intensities of C4A and C4B genes suggested that, while one gene was coding for C4B protein, two were coding for the C4A proteins. Furthermore, C4 allotyping revealed that F23 had three C4 protein variants: C4B1, C4A3 and C4A6. Together the results inferred that the monomodular-long C4 gene coding for C4A3 protein was passed on from the father to the patient, and the other monomodular-short C4 gene coding for C4B1 must be from the mother. Similarly, patient P10 was found to have four functional C4 genes organized in LL/LS that consisted of two C4A genes and two C4B genes. Both C4A genes coded for C4A3, while the two C4B genes coded for C4B1. The mother, M10, had four C4 genes arranged in an LL/LL RCCX configuration. Among them, three C4 genes were C4A and one was C4B. The father, F10, had three C4 genes with an LS/S RCCX configuration that code for C4A3-C4B1 and C4B1. Together, one maternal chromosome 6 with an LL RCCX coding for C4A3C4B1 and the paternal chromosome 6 with an LS RCCX also coding for C4A3C4B1 were transmitted to the patient. In family 16, patient P16 had two C4A genes and two C4B genes arranged in an LS/LS configuration. The C4A genes coded for the C4A3 proteins, while the C4B genes coded for the C4B2 proteins. The mother, M16, had the same RCCX configuration and also two C4A and two C4B genes. However, while the LS on Chromosome 6 were coding for C4A3-C4B2, the LS on the other

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Y.L. WU ET AL.

A: Taq I RFLP RP1-C4L 7.0 kb

P23 F23

P10 M10 F10

P16

M16

RP1-C4S 6.4 kb RP2-C4L 6.0 kb RP2-C4S 5.4 kb

CYP21B 3.7 kb CYP21A 3.2 kb

TNXB 2.5 kb TNXA 2.4 kb

5&&;

L/ S

LS/ L

LL/ LS

LL/ LL

LS/ S

LS/ LS

LS/ LS

2:2

3:1

1:2

2:2

2:2

B: Psh AI- Pvu II RFLP C4B 2.2 kb C4A 1.7 kb C4A:C4B

1:1

2:1

C: C4 Allotyping P10 M10 F10

P23 F23

P16 M16

B1

B1

A3 A6

A3

B2 B5 A3

Figure 5. Genotyping (A and B) and phenotyping (C) analyses of human C4A and C4B in pediatric SLE families. Results from families of the three patients illustrated in Figure 1 are shown. In each panel P stands for the patient, M stands for the mother, and F for the father. Panel A shows genomic TaqI restriction fragment length (RFLP) analysis of the RP-C4-CYP21-TNX (RCCX) modules, which yields data on the number and size of C4 genes. Panel B shows genomic PshAI-PvuII RFLP that yields information on the ratios of C4A to C4B genes in each subject. Panel C shows immunofixation of EDTA-plasma to determine the polymorphism of C4A and C4B proteins.

COMPLEMENT C4 PROFILES IN SLE

241

Chromosome 6 were coding for C4A3-C4B5, and it was the C4A3–C4B2 chromosome that was transmitted to the patient. 4.6. C4 Gene Dosage Is a Determinant of the Maximum Serum C4 Concentrations in Pediatric SLE The total C4, C4A, and C4B gene dosage variations of the SLE patients are listed in Table 4. The copy number of total C4 genes (i.e., C4A and C4B) varied from 2 to 6 among the patients. The C4A and C4B gene copy numbers from the patient cohort ranged from 0 to 4, and from 1 to 3, respectively. Approximately 40% of the SLE patients each had two copies of C4A genes plus two copies of C4B genes in a diploid genome; the other 60% of patients had unequal numbers of C4A and C4B genes. Of the 20 Caucasian patients, 9 (45 %) had two or three structural/functional C4 genes, 10 (50%) had four C4 genes, and one patient had five C4 genes. Homozygous or heterozygous deficiencies of C4A were present in 35% of the Caucasian patients. Six out of seven cases of C4A deficiencies were due to the absence of a C4A gene in an MHC haplotype; the other had homozygous mutations in exon 29 of the C4A gene (i.e., 2-bp insertion at codon 1213; patient no. 12). In addition, two patients (nos. 16 and 20) were found to have the 28-bp deletion at the junction between exon 6 and intron 6 of the C2 23 genes . The relatively small sample size of the pediatric SLE patients did not allow a meaningful statistical analysis of C4 gene dosage variation as a risk factor in this SLE cohort. To assess the effects of C4 gene dosage on C4 and C3 protein levels, we chose to determine whether there was an association between the C4 gene dosage and the maximum serum C4 and serum C3 protein levels in the patients during the course of the study. It is reasoned that the maximum C4 and C3 levels in a patient would reflect the intrinsic power or innate capability to synthesize C4 or C3 proteins. The Pearson correlation coefficient between C4 gene dosages and maximum concentrations of serum C4 was 0.424 (p = 0.014). On the contrary, no significant correlation between C4 gene dosages and maximum concentrations of serum C3 was found (R = 0.095, p = 0.601). The mean maximum serum C4 and C3 levels among the patients with two or three C4 genes were 19.2 ± 5.4 and 125.6 ± 24.3 mg/dL, respectively (Table 2). The mean maximum serum C4 and C3 levels among the patients with four C4 genes were 26 ± 10.0 and 132.4 ± 30.4 mg/dL, respectively. The mean maximum serum C4 and C3 levels among the patients with five or six C4 genes were 28.3 ± 7.1 and 116 ± 20.0 mg/dL, respectively.

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Table 4. Genotypic and Phenotypic Variations of Complement in Pediatric SLE Patient No.

ACR criteria

RCCX Hap 1*

RCCX Hap 2

C4 Gene

C4A Gene

C4B Gene

C4A allotype

C4B allotype

A3A3 A3 A3A3 AQ0AQ0 A3A3 A3 A3A3 A3A3

B1 B1B1 B1B1 B2B2 B1B1 B1 B1B2 B1B1

Group I 6 7 11 12 14 23 27 29

4 6 4 4 4 4 5 6

LL LL LL LS LS L LL LL

L S LL LS LS S LS LL

3 3 4 2 4 2 4 4

2 1 2 0 2 1 2 2

1 2 2 2 2 1 2 2

5 5 5 5 7 5 7 5 8 4 5 6

L LLLL LS LS LL LL LS LL LL LL LS LSS

LL LS LLL S LS S L S L LS LS S

3 6 5 3 4 3 3 3 3 4 4 4

2 4 3 1 2 1 2 1 2 2 3 1

1 2 2 2 2 2 1 2 1 2 1 3

A3A3 B1 A3A3A3A3 B1B2 A3A3A3 B1B2 A3 B1B2 A3A3 B1B1 A3 B1B1 A3A3 B1 A3 B1B1 A3A3 B1 A3A3 B1B2 A3A12A91 B1 A3 B1B2B2

4 6 5 5 5 5 4 4 6 5 5 7 5

LL LL LL LL LL LS LS LS LSL LS LS LS LS

LS S LL LL S LS LS LS S LS LSS LS LSL

4 3 4 4 3 4 4 4 4 4 5 4 5

3 1 2 2 1 2 2 3 2 2 2 2 2

1 2 2 2 2 2 2 1 2 2 3 2 3

A3A3A3 A3 A3A3 A3A3 A3 A3A3 A3A3 A3A12A91 A3A3 A6A3 A3A3 A3A3 A2A3

Group II 2 4 8 9 10 17 18 19 22 26 30 33 Group III 1 3 5 13 15 16 20 21 24 25 28 31 32

B1 B1B1 B1B2 B1B2 B1B1 B2B2 B2B2 B1 B1B1 B1B21 B1B1B1 B1B3 B1B1B2

* RCCX hap1, RCCX haplotype 1; RCCX hap 2, RCCX haplotype 2.

5. DISCUSSION After analyzing serial protein profiles of serum complement C4 and C3 from thirty-three pediatric SLE patients, we observed three major groups of serum C4

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concentration variations. Group I was characterized by persistently low C4 levels (<10 mg/dL) throughout the course of the study but with relatively mild manifestation of disease activities. On average, the serum complement C3 concentrations were 11.1 times greater than those of C4. Group I patients may have 2, 3, or 4 copies of C4 genes in a diploid genome. Group II was characterized by periodic fluctuations of serum C4 protein concentrations above and below 10 mg/dL. These patients tended to have a more variable disease course, with fluctuations in lupus activity. The mean C4 protein levels were between 10 and 15 mg/dL. The serum C3 protein levels were about 7.5 times greater than those of C4. Most Group II patients had an unequal copy number of C4A and C4B genes, and about half of the patients had low C4 gene dosages (three copies of C4 genes in a diploid genome). All except one patient in this group satisfied five or more ACR diagnostic criteria for SLE. Group III patients had relatively high serum C4 levels and a wide spectrum of disease severity. The occasionally low C4 and C3 levels in this group of patients were usually coincident with disease flares. The C3 protein levels were about 5.3 times greater than that of C4. Most patients in this group had four or five copies of C4 genes in a diploid genome. Complement C3 and C4 protein levels are used by rheumatologists as a means to monitor SLE disease activity and as biomarkers for response to medi26-31 cal treatment . C3 and C4 levels within the normal range are suggestive of controlled disease activity. While patients from all C4 gene dosage groups could experience very low serum C4 and C3 levels during their disease courses (especially during disease flares), the patients' capability to respond to medication to normalize their C4 levels may be dependent upon their inborn genetic factors. For example, our data suggested that the maximum serum protein concentration of complement C4 present in a patient is directly related to her/his total C4 gene dosages. Therefore, classifying C4 and C3 protein profiles and elucidating the genotypic and phenotypic variations of C4 may improve prediction of SLE disease flares and severity. This is a new and testable hypothesis for further research. The parallel fluctuations of serum C3 and C4 protein concentrations had 14 been noticed by investigators in lupus nephritis patients . In the current study tight associations of serum C3 and C4 protein concentrations were observed in 31 out of 33 pediatric SLE patients. From 899 sets of C3 and C4 datapoints, a 2 correlation coefficient of 0.854 was obtained (R = 0.730, p < 0.0001). Interestingly, such an association between C3 and C4 protein levels is not obvious in healthy subjects. What underlies the coupled fluctuations of C3 and C4 protein levels in SLE is probably a powerful mechanism that drives the ups and downs of serum C3 and C4 protein levels systemically. Given the evidence that incidences of disease flares often correlate with the dips of both proteins in time, it is plausible that C3 and C4 protein consumption as a result of complement activation may be the responsible driving force. This is probably intuitive, considering that the activation of C3 is downstream of C4 in the antigen–antibodymediated classical pathway of complement activation. An alternative but not

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mutually exclusive explanation is the presence of a disease-specific regulatory mechanism that tied the biosynthesis and catabolism of these two serum proteins together. In this study we extended the previous observation that serum or plasma complement C3 and C4 protein concentrations are associated with an individ25 ual's BMI . Strong associations between BMI and C3, and between BMI and C4, were observed in both healthy Caucasian subjects and pediatric SLE patients from multiple ethnic groups. This phenomenon underscores the relevance of adipose tissues as an extrahepatic site for the biosynthesis of complement C3 32 and C4 . It is worthwhile to point out that the parallel fluctuations of serum C3 and C4 in SLE is independent of their concurrent associations with BMI, or the association of serum C4 with C4 gene dosages. Our earlier analyses of physical parameters determining C4 and C3 concentrations in the pediatric SLE patients also suggested that the age of a subject could be a contributing factor. The relationship between age and serum C4 pro33-35 tein levels had also been observed by other investigators . However, our linear regression analyses revealed that when BMI was included in the association analysis the age factor became irrelevant. One likely explanation is that the influence of age on serum complement C4 levels was probably exerted through changes of BMI with age. In conclusion, the serum complement C3 and C4 levels in an SLE patient at a specific time appear to reflect the interplay among SLE disease activity, the effects of medication and patient compliance to treatment, the effects of genetic factors (including the patient's total C4, C4A, and C4B gene dosages), and the patient's physical parameters such as the body mass index. While not studied here, other environmental factors such as stress or infections may also be in36 volved .

6. ACKNOWLEDGMENTS We thank the SLE patients and family members and healthy subjects for the blood donation, Karla Jones for help in the recruitment of healthy subjects, and Denise Yu for technical assistance. This work is supported by PHS grants R01 AR050078 from NIAMS and P01 DK55546 from NIDDK.

7. REFERENCES 1. 2. 3.

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17 A MINIMUM CR2 BINDING DOMAIN OF C3D ENHANCES IMMUNITY FOLLOWING VACCINATION Joseph F. Bower and Ted M. Ross

1. ABSTRACT The degradation product of the third (C3) complement component, C3d, links innate and adaptive immunity, and the covalent attachment of C3d to an antigen enhances antigen-specific immune responses. C3d has been hypothesized to enhance immunity by direct interaction with complement receptor 2 (CR2/CD21) on immune cells. However, the domains on C3d important for CR2 binding have been controversial, with various studies reaching contradictory conclusions. In addition, the concept of B-cell activation via CR2 by C3d has been questioned, since mice lacking CR2 still elicit C3d-enhanced immunity following vaccination. Therefore, the goal of this study was to determine if a peptide representing one of the proposed CR2 binding domains of C3d could substitute for the entire protein and enhance antigen-specific immunity. Mice (BALB/c) were vaccinated with the HIV-1 gp120 envelope glycoprotein (Envgp120) alone or fused to multiple copies of the murine C3d or a twenty-eight amino-acid peptide (P28) containing a minimum CR2 binding domain. Each immunogen was expressed from DNA plasmid in vivo or injected as purified recombinant protein. The fusion of the P28 peptide to Envgp120 enhanced both humoral and cell-mediated immune responses with similar efficiency as Envgp120 conjugated to C3d. The fusion of C3d or P28 to Envgp120 elicited higher-titer anti-Env specific antibody, enhanced avidity maturation of the elicited antibody, and elicited higher numbers of IFN-γ and IL-4 secreting cells compared to Envgp120 immunizations. This CR2-binding domain specific 28 amino acid peptide can substitute for the entire C3d molecule and enhance immunity. These results indicate that the adjuvant properties of C3d are associated with CR2 interaction.

Joseph F. Bower and Ted M. Ross,* Department of Medicine, Division of Infectious Diseases, University of Pittsburgh, School of Medicine, Pittsburgh, PA, 15261, USA. *Corresponding author.

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2. INTRODUCTION The third complement component, C3, enhances adaptive immune responses to foreign antigens. During the complement cascade, several components of C3 are generated (for reviews please see [1–5]). One of these degradation products, C3d, as part of the innate immune response, stimulates a variety of immune cells, but in particular B lymphocytes and follicular dendritic cells (FDC) via the binding of C3d coated antigens to the cell-surface complement receptor 2 (CR2, also known as CD21) in splenic germinal center follicles. CR2 belongs to the 6 regulators of complement activation family of proteins that is expressed on B cells, follicular dendritic cells, epithelial cells, and a subset of T cells. This receptor has 15 or 16 short consensus repeat (SCR) domains depending on the 7 splice site usage . CR2 is involved in B-cell activation, generation of immunologic memory, immunoglobulin class switching, and B-cell tolerance. During complement activation, antigens are coated with C3d or iC3b, and are directed 8-10 to immune cells, resulting in stimulation of these cells via CR2 . Our laboratory, as well as others, has demonstrated that coupling of an antigen to multiple copies of C3d enhances T-dependent B-cell responses following vaccination 11-23 with either purified proteins or DNA plasmids . This immune enhancement is hypothesized to be a result of enhanced retention of antigen by follicular dendritic cells in germinal centers and enhanced recruitment of the 8-10 CR2/CD19/CD81 coreceptors into the B-cell receptor complex . The number of CR2 binding sites on C3d is controversial. One early study indicated that the CR2 binding site on C3d mapped to residues 1205–1214 (C3 24 sequence) , and this minimum binding domain, contained within a 28-amino acid peptide (P28), activated B cells, albeit with less efficiency than the entire 24-26 C3d molecule . The lower binding affinity of this peptide may be one explana25,26 tion for the necessity of high-affinity ionic interactions between C3d to CR2 . Mutagenesis analysis of this region appeared to indicate that this region was not 27 of importance for CR2 binding and that an acidic pocket was most likely the 28 CR2 binding domain on C3d . However, kinetic binding studies once again indicated that this P28 segment directly interacted with CR2 and was involved in C3d binding to CR2. Despite the lower affinity of P28 for CR2, conjugation of multiple copies of the P28 peptide to the highly immunogenic molecule — hepa29 titis B surface antigen (HBsAg) — enhanced anti-HBsAg antibodies . Therefore, in this study we directly compared the elicitation of humoral and cellular immune responses by a C3d conjugated antigen to a P28 peptide conjugated to a poorly immunogenic antigen expressed from a DNA vaccine or as purified proteins. Multimers of each adjuvant were conjugated to the well characterized 11,12,20,30-32 HIV-1 Envgp120 . When expressed from a DNA plasmid in vivo, Envgp120 elicits little or no anti-Env immunity. Regardless if Envgp120 was conjugated to C3d or P28, or the route of inoculation, vaccinated mice had similar anti-Env humoral and cell-mediated immune responses.

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3. MATERIALS AND METHODS 3.1. Plasmid DNA 12,33

Eukaryotic expression vector pTR600 has been described . Plasmids were constructed to express a soluble form of HIV-1 envelope gp120 (Envgp120) using a 11,12,20,30-32 wild-type gene sequence from HIV-1 isolate ADA . The first 32 amino acids were deleted from the N terminus of gp120 and replaced with a leader sequence from the trypsin plasminogen activator (tpA). In addition, mC3d3 was fused to the C terminus of the gp120 molecule (gp120-mC3d3). Four copies of the synthetic peptide corresponding to the CR2 binding domain of C3d, P28, from amino acids 1187–1214 (KFLTTAKDKNRWEDPGKQLYNVEATSYA) was fused to the 3′ of gp120 through (G4S2)2 linker domains (gp120–P284). Each DNA vaccine was designated for the gene insert that the plasmid expressed (Figure 1). gp160ADA

gp120

gp41

gp120ADA

gp120 gp120ADA-P284

gp120 gp120ADA-mC3d3

gp120 Figure 1. Schematic of the each gene insert representing gp120ADA,: gp120ADA–P284 and gp120ADA– mC3d3. Each gene insert was cloned into pTR60012,33.

Each plasmid was amplified in Escherichia coli strain-DH5α, purified employing anion-exchange resin columns (Qiagen, Valencia, CA) and stored at – 20°C in dH20. Plasmids were verified by appropriate restriction enzyme digestion and gel electrophoresis. Purity of DNA preparations was determined by optical density reading at 260 nm and 280 nm. Each DNA vaccine inoculation contained <50 fg/µg of endotoxin per DNA inoculation. 3.2. Purification of Recombinant Protein Antigens Recombinant proteins (gp120, gp120-P284 and gp120-mC3d3) were purified from the supernatants of 293T cell cultures transiently transfected with each 6 vaccine plasmid. Flasks (T-75) of cells (6 x 10 ), maintained in DMEM media supplemented with 10% fetal bovine serum (FBS), were transfected with DNA

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plasmid expressing each plasmid (5 µg) using lipofectamine reagent (Invitrogen, 10 Carlsbad, CA). Supernatants from T-75 flasks were pooled (100 ml) and centrifuged (2500 rpm) to remove any cellular debris. The clarified supernatants were loaded onto an agarose Galanthus Nivalis lectin column (Vector Laboratories, Burlingame, CA) (5-ml bed volume), which had been pre-eluted with 5 column volumes of elution buffer (Mes Buffer (0.02 M Mes, 0.13 M NaCl, 0.01 M CaCl2) + 1.25 M mannopyranoside). The column was washed with 10 column volumes of Mes wash buffer or until O.D. reached background. The column was eluted with 5 column volumes, collected in 5 ml fractions, of which >90% of the eluted envelope protein was collected in the second and third fractions. Proteins were concentrated to ~1 µg/µl using a 10,000 MWCO Amicon (Millipore, Billerica, MA). The purity of the eluted proteins was verified by silver stain (data not shown) and the concentration was determined by BCA analysis (Pierce, Rockford, IL). 3.3. Protein Expression Protein expression of each DNA vaccine plasmid was determined by Western 30 blot analysis as described previously . Supernatant (1.5%) and lysates (3.0%) were resolved on a 7% polyacrylamide/SDS gel and transferred onto a PVDF membrane (Sigma, Saint Louis, MO). The membrane was incubated with HIVIg (1:5000 dilution in PBS-T for 1 h at 25ºC). Following extensive washing with PBS-T, human antibodies bound to the membrane were detected using horseradish peroxidase-conjugated goat anti-human antiserum (Bio-Rad, Hercules, CA) (1:8000 dilution in PBS-T for 1 h at 25ºC). Antibody complexes bound to the membrane were detected using enhanced chemiluminescence (Pierce Biotechnology Inc., Rockford, IL). 3.4. Animals and Immunizations Six- to 8-week-old BALB/c mice (Harlan Sprague Dawley, Indianapolis, IN) were used for inoculations. Mice, housed with free access to food and water, were cared for under USDA guidelines for laboratory animals. Mice were anesthetized with 0.03–0.04 ml of a mixture of 5-ml ketamine HCl (100 mg/ml) and 1-ml xylazine (20 mg/ml). For DNA vaccinations, mice were inoculated in one of three routes: intradermal, intramuscular, or intranasal. Intradermal gene gun immunizations were performed on shaved abdominal skin using the hand-held 12,13,17,19,20,33 gene delivery system (Bio-Rad, Hercules, CA) as described previously . Mice were immunized with two gene gun doses containing 1.0 µg per 0.5 mg of approximately 1-µm gold beads (Bio-Rad, Hercules, CA) at a helium pressure setting of 400 psi. For intramuscular inoculations, mice were injected in the left and right quadriceps muscles with a total of 50 µg (25 µg in each leg) of purified plasmid DNA suspended in 0.9% saline.

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Some groups of mice were inoculated with purified proteins via intramuscular injection or intranasal instillation. Mice were vaccinated with 10 µg of purified recombinant protein suspended in 0.9% saline into the quadriceps muscles. Mice were vaccinated with 10 µg of protein in a total of 20 µl of 0.9% saline into each nare using a Pipetman for intranasal inoculation and in a total volume of 50 µl intramuscularly. Protein concentration was determined based upon the gp120 portion of each protein. 3.5. ELISA An endpoint ELISA was performed to assess the titers of anti-Env, as de30 scribed . Briefly, plates were coated with gp120 (25 ng/well) overnight at 4ºC. Each plate was blocked with non-fat dry milk (5%) in PBS-T (1 h at 25ºC) and then extensively washed with PBS-T. Serial dilutions of antisera from vaccinated mice were allowed to bind (1 h) to coated plates. Subsequently, the primary antisera were detected by anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA). Following thorough washing, antibody complexes were incubated (1 h at 25ºC) with TMB substrate (Sigma, Saint Louis, MO), and the optical density (O.D.) of each well was determined by spectrophotometry. Endpoint dilution titers from vaccinated mice that were twofold higher than sera from mice vaccinated with vector only were considered positive. To determine the affinity of the raised antibody, a particle disruption ELISA 12,13,17,30 using the chaotropic agent sodium thiocyanate (NaSCN) was employed .A constant dilution of sera from vaccinated mice was incubated in the wells of a 96-well plate (1 h). Each sample was normalized to total anti-Env IgG concentration prior to performing the assay. Varying concentrations of NaSCN (0–3.5 M) in PBS were incubated with mouse antisera (30 min). Plates were washed, and bound antibody was detected by TMB substrate (Sigma, Saint Louis, MO). Specific IgG binding in the presence of NaSCN was recorded as a percent of total IgG bound in the absence of NaSCN. 3.6. ELISpot Splenocytes from immunized mice were collected (week 10) and resuspended (5 million cells/ml) in RPMI-1640 supplemented with 10% FCS. Each well of a 96-well PVDF-coated plate (Millipore, Billerica, MA) was coated with antibodies that detected the anti-mouse cytokines IFN-γ (10 µg/ml) or IL-4 (4 µg/ml) (mAb R4-6A2, mAb BVD4-1D11 , Pharmingen, San Diego, CA). Plates were blocked with complete RPMI (2 h at RT) and then washed with PBS-T (3X). Splenocytes were added to each well (500,000 cells/well) and were stimulated (40 h at 37ºC) with peptides. The C1 pool contained ten overlapping (15 amino acids/peptide) peptides representing a T-helper epitope in the first constant re34 gion of gp120MN (amino acids 40–60) at 2 µg per peptide . The V3 loop pool

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contained two peptides (15 amino acids/peptide) representing the V3 loop of the consensus Clade B and B-subtype, which matches the HIV-1YU2ADA V3 loop sequence. Plates were washed with PBS-T (3X) and incubated (4°C overnight) with biotinylated anti-mouse IFN-γ (2 µg/ml) or anti-mouse IL-4 (2 µg/ml)(mAb XMG1.2, mAb BVD6-24G2; Pharmingen). Following thorough washing, plates were incubated with peroxidase-labeled avidin (Sigma, St. Louis, MO) in PBS (1/1000 dilution). Wells were washed with PBS-T and spots were developed by addition of the substrate 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in Tris–HCl (50 mM, pH 7.5) containing hydrogen peroxide (0.015%). Spots were counted with the aid of a stereomicroscope. 3.7. Statistic Analysis For statistical analysis, a student’s t test was employed. The difference between DNA vaccines expressing Env fused to multiple copies of mC3d or P28 were compared to DNA expressing Env alone. Differences were considered statistically significant when the p value was less than 0.05.

1

2

3

4

5

6

7

250 kDa gp120-mC3d3 150 kDa

gp120-P284 gp120

100 kDa

Figure 2. Expression of DNA vaccine inserts. Supernatants (lanes 1–4) and lysates (lanes 5–7) from transiently transfected 293T cells were resolved by SDS-PAGE. Lane 1: gp120ADA; lane 2: gp120ADA– P284; lane 3: gp120ADA–mC3d3; lane 4: mock; lane 5: gp120ADA; lane 6: gp120ADA–P284; lane 7: gp120ADA–mC3d3.

4. RESULTS 4.1. Expression of Vaccine Plasmids Each vaccine plasmid efficiently secreted protein from 293T cells transiently transfected with DNA plasmids (Figure 2). Greater than 95% of the protein was secreted from cells, and only minimal amounts of each protein were retained in 12,19 the cell lysate. As previously described , the addition of 3 copies of mC3d to Envgp120 decreases the overall expression by approximately 3-fold, due to an in-

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crease in molecular weight of 90 kDa. The addition of 4 copies of the P28 peptide added an additional 16kDa in the fusion protein, and the level of protein expression was therefore similar to Envgp120. 4.2. Cell-Mediated Immune Responses Elicited by Env-mC3d3 Mice (BALB/c) were vaccinated with DNA plasmids intradermally via gene gun or by intramuscular injection. Splenocytes were isolated from mice vaccinated with each of the vaccine plasmids, and regardless of the DNA vaccination regimen, splenocytes from mice vaccinated with gp120–mC3d3–DNA had higher numbers of IL-4-secreting cells following stimulation with peptides specific for MHC class II (C1 pool) than mice vaccinated with gp120-DNA alone (Figure 3A). Similar results were observed using recombinant gp120 stimulation. In addition, splenocytes from mice vaccinated with gp120–P284–DNA had statistically similar numbers of IL-4 secreting cells as mice vaccinated with gp120– mC3d3–DNA following peptide stimulation.

400 IL-4 gp120 350 gp120-P284 300 gp120-mC3d3 250 200 ** 150 * * 100 50 0 C1 Pool Env Intradermal

**

B. * *

Spot Forming Colonies

Spot Forming Colonies

A.

50 INF-γ

* *

40 30 20

**

10 0

C1 Pool

Env

Intramuscular

V3 Pool

Env

Intradermal

V3 Pool

Env

Intramuscular

Figure 3. ELISPots were performed on isolated splenocytes from vaccinated mice. (A) The number of IL-4 spot-forming colonies (SFCs). (B) The number of IFN-Ȗ SFC. *p < 0.05 comparing the number of SFCs in mice vaccinated with gp120-P284 or gp120–mC3d3 and gp120.

Interestingly, these same mice had fewer numbers of IFN-γ secreting cells than IL-4-secreting cells (Figure 3B), regardless of the vaccination regimen. As observed for IL-4 secretion, splenocytes from mice vaccinated with gp120– mC3d3–DNA elicited higher numbers of IFN-γ-secreting cells than mice vaccinated with gp120 alone following stimulation with MHC class I peptides (V3 loop). Once again, gp120–P284–DNA vaccinated mice had similar numbers of INF-γ-secreting splenocytes as gp120–mC3d3–DNA-vaccinated mice. However, unlike IL-4 secretion, whole Envgp120 protein stimulation elicited similar numbers of INF-γ-secreting splenocytes, regardless of the immunogen used for vaccination. Overall, there was little difference in the effectiveness of P28 to stimulate enhanced anti-Env-specific cellular responses compared to C3d.

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4.3. Anti-Env Antibody Responses 11,12,17,20,30-32

, mice vaccinated Consistent with previous studies from our laboratory with gp120–mC3d3–DNA elicited significantly higher titers of anti-Env-specific antibody compared to mice vaccinated with gp120–DNA (p > 0.05) (Figure 4). C3d enhanced anti-Env-specific antibodies, regardless of the route of vaccination. Mice vaccinated with gp120–P284–DNA elicited similar high anti-Envspecific antibody titers as mice vaccinated with gp120–mC3d3–DNA. B.

A.

gp120

100000

gp120-mC3d3 vector

Intradermal

10000

1000

100 0

2

4

6 Week

8

10 12

Endpoint Dilution Titer (1/log)

Endpoint Dilution Titer (1/log)

gp120-P284

100000

Intramuscular

10000

1000

100 0

2

4

6

8

10 12

Week

Figure 4. Anti-Env IgG titers following DNA vaccination. Mice (BALB/c) were vaccinated with DNA vaccines (arrow). Data represent the average of five mice. Pre-immune sera from mice had no detectable specific IgG. Endpoint dilution titers were conducted by diluting the sera until O.D. values reached background. (A) Intradermal inoculation via gene gun. (B) Intramuscular injection.

The avidity of elicited anti-Env antibodies was determined using a sodium thiocyanate (NaSCN) disruption ELISA. Antibodies with tighter binding to gp120 require higher concentrations of NaSCN to disrupt the specific antibody:antigen complex. Similar concentrations of NaSCN (1.5 M) were required to achieve 50% inhibition of gp120-specific antibody to the gp120 protein using antisera collected from mice vaccinated with either gp120–mC3d3– or gp120– P284–DNA, indicating similar levels of avidity for the antigen. Sera from mice vaccinated with gp120–DNA elicited antibodies with little avidity to gp120. Therefore, C3d and the P28 peptide enhance the avidity maturation of anti-Env antibodies with similar efficiency. 4.4. Mucosal Immunizations Previous studies have reported an ability of C3d-fused antigens to enhance mucosal immunity and then protect mice from viral challenge in the absence of an 18 exogenous adjuvant . To determine if P28 peptide could induce immunity following immunization of the mucosa with similar efficiency as C3d, mice were vaccinated with each recombinant protein intranasally. Total anti-Env IgG was

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Endpoint Dilution Titer (1/log)

detected in the serum in all vaccinated mice. However, mice vaccinated with purified gp120–mC3d3 had a sharper rise in anti-Env antibody and at week 9 there was a 7-fold increase in anti-Env serum antibodies compared to mice vaccinated with purified gp120 (Figure 5).

100000 Intranasal

gp120 gp120-P284 gp120-mC3d3 vector

10000

1000

100 0

2

4

6

8 10

Week

Figure 5. Anti-Env IgG serum titers following intranasal inoculation of purified proteins. Mice (BALB/c) were vaccinated with DNA vaccines (arrow). Data represent the average of five mice. Pre-immune sera from mice had no detectable specific IgG. Endpoint dilution titers were conducted by diluting the sera until O.D. values reached background.

Conjugation of P28 to gp120 was not as effective at enhancing serum antibodies as C3d (2.7-fold compared to gp120). A similar pattern of elicited antiEnv IgG antibody titers were detected in lungs of vaccinated mice (Table 1); however, no sIgA was detected. In contrast to intranasal vaccinations, both P28 and C3d conjugated gp120 proteins enhanced serum antibodies with equal efficiency following intramuscular injection (data not shown). There was little difference in the immune enhancement in P28-conjugated or C3d-conjugated Envgp120 mice following intramuscular injection at two doses (10 or 1 µg). Therefore, both P28 peptide and the whole C3d protein enhance mucosal immunity, but C3d appears more effective than P28 following intranasal vaccination.

Table 1. Anti-Env-Specific Antibody

Vaccine

Serum

Lung Wash

1600a

64

gp120-P284

4266

128

gp120-C3d3

11200

384

gp120

a

Anti-Env endpoint IgG dilution titer (1/log) at week 9.

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5. DISCUSSION In previous C3d–CR2 studies, CR2 binding has not been linked to the immune enhancement effects of C3d. In this study, in order to determine the specificity of the interaction between C3d and CR2, we directly assessed the adjuvant effect of a peptide representing the amino-acid sequence of one of the proposed CR2 binding sites on C3d (P28). The HIV-1 Envgp120 was conjugated to multimers of P28 and directly compared to elicitation of immune responses by Envgp120 conjugated to multimers of the full C3d protein. DNA vaccines expressing Envgp120 are poorly immunogenic when expressed from DNA, eliciting low 11,12,17,20,30-32 anti-Env immunity (Figures 3 and 4, as well as ); however, the HIV-1 envelope is highly immunogenic when presented to the immune system as a 30,35-48 purified protein . Fusion of multimers of the whole C3d or the P28 peptide to Envgp120 enhanced the level of immunogenicity, regardless of the route of inoculation of DNA. Fusions of C3d or P28 increased the number of cytokinesecreting cells compared to Envgp120 alone (Figure 3). In addition, both Envgp120– mC3d3–DNA and Envgp120–P284–DNA elicited high-titer anti-Env serum antibodies with higher avidity compared to Envgp120–DNA-vaccinated mice (Figure 4, Table 1). The route of inoculation (intradermal via gene gun or intramuscular injection) did not appear to affect the results. Interestingly, conjugation of C3d was better at eliciting enhanced immune responses compared to P28 conjugation following intranasal inoculation of purified proteins, which may be related to the fewer number of CR2-positive cells in the nasal mucosa and the lower binding efficiency of P28 to CR2 compared to C3d. The number of binding sites on C3d necessary for C3d–CR2 interaction has been controversial. Various studies have reached contradictory conclusions examining the same domains on C3d. Several lines of evidence indicate that this region is important in CR2 binding. One of the earliest studies mapped the CR2 binding site to a region on C3d (amino acids, 1199–1210 (mature C3 number24 ing)) using chemical fragmentation and peptide mapping . The same P28 pep24 tide used in this study binds to CR2 on Raji cells and stimulates B-cell prolif49-52 eration . In addition, this peptide is homologous in sequence to the CR2 53-58 binding region of the EBV surface glycoprotein gp350/220 , once again indicating that this sequence is critical for CR2 interaction. Soluble C3d or iC3b inhibits soluble CR2 binding to iC3b-coated erythrocytes, indicating that the 28 interaction between CR2 and C3 is located on C3d . Other reports have cast doubt on this region as a significant CR2 binding domain of C3d and suggest that an acidic pocket region, identified by x-ray 28 crystallography, is the true CR2 binding domain of C3d . Mutagenesis of the 27 P28 region results in only a marginal reduction in the binding of iC3b to CR2 . 59 Multiple sites on C3 may interact with CR2 , and both the P28 and the acidic pocket domains may cooperate in high-affinity binding of C3d to CR2. Recent studies identified differences in the kinetics of the interaction of CR2 with C3d 60 and iC3b in real time using surface plasmon resonance (SPR) . The binding of

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CR2 to the EBV gp350 follows a simple 1:1 binding model, but the binding kinetics of C3 proteins to CR2 are more complex, indicating that more than one 60 domain on C3d may be involved in CR2 binding . In addition, the synthetic P28 peptide bound CR2, once again demonstrating that this region on C3d is involved in receptor interaction, further supporting the hypothesis that P28 is an important determinant in CR2 interaction Despite the controversy of C3d binding sites and interaction with CR2, the role of these domains in the observed C3d adjuvanticity remain unclear. Linking the binding of CR2 to the adjuvant properties of C3d was first indicated when this binding region was conjugated to anti-idiotype antibody. Vaccination with 61 these conjugated proteins induced strong idiotype antibodies in mice . In this report, P28 not only enhanced antibody responses with similar efficiency as the entire C3d molecule, but also enhanced cellular immunity to the conjugated antigen, thereby linking this CR2 binding region to the adjuvanicity of C3d. Three major hypotheses have been proposed for the effects of C3d enhancement, two of which involve CR2 binding by C3d. (1) Direct stimulation of CR2 and the B-cell receptor complex resulting in proliferation of B lymphocytes. In this hypothesis, C3 molecules covalently bind foreign antigens to B cells via membrane-bound antibody and CR2, which subsequently forms an ac10 tivation complex with CD19 and the TAPA-1 co-receptor . Presentation of antigen to the activation complex most likely enhances the immune responses by lowering the threshold of signal transduction required to active B cells. (2) C3dcoated antigens are targeted to FDCs in the spleens, thereby enhancing their antigen uptake and presentation to effector immune cells. Alternatively, C3d enhances the immunogenicity of conjugated antigens by (3) extending the halflife of these antigens in vivo through a carrier effect. This minimal CR2-binding domain of C3d is as effective at enhancing immune responses, when coupled to a foreign antigen, as the full-length molecule. In addition, these results would argue against the hypothesis that the adjuvant effects associated with C3d are related to a carrier effect. The entire P284 molecule consists of 151 amino acids and is only 22% of the entire conjugated protein, and it is therefore unlikely that the half-life of the Envgp120–P284 protein would be significantly extended in vivo compared to Envgp120 only. The use of P28, which binds to CR2, would support the hypothesis of direct stimulation of B cells by C3d. However, we have reported that C3d enhanced immunity in 21 mice lacking CR2 , and we speculated that the adjuvant effect associated with C3d occurred via both CR2-dependent and CR2-independent mechanisms. Therefore, we have a conundrum, where two sets of independent data indicate that CR2 is and is not important in C3d enhancement, but the CR2-independent mechanism is not likely due to prolonged protein half-life. In addition, C3d immune enhancement has species limitations, which argues for receptor specificity, since no immune enhancement was observed in mice vaccinated with Envgp120–C3d3 or influenza HA–C3d3 DNA vaccines using human, rhesus, feline, or chicken C3d (unpublished observation). Also, the same Envgp120–mC3d3 DNA

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plasmids used in this study did not enhance anti-Env immunity in vaccinated rabbits, even though Env conjugated to the human or rhesus macaque C3d3 did 12 enhance immunity . Therefore, we hypothesize that the adjuvant effect observed with P28 is related to receptor binding and that more than one receptor may be responsible for the C3d/P28 immune enhancement and not an in vivo carrier effect.

