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. Production and characterisation of ovine VCAM-1 monoclonal antibodies 183
6.t.1. Immunisation and screening strategies 183
6.1..2 Protein capping and internalisation with mAbs to VCAM-1 184
6.L.3 VCAM-1 expression in normal and inflamed kidney 187 6.2 Kinetics of ovine VCAM-L induction t9t 6.3 Functional studies of the three anti-ovine VCAM-L mAbs 193
6.3.1. Endothelial cell-leukocyte adhesion assays 193
6.3.2 Inhibition of ovine MNC proliferation in the MLER 194
6.3.3 lnhibition of ovine MNC proliferation in the MLC 194 6.4 Structural studies of ovine VCAM-L using the panel of anti-VCAM-1mAbs 197 Summary 197 vll
CHAPTER 7 Studies of an anti-VCAM-L monoclonal antibody, or combined anti-VCAM-l- & anti- LFA-I monoclonal antibodies, in acute rejection in the ovine model of renal transplantation
Introduction 199 Materials and Methods 200 Results and Discussion 20L 7.1, VCAM-L expression in rejection renal allografts 201, 7.2 Anti-VCAM-l. mAb theraPY 203 7.2.1, Dosage regimen for QE4G9 treated-sheep 203 7.2.2 Monitoring of mAb level in QE4G9 treated-sheep 204 7.2.3 Effect of QE4G9 on renal allografts survival 206 7.2.4 Histopathology of allografts from QE4G9-treated sheep 210 7.2.5 Effects of QE4G9 on peripheral blood leukocytes 21,0 7.3 Combined anti-VCAM-1 and anti-LFA-L mAb therapy 21,0 7.9.1. Dosage regimen for combined QE4G9 artd72-87 in sheep 21.0 7.3.2 Effect of combined QE4G9 and72-87 on renal allograft survival 272 7.9.3 Histopathology of allografts from combination mAb-treated sheep 212 7.3.4 Effects of QE4G9 and72-87 on peripheral blood leukocytes 21.5 7.4 Failure of either single or combined mAb therapy to inhibit cellular infiltration or prolong allograft survival in the sheep model 2t5 Summary 218
CHAPTER I Attempted production of monoclonal antibodies against ovine ICAM-I
Introduction 220 Materials and Methods 220 Results and Discussion 221 Summary 224
CHAPTER 9 Concluding remarks 226
APPENDIX 1. 230
BIBLIOGRAPHY 231.: v111
SUMMARY
This thesis examines the expression of cell adhesion molecules on both vascular endothelial cells (ECs) and circulating leukocytes, and investigates the role of these molecules in renal allograft rejection. The aims of the study were to (i) establish an ovine model of renal allograft transplantation (ii) generate monoclonal antibodies (mAbs) against ovine endothelial cell adhesion molecules, (iii) to examine the efficacy of the mAbs in prolonging the survival of the sheep renal allografts and (iv) to study the kinetics of expression of cell adhesion molecules (CAMs) during cellular activation in aitro and in oirto during the onset of allograft rejection.
The first results chapter (Chapter 3), describes the development of the ovine renal transplantation model. Flow cytometric analysis of cells collected from the lymph draining the renal allograft demonstrated the cells to be predominantly T lymphocytes. A reversal of the CD4:CD8 T lymphocyte ratio was observed during the onset of acute rejection along with increased MHC class II antigen expression. The lymph-borne cells also exhibited a single çp44high phenotype, whereas peripheral blood lymphocytes demonstrated a broader CD44 phenotype, ranging from negative to high.
Chapter 4 examines the ability of an anti-ovine LFA-1 mAb (72-87) Lo inhibit leukocyte-endothelial cell interactions in oitro and in aioo. Significant dose- dependent inhibition of sheep MNC adhesion and proliferation in the presence of sheep ECs was observed in aitro. However, when used as a ProPhylactic immunosuppressive agent in the ovine renal transplantation model, 72-87 was unable to prolong survival of the renal allograft compared to sheep treated with
an irrelevant control mAb, TIB-191. x
Chapter 5 is a description of the isolation and characterisation of sheep umbilical vein endothelial cells (ShUVECs). These cells exhibit many of the classical EC phenotypic markers, including vWF and metabolism of acetylated low density lipoproteins. Studies in parallel with HUVECs demonstrated that
ShUVECs express similar levels of cell surface molecules such as VCAM-1, MHC class I and II, and CD29. Cytokine-stimulation of ShUVECs and HUVECs revealed similar kinetics of induction of CAMs at the mRNA and protein levels.
The production and characterisation of three anti-ovine VCAM-I mAb is d.iscussed in Chapter 6. The specificity of the mAb for ovine VCAM-I was determined by comparison against a mAb known to cross-react with ovine VCAM-L. Studies included analysis of flow cytometric staining profiles on a VCAM-1 transfectant and on stimulated and unstimulated ShUVECs, protein capping and internalisation studies and immunoperoxidase staining for tissue distribution on normal and inflamed kidney. All three mAb displayed significant inhibition of MNC adhesion to ShUVECs in oitro and one mAb (QE4G9) also inhibited MNC proliferation in the mixed lymphocyte endothelial cell reaction .
The ability of QE4G9 to prolong renal allograft survival is discussed in
Chapter 7. Despite inhibition of MNC adhesion and proliferation in aitro, QE4G9 was unable to prolong graft survival nor block mononuclear infiltration in the sheep renal allograft model. Having observed an additive inhibition of MNC adhesion to ShUVECs in aitro inthe presence of both QE4G9 and72-87, combined mAb therapy was also investigated in aiuo. As with single mAb therapy, the combined mAb were also unable to prolong allograft survival. The temporal
expression of VCAM-I- in a rejecting renal allograft was also studied.
In Chapter 8, the final experimental chapter, I have described the attempts to produce an anti-ovine ICAM-1 mAb. Despite the use of several different immunisation strategies, no sheep specific anti-ICAM-L mAb were produced, although numerous potential anti-human ICAM-L mAbs wefe identified. x
DECLARATION
This work contains no material which has been accepted for the award of any other degree or diploma in any University or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying.
Warwick L. 29 /11./e6 xl
ACKNOWLEDGMENTS
This thesis would not be possible without the help of many people. I am sincerely grateful to my two supervisors, Dr Graeme Russ and Dr Ravi Krishnan, for their guidance, encouragement and support throughout my PhD. I'd also like to thank Graeme and Ravi for their suggestions and proof-reading of this thesis.
I am especially grateful to |ulie Johnston for her excellent technical support, especially during the transplantation studies and the cutting of tissue sections. Thank you Julie.
I would also like to express my sincere thanks to the following people for their encouragement, support and advice throughout the difficult times and the good times; Svjetlana Kireta, Geraldine Murphy, Guy Patrick, Peter Laslo, Antiopi Varelias and Henry Betts. Thanks also to Henry for proof-reading much of this thesis and for his assistance in resolving various computing problems during the course of the PhD.
Many thanks also to Mr. Mohan Rao for providing the expert surgical skills necessary to complete the transplantation studies of this project. I would also like to thank Ken Porter and the staff of the QEH Animal House for their help and advice during the animal studies.
I would like to thank Dr Tim Mathew, Director of the Renal Unit, TQEH for his support and encouragement, and the Transplantation Society of Australia and New Zealand for financial support to attend local and international scientific meetings.
