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Studies of Endothelial and Leukocyte Cell Adhesion Molecules in Renal Transplantation

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Studies of Endothelial and Leukocyte Cell Adhesion Molecules in Renal Transplantation ì1-3-r\"1\, Studies of Endothelial and Leukocyte Cell Adhesion Molecules in Renal Transplantation A Thesis submitted to the University of Adelaide as the requirement for the degree of Doctor of PhilosoPhy by Warwick L Grooby BSc (Hons) Department of Medicine, University of Adelaide and Transplantation Immunology Laboratory The Queen Elizabeth Hospital November \996 I TABLE OF CONTENTS Table of Contents I Summary vlll Declaration x Acknowledgments xi Dedication xii Publications and Presentations xtll Abbreviations xv CHAPTER 1 Introduction 1..L Introduction 2 L.2 Cell Adhesion Molecules 3 1,.2.1, Integrin Family J 8 1.2.1,.1. PL integrin subfamilY 'J,.2.1..2 9 P2 integrin subfamilY 11 1,.2.1.3 P7 integrin subfamilY 1,.2.2 Immunoglobulin (Ig) SuperfamilY 12 1,.2.2.1 ICAM-L (Intercellular adhesion molecule-l) 14 t.2.2.2 ICAM-2 (Letercellular adhesion molecule-2) 15 1..2.2.3 ICAM-3 (Intercellular adhesion molecule-3) 15 t.2.2.4 VCAM-L (Vascular cell adhesion molecule-1) 16 1,.2.2.5 PECAM-L (Platelet-endothelial cell adhesion molecule-1') 18 L.2.2.6 MAdCAM-L (Mucosal addressin cell adhesion molecule-L) 19 1,.2.3 Selectin Family 19 1,.2.3.1 E-selectin 20 1,.2.3.2 P-selectin 22 7.2.3.3 L-selectin 23 1.2.4 Vascular Mucins (addressins) 24 L.2.4.L GIyCAM-1 (Glycosylation-dependent cell adhesion molecule-1) 24 1,.2.4.2 CD34 25 1,.2.5 Cadherins 25 1..2.6 CD4O 26 1.2.7 CDM 27 l1 L.3 Vascular Endothelium 28 L.3.1 MorpholoW of Endothelial Cells 29 1.3.1.1 High Endothelial Venules 30 1.3.2 Study of Endothelial Cells in aitro 31 L.3.2J, Large Vessel Endothelial Cells 31 1.3.2.2 Microvascular Endothelial Cells 32 1,.3.2.3 Transformed Endothelial Cells 33 1.3.3 Heterogeneity of Vascular Endothelium 33 1.3.3.1 Weibel-Palade bodies (WPBs) 33 1,.3.3.2 von Willebrand factor (vWF) 34 1.3.3.3 Biosynthetic Heterogeneity 34 L.3.3.4 Antigenic HeterogeneitY 35 1..3.4 Endothelial Cell Functions 36 1..3.4.1. Coagulation 36 1,.3.4.2 Angiogenesis 38 7.9.4.3 Cytokine Productionby Endothelial Cells 39 1..3.4.4 The Role of Endothelial Cells in Antigen Presentation 4l L.3.5 Endothelial Cells and Allograft Rejection 45 1.3.5.1 Hyperacute Rejection 46 1,.3.5.2 Acute Rejection 46 1.3.5.3 Chronic Rejection 47 1.4 Leukocyte-Endothelial Cell Interactions 48 1..4.1. Differential Migration of Naive and Memory T Cells 48 1,.4.2 Lymphocyte-Endothelial Cell Adhesion at HEVs 50 t.4.3 Leukocyte-EndothelialCelllnteractionsDuringlnflammation 5L 1.4.4 Adhesion Cascade 54 I.4.4.1. Step L- Tethering/Rolling 54 1..4.4.2 Step 2- Activation/Triggering 57 L.4.4.3 Step 3- Strong Adhesion to Endothelium 59 1..4.4.4 Step 4- Leukocyte Transmigration 60 7.4.5 "Traffic signals" or "Area codes" 61 1-.5 Adhesion Molecules in Disease 64 1.5.L Anti-adhesion Therapy: Potential Targets and Strategies 64 1.5.1.1 Monoclonal Antibody Therapy 64 1,5.1.2 Soluble Receptor Antagonists 70 1.5.1.3 Antisense Oligonucleotide Therapy 7t 1.5.1.4 Inhibition of Surface Expression of Cell Adhesion Molecules 72 1.6 Aims of Thesis 74 111 CHAPTER 2 Materials and Methods 2.1 Materials 75 2.1..1, Monoclonal Antibodies 75 2.1..2 Cytokines and cDNA temPlates 77 2.L.3 Cell lines 77 2.1..4 Buffers and culture media 78 2.1,.4.L Cell culture buffers 78 2.1,.4.2 Immunoprecipitation and SDS-PAGE buffers 78 2.1,.4.3 Bacteriological culture media, antibiotics and buffers 79 2.1.4.4 RNA preparation buffers 80 2.7.4.5 Northem transfer buffers 81, 2.1.4.6 Miscellaneous buffers 81 2.1,.5 Reagents and supplements for culture media 82 2.L.6 Other chemicals and reagents 82 2.1,.7 Radiochemicals 86 2.2 Methods 86 2.2.7 Cell preparations 86 2.2.1.1. Isolation of ovine mononuclear cells 87 2.2.1..2 Isolation of sheep umbilical cord vein endothelial cells 87 2.2.1.3 Isolation of human umbilical cord vein endothelial cells 88 2.2.2 Leukocyte-endothelial cell adhesion assay 88 2.2.3 Proliferation assays 89 2.2.3.L Mixed lymphocyte-endothelial cell reaction (MLER) 89 2.2.3.2 Mixed lymphocyte culture (MLC) 89 2.2.4 Protein capping and internalisation assays 90 2.2.5 Metabolism of Acetylated-low density lipoprotein 90 2.2.6 Antibody-dependant cellular cytotoxicity 90 2.2.7 Production of monoclonal antibodies 9t 2.2.7.1 Animals 91. 