6. ACKNOWLEDGMENTS This research was supported by National Institutes of Health Grants AI49061 to T.M.R. and AI57630 to J.F.B. The authors thank Douglas Fearon for supplying the murine C3d constructs. Also, we would like to thank Franklin Toapanta, Kelly Young, and Sean McBurney for technical assistance. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 MN peptides; HIV-Ig from NABI and NHLBI.

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18 STRUCTURE AND FUNCTION OF FICOLINS Yuichi Endo, Yu Liu, and Teizo Fujita

1. INTRODUCTION Lectins, carbohydrate-binding proteins, play an important role in innate immunity by recognizing a wide range of pathogens and by aggregating and opsonizing them. This recognition is mediated by a set of pattern-recognition receptors that recognize conserved pathogen-associated molecular patterns (PAMPs) shared by broad classes of microorganisms, thereby successfully discriminating pathogens and self. Among the animal lectins, mannose-binding lectin (MBLs) has a structure consisting a collagen-like domain and a carbohydrate recognition domain (CRD), and it has a unique ability to activate the complements through its association with novel C1s-like serine proteases, designated as MBL1 associated serine proteases (MASPs) . Upon binding of MBL to PAMPs on pathogens, the proenzyme forms of MASPs were converted to the active forms and in turn MASPs activated the complement. This activation pathway for the complement cascade is called “the lectin pathway” and has been verified to be 2 crucial in innate immunity defense . Ficolins, like MBLs, are a group of proteins that contain a collagen-like stem structure. Unlike MBLs, however, ficolin has a fibrinogen-like domain at the C-terminal. In 1993, ficolin was originally identified as a transforming 3 growth factor (TGF)-β1-binding protein on porcine uterus membranes . The physiological importance of the interaction of porcine ficolins with TGF-β remains unresolved. We isolated the first human ficolin, consisting of a 35-kDa subunit from plasma in 1996, which was temporarily designated P35 and after4 ward renamed L-ficolin (FCN-2) . Surprisingly, L-ficolin/P35 was a serum lectin with no CRD. A homology search demonstrated that the primary structure 5 of L-ficolin was identical to elastin-binding protein and corticosteroid-binding

Yuichi Endo, Yu Liu, Teizo Fujita, Department of Immunology, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1295, Japan.

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protein . We also isolated the second human ficolin, designated P35-related pro7 tein and renamed M-ficolin (FCN-1), which was 76% identical to L-ficolin at the primary structure level. The third ficolin, designated as Hakata antigen (Hficolin, FCN-3), was isolated by Sugimoto et al. in 1998 as a serum antigen recognized by an autoantibody present in patients with systemic lupus erythemato8 sus . So far, ficolins have been widely identified in vertebrates — including hu4,7-9 10,11 3 12 13 mans , rodents , pigs , hedgehogs , and Xenopus — and in invertebrates — 14 ascidians . Serum ficolins from humans, mice, pigs, Xenopus, and ascidians are lectins with a common binding specificity for N-acetylglucosamine (GlcNAc). 15 This lectin activity is executed through their fibrinogen-like domains . The functions of the fibrinogen-like domain seen in the proteins are not fully understood. However, accumulating evidence has shown that one of the important functions of the fibrinogen-like domain is to bind carbohydrates, as is seen in ficolins. Recently, we demonstrated that, like MBL, L- and H-ficolin complexed 16,17 with MASPs and activated the complement via the lectin pathway . In this review, we focus on the structure and function of ficolins, including our recent observations, and discuss their possible roles in innate immunity.

2. STRUCTURE OF FICOLIN Ficolin is a multimeric protein consisting of homogenous 34–40-kDa subunits. For example, human L-ficolin is a homooligomer (probably 12-mer) of 35 kDa subunits. Each subunit consists of a short N-terminal region of 13 amino acids having a cysteine residue, a middle collagen-like domain with 2 Gly-X-Y triplets, followed by a gap of six amino-acid residues and 15 Gly-X-Y repeats, a short neck domain of 9 amino acids, and a globular fibrinogen-like domain of 4 209 amino acids . The oligomeric structure of L-ficolin is formed presumably by the crosslinking of three subunits via disulfide bridges in the N-terminal region (Figure 1a). The fibrinogen-like domain forms a globular structure like a CRD. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12–24 mers. Two cysteine residues in the Nterminal domain are thought to mediate multimerization by disulfide bonding. 18 Ohashi and Erickson proposed a model in which symmetric Cys24–Cys24 disulfide bonds between trimers are the basis for multimerization. The overall structure of L-ficolin looks like a “bouquet,” as illustrated in Figure 1a. Human H-ficolin/Hakata antigen forms oligomers consisting of 34-kDa subunits linked by disulfide bonds, like L-ficolin. H-ficolin is 49% identical to L-ficolin at the primary structure level. An octadecameric form of H-ficolin, a hexamer of the 8 trimers, was proposed based on electron microscopy . Recently, we proposed a similar structure for human M-ficolin, based on the molecular size of its mul19 timer, about 610 kDa (18-mer of 35-kDa subunits) . Mouse ficolin A resembles human L-ficolin in that it is a tetramer of the trimers (12-mer). Two types of 20 porcine ficolin with different molecular size were purified from the plasma ,

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Figure 1. Multimeric structures of ficolin and mannose-binding lectin (MBL) and schematic representation of the lectin pathway initiated by ficolin (or MBL)/MASPs/sMAP complexes. (a) Ficolin monomer consists of a short N-terminal region, a middle collagen-like domain with Gly-X-Y triplets, a short neck domain, and a globular fibrinogen-like domain. The monomer subunits assemble the trimer via the collagen-like domain, and four to eight trimers assemble the multimer probably by crosslinking via disulfide bridges in the N-terminal region. The fibrinogen-like domain forms a globular structure like a CRD. MBL assembles in a similar manner to ficolin, where two to six trimers are combined to the multimer via the N-terminal disulfide-bondings. (b) The lectin pathway initiated by ficolin (or MBL). The lectin pathway is initiated by the binding of either MBL or ficolin, associated with MASP-1, MASP-2, MASP-3, and small MBL-associated protein (sMAP), to an array of carbohydrates on the surface of a bacterial cell. As with C1s, MASP-2 is responsible for C4 and C2 activation, leading to generation of the same C3 convertase as in the classical pathway. MASP-1 is able to cleave C3 directly.

named little ficolin and big ficolin, both of which are reactive with antificolin α. Little ficolin is a tetramer of the trimers (12-mer), while big ficolin is a 24-mer, being a dimer of little ficolin. As described below, like MBL, the multimeric ficolins form complexes with MASPs and the complexes are thought to be involved in innate immunity defense via the lectin pathway (Figure 1b). 7 From the view of gene structure, the L-ficolin gene contains eight exons : the first exon encodes the 5′UT signal peptide (25 amino acids) and 9 Nterminal amino acids. The second and third exons encode the collagen-like domain. The fourth exon encodes the neck domain. The fifth to seventh exons encode the upstream portion of the fibrinogen-like domain. The last exon encodes the remaining fibrinogen-like domain and 3′UT. The exon–intron structure was 21 closely similar among the human and mouse ficolins . For example, the exon organization of M-ficolin resembles that of L-ficolin, except that M-ficolin has 7,21 an extra exon encoding an additional segment of four Gly-X-Y repeats .

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3. TISSUE AND CELL TYPE EXPRESSING FICOLIN We previously reported that human L-ficolin was expressed mainly in the liver 4 and present in the circulation as a serum lectin . Using various kinds of cell lines, the expression of L-ficolin mRNAs was detected in lymphoid, myeloid, 22 and non-myeloid cells . H-ficolin mRNA is mainly found in the liver and lungs. In the liver, H-ficolin is produced by bile duct epithelial cells and hepatocytes 23 and is also secreted into the bile duct . In the lung, H-ficolin is produced by ciliated bronchial epithelial cells and type II alveolar epithelial cells and is secreted into the bronchus and alveolus. In addition, we recently reported that the 22 glioma cell line produced H-ficolin . M-ficolin is not considered to be a serum lectin and its mRNA is expressed 7,9 in monocytes, the lung, and spleen . M-ficolin protein was reported to be expressed on the surfaces of peripheral blood monocytes and promonocytic U937 cells. However, we recently reported that M-ficolin was located in secretory granules in the cytoplasm of neutrophils and monocytes and in type II alveolar 19 epithelial cells in the lung, suggesting that M-ficolin is a secretory protein . It was reported that the expression of mRNA of porcine ficolin α is high in the lung, liver, and bone marrow and is low in the uterus, suggesting that ficolin 24 α plays important roles in tissues other than the uterus . Ficolin α is present in the blood as a serum lectin, while ficolin β messages are expressed slightly in the bone marrow, while there is no expression in the uterus. Porcine ficolin β is 25 also expressed and secreted by neutrophils . Two types of ficolins, ficolin A and ficolin B, have been identified in mice. Messages of ficolin A are highly expressed in the liver and spleen, and the pro8 tein is present in plasma . Ficolin B is mainly expressed in the bone marrow and 24 spleen . Recently, we delineated the ontogeny and cell types that express ficolin 26 A and ficolin B . As shown in Figure 2a, RT-PCR analysis showed that expression of ficolin A in the liver progressively increased during development. PCR analysis showed that expression of ficolin A in the liver progressively increased during development, peaked around birth, and declined slightly in the adult phase. This pattern was closely similar to those of MASP-1 and MASP-2 throughout the prenatal and postnatal stages. The cell types that express ficolin A mRNA in both the adult liver and spleen were identified as macrophages by in situ hybridization (Figure 2b). Ficolin B exhibited a distinct ontogeny pattern: the expression increased with development, peaking two or three days before birth. Thereafter, it rapidly declined to an undetectable level prior to the age of 4 weeks in the liver. The expression of ficolin B was switched from the embryonic liver to postnatal bone marrow and spleen. The cells that express ficolin B mRNA were identified as belonging to the myeloid cell lineage, but not to other lineages, including erythroid, and T- and B-cell lineages in bone marrow. The different spatial-temporal expression patterns of ficolins A and B suggest that these molecules play distinct roles in the prenatal and postnatal stages.

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Figure 2. Temporal expression patterns of mouse ficolin A and ficolin B and identification of celltype expressing ficolin A in the liver. (a) Schematic representation of the expression levels of ficolin A, ficolin B, MASP-1, and MASP-2 in the prenatal and postnatal stages in the liver (left) and spleen (right). The expression levels were estimated by RT-PCR. (b) Identification of Kupffer cells as an expression site for ficolin A in mouse liver. In situ hybridization was performed on tissue slices prepared from fixed adult liver using labeled ficolin A cRNA as a probe.

Thus, two types ficolin seem to be present in each species: serum type (plasma type) and non-serum type (non-plasma type). The former is expressed mainly in the liver and is present in the circulation as a serum lectin, including human L-ficolin and H-ficolin, mouse ficolin A, and pig ficolin α, whereas the latter was expressed mainly in non-liver tissues, such as peripheral leukocytes and bone marrow, including human M-ficolin, mouse ficolin B and pig ficolin β. Although these two types of ficolin are expressed in different tissues, the cell types seem to be closely related; for example, macrophages for ficolin A and myeloid cells for ficolin B, since myeloid cells are the precursor of macrophages.

4. PHYLOGENY OF THE FICOLIN FAMILY We cloned ficolin cDNAs from several key animals: a lower vertebrate Xenopus 13 14 laevis and an invertebrate, ascidian Halocynthia roretzi , which is the closest relative of vertebrates. The deduced amino-acid sequences of the cDNA clones, termed Xenopus ficolin 1–4 (XeFCN1–4) and ascidian ficolin 1–4 (AsFCN1–4),

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showed that their fibrinogen-like domains showed 47–62% identity with mammalian ficolins. The ascidian ficolins, AsFCNs, have a shorter collagen-like domain (5 Gly-X-Y repeats), compared with those of mammalian ficolins (11–19 repeats). This feature is similar to XeFCN3, which has as few as 7 Gly-X-Y repeats, suggesting that AsFCNs might be a prototype of the ficolin family and that during evolution they acquired the long collagen-like domain. The phylogenetic relationship among the ficolin family and the related proteins were analyzed by phylogenetic trees constructed based on the entire sequences of ficolins and the sequences of the fibrinogen-like domains. As shown in Figure 3a, in the tree of the entire amino acid sequences, each of four Xenopus and ascidian ficolins formed a respective tight cluster, supported by high bootstrap percentages. Similarly, each group of ficolins in the same species, such as human L- and M- ficolins, and pig ficolins α and β, independently formed monophyletic clusters in this tree. These results suggest that each ficolin might have diverged after the emergence of the respective lineage by gene duplication. This implies that evolutionary pressure has independently executed to generate two types (serum type and non-serum type) of ficolins within each spe21 cies. Our recent detailed analysis based on the gene loci, gene organization, and the phylogenetic trees confirmed that ficolin B is the mouse ortholog of Mficolin and that the genes encoding serum-type ficolins, ficolin A, and L-ficolin were evolved independently from a common ficolin B/M-ficolin gene lineage in mice and primates. Human H-ficolin originated from a root of the mammalian/amphibian ficolins, indicating that it has an ancient origin going back to an evolutionary stage prior to the emergence of Xenopus (amphibia). In the tree of the sequences of the fibrinogen-like domains, mammalian ficolins and XeFCNs had a very similar branching to that in the tree of the entire sequence, in which human Hficolin originated from the root of this branch, again suggesting its ancient origin (Figure 3b). This suggests that Xenopus and higher vertebrates should have the H-ficolin homologs. Recently, we demonstrated that the murine homolog of the 21 H-ficolin gene had converted to the pseudogene in the genome . Proteins with fibrinogen-like domains — such as tenascins, angiopoietins, and horseshoe crab tachylectins — seem to be closely related to the ficolin family, although these families are different in their entire domain structures. As shown in Figure 3b, the amino-acid sequences of the fibrinogen-like domains formed a large cluster independent of the primary fibrinogens. This suggests that all of the fibrinogen-like domains might have evolved from a common ancestor that was derived from the authentic fibrinogen. Among the fibrinogen-like domains, those of the tenascin family seem to be closest to those of the ficolin family, although the domain structure of tenascins is quite different from that of ficolins. This suggests that these families evolved by distinct exon-shuffling using a common ancestral fibrinogen-like sequence. Interestingly, the fibrinogen-like domains of tachylectins 5A and 5B, which are nonself-recognizing 27 lectins isolated from the horseshoe crab , are also related to those of the ficolin

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Figure 3. Phylogenetic trees of ficolins, fibrinogen-like domain-bearing proteins, and fibrinogens. The tree was constructed based on the alignments of the entire amino-acid sequences of the ficolin family (a) and the sequences of the fibrinogen-like domains (b) with Clustal W software. The phylogenetic trees were constructed by neighbor-joining methods. Numbers on branches are bootstrap percentages supporting a given partitioning. The sequence names of tenascin, angiopoietin, and fibrinogen groups were omitted in the tree (b). L-FCN, L-ficolin; M-FCN, M-ficolin; H-FCN, Hficolin; PoFCNα, porcine ficolin α; PoFCNβ, porcine ficolin β; MFCNA, mouse ficolin A; MFCNB, mouse ficolin B; RFCNA, rat ficolin A; XeFCN1-4, Xenopus ficolin 1–4; AsFCN 1–4, ascidian ficolin 14; MFA4, microfibril-associated protein 4.

family. The clustering of ficolins, tenascins and tachylectins are supported by a relatively high bootstrap percentage of 69%. Recently, the crystal structure of tachylectin 5A revealed that the fibrinogen-like structure shares not only a common fold, but also has functional sites related to a γ-chain of mammalian 28 fibrinogens . This may provide evidence for the common origin of the innate immunity and blood coagulation systems.

5. FUNCTION OF FICOLIN 5.1. Carbohydrate Binding of Ficolin We previously reported that L-ficolin bound to GlcNAc in GlcNAc-BSA and complex-type oligosaccharide chains with GlcNAc residues linked to the trimannosyl core, but not to mannose-BSA or high mannose-type or hybrid-type 4 oligosaccharides . Unlike MBL, L-ficolin was able to bind to GlcNAc next to a terminal galactose in oligosaccharides. H-ficolin binds GlcNAc and GalNAc, 8 but not mannose and lactose . It agglutinates human erythrocytes coated with lipopolysaccharides (LPSs) derived from Salmonella typhimurium, Salmonella minnesota, and Esherichia coli (O111). The agglutination of erythrocytes coated

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with LPS from Salmonella typhimurium is inhibited by GlcNAc, GalNAc, and fucose, indicating that the bindings of H-ficolin to LPS are mediated by its lectin activity. The fibrinogen-like domain of M-ficolin had an affinity for GlcNAc and that antibody against this polypeptide inhibited phagocytosis of Esherichia 29 coli by U937 cells . We recently demonstrated that the mature form of recombinant M-ficolin showed a lectin activity for GlcNAc-BSA, GalNAc-BSA, and 19 sialyl N-acetyllactosamine-BSA . The specific binding of M-ficolin to sialic acid was confirmed by its binding to a glycoprotein, fetuin, but not to its asialo derivative. Recently, we also demonstrated that mouse ficolin A and ficolin B showed lectin activities for GlcNAc and GalNAc in common, and ficolin B additionally recognized sialic acid like human M-ficolin (unpublished data). The carbohydrate binding of ficolin is executed by its fibrinogen-like domain. The fibrinogen-like domain of ficolin shows similarity to the C-terminal halves of the fibrinogen β and γ chains. Formerly, Doolittle pointed out that the primary structure of fibrinogen was related to CRD of MBL, especially in terms 30 of coordination of the charged amino-acid residues essential for sugar-binding . As described above, ficolins recognize a common carbohydrate, GlcNAc. It 2+ has been controversial as to whether this recognition requires Ca or not. We 2+ 4 first reported that the GlcNAc-binding of L-ficolin was Ca dependent . How2+ 20,31 ever, the several papers that followed reported it to be Ca independent . Recently, we verified our previous result using an ELISA: the binding of L-ficolin 2+ to GlcNAc-BSA-coated microtiter plate required Ca at less than 50 µM, while the presence of EDTA at 1 mM or more completely inhibited binding (Figure 2+ 4a). Our result might suggest that L-ficolin binds Ca during biosynthesis in 2+ cells, prior to secretion into the circulation, where the Ca concentration is very -6 -7 2+ low, generally being 10 –10 M. A similar Ca dependency was observed in the 27 carbohydrate binding of tachylectin 5A . Recently, the structure of tachylectin 5A complexed with GlcNAc has been solved by x-ray analysis and the binding 28 mechanism has been clarified . The crystal structure of tachylectin 5A demonstrated that the carbohydrate binding site is located adjacent to and coordinated 2+ with the Ca binding site (Figure 4b). Analysis of the ligand-binding pocket explains its acetyl-group specificity where four aromatic side chains (Tyr210, His220, Tyr236, and Tyr248) form a funnel with a methyl side chain of Ala237 at its base. The sugar methyl group lies in the middle of the funnel, surrounded 2+ by the aromatic side chains. The Ca is bound in the loop region located adjacent to the ligand-binding site, and coordinated by seven oxygen ligands in a pentagonal bipyramidal manner. Both carboxylate oxygen atoms of Asp198, the main chain carbonyl oxygen of His202 and Thr204, and one water molecule form the pentagonal base. As shown in Figure 4c, the sequence alignment of ficolins and tachylectin 5A indicates that most of the residues essential for sugar 2+ binding and Ca binding of tachylectin 5A are conserved in ficolins. This may propose the possibility that ficolin binds carbohydrates in a similar manner to tachylectin 5A.

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Figure 4. Carbohydrate-binding properties of ficolin. (a) Ca2+-dependency of binding of L-ficolin to GlcNAc. The binding of L-ficolin to GlcNAc–BSA-coated microtiter plate was performed by incubation of various amounts of recombinant L-ficolin (mature form) in the plate in the presence of 0.05–5 mM CaCl2 or 1–10 mM EDTA or 150 mM GlcNAc. The binding was detected by using rabbit anti-L-ficolin antibody and horseradish peroxidase-conjugated anti-rabbit IgG. The dosedependent binding of L-ficolin to GlcNAc was observed in the presence of 0.05 mM or more of CaCl2, while the binding was completely inhibited in the presence of EDTA or soluble GlcNAc. (b) Three-dimensional model of tachylectin 5A. The original image is deposited in the databases at Accession number 1JC9. The structure surrounded by a white circle represents GlcNAc ligand and the “Ca” under GlcNAc denotes the Ca ion bound to tachylectin 5A. (c) Alignment of L-ficolin, Mficolin, and tachylectin 5A. About 100 C-terminal amino acids were aligned with Clustal W software. Red letters in tachylectin 5A represent the residues essential for GlcNAc-binding and green letters for Ca ion–binding. Blue letters in L-ficolin indicate two positions of the polymorphic site (see text), Thr236Met and Ala258Ser.

5.2. Binding of Ficolin to Bacteria Several reports have shown that ficolins, like MBL, bind to the cell wall components of bacteria and fungi. Human L-ficolin can bind specifically to lipoteichoic acid (LTA), which is a defined cell wall constituent of Gram-positive 32 bacteria . It can also bind to peptidoglycan (PGN), the key component of the bacterial cell wall, and 1,3-β-D-glucan, the major component of yeast and fungal 33 cell walls, which are β-1,3- and β-1,6-linked polymers of glucose . Its porcine homolog, ficolin α, reacts with lipopolysaccharides (LPSs) from different Gramnegative bacteria, such as Esherichia coli, Salmonella typhimurium, Salmonella

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enteritidis, Salmonella abortus equi, Pseudomonas aeruginosa, Shigella flexeneri, and Serratia marcescens, and with LTA from Gram-positive bacteria such as Streptococcus sanguis, Bacillus subtilis, Streptococcus pyogenes, and Staphy34 lococcus aureus . Indeed, accumulated data reveal that ficolins can recognize certain kinds of microbes. Human L-ficolin, being the most investigated, can bind to Grampositive bacteria, such as serotype III group B streptococci, Streptococcus pneumoniae 11F, and Staphyloccus aureus, and Gram-negative bacteria, such as 4,35,36 . Escherichia coli and the rough type of Salmonella typhimurium TV119 Human M-ficolin can also bind to Staphyloccus aureus and Salmonella typhimu19 rium LT2 . In contrast, human H-ficolin binds neither Staphyloccus aureus nor Streptococcus pneumoniae, but it binds to Aerococcus viridans and inhibits its 37 growth . These different binding properties among human ficolins might suggest their distinct roles in facing various invading organisms. Other than humans, ficolins in other species also show the ability to bind to pathogens. For example, porcine ficolins bind to the Actinobacillus pleuropneumoniae serotype 38,39 5B . These bindings of ficolins to bacteria are incompletely inhibited by the presence of sugars such as GlcNAc, suggesting that the bindings are mediated by carbohydrates on the surfaces of microbes. 5.3. Ficolin as a Recognition Molecule in the Lectin Pathway We previously reported that both L-ficolin and H-ficolin activate the lectin 16,17 complement pathway in association with MASPs like MBL . The exact stoichiometry among ficolin (or MBL), MASPs, and sMAP in the complexes is sill unknown, although the overall structure is considered to be similar to the C1 complex in the classical pathway, in which one molecule of C1r (18-mer) is associated with one dimer of C1r and one dimer of C1s. C1q is structurally related to MBL and ficolin in having a collagenous stalk, and serine proteases C1r/C1s are structurally and functionally similar to MASPs, forming the 40,41 MASP/C1r/C1s family . Recently, we demonstrated that recombinant human 19 M-ficolin could also form complexes with MASP-1, MASP-2, and sMAP . The association of L-ficolin with MASPs was confirmed by its binding to recombi42 nant MASP-1, MASP-2, and their synthetic peptides . Ficolin associates to MASPs and its truncated form (sMAP) through the interaction between the C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB) and epidermal growth factor-like (EGF-like) domains of MASPs/sMAP and the collagen-like domain of ficolin. Interestingly, we observed that sMAP competes with MASP-2 in association with MBL and ficolin, and therefore inhibits complement activation by lectin/MASP-2 complexes (manuscript in preparation). This observation suggests that sMAP has a regulatory role in the lectin pathway. L-ficolin enhanced the uptake of Salmonella typhimurium strains by neutrophils in a dose-dependent manner, suggesting that it serves as an opsonin for 4 phagocytosis . The phagocytosis of Escherichia coli by U937 cells could be in-

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hibited by anti-M-ficolin antibody, indicating that M-ficolin also functions as an 29 opsonin . In addition, like MBL L-ficolin and H-ficolin specifically bound to apoptotic Jurkat cells, C3 and C4 were subsequently deposited on apoptotic 22 cells , suggesting that the binding of L-ficolin and H-ficolin to the apoptotic cells leads to activation of the complement. All these results imply that, like MBL, ficolins function as recognition molecules and participate in the clearance of microorganisms and apoptotic cells through the lectin pathway.

6. POLYMORPHISMS OF THE FICOLIN GENE Polymorphisms in the human MBL gene have been reported to be associated with serum concentration and therefore associated with susceptibility to infectious diseases. Two single nucleotide polymorphisms (SNPs) are known to oc43 cur in the promoter region of the human MBL gene. Functional promoter analysis indicated that three haplotype variants in two positions — HY, LY and LX — exhibit high, medium, and low promoter activity, respectively, in accor44 dance with the results of a previous population study . Like MBL, the serum concentration of L-ficolin varies considerably in healthy individuals. To date, about 20 SNPs have been reported in the L-ficolin gene. Three of five SNPs present in the promoter region are significantly associated with the serum con45 centration of L-ficolin . Interestingly, two SNPs in exon 8 of the L-ficolin gene substitute amino acids — Thr236Met and Ala258Ser — in the fibrinogen-like 45 domain and are associated with increased or decreased GlcNAc-binding . As depicted in Figure 4c, these two SNPs are localized around both carbohydrate2+ binding and Ca -binding sites. The M-ficolin gene also contains more than 10 SNPs in the promoter and structural part of the gene. In the H-ficolin gene, only a single frame-shift deletion in exon 5 was detected. These results show that the ficolin genes are polymorphic and that, particularly, L-ficolin harbors functional polymorphic sites that regulate both expression and recognition ability, which may have pathophysiological implications for innate immunity.

7. CONCLUSIONS Ficolins are proteins that consist mainly of two domains: an N-terminal collagen-like domain and a C-terminal fibrinogen-like domain. This domain structure is related to MBL or C1q, a subcomponent of the complement C1 complex in having a collagenous stalk. Like MBL and C1q, the ficolin monomer assembles the multimeric form via the trimers. Accumulating data indicate that one of the most important functions of ficolin is to bind carbohydrates, which are responsible for the fibrinogen-like domain. The carbohydrate specificities have suggested that ficolin is able to distinguish between pathogens and self. Indeed, ficolin binds to certain kinds of bacteria through its lectin activity. Interestingly, like MBL, some of the ficolins form complexes with MASPs and the complexes

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can activate complements through the lectin pathway. All these results suggest that ficolin functions to eliminate the non-self in innate immunity as a recognition molecule.

8. ACKNOWLEDGMENTS The authors thank Prof. M. Matsushita for his constant advice and discussion and Ms. N. Nakazawa for her technical assistance. Most of our work introduced in this review was supported by grants from the Ministry of Education, Science, Sports, and Technology of Japan, and CREST, the Japan Science and Technology Agency.

9. REFERENCES 1.

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19 ROLE OF MANNOSE-BINDING LECTIN (MBL2) GENOTYPING IN PREDICTING THE RISK OF RECURRENT OTITIS MEDIA (ROM) Lieve Nuytinck, Els De Meester, Martine Van Thielen, and Paul Govaerts

1. INTRODUCTION Otitis media (OM) is the most common childhood disease. Almost all children experience at least one episode of otitis media with effusion (OME) or acute otitis (OMA) during childhood, and a substantial subgroup is confronted with recurrent episodes. If not treated adequately, OM may lead to temporary or permanent hearing loss. It is well known that bacteria and viruses cause OMA, and may play a role in OME. Bacteria such as Streptococcus pneumoniae (pneumococcus) and Haemophilus influenzae account for about of 85% of OMA cases, with viruses making up the remaining 15%. The chronic and recurrent forms are thought to be superimposed on an underlying middle ear ventilation problem, sometimes expressed as OME. Although the complete pathophysiological picture still remains to be fully elucidated, bacteria and viruses are certainly thought to play an important role in both chronic and recurrent otitis forms. The treatment for acute otitis media is antibiotics, usually for 7 to 10 days. In the case of rOM one may elect for surgery (adenotomy or tympanotomy with placement of grommets) in order to prevent future remissions. It has been widely accepted that environmental and infectious factors trigger the development of OM in general. However, there is significant evidence from epidemiologic, anatomic, physiologic, and immunologic studies that susceptibility to recurrent episodes of OM is also partially genetically 2,3 12 determined , although unlikely to be due to a single major gene . The specific

Lieve Nuytinck, Els De Meester, Martine Van Thielen, Innogenetics NV, Ghent, Belgium. Paul Govaerts, The Eargroup, Antwerpen-Deurne, Belgium.

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genes conferring susceptibility to OM are largely unknown, but ongoing genome-wide linkage studies may provide new insights into susceptibility and the 4,3 pathogenesis of OM in the near future . One such approach seeks to understand the genetic links between innate immunity and OM. The innate immune system represents the first line of host defense against infectious agents, permitting an immediate response by a constellation of sensing and effector mechanisms (antimicrobial peptides, phagocytic cells, and complement pathways) that recognize, contain, and usually remove pathogens swiftly. It relies on a pool of germline-encoded pattern-recognition receptors (PRRs) that can bind to highly conserved structures known as pathogenassociated molecular patterns (PAMPs), which are commonly present across large groups of invasive microorganisms. The PRRs may be divided into three classes: signalling, endocytic, and secreted. The latter class functions as opsonins by binding to microbial and viral cell walls and activating the complement system. The most important receptor is mannose-binding lectin (MBL), which is a soluble protein, synthesized mainly by the hepatocytes. MBL not only binds to microbial carbohydrates but also to phospholipids, nucleic acids, and nonglycosylated proteins, properties that may be relevant to the clearance of apoptotic cells and avoidance of autoimmunity. Upon binding of MBL to the surface of microorganisms, MBL undergoes a conformational change leading to activation of two MBL-associated serine proteases (MASPs). In a next step, the complement factor C4 is activated by the MBL/MASP complex, resulting in formation of the C3 convertase, the key reaction leading to terminal complement pathway initiation. During the past decade, a steadily increasing number of clinical studies aimed at elucidating the role of MBL in health and disease has been pub11,16 lished . From these data, it became apparent that MBL deficiency is selectively associated with an increased risk for infections and autoimmune conditions, and influences the severity and course of several diseases. Initially, quantification of MBL in serum or plasma using ELISA technologies were used. However, as it becomes more apparent that the serum levels of MBL are influenced by genetic polymorphisms in the gene encoding MBL, i.e., the MBL2 gene, genotying seems to be the method of choice due the simplicity of the test. From the literature, there are strong suggestions that innate immunity may play a role in the development of rOM. In view of the importance of bacterial or viral agents in OM, the gene encoding mannose-binding lectin (MBL2) was chosen as a first focus of attention for the following reasons: •

•

MBL is a key component of innate immunity as a molecular sen10 sor for bacteria or viruses . It activates the complement cascade via the lectin pathway using the MASPs as co-activators. Association studies have shown that individuals with diminished MBL activity show a higher susceptibility to sepsis or recurrent 17,11 infection .

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•

•

•

283

It has been shown that the MBL protein is present in both nasopharyngeal secretions and middle ear effusions, suggesting a de6 fensive role for this protein in the middle ear . The incidence of OM is highest in young children where the adaptive immune system has not yet fully developed, and where infections are recognized mainly by the receptors of the innate immunity system. From earlier studies, a possible role of MBL in the development of OM has been suggested, although contradictory results have 6,15,7,13 been reported .

An exploratory pilot study was therefore conducted to investigate the occurrence of MBL2 gene polymorphisms in rOM cases.

2. MBL2 GENE AND POLYMORPHISMS The functional human MBL2 gene is located on chromosome 10q11.2-q21 and consists of four exons. Six key variations (Figure 1) affecting expression and functionality of MBL have been described. The polymorphisms are localized in the promotor and 5′ untranslated region [–550G>C, –221G>C, and +4C>T], and affect expression of the MBL2 gene. In exon 1, encoding the collagen-like region necessary for correct multimer formation, three polymorphisms [R52C, G54D, and G57E] influencing the functionality of the protein have been identified (Table 1).

Figure. 1. Structure and organization of the MBL2 gene. The localization of the principal structural and promoter polymorphisms with respect to the four domains of the MBL molecule is indicated.

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Table 1. Overview of the 6 Polymorphisms in the MBL2 Gene Detected by the INNO–LiPA MBL2 Kit Common name

Abbreviation commonly used in the literature

Nomenclature according to HGVS

Minor allele frequency n=172

–550G>C

H/L

–619C>G

0.41

–221G>C

Y/X

–290C>G

0.19

+4C>T

P/Q

–66C>T

0.23

R52C - CGT>TGT

A/D

154C>T

0.06

G54D - GGC>GAC

A/B

G57E - GGA>GAA

A/C

0.14 161G>A 170G>A

0.03

The nomenclature for the MBL2 polymorphisms is generally accepted by the scientific community8,9. To avoid confusion, a conversion table is given showing the nomenclature using the guidelines as recommended by the Human Genome Variation Society (HGVS)5.

The characterization of the promotor polymorphisms led to identification of four commonly found haplotypes: LXP, LYP, LYQ, and HYP. Since each of the three exon 1 mutations is in strong linkage disequilibrium with a different promotor haplotype, seven common haplotypes have been described: HYPA, LYPA, LXPA, LYQA, HYPD, LYPB, and LYQC. Two very rare haplotypes, 14 1 HXPA and LYPD , have also been reported in the literature. An abbreviated nomenclature, taking into account only one promotor polymorphism (X/Y) and the presence of an exon 1 polymorphism (normal exon 1 sequence is expressed as A), are written as YAYA, YAYO, YAXA, YOYO, YOXA, and XAXA.

3. MATERIALS AND METHODS 3.1. Patients and Controls Seventeen patients who presented at an ear, nose, and throat (ENT) clinic with OM and showing clinical evidence of recurrent or persistent disease took part in the study. Recurrence/persistence was judged based on the patient’s history. All patients were under 10 years of age at entry, and all were scored as having “severe” rOM according to the clinician’s judgement of the recurrence/persistence rate. The score “severe” was given in case of 3 successive

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ENT visits with positive otitis findings (OME or OMA) or in the case of a history of three or more otitis episodes with antibiotic treatment during the preceding year. As a control population, 172 healthy volunteers were included. Informed consent was obtained from all control subjects and patients. 3.2. MBL2 Genotyping Genomic DNA was isolated from buccal cells from children with recurrent OM or leukocytes from healthy volunteers using standard procedures. Genotyping was caried out using the INNO-LiPA MBL2 assay (Innogenetics, Belgium). In total, 17 patient and 172 control samples were tested; for all samples both haplotypes could be identified. The INNO-LiPA MBL2 assay is a multi-parameter assay allowing simultaneous detection of the 6 relevant DNA variants in the MBL2 gene using reverse hybridization technology (Figure 2). The test is easy to perform and takes about two hours starting from amplified product.

Figure 2. The INNO-LiPA principle.

Specific probes designed to hybridize with their complementary sequences are coated as parallel lines on a nitrocellulose membrane. The hybridized probes can be visualized as purple lines on the strip (Figure 3). Interpretation of the results can be performed using the LiRAS£ for LiPA MBL2 software or manually using a typing table.

4. RESULTS An overrepresentation of the MBL2-G54D variant (B-allele) [OR 2.9 (95% CI 1.35–6.44)] was found in the rOM patients compared to a healthy control population (Figure 4A). When the combined genotypes of rOM patients and healthy controls were compared, the genotype comprising one exon1 polymorphism in combination with the minor allele of the promotor polymorphism in position

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–221G>C was found to be significantly overexpressed in the patient group [OR 14.35 (95% CI 4.3-47.6)] (Figure 4B).