Thanks also to the staff of the Clinical Photography Department, Medical Llbrary,Electron Microscopy Unit and the Immunopathology laboratory, TQEH.
Many thanks also to my wife Sue and chilclren, Leah and Matthew for their help in preparing diagrams and typing this thesis.
Finally, I'd like to thank my friends for their patience, support and understanding over the years.
The work presented in this thesis was undertaken whilst in receipt of a Queen Elizabeth Hospital Research Foundation Supplementary Postgraduate scholarship, for which I am most grateful. xl1
DEDICATION
I wish to dedicate this thesis to my family
Firstly, to my wife Sue, who's love, patience and encouragement has been immeasurable and invaluable. These words seem inadequate to express my appreciation for the unending support you have given me over man/, many years.
To my children Leah and Matthew, who have shown remarkable patience and understanding for people so young. Waiting for me to complete this project has been like waiting for Christmas to come. We've got there at last kids!
To my parents, who have also provided me with encouragement and support through their continual interest in my work and the well being of myself and my family. Thank you very much. x111
PUBLICATIONS
Grooby WL, Lin Y, Rao MM, Russ GR: Immunophenotypic analysis of cells in lymphatic drainage from rejecting sheep renal allografts. Trønsplant. Proc. t992;24:248-249.
Grooby WL, Carter JK, Rao MM, Dewan P, Seymour AE, Brandon M, Russ GR: Use of an anti-LFA-L antibody in renal allograft rejection in sheep. Transplønt Proc. 1992;24:2305.
Krishnan & Kireta S, Carter JK, Grooby WL, Rao MM, Russ GR: Expression of alternatively spliced CD mRNA in sheep renal allografts. Trønsplønt. Proc. t995;27:2170-2171'.
Grooby WL, Krishnan R, Russ GR: Characterisation of ovine umbilical vein endothelial cells and their expression of cell adhesion molecules: comparative study with human endothelial cells. lmmunol. Cell Biol. 1997; (in press).
Grooby WL, Krishnan R, Russ GR; Production and characterisation of ovine anti- VCAM-1 monoclonal antibodies. (manuscript in preparation).
Grooby WL, Krishnan R, |ohnston fK, Rao MM, Russ GR; Inability of an anti- VCAM-L mAb, or combined anti-VCAM-1 and anti-LFA-L mAbs, to prolong renal allograft survival in an ovine model. (manuscript in preparation).
PRESENTATIONS
Grooby WL, Yuan L, Rao MM, Russ GR. and Brandon M. Immunophenotypic analysis of cells in lymphatic drainage from rejecting sheep renal allografts. Transplantation Society of Australia & New Zealand 9th Annual Scientific Meeting, Canberra, 1991'.
Grooby WL, Carter ]K, Rao MM, Dewan P, Seymour AE, Russ GR and Brandon M. Use of an anti-LFA-L antibody in renal allograft rejection in sheep. xlv
Transplantation Society of Australia & New Zealand LOth Annual Scientific Meeting, Canberra, L992.
Grooby WL, Carter JK, Rao MM, Dewan P, Seymour AE, Russ GR and Brandon M. Use of an anti-LFA-L antibody in renal allograft rejection in sheep. Australian Society for Medical Research, Adelaide,1992.
Grooby WL, Carter fK, Rao MM, Dewan P, Seymour AE, Russ GR and Brandon M. Use of an anti-LFA-L antibody in renal allograft rejection in sheep. The Queen Elizabeth Hospital Research Day,L992.
Grooby wL, Carter fK, Rao MM, Dewan P, Seymour AE, Russ GR and Brandon M. Failure of an anti-LFA-L antibody to prevent renal allograft rejection in sheep. 22ndAustralasian Society for Immunology Annual Scientific Meeting, Auckland N2,1992.
Grooby WL, Krishnan R and Russ GR. Kinetics of cell adhesion molecule expression by human & sheep umbilical vein endothelial cells. Transplantation Society of Australia & New Zealand L2th Annual Scientific Meeting, Canberra, 7994.
Grooby WL, Krishnan R and Russ GR. Production of a panel of monoclonal antibodies recognising Ovine VCAM-1. Transplantation Society of Australia & New Zealand L3th Annual Scientific Meeting, Canberra ,1995.
Grooby WL, Krishnan R, fohnston JK, Rao MM and Russ GR. Inability of an anti- VCAM-I mAb to prolong renal allograft survival in an ovine model. Transplantation Society of Australia & New Zealand L4th Annual Scientific Meeting, Canberra, 1996.
Grooby WL, Krishnan R, |ohnston JK, Rao MM and Russ GR. Inability of an anti- VCAM-L mAb to prolong renal allograft survival in an ovine model. The Queen Elizabeth Hospital Research Day, October 1996. XV
ABBREVIATIONS
Ac-LDL acetylated-low density lipoproteins APC(s) antigen presenting cell CAM(s) cell adhesion molecule CHO cell Chinese hamster ovary cell OvI conditioned medium Con A concanavalin A type IV EC(s) endothelial cell ECGF endothelial cell growth factor ECM extra cellular matrix FACS flow cytometric analysis FCS foetal calf serum HAT - Media Supplement hypoxanthine, aminopterin, thymidine FIEV high endothelial venule hr hour(s) HT - Media Supplement hypoxanthine, thymidine HUVEC(s) human umbilical vein endothelial cell i.p. intraperitoneal i.v. intravenous LPS lipopolysaccharide mAb(s) monoclonal antibody MHC major histocompatibility complex MLC mixed lymphocyte culture MLER mixed lymphocyte-endothelial cell culture MNC(s) mononuclear cell n number in study or group p probability PAGE polyacrylamide gel electrophoresis PBL peripheral blood leukocyte PP Peyer's patch PLN Peripheral lymph node PMA phorbol myristate acetate PMN (s) polymorphonuclear cell t recombinant rh recombinant human ro recombinant ovine SDS sodium dodecyl sulphate ShUVEC(s) sheep umbilical vein endothelial cell sLea slewisa antigen sLex slewisx antigen TEMED N,N,N',N'-tetramethyl-etþlenediamine v\AIF von Willebrand factor WPB(s) Weibel-Palade body 1
Ch"pter 1
INTRODUCTION
", . .Cfr¿tflotfrcrøpeutíc øgents øre rwt or0 enls in tfr¿msefou 6ut øßo seft)e øs tooß for unbcfrng loors ønl proííng 9{øtttre's mysteriu , , ,
, , . Sefectiüíty rerrunns our øLrn ønl unlerstønling its 6asß ourguile to tfrzfutura"
Gertrude B. Elion L988
(Lecture given upon receiving the Nobel Prize for Physiology/Medicine) 2
1..1. INTRODUCTION
The evolution of specific recirculation and homing characteristics of leukocytes enables efficient immunosurveillance of tissues and allows rapid recruitment at sites of inflammation. The distinct migratory properties of leukocytes depend upon the expression of specific complementary adhesion molecules on both leukocytes and endothelial cells (ECs). Expression of these molecules is finely controlled according to the cell type and activation state, and the tissue site at which antigens are encountered. The nature of the inflammatory stimulus determines which group (or groups) of leukocytes respond. At sites of inflammation activated ECs present a different array of cell adhesion molecules (CAMs) compared to those expressed by resting ECs and therefore provide "traffic signals", assisting and directing the migration and homing of leukocytes
[Springer, t994].
