REGULATION AND FUNCTION OF THE LEUKOCYTE IMMUNOGLOBULIN-LIKE RECEPTORS (LILRS) IN RHEUMATOID ARTHRITIS. A thesis submitted to the University of New South Wales by OWEN ANTHONY HUYNH For the degree of Doctor of Philosophy (Ph.D) Inflammatory Diseases Research Unit, School of Medical Sciences University of New South Wales 2008 ORIGINALITY / COPYRIGHT / AUTHENTICITY STATEMENTS ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. 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No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ th Owen Anthony HUYNH 24 November, 2008 B.MedSci (Hons) Date TABLE OF CONTENTS ORIGINALITY / COPYRIGHT / AUTHENTICITY STATEMENTS ...................................................... i TABLE OF CONTENTS ................................................................................................................................ ii ABSTRACT .................................................................................................................................................... vi ACKNOWLEDGEMENTS .........................................................................................................................viii PERSONAL THANKS .................................................................................................................................. ix PUBLICATIONS ARISING FROM CANDIDATURE.............................................................................. xi CONFERENCES ATTENDED DURING CANDIDATURE..................................................................... xi LIST OF IMPORTANT ABBREVIATIONS ............................................................................................. xii LIST OF TABLES........................................................................................................................................ xvi LIST OF FIGURES..................................................................................................................................... xvii CHAPTER 1. LITERATURE REVIEW....................................................................................................... 1 1.1 THE IMMUNE SYSTEM AND ITS REGULATION........................................................................................... 1 1.2 PATHWAYS FOR NEGATIVE AND POSITIVE SIGNALLING BY IMMUNE CELLS............................................ 3 1.2.1 Immunoreceptor Tyrosine-based Activation Motifs (ITAMs)........................................................ 3 1.2.2 Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs) .......................................................... 4 1.3 THE LEUKOCYTE IMMUNOGLOBULIN-LIKE RECEPTORS (LILRS)............................................................. 8 1.3.1 Nomenclature of LILRs ................................................................................................................ 11 1.3.2 Structure of LILRs ........................................................................................................................ 13 1.3.3 Expression of LILRs ..................................................................................................................... 16 1.3.4 Ligands of LILRs.......................................................................................................................... 20 1.3.5 Function of LILRs......................................................................................................................... 25 1.3.6 Pathological Role of LILRs .......................................................................................................... 34 1.4 LILR-RELATED RECEPTORS .................................................................................................................. 41 1.4.1 Killer Cell Immunoglobulin-like Receptors (KIRs)...................................................................... 41 1.4.2 LAIR-1 (CD305)........................................................................................................................... 44 1.4.3 Murine Paired Immunoglobulin-like Receptor-B (PIR-B)............................................................ 45 1.4.4 Murine LILRB4 (mLILRB4 or gp49B) ........................................................................................ 45 1.5 TOLL-LIKE RECEPTORS (TLRS) ............................................................................................................. 48 1.6 RHEUMATOID ARTHRITIS ...................................................................................................................... 52 1.6.1 Background................................................................................................................................... 52 ii 1.6.2 Etiology......................................................................................................................................... 52 1.6.3 Pathology ...................................................................................................................................... 54 1.6.4 Treatments..................................................................................................................................... 61 1.6.5 LILRs in Rheumatoid Arthritis ..................................................................................................... 70 1.7 HYPOTHESIS AND AIMS ......................................................................................................................... 71 CHAPTER 2. REGULATION OF LILRA2 AND LILRB4 BY CYTOKINES AND LPS ..................... 72 2.1 INTRODUCTION ...................................................................................................................................... 72 2.2 MATERIALS AND METHODS................................................................................................................... 75 2.2.1 Antibodies and Reagents............................................................................................................... 75 2.2.2 Cell Culture................................................................................................................................... 76 2.2.3 Peripheral Blood Mononuclear Cells (PBMCs) Extraction .......................................................... 76 2.2.4 Differentiation of THP-1 Cells using Vitamin D3......................................................................... 77 2.2.5 Cell Stimulation ............................................................................................................................ 77 2.2.6 Flow Cytometry ............................................................................................................................ 77 2.2.7 RNA Extraction ............................................................................................................................ 78 2.2.8 DNase-Treatment and Reverse Trascription (RT) ........................................................................ 78 2.2.9 Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) ................................................ 79 2.2.10 Statistical Analyses ..................................................................................................................... 81 2.3 RESULTS ................................................................................................................................................ 82 2.3.1 Regulation of LILRA2 and LILRB4 Expression upon Differentiation of Monocytes to Macrophages.......................................................................................................................................... 82 2.3.2 Regulation of LILRA2 and LILRB4 Expression by Cytokines and LPS on Monocytes and Macrophages.........................................................................................................................................