Figure 3. The INNO-LiPA MBL2 assay is a validated, CE-marked multiparameter assay allowing simultaneous genotyping of 6 DNA variants in the human MBL2 gene using reverse hybridization technology. Specific probes designed to hybridize with their complementary sequences are coated as parallel lines on a nitrocellulose membrane. The hybridized probes can be visualized as purple lines on the strip. Interpretation of the results can be performed using the LiRAS™ for LiPA MBL2 software, or manually using a typing table. The test is easy to perform and takes about two hours starting from amplified product.

5. DISCUSSION OM and OM-related upper respiratory problems are encountered with great frequency in pediatric and ENT practices. Most children experience at least one episode of OM during their first years of life. Many different types of OM have been defined, mainly on clinical grounds rather than based on pathophysiological criteria. The most clear-cut form is OMA. This results from a microbial infection of the middle ear, leading to a purulent collection in the middle ear cavity. The spontaneous course of this infection is almost always uncomplicated, with healing occurring either spontaneously or after antibiotic treatment. Some

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35 30 25 20

Control n=172

%

15

rOM n=17

10 5 0 HYPA LYQA LYPA LXPA HYPD LYPB LYQC

45 40 35

%

30 25

Control n=172

20

rOM n=17

15 10 5 0 YAYA YAYO YAXA YOYO YOXA XAXA

Figure 4. Haplotyping results and comparison of haplotype frequency. (a) Haplotyping results of 17 OM patients and 172 healthy subjects using the INNO–LiPA MBL2 kit. A significant overrepresentation of the LYPB genotype in the rOM group was detected [OR 2.9 (95% CI 1.35–6.44)]. (b) Comparison of the frequency of the combined haplotypes using the two-letter code expression showed a significant overrepresentation of the YOXA genotype in the patient group [OR 14.35 (95% CI 4.3–47.6)].

children, however, develop chronic or recurrent forms of otitis and an underlying chronic middle ear ventilation problem (often controversially called “eustachian tube dysfunction”) is believed to be a basic factor in both forms of OM. In addition, microbial factors have been found to contribute to OM pathogenicity, as have genetic factors, in view of the familial occurrence that is often seen. These chronic or recurrent forms cause important morbidity with multiple episodes of pain, fullness, hearing loss, fever, behavioral problems, language developmental delays, etc., resulting in significant drug consumption and surgical interventions.

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A remarkable feature of all forms of otitis media is its age-related occurrence. Under the age of 6–7 years, many children seem to be very prone to OM, whereas its incidence dramatically drops beyond this age. Since the adaptive immune system in this population is not yet fully matured, innate immunity thereby bears the burden of protecting the child against upper respiratory infections. This could explain the age-specific proneness of this kind of infections. A deficient or inadequate level of innate immunity in a subpopulation of these children would undermine this sole protective shield, and could be associated with more severe forms of upper respiratory infections, such as rOM. Given the importance of innate immunity and, more particularly, complement pathway activation as the first line of defense in young children, significantly decreased MBL functionality in serum may be a clinically relevant factor for susceptibility to upper airway infection in this age group. In general, the majority of individuals in the general population with low MBL levels do not suffer from diseases directly related to decreased MBL functionality. However, hypersensitivity to infection becomes particularly apparent when constitutively low MBL levels, due to the presence of one or two mutations affecting the expression rate or functionality of MBL, occur in the context of coexisting primary (e.g., C4-null alleles) or secondary immune deficits (e.g., SLE). The involvement of MBL in otitis media has been studied previously, but inconsistent results were obtained. This may be due to several reasons, such as low numbers of patients included, differing inclusion/exclusion criteria, alternative approaches to measurement of MBL (serum levels vs. genotyping), etc. A possible association between recurrence of otitis media and low concentrations of MBL in plasma and upper airway secretions was investigated in 89 children 6 with several presentations of OM . Their results did not support the assumption that low MBL levels alone predispose to recurrence of otitis media in Caucasian 15 children. Subsequently, Tezcan et al. investigated 51 children between 6 and 48 months with OMA. They observed a defective opsonization in the majority of 7 patients, and indirectly concluded that an MBL deficiency might be involved. 7 Homoe et al. studied a group of Greenlandic children, although no association 13 with low MBL levels was found. Straetemans et al. recently showed a role of serum MBL levels together with the FcγRIIa–R/R131 polymorphism in the pathogenesis of recurrent OM. Our results, performed on a limited group of children with severe rOM, found that MBL2 genotyping may be useful for patient stratification, especially in identifying children at high risk for severe recurrent otitis media. These results confirm the findings that innate immunity, especially MBL and complement activation, could well play an important role in first-line defense in the middle ear. The presence of the MBL2–G54D variant or the combination of an exon 1 variant on one allele and the C-allele of the MBL2–221G>C variant on the second allele, is associated with susceptibility to recurrent otitis media. The overexpression of genotypes leading to diminished production of MBL in our study group provides a possible causative explanation for the phenotypic

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trait of rOM. This seems to be a particularly promising line for further research. If confirmed, these results may lead to the development of new molecular diagnostic tests enabling the clinician to identify children at risk for OM and thereby help in preventing the possible consequences of the disease, such as hearing loss and developmental problems. Furthermore, these data might be used in the future for the development of more focused treatments. At present, no specific therapy is available, but it might become so in the future in the form of MBL supplementation therapy. Alternatively, the subgroup of children with rOM and deficient MBL may become candidates for new forms of personalized therapies (such as more aggressive antibiotic therapy), coupled with more intensive follow-up.

6. REFERENCES 1.

Boldt AB, Petzl-Erler ML. A new strategy for mannose-binding lectin gene haplotyping. Hum Mutat 19(3):296–306 (2002). 2. Casselbrant ML, Mandel EM. The genetics of otitis media. Curr Allergy Asthma Rep 1(4):353–357 (2001). 3. Casselbrant ML, Mandel EM. Genetic susceptibility to otitis media. Curr Opin Allergy Clin Immunol 5(1):1–4 (2005). 4. Daly KA, Brown WM, Segade F, Bowden DW, Keats BJ, Lindgren BR, Levine SC, Rich SS. Chronic and recurrent otitis media: a genome scan for susceptibility loci. Am J Hum Genet 75(6):988–997 (2004). 5. den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15(1):7–12 (2000). 6. Garred P, Brygge K, Sorensen CH, Madsen HO, Thiel S, Svejgaard A. Mannanbinding protein--levels in plasma and upper-airways secretions and frequency of genotypes in children with recurrence of otitis media. Clin Exp Immunol 94(1):99– 104 (1993). 7. Homoe P, Madsen HO, Sandvej K, Koch A, Garred P. Lack of association between mannose-binding lectin, acute otitis media and early Epstein-Barr virus infection among children in Greenland. Scand J Infect Dis 31(4):363–366 (1999). 8. Madsen HO, Garred P, Kurtzhals JA, Lamm LU, Ryder LP, Thiel S, Svejgaard A. A new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein. Immunogenetics 40(1):37–44 (1994). 9. Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, Svejgaard A. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol 155(6):3013–3020, 1995. 10. Medzhitov R, Janeway C Jr. Innate immunity. N Engl J Med 343(5):338–344 (2000). 11. Nuytinck L, Shapiro F. Mannose-binding lectin: laying the stepping stones from clinical research to personalized medicine. Personalized Med 1(1):35–52 (2004) 12. Rich SS, Daly K, Levine SC. Familial aggregation and risk factors for chronic recurrent otitis media. In: Recent advances in otitis media: proceedings of the sixth international symposium 1995 BCDecker (Hamilton), 65–68 (1996).

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13. Straetemans M, Wiertsema SP, Sanders EA, Rijkers GT, Graamans K, Van Der Baan B, Zielhuis GA. Immunological status in the aetiology of recurrent otitis media with effusion: serum immunoglobulin levels, functional mannose-binding lectin and fc receptor polymorphisms for IgG. J Clin Immunol 25(1):78–86 (2005). 14. Sullivan KE, Wooten C, Goldman D, Petri M. Mannose-binding protein genetic polymorphisms in black patients with systemic lupus erythematosus. Arthritis Rheum 39(12):2046–2051 (1996). 15. Tezcan I, Yilmaz Y, Oner F, Yel L, Sanal O, Ersoy F, Onerci M, Berkel AI. Defective serum opsonization activity in children aged 6-48 months having acute purulent otitis media. Turk J Pediatr 39(4):453–457 (1997). 16. Thiel S, Frederiksen PD, Jensenius JC. Clinical manifestations of mannan-binding lectin deficiency. Mol Immunol 43(1–2):86–96 (2006) 17. Turner MW. The role of mannose-binding lectin in health and disease. Mol Immunol 40(7):423–429 (2003).

20 CONFORMATIONAL COMPLEXITY OF COMPLEMENT COMPONENT C3 Bert J.C. Janssen and Piet Gros

1. INTRODUCTION The complement system is an important component of the humoral immune response in vertebrates. The complement system consists of 30–40 soluble plasma proteins and cell-surface receptors that form a complex set of regulatory 1 pathways (reviewed by Walport ). It enables the host to recognize invading microorganisms. Activation of the complement system may result in a range of effector functions: bacterial lysis, initiation of inflammatory responses, phagocy2 tosis, and stimulation of B-cell response (reviewed by Carroll ). Regulation of this defense system in blood plasma is of critical importance for the homeostasis of the host, as indicated by a wide range of (auto-)immune disorders that are associated with uncontrolled complement response. The molecule C3 plays a central role in the complement activation path3 ways (reviewed by Sahu and Lambris ). Three pathways of complement activation exist: (i) the antibody-mediated classical pathway, (ii) the lectin-mediated pathway and (iii) the alternative pathway. These pathways are characterized by protein–protein complex formation and proteolytic activation of the large multidomain complement proteins. The first two pathways involve antibodies or lectins that bind to the surface of microorganisms. The third pathway, the alternative pathway, reacts intrinsically onto any, host or foreign, surface. All three pathways converge in the proteolytic activation of C3, which generates the biologically active fragments C3a and C3b. The small fragment C3a functions as an anaphylatoxin, initiating inflammatory responses. The large fragment, C3b, plays a central role in localization and amplification of complement response

Bert J.C. Janssen and Piet Gros, Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Dept. of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. E-mail address [email protected].

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and initiation of the terminal phase of complement activation. Localization is achieved by covalent attachment through a highly reactive thioester moiety in C3b. Surface-bound C3b serves multiple purposes. It acts as an opsonin providing a strong signal for phagocytosis by macrophages, and it provides a molecular platform for formation of the convertases of the complement system. The convertases induce amplification of the response and initiate the terminal complement phase. The level of complement response is primarily determined by the lifetime of the convertase complexes, which are inherently instable with half-life 4 times of 90 seconds . One complement protein has been identified that enhances the lifetime of the convertases, i.e. properdin, whereas various “decayaccelarating and cofactor activity” regulators are known that induce dissociation of the convertases and proteolytic degradation of C3b by factor I. The cleavage products iC3b, C3dg, and C3d have important signaling roles. iC3b acts as an opsonin-facilitating phagocytosis of foreign material by leukocytes. Next to that, iC3b, C3dg, and C3d amplify B-cell responses and thereby provide a key interface between innate and adaptive immunity. Structural data are instrumental in understanding the molecular mechanisms of complex formation and activation of the large multi-domain complement proteins. Resolving these structural details, however, is technically challenging. The proteins involved are typically large, multi-domain, and flexible molecules, and they associate into large and, in some cases inherently, unstable complexes. Moreover, due to size and post-translation modifications, these proteins are often hard to produce by recombinant protein expression techniques. Nonetheless, in the last decade significant advances have been made in the structure determination of complement proteins and protein domains (see Table 1). Most recently, we published the structures of native C3 and its major proteolytic frag5 ment C3c . These structures provide insight into the domain organization, structure, and dynamics of the central C3 protein of the complement system. Here, we present an overview of these new structural insights and discuss the implications for complement convertase formation, decay acceleration, and cofactor activity, and the signaling roles of the proteolytic fragments iC3b, C3dg, and C3d.

2. STRUCTURAL ORGANIZATION OF C3 The protein molecule C3 is the most abundant complement protein in human blood plasma with levels of 1–1.5 g/l in healthy individuals. It is synthesized as a single poly-peptide chain of 1641 amino-acid residues and secreted into the blood as a glycosylated two-chain protein. Residues 1–645 form the β-chain and residues 650–1641 form the α-chain with N-linked glycans on positions Asn-63 42,43 and on Asn-917 of the β- and α-chain, respectively . Residues Arg-646–Arg649 are proteolytically removed during post-translational processing. A remarkable aspect of its post-translational modifications is the formation of a thioester

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Table 1. Solved Structures in the Complement System Protein Central complement components

C1r C1s C1q C3

C4 C5 C8 Factor B

Factor D MASP2

Regulators

Map19 Factor H

DAF (CD55)

MCP (CD46) CR1 (CD35)

In complex Foreign

CR2 (CD21) CR3 (CD11b/CD18) CR4 (CD11c/CD18) CD59 C3d-CR2 VCP

BbCRASP-1 CHIPS

Domain/fragment EGF-like CCP1-CCP2-SP CCP2-SP CUB1-EGF Globular head C3a C3d C3 C3c C4d C5a C345C C8 SP vWA factor Bb Factor D CUB1-EGF-CUB2 CCP1-CCP2-SP Map19 CCP16 CCP5 CCP15-CCP16 CCP3-CCP4 CCP2-CCP3 DAF (CCP1,2,3,4) CCP1-CCP2 CCP15-CCP16 CCP16-CCP17 CCP1-CCP2 I-domain I-domain CD59 (soluble form) C3d CR2-CCP1,2 CCP3-CCP4 VCP CCP2-CCP3 BbCRASP-1 CHIPS

Method NMR X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray NMR NMR X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray NMR NMR NMR X-ray NMR X-ray X-ray NMR NMR X-ray X-ray X-ray NMR X-ray NMR X-ray NMR X-ray NMR

Ref. 6 7 8 9 10 11 12 5 5 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 31 32 33 34 35 36 37 38 39 40 41

bond in the protein. The side chains of residues Cys-988 and Gln-991 form a 44 Cα–Cβ–Sγ–(Cδ=Oε)–Cγ–Cβ–Cα thioester linkage . The length of the polypeptide chain and the extensive post-translational modifications (twofold glycosylation, 11,45 formation of thirteen disulfide bridges , removal of the tetra arginine (646–

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649), and formation of a thioester moiety) indicate a complicate folding process of this intricate protein molecule (Figure 1). a

b

1615-1624

1615-1624 1515-1639

1496-1568

1515-1639

1336-1467 1496-1568

1484-1489 851-1491

1367-1436

1367-1436

1484-1489

Asn917

851-1491 1336-1467

685-706 thioester 672-705

671-698

1079-1136

537-749 605-640

Asn63

537-749

605-640

Asn63

Figure 1. Structures of C3 and C3c. (a,b) Ribbon representation of C3 and C3c, respectively. Disulfide bonds and the thioester are shown as black spheres. Glycan moieties are shown as black sticks.

We recently published the crystal structure of native C3 and its major pro5 teolytic fragment C3c . Both proteins were purified from human blood plasma: 46 native C3 was purified from fresh plasma ; whereas C3c was purified from outdated plasma stored at 4°C. First we determined the crystal structure of C3c, which is a proteolytic fragment representing the core structure of C3 and is conceivably less flexible and hence possibly easier to crystallize. Indeed, in our hands C3c crystallizes more readily than C3. C3c crystals typically diffracted to approx. 3-Å resolution and best diffraction data was collected up to 2.4-Å resolution. Since the protein was isolated from a natural source and no significant structural models were a priori available, we obtained phase information through the classical method of multiple-isomorphous replacement using heavy-atom compounds. Overall, the process involved many crystals of varying diffraction quality (mostly 3–3.5 Å resolution) in three different space groups. An interpretable electron-density map was obtained after multi-crystal averaging, combining data from multiple crystal forms after extensive optimization of the operators and masks for mapping corresponding structural fragments in the various crystal

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forms. This experimental electron-density map was used to build an atomic model of C3c. The structure of C3c consisting of ten domains was instrumental in solving the structure of native C3. Diffraction data of C3 was collected from a single crystal diffracting to 3.3-Å resolution. The structure was solved by maximum-likelihood molecular replacement placing known structures of domains 12 (derived from the structures of C3c and C3d ) into the unit cell. After successfully placing 10 domains, the model was completed by model building and refinement. The resulting final model of C3 consisted of a total of 13 domains. The thioester is intact in the crystals, as indicated by the electron density at the thioester linkage, and is occluded in the structure of C3 as expected for the native conformation of C3. The structure of C3 is characterized by an intricate arrangement of 13 domains (see Figure 1; domain names and secondary structure labeling as defined in [5]). The core of the structure is formed by eight homologous domains from both the β- and α-chains with an additional five domains appearing as inserts and a C-terminal extension. We named the core domains macroglobulin (MG) domains 1 through 8 (referring to the α2-macroglobulin protein family). The MG domains display a fibronection type-III like fold that is part of the superfamily of immunoglobulin folds. Domains MG1–MG5 are formed by residues of the β-chain, whereas domains MG7 and MG8 are formed by the α-chain. Surprisingly, domain MG6 is formed by residues of both the β-chain (res. 535– 577) and the α-chain (res. 746–806). This reflects that C3 is a single gene product that should not be considered a gene fusion of a β- and α-part. C3 is rich in disulfide linkages. One disulfide bridge covalently links the β- and α-chains in domain MG6 (537-794), one disulfide bridge is inter-domain (from MG7 to the anchor region; 851–1491), and the eleven remaining are intra-domain disulfide bonds. C3 has two N-linked glycosylation sites — on Asn-63 and Asn-917. At both positions electron density confirms the presence of glycan moieties. The glycan on Asn-917 has been implicated in folding and correctly predicted to be 47 concealed . The base of the glycan, i.e., the first two N-acetylglucosamines is well protected in C3 by the CUB, TED, and MG8 domains. This glycan is close to the factor I cleavage sites (it is 13 Å away from cleavage site, 1298–1299); proteolysis at these sites induce conversion of C3b into iC3b that is accompanied by conformational changes exposing the glycan at Asn-917 for conglutinin 48 binding . A central feature of the C3 molecule is the reactive thioester moiety. In native C3 the thioester is protected from the surrounding solvent by close packing of the TED domain, which harbours the thioester, against domain MG8. This arrangement appears to be stabilized by the overall domain—domain arrangement in native C3, where the ANA domain plays a decisive role by holding MG8 in place with respect to domain MG3 of the β-ring. Residues Phe-1047 from the TED domain and Met-1378, Tyr-1425, and Tyr-1460 from the MG8 domain form a shield around the thioester, limiting access of small amino and hydroxyl nucleophiles. High reactivity toward hydroxyl nucleophiles requires a transformation of the thioester (Cys-988–Gln-991) to a free thiolate anion (Cys-

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988) and an acylimidazole (Gln-991–His-1104) . Comparison of the native C3 12 and C3d structures shows significant structural differences that can be correlated with this transformation from thioester to thiolate anion and acylimidazole 12,52 13 intermediates. The fact that the structures of C3d and C4d correlates best with the thiolate/acylimidazole form indicates that this is the most stable conformation. Consequently, the domain–domain interactions observed in native C3 function to maintain the thioester state of the protein. Activation of C3 induces large conformational changes. Comparison of the structures of C3 and C3c shows that the arrangements of domains in the β-chain are structurally relatively stable and that the domains of the α-chain (as exemplified by MG7 and MG8) undergo large rearrangements.

3. CONVERTASE FORMATION 53

C3 undergoes profound conformational changes upon proteolytic activation generating C3a and C3b. C3b, the activated form of C3, exposes various pro54 tein-binding sites among which is that for factor B. Factor B binding to C3b results in formation of the bimolecular complex C3bB, followed by cleavage of factor B by factor D and formation of the C3 convertase C3bBb. C3b is a large 1560-residue protein fragment (with MW = 176 kDa) for which its 12-domain organization may be inferred from the structure of full-length C3 and C3c. Factor B consists of 739 residues (MW 90 kDa) that form five domains: three Nterminal short complement-control-protein (CCP1-3) domains consisting of approx. 60 residues each, a Von Willebrand-factor type A (VWA) domain of 215 residues, and a C-terminal trypsin-like serine protease (SP) domain of 297 residues. Factor B shows no proteolytic activity toward its substrate C3 in solution. It requires complex formation with C3b and proteolytic activation for its activity. First, factor B associates with C3b. Factor B then becomes sensitive to proteolysis by factor D, which cleaves the Arg-234–Lys-235 peptide bond generating fragments Ba (consisting of the three N-terminal CCP domains) and Bb (consisting of the VWA and SP domains). Ba then dissociates from the complex. This yields the active (and short-lived) convertase complex of the alternative pathway C3bBb that will amplify the complement response by cleaving additional C3 into C3a and C3b. Binding sites for C3b on factor B have been re55 viewed previously . The proposed C3b-binding sites include peptide stretches 56 57-61 on CCP2 and CCP3 , the MIDAS site on VWA , and a possible binding site 62 on the SP domain . The resulting complexes, C3bB and C3bBb, are labile. When dissociated, factor B may re-associate with C3b, yielding a new complex; whereas fragment Bb cannot re-associate. This would suggest that dissociated Bb has undergone conformational changes, making it unsuitable for reassociation. However, conformational changes in C3b induced by binding of factor B, and not by fragment Bb, cannot be excluded at this moment. In the

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following section we discuss the putative binding sites for factor B onto C3b in the formation of the C3 convertase. Four separate sites have been identified that either present putative binding sites for factor B or are indirectly involved in binding factor B, possibly through conformational changes. One site of importance has been identified by peptide63 binding and mutagenesis studies. Peptide binding studies have shown that residues 727–745, which form the N-terminal region of the α′ chain (α′NT) in C3b, are involved in the interaction of C3b with factor B. Mutation studies in this region have pinpointed four acidic residues, i.e., Asp-730, Glu-731, Glu-736, 64 65 and Glu-737, to be involved in the convertase formation . Another study showed that cleavage fragment C3o of C3 was able to form a C3 convertase complex with factor B. In contrast, fragment C3c, which resembles C3o, cannot form a convertase complex. C3o and C3c differ by the presence of residues 933– 942 in C3o. Thus, the binding data suggest that this residue stretch (forming 5 strand β5 and its flanking loops in the CUB domain in C3) is involved in factor B binding. One possible binding site for factor B located on the β-chain of C3 has been suggested based on sequence homology. Complement C2 receptor in66 hibitor trispanning (CRIT) binds C2 . The C2 binding site was shown to reside in a short segment of the extracellular domain of CRIT. This segment has high sequence homology with a segment of C4. It was concluded that this segment in 66,67 C4 may be directly involved in binding C2 . The analogous site in C3 is formed by residues 200–220 of the β-chain. They are located on the last β-strand (βG) of MG2, the first strand (βA) of MG3, and the connecting loop. Recently, it has been shown that the C-terminal C345C domain of C3 is also involved in 68,69 the binding of factor B . In these studies various parts of the C terminus of C3 have been replaced by the corresponding parts of the snake venom homolog cobra-venom factor. The resulting chimaeras yielded enhanced lifetimes of the convertase complex, suggesting that there is at least one binding site for factor B on the C345C domain of C3. As shown in Figure 2, these data indicate four separate regions located in the top half, primarily α-chain, of the C3 molecule. To date, structures have been published of C3, C3c, and of a disulfide19 bridge engineered construct of fragment Bb . Structures of C3b and B, are not available yet. Discussions on structural implications of the putative factor B binding sites must consider the possibility of conformational changes. The C345C domain is surface exposed in both C3 and C3c. Its outward position in the molecule suggests it will be exposed in C3b as well and accessible for factor B binding. The “C3o segment” formed by residues 933–942 is surface exposed in C3 and is absent in C3c due to proteolytic processing by factor I. This segment is structurally close to the C345C domain, suggesting that, possibly even after conformational changes, factor B might bind both regions simultaneously. The “CRIT segment” (formed by residues 200–220), however, is exposed to the solvent but inaccessible to proteins in both the C3 and C3c structures. Direct involvement in factor B binding here would imply large conformational changes of the β-ring in the conversion of C3 to C3b. These changes are not observed

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a

b C345C

C345C

fB

fB

“C3o”

“C3o”

properdin

1187-

fH, 1249 CR2 1205-

1402-1435

1223

fB, CR1, CR3

1008+1010

CR2

“C3d-CR2 structure”

fH 744+ 747

730-737

fB

fH 744+747

“CRIT” 744-758

CR1 744-758

CR1

Figure 1. Possible binding sites mapped on the structure of C3. (a,b) Possible binding sites for factor B, receptors CR1, CR2, CR3, and soluble regulators factor H and properdin are indicated in black cartoon drawing, by arrows and residue numbering or “keywords” of possible binding sites. (b) C3 rotated 180° with respect to the view shown in (a).

when comparing the structures of C3 and C3c. Interestingly, the α′NT that carries the four acidic residues important for factor B binding is buried in C3 but exposed in C3c. In C3 the acidic residues occupy a cone formed by the ANA, MG3, and MG8 domains, where they are loosely structured as judged by the quality of the electron density. At Glu-737 the chain exits this cone and makes a 90º turn at 737–738 and runs through the β-ring passing residues Glu-202–Pro206 (these latter residues are part of the CRIT segment and hence implied in 66,67 745 748 factor B binding ). At FPES the chain kinks and starts MG6α of the intertwined MG6β/MG6α domain. In C3c the α′NT resides fully on the opposite, MG6 side of the molecule. In the structure of C3c residues Asp-730–Arg-742 interact with MG7. The kink 745–748 is reoriented and the chain continues into MG6. Thus, the α′NT has slid through the ring formed by the β-chain in the conversion of C3 into C3c. Consequently, residues 727–768 (as studied by anti70 body binding ) form one continuous solvent exposed region in C3c, whereas this region is on two separate sides of the molecule and partially shielded in C3. The large structural relocation of α′NT from C3 to C3c poses the question of where the α′NT region resides in C3b. In other words, on which side of C3b does factor B bind, assuming that α′NT is directly involved in factor B binding? The C3 convertase can be formed either with C3b or C3(H2O) that still has the

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ANA domain covalently attached. This observation argues for factor B binding to α′NT on the ANA side of the molecule. However, this argument is not conclusive when the possible flexibility of C3 is taken into account. The α′NT is poorly structured and its preceding loop 720–729 is not structured in C3. Thus, it is possible that ANA may reorient in the activated C3(H2O), yielding enough leverage for the α′NT to slip under the bridge. This is supported by deletion mutants of C3 lacking residues 759–765, 759–762, or 762–765 of MG6 that result in threefold or more increased concentrations of C3(H2O) in COS cell 71 supernatants . On fragment Bb three positively charged regions have been pro19 posed to bind the acidic residues of the α′NT . These regions are located on the SP domain (residues 701–708) and two patches at the VWA–SP domain intersection. These data, taken together, do not yet provide a conclusive model of factor B or fragment Bb binding to C3b. Since the C3bB and C3bBb complexes are labile, a more achievable goal is to obtain a structure of C3b that will provide the arrangements of the multiple binding sites for factor B. The labile C3bB and C3bBb complexes are stabilized by properdin. Properdin consists of 410 residues that form six or seven trombospondin (TSR) do72 73 mains and exists as either dimers, trimers, or tetramers . It increases the affinity of factor B for C3b, prevents cleavage of C3b to iC3b by factor I, and 74 enhances the convertase lifetime to approx. 30 minutes . A binding site for 75 properdin on C3b has been identified on residues 1402–1435 . This site is situated on MG8, partly on an insertion between strands βC and βC′, which changes conformation in the conversion of C3 to C3c from β–α–β to β–α–α. This site is hidden in C3 and exposed in C3c. Properdin binding indicates that this site is probably exposed in the C3bB and C3bBb complexes. In addition, the properdin–C3bBb interaction has been described to be dependent mainly on ionic 76 strength . Properdin is predominantly positively charged, especially the domains 72 TSR-3, 5, and 6 (TSR-4, 5, and 6 have been shown to be involved in C3b bind72,77 5 ing ). C3 and C3c are mainly negatively charged , and factor Bb has dispersed 19 positive- and negative-charged patches . Possibly some of the positive patches of Bb are involved in electrostatic interaction with C3b, resulting in a mainly negatively charged C3bBb complex in good agreement with ionic strengthdependent binding of the positively charged properdin. A new and totally different mode of convertase stabilization has recently been described for a comple78 ment inhibitor from S. aureus, called SCIN . This 85-residue protein not only stabilizes but also inhibits the C3bBb convertases, indicating that these two proteins probably interact in distinct ways to stabilize the convertase complex.

4. DECAY ACCELERATION When complement is not properly regulated it may cause severe damage to host tissues. To prevent self tissue from complement-mediated destruction, host cells express various regulators that downregulate convertase activity. These

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regulators form a family of related proteins, called “regulators of complement activation” (RCA), which consist almost entirely of CCP domains arranged in a beads-on-a-string fashion. They act on convertases by accelerating the dissocia79 tion of the complexes, termed “decay-acceleration activity,” and function as markers to discriminate between self and non-self. Factor H, decay-accelerating factor (DAF, CD55), and complement receptor 1 (CR1, CD35) are three important members of this family that are composed of 20, 4, and 30 CCP domains respectively. Whereas DAF and CR1 are cell-surface proteins, factor H is a soluble regulator and thus must possess additional binding sites to distinguish self from foreign. The tick-borne pathogen Borrelia burgdorferi utilizes this regulator from the infected host; it hijacks factor H by binding it to its Bb80 CRASP surface protein, thereby avoiding complement activation on its surface . In addition, other pathogens protect themselves from complement activation by expressing CCP containing proteins that have similarly decay-accelerating and 81 cofactor activities . Thus, strings of CCP domains provide an important framework for regulating convertase activity. Although the regulators differ enormously in number of CCP domains, it has been shown that typically three CCP 82 domains suffice to achieve decay-accelerating activity: e.g., CCP2-4 of DAF , 83 84 CCP1-3 of CR1 , and CCP1-4 of factor H . Various structural data have 28 27 been reported of CCP domains of these proteins: CCP2-3 , CCP3-4 , and more 29 31 recently all four CCP’s of DAF have been solved; CCP15-17 of CR1 ; 26 25 and, CCP15-16 and CCP5 of factor H (see Table 1). Many questions, however, remain unanswered with respect to multiple binding sites and conformational flexibility of the CCP containing molecules. Understanding the molecular mechanisms that underlie the decay-accelerating activities is expected to have a significant impact in developing inhibitors of complement for therapeutic pur85 poses . 63,64,70,86,87 Multiple studies indicate that residues 727–767 of C3b form an important interaction site for CR1 and factor H. Moreover, factor B, factor H, and CR1 compete for interaction with C3b or C3(H2O). As for factor B, mutagenesis studies have identified various acidic residues important for binding. Factor H binding depends on Glu-744 and Glu-747 located in domain MG6. For CR1 binding Asp-730, Glu-731, Glu-736, Glu-737, Glu-747, Glu-754, and Glu-755 of C3b are important. The latter residues are part of the α′NT region (729–745) and MG6 domain (535–577 and 746–806). These data possibly indicate that both factor H and CR1 have a primary binding site on MG6 (Figure 2). Two possible modes of action exist for decay-accelerating activity of factor H and CR1. If the factor B binding site lies on the MG6 side on C3b, then factor H and CR1 might at least in part act through steric hindrance. If, in contrast, the factor B binding site is on the ANA side of C3b, then factor H and CR1 may affect the α′NT by binding to MG6 and altering the conformation of α′NT by pulling these residues completely or in part through the β-ring. The effect of factor H, however, must be reversible, since C3b can bind factor B after factor H is re88 moved from a C3b–H complex . Furthermore, factor H has at least one addi-

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tional interaction site on C3b, i.e., helices α9–α11 and neighboring loops in the 89,90 TED domain (1187–1249) . For DAF the binding site on C3b is unknown, 91 though it has been shown that DAF can bind to C3b without Bb present . Important sites for interaction with factor H, DAF, and CR1 on fragment Bb have 60 been identified on the VWA domain . Mutations around helix α1 and neighboring loops were shown to be important for resistance to decay acceleration by all three regulators. A second site important for interaction with DAF and CR1, but not factor H, was shown to reside on helices α4 and 5. The interactions of the regulators with C3b, and its homolog C4b of the classical pathway, have been found to be dominated by electrostatic interactions, as demonstrated by the salt 76,92 dependency . Furthermore, mutation studies on the regulators have indicated 28,31,83,93-95 that positive charges are favorable in binding to C3b (and C4b) . This is in good agreement with the predicted overall negative charge of the C3bBb complex, as discussed above. Although the recently solved DAF structure indicates that there is also a negatively charged region on CCP3 and 4 that might be 29 important for the interaction with the C3bBb convertase . Possibly the positively charged region on MG1 and MG5 of C3b or one of the positive patches in factor Bb is involved in this interaction. As of yet it is not clear what the mode of action for these regulators is; however, as factor Bb cannot bind C3b after its dissociation from the convertase, it is possible that the regulators induce a conformational change in factor Bb that is unfavorable for binding to C3b and thus accelerate the decay.

5. COFACTOR ACTIVITY Next to decay-accelerating activity, factor H and CR1 serve, as does membrane96-99 cofactor protein (MCP), as cofactors in the cleavage of C3b by factor I , i.e., these proteins exhibit so-called “cofactor activity.” Like factor H and CR1, MCP is a member of the RCA family of proteins. It consists of four N-terminal CCP domains, an O-glycosilated serine, threonine and proline-rich region, a trans100 membrane region, and a C-terminal intracellular domain . The structure of the 30 CCP1-2 pair has been solved . CCP3–4 are sufficient for binding to C3b; how101 ever, CCP2 is required for cofactor activity . The cofactor-mediated factor I proteolytic inactivation of C3b involves three cleavages in the CUB domain of C3b. The first cleavage, between residues Arg-1281–Ser-1282 in loop β6–β7 f (i.e., the loop connecting strands β6 and β7) of the CUB part of CUB, generates iC3b1. The second cleavage occurs between Arg-1298–Ser-1299, situated in f 102 strand β8 of CUB and generates fragment C3f (2 kDa) and iC3b2. The third g cleavage, between Arg-932–Ser-933 in loop β3′–β4 of the CUB part of CUB, 103 results in the formation of C3dg (40 kDa) and C3c (135 kDa). This indicates variable binding modes of the protease, factor I, with respect to the CUB domain that carries the three scissile bonds. Residues of the TED domain that are impor89 tant for factor H binding (1187–1249) lie adjacent to CUB (Figure 2), suggest-

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ing that factor H binding to this site may be involved directly in orienting factor I with respect to cleavage sites in CUB. Moreover, factor H, CR1, and MCP are possibly involved in unraveling of the CUB domain, enabling factor I to bind and cleave CUB three times. So far no interaction sites on C3b for MCP are known. Mutations in C3(H2O), however, that affect factor H and CR1 binding have no effect on MCP binding, indicating a different mode of interaction for 87 MCP . Taken together, the available data suggest a model in which the compact arrangement of the TED domain in C3 is relaxed and opened up in C3b (with TED attached to the target surface), while the CUB domain is gradually unfolded in iC3b due to cleavages, until the bond between C3dg and C3c is finally severed.

6. SIGNALING ROLES OF C3B FRAGMENTS The factor I- and factor H-, CR1- or MCP-mediated conversion of C3b to 104 iC3b induces large structural changes and alters the binding properties of the 3 molecule . Cleavage of C3b into iC3b results in loss of factor B and properdin binding; and gain of complement receptor 2 (CR2, CD21), 3 (CR3, αMβ2, CD11b/ CD18, Mac-1) and 4 (CR4, αXβ2, CD11c/CD18, p150,95); and conglu54 tinin binding . At the same time, the binding modes for factor H and CR1 change, since different residues of the α′NT and the MG6 domain become im64,87 portant for interaction with factor H and CR1 . Finally, proteolysis leads to two separate products — C3c released into the medium and C3dg fragment attached to the surface. iC3b is the opsonin that facilitates phagocytosis of antigens by leukocytes. 105-107 Recognition of iC3b by leukocytes is mediated by the integrin CR3 . The inserted (I) domain plays a critical role in ligand binding to CR3, similar to other integrins, e.g., CR4. In both cases, the binding site involves a metal-ion108 dependent adhesion site (MIDAS) in the I domain . Various structural stud109,110 ies have shown that ligand binding to a MIDAS site involves an acidic residue of the ligand that completes the coordination sphere of the divalent ion bound at the MIDAS. In the integrins the ligand binding induces a large structural change of the C-terminal α-helix in the I domains, which activates the in109-111 tegrin . These I domains are structurally homologous to the VWA domain of 33,34 factor B . Also in the case of factor B, the MIDAS site is important for the 112 metal-dependent interaction with C3b . This homology between I and VWA domains have prompted others to propose a similar activation mechanism for 55,113 factor B in the formation of the C3bBb convertase . Nonetheless, CR3 and CR4 bind iC3b, whereas factor B binds C3b. A possible binding site for both 64 factor B and CR3 has been identified on the α′NT region of C3b and iC3b . This would suggest that there are differences in or near this interaction site between C3b and iC3b or that both factor B and CR3 bind multiple regions in C3b

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and iC3b respectively. The latter is supported by various studies that show that 114,115 regions outside the I domain contribute to iC3b interaction . Surface-bound iC3b and C3dg play critical roles in B-cell stimulation and the initiation of adaptive immune responses. Coligation of C3dg or iC3b with CR2 and the B-cell antigen receptor complex amplifies a signal transduction 2 cascade through the CR2/CD19/CD81 co-activation complex . CR2 is a member of the RCA family. It consists of 15 or 16 N-terminal CCP modules, a 24 residue transmembrane domain, and a C-terminal 34-residue intracellular part. Only 116 CCP1 and 2 are necessary for binding to C3dg or iC3b . The CR2 binding site is located on the TED domain. Various studies have provided information on the 117-124 interaction and binding sites in the iC3b–CR2 and C3d–CR2 complexes .A few years ago, the structure of C3d in complex with CCP1-2 of CR2 was 36 solved . This structure shows extensive main-chain interactions between C3d and CCP2 of CR2 and no direct interaction between CCP1 of CR2 and C3d. However, the observed structural arrangement of the complex is controversial. Very recently it was shown that, in addition, the CCP1 domain of CR2 probably 125,126 makes direct contacts to C3d . Nevertheless, the exact site of interaction of CCP1 on C3d still remains unknown. Though significant structural differences are apparent between the structures of C3d in the CR2–C3d complex and the TED domain in C3, the CR2–CCP2 binding site is very similar in the two structures — ruling out that conformational changes play a role for this subsite. Moreover, this site is completely exposed in native C3 (Figure 2). This indicates that CCP1 of CR2 discriminates between the conformational states of C3 vs. iC3b and C3dg, because its binding site is either inaccessible in C3 or has changed its conformation.