The past 10-15 years has seen a wealth of information accumulate regarding the expression, distribution and function of CAMs. During the same time span, the understanding of the nature ECs and their role in a range of immunological and haemostatic functions has also changed dramatically. In this chapter the families of CAMs involved in leukocyte-endothelial cell adhesion will be reviewed, including their role in recirculation, inflammation and signalling. This will be followed by a discussion on the endothelium as an active participant in several patho-physiological processes, with particular emphasis on its role in allograft rejection. The third component of this introduction will briefly examine the expression of CAMs in a number of diseased states. This will be followed by a review of the development of new therapeutic agents designed to ameliorate unwanted inflammatory responses, such as allograft rejection, by targeting leukocyte-endothelial adhesion mechanisms. I.2 CELL ADHESION MOLECUTES
Leukocyte extravasation into inflamed sites and migration through the extra cellular matrix (ECM) are mediated by various families of CAMs. There are five main families of adhesion molecules; the integrins, the immunoglobulin superfamily, selectins, cadherins and mucin-like molecules. The grouping of these molecules has been defined on the basis of both protein structure and sequence homology. A summary of the members of each family and their ligands is shown in Table 1.L. There are also several other molecules, such as CD44, CD40, CD15 and CD38, which have been shown to play a role in lymphocyte migration and signalling, but do not belong in any of the major family SrouPs.
It has become increasingly apparent that many of the molecules originally identified based upon their adhesion properties may actually serve a multiplicity of functions. In addition to acting as adhesion receptors, many CAMs also play a role in signal transduction, both into and out from cells, and in some cases the adhesion function may be of secondary importance.
The distribution and function of most of these molecules have been comprehensively discussed in several excellent reviews [Pardi et n1.,1992; Shimizu et ø1.,1992; Shaw, 1993; Carlos and Harlan,1994; Springer, L9941. Therefore this introduction will concentrate primarily on CAMs involved in leukocyte- endothelial cell interactions, with particular reference to those expressed at sites of inflammation.
L.2.1 Integrin Family
The integrin family comprises the largest group of adhesion molecules,
each consisting of non-covalently linked, transmembrane heterodimeric cr and B
chains [Hynes, L992]. Eight B chains and L6 cr chains have been described to date, including the recently identified øD82, found on a subset of T lymphocytes and 4
Tøble 1.1 Adhesion molecules and their ligands
Adhesion molecule Ligand
Integrins olp1(VLA-l) laminin, collagen CI2p1(VLA-2) laminin, collagen cr3p1(VLA-3) laminin, collagen, fibronectin o4p1(VLA-4) VCAM-L, fibronectin, MAdCAM (weakly) üsp1(VLA-5) fibronectin û6p1(VLA-6) laminin s7þ1 ? û8P1 ? cr9p1 fibronectin txlp2 (LFA-l) ICAM-]., ICAM-2, ICAM-3 dMP2 (Mac-l) ICAM-I, C3bi, fibrinogen, Factor X oxp2 (p150,95) C3bi, fibrinogen o;Dþ2 ICAM-3 qIIb p3 fibrinogen, fibronectin, vWF, vitronectin aVp3 fibrinogen, fibronectin, vWF, vitronectin laminin, thrombosp ondin
d6þ4 laminin crVp5 vitronectin, fibronectin aVp6 fibronectin uEþ7 E-cadherin a4þ7 MAdCAM-1., VCAM-I, fibronectin aVps ? Selectins L-selectin GlyCAM, MAdCAM-L,CD34 P-selectin Sialyl-Lewisx, PSGL-L E-selectin Sialyl-Lewisx Ig superfamily ICAM-1 LFA-I, Mac-1, CD43 ICAM-2 LFA-]. ICAM-3 LFA-1. VCAM-]. YLA-A,a4þ7 MAdCAM-1 L-selectin, a4þ7, a4þ1. (weakty) cD31(PECAM-I) CD3l., proteoglycans CDz LFA-3, CD48, CD59 LFA-3 CDz Vascular Mucins (addressins) GIyCAM-1 L-selectin CD34 L-selectin PSGL-1. P-selectin MAdCAM-1 o4p 7, L-selectin, 44p1. (weakly) Unclassified CD44 hyaluronic acid, collagen, fibronectin CDTO CD4OL 5
some tissue macrophages in dog [Danilenko et ø1.,1995]. With only a few exceptions, most cr chains associate with one specific B chain, giving rise to a total ol22different integrin combinations (Figure l-.L and Table 1.1).
The integrins are involved in cell-cell communication and mediating firm ad,hesive interactions with other cells [Hemlet, 1990; Hynes, 1992; Pigott and power, 1gg3l. Many integrins are also ECM receptors, recognising specific amino acid sequence motifs such as the highty conserved arginine-glycine-aspartic acid (RGD) sequence found in a number of ECM proteins, including fibronectin, fibrinogen, vitronectin and the thrombospondin [Ruoskahti and Pierschbacher,
I9B7; Hynes, tggz). Although expression of þ2 and þ7 molecules is restricted to leukocytes, the integrins are generally expressed on most cells in the body, with many cells expressing several types of integrins.
The cr subunits possess multiple divalent cation binding sites [Corbi et øl',
L987; Corbi et ø1.,1988; Larson et ø1.,19891. These sites are essential for integrin function and the nature of the cation can effect the specificity and affinity for ligands [Kirchhof er et a\.,1990; van Kooyk et ø1., 1991; Dransfield et al', 7992; Masumoto and Hemler, 1993].
In addition some o subunits, ü1, a2, A4, crM, crE and CrX, contain an extra inserted domain, or "I" domain, of approximately 180-200 amino acids [Kishimoto et aL,19S9bl. The I domains belong to the superfamily of type A domain proteins and are homologous to domains found in von Willebrand factor, cartilage matrix protein and complement proteins [Colombatti and Bonaldo, I99t]. This domain is also thought to provide specificity for ligands as the VLA-1 binding site for
collagen and laminin [Kern et al., 1994], the VLA-2 binding site for collagen
[Kamata et ø1., 1994], the Mac-1 binding site for C3bi [Ued a et ø1. , L994] and the LFA-I binding site for ICAM-1 and ICAM-3 are all within the I domain [Landis ef
al., !994; Randi and Hogg, L994; Edwards et ø1.,1995; Huang and Springer, 19951.
Evidence that the I domain may be involved in regulating integrin ligand binding 6 affinity comes from studies using mAbs directed at the I domain. Binding of the mAbs induced a conformational change in LFA-1 to the activated, ICAM-L and ICAM-3 binding state [Springer, 1990; Landis et øl', t993; Landis et ø1., 1994; Huang and Sprin ger, 19951.
The rapid conversion of integrins from a non-adhesive, low affinity state to a transient high affinity state is one of the most important aspects of integrin function [Dustin and Springer, t989; Hynes, 1992]. For instance, crosslinking of the T-cell antigen receptor (TCR) triggers a transient increase in LFA-L avidity, peaking at 5-10 minutes and returning to the basal, low avidity state by 30 minutes to 2 hours [Dustin and Springer,1989; Dustin, 1990]. As this Process can be inhibited by increasing cytoplasmic cyclic AMP, or by blocking protein kinase C, it indicates that cytoplasmic signals influence the activation state of the integrins.
