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The Complement System The Role of Intravenous Immunoglobulin Anti-A and Anti-B in Complement Activation and Red Blood Cell Phagocytosis By Daniella Perri A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Daniella Perri 2009 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-59437-7 Our file Notre référence ISBN: 978-0-494-59437-7 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L’auteur conserve la propriété du droit d’auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author’s permission. In compliance with the Canadian Conformément à la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thèse. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n’y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. The role of IVIg Anti-A and Anti-B in Complement Activation and Red Blood Cell Phagocytosis Daniella Perri Master of Science Department of Laboratory Medicine & Pathobiology University of Toronto 2009 Abstract Intravenous immunoglobulin is a human blood derived product that is used to treat immunodeficiencies and autoimmune disorders. An adverse side effect of IVIg therapy is hemolysis. Patients who experience hemolysis are mainly blood group A or AB. Clinical laboratory studies have demonstrated that IVIg contains ABO blood group antibodies, which can bind complement proteins. This study hypothesizes that anti-A/B in IVIg will bind to A/B antigens and activate complement in a dose dependant manner, which may lead to enhanced RBC phagocytosis. This study observed that the quantity of ABO antigens does not affect the in vitro binding of IVIg to RBCs. IVIg induced C3b deposition at high doses; however, the amount of complement deposition was insufficient to enhance phagocytosis of IVIg-sensitized RBCs by monocytic THP-1 cells in vitro. These studies emphasize that hemolytic reactions involve many factors in conjunction with antibodies and complement proteins. ii Acknowledgements I would like to thank my supervisor, Dr. Greg Denomme, for supporting and guiding me through the triumphs and tribulations of scientific research, experimental planning, and manuscript writing. I would also like to extend my gratitude to my committee members, Dr. J. Pendergrast, Dr. J. Semple, and Dr. D. Branch, for their expertise and advice. I would also like to thank the members of the Denomme and Semple lab who have supported and encouraged me through the duration of this project. Also, many thanks go to Igor, Dipen, Rukshana, and Mike for their technical support. My sincerest gratitude goes towards the Canadian Blood Services employees who have contributed to my project by either donating blood samples or providing technical support. Your willingness and enthusiasm to contribute to my project was greatly appreciated. Last, but not least, I would like to thank my parents, Marianne and Joe for providing me with the support and determination I needed to reach my potential and beyond during my masters program. iii Table of Contents Abstract ii Acknowledgements iii Table of Contents iv List of Tables viii List of Figures ix List of Appendices x Abbreviations xi 1.0 Introduction 1 1.1 Blood Transfusion Reactions 2 1.1.1. Acute Immunologically Mediated Reactions 2 1.1.2 Delayed Immunological Reactions 3 1.1.3 Passive Antibodies in Transfusion Products 4 1.1.3.1 Anti-A and Anti-B in Transfusion Products 4 1.2 Transfusion of Blood Components: Intravenous Immunoglobulin (IVIg) 5 1.2.1 Biochemical Characteristics of IVIg 5 1.2.2 Clinical Use of IVIg 6 1.2.3 Mechanisms of Action of IVIg 6 1.2.3.1 IVIg Contains Anti-A/B That Can Cause Erythrophagocytosis 7 1.2.3.2 Fc-Dependant Mechanisms 7 1.2.3.3 Formation of Immune Complexes 9 1.2.3.4 Immunomodulatory Mechanisms 10 1.2.4 Hemolysis Associated with High Dose IVIg: In vivo Studies 10 1.3 The Classical