7. CONCLUDING REMARKS The structures of native C3 and its major fragment C3c have provided a wealth of structural insights into the central protein of the complement system. The proteolytic activation steps, generating the important fragments C3b and iC3b, are thought to induce significant conformational changes in the molecule, yielding protein molecules with distinct binding properties. The structures of C3 and C3c reveal the extent of conformational changes that may be expected. These structures together with the large amount of biochemical, mutagenesis, and binding data available on C3, its fragments, and the various interacting partners provide for the first time a detailed map of the various proposed binding sites. Still, many questions remain unanswered and additional structural data and sitedirected mutagenesis experiments, now made possible in a more rational way, are required to elucidate the complete complexity of the central component of the complement system.

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8. ACKNOWLEDGMENTS We are grateful to Fin Milder, Michael Hadders and Lucio Gomes (Utrecht), and John D. Lambris (Philadelphia) for critically reading the manuscript. We thank the members of the laboratory for Crystal and Structural Chemistry for support and discussions. Financial support by the Council for Chemical Sciences of the Netherlands Organization of Scientific Research (NWO/CW) is gratefully acknowledged.

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T. S. Jokiranta, J. Hellwage, V. Koistinen, P. F. Zipfel and S. Meri, Each of the three binding sites on complement factor H interacts with a distinct site on C3b, J Biol Chem 275, 27657–27662 (2000). C. L. Harris, R. J. Abbott, R. A. Smith, B. P. Morgan and S. M. Lea, Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55), J Biol Chem 280, 2569–2578 (2005). M. Krych, L. Clemenza, D. Howdeshell, R. Hauhart, D. Hourcade and J. P. Atkinson, Analysis of the functional domains of complement receptor type 1 (C3b/C4b receptor; CD35) by substitution mutagenesis, J Biol Chem 269, 13273–13278 (1994). M. Krych-Goldberg, R. E. Hauhart, V. B. Subramanian, B. M. Yurcisin 2nd, D. L. Crimmins, D. E. Hourcade and J. P. Atkinson, Decay accelerating activity of complement receptor type 1 (CD35). Two active sites are required for dissociating C5 convertases, J Biol Chem 274, 31160–31168 (1999). A. M. Blom, A. F. Zadura, B. O. Villoutreix and B. Dahlback, Positively charged amino acids at the interface between alpha-chain CCP1 and CCP2 of C4BP are required for regulation of the classical C3-convertase, Mol Immunol 37, 445–453 (2000). L. A. Kuttner-Kondo, L. Mitchell, D. E. Hourcade and M. E. Medof, Characterization of the active sites in decay-accelerating factor, J Immunol 167, 2164–2171 (2001). M. K. Pangburn, R. D. Schreiber and H. J. Muller-Eberhard, Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution, J Exp Med 146, 257–270 (1977). G. D. Ross, J. D. Lambris, J. A. Cain and S. L. Newman, Generation of three different fragments of bound C3 with purified factor I or serum. I. Requirements for factor H vs CR1 cofactor activity, J Immunol 129, 2051–2060 (1982). T. Seya, J. R. Turner and J. P. Atkinson, Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b, J Exp Med 163, 837–855 (1986). R. G. Medicus, J. Melamed and M. A. Arnaout, Role of human factor I and C3b receptor in the cleavage of surface-bound C3bi molecules, Eur J Immunol 13, 465– 470 (1983). M. K. Liszewski, T. W. Post and J. P. Atkinson, Membrane cofactor protein (MCP or CD46): newest member of the regulators of complement activation gene cluster, Annu Rev Immunol 9, 431–455 (1991). E. M. Adams, M. C. Brown, M. Nunge, M. Krych and J. P. Atkinson, Contribution of the repeating domains of membrane cofactor protein (CD46) of the complement system to ligand binding and cofactor activity, J Immunol 147, 3005–3011 (1991). R. A. Harrison and P. J. Lachmann, Novel cleavage products of the third component of human complement, Mol Immunol 17, 219–228 (1980). P. J. Lachmann, M. K. Pangburn and R. G. Oldroyd, Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies, J Exp Med 156, 205–216 (1982).

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104. D. E. Isenman, Conformational changes accompanying proteolytic cleavage of human complement protein C3b by the regulatory enzyme factor I and its cofactor H. Spectroscopic and enzymological studies, J Biol Chem 258, 4238–4244 (1983). 105. T. Ueda, P. Rieu, J. Brayer and M. A. Arnaout, Identification of the complement iC3b binding site in the beta 2 integrin CR3 (CD11b/CD18), Proc Natl Acad Sci USA 91, 10680–10684 (1994). 106. T. Kamata, R. Wright and Y. Takada, Critical threonine and aspartic acid residues within the I domains of beta 2 integrins for interactions with intercellular adhesion molecule 1 (ICAM-1) and C3bi, J Biol Chem 270, 12531–12535 (1995). 107. S. L. McGuire and M. L. Bajt, Distinct ligand binding sites in the I domain of integrin alpha M beta 2 that differentially affect a divalent cation-dependent conformation, J Biol Chem 270, 25866–25871 (1995). 108. V. A. Ustinov and E. F. Plow, Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2, Biochemistry 44, 4357–4364 (2005). 109. J. Emsley, C. G. Knight, R. W. Farndale, M. J. Barnes and R. C. Liddington, Structural basis of collagen recognition by integrin alpha2beta1, Cell 101, 47–56 (2000). 110. M. Shimaoka, T. Xiao, J. H. Liu, Y. Yang, Y. Dong, C. D. Jun, A. McCormack, R. Zhang et al., Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation, Cell 112, 99–111 (2003). 111. J. Emsley, S. L. King, J. M. Bergelson and R. C. Liddington, Crystal structure of the I domain from integrin alpha2beta1, J Biol Chem 272, 28512–28517 (1997). 112. Z. Fishelson, M. K. Pangburn and H. J. Muller-Eberhard, C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3b, Bb (Ni) complex, J Biol Chem 258, 7411–7415 (1983). 113. J. Hinshelwood and S. J. Perkins, Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: a solution study by circular dichroism, fourier transform infrared and (1)H NMR spectroscopy, J Mol Biol 298, 135–147 (2000). 114. Y. M. Xiong and L. Zhang, Structure-function of the putative I-domain within the integrin beta 2 subunit, J Biol Chem 276, 19340–19349 (2001). 115. Y. Li and L. Zhang, The fourth blade within the beta-propeller is involved specifically in C3bi recognition by integrin alpha M beta 2, J Biol Chem 278, 34395– 34402 (2003). 116. C. A. Lowell, L. B. Klickstein, R. H. Carter, J. A. Mitchell, D. T. Fearon and J. M. Ahearn, Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2, J Exp Med 170, 1931–1946 (1989). 117. L. Clemenza and D. E. Isenman, Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21), J Immunol 165, 3839– 3848 (2000). 118. H. Molina, S. J. Perkins, J. Guthridge, J. Gorka, T. Kinoshita and V. M. Holers, Characterization of a complement receptor 2 (CR2, CD21) ligand binding site for C3. An initial model of ligand interaction with two linked short consensus repeat modules, J Immunol 154, 5426–5435 (1995). 119. J. D. Lambris, V. S. Ganu, S. Hirani and H. J. Muller-Eberhard, Mapping of the C3d receptor (CR2)-binding site and a neoantigenic site in the C3d domain of the third component of complement, Proc Natl Acad Sci USA 82, 4235–4239 (1985).

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120. I. Esparza, J. D. Becherer, J. Alsenz, A. De la Hera, Z. Lao, C. D. Tsoukas and J. D. Lambris, Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21), Eur J Immunol 21, 2829–2838 (1991). 121. R. J. Diefenbach and D. E. Isenman, Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2, J Immunol 154, 2303–2320 (1995). 122. K. R. Kalli, J. M. Ahearn and D. T. Fearon, Interaction of iC3b with recombinant isotypic and chimeric forms of CR2, J Immunol 147, 590–594 (1991). 123. M. R. Sarrias, S. Franchini, G. Canziani, E. Argyropoulos, W. T. Moore, A. Sahu and J. D. Lambris, Kinetic analysis of the interactions of complement receptor 2 (CR2, CD21) with its ligands C3d, iC3b, and the EBV glycoprotein gp350/220, J Immunol 167, 1490–1499 (2001). 124. D. Morikis and J. D. Lambris, The electrostatic nature of C3d-complement receptor 2 association, J Immunol 172, 7537–7547 (2004). 125. J. P. Hannan, K. A. Young, J. M. Guthridge, R. Asokan, G. Szakonyi, X. S. Chen and V. M. Holers, Mutational analysis of the complement receptor type 2 (CR2/ CD21)–C3d interaction reveals a putative charged SCR1 binding site for C3d, J Mol Biol 346, 845–858 (2005). 126. H. E. Gilbert, J. T. Eaton, J. P. Hannan, V. M. Holers and S. J. Perkins, Solution structure of the complex between CR2 SCR 1-2 and C3d of human complement: an X-ray scattering and sedimentation modelling study, J Mol Biol 346, 859–873 (2005).

21 DISEASE-ASSOCIATED SEQUENCE VARIATIONS IN FACTOR H: A STRUCTURAL BIOLOGY APPROACH Andrew P. Herbert, Dinesh C. Soares, Michael K. Pangburn, and Paul N. Barlow

1. INTRODUCTION The complement system eliminates infectious and unwanted particles from the 1, 2 body. It has destructive potential and must be regulated so that it can carry out its role with minimum damage to host tissue. A failure to properly control complement underlies the debilitating symptoms of a long list of autoimmune, degenerative, and iatrogenic disorders. A subset of the 30 or so proteins that make up the complement system is dedicated to regulating the others. Membrane-associated regulators of complement activation (RCA) are present on all host cells, where they have a protective function. Soluble regulators augment the membrane-bound ones and, additionally, act to prevent complement activation on self-surfaces not enclosed by a cell membrane. Because there is functional degeneracy amongst the RCA proteins, individuals with variant forms may escape consequences for most of their lives, but they may be predisposed to medical conditions that arise from inappropriate or misdirected complement activation. Thus, some mutations in factor H (fH) — 3, 4 a soluble RCA — predispose to atypical hemolytic uremic syndrome (aHUS),

Andrew P. Herbert,* School of Chemistry/Institute of Structural and Molecular Biology, University of Edinburgh, King’s Buildings, Edinburgh EH9 3JJ, UK, and Department of Biochemistry, University of Texas Health Science Center, 11937 U.S. Hwy 271, Tyler, Texas 75703, USA. Dinesh C. Soares,* School of Chemistry/Institute of Structural and Molecular Biology, University of Edinburgh, King’s Buildings, Edinburgh EH9 3JJ, UK. Michael K. Pangburn Department of Biochemistry, University of Texas Health Science Center, 11937 U.S. Hwy 271, Tyler, Texas 75703, USA. Paul N. Barlow,** Joseph Black Chemistry Building, University of Edinburgh, West Mains Road, Edinburgh EH9 3JR, UK. *These authors contributed equally to this publication. **Corresponding author.

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a rare often fatal kidney disease. A different amino-acid substitution in fH increases the risk of contracting the eye condition, age-related macular degenera5-8 tion (AMD), a common cause of irreversible sight loss in people over 60. 9-11 We have determined structures for 5 of the 20 component modules of fH (and unpublished data). Our understanding of fH structure has now reached a point where we can model by homology the other modules, and predict structural and functional consequences of disease-linked amino-acid substitutions. Here we describe the results of such an exercise. The sequence data and the 3Dstructural models used are all available online at http://www.bru.ed.ac.uk/ ~dinesh/ccp-db.html. First, to place this work in context, background information on complement, fH, and its associated diseases is presented.

2. REGULATION OF THE COMPLEMENT SYSTEM Key to activation and regulation of complement are convertases — proteolytic enzymes that drive the amplification cascade at the heart of the complement system. The bimolecular alternative pathway (AP) C3 convertase consists of C3b plus factor Bb (i.e., C3b.Bb). The equivalent classical pathway (CP) convertase is C4b.2a. The relative rates of convertase assembly and decay dictate the extent and duration of a complement response. The generation of C3b and C4b determines the rate at which the population of convertase complexes accumulates. On the other hand, the convertases are unstable, with a lifetime in the region of 70–90 seconds; and once the components have separated they cannot 12 reassemble. Five human proteins (the founding members of the RCA family) act by shortening the lifetime of the convertases still further, or by inhibiting their assembly. The best-characterized modes of action are: cofactor activity (CA) — some regulators are cofactors for factor I-catalyzed destruction of C3b and/or C4b; and decay accelerating activity (DAA) — through an unknown mechanism, some regulators catalyze the disassembly of the convertase into its components. The five RCAs have overlapping functional profiles — for example, fH has both CA and DAA but acts only on the AP convertase, whereas membrane cofactor protein (MCP) has only CA but acts on both pathways. Of the five regulators, fH and C4b-binding protein are circulating, fluid-phase proteins, while MCP, decay-accelerating factor (DAF), and complement receptor type 1 are all membrane-associated. All five proteins are composed entirely, or almost entirely, from multiple examples of a domain-type known as the complement control protein (CCP) module (also known as the short consensus re12 peat). The remainder of this article will focus on fH, although genetic varia13 14 tions in MCP and in factor I have also been linked to aHUS.

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3. FACTOR H 15

Mature factor H is a 155-kDa plasma glycoprotein (~250–800 µg/ml) consisting of 1213 residues that form 20 tandemly arranged CCP modules (fH~1–20). It is encoded by the gene FH1 within the RCA gene cluster that is located on chromosome 1 at position 1q32. FH1, along with genes for fH-related (fHR-) proteins 1, 2, 3, 4a, 4b, and 5, and the gene for blood-clotting factor FXIIIb, lies 16 within a 650 Kb–2.2 Mb subcluster that is separated by a genetic distance of 7 17, 18 There is a high degree to 22.5 centiMorgans from the remaining RCA genes. of similarity between the CCP modules of fHR-proteins and those of fH. The fH-splice variant, fH-like protein, contains seven N-terminal CCP modules identical to those of factor H, and a four-residue C-terminal extension. Most of the modules in the factor H family belong to cluster-B and cluster-I according to 19 Soares et al. Clusters-B and –I are not represented in other RCA proteins. Factor H probably diverged from the other RCAs early in the evolution of the com20 plement system. The fH modules are mostly encoded by discrete exons, with exon boundaries at random positions within the linkers — the exception is fH CCP 2, which is encoded by a split exon. Nearly all exons in RCA genes have phase 1 introns, a signature of domain shuffling. There are eight potential or known N-glycosylation sites in fH (two on CCP 15, one each on CCPs 9, 12, 13, 14, 17, and 18). Each CCP module contains four Cys residues, forming two disulfides, and 18 of the modules have a Trp residue between Cys III and IV that also forms part of the consensus sequence for a CCP module (fH~10 and fH~20 have a Leu residue instead). The CCP modules in fH range in length from 51 amino acids to 62 (inclusive of their Nand C-terminal Cys residues), and the linkers between them vary from three to eight residues. The 3D structures for 5 of the 20 CCP modules in factor H have been determined. The dominant structural features of CCP modules consist of five extended segments aligned approximately with the long axis of the oblate module. They run up-down-up-down-up so that N and C termini are at opposite ends of the module. For all or part of their lengths these extended segments form βstrands, arranged in small antiparallel sheets. The segments or strands are joined by loops and turns that are sites of insertions or deletions. Most loops and turns lie close to the N and C termini, but they may also form lateral bulges. Particularly prominent in many structures is the “hypervariable loop” (Figure 1) corresponding to a region of high sequence, length, and structural variation. Conservatively replaced residues in the extended segments contribute side-chains to a compact hydrophobic core that is bounded at either end by the disulfide bridges. Some Gly and Pro residues are well conserved, but levels of sequence identity across the family are low — the scaffold-like structure can support variation at most amino-acid positions, in addition to insertions and deletions in loops. Module 20 differs from the other CCP module structures solved to date, consistent with its divergent sequence that lacks the consensus Trp residue.

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Figure 1. 3D-model of factor H CCP module 7. Cartoon schematic PyMol (http://www.pymol.org) representation of module depicting a typical CCP-module fold. β-strands numbered and disulfides and tryptophan are drawn as sticks. The AMD-associated SNP, 402, and aHUS-implicated variant, 400, located in the hypervariable loop are shown as sticks.

The overall structure and flexibility of fH depends upon the nature of the junctions between consecutive modules and any higher-order effects involving contacts between non-neighboring modules. Two of the solved structures in10 volve module pairs (and unpublished data). The fH 15–16 pair appears flexible at the linker between modules, while the fH 19–20 pair is significantly less flexible — its comparative rigidity stems from the shorter linker between modules 19 and 20, and the bulky side-chains of the three linker residues. CCP mod21 ule pairs from other proteins also display a range of flexibilities, so it is probable that along its length fH has some rigid sections and others that are flexible. 22 Such a notion is consistent with electron microscopy and other evidence, in23 cluding small-angle X-ray scattering and analytical ultracentrifugation. Of note in factor H are the longer linkers (up to eight residues) between its central (CCPs 12–14) modules. These could represent sites where the chain of modules bends back upon itself bringing non-consecutive modules into proximity, and there is 23 some low-resolution structural data to support this — but they could equally well correspond to sites of extreme flexibility.

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4. FUNCTIONAL SITES OF FACTOR H Despite the apparent simplicity of its repetitive structure, factor H is functionally complex. Along its length it carries numerous binding sites (Figure 2) for both 24 25 26 host and pathogen-borne protein, and carbohydrate, ligands. The four Nterminal modules harbor a C3b-binding site and this region is the locus of the CA and DAA of factor H, while the two C-terminal modules form another C3bbinding site (specifically for the C3d component of C3b) and are crucial for self and non-self discrimination. A third set of modules involving CCPs 12–14 are thought to bind to another region of C3b. Three sites recognize polyanions, such as glycosaminoglycans (GAGs) and sialic acid (CCPs 7; 12–14; 19,20). Factor 27 28 H binds additional ligands — adrenomedullin, L-selectin, and C-reactive pro29 tein — and numerous pathogen proteins as shown in Figure 2.

Figure 2. Factor H, a multiadhesive protein. Arrangement of the 20 CCP modules of fH taken from SMART (http://smart.embl-heidelberg.de/). Vertical lines indicate sites of interaction with ligand. Abbreviations used in figure; CRP = C-reactive protein, CRASP = Complement regulator-acquiring surface protein, Osp = Outer surface protein, Hic = factor H-binding inhibitor of complement.

Given the inherent flexibility of some intermodular junctions, it has been suggested that modules contribute cooperatively or in a combinatorial fashion to the biological role of factor H, so that binding at one site could modulate binding or function at other sites.

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5. ATYPICAL HEMOLYTIC UREMIC SYNDROME Hemolytic uremic syndrome (HUS) causes the majority of kidney failures in children and is typically associated with bacterial infections. It is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal fail30,31 A related disorder of the microvasculature is thrombotic thrombocytoure. penic purpura (TTP). TTP occurs mainly in adults, and is normally accompanied by neurological deficits; but TTP and HUS are regarded by many clinicians as a different manifestation of the same pathophysiological process. The rare atypical HUS is not linked to infection, but is sporadic or familial. While full recovery from typical HUS is the norm, the long-term diagnosis for sufferers of aHUS — 5–10% of cases of HUS — is unfavorable. A growing list of genetic mutations and SNPs (Table 1, see below) within the 3,32-36 These include both misRCA gene cluster has been associated with aHUS. sense mutations yielding normal plasma levels of factor H and mutations leading to fH deficiency. Factor H deficiency has also been linked to membranoproliferative glomerulonephritis (MPGN), and (in one case) to type III collagen 37 glomerulopathy. 38,39 Missense mutations associated with aHUS cluster in fH CCPs 19 and 20, 39 and a haplotype block involving alleles spread across the RCA gene cluster is strongly linked to disease. A reasonable biological model would involve a deficit in fH-mediated complement regulation on the basement membrane exposed by the large fenestrations of the capillary endothelium within the glomerulus. If accompanied by a deficit of one or more cell-surface complement regulators, the defensive capacity of nearby cells might be overwhelmed. Alternatively, inadequate protection of endothelial cells against complement activation might lead to compromise of the endothelial cell layer and exposure of the underlying basement membrane that is not protected by a fully functional fH. Environmental triggers, such as immunosuppressive drugs, cancer therapies, and oral contraceptives, are also involved. Thus, the disease is probably multifactorial, with fH dysfunction playing a central role. Intriguingly, a factor H mutation was identified in a TTP patient who developed renal failure — the mutation was absent in a sibling who also had TTP but presented with exclusively neurological symp40 toms. Overall, the correlation of mutations/SNPs with disease penetrance, disease severity, prognosis, and choice of treatments remains obscure in the case of aHUS.

6. AGE-RELATED MACULAR DEGENERATION As a disease of the macula, a region of densely packed photoreceptors toward the center of the retina, AMD affects central-field vision, leading to significant loss in quality of life for up to 50 million sufferers worldwide. AMD is a complex disease, probably involving a combination of genetic factors and environmental triggers, such as diet and (especially) smoking. The characteristic feature

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of early AMD is the presence of large fatty yellow deposits — drusen — between the retinal pigment epithelium (RPE) that underlies and nourishes the photoreceptors, and the layer of extracellular matrix known as Brüch’s membrane (BM) that separates the RPE from its blood supply in the choroid. The drusen intrude into the RPE and contain cell debris in addition to copious quantities of immune-associated molecules. In particular, they contain both comple41 ment regulators and the C5b–C9 membrane attack complex. Hageman et al. cleverly noticed a connection between AMD and MPGN type II, a rare kidney 42 disease. Some MPGN II patients developed ocular drusen that closely resembled those of AMD. Glomerulonephritis had been connected to fH dysfunction or deficiency, leading Hageman et al. to perform an SNP analysis of FH1 in two 7 large cohorts of AMD patients and matched control sets. This led to identification of both risk and protective haplotypes clustered in the N-terminal half of factor H (Table 1, see below). The most common at-risk haplotype — present at a frequency of 50% in AMD cases and 29% in controls — included the SNP Y402H. Simultaneously, three other groups had independently identified Y402H 5,6,8 of fH as a susceptibility-SNP. A plausible disease model would involve an instigating, localized, release of debris from necrotic RPE cells that is deposited in the space between the RPE and BM. The deposits would include potent stimulators of the alternative pathway. In the absence of fully functional fH, they could trigger a self-perpetuating cycle of inflammatory events involving bystander cell damage, further release of cell debris, and accumulation of immune molecules. This process would lead over time to formation and then expansion of drusen to the point where they compromise the RPE and incite phenomena associated with the later stages of AMD — geographic atrophy and choroidal neovascularization. Several other genes have also been linked to AMD, and it may well be that disease instigation and progression depends upon additional proteins malfunctioning, as well as environmental factors. Knowledge of the likely functional and structural consequences of sequence variations in fH will thus help to unravel the complex relationship between genotype and phenotype for both aHUS and AMD. The remainder of this paper deals with the likely structural ramifications of polymorphisms in fH.

7. MODELED MODULES OF FACTOR H The 3D structure of fH has not been determined. Therefore, the structural outcomes of amino-acid substitutions must be considered at the level of its 20 individual CCP modules and the interfaces between them. Experimentally deter9 mined 3D structures are available for fH module numbers: 5 (coordinates 10 available at http://www.bru.ed.ac.uk/~dinesh/ccp-db.html), 15 and 16 (PDB ID: 1HFH), and 19 and 20 (unpublished, PDB ID: 2BZM). Additionally, homology-derived models for eight modules — numbers 2, 3, 4, 11, 12, 14, 17, and 18 — are downloadable from the CCP-module model database (http://www.

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bru.ed.ac.uk/~dinesh/ccp-db.html). This was created using a large-scale model19 ing procedure described in Soares et al. 2005. Two other modules — numbers 1 and 7 — were homology-modeled individually for the purposes of the current paper.

Figure 3. Disease-associated variants mapped on the multiple sequence alignment of the 20 factor H CCP modules. β-strands (arrows) indicated above sequences along with hypervariable loop (HVloop). Consensus residues for each position in the alignment (the greater the height of the residue, the greater the conservation) are shown below (http://weblogo.berkeley.edu/). Variants (Table 1) highlighted as follows: bold letters indicate the variant is expressed at normal or high levels in plasma; underlined bold residues indicate the variant is expressed at low levels or is absent; italicized bold residues indicate variants where no information is available.

Modeling of each of these two CCP modules was based upon the structure 43 with the most similar sequence as identified in the PDB (http://www. 44 rcsb.org/pdb) with a BLAST search, and employed the program Modeller re45 lease 8 version 1. Modules 1 and 7 were based upon templates DAF~2 (PDB ID: 1OK3) and VCP~4 (PDB ID: 1G40), respectively. The sequence alignments between target and template sequences were created based on initial multiple sequence alignments, which were further manually edited to ensure the most plausible alignment of conserved amino-acid residues and secondary structure elements between the templates and those strongly predicted by PsiPred version 46 2.4 in the target sequences. Disulfide bridges were automatically restrained during model building. Twenty models were generated in each case, and the one

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with the lowest objective function score was selected as the representative model. For each representative model, non-identical side-chains between template and target were optimized using the side-chain replacement program, SCWRL 47 version 3. The models were then protonated, and subjected to energy minimization carried out in SYBYL version 6.9 (Tripos Associates, St. Louis, MO, USA) using the Tripos forcefield (30 steps steepest descent followed by 30 steps conjugate gradient). The models were finally checked for valid stereochemistry 48 using PROCHECK version 3.5.4. The accuracy of these two models may be inferior relative to those created previously by the large-scale modeling strategy because of lower sequence similarity between targets and templates. They represent, however, the product of the best available modeling techniques and are available for download at the CCP module model database (http://www.bru.ed.ac.uk/~dinesh/ccp-db.html).

8. PREDICTED STRUCTURAL CONSEQUENCES OF AMINO-ACID SUBSTITUTIONS Structural predictions (summarized in Table 1, below) can be combined with knowledge of phenotype and of the locations of binding sites on factor H, to help dissect the contribution of the mutation or SNP (Figure 3) to the disease process. For example, our model suggests that the SNP involving residue position 402 — a His in the population at risk of AMD, otherwise a Tyr — is not likely to disrupt the structure of module 7 since its side-chain is largely solventexposed (Figure 1). Nor is it likely to affect the orientation or flexibility of module 7 with respect to its neighboring modules. A local effect on the structure or flexibility of the hypervariable loop cannot be ruled out. Module 7 is known to form part of a binding site for polyanions (one of three such sites in factor H), and also to contribute to a binding site for C-reactive protein. It is therefore likely that its hypervariable loop is involved in direct contact with ligand, and that substitution with His perturbs one or other interaction so as to compromise affinity. Even a minor effect on binding could be critical since incomplete shutdown of the AP, over a time course of years, could promote gradual enlargement of ocular drusen. Many individuals with the at-risk allele do not have AMD, so the disease process may require some triggering event — for example compromise of cells in the RPE due to another disease or an environmental insult — or there might be additional gene products involved. The occurrence of another structurally neutral (according to our predictions) mutation in an adjacent residue, Q400K (Figure 1), in an aHUS patient reinforces the hypothesis that there is a binding site in this hypervariable region of module 7. Table 1 summarizes the predicted structural consequences for a selection of disease-linked mutations and SNPs of factor H. Many of these involve a consensus Cys; or (not shown) they involve the introduction of stop codons/deletions.

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Table 1. Disease-Associated Factor H Variants Variant I62V

7

R78G

33

CCP

Predicted effect

Pathology

fH level

1

No effect on structure

AMD

High n.r.

1

Structural disruption

HUS

R127L34

2

Protein more “sticky?”

MPGN

Low

Q400K34

7

No effect on structure

HUS

n.r.

Y402H7

7

No effect on structure

AMD

High

C431S34

7

Structural disruption

MPGN

Low

C536R, C959Y37,50

9,16

Structural disruption

CIIIG

Absent

C630W51

11

Structural disruption

HUS

Normal

C673Y34

11

Structural disruption

HUS

Low/Normal

C673S34

11

Structural disruption

MPGN

Low

V835L52

14

Structural disruption

HUS

n.r.

E850K51

14

No effect on structure

HUS

High Normal

S890I40

15

Protein more "sticky?"

HUS

H893R34

15

Disrupts junction

HUS

Low

C915S34

15

Structural disruption

HUS

Low

E936D33

16

No effect on structure

HUS

Normal

Q950H33

16

No effect on structure

HUS

n.r.

Y951H33

16

Disrupts junction?

HUS

n.r.

T956M38,39

16

No effect on structure

HUS

Normal/High

C973Y52

16

Structural disruption

HUS

Low/Absent

W978C51

16

Structural disruption

HUS

Normal

T987A52

16/17

Disrupts junction

HUS

n.r.

V1007C52

17

Protein cross-linked?

HUS

Normal

Y1021F51

17

Disrupts junction?

HUS

Normal

C1043R51

17

Structural disruption

HUS

Normal

Q1076E35,51

18

No effect on structure

HUS

Normal

D1119G35

19

Disrupts GAG-binding sitea

HUS

Normal

V1134G51

19

Structural disruption

HUS

High

Y1142D51

19

Disrupts junction

HUS

High

W1157R51

19

Structural disruption

HUS

High

C1163W33

19

Structural disruption

HUS

n.r.

R1182S52

20

Disrupts GAG-binding site

HUS

n.r.

W1183R51,53

20

Disrupts GAG-binding site

HUS

Normal

W1183L34,38,39

20

Disrupts GAG-binding site

HUS

Normal/High

T1184R35

20

Disrupts GAG-binding site

HUS

Normal

L1189F39,54

20

C3b-binding?

HUS

Normal

L1189R38,39

20

C3b-binding?

HUS

Normal/High

S1191L35,51

20

Disrupts GAG-binding site

HUS

n.r.

S1191W39,54

20

Disrupts GAG-binding site

HUS

Normal

G1194D33,55

20

Structural disruption

HUS

n.r.

V1197A33,35,38,39,56

20

Structural disruption

HUS

Low/Normal

E1198A33

20

Disrupts GAG-binding site

HUS

n.r.

DISEASE-ASSOCIATED FACTOR H SEQUENCE VARIATIONS 34

323

F1199S

20

Structural disruption

HUS

Low

R1210C33,39,51,55-57

20

Protein cross-linked?

HUS

Normal/High

R1215G3,35

20

Disrupts GAG-binding site

HUS

Normal

R1215Q56 P1226S51

20 20

Disrupts GAG-binding site Structural disruption

HUS HUS

Normal High

a The GAG-binding site on fH~19,20 has been delineated experimentally (AP Herbert, D Uhrin, M Lyon, MK Pangburn and PN Barlow, unpublished). Abbreviations used in Table 1: CIIIG = collagen type III glomerulopathy; n.r. = not reported/known; other abbreviations explained in text.

Variants in either of these categories are likely to have serious consequences for expression, and structural integrity and stability — indeed, many are known to be associated with factor H deficiencies. Another class of mutations are those that are predicted to disrupt the interface between two modules. In this case, it is not likely that expression will be adversely affected, although a more open interface could increase susceptibility to proteolysis. More likely, these mutations illustrate the functional importance of defined levels of flexibility between modules of the intact protein. Of the remaining variations, several involve buried hydrophobic residues (V835, W978, W1157, V1197, and F1199) likely to destabilize individual module structures. One of these (W978) affects the stability of module 16, not previously shown to interact directly with other complement proteins, and therefore having a potential role in the overall protein architecture of fH. On the other hand, this CCP module, along with modules 1, 7 (see above), 14, 18, 19, and 20, harbor disease-associated residues that are solvent-exposed, suggesting the involvement of ligands, some of which may not have been identified so far. The majority of nonstructural residues, however, lie in or close to the GAGbinding site. It is interesting that several such mutants are nonetheless pulled out 49 on a heparin column. On the other hand, heparin is not regarded as a physiological ligand for GAG-binding proteins. The data in Table 1 provide an intriguing indication that it is the inability of variant forms of factor H to distinguish, properly, self- from non-self surfaces via their carbohydrates, which is the dominant disease-causing factor.

9. REFERENCES 1. 2. 3.

M. J. Walport, Complement. First of two parts, N Engl J Med 344(14), 1058–1066 (2001). M. J. Walport, Complement. Second of two parts, N Engl J Med 344(15), 1140– 1144 (2001). P. Warwicker, T. H. Goodship, R. L. Donne, Y. Pirson, A. Nicholls, R. M. Ward, P. Turnpenny, and J. A. Goodship, Genetic studies into inherited and sporadic hemolytic uremic syndrome, Kidney Int 53(4), 836–844 (1998).

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17. J. Rey-Campos, P. Rubinstein, and S. Rodriguez de Cordoba, A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP, J Exp Med 167(2), 664–669 (1988). 18. D. Heine-Suner, M. A. Diaz-Guillen, F. P. de Villena, M. Robledo, J. Benitez, and S. Rodriguez de Cordoba, A high-resolution map of the regulator of the complement activation gene cluster on 1q32 that integrates new genes and markers, Immunogenetics 45(6), 422–427 (1997). 19. D. C. Soares, D. L. Gerloff, N. R. Syme, A. F. Coulson, J. Parkinson, and P. N. Barlow, Large-scale modelling as a route to multiple surface comparisons of the CCP module family, Protein Eng Des Sel 18(8), 379–388 (2005). 20. J. Krushkal, O. Bat, and I. Gigli, Evolutionary relationships among proteins encoded by the regulator of complement activation gene cluster, Mol Biol Evol 17(11), 1718– 1730 (2000). 21. D. C. Soares, and P. N. Barlow, Complement control protein modules in the regulators of complement activation, in: Structural biology of the complement System, edited by D. Morikis and J. D. Lambris (Taylor & Francis, Boca Raton, Florida, 2005), pp. 19–62. 22. R. G. DiScipio, Ultrastructures and interactions of complement factors H and I, J Immunol 149(8), 2592–2599 (1992). 23. M. Aslam, and S. J. Perkins, Folded-back solution structure of monomeric factor H of human complement by synchrotron X-ray and neutron scattering, analytical ultracentrifugation and constrained molecular modelling, J Mol Biol 309(5), 1117–1138 (2001). 24. T. S. Jokiranta, J. Hellwage, V. Koistinen, P. F. Zipfel, and S. Meri, Each of the three binding sites on complement factor H interacts with a distinct site on C3b, J Biol Chem 275(36), 27657–27662 (2000). 25. P. F. Zipfel, C. Skerka, J. Hellwage, S. T. Jokiranta, S. Meri, V. Brade, P. Kraiczy, M. Noris, and G. Remuzzi, Factor H family proteins: on complement, microbes and human diseases, Biochem Soc Trans 30(Pt 6), 971–978 (2002). 26. M. K. Pangburn, M. A. Atkinson, and S. Meri, Localization of the heparin-binding site on complement factor H, J Biol Chem 266(25), 16847–16853 (1991). 27. R. Pio, A. Martinez, E. J. Unsworth, J. A. Kowalak, J. A. Bengoechea, P. F. Zipfel, T. H. Elsasser, and F. Cuttitta, Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners, J Biol Chem 276(15), 12292–12300 (2001). 28. R. Malhotra, M. Ward, R. B. Sim, and M. I. Bird, Identification of human complement Factor H as a ligand for L-selectin, Biochem J 341(Pt 1), 61–69 (1999). 29. H. Jarva, T. S. Jokiranta, J. Hellwage, P. F. Zipfel, and S. Meri, Regulation of complement activation by C-reactive protein: targeting the complement inhibitory activity of factor H by an interaction with short consensus repeat domains 7 and 8–11, J Immunol 163(7), 3957–3962 (1999). 30. C. M. Taylor, Hemolytic-uremic syndrome and complement factor H deficiency: clinical aspects, Semin Thromb Hemost 27(3), 185–190 (2001). 31. D. P. Blackall, and M. B. Marques, Hemolytic uremic syndrome revisited: Shiga toxin, factor H, and fibrin generation, Am J Clin Pathol 121(Suppl), S81–88 (2004). 32. M. R. Buddles, R. L. Donne, A. Richards, J. Goodship, and T. H. Goodship, Complement factor H gene mutation associated with autosomal recessive atypical hemolytic uremic syndrome, Am J Hum Genet 66(5), 1721–1722 (2000).