Ligand binding fegron
p cr subunit p subunit
a7 cr8 p3 oIIÞ Extracellular a9 domain ps 0(v - I p6 \ p8 Cytoplasmic domain þ2
Eigure 1-.1. Integrin family structure and combinations of cr and B subunits. The divalent cation binding sites in the cr chain are shown as M++' 7
The rapid conversion of integrins from a non-adhesive, low affinity state to a transient high affinity state is one of the most important aspects of integrin function [Dustin and Springer, !989; Hynes, t992]. For instance, crosslinking of the T-cell antigen receptor (TCR) triggers a transient increase in LFA-1 avidity, peaking at 5-10 minutes and returning to the basal, low avidity state by 30 minutes to 2 hours [Dustin and Springer,!989; Dustin, 1990]. As this Process can be inhibited by increasing cytoplasmic cyclic AMP, or by blocking protein kinase C, it indicates that cytoplasmic signals influence the activation state of the integrins.
The modulation of integrin affinity by events occurring within the cytoplasm have been described as "inside-out signalling" [Dustin and Springer, t989;Dustin and Springer,1991,; Hynes, 1992]. This process is usually observed as an increase in ligand affinity binding with no change in the number of cell surface receptors and is thought to be due to a change in conformation of the integrin in the plasma membrane [Dustin and Springer, 1989; Ds et ø1., 1991'; Diamond and
Springer, 1993; Landis et a1.,1993; Faull et ø1., t9941. The cytoplasmic tail of both cl and B chains interact with the cytoskeleton and are important for integrin function [Williams et ø1., 1994; Lub et ø1., L995]. The B subunit interacts with cytoskeletal actin filaments through the proteins talin and cr-actinin. Evidence for the important role of the cytoplasmic region of the B chains came from studies demonstrating the loss of adhesiveness of B 1 and B 2 integrins following truncation of the cytoplasmic domains [Hayashi et ø1.,1990; Hibbs et a\,,1,991]. In contrast, there was an increase in a4B7 integrin adhesiveness after removal of the
B7 cytoplasmic domain.
The cr chain also plays a role in affinity modulations through the GFFKR motif, present in all a chain cytoplasmic domains [Sastry and Horwitz, 1993; Williams et ø1., 1,9941. Deletion of this sequence from LFA-1 resulted in the transformation to a high affinity state [O'Toole eú a1.,1994f, while truncation of 8 sequences in the u2, a4 and cr6 cytoplasmic domains also influenced the
activation state of the B1 integrins [Williams et ø1.,1994].
In addition to responding to intracellular signals, studies have also demonstrated that integrins themselves can transmit signals into the cells, representing "outside-in signalling" [Damsky and Werb, 1'992; Sastry and Horwitz, 19931. Ligation of integrin receptors by antibodies can induce
intracellular signals [Kanner et nL, 1993; Arroyo et al., L994] resulting in a variety of cellular responses including differentiation, proliferation, cytokine production,
cytotoxicity and migration [Figdor et ø1.,1990; Hynes, 1992; Moy and Brian, L992;
Juliano and Haskill,1993; Koopman et ø1.,19941.It has been proposed that ligation of different binding sites of particular integrins may trigger transduction of
different intracellular signals [Hynes, 1992].
L.2.1,.7 þL integrin subfømily
The Bl subfamily consists of combinations of the B1 chain (CD29) with the
cr subunits (CDa9) (see Figure 1.1). These receptors are often referred to as the very late antigens (VLA) following the identification of the first numbers of this group ryLA-1 & VLA-2) on T cells afler 2 weeks of in aitro stimulation [Hemler ef ø1., 1.984; Hemler et ø1., 1935]. The B1 integrins are Present at low levels on lymphocytes and increase in number after cell stimulation [Hemler et a\.,1987a;
Hemler et aI.,19S7bl. With the exception of craBl ffLA-4) and cr6B1 (VLA-6), the
B1 integrins function as receptors for ECM molecules and are expressed in most tissues.
cr4Bl and a6p1 are unique in that they are also involved in cell-cell interactions. a4Bl is expressed on all leukocytes [Hemler et al., t987b], with the exception of neutrophils [Dobrina et ø1., l99L; Walsh et ø1., 1991; Bochner et al.,
19951. It has been reported that cr4B1 possesses three distinct epitope binding sites 9
[Pulido et al., 1991] for its ligands, vascular cell adhesion molecule-L (VCAM-l, CD106) [Elices et a\.,1990] and fibronectin. Furthermore, u4B1 recognises two distinct domains within fibronectin, one containing an RGD sequence located in the centre of the molecule [Humphries et al., 1987] and the second is the connecting segment-1 (CS-1) [Wayner et a\.,1989]. Binding of cr4B1 to either VCAM-L or fibronectin in aitro results in transduction of activation signals and [Garcia-Pardo and Ferreira, 1989; Wayner et ø1., t989; Davis et a\.,1990; Guan Hynes, 1990;Nojima et a\.,1990; Shimizu et al.,l990a; Mould et aI',t991) involving a tyrosine phosphorylation pathway [Nojima et øl',1992].
o6Bl is expressed by polymorphonuclear cells (PMNs) and ECs. On PMNs, it is involved in adhesion to laminin [Bohnsack et a1.,1990], while expression by
ECs is associated with lymphocyte adhesion to non-inflamed tissue [Lenter et ø1.,
I9g3l and homing of T cell progenitors to the thymus [Dunon and Imho1,1993].
7.2.1..2 þ2 integrin subfømily
The B2 subfamily, which has been well characterised in humans and mice, represents the predominant integrin on leukocytes. These four molecules consist of unique cr chains (CD11) in combination with the shared B2 (CD18) chain (see
Figure 1.L). Although integrins generally display a broad tissue distribution, uLþ2
(LFA-1 or CD1.La) is restricted to white blood cells, while ulvlB2 (Mac-1 or CD1Lb), and uXB2 (p150,95 or CD1Lc) are more restricted, being expressed on monocytes, macrophages, neutrophils and NK cells. The recently described ctDB2, is also limited in its expression, having been identified on tissues macrophages and a subset of T lymphocytes [Danilenko et ø1.,1995i.
The members of the B2 subfamily play a number of important roles in the immune response, including the promotion of strong adhesion of rolling leukocytes to ECs, the transmigration of leukocytes through endothelium and the 10 migration of leukocytes within tissues [Arnaout, 1990]. The functional significance of þ2integrins in leukocyte adhesion was revealed by the identification of a SrouP of patients with an immunodeficiency disorder called leukocyte adhesion deficiency-I (LAD). LAD-I is an autosomal recessive disease caused by mutations in the common B2 subunit [Rodriguez et ø1.,1993]. As a result, LFA-1, Mac-1 and p150,95 are ineffective or absent and patients are susceptible to recurrent life- threatening infections, neutrophilia, delayed separation of umbilical stump and developmental abnormalities [reviewed in Anderson and Springer,l997;Etzioni, lgg4l. Neutrophils from these patients are unable to adhere and migrate through the endothelium at sites of infection [Anderson and Springer, 1987], whereas normal leukocytes transferred into these patients are able to infiltrate inflammatory sites [Bowen et ø1.,1982].