2,2.7.2 Immunisation 91. 2.2.7.3 Fusion partner cell line 92 2.2.7.4 Cell fusion 92 2.2.7.5 Screening of fusions 93 2.2.7.6 Cloning of hybridomas 93 2.2.7.7 Cryopreservation of cells 94 2.2.7.8 Preparation of immrrne ascites 94 2.2.7.9 Isotyping of antibodies 94 2.2.8 Ammonium sulphate precipitation of monoclonal antibodies 94 lV 2.2.9 FlTC-conjugation of monoclonal antibodies 95 2.2.10 Monoclonal antibody concentration determination - ELISA 96 2.2.11, Staining techniques 96 2.2.11..1. Indirect immunofluorescence for flow cytometric analysis (FACS) 96 2.2.71.1. Indirect imrnunoPeroxidase 97 2.2.12 lmmunoprecipitation of surface antigens 98 2.2.12.7 Lactoperoxidase catalysed cell surface iodination 98 2.2.12.2 Detergent solubilisation of labelled cell surface molecules 99 2.2.12.3 Immunoprecipitation ustng Stnphylococctts atfi eus 99 2.2.13 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) L00 2.2.1.4 cDNA insert subcloning and linearisation 101 2.2.1,4.1, Digestion of vector and insert 102 2.2.14.2 Recovery of ICAM-I insert 702 2.2.L4.3 Dephosphorylation of pGEM-7 Z vector 102 2.2.1,4.4 Ligation of vector and insert 102 2.2.14.5 Preparation of comPetent cells 103 2.2.1.4.6 Bacterial transformation 103 2.2.1,4.7 Small-scale bacterial cultures 104 2.2.14.8 Large-scale bacterial cultures 105 2.2,1.4.9 Linearisation of ICAM-L and VCAM-1 cDNA vectors 105 2.2.15 Northern blot analysis 1,06 2.2.15.1, RNA preparation 106 2.2.L5.2 RNA gel electrophoresis 106 2.2.15.3 20X SSC transfer 107 2.2.L5.4 32P-lub"llit g of cRNA probes 107 2.2.15.5 Prehybridisation 108 2.2.t5.6 Hybridisation 108 2.2.15.7 Autoradiography 108 2.2.16 Data Analysis 108 CHAPTER 3 Establishment of a sheeP renal allograft model: Immunophenotypic comparison of cells in the lymphatic drainage and peripheral blood Introduction L09 Development of the Model 112 Results and Discussion L1.5 3.1 Graft function 115 3.2 Cellular infiltration and graft histopathology lt6 V 3.3 Lymph and urine outPut 119 3.4 Immunophenotypic analysis of lymphatic drainage 125 g.4.L CDs+ T lymphocytes in draining lymph and peripheralblood 125 3.4.2 CD4:CD8 ratio and MHC class II expression by cells in draining lymph and peripheral blood 125 3.5 CD44expression by cells in draining lymph and peripheral blood 131. Summary 136 CHAPTER 4 Studies of an anti-ovine LFA-L monoclonal antibody in ztitto and in aizto Introduction L38 Materials and Methods 140 Results and Discussion 141. 4.1, Characterisation of 72-87 inaitro 141, 4.1,.1. mAb 72-87 recognises ovine LFA-L 1.4L 4.L.2 Inhibition of proliferationby 72-87 142 4.1'3 Irùribition of leukocyte-endothelial cell adhesionby 72-87 1.42 4.1.4 CytotoxicityoÍ72-87 inuitro 1,45 4.2 In aiao studies 148 4.2.1. mAb dosage and safetY 1.48 4.2.2 Effect of 72-87 onrenal allograft survival 150 4.2.3 Effects of.72-87 on graft infiltrate 150 4.2.4 Effect of 72-87 on peripheral blood leukocytes 154 4.2.4.L \¡Vhite cell counts 154 4.2.4.2 CD4 and CD8 subpopulations 754 4.3 Side effects of 72-87 theraPY 156 4.3.1 Endotoxin levels in monoclonal antibody preparations 157 Summary L60 CHAPTER 5 Characterisation of ovine umbilical vein endothelial cells and their expression of cell adhesion molecules: comParative study with human endothelial cells Introduction 162 Materials and Methods 164 Results and Discussion 164 V1 5.1 Isolation of ShUVECs 1.64 5.2 Phenotypic characterisation of ShUVECs 166 5.3 Metabolism of acetylated-low density lipoprotein (AC-LDL) 166 5.4 Endothelial cell phenotypic markers t68 5.4.1, Expression of von Willebrand Factor 168 5.4.2 Expression of P-selectin 168 5.4.3 Weibel-Palade bodies 168 5.5 Stimulation of endothelial cells- Expression of cell surface molecules 171 5.5.1 Expression of MHC molecules 771 5.5.2 Expression of p1 integrin molecules 173 5.5.3 Cell surface expression of VCAM-1 175 5.5.4 Cell surface expression of ICAM-I' 177 5.6 Northern blot analysis of VCAM-1 and ICAM-L mRNA induction 177 5.6.1, ICAM-L mRNA expression 177 5.6.2 VCAM-1 mRNA expression 778 Summary 778 CHAPTER 6 Production and characterisation of monoclonal antibodies against ovine VCAM-I- Introduction 182 Results and Discussion L83 6.1.
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