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33. J. Caprioli, F. Castelletti, S. Bucchioni, P. Bettinaglio, E. Bresin, G. Pianetti, S. Gamba, S. Brioschi, E. Daina, G. Remuzzi, and M. Noris, Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C-257T, the A2089G and the G2881T polymorphisms are strongly associated with the disease, Hum Mol Genet 12(24), 3385–3395 (2003). 34. M. A. Dragon-Durey, V. Fremeaux-Bacchi, C. Loirat, J. Blouin, P. Niaudet, G. Deschenes, P. Coppo, W. Herman Fridman, and L. Weiss, Heterozygous and homozygous factor h deficiencies associated with hemolytic uremic syndrome or membranoproliferative glomerulonephritis: report and genetic analysis of 16 cases, J Am Soc Nephrol 15(3), 787–795 (2004). 35. A. Richards, M. R. Buddles, R. L. Donne, B. S. Kaplan, E. Kirk, M. C. Venning, C. L. Tielemans, J. A. Goodship, and T. H. Goodship, Factor H mutations in hemolytic uremic syndrome cluster in exons 18–20, a domain important for host cell recognition, Am J Hum Genet 68(2), 485–490 (2001). 36. P. Sanchez-Corral, C. Gonzalez-Rubio, S. Rodriguez de Cordoba, and M. LopezTrascasa, Functional analysis in serum from atypical hemolytic uremic syndrome patients reveals impaired protection of host cells associated with mutations in factor H, Mol Immunol 41(1), 81–84 (2004). 37. B. H. Ault, Factor H and the pathogenesis of renal diseases, Pediatr Nephrol 14(10– 11), 1045–1053 (2000). 38. D. Perez-Caballero, C. Gonzalez-Rubio, M. E. Gallardo, M. Vera, M. LopezTrascasa, S. Rodriguez de Cordoba, and P. Sanchez-Corral, Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome, Am J Hum Genet 68(2), 478–484 (2001). 39. J. Esparza-Gordillo, E. Goicoechea de Jorge, A. Buil, L. Carreras Berges, M. LopezTrascasa, P. Sanchez-Corral, and S. Rodriguez de Cordoba, Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32, Hum Mol Genet 14(5), 703–712 (2005). 40. M. Noris, S. Bucchioni, M. Galbusera, R. Donadelli, E. Bresin, F. Castelletti, J. Caprioli, S. Brioschi, F. Scheiflinger, and G. Remuzzi, Complement factor H mutation in familial thrombotic thrombocytopenic purpura with ADAMTS13 deficiency and renal involvement, J Am Soc Nephrol 16(5), 1177–1183 (2005). 41. L. V. Johnson, W. P. Leitner, M. K. Staples, and D. H. Anderson, Complement activation and inflammatory processes in Drusen formation and age related macular degeneration, Exp Eye Res 73(6), 887–896 (2001). 42. R. F. Mullins, N. Aptsiauri, and G. S. Hageman, Structure and composition of drusen associated with glomerulonephritis: implications for the role of complement activation in drusen biogenesis, Eye 15(Pt 3), 390–395 (2001). 43. H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, and P. E. Bourne, The Protein Data Bank, Nucleic Acids Res 28(1), 235–242 (2000). 44. S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, Basic local alignment search tool, J Mol Biol 215(3), 403–410 (1990). 45. A. Sali, and T. L. Blundell, Comparative protein modelling by satisfaction of spatial restraints, J Mol Biol 234(3), 779–815 (1993). 46. L. J. McGuffin, K. Bryson, and D. T. Jones, The PSIPRED protein structure prediction server, Bioinformatics 16(4), 404–405 (2000).

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47. M. J. Bower, F. E. Cohen, and R. L. Dunbrack, Jr., Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: a new homology modeling tool, J Mol Biol 267(5), 1268–1282 (1997). 48. R. A. Laskowski, M. W. MacArthur, D. S. Moss, and J. M. Thornton, PROCHECK: a program to check the stereochemical quality of protein structures, J Appl Cryst 26, 283–291 (1993). 49. M. J. Lehtinen, H. Hagglund, and T. S. Jokiranta, Reason for the association between factor H mutations and atypical hemolytic-uremic syndrome is not in all cases impaired heparin binding of the mutant protein. Mol Immunol 43, 166 (2006). 50. B. H. Ault, B. Z. Schmidt, N. L. Fowler, C. E. Kashtan, A. E. Ahmed, B. A. Vogt, and H. R. Colten, Human factor H deficiency. Mutations in framework cysteine residues and block in H protein secretion and intracellular catabolism, J Biol Chem 272(40), 25168–25175 (1997). 51. H. P. Neumann, M. Salzmann, B. Bohnert-Iwan, T. Mannuelian, C. Skerka, D. Lenk, B. U. Bender, M. Cybulla, P. Riegler, A. Konigsrainer, U. Neyer, A. Bock, U. Widmer, D. A. Male, G. Franke, and P. F. Zipfel, Haemolytic uraemic syndrome and mutations of the factor H gene: a registry-based study of German speaking countries, J Med Genet 40(9), 676–681 (2003). 52. R. E. Saunders, and S. J. Perkins, A website for factor H-associated haemolytic uraemic syndrome and disease-associated mutations. Session 14: Novel roles of complement regulators and receptors. Immunology 113(s1), 73–79 (2004). 53. G. Remuzzi, P. Ruggenenti, D. Codazzi, M. Noris, J. Caprioli, G. Locatelli, and B. Gridelli, Combined kidney and liver transplantation for familial haemolytic uraemic syndrome, Lancet 359(9318), 1671–1672 (2002). 54. S. Rodriguez de Cordoba, J. Esparza-Gordillo, E. Goicoechea de Jorge, M. LopezTrascasa, and P. Sanchez-Corral, The human complement factor H: functional roles, genetic variations and disease associations, Mol Immunol 41(4), 355–367 (2004). 55. S. J. Perkins, and T. H. Goodship, Molecular modelling of the C-terminal domains of factor H of human complement: a correlation between haemolytic uraemic syndrome and a predicted heparin binding site, J Mol Biol 316(2), 217–224 (2002). 56. J. Caprioli, P. Bettinaglio, P. F. Zipfel, B. Amadei, E. Daina, S. Gamba, C. Skerka, N. Marziliano, G. Remuzzi, and M. Noris, The molecular basis of familial hemolytic uremic syndrome: mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20, J Am Soc Nephrol 12(2), 297–307 (2001). 57. P. Sanchez-Corral, D. Perez-Caballero, O. Huarte, A. M. Simckes, E. Goicoechea, M. Lopez-Trascasa, and S. R. de Cordoba, Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome, Am J Hum Genet 71(6), 1285–1295 (2002).

22 TRANSDERMAL PHARMACOLOGY OF SMALL MOLECULE CYCLIC C5a ANTAGONISTS Lavinia M. Proctor, Trent M. Woodruff, Prakirti Sharma, Ian A. Shiels and Stephen M. Taylor

1. ABSTRACT Overproduction or underregulation of the proinflammatory complement component C5a has been implicated in numerous immune and inflammatory conditions. Therefore, targeting the C5a receptor (C5aR) has become an innovative strategy for antiinflammatory drug development. The novel cyclic peptide C5aR antagonist, AcF-[OP(D-Cha)WR] (PMX53), attenuates injury in numerous animal models of inflammation following intravenous, subcutaneous, intraperitoneal, and oral administration. In the present study the transdermal pharmacology of PMX53 and three analogs designed with increased lipophilicity, hydrocinnamate-[OP(D-Cha)WCit] (PMX200), AcF-[OP(D-Cha)WCit] (PMX201) and hydrocinnamate-[OP(D-Cha)WR] (PMX205), have been examined in order to assess their transdermal permeability and inhibitory effect on C5a-mediated lipopolysaccharide (LPS)-induced systemic responses. In the rat, PMX53, PMX201, and PMX205, were bioavailable following topical dermal administra2 tion (10 mg/50 cm site/rat). All analogs functionally antagonized neutropenia and hypotension induced by systemic challenge with LPS (1 mg/kg i.v.). Interestingly, PMX200 attenuated LPS-induced neutropenia more effectively than other analogs, despite undetectable (<5 ng/ml) circulating levels following topical administration. In conclusion, we have demonstrated that cyclic peptide C5aR antagonists can penetrate transdermally sufficiently to have systemic effects. However, increasing lipophilicity in these compounds did not result in

Lavinia M. Proctor, Promics Ltd., Trent M. Woodruff, Promics Ltd. Prakirti Sharma, School of Biomedical Sciences, University of Queensland, QLD 4072, Australia. Ian A. Shiels, School of Biomedical Sciences, University of Queensland, QLD 4072, Australia. Stephen M. Taylor, Promics Ltd. and School of Biomedical Sciences, University of Queensland, QLD 4072, Australia.

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increased blood levels. Nonetheless, topical application of C5aR antagonists produced circulating levels of the drugs that antagonized the LPS-induced systemic responses of neutropenia and hypotension. This suggests that these smallmolecule C5aR antagonists may be developed for topical administration for the treatment of local and systemic inflammatory conditions in the human and veterinary pharmaceutical markets. 2. INTRODUCTION The complement system is part of the normal innate host defense in response to 1 infection or injury . Activation of the complement cascade generates C5a, a potent proinflammatory peptide that interacts with its specific G protein-coupled C5a receptor (C5aR) located on numerous myeloid and non-myeloid cell types to induce the recruitment, infiltration, and activation of leukocytes at sites of 2 inflammation . Overexpressed or underregulated C5a is implicated in the pathogenesis of 3 numerous acute and chronic immune and inflammatory diseases , and, consequently, the C5aR has been targeted by numerous groups for therapeutic inter4 vention. Our group has developed a series of cyclic peptide C5aR antagonists , 5,6 with the lead compound, AcF-[OP(D-Cha)WR] (PMX53 ), now being advanced commercially by the Australian company, Promics Ltd. (www.promics. com). Following oral, intravenous, intraperitoneal, and subcutaneous administration, PMX53 (or the closely related analog F-[OP(D-Cha)WR]) have demonstrated clear therapeutic efficacy in the treatment of numerous experimental in7,8 9 flammatory conditions — including endotoxic shock , sepsis , monoarticular 10 11 antigen-induced arthritis , inflammatory bowel disease , ruptured abdominal 12 aortic aneurysm , and intestinal, hepatic, limb, and renal ischemia/reperfusion 13-16 17 18 injury , aPL antibody-induced fetal loss , experimental lupus nephritis , and 19 traumatic brain cryoinjury . Recently, a lipophilic analog of PMX53, hydrocinnamate-[OP(D-Cha)WR] (PMX205), with similar in-vitro activity as PMX53 but superior resistance to intestinal metabolism, has demonstrated greater oral 20 potency than PMX53 in a rat model of TNBS-induced colitis . A clear advantage of these cyclic peptide C5aR antagonists is their ability to be delivered via both oral and parenteral routes of administration. However, for some indications, particularly inflammatory skin conditions where both localized and systemic inflammation are present, there may be an advantage for a therapeutic agent that can be administered topically. Transdermal delivery offers an attractive, noninvasive alternative to parenteral drug administration that avoids the gastrointestinal tract and hepatic first-pass biotransformation and me21 tabolism . However, human skin, and in particular the stratum corneum, is a very efficient physiochemical barrier that has evolved to impede the transdermal absorption of exogenous compounds, particularly for larger drugs and macro22 molecules (>500 Da) . As an aid to drug penetration, chemical penetration enhancers have been 23 used to perturb the skin barrier and facilitate drug delivery . One of the earliest

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and most widely applied chemical penetration enhancers for both lipophilic and 23,24 hydrophilic drugs was aprotic solvents such as dimethylsulfoxide (DMSO) . DMSO acts to enhance permeation by altering the secondary conformation of intracellular keratin and also through interaction with the intracellular lipid do23 mains of the stratum corneum, producing distorted packing of the lipid bilayer . Due to the drawbacks of local irritation and toxicity, DMSO is now mainly used 24 as a permeation enhancer in veterinary pharmaceuticals . Propylene glycol (PG) is also a mild penetration enhancer for lipophilic and polar drugs and has also 23 been widely used as a vehicle for penetration enhancers, e.g., azole . Propylene glycol is an excipient in many pharmaceuticals, including topical and cosmetic 25 formulations . In addition to the use of chemical permeation enhancers, the PMX compounds used in the present study were specifically designed for increased lipophilicity compared to PMX53, which may result in enhanced topical bioavailability. In order to define a pharmacophore for C5a antagonism, various analogs 4 of PMX53 have been assessed for in-vitro activity at human PMN C5aRs . This study demonstrated that the extracyclic region of PMX53 could be conservatively substituted without loss of C5a receptor affinity; however, substitutions at 4 several critical positions attenuated C5aR affinity . The arginine residue is present in linear and cyclic C5aR antagonists derived from the active C-terminal 4,6,26 region of C5a and is considered important for activity ; however, this positively charged residue may hinder transport of PMX53 across lipidic barriers. In an effort to increase the lipophilicity of PMX53, limit the influence of the charged guanidino group and possibly enhance topical penetration, three PMX53 analogs have been utilized. PMX205 (hydrocinnamate-[OP(DCha)WR]), which incorporates an extracyclic hydrocinnamate moiety; PMX201 (AcF-[OP(D-Cha)WCit]), in which Arg is substituted with an uncharged citrulline residue; or PMX200 (hydrocinnamate-[OP(D-Cha)WCit]), combining a citrulline residue with a extracyclic hydrocinnamate moiety (Figure 1). In the present study the chemical permeation enhancers DMSO and PG have been utilized to study the transdermal pharmacology of PMX53 and three lipophilic analogs, i.e., PMX201, PMX205, and PMX200. Specifically, the systemic bioavailability of each compound and their effect on systemic responses of neutropenia and hypotension following infusion lipopolysaccarhride (LPS)inducing endotoxic shock. The pathogenic role of C5a in experimental sepsis and endotoxic shock has been well established. Infusion of C5a produces many 27-29 of the same effects as Gram-negative endotoxemia , and the antiinflammatory effect of C5 deficiency may be attributed to the absence of C5a. Anti-C5a mAb treatment has been protective in several animal models of sepsis and septicemia 30,31 32,33 and in the rat . Furthermore, administration of our cyclic C5aR in the pig antagonist, F-[OP(D-Cha)WR], increased survival in cecal ligation puncture9 treated septic mice , and PMX53 attenuated neutropenia and elevation of serum 8 TNF-α following endotoxic shock in the rat .

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H N

O

O

O N

N

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O HN

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NH

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O H N N

N H

H N

O N

O

O

N

NH

NH+

N

N H

O

O

NH2

N

PMX200

PMX53 O H N

H N

O

O

O

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N O

HN

O HN

NH

O

O H N

N O

NH

N H

H N

O N

O N

NH2

N

PMX201

N H

O

O

NH+

N

PMX205

Figure 1. Chemical Structure of Cyclic C5a Receptor Antagonists. Boxed areas show structural changes present in PMX analogs of the lead compound, PMX53.

3. MATERIALS AND METHODS 3.1. Materials The four C5aR antagonists AcF-[OP(D-Cha)WR] (PMX53), hydrocinnamate[OP(D-Cha)WCit] (PMX200), AcF-[OP(D-Cha)WCit] (PMX201), and hydrocinnamate-[OP(D-Cha)WR] (PMX205) were all synthesised by solution phase 4 methods, purified, and characterized essentially as described . 3.2. Isolation of Polymorphonuclear Leukocytes Human polymorphonuclear leukocytes (PMNs) were isolated from heparinized whole blood by density-gradient separation over a Ficoll-Hypaque solution 6 (Histopaque 1077, Sigma, USA) as previously described .

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3.3. Receptor Binding Assay 4

PMN C5aR binding assays were preformed as previously described using a 96well multiscreen filtration plate (GV 0.22 µM; Millipore, Australia) system. 3.4. Myeloperoxidase Release from PMNs Functional antagonism of the PMN C5aR was assessed using the myeloperoxi4 dase release assay as previously described in detail . 3.5. In-Vivo Studies Female Wistar rats (200–230 g) were anaesthetized with an intraperitoneal injection of zolazepam and tiletamine (Zoletil; 50 mg/kg; Virbac, Australia) and xylazine (10 mg/kg, Ilium, Australia). Rats were administered (200 µL) the C5a receptor antagonists (1 mg/kg) or vehicle (10% ethanol/saline) intravenously 15 min prior to intravenous infusion of LPS (1 mg/kg, Sigma, USA) or vehicle (saline). Alternatively, vehicle (500 µL of 50% DMSO/water) or C5aR antagonists 2 (500 µL) were applied topically (10 mg/50 cm site/rat) 60 min prior to intravenous infusion of LPS (1 mg/kg) or vehicle (saline). 3.5.1. Circulating Neutrophils Serial blood samples (100 µL) were collected from the tail vein into heparinized tubes (50 IU/mL), layered over Histopaque-1083 (200 µL; Sigma, USA), and centrifuged (400 x g, 25 min at 25°C). The supernatant was removed, distilled water (4°C) added to the remaining pellet, and the tube shaken for 40 s to lyse residual red blood cells. Dulbecco’s phosphate buffered saline (10x) (Sigma, USA) was added at a volume 10% of the distilled water to restore isotonicity before centrifugation (700 x g for 10 min at 4°C). The pellet was washed, centrifuged (700 x g for 10 min at 4°C), resuspended in saline, and cells counted on a hemocytometer. PMN numbers were presented as mean percentage ± SE of the values obtained immediately prior to administration of the test compound. 3.5.2. Blood Pressure Measurements The noninvasive blood pressure of female Wistar rats was recorded throughout the experiment using a pressure transducer (ADI Instruments) and an inflatable 34 tail cuff as previously described . Results were expressed as a percentage of the resting blood pressure ± SE.

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3.5.3. Pharmacokinetics Female Wistar rats (200–230 g) were anaesthetized with an intraperitoneal injection of zolazepam and tiletamine (Zoletil; 50 mg/kg; Virbac, Australia) and xylazine (10 mg/kg, Ilium, Australia), and body temperature was maintained throughout the experiment using a heating pad. PMX53 (500 µL of a 20 mg/mL solution in either 10% or 50% PG or 10% or 50% DMSO) was applied to a 2 shaved site (50 cm ) on the lower abdominal region of the rat. PMX200, 201, and 205 (500 µL of a 20-mg/mL solution in 50% DMSO) were applied to an identical abdominal region. Serial blood samples were collected into heparinized tubes (50 IU/mL) over the experimental period. Heparinized samples were centrifuged (11000 x g, 4 min) and plasma (100 2 µL) collected. A deuterated internal standard ( H3CO-F-[OP(D-Cha)WCit], 250 pg, 50% ACN/water) was added and the sample vortexed. The samples were diluted 1:3 with HPLC grade acetonitrile, rapidly vortexed and centrifuged (11000 x g for 4 min). This process produced precipitation of plasma proteins and complete extraction of the drug from the plasma. The supernatant (100 µL) was collected and stored at –20°C until analysis. Pharmacokinetic analysis was 35 performed substantially as described previously in detail . Samples were analyzed by LCMS and concentrations determined from a standard curve of drug:internal standard peak area ratios. Standards were prepared by adding an appropriate amount of the drug and internal standard to plasma from an untreated rat and were extracted and prepared by the same method as the experimental samples. Additionally, standard solutions made up in mobile phase were compared to the plasma-extracted standards to demonstrate that the drug was being extracted efficiently. The instrument limit of detection in plasma was ~5 ng/mL. 3.6. Statistical Analysis For both receptor binding and antagonism assays, nonlinear regression was performed using Prism 4 (Graph Pad Software, USA), inhibition curves produced and IC50 and –logIC50 values determined. Receptor affinities and antagonist potencies (–log IC50) were compared statistically using a one-way ANOVA and Tukey post test. Standard curves of cyclic peptide in acetonitrile and plasma were plotted as ratios of the drug relative to a deuterated analogs internal standard versus concentration. These curves were used to determine the concentrations of C5aR antagonists. The AUC0-∞, elimination half-life, Cmax, and Tmax were calculated for individual experiments for each compound using standard methods and results expressed as mean (range) for AUC0-∞, elimination half-life, and mean ± SE for Cmax and Tmax. Results for blood pressure and neutropenia experiments were statistically analyzed with a one-way ANOVA test (nonparametric) coupled with a Dunnett’s post test. For all experiments statistical significance was defined as p ≤ 0.05.

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Table 1. C5a Receptor Affinity and Antagonist Activity of PMX Compounds C5a receptor affinity

Compound

a

Antagonist activity b

a

n

–log IC50

IC50 (µM)

n

–log IC50

IC50 (µM)

PMX53

22

6.43 ± 0.07

0.38Ω

22

7.48 ± 0.12

0.03

PMX200

4

4.72 ± 0.06*

19.2

3

4.87 ± 0.21*

13.4

PMX201

3

5.22 ± 0.06*

6.0Ω

3

5.05 ± 0.24*

8.8

PMX205

3

6.65 ± 0.06

0.22Ω

4

7.39 ± 0.17

0.04

c

Ω, Data published previously in March et. al. (2004); a, –log IC50 expressed as mean ± SE; b, concentration of peptide resulting in 50% inhibition of the maximum binding of 125I-C5a; c, concentration of peptide producing 50% of the maximal-induced activity; n, represents the number of separate experiments performed; *p < 0.05 vs. PMX53.

4. RESULTS 4.1. In-Vitro Activity of PMX Compounds We have previously reported the receptor affinity of PMX53, PMX201 and 4 PMX205 (see Table 1). In this study, PMX205 had similar affinity to PMX53, while PMX201 demonstrated a significant (p ≤ 0.05) ~16 fold lower affinity for 4 the C5a receptor than PMX53 . In the present study we report that PMX200 (hydrocinnamate-[OP(D-Cha)WCit]) has ~50-fold lower affinity than PMX53 at C5aRs (p ≤ 0.05; Table 1). We have also assessed antagonism of C5a-mediated 4 myeloperoxidase release from isolated human PMNs. As previously described , both PMX53 and PMX205 have similar antagonist activity. In contrast, PMX200 and PMX201 have significantly (p ≤ 0.05) lowered antagonist potency (~ 500 and 300-fold, respectively) compared to the lead compound, PMX53 (Table 1). 4.2. Transdermal Pharmacokinetics of Cyclic C5a Receptor Antagonists 4.2.1. Effect of Delivery Vehicle on the Topical Bioavailability of PMX53 2

Circulating levels of PMX53 following topical administration (10 mg/50cm site/rat or ~50 mg/kg) to the rat in 10% or 50% DMSO/water or 50% PG are shown in Figure 2. Application of PMX53 in 10% PG/water resulted in plasma levels below the assay limit of detection (<5 ng/mL). Following application of

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Plasma Conc (ng/mL)

64 32 16 8 4 2 0

30

60

90

120

150

180

210

Time (min) PMX53 (10 mg/site/rat) in 10% DMSO/water PMX53 (10 mg/site/rat) in 50% DMSO/water PMX53 (10 mg/site/rat) in 50% PG/water Figure 2. Transdermal Pharmacokinetics of PMX53. PMX53 was administered topically (10 mg/site/rat) in various vehicles (10 or 50% DMSO or 50% PG) to a single 50-cm2 shaved abdominal site on each rat (n = 4–5).

Table 2. Pharmacokinetic Parameters for PMX53 in Various Vehicles Administered Topically a

t½ elimination (min)

b

Cmax c (ng/mL)

d

Tmax (min)

Treatment

AUC0-∞

PMX53 (10%DMSO)

3.10 (2.90-3.45)

189.9 (28.55-326.4)

10.05 ± 5.60

PMX53 (50% PG)

3.36 (0.95-5.80)

75.7 (34.4-117.5)

15.4 ± 2.2

60

PMX53 (50%DMSO)

26.3 (2.29-54.9)

929.3 (58.9-2059.5)

22.3 ± 6.7

60

60

a, calculated area under the curve from 0-infinity; b, half-life of elimination; c, maximal concentration detected; d, time when maximal concentration was achieved.

PMX53 in 10% DMSO, 50% DMSO, or 50% PG, the plasma concentration increased to various maximal concentrations at Tmax of 60 min (n = 4–5; Table 2). Similar AUC0-∞ and Cmax values (µg/mL) were determined for PMX53 in both 10% DMSO/water and 50% PG/water, although the former did exhibit trends toward a more prolonged elimination half-life (Table 2). By contrast, the elimi-

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337

nation half-life and calculated AUC0-∞ of PMX53 in 50% DMSO/water was significantly greater than for the other delivery vehicles (Table 2). The transdermal pharmacokinetics of all PMX53 analogs were subsequently performed using 50% DMSO/water as a vehicle. 4.2.2. Topical Bioavailability of PMX200, PMX201 and PMX205 Topical administration of PMX200 in 50% DMSO/water did not produce circulating levels at or above the assay limit of detection (5 ng/mL). The plasma concentrations of PMX53, PMX201, and PMX205, applied topically in 50% DMSO/water, were determined (Table 3, Figure 3). Comparative elimination half life, Cmax, Tmax and AUC0-∞ values for PMX53, PMX201 and PMX205 were calculated (n = 4–5; Table 3). Both PMX201 and PMX205 (Tmax = 90 min) reached maximal concentrations 30 min later than PMX53. However, plasma levels of both compounds also surged 15 min after topical administration and reached concentrations of 14.2 ± 3.4 ng/ml and 13.2 ± 4.3 ng/mL, respectively. These levels were only slightly lower than Cmax concentrations (Table 3). Elimination half-lives, Cmax, and AUC0-∞ values for both PMX201 and PMX205 were lower than comparative values for PMX53 in 50% DMSO/water.

Table 3. Pharmacokinetic Parameters of Transdermally Administered PMX Compounds Treatment groups

AUC0-∞

a

t½ elimination (min)

b

d

Cmax c (ng/mL)

Tmax (min)

PMX200

ND

ND

ND

ND

PMX201

3.21 (2.37-4.83)

100.8 (85.5-121.9)

14.6 ± 2.1

90

PMX205

5.53 (2.12-10.65)

480 (77.0-539.4)

13.3 ± 5.9

90

a, calculated area under the curve from 0 to infinity; b, half-life of elimination; c, maximal concentration detected; d, time when maximal concentration was achieved. ND, not detected;

4.3. Effect of Administration of C5a Antagonists on LPS-Induced Neutropenia and Hypotension 4.3.1. Vehicle and Drug-Only Controls Intravenous infusion of vehicle (saline and 10% ethanol/saline, n = 6-8) or PMX53, PMX200, PMX201 or PMX205 alone (1 mg/kg i.v., n = 6-9) did not

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significantly (p ≤ 0.05) affect the number of circulating PMNs or resting blood pressure (data not shown). Similarly, topical administration of vehicle (50% DMSO; 500 µL/site, n = 6), PMX53, PMX200, PMX201, or PMX205 (10 mg/site/rat, n = 3–4) did not significantly affect circulating PMNs and resting blood pressure (data not shown).

Plasma Conc (ng/mL)

64

32

16

8

4 0

30

60

90

120

150

180

210

Time (min) PMX53 (10 mg/site/rat) in 50% DMSO/water PMX205 (10mg/site/rat) in 50% DMSO/water PMX201 (10 mg/site/rat) in 50 % DMSO/water Figure 3. Transdermal Pharmacokinetics of PMX201, PMX205, and PMX53. PMX compounds were administered topically (10 mg/site/rat) in 50% DMSO/water to a single 50-cm2 shaved abdominal site on each rat (n = 4–5).

4.3.2. Circulating Neutrophil Numbers Intravenous administration of LPS (1 mg/kg) caused a rapid and sustained systemic neutropenia (Figure 4, n = 8, P ≤ 0.05). Circulating PMNs declined to 24.3 ± 5.4% (n = 7) of pre-LPS numbers 30 min following challenge with intravenous LPS. Numbers of circulating PMNs partially recovered to 63.7 ± 28.4% (n = 7) over the experimental period. Intravenous administration of PMX53 (1 mg/kg) or topical application of PMX53 (10 mg/site/rat) 10 or 60 min prior to LPS infusion, respectively, significantly attenuated LPS-induced neutropenia at 60 and 90 min following LPS challenge (Figure 4A, n = 5-6). PMX200, incorporating both an extracyclic hydrocinnamate group and citrulline for arginine substitution, significantly inhibited LPS-induced neutropenia over the majority of the experimental period (10–90 min) when administered both intravenously and

120 110 100 90 80 70 60 50 40 30 20 10

A [PMN] (% resting)

[PMN] (% resting)

SMALL MOLECULE CYCLIC C5A ANTAGONISTS

-30

0

30

60

90

120

120 110 100 90 80 70 60 50 40 30 20 10

150

339

B

-30

0

Time (min)

C [PMN] (% resting)

[PMN] (% resting)

120 110 100 90 80 70 60 50 40 30 20 10

-30

0

30

60

90

120

150

Time (min) LPS (1mg/kg i.v.) PMX201 (1mg/kg i.v.) + LPS (1 mg/kg i.v.) PMX201 (10 mg/site/rat) + LPS (1 mg/kg i.v.)

30

60

90

120

150

Time (min) LPS (1 mg/kg i.v.) PMX200 (1 mg/kg i.v.) + LPS (1 mg/kg i.v.) PMX200 (10 mg/site/rat) + LPS (1 mg/kg i.v.)

LPS (1 mg/kg i.v.) PMX53 (1 mg/kg i.v.) + LPS (1 mg/kg i.v.) PMX53 (10mg/site/rat) + LPS (1 mg/kg i.v.)

120 110 100 90 80 70 60 50 40 30 20 10

D

-30

0

30

60

90

120

150

Time (min) LPS (1 mg/kg i.v.) PMX205 (1 mg/kg i.v.) + LPS (1 mg/kg i.v.) PMX205 (10 mg/site/rat) + LPS (1 mg/kg i.v.)

Figure 4. Effect of PMX Compounds on LPS-Induced Neutropenia. Timepoints where a statistically significant difference (P < 0.05) is achieved between animals challenged with LPS alone and those treated intravenously or topically with (A) PMX53, (B) PMX200, (C) PMX201, and (D) PMX205 prior to LPS challenge are shown with either a solid line (intravenous) or dotted line (topical) below the data points.

topically (Figure 4B, n = 5–6). PMX201, in which the positively charged arginine is absent, when administered intravenously (1 mg/kg) resulted in a significant reduction in LPS-induced neutropenia at 10–90 min following LPS infusion (Figure 4C, n = 5). Topical administration of PMX201 (10 mg/site/rat) also significantly inhibited LPS-induced neutropenia at 10-30 min (n = 5; Figure 4C). Intravenous administration of PMX205 (1 mg/kg) produced a significant reduction in neutropenia 10–30 min following LPS challenge (n = 6; Figure 4D). The LPS-induced decline in numbers of circulating PMNs was significantly attenuated at only 30 and 60 min in rats treated topically with PMX205 (n = 6; Figure 4D). 4.3.3. Blood Pressure Changes Intravenous administration of LPS (1 mg/kg) caused a relatively rapid and prolonged hypotensive effect in the rat (Figure 5A–D, n = 8). LPS challenge resulted in rapid hypotension causing blood pressure to fall to 61.4 ± 9.8% of con-

340

L.M. PROCTOR ET AL.

110

Blood Pressure(% resting)

Blood Pressure(% resting)

trol levels after 90 min (n = 8), which was not significantly different from LPSinduced blood pressure changes following topical pretreatment with vehicle (50% DMSO, 59.3 ± 9.8%, n = 6). The declining blood pressure following LPS infusion did not recover substantially over the remainder of the experimental period (Figure 5).

A 100 90 80 70 60 50 -30

0

30

60

90

120

110

B 100 90 80 70 60 50 -30

150

0

30

LPS (1 mg/kg i.v.) PMX200 (10mg/site/rat) + LPS (1 mg/kg i.v.)

Blood Pressure(% resting)

Blood Pressure(% resting)

*

90

*

*

80 70 60 50 0

150

PMX200 (1 mg/kg i.v.) + LPS (1 mg/kg i.v.)

100

-30

120

LPS (1 mg/kg i.v.)

PMX53 (10/mg/site) + LPS (1 mg/kg i.v.)

C

90

PMX53 (1 mg/kg i.v.) + LPS (1 mg/kg i.v.) 120 110

60

Time (min)

Time (min)

30

60

90

120

150

Time (min) LPS (1 mg/kg i.v.) PMX201 (1 mg/kg i.v.) + LPS (1 mg/kg i.v.) PMX201 (10mg/site/rat) + LPS (1 mg/kg i.v.)

120 110

D

100 90 80 70 60 50 -30

0

30

60

90

120

150

Time (min) LPS (1 mg/kg i.v.) PMX205 (1 mg/kg i.v.) + LPS (1 mg/kg i.v.) PMX205 (10 mg/site/rat) + LPS (1 mg/kg i.v.)

Figure 5. Effect of PMX Compounds on LPS-Induced Changes Resting Blood Pressure. Timepoints where a statistically significant difference (p < 0.05) is achieved between animals challenged with LPS alone and those treated intravenously or topically with (A) PMX53, (B) PMX200, (C) PMX201, and (D) PMX205 prior to LPS challenge are shown with either a solid line (intravenous) or dotted line (topical) below the data points.

Intravenous administration of PMX53 (1 mg/kg) resulted in a significant inhibition of LPS-induced hypotension 10, 30, and 60 min after LPS infusion (Figure 5A, n = 6). Topical administration of PMX53 (10 mg/site/rat) significantly inhibited the LPS-mediated fall in blood pressure at 10–60 min following LPS challenge (Figure 5A). Interestingly, despite the lack of protection afforded by intravenous infusion of PMX200 (n = 5) and the inability to detect circulating blood levels of PMX200 following topical application, topical PMX200 signifi-

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341

cantly attenuated LPS-induced hypotension 10-60 min after LPS infusion (p < 0.05, n = 5, Figure 5B). Intravenous and topical administration of PMX201, 10 and 60 min prior to LPS challenge, respectively, poorly inhibited LPS-induced hypotension. Intravenous infusion (n = 6) of PMX201 significantly inhibited LPS-mediated hypotension at 60 and 120 min following LPS challenge, while topical administration (n = 6) only significantly attenuated blood pressure changes 10 min following LPS-infusion (Figure 5C). Intravenous administration of PMX205 led to significant inhibition of LPS-induced hypotension early (10– 30 min) in the experimental period (n = 6; Figure 5D). Topical administration of PMX205 significantly inhibited LPS-induced hypotension later (30–60 min) than intravenous treatment (n = 6; Figure 5D).

5. DISCUSSION The PMX compounds used in the present study were specifically designed with increased lipophilicity compared to PMX53, which we hypothesized may result in enhanced topical bioavailability. Of these compounds, only PMX205 (hydro4 cinnamate-[OP(D-Cha)WR]) has similar C5aR affinity as PMX53 in vitro ; indeed, both PMX200 (hydrocinnamate-[OP(D-Cha)WCit]) and PMX201 (AcF[OP(D-Cha)WCit]) have ~16 and ~50-fold less affinity than PMX53 for the PMN C5aR, respectively. An even greater divergence is observed between the ability of PMX200 or 201 and PMX53 or 205 to antagonize C5a-mediated enzyme release in vitro. However, we have recently demonstrated that the in-vitro activity of this class of compounds may not necessarily reflect their potency and efficacy in vivo. Following oral administration, PMX205 has demonstrated 10– 20 30-fold greater potency than PMX53 in a rat model of TNBS-induced colitis . These differences may be due to changes in drug metabolism and/or pharmacokinetic factors that may make these analogs particularly suitable for specific 20 routes of administration and inflammatory conditions . Particularly, for the treatment of inflammatory skin conditions, such as psoriasis, where both local36-38 ized and systemic inflammation are present , there may be an advantage for a therapeutic that can be administered topically. In order to investigate the transdermal pharmacology of lipophilic PMX compounds we examined their plasma pharmacokinetic profiles following topical administration and their ability to inhibit some effects of systemic complement activation following induction of endotoxic shock in the rat. In the present study we have demonstrated that in the rat, cyclic peptide C5aR antagonists can penetrate transdermally producing detectable systemic blood levels. However, increasing lipophilicity in these compounds did not increase circulating blood levels. In fact, topical administration of the PMX compound designed with greatest lipophilicity, PMX200, did not produce detectable circulating levels. However, topical application of all C5a cyclic antagonists investigated did result in antagonism of systemic responses induced by comple-

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ment activation during endotoxic shock. These results suggest that these small molecule C5aR antagonists may be developed for topical administration for the treatment of local or systemic inflammatory conditions and reinforce our belief that the in-vitro activity of this class of cyclic peptide C5a receptor antagonists does not necessarily reflect their ability to effectively attenuate C5a-mediated pathophysiology in vivo. Despite the absence of detectable (< 5 ng/ml) plasma levels, the most lipophilic analog, PMX200, attenuated LPS-induced pathophysiological changes. Indeed, topically administered PMX200 significantly inhibited LPS-induced neutropenia over a longer experimental period than any other PMX compound and attenuated LPS-induced hypotension to a similar extent as PMX53. Given the ability of PMX200 to attenuate C5a-mediated LPS-induced responses, it can be inferred that PMX200 is bioavailable following topical administration. The reasons for this effect are unclear; however, the highly lipophilic nature of this compound may provide an explanation for this apparent paradox. Due to its lipophilic nature, PMX200 may more readily penetrate the stratum corneum, enter the bloodstream, and rapidly distribute into tissues lowering the blood levels to undetectable levels. The pseudo-irreversible nature of these compounds can also result in the divergence between blood levels and pharmacologic ef8,39 fects . Lipophilicity is an important property that contributes to pharmacological activity following topical administration and can counterbalance lower receptor affinity and antagonist activity in vitro. These results may indicate a basis and a strategy for developing topically active C5a antagonist therapeutics.