LFA-1 rn¡as first described as a CAM in mice during the screening for mAbs that block cytotoxic T lymphocyte (CTL) mediated killing of target cells
[Davignon et a1.,1981]. Mac-L was identified as a receptor for the C3bi fragment of complement following the observation that antibodies against Mac-L blocked adhesion of myeloid cells to C3bi-coated particles [Beller et al., 7982]. Mac-L also binds fibrinogen, factor X and lipopolysaccharide (LPS) [Arnaout, 1'990].
Although p150,95 is the least characterised of the original B2 integrins, it is similar to Mac-l- in binding C3bi [Myones et ø1., 1988] and having a limited tissue distribution [Arnaout, 1990).
Whereas the cellular ligands for p150,95 are yet to be confirmed, both LFA-
L and Mac-1, interact with the intercellular adhesion molecules (ICAMs), members of the immunoglobulin super family (see below). LFA-1 is capable of binding to ICAM-L [Marlin and Springer,l987f,IcAM-2 [Staunton et a\.,1989a] and ICAM-3
[de Fougerolles and Springer,1992]. ICAM-1 is also the ligand for Mac-L [Smith ef øt., 1989; Diamond et ø1., 19901, although the binding site is different to that of
LFA-1 [Diamond et ø1.,199L]. L1
Although not present in tissue macrophages, circulating monocytes and granulocytes contain intracellular storage pools of Mac-L and p150,95 which can be translocated to the cell surface within minutes of activation [Miller et ø1. ,1987]. In contrast, lymphocytes and neutrophils have no significant intracellular stores of LFA-1 [Anderson and Springer,l987). Although monocytes rapidly upregulate LFA-1 upon stimulation, it is unclear whether this comes from an intracellular pool fMiller et a\.,1987].
The newest member of the B2 subfamily, uDþ2, has recently been isolated from canine splenocytes [Danilenko eú ø1.,1995]. As stated earlier, the distribution of this molecule is very restricted and at this stage its function is yet to be defined.
Flowever, studies with an uDþ2 human counterpart indicate the main ligand to be ICAM-3 lcited in Danilenko eú ø1.,1995], suggesting an intercellular role in lymphoid tissue such as spleen and lymph nodes, sites where aDB2 showed high levels of expression.
1.2.7.3 B7 integrin subfømily
The CAM u4þ7, previously designated u4BP, was first identified on a subset of murine and human lymphocytes which selectively migrate to the gut and gut-associated lymphoid tissues [Holzmann et a1.,1989; Schweighoffer et ø1.,
19931. Recent studies have demonstrated a broader cellular distribution of. a4þ7, including activated T cells [Ruegg et ø1.,1992], B cells, eosinophils and NK cells
[Erle eú øL,1994].
Monoclonal antibodies against a4þ7 block adhesion of murine lymphocytes to Peyer's patch (PP) high endothelial venules (HEV), but had no effect on adhesion to peripheral lymph node (PLN) HEV [Holzmann et ø1.,1989] (structure and function of HEV are discussed in detail below). Ligands lor u4þ7 include MAdCAM-1 (mucosal addressin cell adhesion molecule-1; see below)
[Berlin et a1.,1993; ErIe et ø1.,19941, VCAM-I and the CS-L region with fibronectin 12
[Ruegg et a\.,1992; Postigo et ø1.,L993; Crowe et ø1.,1994]. The interaction of a+þZ with VCAM-L involves similar but nonidentical amino acid residues on VCAM-1 which are also required for binding of VLA-4 [Chiu et ø1.,19951. Studies with a panel of anti-a487 mAbs suggest that a4B7 interacts with VCAM-I, MAdCAM-1 and fibronectin through distinct but overlapping epitopes [Andrew et aL,1994].It has been proposed that the regulated expression of u4þ7 determines the level of interaction with MAdCAM-L and may influence lymphocyte recirculation, through preferential binding of CD4+ memory cells compared to naive cells [Rott et ø1.,19961.
The integrin aEBT (also known as cIIELBT or crM290Þ7) is involved in lymphocytes interactions with the gut, in particular the intestinal epithelium [Cerf
Bensussan et ø1., L992; Cepek et ø1., 1993; Roberts and Kilshaw, 1,9931.
Lymphocytes expressing uEBT bind to the epithelial cells through the ligand E- cadherin [Cepek et al., t994].
T.2.2 Immunoglobulin (Ig) Superfamily
The immunoglobulin (Ig) superfamily consists of more than 70 molecules (Table 1.L) involved in adhesion and signal transduction in leukocytes. The molecules contain Ig domains, consisting of 70 to 1L0 amino acids which are fotded and held together by disulphide bonds (Figure 1.2).
These adhesion molecules consist of domains which are designated V and
C depending upon their structural homology with variable (V-type) and constant (C-type) Ig domains. The C-type domains are further subdivided into CL and C2 subtypes according to sequence similarities. Within the C2-subtype there are five members of the Ig gene superfamily involved in leukocyte-endothelial cell interactions; intercellular adhesion molecule-L (ICAM-l; CD54),ICAM-2 (CD102), VCAM-I (CD106), platelet endothelial adhesion molecule-L (PECAM-I-, CD31) L3 and MAdCAM-L. Although ICAM-3 appears to be primarily associated with adhesion between resting leukocytes [de Fougerolles and Springer, 1992; Fawcett et al., L992;Vazeux et a1.,19921, a limited expression has also been demonstrated on endothelial cells [Doussis Anagnostopoulou et aI., t993]. ICAM-L, ICAM-2, VCAM-L, PECAM-I and MAdCAM-1 are all expressed on ECs and are involved in leukocyte adhesion, activation and migration [Marlin and Springer, 1987;
Osborn et ø1.,L9ï9;Staunton et al.,L989a;Newman et ø1.,1990;Erleet ø1.,19941.
vcAM-1
N cD31
NH 2 tcAM-1 tcAM-3
NH NH 2 2 domain spliced in VCAM-1 6D form
tcAM-2 LFA-3
NH 2
cooH cooH cooH cooH cooH cooH
Eigure 7.2 Tlne structure of the immunoglobulin superfamily molecules involved in leukocyte-endothelial cell interactions. LFA-3 (CD58) can be expressed in transmembrane and phosphatidylinositol-linked forms. 1,4
L.2.2.L |CAM-|- (lntercellulør Adhesion Molecule '7)
ICAM-1 was originally identified by a monoclonal antibody, RR1/1, which recognised a cell surface protein expressed at low levels on unstimulated ECs and leukocytes [Rothlein et al., 1986].ICAM-1 has five lg-like domains. A 3 Kb mRNA transcript gives rise to a 95-105 kDa protein (depending upon the cell type) which is expressed on ECs, mononuclear cells (MNCs), fibroblasts [Dustin et ø1., L986;
Rothlein et a\.,19861and epithelial cells [Faull and Russ, 1989; Gundel et ø1.,1991']. Constitutively expressed low levels of ICAM-I are found on ECs and lymphocytes and moderate levels on monocytes [Dustin et ø1.,1986; Rothlein et al',
19861. In response to inflammatory mediators such as TNF-c, IL-L,IFN-y or LPS,
ICAM-I expression is upregulated [Dustin et al., 1986]. The increase in ICAM-L expression requires mRNA transcription and de noao protein synthesis, with increased surface expression evident 6-8 hours after stimulation and remaining elevated beyond 48 hours [Dustin et øL, 1986]. The constitutive exPression of ICAM-I on cultured human umbilical vein endothelial cells (HUVECs) is inhibited by IL-4 [Thornhill and Haskard, L990]. L-10 has been shown to down- regulate the expression of ICAM-I on ll.-1-stimulated HUVECs [Krakauer,1995] and lFN-lstimulated monocytes [Willems et ø1.,1994] Similarly, IL-13 has been reported to cause down-regulation of IL-1 induced ICAM-L [Sironi et ø1.,1994).