Complement System 12 1.3.1 Complement Components and the Classical Complement Cascade 12 1.3.2 Complement Receptor 1 (CR1/CD35): C3b Receptor 13 1.3.2.1 Biochemical Properties 13 1.3.2.2 Genetic Polymorphisms of CR1 14 1.3.2.2.1 Structural Polymorphism 14 iv 1.3.2.2.2 Expression Levels 15 1.3.2.2.3 Knops Antigens 15 1.3.3 Complement Receptor 2 (CR2) 16 1.3.4 Inhibition of the Classical Complement Pathway 16 1.3.5 Decay Accelerating Factor (DAF) 17 1.3.5.1 Cromer Blood Group System 19 1.4 Fc gamma receptor (FcγR)-mediated Immune Response 19 1.4.1 FcγR Biology 19 1.4.1.1 FcγRI (CD64) 20 1.4.1.2 FcγRII (CD32) 20 1.4.1.3 FcγRIII (CD16) 21 1.4.2 Macrophage Phagocytosis of Sensitized Red Blood Cells (RBCs) 21 1.4.2.1 Role of FcγRs and CRs 22 1.5 ABO Blood Group System 23 1.5.1 The ABO gene: Glycosyltransferases 23 1.5.1.1 The Structure of the ABO Glycosyltransferases 24 1.5.2 Differences Between Blood Groups A1 and A2 25 1.6 Lewis Blood Group System 25 1.6.1 Soluble ABO Substances 26 1.7 Rationale 27 1.7.1 Objectives 27 1.7.2 Hypothesis 27 2.0 Materials and Methods 28 2.1 Cell Line: THP-1 29 2.2 Blood Sample Donors 29 2.3 Intravenous Immunoglobulin (IVIg) 29 2.4 Effect of IVIg Dose on Anti-A/B Binding to RBCs 29 2.4.1 ABO Genotyping: DNA Extraction and Quantification 30 2.4.1.1 ABO PCR 31 2.4.1.2 Exon 6 and 7 Restriction Enzyme Digestion 31 2.4.2 Assessing the Level of Membrane-bound A-antigens 32 v 2.4.3 Lewis Blood Group Serology Testing 32 2.5 Complement Activation with IVIg 33 2.5.1 Flow Cytometric Analysis of Complement Proteins 33 2.5.2 Absorption of Anti-A/B from IVIg 33 2.6 Inhibition of Rosette Formation by IVIg 34 2.7 RBC Phagocytosis Bioassay 34 2.7.1 Setting the Phagocytosis Assay Controls 35 2.7.2 Flow Cyometric Analysis of THP-1 Cell Viability 36 2.8 Statistical Analysis 36 3.0 Results 37 3.1 Characteristics of the Antibodies in IVIg: Clinical Laboratory Studies 38 3.1.1 ABO Antibodies in IVIg 38 3.1.2 Antibody Titres in IVIg 39 3.1.3 IVIg Anti-A/B and Complement Activation 40 3.2 Studies on IVIg with Whole Blood 40 3.2.1 The Effect of IVIg Concentration on Whole Blood 41 3.2.1.1 The Relationship Between Blood Group and IVIg Dose on 41 Anti-A/B Binding 3.2.1.2 The Effect of A-antigen quantity on IVIg 41 Anti-A binding 3.2.1.3 The Effect of Membrane-bound A/B Antigens on 44 Anti-A/B Binding 3.2.1.4 The Effect of Soluble A Substance on Anti-A Binding 46 3.2.1.5 The Effect of IVIg Dose on the Presence of Anti-A/B 47 in the Plasma 3.3 The Ability of IVIg anti-A/B to Activate the Classical Complement Cascade 48 3.4 The Interaction Between Red Blood Cells and Mononcytes 50 3.4.1 THP-1 Cell and IgG-sensitized RBC Interaction: Inhbition of 50 Rosette Formation 3.4.2 THP-1 Cell and RBC Interaction is Fcγ-receptor Dependent 53 3.4.3 IVIg-induced RBC Phagocytosis with and without 56 Complement Activation 4.0 Discussion 60 vi 4.1 Reviewing ABO Blood Group Antibodies and Hemolysis 61 4.2 Assessment of IVIg and Complement Activation: Clinical Studies 61 4.3 The Effect of IVIg Dose and ABO Genotype on Anti-A/B Binding 62 4.3.1 The Effect of IVIg Binding in the Presence of Soluble A Substance 62 4.4 Activation of the Classical Complement Cascade by IVIg 63 4.4.1 The Effect of Soluble A Substance on Complement Activation 63 4.4.2 Complement Activation on Group O RBCs 63 4.4.3 The Role of Complement Regulators on C3b Deposition 64 4.5 The Interaction Between Monocytes and RBCs is FcγR-dependant 64 4.6 Studies on Complement-mediated Synergism of IgG Sensitized RBC 65 Phagocytosis 4.6.1 The Effect of Receptor Expression on THP-1 cells on RBC 66 Phagocytosis 4.6.2 The Presence of Complement Regulators and Their Influence on 67 RBC Phagocytosis 4.7 Summary 67 4.8 Future Directions 69 5.0 References 72 6.0 Appendix 91 vii List of Tables Table 1.
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