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21. J.J. Berti and J.J. Lipsky, Transcutaneous drug delivery: a practical review, Mayo Clin Proc 70(6), 581–586 (1995). 22. J.D. Bos and M.M. Meinardi, The 500 Dalton rule for the skin penetration of chemical compounds and drugs, Exp Dermatol 9(3), 165–169 (2000). 23. A.C. Williams, B.W. Barry, Penetration enhancers, Adv Drug Deliv Rev 56(5), 603– 618 (2004). 24. B.M. Magnusson, K.A. Walters and M.S. Roberts, Veterinary drug delivery: potential for skin penetration enhancement, Adv Drug Deliv Rev 50(3), 205–227 (2001). 25. J. Hadgraft and W.J. Pugh, The selection and design of topical and transdermal agents: a review, J Investig Dermatol Symp Proc 3(2), 131–135 (1998). 26. J.A. DeMartino, Z.D. Konteatis, S.J. Siciliano, R.G. Van, D.J. Underwood, P.A. Fischer and M.S. Springer, Arginine 206 of the C5a receptor is critical for ligand recognition and receptor activation by C-terminal hexapeptide analogs, J Biol Chem 270(27), 15966–15969 (1995). 27. T. Hoffmann, E.C. Bottger, H.P. Baum, R. Dennebaum, U. Hadding and D. BitterSuermann, Evaluation of low dose anaphylatoxic peptides in the pathogenesis of the adult respiratory distress syndrome (ARDS). Monitoring of early C5a effects in a guinea-pig in vivo model after i.v. application, Eur J Clin Invest 16(6), 500–508 (1986). 28. C. Lundberg, F. Marceau and T.E. Hugli, C5a-induced hemodynamic and hematologic changes in the rabbit. Role of cyclooxygenase products and polymorphonuclear leukocytes, Am J Pathol 128(3), 471–483 (1987). 29. G. Smedegard, L.X. Cui and T.E. Hugli, Endotoxin-induced shock in the rat. A role for C5a, Am J Pathol 135(3), 489–497 (1989). 30. M. Mohr, U. Hopken, M. Oppermann, C. Mathes, K. Goldmann, S. Siever, O. Gotze, H. Burchardi, Effects of anti-C5a monoclonal antibodies on oxygen use in a porcine model of severe sepsis, Eur J Clin Invest 28(3), 227–234 (1998). 31. U. Hopken, M. Mohr, A. Struber, H. Montz, H. Burchardi, O. Gotze and M. Oppermann, Inhibition of interleukin-6 synthesis in an animal model of septic shock by anti-C5a monoclonal antibodies, Eur J Immunol 26(5), 1103–1109 (1996). 32. M. Huber-Lang, V.J. Sarma, K.T. Lu, S.R. McGuire, V.A. Padgaonkar, R.F. Guo, E.M. Younkin, R.G. Kunkel, J. Ding and R. Erickson, J.T. Curnutte, P.A. Ward, Role of C5a in multiorgan failure during sepsis, J Immunol 166(2), 1193–1199 (2001). 33. I.J. Laudes, J.C. Chu, S. Sikranth, M. Huber-Lang, R.F. Guo, N. Riedemann, J.V. Sarma, A.H. Schmaier and P.A. Ward, Anti-c5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis, Am J Pathol 160(5), 1867–1875 (2002). 34. A.J. Short, N.J. Paczkowski, S.M. Vogen, S.D. Sanderson and S.M. Taylor, Response-selective C5a agonists: differential effects on neutropenia and hypotension in the rat, Br J Pharmacol 128(3), 511–514 (1999). 35. A.J. Strachan, I.A. Shiels, R.C. Reid, D.P. Fairlie and S.M. Taylor, Inhibition of immune-complex mediated dermal inflammation in rats following either oral or topical administration of a small molecule C5a receptor antagonist, Br J Pharmacol 134(8), 1778–1786 (2001). 36. H. Tagami, The role of complement-derived mediators in inflammatory skin diseases, Arch Dermatol Res 284, S2–9 (1992).

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23 INACTIVATION OF COMPLEMENT BY RECOMBINANT HUMAN C3 DERIVATIVES Edzard Spillner, Johanna Kölln, and Reinhard Bredehorst

1. INTRODUCTION The complement system is a highly sophisticated host-defense system playing an important role in both innate and antibody-mediated immunity. Pathophysiological disregulation of the complement system, however, contributes to tissue damage in many clinical conditions, including ischemia-reperfusion injuries after myocardial infarction, interventional activation of complement in patients 1-4 undergoing cardiopulmonary bypass, and immune-mediated diseases . From these implications an urgent need for therapeutics effecting reduced complement activity in vivo has emerged. Approaches for inhibition of complement developed so far employ the blockade of essential cascade components using inhibi5-7 tory proteins, small compound inhibitors, or solubilized receptor molecules . However, these reagents exert their activity by blocking complement components at a 1:1 stoichiometry and, therefore, need to be present in relatively high concentrations. An alternative strategy relies on the design and application of a stable C3 convertase capable of effecting inhibition by catalytic depletion of 8,9 complement components . Such an enzyme-mediated approach is likely to enhance inhibitory efficacy significantly. The C3 convertases of the classical and lectin pathways, C2bC4a, and the alternative pathway (AP), C3bBb, converge in proteolytic activation of C3 into the non-catalytic subunit of the C3 convertase C3b and the anaphylatoxin C3a. The alternative pathway is continuously activated at a low rate in human plasma

Edzard Spillner, Johanna Kölln, Reinhard Bredehorst, Institut für Biochemie und Lebensmittelchemie, Abteilung für Biochemie und Molekularbiologie, Universität Hamburg, Germany.

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Figure 1. Schematic representation of the binding and cleavage sites of regulatory proteins in the human C3 molecule.

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due to slow spontaneous hydrolysis of plasma protein C3 to C3(H2O) . Upon binding of factor B and cleavage of C3b-bound factor B by factor D, the actual AP C3 convertase, C3bBb, is formed, which initiates amplification of the complement cascade by increased deposition of C3b and subsequent formation of 11 new C3 convertases . Following association of both types of C3 convertases with a generated C3b molecule, the C5 convertases are formed by a switch in specificity. The C5 convertases then activate C5 by generation of C5b and subsequently the downstream components of the terminal pathway of complement, causing formation of the membrane attack complex, allowing rupture of bilipid membranes. The natural AP C3 convertase, C3bBb, similar to the C3 convertase of the classical pathway, is tightly controlled by a complex network of regulatory proteins that irreversibly dissociate the convertase subunits and serve as 12 cofactors for the proteolytic cleavage of C3b by factor I . In most of these regulation events the N terminus of the C3 alpha chain and the C3d region are impli13-20 cated (Figure 1). In addition to the regulatory proteins, the C3 convertase is 21 controlled by its own limited half-life of less than 2 min .

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Cobra venom factor (CVF), a 149-kDa C3b homolog from snake venom , forms CVF-dependent C3 and C5 convertase that are active in fluid phase, escape regulation by regulatory proteins, and exhibit an increased half-life of 7 23 hours . The CVFBb convertase obliterates functional complement in vivo for 23 days by continuous consumption of complement components . CVF has been shown to be highly effective in many experimental models of complementmediated pathologies and has provided the proof-of-concept for the beneficial 26 effect of catalytic complement depletion . For therapeutic applications, however, CVF is not suitable due to its immunogenic properties. To generate a stable, non-immunogenic C3 convertase we produced various chimeric proteins comprising human C3 domains and stabilizing elements of CVF. Following this strategy, derivatives were generated that exhibit characteristics similar to those of CVF. The obtained results allow insights in the structure/function relationships of convertase formation of the complement system, and, moreover, emphasize the feasibility of this approach in developing novel therapeutics for catalytic reduction of complement activity in complementmediated pathologies.

2. GENERATION OF CVF CHIMERAS AND C3 DERIVATIVES In a first approach, the regions in CVF corresponding to particular binding sites in C3 were substituted to dissect the mechanisms by which the CVF-dependent C3/C5 convertase is stabilized. Important properties determining the stability of the CVF-dependent convertase include a stable interaction with factor B and resistance to cleavage by factors I and H. On the basis of competitive approaches employing synthetic peptides and antibodies, binding sites for factor B and factor H have been postulated in the more N-terminal region of the alpha 13-20 chain of human C3 . To elucidate the importance of corresponding regions in CVF, two CVF-derived constructs were designed in which short sequences in the gamma chain were substituted by sequences of human C3 (Figure 2, rCVF χ1 and χ2). In a second approach, various regions in human C3 were substituted by corresponding CVF-derived sequences to dissect the mechanism(s) responsible for the short half-life of the C3-dependent convertase (Figure 2, hC3 derivatives DIII-DVI). Since the majority of binding and cleavage sites are located in the alpha chain of human C3 (Figure 1), we focused on the design of C3 derivatives comprising CVF substitutions in this region. Recombinant human C3 (hC3) and recombinant CVF (rCVF) were generated for control. Appropriate expression systems are crucial for production of functionally active CVF chimeras and hC3 derivatives. For CVF, homologous production in immortalized cells from venom glands of cobra appears to be the most appropriate, but a difficult, approach. However, CVF recently has been produced successfully in different eukaryotic expression systems including baculovirus27 mediated transfection of Spodoptera frugiperda insect cells , stably transfected

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Figure 2. Design of recombinant CVF chimeras and human C3 derivatives. Indicated in the primary structure are the binding or cleavage sites of relevant regulatory proteins as well as the nomenclature of the chains of the resulting proteins. At the C terminus the C345C domains of hC3 (grey) and CVF (hatched) are depicted.

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Drosophila S2 cells , and mammalian cells . Apparently, CVF can be produced in these systems without significant impact on its decomplementing activity, indicating that neither glycosylation nor post-translational processing of cells from venom glands are essential requirements. For the expression of human C3 and derivatives, the human cell line HEK293 was utilized. As shown in previous studies, mammalian cells are suitable for 19,29 expression and analysis of human C3 and C3 mutants . Although natural CVF is composed of three chains, rCVF expressed in insect or mammalian cell exhibits a two-chain structure, like human C3 (Figure 3). Since the addition of venom to human C3 results in its cleavage into a CVF-like 30 three-chain structure , the type of chain structure arises most likely due to the lack of venom-specific proteases.

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Figure 3. Depicted are the chain structures of hC3, CVF, and an hC3 derivative recombinantly produced in mammalian cells. In native CVF the C3a and C3d region are proteolytically removed, resulting in a C3c-like three-chain structure.

Heterogeneity of secreted rCVF, e.g., the presence of single-chain pro-CVF 29 as found in supernatants of Sf9 insect cells , is detectable only to a minor extent upon expression in mammalian cells. Apparently the derivatives shown in Figure 2 also exhibit a two-chain structure, since their migration behavior in SDS9 PAGE is comparable to that of hC3 and rCVF .

3. FUNCTIONAL CHARACTERISTICS OF THE C3 DERIVATIVES The characterization of anti-complementary properties of the recombinant derivatives expressed in mammalian systems is hampered by the presence of interfering serum components. Therefore, several strategies were developed for affinity purification of the recombinant derivatives. Previous experiments have demonstrated that by addition of a C-terminal affinity-tag the C5 convertase 27 activity of rCVF is impaired , whereas the functional activities of rCVF are not 8 affected by N-terminal affinity-tags . Utilizing an N-terminal his-tag for affinity purification, functionally active derivatives could be isolated from supernatants of mammalian cells. Purification approaches utilizing strep-tag II, a streptavidin-

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binding peptide tag , failed since mild elution by desthiobiotin resulted in partial reduction of the derivatives. As an alternative strategy, recombinant antibody fragments with specificity 32 for CVF or C3, isolated by phage display technology and immobilized to a nickel resin via a C-terminal his-tag, were utilized for immunoprecipitation to 8 remove interfering serum components . The his-tagged recombinant antibody fragments allowed elution of the complexes from the resin under mild conditions, which is a prerequisite for subsequent functional analyses. Since the employed recombinant antibody fragments were found not to affect the functional activity of the derivatives, they had not to be removed prior to the analyses. Both chimeric CVF molecules with the introduced short sequences of human C3 were found to exhibit an anti-complementary effect, suggesting that the substituted regions do not contribute to the stability of the CVF-dependent con8 vertase . In a subsequent approach, entire chains instead of short sequences were substituted. Proof of concept for this approach was demonstrated by the observation that substitution of the entire alpha chain by the beta/gamma chain of CVF (Figure 2, hC3 derivative DI) resulted in a construct exhibiting an anticomplementary effect comparable to that of CVF. In the ongoing reduction of the beta/gamma chain of CVF, it became evident that the C-terminal region of 9 the C3 alpha chain is essential for the complement-consuming activity . Reduction of CVF sequences to the ultimate C terminus points to a sequence of approx. 120 amino acids in length as the essential feature in providing complement consuming activity. Since the anti-complementary activity has been conferred to human C3 by grafting of CVF domains into the scaffold of human C3, the mechanism most likely corresponds to that of CVF. CVF itself forms a stable fluid-phase C3 and C5 convertase with factor B upon cleavage by factor D. This complex has been shown to exhibit a half-life in the range of 6–7 h. Moreover, it escapes regulation by factor H and I. To compare the functional characteristics of the hC3 derivatives with those of CVF, his-tag-derivatized proteins were purified from 2+ culture supernatants of stable transfectants by means of Ni -chelate chromatography and were assessed for their fluid-phase C3 and C5 convertase activity, the half-life of the formed convertase, and cleavage of factor B by factor D upon 9,33 convertase complex formation . The derivatives DIII and DIV were found to exhibit a fluid phase C3 convertase activity comparable to that of CVF (Figure 4a). As shown for DIII, the DIII-dependent C3 convertase is formed by association with Bb upon cleavage by factor D. Once formed, the complex exhibits a half-life in the range of 5–6 h, corresponding to the half-life of the CVF-dependent convertase. The derivatives exhibit also an enhanced fluid C5 convertase activity (Figure 4b). The DIIIdependent convertase also exhibited fluid-phase C5 convertase activity, though this activity is strongly reduced when compared to that of CVF. Apparently, the molecular basis for fluid-phase C5 convertase activity is located in the C terminus of the alpha chain of the C3 derivatives (corresponding to the C terminus of

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the CVF beta chain, Figure 3). This is supported by the observation that C27 terminal fusion of affinity tags to rCVF affects its C5 convertase activity .

Figure 4. C3 and C5 convertase activity of stably expressed derivatives DIII and DIV. (a) Complement consumption by C3 convertase activity was assayed by hemolysis of sensitized sheep erythrocytes after incubation of human serum as a complement source in the presence of purified hC3 (dots), nCVF (squares), hC3-DIII (up triangles), or hC3-DIV (down triangles). (b) C5 convertase activity was assayed by incubation of non-sensitized sheep erythrocytes in human serum in the presence of purified hC3, nCVF, hC3-DIII, or hC3-DIV (bystander lysis assay).

4. THE C345C DOMAIN IN COMPLEMENT The C-terminal domain of the alpha chain intriguingly is formed by a singular 34 structural motif that spans the last approx. 120 amino acids . The rest of the chain is composed of the N-terminal anaphylatoxin domain and the alpha2

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macroglobulin domains. The motif found in the C terminus of C3 itself is called the C345C domain since it is also present in the C terminus of complement C4 and C5. Homologous modules have been found in other proteins, including netrins and tissue inhibitors of metalloproteinases (TIMPs) throughout a variety 35-38 of species . As revealed by structural analyses the C345C module is characterized by a high content of beta-sheets, which in C3 and C5 additionally are stabi39 lized by three internal disulfide bonds . The presence of the C345C domains in C3b and C4b, the non-catalytic subunits of both C3 convertases, suggests a functional relevance for stabilization or regulation of the convertases. The importance of amino-acid residues within the C345C domain of C5 for proteolytic 40,41 activation by the classical pathway C5 convertase indicate that these residues in C5–C345C contribute to contact between C5 and the convertase. Moreover, in activated C5 the C345C domain has been shown to interact with C6 and C7, 40,41 preforming the membrane attack complexes . The C345C module also is present in CVF and exhibits a sequence identity to human C3 is in the range of 44% (Figure 5). Disulfide bonds most likely are retained since all relevant cystein residues are conserved. Deletion or chimerization of this domain in the hC3 derivatives DV and DVI resulted in abolishment 33 of complement-consuming activity . Obviously, the integrity of the CVFderived C345C module is associated with the capability of stable convertase formation.

Figure 5. Shown is the sequence alignment of the C-terminal regions in hC3 and CVF. The C345C domains of hC3 and CVF including the disulfide linkages are indicated as well as the boundaries of the constructs DIV, DV, and DVI.

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5. THERAPEUTICAL IMPLICATIONS Currently available complement inhibitors are blocking specific components of the complement cascade and avoid or reduce activation on defined steps. Such inhibitors include complement regulatory proteins from different species, humanized or fully human antibodies, as well as low-molecular-weight compounds of peptidic and non-peptidic origin, which interfere with binding of activation products to receptors or with processing of complement components into their active form. Consequently, these types of inhibitors need to be administered in stoichiometric amounts. In contrast to the inhibitors mentioned above, the hC3 derivatives probably will require much lower concentrations for effective decomplementation. Comparative animal studies with CVF and selected blocking agents revealed an effective dose of CVF that is 1 to 2 orders of magnitude 42,43 lower than those of blocking agents . Although widely employed for decomplementation, CVF as well as the hC3 derivatives clearly are not complement inhibitors in classical understanding. However, the catalytic mode of action can provide important advantages. The exact risks associated with catalytical complement depletion in man via the novel hC3 derivatives are not known. The anti-complementary effect of purified CVF led to in vivo studies in which CVF was administered to laboratory 44,45 46 animals . These and later investigations demonstrated that CVF can be safely administered to laboratory animals, with its only effect being apparently a temporary suppression of complement activity. Potentially, complement activation that accompanies decomplementation by CVF can induce iatrogenic shock syn47 dromes, as observed in some animal models . However, this report explicitly states the injection of high doses of CVF intravenously. At moderate and low doses, CVF can be safely administered to laboratory animals without inducing iatrogenic shock syndromes as demonstrated by numerous studies and diverse 26 animal models in which CVF has been used for complement depletion . Consequently, the generation of the anaphylatoxic peptides C3a (and C5a) accompanying complement activation could be controlled by repeated injections of low doses of these derivatives. Furthermore, some of the derivatives exhibit only low C5 convertase activity, and the relative biological activity of C3a for certain responses has been reported to be at least 10-fold lower than that of 48 C5a . Ongoing engineering of the derivatives may enable complete elimination of C5 convertase activity while retaining C3 convertase activity. Even the level of C3a generated by application of C3 derivatives may be further reduced, since 60% inhibition of complement is sufficient to provide therapeutic benefit in col49 lagen-induced arthritis . Limitation of complement depletion may be necessary anyway. On one hand, complement deficiencies are associated with increased 50 51 susceptibility to infection and with autoimmunity . On the other hand, different noninflammatory types of processes — including differentiation, tissue regeneration, and signal transduction — have been linked to complement, including 52 its activation products .

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One potential risk is the immunogenicity of CVF-derived sequences in the hC3 derivatives. However, due to the small degree of amino-acid substitution (66 out of 1653 or 96% sequence identity), it is expected that the immunogenicity of the hC3 derivative will be very low to negligible. Therapeutic antibodies 53-55 generally provide between 65 and 95% human origin depending on degree of humanization. Even administration of an anti-C5 scFv-containing murine CDR and artificial linker sequences was found to be well tolerated and did not pro56,57 voke any immune response . To what extent the further reduction of nonhuman sequences in the C3 derivatives can be performed without loss of function remains to be determined. Furthermore, the high identity of the derivatives to the high abundance serum protein hC3 provides additional important advantages with regard to pharmacokinetics and safety. The short C-terminal sequence derived from the non49,50 toxic CVF itself is unlikely to affect either the safety or the biochemical characteristics of the molecule. Utilizing mammalian cells for recombinant production, the glycosylation pattern can be assumed to be similar to that of hC3. 58,59 Since glycosylation plays an important role in the half-life of serum proteins , hC3 derivatives are likely to provide a half-life sufficient for effective complement depletion in vivo.

6. CONCLUSIONS In summary, the design of recombinant C3 molecules with tailor-made anticomplementary properties represents a novel approach for depletion or modulation of complement, the mechanism of which is based on the well-established anti-complementary reagent CVF. Moreover, the characteristics of the derivatives can enable further molecular analyses and insights into the mechanisms of convertase formation.

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37. J. Stetefeld, M. Jenny, T. Schulthess, R. Landwehr, B. Schumacher, S. Frank, M.A. Ruegg, J. Engel, and R.A. Kammerer, The laminin-binding domain of agrin is related to N-TIMP-1, Nat Struct Biol 8, 705–709 (2001). 38. E. Liepinsh, L. Banyai, G. Pintacuda, M. Trexler, L. Patthy, and G. Otting, NMR structure of the netrin-like domain (NTR) of human type I procollagen C-proteinase enhancer defines structural consensus of NTR domains and assesses potential proteinase inhibitory activity and ligand binding, J Biol Chem 278, 25982–25989 (2003). 39. K. Dolmer, and L. Sottrup-Jensen, Disulfide bridges in human complement component C3b, FEBS Lett 315, 85–90 (1993). 40. P.J. Low, R. Ai, and R.T. Ogata, Active sites in complement components C5 and C3 identified by proximity to indels in the C3/4/5 protein family, J Immunol 162, 6580– 6588 (1999). 41. A. Sandoval, R. Ai, J.M. Ostresh, and R.T. Ogata, Distal recognition site for classical pathway convertase located in the C345C/netrin module of complement component C5, J Immunol 165, 1066–1073 (2000). 42. D. Candinas, B.A. Lesnikoski, S.C. Robson, T. Miyatake, S.M. Scesney, H.C. Marsh Jr, U.S. Ryan, A.P Dalmasso., W.W. Hancock, and F.H. Bach, Effect of repetitive high-dose treatment with soluble complement receptor type 1 and cobra venom factor on discordant xenograft survival, Transplantation 15, 336–342 (1996). 43. F.J. Vriesendorp, R.E. Flynn, M.A. Pappolla, Soluble complement receptor 1 (sCR1) is not as effective as cobra venom factor in the treatment of experimental allergic neuritis, Int. J Neurosci 92, 287–298 (1997). 44. C.G. Cochrane, H.J. Müller-Eberhard, and B.S. Aikin, Depletion of plasma complement in vivo by a protein of cobra venom: its effect on various immunologic reactions, J Immunol 105, 55–69 (1970). 45. R.A. Jr Nelson, A new concept of immunosuppression in hypersensitivity reactions and in transplantation immunity, Surv Ophthalmol 11(4), 498–505 (1966). 46. C.W. Vogel in: Handbook of Natural Toxins edited by A.T. Tu (Marcel Dekker Inc., New York, 1991), vol. 5, pp. 147–188. 47. Younger, J.G., et al., Systemic and lung physiological changes in rats after intravascular activation of complement. J Appl Physiol 90, 2289–2295 (2001). 48. R.A. Wetsel, and W.P. Kolb, Expression of C5a-like biological activities by the fifth component of human complement (C5) upon limited digestion with noncomplement enzymes without release of polypeptide fragments, J Exp Med 157, 2029–2048 (1983). 49. Y. Wang, S.A. Rollins, J.A. Madri, and L.A. Matis, Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease, Proc Natl Acad Sci USA 92(19), 8955–8959 (1995). 50. J.E. Figueroa and P. Densen, Infectious diseases associated with complement deficiencies, Clin Microbiol Rev 4, 359–395 (1991). 51. M.C. Carroll, The role of complement and complement receptors in induction and regulation of immunity, Ann Rev Immunol 16, 545–568 (1998). 52. D. Mastellos, and J.D. Lambris, Complement: more than a “guard” against invading pathogens? Trends Immunol 23, 485–491 (2002). 53. M. Bruggemann, G. Winter, H. Waldmann, and M.S. Neuberger, The immunogenicity of chimeric antibodies, J Exp Med 170, 2153–2157 (1989).

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54. Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy, Nature 332, 323–237 (1988). 55. G. Hale, M.J. Dyer, M.R. Clark, J.M. Phillips, R. Marcus, L. Riechmann, G. Winter, and H. Waldmann, Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H, Lancet 2, 1394–1399 (1988). 56. T.C. Thomas, S.A. Rollins, and R.P. Rother, M.A. Giannoni, S.L. Hartman, E.A. Elliott, S.H. Nye, L.A. Matis, S.P. Squinto, and M.J. Evans, Inhibition of complement activity by humanized anti-C5 antibody and single chain Fv, Mol Immunol 33, 1389–1401 (1997). 57. J.C. Fitch, S. Rollins, L. Matis, B. Alford, S. Aranki, C.D. Collard, M. Dewar, J. Elefteriades, R. Hines, G. Kopf, P. Kraker, L. Li, R. O’Hara, C. Rinder, H. Rinder, R. Shaw, B. Smith, G. Stahl, S.K. Shernan, Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. Circulation 100, 2499–2506 (1999). 58. A. Wright, and S.L. Morrison, Effect of C2-associated carbohydrate structure on Ig effector function: studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells, J Immunol 1607, 33393–33402 (1998). 59. X. Dong, W.J. Storkus, and R.D. Salter, Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells, J Immunol 16310, 5427–5434 (1999).

24 COMPLEMENT ANALYSIS IN CLINIC AND RESEARCH Tom E. Mollnes and Michael Kirschfink

1. INTRODUCTION A normally functioning complement system is required for effective protection against invading microorganisms, for immune complex elimination, and for 1-3 physiological tissue regeneration and repair . As a key mediator of inflammation, complement has the potential to damage host tissue as well. Protection against such inappropriate complement-mediated tissue destruction is provided by various soluble and membrane-bound regulatory proteins. Thus, defects in complement components increase susceptibility to infection and autoimmune disorders, whereas defects in complement regulatory proteins may lead to serious disorders due to unrestricted activation. In recent years, great progress has been made in complement analysis to better define disease severity, evolution, and response to therapy. Modern diagnostic technologies that focus on quantification of complement-derived split products or protein complexes now provide a comprehensive insight into the activation state of the system. The present paper briefly reviews the most frequent clinical conditions caused by complement defects and the methods available for complement system analysis in clinical medicine and in research. 1.1. The Complement System Complement is activated through three pathways, the classical, the lectin and the alternative pathway, all of them converging at the C3 activation step through the C3 convertase. The ensuing C5 cleavage leads to the formation of the C5b-9 membrane attack complex (Figure 1). Activation of C3 and C5 results in the

Tom E. Mollnes, Institute of Immunology, Rikshospitalet University Hospital and University of Oslo, Norway. Telephone: +4790630015, Telefax: +4723073510, E-mail: t.e.mollnes@medisin. uio.no. Michael Kirschfink, Institute of Immunology, University of Heidelberg, Germany,

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Classical pathway

Lectin pathway

C1q,C1r,C1s

MBL, MASPs C4

C4a

Alternative pathway C3(H2O)

C2b

C4bC2 C4b2a

C3a

C3

C3b

C4b2a3b

D

C3(H2O)B D C3(H2O)Bb C3bB P C3bBbP

Ba

C3b2BbP

C5 C5b C6+C7+C8+C9

C5a

Fluid-phase

SC5b-9

C5b-9(m) MAC

TCC Terminal complement complex

Membrane

Figure 1. Activation of the complement system starts through the classical, lectin, or alternative pathway and converges at C3. This initial part of the activation is characterized by proteolytic cleavage of components by proteins or complexes with serine protease activity (red ink), leading to release of small biologically active fragments like the anaphylatoxins (green ink). Activation of the terminal sequence after the last enzymatic cleavage (C5) is followed by assembly of a macromolecule consisting of C5b, C6, C7, C8, and C9 (blue ink). This terminal complement complex (TCC) may either insert into a membrane as the sublytic or lytic membrane attack complex, or be released in a soluble form to the fluid phase (SC5b-9).

generation of C3b and C5b, and of small split products C3a and C5a. These socalled anaphylatoxins, in particular C5a, elicit a number of proinflammatory effects, such as chemotaxis of leukocytes, degranulation of phagocytic cells, mast cells and basophils, smooth muscle contraction, and increased vascular 4 permeability . The anaphylatoxins can activate cells so that the inflammatory response is further amplified by subsequent generation of toxic oxygen radicals and induction of synthesis and release of arachidonic acid metabolites and cytokines. Consequently, activation of complement presents a considerable risk of harming the host by directly and indirectly mediating inflammatory tissue destruction.

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Under physiological conditions activation of complement is effectively controlled by the coordinated action of soluble and membrane-associated regulatory proteins (Table 1). Soluble complement regulators, such as C1 inhibitor, C4bbinding protein (C4bp), factors H and I, clusterin and S-protein (vitronectin), and serum carboxypeptidase N (SCPN) restrict the action of complement in body fluids at multiple sites of the cascade reaction. In addition, each individual cell is protected against the attack of homologous complement by surface proteins, such as complement receptor 1 (CR1, CD35), membrane cofactor protein (MCP, CD46), as well as by the glycosylphosphatidylinositol (GPI)-anchored proteins, decay-accelerating factor (DAF, CD55), and CD59. Altered surface expression of complement regulators may have important consequences for the 5,6 fate of a cell, e.g., when transformed to a malignant or apoptotic phenotype . One of the best-described clinical conditions of a defect in complement regula7 tory proteins is paroxysmal nocturnal hemoglobinuria (PNH) .

Table 1. Main Complement Regulatory Proteins Protein Soluble regulators C1-inhibitor Carboxypeptidase N Factor I C4b-binding protein Factor H Properdin Vitronectin Clusterin Membrane regulators CR1 (CD35) DAF (CD55) MCP (CD46) CD59

Function Inactivates C1r, C1s and MASP-2 Anaphylatoxin inactivator; des-arginates C3a, C4a and C5a Inactivates C3 and C4 by proteolytic cleavage Cofactor for factor I in the classical pathway Cofactor for factor I in the alternative pathway Stabilises the alternative pathway convertase; only known regulator of complement with enhancing effect on activation Binds to C5b-7, diverging it from the membrane to soluble SC5b-9 As for vitronectin Co-factor for factor I and decay accelerator for C3 and C5 convertase Decay accelerator for C3 and C5 convertase Co-factor for factor I Inhibits insertion of C5b-9 into the membrane

2. CLINICAL INDICATIONS FOR COMPLEMENT ANALYSIS Clinical and experimental evidence underlines the prominent role of comple8 ment in immunodeficiency disorders and in the pathogenesis of numerous inflammatory diseases including immune complex and autoimmune disorders, 9 10 such as systemic lupus erythematosus and autoimmune arthritis . Complement has also been implicated in neurodegenerative disorders, such as Alzheimer's

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12

13

disease , multiple sclerosis , and Guillain-Barré syndrome . Complement activation after polytrauma substantially contributes to the development of systemic 14,15 inflammatory response syndrome (SIRS) and multiple organ failure . In recent years complement has been recognized as a major effector mechanism of reper16 fusion injury . Furthermore, activation of complement is a critical event in the 17 pathogenesis of sepsis and septic shock . The inflammatory response induced by artificial surfaces in hemodialysis and cardiopulmonary bypass may lead to organ dysfunction. Here, complement activation has been shown to be associated with transient neutropenia, pulmonary vascular leukostasis, and occasion18,19 ally with anaphylactic shock of variable severity . In fact, the list of conditions where complement is involved in pathogenesis is so rapidly increasing that it may be appropriate to rather ask in which conditions complement is not involved. 2.1. Recurrent Infections Complement defects may be the cause of immunodeficiency-mediated recurrent 8 infections . The total complement activity test is a useful initial screening for complement deficiency when examining patients with suspected immunodeficiency. Recurrent Neisseria infections, particularly if systemic and with rare serotypes, is associated with complement defects, preferentially in the terminal C5–9 pathway. The frequency of meningococcal disease reported in C5–C9deficient individuals reflects a 1000- to 10,000-fold higher risk in this popula20 tion . Late component-deficient patients, which are readily identified by assays of total complement activity and further identified by selective C5–C9 determination, should be vaccinated with tetravalent vaccines against all four strains of 21 Neisseria meningitidis (A,C,W135 and Y) . There is also a high prevalence of properdin deficiency in patients with meningococcal disease, mainly caused by the uncommon serogroups W135 and 22 Y with a fatality rate of more than 25% . The most common type 1 deficiency is characterized by the absence of properdin in plasma, whereas in type 2 deficiency properdin is low but detectable (<10% of normal). Notably, properdin defects are not always detected in conventional hemolytic assay (AH50), but a novel ELISA-based total complement activity test shows a low alternative path23 way activity in sera with properdin deficiency . Mannose binding lectin (MBL) is a key protein of the lectin pathway of 24 complement. MBL variant genes can be found in 20–30% of the population . Most individuals with deficiency of MBL are completely healthy, a fact that emphasises the vital redundancy of innate host defence against infection. However, variant MBL genes are to some extent associated with increased infection risk. It has been suggested that this is particularly the case in early childhood 25 between the age of 6 and 18 months pointing to the important role of innate immunity before the establishment and development of the infant’s own adap-

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tive immune system. Thus, it is recommendable to add MBL to the list of parameters to be tested when immunodeficiency is suspected.

2.2. Autoimmune Diseases Complement defects, particularly of the classical pathway, are frequently asso26-28 ciated with systemic lupus erythematosus-like autoimmune disease . The strength of the association of a complement deficiency with SLE increases from C2 (10% prevalence) over C1r/s (57% prevalence), C4 (75% prevalence) to C1q 29 (90% prevalence) . Although complement deficiencies are frequently associated with autoimmunity, autoimmunity is only occasionally associated with complement deficiency. Therefore, in active SLE, particularly with renal involvement, low total complement activity and low C4 are more often due to increased in vivo activation, which can be verified by detection of complement activation products. Fluctuation in C4 levels is probably the best complement indicator of disease activity. In patients with severe clinical outcome, such as lupus nephritis, 30 autoantibodies to C1q are often found and may be of prognostic value .

2.3. Membranoproliferative Glomerulonephritis (MPGN) and Hemolytic Uremic Syndrome (HUS) In certain vasculitides and renal diseases a substantial activation and consumption of C3 can be observed, which is due to defective alternative pathway regulation. Patients suffering from membranoproliferative glomerulonephritis 31 (MPGN) , especially of the histologically defined type 2, often show low C3 values and consequently low activity in all pathways in the total complement activity test. This results from a continuous C3 activation either triggered by the 32 presence of a C3-convertase stabilizing autoantibody, termed nephritic factor , 33 or by factor H deficiency . These two factors should be analyzed when a patient presents with a very low C3 value. This disease can also be caused by other 34 complement disorders, like the rare genetic C3 deficiency , and by a unique 35 light-chain antibody that neutralizes factor H . Hemolytic uremic syndrome is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. There is compelling evidence that the atypical form of this fatal disease is often associated with a mutation in the C terminus of factor H, resulting in suboptimal regulator activity 36 and consequently leading to hypocomplementemia .

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2.4. Hereditary Angioedema Hereditary angioedema (HAE) is an autosomal dominant condition with reduced 37,38 concentration (type 1) or function (type 2) of C1 inhibitor . Considering the life-threatening consequences of edema formation, early diagnosis of C1 inhibor deficiency in these patients is extremely important. As spontaneous mutations can occur, a negative family history does not exclude the diagnosis. Furthermore, the penetrance differs significantly and minor symptoms or subclinical cases can be found. The pathophysiology of HAE is complex, but it is now generally accepted that formation of bradykinin through activation of the kallikrein– kinin system, which is also controlled by C1 inhibitor, is the major inductor of 39 the edema . The diagnosis is based on C1 inhibitor and C4 quantification. It is important to include both antigenic and functional assays for C1 inhibitor, since 15% of the patients have type 2 HAE with normal or even increased antigen concentration of C1 inhibitor. C4 is usually low in both cases even between the attacks and serves as a valuable supplement to C1 inhibitor analysis. Concentration of C3 is usually normal in HAE. Acute treatment requires purified C1 inhibitor or plasma transfusion, whereas androgens can be given prophylactically. An acquired condition with a clinical picture identical to HAE may occur in patients who develop autoantibodies to C1 inhibitor, in some cases related to 40 hematologic malignancies . In addition to reduced C1 inhibitor and C4 levels, these patients frequently have a low C1q concentration in contrast to the HAE patients. Identification of this form of acquired angioedema is important as its acute treatment requires higher doses of C1 inhibitor. 2.5. Paroxysmal Nocturnal Hemoglobinuria (PNH) A somatic mutation in the gene coding for the phosphatidylinositol (PI) anchor (PIG-A) results in decreased expression of membrane proteins linked to this 41 structure , including decay-accelerating factor (DAF, CD55) and CD59. Decreased expression of DAF and/or CD59 renders the red cells susceptible to complement-mediated lysis, which is the hallmark of this condition. Diagnosis was traditionally made by Ham’s test (acid lysis test) but is now specifically 42 assessed by flow-cytometric analysis of the respective cell-surface proteins .