As described earlier, although ICAM-L is a ligand for LFA-L and Mac-L, the binding sites are distinct with LFA-1 recognising domains L and 2 [Staunton et ø1,,
19901, while Mac-L binds to domain 3 [Diamond et al., 1991]. ICAM-L also functions as a receptor for two pathogens, Plasmodium følciparum and Rhinoviruses. The binding of. Phsmodium følciparum-infected erythrocytes to ICAM-I expressed on the endothelium of small blood vessels may facilitate the sequestration of the infected cells, thereby avoiding removal by the spleen
[Berendt et al.,1989]. The binding of infected erythrocytes occurs at a site distinct from that used by LFA-L, Mac-L or rhinoviruses [Berendt et aL,1992; Ockenhouse et ø1., 1992a1. Rhinoviruses are a major cause of common colds and ICAM-1 is a 15 ligand for approximately 90% ol human rhinovirus serotypes [Greve et ø1., L989;
Staunton et ø1.,1989b]
1.2.2.2 ICAM-2 (Intercellulør Adhesion Molecule'2)
ICAM-2 (a6 kDa) contains only 2 Ig-like domains and is expressed on ECs, lymphocytes and monocytes, but not on neutrophils [de Fougerolles et ø1., l99t;
Nortamo et al., 199t1. Constitutively high levels of ICAM-2 arc expressed by resting ECs, but unlike ICAM-I it is not inducible by activation of the ECs with inflammatory stimuli [Staunton et ø1., 1989a; de Fougerolles ¿ú aI., 1991'; Nortamo et ø1.,19911. This suggests that ICAM-2 is less important in acute inflammatory or immune responses, but may have a role in leukocyte trafficking across resting endothelium or early in inflammation as demonstrated in ICAM-L deficient mice
[Sligh et a\.,1993]. Nonetheless,ICAM-2 has been shown to provide costimulatory signals during TCR-mediated activation of human T cells [Damle et ø1.,\992a].In addition to functioning as a major receptor for CDt1,/CD18 molecules, ICAM-2 has also been reported to bind CD49d/CD29 (VLA-4) [Seth ef a1.,199t).
1..2.2.3 ICANI-3 (lntercellulør Adhesion Molecule -3)
Observations that combinations of mAbs against ICAM-L and ICAM-2 were unable to completely inhibit LFA-1 dependent adhesion between lymphoid cells indicated the existence of additional ligands for LFA-1 [de Fougerolles and
Springer, t9921. This was confirmed by both the production of a new mAb, which in combination with ICAM-1 and ICAM-2 mAbs, completely inhibited adhesion to purified LFA-L [de Fougerolles and Springer,1992], and the isolation of cDNA for a protein identified as ICAM-3 [Fawcett et ø1.,1992; Vazeux et ø1.,1992].
Initially, ICAM-3 tissue distribution was thought to be restricted to haematopoietic cells, including monocytes, lymphocytes and granulocytes [de 1.6
Fougerolles and Springer, 1992]. However, it has recently been detected on human epidermal Langerhans cells [Staquet et ø1., 1'995]. High levels of constitutive ICAM-3 expression were observed on all of these cell populations. ICAM-3 contains five Ig-like domains exhibiting a high degree of homology to those found in ICAM-L and ICAM-2 and has a molecular weight of betweenl20-
160 kDa, depending upon cell type [Fawcett et nl., 1992; Vazeux et ø1., 1992; de Fougerolles et ø1., 1993). Studies of LFA-l-dependent cell adhesion to ICAM-3 mutants demonstrated that LFA-I binds to the amino-terminal of ICAM-3 [Sadhu et ø1,,19941.
ICAM-3, recently*ee* confirmed to be the same molecule previously identified as CD50 []uan et ø1., 19941, has been implicated to play an important role in the initiation of the immune response through both cell-cell adhesion and signal transduction. Monoclonal antibodies against ICAM-3 inhibit alloimmune responses [Vilella et a\.,1990], stimulate both resting and activated T lymphocytes through modulation of Bl and þ2 integrin function [Campanero et aL.,1993;
Hernandez-Caselles et ø1.,1993; Campanero et ø1. ,1994; Cid et ø1., \9941 and induce calcium mobilisation and tyrosine phosphorylation in T cell lines and neutrophils
[]uan et ø1., 1994; Skubitz et ø1., 19951. The accumulation of ICAM-3 in cellular uropods of activated, adherent lymphocytes suggests a further role for this molecule in the recruitment of additional cells to sites of inflammation [del Pozo et ø1.,1995; del Pozo et a\.,19961.
7.2.2.4 VCAM-7 (Vøsculør Cell Adhesion Molecule-7)
VCAM-1 (CD106), previously designated INCAM-110, is a single chain glycoprotein of approximately 110 kDa. VCAM-I was first identified by a mAb that inhibited adhesion of melanoma cells to cytokine-activated endothelial cells
[Rice and Bevilacqua, 1989] and also by functional cloning from interleukin-1- stimulated endothelial cells [Osborn et ø1., 1989]. Either a six or seven Ig-like t7 domain form, the result of alternative splicing of mRNA, are exPressed by activated ECs [Polte et ø1.,1990; Cybulsky et ø1.,199L; Hession et a1.,1991'; Polte ef
øt., t9911. The seven domain form is predominant in humans in which domains l'-
3 are homologous to domains 4-6 [Hession ef ø1.,1991'; Polte et ø1.,1991]. Domain 4 is deleted through alternative splicing in the six domain form lOsborn et ø1,,1992]. A third isoform of VCAM-1- has been identified in mice which contains only 3 domains and. is bound to the cell membrane by a phosphatidylinositol linkage
[Cybulsky et a\,,1993;}r/roy et n1.,1993; Terry et ø1.,1993; Kumar et a1.,1994]. Pigs have been reported to express yet another form of VCAM-L, containing five immunoglobulin domains with approximately 77% homology to the human protein [Tsang et a1.,1994].
In addition to activated vascular endothelium, VCAM-I expression occurs on bone marrow stromal and fibroblast cells [Miyake et ø1.,1991'; Ryan et ø1., t991';
Dittel et aI., lgggj, follicular DCs [Rice et al., 1991'; Huang et ø1,, t995] and tissue macrophages and renal tubular epithelial cells [Freedman et ø1., 1990; Rice et ø1.,
L99l;Briscoe et ø1.,t992;Alpers et ø1.,1993;Fuggle et ø1.,19931.