3. COMPLEMENT TESTS 3.1. Functional Assays The main complement assays are summarized in Table 2.

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Table 2. Main Complement Assays Function

Proteins

Genetic mapping Surface proteins

Activation products

Total complement activity (screening for complement deficiency): a) CH50 and AH50 hemolytic assays for CP and AP activity b) ELISA for specific evaluation of CP, LP and AP activity using C5b-9 as readout Functional activity of single components: a) Hemolytic assays for single components (e.g. C3) using corresponding deficient sera as test system b) ELISA for MBL/MASP functional activity using deposition of C4 as readout c) C1 inhibitor assay (chromogenic substrate or ELISA) for diagnosis of hereditary angioedema (HAE) type 2 Measuring the concentration of single components by immunoprecipitation (RID, nephelometry), ELISA or Western Blot, e.g.: a) C3 and C4 to detect “hypocomplementemia” b) Follow-up of a low activity detected in total complement activity screening (any component) c) C1 inhibitor for diagnosis of HAE type 1 and acquired angioedema Detection of disease associated genetic variants; e.g factor H, MCP and factor I in hemolytic-uremic syndrome Flowcytometric quantification of CD55 (DAF) and CD59 for diagnosis of paroxysmal nocturnal hemoglobinuria and of CD11/CD18 (incl. CR3 and CR4) for leukocyte adhesion deficiency ELISAs preferentially based on antibodies to neoepitopes expressed selectively on the activation products: a) Split products from components after proteolytic cleavage (e.g. C3a, C4a, C5a) b) Complexes between the activated component and its inhibitor (e.g. C1rs-C1 inhibitor) c) Macromolecular complexes (e.g. the AP convertase C3bBbP and the terminal SC5b-9 complex)

3.1.1. Total Complement Activity of the Classical, the Lectin, and the Alternative Pathways Hemolytic assays performed either in tubes or in agarose plates have traditionally been used to assess the functional activity of the complement system. They provide insight into the integrity of the entire cascade reaction. These tests are particularly useful in the investigation of suspected complement deficiencies. Serial dilutions of the sample to be analyzed are incubated with antibody-sensitized sheep erythrocytes at a defined temperature. Results are usually expressed as a reciprocal dilution of the sample required to produce 50% (100%) lysis (CH50, CH100). Tests evaluating the functional activity of the alternative pathway (APH50) use guinea pig, rabbit, or chicken erythrocytes as target cells. Here, activation of the classical pathway has to be blocked by adding

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2+

EGTA to chelate Ca , and an optimal concentration of Mg is required. A disadvantage with the hemolytic assays is their dependency on stable red cells. Furthermore, APH50 is frequently normal or only slightly reduced in properdin deficiencies and thus not suitable for detection of the most frequent alternative pathway deficiency.

Total Complement System Activity

C7 C6 C5 C3

C8

C9 C7 C6 C5 C3

C4 C2

C4 C2

C1qrs

MASP-2

C8

C9

MBL

IgM

Classical pathway

Mannan

Lectin pathway

C7 C6 C5 C3 P FB

C8

C9

FD

LPS

Alternative pathway

Figure 2. Enzyme immunoassay for detection of total complement activity. The three initial pathways can be evaluated separately by coating the microwell with different agents and using different buffer conditions. The final read-out is deposition of C5b-9, detected by a monoclonal antibody with specificity for the incorporated C9, implying that all components need to be present in order to get a positive result in the test.

A novel methodological approach, an enzyme immunoassay, has been developed and is about to replace the hemolytic assays for specific evaluation of 23 the classical, lectin, and alternative pathway activity . The three pathways are tested in microtiter wells coated with IgM, mannan, and lipopolysaccharide, respectively. When adding serum, the activation will continue until assembly of C5b–C9, which can then be quantified by an antibody specific for activated C9 (Figure 2). Thus, the principle is very similar to the hemolytic assays, but this assay is more robust and suitable for large-scale complement deficiency screening. A low classical pathway activity with normal lectin and alternative pathway function indicates C1q, C1r, or C1s deficiency. Similarly, a low lectin pathway function with normal classical and alternative pathway activity indicates MBL

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or MASP-2 deficiency, whereas a low alternative pathway activity is consistent with properdin, factor B, or factor D deficiency. C2 and C4 deficiencies give low classical and lectin pathway activity with normal alternative activity, whereas C3 or C5 through C9 deficiencies will result in low activity in all three pathways. From the initial screening it is thus possible to select for candidate complement protein deficiencies, which should then be verified by quantifying the individual components. Total complement activity, whether analyzed by hemolytic or enzyme immunoassay, is sensitive to in-vitro activation of complement in serum. If serum is heat inactivated, has been stored for a long time at room temperature, or contains complement-activating agents (e.g., immune complexes, cold agglutinins), activity is reduced and may even be zero. Thus, fresh serum (< 48 hrs) should be o used or samples should be stored at –70 C until tested. 3.1.2. Activity of Individual Components The most convenient way to detect the functional activity of individual complement components is to test for the sample's capability to reconstitute the activity of a serum that is deficient for that particular protein in hemolytic assays designed like the CH50. With the availability of complement-deficient or depleted human sera, titration of individual complement proteins became much easier to perform. To finally prove that only one single component is lacking, the respective purified functionally active component needs to be added to the serum to restore the hemolytic activity. It is hereby possible to use the functional enzyme immunoassay as described above for identification of single-component functional activity. 43 A special assay has been described for MBL functional activity . It is based on the same principle as for lectin pathway screening in the enzyme immunoassay described above, i.e., serum is added to a microwell coated with mannan. However, this is done in high salt concentrations in order to prevent classical pathway activation. After washing, exogenous C4 is added in PBS and the amount of bound C4 is then detected, reflecting the activity of the MBL– MASP-2 complex. 3.1.3. C1 Inhibitor Activity 15% of patients with hereditary angioedema (HAE) have a dysfunctional protein (type 2 HAE) with normal or increased concentrations of C1 inhibitor antigen. To diagnose this form of HAE it is mandatory to include a functional C1 inhibi44 tor test , which can be done by commercially available quantitative chromogenic assays or novel enzyme immunoassays based on complex formation between the serine protease inhibitor and its substrate(s).

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3.2. Protein Quantification of Individual Components Individual complement components, irrespective of their functional activity, can be measured by immunoprecipitation tests (radial immunodiffusion (RID) or nephelometer techniques), by enzyme-linked immunosorbent assays (ELISA), or by Western blot. In routine diagnosis, C3 and C4 are most frequently measured to reveal hypocomplementema particularly associated with autoimmune vasculitis and glomerulonephritis. C1 inhibitor concentration is measured to verify the diagnosis of HAE type 1 and acquired angioedema. When a screening test for total complement activity indicates a deficiency, protein quantification of candidate components should be performed — e.g., C1, MBL, and properdin when CP, LP, and AP activity is defective, respectively, C2 and C4 when both CP and LP activity is defective, and C3 as well as terminal components if all pathways are defective. If a defect is verified by protein quantification, further functional assays are not required. On the other hand, if immunochemical assays do not reveal any deficiency, the actual component may be functionally inactive and a functional assay can then verify the diagnosis. 3.3. Genetic Analysis Genetic variants of complement components may predispose to certain clinical conditions. In particular, factor H mutations have been associated with atypical 45 46 47 forms of hemolytic-uremic syndrome . Recently also, factor I and MCP have been attributed roles in the development of this disease. Thus, the molecular study of these proteins may be helpful in diagnosis of HUS. 3.4. Cell Surface Expression of Complement Proteins PNH is a disease associated with increased susceptibility of red cell lysis due to reduced expression of the complement inhibitors DAF (CD55) and CD59. These are non-transmembrane proteins, and genetic deficiency affects their common GPI anchor. Flow-cytometric quantification of DAF and CD59 is a valuable diagnostic tool of PNH. Analysis of CD11/CD18 (including CR3 and CR4) needs to be performed in cases of leukocyte adhesion deficiency type 1(LAD-1). 3.5. Analysis of Complement Activation Products Reduced total hemolytic activity and levels of individual complement components may indicate ongoing complement activation, e.g., low C4 and CH50 in active SLE. However, the same results may be obtained in cases with an underlying genetic deficiency of C4, as frequently observed in SLE. Furthermore, in vivo complement activation going along with an acute-phase reaction may leave the individual components within the normal range despite ongoing consumption, since most of the complement components are acute-phase proteins. In

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general, total hemolytic activity and individual component measurements are useful as first-level screening techniques but are not appropriate to detect with enough sensitivity pathologically increased complement activation in vivo. During the last two decades highly specific monoclonal antibodies have been produced which recognize neoepitopes exclusively exposed upon activation48 induced conformational changes . This enables direct capture of the activation fragment without interference of the non-activated component. Quantification 49 can be done as traditional ELISA , upon binding to high-capacity immunosor50 bents with subsequent elution , or to microbeads applied in multiplex flowcytometric technology (Figure 3). Today neoepitope-based assays have replaced the older generation of tests, which were hampered by pre-assay precipitation or fractionation steps, and were time-consuming or of low capacity. Furthermore, neoepitope-specific antibodies are particularly suitable to detect in situ complement activation applying immunohistochemistry since they discriminate between activation products and passively trapped native components.

Complement activation assays based on neoepitope-specific antibodies anti-C6

Av-Px

C3a

C4a

Biotin

A

B

SC5b-9

anti-C9neo mAb aE11 ELISA

C

C5a

Cytometric beads

Figure 3. Detection of complement activation products is preferentially done by using enzyme immunoassays based on monoclonal antibodies to neoepitopes expressed selectively in the activated form of the protein. To the left is illustrated the principle of an ELISA for quantification of TCC, and to the right is the corresponding principle for a cytometric bead assay to quantify the three anaphylatoxins in one single sample.

Complement activation products may be either split fragments after enzymatic cleavage of certain components, e.g., C4 (C4a, C4b/c, C4d), C3 (C3a, C3b/c, C3d), factor B (Ba, Bb), and C5 (C5a), or protein complexes where activated components are bound to their respective regulators, like C1rs–C1 inhibi-

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tor, the properdin-containing alternative pathway convertase C3bBbP, and SC5b-9 (soluble terminal complement complex). Neoepitope-based assays for 51 specific detection of the classical (e.g., C1rs-C1 inhibitor) and the alternative 52 (e.g., C3bBbP) pathways have been established , whereas specific assays for activation products of the lectin pathway are still missing. Indirectly, lectin pathway activation can be suspected if C4 activation products are detected without evidence of classical pathway activation. Complement activation products are usually present only in trace amounts 53 in vivo, but they are rapidly generated in vitro . Therefore, it is crucial that the samples are collected and stored properly in order to avoid in-vitro activation. Blood should be drawn directly into EDTA-containing tubes at a final EDTA concentration of at least 10 mM. Alternatively, nafamostat mesilat can be used as anticoagulant, whereas citrate and heparin do not block complement activation sufficiently. The sample should be cooled, and plasma should be prepared immediately and stored at –70°C. The various complement activation products have different half-lives in vivo. This is important for the activation product(s) of choice to be measured. Due to rapid receptor binding, the biologically highly active and important C5a 54,55 fragment has a half-life of approximately one minute and is difficult to detect in samples obtained in vivo, whereas the various C3 activation products are 56 readily detectable due to half-lives of a few hours . The half-life of SC5b-9 is 57,58 50–60 minutes . SC5b-9, in contrast to C3 activation products, is relatively stable in vitro, and since it reflects activation to the end of the terminal sequence irrespective of the initial pathway involved, it is a particularly good candidate for general evaluation of complement activation. If measuring an activation product from a single component, the amount should be related to the concentration of the native component since a low level of native component would yield smaller amounts of activation products during in-vivo activation. Thus, it has been postulated that the ratio between the activation product and the native component is a more sensitive indicator of in-vivo 59,60 activation than the activation product alone .

4. COMPLEMENT ANALYSIS IN EXPERIMENTAL SETTINGS 4.1. In-Vitro Experiments with Human Serum and Blood Activation mechanisms and intervention of complement activation can readily be studied in vitro using human serum. Since there are no cells present and there is no biological turnover, any activation product including C5a can be detected. A more physiological approach, however, is to use whole human blood. In this case anticoagulants needed to be added. However, most of the anticoagulants, such as EDTA, citrate, and heparin, interfere with complement activation and should be avoided. From our own experience recombinant hirudin (lepirudin), a highly specific thrombin inhibitor, does not affect complement and seems par-

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ticularly useful for complement studies in whole human blood . To avoid further activation during preparation and storage after the experiments have been performed, it is necessary to add EDTA or nafamostat mesilat at the end of the experiments. In vivo, experimental studies of human complement are limited for obvious reasons. However, certain clinical conditions can be mimicked by experimental settings, e.g., extracorporeal circulation (hemodialysis or cardiopul62 monary bypass), and proved to be useful to study complement activation and 63 possible effects of complement intervention . 4.2. Animal Experiments 4.2.1. Application of Genetically Deficient or Knockout Animals A number of animals with genetic deficiencies in individual complement com64 ponents have been described and are commercially available, e.g., C2- , C4-, or 65 66 C3-deficient guinea pigs, C3-deficient dogs , C5-deficient mouse , and C667 deficient rats and rabbits . During the last few years an additional number of 68,69 knockout mice have been generated both for cascade components , regulatory 70 71,72 proteins , and complement receptors . These animals are particularly useful to study the importance of complement in various diseases and to delineate activation mechanisms and the pathways of activation involved. In principle, reconstitution experiments using the respective purified component should be included, but for practical reasons this is not always possible since limited numbers of components have been purified from the different species. Provided functional crossreactivity, addition of a component from another species may be possible. Administration of a purified human component to a genetically deficient mouse may succeed regarding some complement effects, such as lysis, but molecular incompatibility may imply that different receptor mediated effects can not be studied. Reconstitution with whole serum of the same species may serve as an alternative. 4.2.2. Complement Depletion Cobra venom factor (CVF) has been used for decades to “de-complement” animals. CVF is analog to C3b and activates C3 but is resistant to regulation by 73 human factors I and H . CVF treatment is frequently and misleadingly called inhibition of complement; in fact, an extreme activation with subsequent complement consumption takes place. The animals usually tolerate this activation fairly well since only a limited activation of the terminal pathway occurs. Never74,75 theless, adverse effects of CVF have been reported , and one should be very cautious with interpretation of the data from such experiments. A direct inhibi76 tion of complement at a certain level is recommended instead of CVF treatment when mechanisms of complement activation are to be studied.

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4.2.3. Measuring Animal Complement Hemolytic assays can be designed for any species. They can be used to monitor the effect of complement inhibition by a certain substance or as a semiquantitiative measure of complement activation under controlled conditions, since hemolysis will be lowered proportionally to the consumption of complement. In many animal species a comprehensive analysis of complement is still hampered by the lack of suitable reagents and assays. A major factor affecting the evaluation of complement activity in the serum of previously unstudied species is the choice of an appropriate target cell. Low or non-detectable classical pathway activity of mouse or bovine complement, for example, results from applying conventional methods using sensitized sheep erythrocytes. Comparison of classical pathway activity between different species is difficult to evaluate since hemolytic titers are also influenced by the source and quality of erythro77 cyte-specific antibodies . Applying optimal test conditions, it can be demonstrated that virtually all species of laboratory and domestic animals possess clas78-80 sical as well as alternative pathway activity . Compatibility of components within the cascade sequence between different species and guinea pig or man 81 often allows titration of individual components . To evaluate the pathophysiological contribution of complement to certain diseases, assessment of complement activation by immunochemical means is indispensable. However, as measurement of components is not a sensitive and reliable indicator of a slight activation, activation products should be tested. Whereas a number of neoepitope specific assays have been described for human complement activation, only a few are available for animal studies. These are either assays for human activation products that crossreact with other species or assays particularly designed for animal studies. The latter include assays based 82 on monoclonal antibodies to neoepitopes of guinea pig C5a and C3 activation 83 84 85 86 products , rabbit C5a , rat TCC and C3a , and murine C3a and C3b/iC3b/C3c . Several of the human neoepitope-specific assays crossreact sufficiently well 87 88 with other species, like baboon C3bc , baboon C3bc, C5a and C5b-9 , baboon 89 90 C4d, C3a, Bb, and C5b-9 , and porcine C5b-9 . The possible species crossreactivity of a certain assay made for human complement activation can easily be checked by activating the serum from the respective species by heat-aggregated immunoglobulin (classical pathway) or zymosan (alternative and lectin pathway) and compare with the non-activated control. If the signal in activated serum is stronger than in the non-activated, the assay crossreacts with this species and the degree of crossreactivity can be estimated. For example, a test for the mannan-binding lectin pathway activity in humans was used in the mouse sys91 tem only with minor changes . On the other side, old methods are constantly 92 being improved , so the field of complement analysis remains dynamic.

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5. OUTLOOK The plethora of settings in which complement plays a role underscores the importance of this ancient system. The implementation of new analysis techniques and the development of new diagnostic tools will help to further elucidate its mechanisms of action and effects. Using these new approaches in a collaborative way between research and clinic, it will be ultimately possible to find therapeutic strategies for diseases in which complement is involved either during pathogenesis or during convalescence.

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25 CELL-BOUND COMPLEMENT ACTIVATION PRODUCTS (CB-CAPS) AS A SOURCE OF LUPUS BIOMARKERS Sarah J. Calano, Pei-an B. Shih, Chau-Ching Liu, Amy H. Kao, Jeannine S. Navratil, Susan Manzi, and Joseph M. Ahearn

1. INTRODUCTION Systemic lupus erythematosus is a clinically and serologically diverse autoimmune disease, primarily affecting women, with a female to male ratio of 6–10:1 and a peak incidence during childbearing ages. Although the etiology and pathogenesis of SLE are elusive, the presence of autoantibody production against “self” antigens and deposition of immune complexes are thought to be 1 evidence of immune dysregulation . Nearly any organ in the body can be affected by SLE, and manifestations vary widely from subtle symptoms to lifethreatening multiorgan failure. The role of complement in SLE is intriguing. On the one hand, complement system activation as a consequence of the deposition of immune complexes may 2,3 be a fundamental part of tissue inflammation and damage . Conversely, a hereditary deficiency of classical pathway components (C1, C2, or C4) has been 4, 5 associated with the development of SLE . These seemingly disparate roles for complement have been explained by investigators suggesting that, while the complement system maintains immune tolerance to prevent the SLE develop4,6,7 ment , it also participates in tissue-destructive inflammatory processes once an 2,3,6,8 individual has developed the disease .

Sarah J. Calano, Pei-an B. Shih, Chau-Ching Liu, Amy H. Kao, Jeannine S. Navratil, Susan Manzi, and Joseph M. Ahearn, Lupus Center of Excellence, University of Pittsburgh School of Medicine and the University of Pittsburgh Graduate School of Public Health.

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2. MEASUREMENT OF COMPLEMENT IN SLE Because complement activation occurs during disease flares in SLE, one might expect complement proteins to be consumed and activation-derived products to be generated at a rate proportional to the degree of disease activ9 10 ity . Since Vaughan et al. first reported an association between decreased complement proteins and active SLE, numerous studies have been conducted to evaluate the worth of measuring serum complement levels in monitoring SLE disease activity. 2.1. Serum C3 and C4 and SLE Disease Activity While some investigators have found serum C4 and C3 levels to be valuable as 11-21 markers of SLE activity, others have found them minimally useful . For ex11 ample, Lloyd and Schur reported that significantly decreased levels of CH50 and serum C3 and C4 were associated with increased SLE disease activity. However, decreased serum C4 levels were also found in a large portion of pa11 tients with stable SLE disease activity . Using either cross-sectional or prospective studies, several investigators have subsequently shown that fluctuations in serum C3 levels correlated more significantly with the changes in activ11,12,14 ity/severity of SLE than serum C4 levels (Table 1) . These studies support the notion that C3 may be superior to C4 as a serological marker in the assessment of ongoing SLE disease activity, specifically renal involvement. In contrast, other investigators have found that serum C4 is a superior assay 9,15 for monitoring SLE . Some studies have suggested that serum C3 levels have 11,15,16,17 no correlation with disease activity (Table 1) , while others observed that 9,11 serum C4 appeared to decrease prior to clinical exacerbation . These studies support the role of serum C4 as a useful marker of SLE disease activity and a predictor for future SLE flares. These observations reflect a more general disagreement regarding the role of C3 and C4, separately or together, as reliable markers of SLE disease activity. 2.2. Issues Associated with Measuring Soluble Complement Components Several factors may be responsible for the inadequate utility of soluble C3, C4, and complement activation products in monitoring patients with SLE. First, the wide range of serum C3 and C4 levels among healthy individuals overlaps with those observed in SLE patients. Second, standard laboratory tests measure the concentration of C3 and C4 molecules in circulation irrespective of their functional status. Third, the consumption of C3 and C4 during activation may be counteracted by an increase in C3 and C4 synthesis during the inflammatory 34 28,29 acute phase response . Fourth, alterations in synthesis and catabolism of C3 35-37 and C4 , reported in SLE patients, can interfere with static measures of C3 and C4 levels. Fifth, lower than normal C4 levels may result from partial deficiency

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Table 1. Selected Studies of Serum Complement Measures in SLE Study Lloyd and Schur11

Valentijin et al.12

Milis et al.14

Study Lloyd and Schur11

Studies supporting the value of serum C3 measures Study design Results/conclusions Prospective study; Serological tests: CH50, C1q, C3, C4, anti-dsDNA, and immune complex (C1q binding activity) levels; Disease activity: physician assessment Retrospective study; Serological tests: CH50, C3, C4, immune complexes (C1q binding); Disease activity: scoring system designed by authors Prospective study; Serological tests: C1q, C4, C4a, C2, C3, C3a, C5, CH50, C4a:C4 ratio, C3:C3a ratio; Disease activity: clinical and serological assessment

CH50, C3, C4 levels lower in patients with active renal disease than in patients with extra-renal involvement Significant correlation between overall disease activity, decreased C3/CH50, and increased immune complex levels; Reduced C2 and C3 levels distinguishing patients with active disease from those with inactive disease; C1q, C4, C5, and CH50 less useful

Studies supporting the value of serum C4 measures Study design Results/conclusions Prospective study; Serological tests: CH50, C1q, C3, C4, anti-dsDNA, and immune complex (C1q binding activity) levels; Disease activity: physician assessment

Decreasing C4 levels preceded disease flares

Cameron et al.15

Prospective study; Serological tests: C3, C4, anti-dsDNA; Disease activity: a 7-point scoring system

Swakk et al.16

Prospective study; Serological tests: C1q, C3, C4, C5, C9, anti-dsDNA:

Ting and Hsieh17

Long term study; Serological tests: C3, C4, ANA, anti-dsDNA; Disease activity: Lupus activity criteria count (LACC)

No correlation between C3 levels and disease activity; Decreased C4 levels detected in both patients with active and inactive disease; Normal C4 not found in patients with active disease Decreasing C4 detectable 20-25 weeks before the flare; Decreased C1q and C3 detected during but not before the flare Decreased C3 levels detected during, but rarely before, the active stage of disease; Decreased C4 levels detected prior and during active disease

Study Abrass et al.18

Morrow et al.19

Esdaile et al.20,21

Studies arguing against the value of serum C measures Study design Results/conclusions Prospective study; Serological tests: C3, antidsDNA, immune complexes; Disease activity: clinical record of disease manifestations and medications Prospective study; Serological tests: CH50, C3, C4, anti-dsDNA, immune complexes; Disease activity: graded as severely active, moderately active, and inactive using the UCH/Middlesex criteria Retrospective analysis of patient serum samples; Serological tests: C3, C4, antidsDNA, immune complexes; Disease activity: SLEDAI, a rise of > or = 6 defined as a flare

C3 and anti-dsDNA neither associated with nor predictive of changes in disease activity None of the serological tests reliably distinguished the three clinical groups

All serological parameters tested poor predictor of SLE flares; Some values of patient-based serial measures in association with specific types of flares (e.g., decreased C3 and renal involvement)

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of C4 rather than from increased complement consumption during disease flares. Sixth, local complement activation from deposition of immune complexes may not be consistent with systemic levels of complement activation products. Besides the influence of genetic and biochemical factors on measures of complement, clinical and study design variability may also contribute to inconsistencies in the data generated to date. 2.3. Complement Activation Products and SLE Disease Activity Given the inadequacy of serum C3 and C4 measures in the diagnosis and monitoring of SLE, many investigators have turned their attention to complement activation products. A number of complement activation products from the classical pathway (C1rs–C1 inhibitor complex, C2a, C4d), alternative pathway (Ba, Bb, C3b(Bb)P), and the common terminal pathway (C3a, iC3b, C3d, C5a, C5b-9 complex), have been investigated as potential disease markers or predictors in SLE (Table 2). Although studies have suggested that these soluble complement activation products may have clinical utility, none of them have replaced measurement of serum C3 and C4.

3. CELL-BOUND COMPLEMENT ACTIVATION PRODUCTS The inconsistent results obtained from measures of soluble complement proteins and activation products have led us to consider measurement of cell-bound complement activation products (CB-CAPs) as biomarkers for SLE diagnosis and monitoring. This hypothesis was based upon the following rationale. First, serum C3 and C4 levels have no diagnostic utility and limited monitoring utility, and the inherent flaws in these measurements have been well documented, as described above. Second, measurement of soluble complement activation products has been shown to have utility in certain clinical situations; however, these assays have not replaced measurement of serum C3 and C4 in clinical practice. Furthermore, the half-lives of these fragments in the circulation are likely to be short. Third, cell-surface receptors for complement activation products are present on all circulating cells and may confound accurate and reliable measurement of the soluble activation products. Fourth, C3- and C4-derived complement activation products are capable of covalent attachment to cell surfaces via thioester bonds, and this property of the peptides may increase longevity in the circulation. Fifth, C4-derived complement activation products are known to be present on surfaces of normal erythrocytes, although the physiologic signifi38-40 cance of this phenomenon is unknown . Sixth, CB-CAPS on specific cell types might reflect additional disease information by unique cellular properties such as the lifespan of erythrocytes and reticulocytes.

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Table 2. Select Studies of Complement Activation Products in SLE

Study Sturfelt et al.22

Comp. activ. product(s) tested C1rs-C1inh, C3d

Nagy et al.23

C1rs-C1inh, C3(Bb)P, C5b-9

Wild et al.24 Belmont et al.25

C4a, C3a C3a, C5a

Hopkins et al.26

C3a, C5a

Porcel et al.27

C3a, C4a, iC3b, C5b-9

Senali et al.28

C4d, C3d

Kerr et al.29

Ba, Bb, C4a, C3d

Buyon et al.30

Ba, Bb, C4d, C5b-9

Manzi et al.31

Bb, C4d, C5b-9

Falk et al.32

C5b-9

Gawryl et al.33

C5b-9

Results/conclusion Increased C1rs-C1inh consistently found during flares; Increased C1rs-C1inh detected before flares, especially extra-renal flares; Increased C3d associated with severe disease flares All 3 activation products elevated in SLE patients with inactive disease compared to healthy controls; C3(Bb)P and C5b-9 but not C1rs-C1inh, distinguishing active disease from inactive disease C4a levels correlated with disease activity; C3a significantly elevated in patients with severe or moderate disease activity and quantitatively correlated with disease severity; C5a significantly elevated in patients with severe disease activity C3a levels significantly higher in patients with a flare than in those with stable disease; Rising C3a levels predictive of disease flares; Highly elevated C3a and C5a in patients with CNS involvement; C3a, C4a, and C5b-9 significantly elevated in patients with active disease, with a positive correlation with disease activity scores; C5b-9 most sensitive and specific; Elevated C4d levels correlated with disease activity in a linear fashion; C3d levels elevated but not linearly correlated with disease activity Increase of Ba, C4a, and C3d in the same patients associated with severe disease activity Ba levels significantly elevated and positively correlated with disease activity; Elevated C4 and increased Bb predictive of subsequent flares C4d and Bb sensitive indicator of moderate-to-severe disease activity; C4d and Bb sensitive at predicting increasing disease activity C5b-9 levels significantly elevated and positively correlated with disease activity Elevated C5b-9 levels correlated with disease exacerbation

3.1. Erythrocyte-Bound C4d as a Diagnostic Assay for SLE As the most abundant cells in the circulation, erythrocytes are potential “magnets” for products of complement activation generated systemically or locally. Some investigators have previously attempted to explore the phenomenon of

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erythrocyte-bound complement fragments in disease utilizing radioimmunoassay 41,42 42 or enzyme-linked immunosorbent assays . For example, Ross et al. demonstrated that SLE patients expressed significant numbers of C3dg/C3d fragments on their erythrocytes, while healthy individuals did not. We have recently used flow-cytometric assays to measure the levels of C4d on the surface of erythrocytes (E-C4d) (Figure 1), which allows quantitative measurement of cell-bound C4d levels to sensitively and accurately represent the degree of complement activation in SLE patients.

CR1 C4d C4d C4d

C4d

C4d CR1

RBC C4d CR1

C4d

C4d

C4d C4d CR1

Figure 1. Schematic illustration of immunodetection of cell-bound complement C4d and CR1. (left) C4d and CR1 present on the surface of blood cells are labeled using anti-C4d and anti-CR1 antibody, respectively, followed by a fluorochrome-conjugated secondary antibody. Labeled cells are analyzed using a flow cytometer. (right) Representative flow cytometric data of C4d and CR1 levels on erythrocytes from a healthy individual and a patient with SLE. Note that erythrocytes from the healthy individual express high levels of CR1 and low levels of C4d, whereas erythrocytes from the SLE patient express low levels of CR1 and high levels of C4d.

Erythrocyte-bound C4d (E-C4d) levels were measured using flow cytometry in a cross-sectional study including patients with SLE, patients with other 43 inflammatory and immune-mediated diseases, and healthy controls . Given the known role of complement receptor 1 (CR1) in the clearance of complementbearing immune complexes, levels of complement receptor 1 (CR1) on erythrocytes were simultaneously determined with E-C4d (Figure 1). The results were used to determine whether SLE-specific pathophysiologic defects might be reflected by abnormal patterns of complement activation products and receptors on erythrocytes of patients with SLE.

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Our results highlight several important points. First, erythrocytes from all three groups have surface-bound C4d. Second, E-C4d and E-CR1 levels remain nearly constant in healthy individuals over time. Third, patients with SLE display significantly higher E-C4d levels than patients with other diseases and healthy individuals. Fourth, abnormally high E-C4d levels occurring with abnormally low E-CR1 levels is a pattern with high diagnostic sensitivity and specificity for SLE with respect to healthy controls (81% sensitive; 91% specific) and patients with other inflammatory diseases (72% sensitive; 79% spe43 cific) . These data suggest that E-C4d measurement may serve as a diagnostic biomarker for SLE. 3.2. Reticulocyte-Bound C4d as an “Instant Messenger” of Disease Activity in SLE A biomarker reflecting the extent of complement activation and SLE disease activity in a timely and quantitative manner would be particularly valuable. The precursor of the erythrocytes, the reticulocyte, develops from hematopoietic stem cells in the bone marrow, and remains distinct for 1–2 days before fully maturing into erythrocytes. Reticulocytes are readily exposed to C4-derived fragments if they enter the peripheral circulation during active disease. Because erythrocytes have a lifespan of approximately 120 days and may bind and accumulate activation-derived C4d throughout that period, the E-C4d level is likely to be the collective result of complement activation and disease activity over a 120-day period. Because of the brevity of the reticulocyte stage, the levels of C4d attached to reticulocytes (R-C4d) are more likely the result of current activation rather than past events. Therefore, we hypothesized that R-C4d levels may serve as "instant messengers" by accurately reflecting SLE disease activity at a specific time point. Measurement of R-C4d levels in patients with SLE, patients with other dis44 eases, and healthy controls led to the following observations. First, a wide range of R-C4d was detected in SLE patients, but not in patients with other diseases and healthy controls. Second, the mean R-C4d level of SLE patients was significantly higher than that of patients with other diseases or healthy controls. Third, R-C4d levels correlated with clinical disease activity measured by the Systemic Lupus Erythematosus Disease Activity Index and Systemic Lupus Activity Measurement. These results support the hypothesis that C4d-bearing reticulocytes may serve as “instant messengers,” and provide clues about present and potential disease flares in SLE patients.

4. SUMMARY Measurement of serum C3 and C4 has been used for several decades to monitor disease activity in patients with SLE. Despite the limited utility and recognized

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weaknesses of these assays, they have remained the gold standard during an era of unprecedented discovery in the complement field. The current urgent need for lupus biomarkers warrants efforts to mine the complement system for assays superior to serum C3 and C4. Recent studies of soluble and cell-bound complement activation products hold promise for achieving this goal.

5. ACKNOWLEDGMENTS We thank our colleagues in the Lupus Center of Excellence and Division of Rheumatology and Clinical Immunology for their support. The studies mentioned in this article were supported by grants from the National Institutes of Health (RO1 AR-4676402, RO1 HL074335, RO1 AR-46588, NCRR/GCRC MO1-RR-00056, K24 AR02213), the Lupus Foundation of Pennsylvania, the Alliance for Lupus Research, the Lupus Foundation of Southeastern Pennsylvania, the American College of Rheumatology Research and Education Foundation, and the Pennsylvania Department of Health.

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Author Index

Ahearn, Joseph M., 381 Babiker, Adil A., 107 Barlow, Paul N., 313 Birmingham, Dan J., 227 Bora, Nalini S., 53 Bora, Puran S., 53 Bower, Joseph F., 249 Bredehorst, Reinhard, 347 Brueckner, Uwe, 217 Calano, Sarah J., 381 Cebada-Mora, Claudia, 95 Chung, Erwin K., 227 Connolly Jr, E. Sander, 189 Cudrici, Cornelia, 139 DeAngelis, Robert A., 17 Del Rio-Tsonis, Katia, 63 De Meester, Els, 281 Ducruet, Andrew F., 189 Endo, Yuichi, 265 Erdei, Anna, 177 Flierl, Michael, 217 Fonseca, Maria I., 153 Fujita, Teizo, 265 Gebhard, Florian, 217 Ghebrehiwet, Berhane, 95 Govaerts, Paul, 281 Gros, Piet, 291 Hack, Eric, 203 Hebert, Lee A., 227 Herbert, Andrew P., 313 Higgins, Gloria C., 227 Huber-Lang, Markus, 217 Janssen, Bert J.C., 291 Jesty, Jolyon, 95 Jha, Purushottam, 53 Kao, Amy H., 381 Kaplan, Henry J., 53 Kato-Unoki, Yoko, 121 Kirschfink, Michael, 361

Köhl, Jörg, 71 Kölln, Johanna, 347 Komotar, Ricardo J., 189 Lambris, John D., 1, 17, 35, 63 Liu, Chau-Ching, 381 Liu, Yu, 265 Lupu, Florea, 203 Manzi, Susan, 381 Markiewski, Maciej M., 17 Mastellos, Dimitrios, 1 Mocco, J., 189 Mollnes, Tom E., 361 Mutsuro, Junichi, 121 Nagaraja, Haikady N., 227 Nakahara, Makiko, 121 Nakao, Miki, 121 Navratil, Jeannine S., 381 Niculescu, Florin, 139 Nilsson, Bo, 107 Nilsson-Ekdahl, Kristina, 107 Nuytinck, Lieve, 281 Pangburn, Michael K., 313 Peerschke, Ellinor I. B., 95 Prechl, József, 177 Proctor, Lavinia M., 329 Ratajczak, Mariusz Z., 35 Reca, Ryan, 35 Rennebohm, Robert M., 227 Rittirsch, Daniel, 217 Ronquist, Gunnar, 107 Ross, Ted M., 249 Rovin, Brad H., 227 Rus, Horea, 139 Schneider, Marion, 217 Schreiber, Heike, 217 Sharma, Prakirti, 329 Shiels, Ian A., 329 Shih, Pei-an B., 381 Soares, Dinesh C., 313 391

392 Sohn, Jeong-Hyeon, 53 Somamoto, Tomonori, 121 Sosunov, Sergei A., 189 Spillner, Edzard, 347 Sughrue, Michael E., 189 Tantral, Lee, 95 Taylor Jr., Fletcher B., 203 Taylor, Stephen M., 329 Tenner, Andrea J., 153 Terényi, Nóra, 177

AUTHOR INDEX Tsonis, Panagiotis A., 63 Van Thielen, Martine, 281 Weiss, Manfred, 217 Woodruff, Trent M., 329 Wu, Yee-Ling, 227 Wysoczynski, Marcin, 35 Yan, Jun, 35 Yang, Yan, 227 Yu, C. Yung, 227 Zhou, Bi, 227

Subject Index

A AcF-[OPdChaWR], 165 Acute-phase proteins, 22 Acylation-stimulating protein (ASP), 80 Adaptive immunity, 74, 83 Adhesion molecules, 21 Adipose tissue and C3, 244 Adrenergic receptors and natural killer T cells, 22 Adrenomedullin, 317 Age-related macular degeneration (AMD), 55–56, 161, 314, 318–319 Akt, 20, 142, 143 Alternative pathway, assay of, 367–369 α'NT, 298–299 Alzheimer's disease, 153, 163 and complement activation, 191 murine models of, 157–162 Amino acid substitutions in factor H, 321–323 Amphibians, regenerative abilities of, 64–68 Amyloid peptide and activation of C1q, 163 in Alzheimer's disease, 154 fibrillar, 155–157 Amyloid precursor protein (APP), 153, 154 ANA domain, 298–299 Anaphylatoxins, 21–22, 24, 38, 78–81, 110, 362 in demyelinization, 183–185 during sepsis, 222–223 receptors in non-myeloid cells, 81 Angioedema, 102, 366 Angiogenesis, 21 and prostasomes, 109 Anterior chamber-associated immune deviation (ACAID), 56–58 Antibodies, natural, 76 Antibody-mediated demyelinating EAE (ADEAE), 183 Anti-Env antibody responses, 256–258 Antigen-presenting cells (APCs) for T cell activation, 71–72

Antigens and dendritic cells, 82 and immune response in the eye, 56–58 Antimicrobial peptides, 80–81 Anti-myelin antibodies, 140 Anura and limb regeneration, 64–65 APH50, 367–368 Apoptosis and C5, 145–146 C5b-9 protection, 146–148 inhibition of, 142–145 Apoptotic cells, 75, 82 and clearance by complement, 194 APP mice, 158–160 Arachidonic acid metabolism, 20 Ascidian ficolin, 269–270 Astrocytes, 180 ATP, 114 ATPase activity, 114–115 Atypical hemolytic uremic syndrome (aHUS), 313–314, 318 Autoimmune demyelination, 139–148 Autoimmune diseases, 365. See also specific diseases Autoimmune uveitis, 55 Autologous cells and CD59 transfer, 112–113 Axonal damage, 142 Axonal outgrowth, 63

B Ba, 129 Baboon model for sepsis, 204 Bacteria and binding of ficolin, 273–274 BAD, 142 Bb, 129 B cells activation by CR2, 250, 259 and C3dg binding, 76 BCL-XL, 142 BDNF, 140 Bf-1, 129