VCAM-1 is generally not detectable on resting HUVECs, however like
ICAM-1, it can be upregulated in response to LPS, TNF-cr, IL-L and IL-4, but not by IFN-1 [Osborn et ø1., 1989; Rice and Bevilacqua, 1989; Carlos et ø1., 1990b; Masinovsky et ø1., L990; Thornhill and Haskatd, 1990; Wellicome et al', L990;
Thornhill et ø1.,19911. In contrast, IL-Lcr- and ll-4-stimulation of human dermal microvascular endothelial cells failed to induce VCAM-L expression while TNF-cr produced only a transient increase in VCAM-L protein and mRNA levels, peaking
at1.6 hours [Swerlick et ø1.,7992]. Recent studies have demonstrated that IL-13 also selectively induces the expression of VCAM-1 (but not ICAM-1 or E-selectin) on HUVECs and enhances the induction caused by IL-1., similar to IL-4 [Sironi ef al., 1994; Bochner et al., 19951. In contrast, IL-l-0 down-regulates VCAM-1 expression on Il-l--stimulated HUVECs [Krakauer, 19951. Increased VCAM-I expression requires mRNA synthesis and protein production with maximum 18 surface expression occurring t}-lzhours after stimulation and remaining elevated for at leastTlhours [Osborn et ø1., L989; Carlos et a1.,1990b; Wellicome et ø1.,1990].
VCAM-1 is the counter-receptor for cr4p1 (VLA-4) which is expressed by all leukocytes, with the exception of neutrophils [Osborn et ø1., 1989; Elices et øl',
I991;Schwartz et ø1.,1990; Carlos et a\.,1991.; Schleimer et n\.,1992; Bochner et ø1., Igg5l. As described earlier, VCAM-I is also able to interact with leukocytes expressin g uaþ7 [Ruegg et ø1.,1992; Crowe et ø1., 1994; Chiu et ø1., 1995]. Studies with Pløsmodium følcipørum-infected erythrocytes isolated from malaria patients demonstrated that the binding of infected erythrocytes to TNF-cr-stimulated HUVECs was partially mediated by VCAM-I [Ockenhouse et ø1., t992b]. In addition to its role in cell adhesion, VCAM-L is also capable of delivering costimulatory signals to resting T cells via its counter-receptor, cr4B1 [BurkIy et ø1.,
1991;van Seventer et nl.,199La; Damle et ø1.,1992b1.
1.2.2.5 PECANI-L (Pl øtelet-Endotheli al C ell Adhe sion MoI ecule-L)
PECAM-1 (CD31) is a six domain molecule, constitutively expressed on ECs, platelets and most leukocytes [Albeld a et ø1., 1990; Newman et ø1., 1990;
Albelda et ø1.,19911. EC expression of PECAM-L is not increased by stimulation of
ECs with TNF-cr, IL-L, or combined TNF-cr and IFN-1 [Simmons et ø1.,1990]. PECAM-L expression is concentrated at the intercellular junctions lAlbelda et ø1.,
L9901 and mAb blocking studies indicate a role in leukocyte transmigration
[Mu|]er et ø1.,L993;Muller, t995]. PECAM-L may also facilitate signal transduction through the Bl and þ2 integrins, regulating leukocyte adhesion and migration through the endothelium [Tanaka et al., t992; Piali et aI., t993; Muller, 1995]. PECAM-1 is also thought to play a role in forming or stabilising the vascular bed
[Muller et a\.,1989; D'Amore, 1992] limiting vascular permeability [Albelda et ø1.,
7991,1. T9
7.2.2.6 MAilCAM-7 (Muc o s øl Addt es sin C ell Adhe si on Molecule-L)
MAdCAM-L is a 58-66 kDa protein expressed mainly on HEV of Peyer's patches, venules in small intestinal lamina propria and on the marginal sinus of the spleen [Berlin et a\.,1993j. MAdCAM-L is capable of performing a dual role in leukocyte adhesion through the presence of both lg-like and mucin domains
[Briskin et ø1., 1993]. The main ligands for MAdCAM-L are the integrin cr4B7 [Berlin et al., L993;ErIe et al. ,1994] and L-selectin [Berg et øl' ,1993].
1.2.3 Selectin Family
The selectin family consists of three molecules designated E-selectin, P- selectin and L-selectin [Bevilacqua et al,, 1,99L]. These receptors are all transmembrane molecules sharing a common structure including an N-terminal lectin domain, a single epidermal growth factor (EGF) like domain, a variable number of short consensus repeats (SCR) which are related to the complement regulatory proteins, a transmembrane domain, and a short cytoplasmic domain
(Figure 1.3). The name "selectins" refers to their lectin domain and their role in selective cell trafficking [Bevilacqua et al., 1989; Tedder et al., 1990b]. Although these molecules display structure and functional similarities, their cellular distribution and regulation of expression differs for each selectin. However despite these differences, the selectins are capable of interacting with one or more common ligands, as discussed below [Foxall et ø1., 1992; Mebius and Watson,
19931. In addition, the role of selectins as adhesion molecules involved in transient, calcium dependent, low strength adhesion and rolling of leukocytes is also examined. 20
L-selectin
cooH NHz
E-selectin
cooH NHz P-selectin
cooH NHz
Figute 1.3 Schematic structures of the selectin family of adhesion molecules. CR; complement regulatory-like domains, ECGF; epiderrnal growth factor-like domain
7.2.3.7 E-selectin
E-selectin (CD62E, ELAM-1) is a LL2 kDa single chain glycoprotein with 6 SCR domains. E-selectin is restricted to ECs and is only expressed after stimulation with inflammatory mediators sudr as IL-1, TNF-a and LPS [Pober ef aI., !986a; Bevilacqua et al.,I98n.IFN-f does not induce E-selectin e>rpression, but does enhance and prolong its expression in resPonse to IL-1. and TNF
fi.eeuwenberg et aI., t990f. In contrast, IL4 and transforming growth factor-B (TGF-P) inhibit the induction of E-selectin [Thornhitt and Haskard,1990; Gamble
et al., t9931. Similar to the effect on ICAM-I, IL-13 does not induce E-selectin
expression [Sironi et ø1.,19941. Surface expression in uitro is maximal4-6 hours after stimulation, with a return to basal levels within 24 hours, even in the
presence of continued stimulation [Bevilacqua et a1.,1987; Bevilacqua et al., 7989; Gamble et al., 1993]. Flowever, studies with cultured human dermal microvascular ECs subjected to repetitive doses of IL-1cr demonstrated persistent
expression of E-selectin [Sepp et a\.,1994]. Induction of E-selectin expression is
dependent on de nouo mRNA and protein synthesis. 21.
Sustained high levels of E-selectin expression have also been detected in several chronic inflammatory skin disorders [Nickoloff et ø1., 1990; Groves et al. ,
1991; Picker et al., L991,al and in rheumatoid arthritis synovial tissue [Koch et øL,
ß911. Multiple mRNA transcripts exhibiting different degrees of stability have
been identified for human E-selectin indicating the E-selectin gene expression is
regulated post-transcriptionally [Chu et ø1., 1994]. The mRNA transcripts were shown to be differentially expressed in cells obtained from different tissue and the
authors suggest this may provide an explanation for the persistent expression of E-selectin in aiao . Two unique rat E-selectin cDNA fragments have also been isolated which differ in the presence of sequences that encode the complement
regulatory domain-S (CR-s) [Fries et ø1,, 1993; Billups et ø1., 1995]. Both mRNA forms were significantly induced in the hearts of rats treated with LPS [Billups eú
ø1., 1995). E-selectin has also been reported to partially mediate binding of
Plasmodium følcipørum-infected erythrocytes to TNF-cr-stimulated HUVECs [Ockenhouse eú ø1., 1992b].