393

394 Bf-2, 129 BfA, 130 BfB, 130 BID cleavage, 143, 144, 145 Big ficolin, 267 Binding energetics, 2–3 Binding reactions, 3 Biotinylation of proteins, 96–97 Blastema, 64 Blood, human and complement analysis, 372–373 Blood-brain barrier, 177 Blood pressure changes induced by lipopolysaccharide, 339–340 Body mass index (BMI) and C3, 244 and serum C3 and C4 levels, 238–239 and serum complement proteins, 228 Bone marrow complement activation in, 37–39 and hematopoietic stem/progenitor cells, 35 regeneration and complement deficiency, 41–42 Bony fish classical complement pathway, 123–125 complement receptors in, 131–132 complement system of, 121–134 lectin complement pathway, 125–126 lytic complement pathway, 131 Borrelia burgdorferi, 300 Bradykinin (BK), 102, 366 Brain and complement system, 155–157 Brain-derived neurotrophic factor. See BDNF

C C1, 53, 370 and myelin damage, 182 C1-esterase inhibitor, 192 C1Inh, 132 C1 inhibitor, 363, 366, 369 C1q, 73, 78, 123, 190, 274, 365, 366, 368 and binding of gC1qR, 95–96 in the brain, 155–157 in central nervous system, 163–165 and clearance of apoptotic cells, 194 and inflammation, 102–103 in murine models of Alzheimer's disease, 158–160 and myelin damage, 182 in neurodegenerative diseases, 153–166

SUBJECT INDEX C1q receptors, 75, 131 C1q-Rp, 75 C1r, 123, 163, 368 C1r/s-A, 123 C1r/s-B, 123 C1s, 123, 163, 274, 368 C2, 53, 123, 297, 365, 369, 370 in bony fish, 125 C2a, 123 C2b, 123 C2bC4a, 347 C3, 53, 80, 361–362, 369, 370 activation of, 53 in alternative pathway, 126–130 and body mass index, 244 and clearance of apoptotic cells, 194 cleavage fragments, 74, 75–78 cleavage of, 38–39, 73–75 in complement activation, 291 conformation of, 4, 6 deficiency in hematopoiesis, 41–42 genetic deficiency of, 365 and immune responses, 74 inhibition of, 5, 165 interaction with compstatin, 4 levels of, 233–238 in limb regeneration, 66–68 and liver regeneration, 9, 20–21, 23–24 and myelin damage, 182 and neuropathology, 162 phosphorylation of, 113–114 protein profiles of, 232–233 tight association of, 235–238 sources of in the central nervous system, 180 structural organization of, 292–296 as substrate for prostasomal phosphokinases, 115 in systemic lupus erythematosus (SLE), 227–228, 382 thioester of, 73–74 C3a, 10, 78, 123, 126, 128, 291–292, 347, 355, 362 and antimicrobial activity, 79–80 in bony fish, 128 during complement activation, 38–39 and engraftment, 195 in experimental autoimmune encephomyelitis, 184 in HSPC engraftment, 42–45 and liver regeneration, 20, 24–26 and malignant cells, 110–111 phosphorylation of, 114

SUBJECT INDEX regulating hematopoietic stem/progenitor cells, 39 and sepsis, 218 during septic shock, 220–223 and tissue recovery, 194 C3ades-Arg, 38–39, 195 in HSPC engraftment, 42–45 regulating hematopoietic stem/progenitor cells, 39 C3aR, 10, 39, 79, 132, 183–184 C3b, 53, 74, 123, 126, 128, 129, 195, 291–292, 296, 300–301, 314, 347–348, 354, 362 and autoimmune uveitis, 55 binding to CR1, 75–76 binding to membrane cofactor protein, 77–78 cleavage of, 76–77 degradation of, 132–133 and neuropathology, 162 signaling roles of, 302–303 C3bBbC3b, 129 C3bBbP, 372 C3b-binding site, 317 C3b/iC3b deposition in the liver, 24–26 C3b/SPICE/VCP interaction, 6–7 C3c, 294–295, 297, 301 C3 convertase, 43–45, 53, 54, 73, 129, 132–133, 282, 296, 299, 301, 314, 347–349, 355, 361, 365 formation, 296–299 C3d, 38, 126, 128, 147, 292, 303 binding sites on, 258 and CR2 binding, 250 enhancing immunity, 249–260 C3d-CR2 interaction, 3, 258–259 C3-derived complement activation products, 384 C3dg, 38, 126, 292, 301, 302–303 binding to CR2, 76 C345C domain, 297, 353–354 C3-H1, 128 C3-H2, 128 C3 isoforms, 127–130 binding efficiency, 128 C3o, 297 C3-S, 128 C4, 53, 123, 282, 354, 365, 369, 370, 371 in bony fish, 123–125, 126 and clearance of apoptotic cells, 194 gene dosage, 241–242 genotypic and phenotypic variations in, 229, 239–241

395 levels of, 233–238 and myelin damage, 182 protein profiles of, 232–233 and systemic lupus erythematosus (SLE) activity, 227–228, 382 C4-1, 124–125 C4-2, 124–125 C4A, 228, 239–241 C4a, 123 in bony fish, 126 during sepsis, 223 C4A3, 239 C4B, 228, 239–241 C4b, 53, 123, 314, 354 and autoimmune uveitis, 55 in bony fish, 126 degradation of, 132–133 C4B1, 239 C4b.2a, 314 C4b-binding protein, 363 C4b/c, 206–207 C4bC2a, 123 C4d reticulocyte-bound, 387 and systemic lupus erythematosus (SLE) activity, 385–387 C4-derived complement activation products, 384 C5, 66, 67, 131, 354, 361–362, 369 and dendritic cells, 83 in experimental autoimmune encephomyelitis, 145–146 and IL-12 production, 84–85 and immune responses, 74 and liver fibrosis, 81 and liver regeneration, 9, 23–25 and myelin damage, 183 and neuropathology, 162–163 and neuroprotection, 195 C57BL/6 mouse line, 178 C5-9 pathway, 364 C5a, 66, 78, 131, 184–185, 329, 362, 372 and acute phase proteins, 22 and adhesion, 21–22 in angiogenesis, 21 in coagulation process, 10–11 and liver regeneration, 20, 24–26 and malignant cells, 110–111 and neuropathology, 162–163 and neuroprotection, 195 and sepsis, 217 and septic shock, 221, 222, 223–224 and tissue recovery, 194

396 C5adesArg, 80 C5aR, 10, 79, 81–82, 131, 132, 165, 329 in experimental autoimmune encephomyelitis, 141 and sepsis, 217–218 loss of, 221–222, 224 signaling in dendritic cells, 82–84 signaling on APCs, 84–86 C5aR antagonists, 329–342 transdermal pharmacokinetics of, 335–337 C5a antagonists with neutropenia and hypotension, 337–341 C5a receptor. See C5aR C5b-9, 66, 156, 157, 183, 361 and E. coli challenge, 206, 207, 208 in multiple sclerosis, 146–148 in neuroinflammation, 140–142 in neuroprotection, 142–146 C5 convertase, 54, 131, 132, 355 activity of, 352–353 C5-I, 131 C5-II, 131 C5 isotypes, 131, 132 C5L2, 43, 80, 163. 195 C6, 56, 131, 142 and myelin damage, 183 C7, 131 C8, 131 C9, 131 C9neo antigen, 178 Calcium and carbohydrate binding of ficolin, 272–273 Calreticulin, 75 caMASP, 125 CAMs, 102 Cancer and gC1qR, 102 and prostasome production, 109–110 Carbohydrate binding of ficolin, 271–273 Carbohydrate recognition domain (CRD), 265 Carp complement receptors of, 132 complement system of, 123–125 and lectin pathway, 125 Casein kinases, 114 Caspase, 142 Caspase-8, 144 Catfish, complement receptors of, 132 Cathelicidins, 81 cC1q-R/CR, 75 CCP, 161, 300

SUBJECT INDEX CD1d, 22 CD4+ lymphocytes, 83 generation of, 100 CD4 T cells, 139 CD11, 132 CD11b, 132 CD11b/CD18, 39 CD18, 132 CD34+ hematopoietic progenitor cells, 10 CD40-CD154 interaction, 84 CD44, 43 CD46, 54, 74, 77–78, 84, 86 CD55, 54, 146 CD59, 54, 185, 363, 366, 370 expression of by prostasomes, 108, 109 transfer of by prostasomes, 111–113 CD88, 80 Cell-bound complement activation products), 384–388 Cell-cycle reentry in liver, 9 Cell-mediated immunity, 77 Cellular immunity, 71 Central nervous system complement regulation in, 185 protective roles of complement, 162–165 and sources of complement, 180 c-FLIPL, 144 CFU-S cells, 41–42 CH50, 382 Chemokines, 35–36 α-chemokine stromal-derived factor-1, 10 Chemotactic factors, 157 Choroidal neovascularization (CNV), 56–57 Circulating tissue factor antigen, 209, 213 Classical complement pathway, 156–157, 291 assay of, 367–369 Coagulation and complement system, 75 modulation by complement, 10–11 Cobra venom factor (CVF), 180–181, 192, 349, 355, 373 Cobra venom factor (CVF) chimeras, 349–351 functional characteristics of, 351–353 Cofactor activity (CA), 301–302, 314 Colitis, 330, 341 Combinatorial protein design, 5–6 Complement and autoimmune uveitis, 55 blocking activation of to treat stroke, 190–193

SUBJECT INDEX cell surface expression of, 370 and cerebral ischemia/reperfusion, 192–193 and clearance of apoptotic cells, 194 and corneal disease, 54–55 deposition and myelin damage, 180–183 in developmental processes, 7 in E. coli sepsis, 203–215 inhibition of, 185–186, 192–195 measurement of, 382–384 modulating coagulation, 10–11 and ocular tolerance, 56–58 protective factor in central nervous system, 162–165 regulation of in central nervous system, 185 role of in liver regeneration, 23–26 sources of in the central nervous system, 180 structure elucidation, 2–3 in systemic lupus erythematosus (SLE), 381–388 systems biology perspective of, 6–9 and therapy of experimental autoimmune encephomyelitis, 185–186 and tissue recovery/repair, 194–195 Complement activation, 1, 37–39, 291 in nervous system diseases, 191 and neurons, 191–192 neuroprotective effects of, 140–148 during sepsis, 217–224 Complement activation products analysis of, 370–372 and system lupus erythematosus (SLE) activity, 384 Complement assays, 366–369 Complement C2 receptor inhibitor trispanning (CRIT), 297 Complement C3. See C3 Complement cascade, 1, 179 activation, 157 Complement components activity of, 369 genetic analysis of, 370 protein quantification of, 370 Complement defects and autoimmune diseases, 365 and recurrent infections, 364–365 Complement depletion, 373–374 Complement hemolytic activity, 218, 221, 222 Complement inhibition, 165–166

397 Complement inhibitors, 5–6 Complement lytic activity, 161 Complement proteins binding thermodynamics, 3–4 interactions, 2 mass spectrometry of, 4 Complement receptor 1. See CR1 Complement receptor 2. See CR2 Complement receptor-mediated pathways interacting with cytokine-driven regenerative network, 9 Complement receptors, 74 in bony fish, 131–132 Complement regulatory proteins. See CRPs Complement system activation of, 53–58, 66, 314, 361–363 alternative pathway, 126–130, 291, 367–369 classical pathway, 123–125, 156–157, 291, 367–369 lectin pathway, 125–126, 291, 367–369 and age-related macular degeneration, 55–56 of bony fish, 121–134 in the brain, 155–157 and coagulation, 75 in experimental autoimmune encephalomyelitis, 179 and the eye, 53–58 inactivation of, 347–356 in innate immunity, 72–74 lytic pathway, 131 mammalian, 122 in multiple sclerosis, 177–178 and ocular diseases, 54–56 regulatory factors, 132–133 and stroke therapy, 189–195 Compstatin, 5, 11, 165 analogs, 5 interaction with C3, 4 Convertases, 110, 292, 314 and C345C domain, 354 formation, 296–299 Cornea and complement system, 54–55 Corneal disease and complement system, 54–55 CR1, 56, 74, 131, 132, 185, 300–301, 363, 386–387 bound by C3b, 75–76 cofactor activity of, 301–302

398 CR2, 75, 131, 303 binding domain and immunity enhancement by C3d, 249–260 binding to C3d, 3, 250 binding to C3dg, 76 CR2-C3d interaction, 3, 250, 258–259 CR3, 24–25, 39, 44–45, 55, 131, 132, 302 activation of, 84 and adhesion of hematopoietic stem/progenitor cells, 40 bound by iC3b, 76–77 C3aR signaling and TLRs, 84–85 CR4, 302 bound by iC3b, 76–77 CraR signaling and TLRs, 84–85 Cross-reactive protein. See CRPs Crosstalk in complement system, 7–9 regulating hematopoietic stem cell engraftment, 10 between TLR4 and C5aR, 85 CRPs, 6, 22, 53–55, 73, 185, 317, 366 in prostate cancer, 111 Crry, 54, 162, 185–186 inhibition, 165 CUB domain, 301–302 CXCR4, 35–36, 42–44, 46 and lipid raft formation, 36–37 regulation of, 36–37 Cyclin D1, 19 Cytochrome C release, 142, 143 Cytokines, 194 after endotoxin challenge, 208–210 IL-12 family, 85 and liver regeneration, 19–20, 26 production of, 102 signaling network interacting with complement receptor-mediated pathways, 9

D DAF. See Decay-accelerating factor (DAF) Danger model of immunity, 72 Decay-accelerating factor (DAF), 54, 132, 185, 192, 300–301, 314, 363, 366, 370 Decomplementation by cobra venom factor (CVF), 180–181, 351–353 Dedifferentiation, 63 during limb regeneration, 64–65 Delayed type hypersensitivity in the eye, 56–58

SUBJECT INDEX Demyelination anaphylatoxin effects in, 183–185 autoimmune, 139–148 by C5b-9, 140–142 in multiple sclerosis, 178 Dendritic cells (DCs) and C5a receptor signaling, 82–84 Dimethylsulfoxide (DMSO), 331 Disease-associated factor H variants, 321–323 Disulfide bonds in C3, 295 in ficolin, 266 DNA synthesis, 66 Dorsal iris in lens regeneration, 65 Down's syndrome, 156 Drug delivery, 330–331, 335–337 Drusen, 55–56, 319 DU145 cell line, 112, 114

E EAE. See Experimental autoimmune encephalomyelitis (EAE) Ectokinases, 111 EDTA-plasma, 229 EGF, 79 Elastase/alpha 1 AT complex, 208 Electrostatic forces, 3, 6 ELISA. See Enzyme linked immunoassay (ELISA) ELISpot, 253–254 Endothelial cells, 102 activation, 19 Endotoxic shock, 331 Endotoxin challenge and complement activation, 208–210 Endotoxin model, human, 204–206 Engraftment of hematopoietic stem/progenitor cells, 42–45 Enlimomab Acute Stroke Trial, 192 Enoxaparin, 165 Enthalpy, 4 Entropy, 4 Env-mC3d3 and cell-mediated immune responses, 255 Enzyme-linked immunoassay (ELISA), 253, 368, 370, 371 Epidermal growth factor, 79 ERK, 20, 84 Erythrocyte-bound C4d (E-Cd4) and systemic lupus erythematosus (SLE) activity, 385–387

SUBJECT INDEX Erythrocyte-bound CR1, 386–387 Erythrocytes and ficolin binding, 271–272 and systemic lupus erythematosus (SLE) activity, 385–387 and transfer of CD59 from prostasomes, 111–113 Escherichia coli and ficolin binding, 273–274 Exons in ficolin, 267 Experimental autoimmune anterior uveitis (EAAU), 55 Experimental autoimmune encephalomyelitis (EAE) as animal model for multiple sclerosis, 178 C5 and neuroprotection in, 145–146 C5b-9 in demyelination during, 141–142 component and therapy of, 185–186 and role of complement system, 179 Extracellular matrix during limb regeneration, 64 Extracellular phosphorylation of plasma proteins, 113–114 Extracellular signal-regulated kinase (ERK), 20, 84 Exudative age-related macular degeneration, 55, 56 Eye and complement system, 53–58

F Factor B, 129, 141, 180, 296–297, 302, 369, 371 isotypes, 130 Factor D, 352, 369 Factor H, 56, 132, 161, 300–301, 370 associated with diseases, 313–314 cofactor activity of, 301–302 deficiency of, 365 fH-related proteins, 315 functional sites of, 317 modeled modules of, 319–321 predicted structural consequences of amino acid substitutions, 321–323 structure of, 315–316 Factor I, 301–302, 370 isotypes, 133 Factor XII. See FXII Fas, 146 FasL, 146 Fas L inducing apoptosis, 144 Fibrillar amyloid peptide, 155–157

399 Fibrillar plaques, 159–160 Fibrinogen, 37, 42, 114 as substrate for prostasomal phosphokinases, 115 Fibrinogen-like domains, 270–271 and carbohydrate binding, 272 Fibrinolysis, 20, 115 Fibrinonectin, 37 Fibroblast growth factors (FGFs) in regeneration, 64, 65 Fibrogenesis, 81–82 Fibronectin, 42, 66 Ficolin, 125, 265–276 binding to bacteria, 273–274 carbohydrate binding of, 271–273 function of, 271–275 non-serum type, 268–269 phylogeny of, 269–271 polymorphisms in gene, 275 as recognition molecule in lectin pathway, 274–275 serum type, 268–269 structure of, 266–267 tissue and cell type expressing, 268–269 Ficolin A, 266, 268, 269, 270 and carbohydrate binding, 273 binding to bacteria, 273 Ficolin B, 268, 270 and carbohydrate binding, 273 Follicular dendritic cells, 250 Frogs and limb regeneration, 64 Fugu, complement system of, 123, 125 Functional C1 inhibitor test, 369 FXII, 95, 102

G Gα16, 79 GAG-binding site, 323 Galactose, 125 GalNAc and binding of ficolin, 271–272 gC1qR, 75 catalyst in inflammation, 95–103 and complement activation, 100–101 expression of recombinant, 98 hemolytic activity of, 99–100 gC1qR/p33. See gC1qR G-CSF, 44 Genetic deficient animals, 373 Genotyping of MBL2, 285 GlcNAc and binding of ficolin, 271–272 Glia in murine models of Alzheimer's disease, 160 Glial cells, 156

400 Glial fibrillary acidic protein (GFAP), 184 Gliotoxin, 19 1,3-β-D-glucan, 273 Glutathione-S-transferase (GST) gene, 98 Glycosaminoglycans, 317 N-glycosylation sites in factor H, 315 Glycosylphosphatidylinositiol (GPI)anchored proteins, 112, 363 gp120, 249, 251 gp120-mC3d3-DNA, 255 and anti-Env antibody responses, 256–258 gp120-P284, 251 GPI-anchored proteins, 112, 363 G-protein-coupled receptors (GPCRs), 74, 79 G-proteins, 79 G-protein signaling (RGS) proteins, 36 Gram-negative bacteria and ficolin binding, 273–274 Gram-positive bacteria and ficolin binding, 274 Growth factors in age-related macular degeneration, 56–57 in liver regeneration, 20–22 and remyelination, 140

H Haemophilus influenzae and otitis media, 281 Hageman factor, 20 Hakata antigen, 266 Halocynthia roretzi, 269 Haptoglobin precursor, 22 Hematopoiesis and complement deficiency, 41–42 Hematopoietic precursors, 10 Hematopoietic stem cell engraftment, 10 Hematopoietic stem/progenitor cells (HSPC) into bone marrow, 35–46 engraftment in complement C3 deficiency, 42–45 regulating biology of, 39–41 Hematopoietic transplantation, complement activation in, 37–39 Hemolytic activity of gC1aR, 99–100 Hemolytic assay for complement system activity, 368–369, 374 Hemolytic uremic syndrome (HUS), 313–314, 318, 365, 370 Hemopexin, 22

SUBJECT INDEX Hemostatic responses to endotoxin challenge, 208–210 Hepatocyte growth factor (HGF), 20–21 Hepatocytes proliferation, 20, 24 Hepatoprotection, 19 Hereditary angioedema (HAE), 366, 369 Herpes virus, 6 H-ficolin, 266, 270 and binding to bacteria, 274 carbohydrate binding of, 271–272 expression of, 268, 269 in lectin pathway, 274–275 polymorphisms of, 275 HGF-Met pathway, 21 High-molecular-weight kininogen. See HK Histidine, 125 in C3 isoforms, 127–128 HK, 95, 102 Homeostasis transmitters, 83, 86 Homing of HSPC in complement C3 deficiency, 42–45 Horseshoe tachylectins, 270–271 HSPC. See Hematopoietic stem/progenitor cells (HSPC) Human endotoxin model, 204–206 Huntington's disease, 163 HUVECs, 21 Hyaluronic acid, 37 Hydrogen/deuterium exchange of complement proteins, 4 Hydrophobic residues, 323 Hypervariable loop in factor H, 315, 321 Hypocomplementemia, 365 Hypotension, 329–330, 331 and C5a antagonists, 337–341

I Iatrogenic shock syndrome, 355 iC3b, 38–39, 55, 78, 126, 195, 258, 292, 302–303 and adhesion of hematopoietic stem/progenitor cells, 40 binding to CR3 and CR4, 76–77 in homing of HSPC to bone marrow, 43–46 in HSPC engraftment, 42–45 in ocular tolerance, 57–58 ICAM-1, 55 IFN consensus sequence binding protein (ICSBP), 84

SUBJECT INDEX IFN-γ, 55, 77 and cell-mediated immune responses, 255 and natural killer T cells, 23 IFN regulatory factor 1 (IRF-1), 84 IκBα, 19 IL-4 activation by natural killer T cells, 22 and cell-mediated immune responses, 255 IL-6, 19, 80, 81 and liver regeneration, 23 IL-10, 55, 57 IL-12, 77, 85 production of by C5a, 84–85 Immune complexes, 75 Immune responses, 74–78 in the eye, 56–58 Immunity adaptive, 74, 83 cell-mediated, 77 cellular, 71 danger model of, 72 enhanced by C3d, 249–260 Immunization mucosal, 256–257 Immunopreciptation tests, 370 Infections homeostatic responses to, 10–11 recurrent and complement defects, 364–365 Infectious non-self (INS) model, 71–72 Inflammation and C5aR antagonists, 330 catalyzed by gC1qR, 95–103 and cytokines and transcription factors, 19–20 and endotoxin challenge, 208–210 in stroke, 190–191 Inflammatory mediators and liver regeneration, 18–23 Inhalation tolerance, 82–84 Innate immunity, 7–9, 74 and complement system, 72–74 by malignant cell-derived prostasomes, 109–110 and MBL, 282 in regenerative response of liver, 9 INNO-LiPA MBL2 assay, 285–286 In silico protein design, 5 Integrins, 43, 66 IP3, 79

401 IP-10, 55 Ischemic stroke and complement system, 189–195 Isothermal titration calorimetry, 3–4

K Kallikrein, 95 Kininogens, 75 Kupffer cells, 19

L Lectin complement pathway, 53, 73, 265 activation of, 125–126, 291 assay of, 367–369 and ficolin as a recognition molecule, 274–275 Lectins, 265 Leishmanial phosphokinase, 114 Leishmania major, 85 Lens regeneration, 65–68 Lepirudin, 372 L-ficolin, 265, 267, 270 bacteria binding of, 273–274 carbohydrate binding of, 271, 272 expression of, 268, 269 in lectin pathway, 274–275 polymorphisms of, 275 structure of, 266 Limb regeneration, 64–65 Lipid raft and CXCR4 formation, 36–37 in engraftment of hematopoietic stem/progenitor cells, 42–44 Lipophilicity of C3aR antagonists, 341–342 Lipopolysaccharides and ficolin binding, 271–272, 273 inducing neutropenia, 337–341 in liver regeneration, 19, 20 Lipoteichoic acid (LTA), 273, 274 Liver fibrosis, 81 and ficolin expression, 268 regenerative response of, 9 Liver regeneration, 17–18, 81 and inflammatory mediators, 18–23 role of complement, 23–26 L-selectin, 317 Lung, inhalation tolerance, 82–84 Lupus. See Systemic lupus erythematosus (SLE) Lupus nephritis, 365

402 Lysis, complement-mediated, 112–113 Lytic pathway, 131

M MAC, 53, 74, 110, 131, 179 in age-related macular degeneration, 55–57 and sepsis, 218, 221, 223 Macroglobulin domains (MGs), 73–74 in C3, 295 Macrophages, 180 and growth factors, 140 responding to endotoxin challenge, 210, 212 Macular degeneration age-related, 55–56, 314, 318–319 and factor H, 161 Malaria, 76 Mannose, 53 Mannose-binding lectin. See MBL MAP-2, 160 MASP1, 73, 125 and ficolin, 274 MASP2, 73, 125–126, 369 and ficolin, 274 MASP3, 125 MASPs, 265, 282 and ficolin, 267, 274 Mass spectrometry of complement proteins, 4 Matrix metalloproteinase-3, 81 MBL, 53, 73, 125, 265, 369, 370 deficiency of, 364–365 in predicting recurrent otitis media, 281–289 MBL2 genotyping, 285 in otitis media, 282–289 and polymorphisms, 283–284 MBL2-221G variant, 299 MBL2-G54D variant, 288 MBL-associated serine protease-1. See MASP1 MBL-associated serine protease-2. See MASP2 MBL-associated serine protease-3. See MASP3 MBL-associated serine proteases (MASPs). See MASPs MBL-MASP2, 126 MCP, 54, 56, 185, 192, 301–302, 314, 363, 370

SUBJECT INDEX Measles virus, 78 MEK/ERK cascade, 79 Membrane attack complex. See MAC Membrane cofactor protein. See MCP Membrane inhibitor of reactive lysis (MIRL), 54 Membranoproliferative glomerulonephritis (MPGN), 318, 319, 365 Meningococcal disease, 364 Metal-ion-dependent adhesion site (MIDAS), 302 Metalloproteases (MMP), 21 M-ficolin, 266, 267, 270 and binding to bacteria, 274 carbohydrate binding of, 273 expression of, 268 in lectin pathway, 274–275 polymorphisms of, 275 MG6 domain, 300 Microglia, 162 Mitogen-activated protein kinase (MAPK), 19, 217–218 MMP-2, 21 MMP-9, 21 MOG. See Myelin oligodendrocyte glycoprotein (MOG) Mucosal immunization, 256–257 Mucosal tolerance, 82–84 Multimerization, 266 Multiple sclerosis, 139 and C5b-9, 146–148 and role of complement system, 177–178 Murine models of Alzheimer's disease, 157–162 Muscle dedifferentiation, 65 Mutations, missense, 318 Myelin associated glycoprotein (MAG), 178 Myelin basic protein (MBP), 178 Myelin damage and complement deposition, 180–183 Myelinogenesis, 140 Myelin oligodendrocyte glycoprotein (MOG), 141, 178 and myelin damage, 181 Myelin proteins, 178 Myeloblative conditioning, 37–39 Myeloid dendritic cells (mDC), 83–84 Myeloid related protein (MRP-14), 25, 77 Myocardial ischemia, 191 Myotubes, 65

SUBJECT INDEX

N N-acetyl glucosamine, 53 Natural antibodies, 76 Natural killer T cells and liver regeneration, 22–23 Neisseria infections, 364 Neisseria meningitidis, 364 Neoepitope-based assays, 371, 372, 374 Nervous system diseases and complement activation, 191 Neurodegenerative diseases and C1q, 153–166 Neurofibrillary tangles, 156–157 Neuroinflammation and role of C5b-9, 140–142 Neurons and complement activation, 191–192 Neuroprotection, 140 and C5b-9, 142–146 of penumbra, 190 Neutropenia, 329–330, 331 and C5a antagonists, 337–341 Neutrophils, 333 and C5aR, 218 and inflammation, 11 responding to endotoxin challenge, 210, 212 Newts and limb regeneration, 64 NF-κB, 23, 25, 66 NKT. See Natural killer T cells Nonexudative age-related macular degeneration, 55 N-terminal affinity tags, 351–352 Nuclear factor-kappa B (NF-κB), 19

O Ocular diseases and complement system, 54–56 Oleic acid, 77 Oligodendrocytes, 139 apoptosis, 142–146 protection from apoptosis by C5b-9, 146–148 Opsonin, 274–275 Organ dysfunction, 364 Orthopox virus, 6 Otitis media and role of mannose-binding lectin (MBL), 281–289

403

P P28 peptide, 249, 250 and binding to CR2, 258–259 and mucosal immunization, 256–258 Paroxysmal nocturnal hemoglobinuria (PNH), 111, 363, 366, 370 Partial hepatectomy, 18 Pathogen-associated molecular patterns (PAMPs), 265, 282 Pathological threats to immune system, 74–78 Pattern-recognition receptors (PRRs), 72, 282 Pax-6 gene, 65 PC-3 cell line, 112 Penumbra, therapy of after ischemic stroke, 189–190 Peptidoglycan (PGN), 273 Permeation enhancers, 330–331 Phagocytosis, 162 and C3 deposition, 182 and CR1, 76 and ficolin, 274–275 Phosphatidylinositol (PI) anchor (PIG-A), 366 Phosphoinositide-3-kinase (PI3K). See PI3K Phosphokinases, 113–114 Phospholipase C, 79 Phosphorylation of plasma proteins, 113–114 Physiological threats to immune system, 74–78 PI3K, 19, 20, 84, 142, 143, 144 Pick's disease and complement activation, 191 Pigment epithelial cells (PECs), 65 PKA, 114 PKC, 79, 114 Plaques and C1q, 155–156, 159–160 Plasmacytoid dendritic cells (pDC), 83–84 Plasma proteins, extracellular phosphorylation of, 113–114 Plasminogen, 20, 21 PMX53, 329–331 C5aR affinity of, 335 effect on neutropenia and hypotension, 337–341 topical bioavailability of, 335–337 PMX200, 331 bioavailability of, 342 C5aR affinity of, 335

404 PMX200, 331 [cont'd] effect on neutropenia and hypotension, 337–341 topical, 337 PMX201, 329–331 C5aR affinity of, 335 effect on neutropenia and hypotension, 337–341 topical bioavailability of, 337 PMX205, 329–331 C5aR affinity of, 335 effect on neutropenia and hypotension, 337–341 topical bioavailability of, 337 Polyanions, 317 Polymorphisms in ficolin gene, 275 and MBL2 gene, 283–284 Polymorphonuclear leukocytes (PMNs), 338 isolation of, 332 myeloperoxidase release from, 333 Porcine ficolin, 268 and bacteria binding, 274 Pro/anti-fibrinolytic protein cascades, 10 Proinflammatory properties, 78–79 Properdin, 292, 299, 369, 370 deficiency, 364 Prostasomal phosphokinases, 115–116 Prostasomes malignant cell-derived, 109–110, 112–113 mapping of protein kinases, 114–115 and prostate cancer, 107–116 seminal, 111–113 transferring CD59, 111–113 Prostate cancer, incidence of, 107 Proteases and CXCR degradation, 36–37 in liver regeneration, 21 Protein kinase C, 79, 114 Protein kinases (PKs) on prostasomes, 109, 114–115 Protein–protein association, 3 measuring conformational changes in, 4 Proteolipid protein (PLP), 178 Prox-1 gene, 65 Psh-AI-PvuII restriction fragment length polymorphism, 229

R Rac-1, 37, 42 Rac-2, 37

SUBJECT INDEX Ras, 79 Recombinant harridan, 372 Recombinant human C3 (hC3), 349–351, 355 functional characteristics of, 351–353 Recombinant protein antigens, purification of, 251–252 Recurrent otitis media. See Otitis media Regeneration of lens, 65–68 of limbs, 64–65 Regulator protein C hemostatic factor, 208 Regulators of complement activation (RCA), 300, 313 Remyelination, 140 and C5, 145 Reperfusion injury, 190, 364 Reticulocyte-bound C4d, 387 Reticulocytes and systemic lupus erythematosus (SLE) activity, 387 Retinal pigment epithelium (RPE), 319 Retinoblastoma protein, 65 Retinoid receptors, 65

S Salamanders and limb regeneration, 64 Salmonella typhimurium and ficolin binding, 273–274 Sand bass cofactor, 133 Sanfilippo syndrome, 163 SAP protein, 73 SBP1, 133 SC5b-9, 372 Sca-1+ cells, 40 SCD-1/CXCR4 axis, 36 sCR1, 185, 191 in stroke, 192–193 sCrry, 191 SDF-1, 35–36, 37 responsiveness of HSPC to, 42–45 SDF-1-CXC4 axis, 39–40, 46 Selectins, 43 Self and non-self discrimination, 71–72 Senile plaques, 154 Sepsis, 331, 364 baboon model for, 204 complement activation during, 217–224 Septic shock, 364 and sepsis-induced complement activation, 220–221 Serine proteases, 125

SUBJECT INDEX Serum amyloid A, 22 Serum amyloid A2, 22 Serum amyloid P, 73 Serum amyloid protein, 22 Serum C3, C4 and body mass index (BMI), 238–239 Signaling by C5aR in dendritic cells, 82–84 by CXCR4, 36–37 factors in liver regeneration, 21 Signal transducer and activator of transcription (STAT) 3. See STAT3 Signal transduction, 66 of C3b fragments, 302–303 SJL/J mouse line, 178 sMAP, 125 and ficolin, 274 SNPs. See Single nucleotide polymorphisms (SNPs) Sodium alginate, 130 Soluble complement receptor-1. See sCR1 Soluble fibrin monomer (sFM), 209 Soluble thrombomodulin (sTM), 209 Species crossreactivity of assays, 374 Spermatozoa, 109 Splenocytes, 255 Staphylococcus aureus and ficolin binding, 274 STAT3, 19, 23, 25, 79 Stem cell differentiation, 10 Streptococcus pneumoniae and otitis media, 281 Stroke therapy and complement system, 189–195 Stromal cell-bound C3b, 38 Stromal derived factor-1. See SDF-1 Sublytic C5b-9, 142–145 Surface plasmon resonance (SPR), 258 Synaptophysin (AP), 160 Systemic inflammatory response syndrome (SIRS), 364 Systemic lupus erythematosus (SLE), 365 clinical features of study population, 230–231 role of complement in, 381–388 and serum complement proteins, 227–228

T Tachylectin 5A, 272 Tachylectins, 270–271 Tau in Alzheimer's disease, 157, 160 T cell-dependent antigens, 76

405 T cells activation, 71–72, 82 and C3b binding, 78 TED domain, 302, 303 Tenascins, 270–271 Terminally differentiated tissues during regeneration, 64 Tetrachloride, 18 Tg2576 mice, 157–159 TGF-β1, 81 TGF-β2, 57 Th1 polarization, 85 Th2 adaptive immune response, 83 Therapeutics, complement-based, 165–166 Thermodynamics of complement protein binding, 3–4 Thioester moiety in bony fish C3 isoforms, 127 in C3, 295 Third complement component. See C3 Thrombin, 65 Thrombin-antithrombin (TAT), 208 Thrombomodulin, 213 Thrombotic thrombocytopenic purpura (TTP), 318 Tissue factor, 10–11 Tissue inhibitor of metalloprotease (TIMP), 1, 3, 21 Tissue integrity, 72 Tissue regeneration and complement, 194–195 TNF-α, 81 inducing apoptosis, 144 in inflammation, 19–20 in liver regeneration, 21, 23 TNF-α-converting enzyme (TACE), 21 TNF receptor 1 (TNFR1), 19 Toll-like receptors (TLR) 4, 25, 71, 84–85 Transcription factors in liver regeneration, 19–20 Transdifferentiation, 63 Transforming growth factor (TGF)-β1, 265 Transplantation, complement activation in, 37–39 Trout, complement system of, 123–124 Type III collagen glomerulopathy, 318 Tyrosine kinase receptor-c-kit, 43

U uPA, 20–21 Urodeles and limb regeneration, 64–65

406

SUBJECT INDEX

V

W

Vaccination and C3d enhancing immunity, 249–260 VCP, 6–7 VCP/C3b interaction, 6 VEGF, 21 Vertebrates, regenerative abilities in, 63–68 Viral immune evasion, 6–7 Vitronectin, 56, 115 VWA domain, 302

Wound epithelium, 64

X Xenopus ficolin, 269–270 Xenopus laevis, 269 X-ray crystallography, 3

Figure 5.1. Expression of C3 (A,B) and C5 (C,D) during limb regeneration, 3 weeks after amputation. Note expression of C3 in the blastema cells (bl; arrow) and of C5 in the wound epithelium (we; arrow). B and D have been counter-stained with DAPI.

Figure 5.2. Expression of C3 (A–C) and C5 (D–F) during lens regeneration 20 days post-lentectomy. Note expression of C3 on the iris (B,C; arrow) and of C5 in the regenerating lens (E,F; arrow). B and E have been counter-stained with DAPI.

Figure 11.1. C1q (red) colocalizes with thioflavine (green) in cortex of Alzheimer’s disease brain. Scale bar: 100 µm.

Figure 11.5. Neurons in rat hippocampal organotypic slice cultures that have ingested exogenously added Aβ are immunoreactive for C1q.

Figure 11.4. Representative images from APP (left) and APPQ-/- (right) mice at 12 (middle) or 16 (top, bottom) months of age. Comparable levels of thioflavine staining are seen in APP and APPQ-/mice (top panel). Scale bar: 100 µm. Decreased astrocytic reactivity (GFAP,red) around fibrillar Aß (thioflavine, green) plaques in APPQ-/- mice relative to APP C1q-sufficient mice is evident (middle panel). Higher density of synaptophysin staining is seen in CA3 area of hippocampus of APPQ-/mice relative to APP mice (bottom panel). Scale bar: 20 µm (middle and bottom panels).