E-selectin is primarily involved in the adhesion of monocytes and
neutrophils to activated endothelium [Leeuwenberg et ø1.,1990; Leeuwenberget a\.,1992), although in aitro and in aiao studies have also indicated the ability to
interact with a subset of memory T cells [Berg et a\.,1991; Picker et al., L991.a;
Shimizu et al., 1991b;|utila et al., 19941.
The ligands for E-selectin include the sialylated oligosaccharides, slewisx
(sl.ex) and slewira (sl-ea) (CD15) [Phillips et ø1.,1990;Walz et øl',7990; Polley eú aL, l99L; Tyrrell et al., L991), and cutaneous leukocyte antigens (CLA), a carbohydrate molecule expressed by a population of skin-associated memory T
lymphocytes [Berg et al., 199L; Shimizu et ø1., 1991b].In addition, studies have demonstrated that soluble E-selectin and COS cells transfected with E-selectin adhere to immobilised CD71,a/CDL8 and CDtIb/CD18 [Kotovuori et a1.,L993]. 22
7.2.3.2 P-selectin
P-selectin (CD62P) expression is restricted to the surface of activated endothelial cells [McEver et a\.,1987; Bonfanti et ø1.,1989] and platelets [Hsu Línet
ø1., 1984; McEver and Martin, 1984; Stenberg et a\.,1985]. The molecule is a single chain glycoprotein of L40 kDa and is stored in the a-granules of platelets
[Stenberg et a1.,1985; Berman et ø1.,1986] and the Weibel-Palade bodies (WPB) of
ECs [Bonf.antiet ø1.,1989; Hattori et ø1.,1989; McEver et ø1.,1989]. Activation of platelets or stimulation of ECs with thrombin, histamine , CSa, LTB¿ or phorbol esters results in the rapid translocation to the cell surface [Hattori et ø1.,L989; Patel et ø1., t99L; Foreman et a\.,19941. Endothelial surface expression occurs rapidly (within minutes), but is transient and returns to basal levels within 30-60 minutes
[Hattori et a1.,1989]. It has been shown that down regulation of P-selectin involves internalisation of the molecule followed by eventual transportation back to the
WPBs for re-use [Subramaniam et aI., 1993]. P-selectin induction has also been reported to occur in the presence of LPS or TNF-cr, both in aitro and in aiao, with kinetics similar to those of E-selectin [Sanders et ø1.,1992; Weller et ø1.,1992;
Gotsch et a1.,t9941.
Analysis of cDNA indicates two forms of P-selectin exist, a transmembrane form or a soluble form with a deleted transmembrane segment []ohnstonet al., 1989]. The complete form of P-selectin has been shown to mediate rapid neutrophil adhesion to activated endothelium [Geng et ø1.,1990] and participate in the initial "rolling" of leukocytes on endothelium prior to firm adhesion
[Lawrence and Springer, 1991.; ]utila et ø1., 1994; Springer, 19941. Although the physiological significance of soluble P-selectin is not known, it may have a regulatory function by binding to activated neutrophils and preventing their adhesion to endothelium [Gamble et ø1., 1990]. Evidence of neutrophilia, and diminished rolling and transmigration of neutrophils at sites of inflammation have been reported in studies using a P-selectin deficient mouse [Mayadas et ø1.,
Lee3l" 23
P-selectin, like E-selectin, binds to sl-ex and sl-ea antigens [Polley et ø1.,
199L;Zkrou et a1.,19911. Another ligand for P-selectin has recently been cloned, the P-selectin glycoprotein ligand-1 (PSGL-L) which is expressed on leukocytes
[Moore et al.,1992; Sako ef ø1.,19931and is a member of the mucin-like adhesion molecules (Table 1.1). The involvement of PSGL-L in leukocyte rolling was shown in aiao where an anti-PSGL-l mAb blocked the ability of cells to ro11 on venules of exteriorised rat mesentery [Norman et aI.,\995).
1.2.3.3 L-selectin
L-selectin (CD62L) contains only 2 SCR domains and is constitutively expressed by all leukocytes, except for activated memory lymphocytes [Picker eú al., 1990; Bradley et al., L9921. Although initially described as a lymphocyte homing receptor involved in the adhesion of lymphocytes to HEV of PLNs
[Gallatin et nl., L983], L-selectin is also involved in leukocyte adhesion to endothelium at sites of inflammation [Ley et ø1., 1991,; Spertini et ø1., 1991b;
Bargatze et a\.,1994).
Whereas neutrophils in circulation express high levels of L-selectin, cells isolated from inflammatory sites express little surface L-selectin. L-selectin expression is rapidly (within minutes) down-regulated by shedding from the surface of activated neutrophils [Kishimoto et øL,1989a; Kishimoto et ø1., L990] or lymphocytes [fung et n\.,1988], and high levels of a soluble form of L-selectin can be found in human plasma [Schleiffenbaum et al., 1992]. The functional significance of soluble L-selectin is not known, however as with soluble P-selectin, it may serve to regulate leukocyte adhesion [Kishimoto et ø1.,I989a].
L-selectin has a number of ligands including sl-ex and sl-ea [Berget øL,
1992; Foxall et ø1., 1992; von Andrian et a1.,1993). Furthermore, it has been demonstrated that L-selectin expressed on neutrophils (but not lymphocytes) is able to interact with E-selectin and P-selectin on activated endothelium lPicker eú 24 at.,lgg¡b]. In addition, at least three mucin-like ligands (discussed below) expressed by HEV have been identified for L-selectin: GIyCAM-1, CD34 and
MA{CAM-I [Lasky et ø1. , t992; Baumheuter et ø1.,1993;Betg et ø1.,1993]. A recent study has demonstrated that the cytoplasmic domain of L-selectin interacts with the cytoskeleton to regulate leukocyte adhesion and rolling on endothelium
[Kansas et aL, t993]. However, this interaction was not necessary for ligand recognition.
1..2.4 Vascular Mucins (addressins)
The mucin-like molecules are glycoproteins which primarily function as receptors for the selectins. They are heavily O-glycosylated, serine and threonine- rich proteins with short oligosaccharides [Springer, 1994]. Four mucins involved in leukocyte-endothelium interactions have so far been identified; GIyCAM-L,
CD34, PSGL-1 and MAdCAM-1. As described earlier, MAdCAM-1 is somewhat unusual in that it also contains lg-like domains and therefore belongs to both CAM families. The role for both MAdCAM-L and PSGL-1 as ligands for the selectins has been discussed and therefore they will not be considered any further in this section.
7.2.4.1 Gly GAM-I (gly co syløtion- dep endent cell adhesion molecule-l.)
GIyCAM-1 is expressed by HEV of PLNs and mesenteric lymph nodes
[Lasky et ø1.,1992].In addition to serving as a ligand for L-selectin, GIyCAM-L has also been reported to bind E-selectin [Mebius and Watson,t993; Lenter et øL,
1994). However, as GIyCAM-L is a soluble 50 kDa protein, which does not contain a transmembrane domain [Brustein et ø1., 1992; Lasky et al., 1992], its role as a vascular addressin is unclear. 25
L.2.4.2 CD34
CD34, a 90 kDa transmembrane sialomucin-like glycoprotein is also a ligandforL-selectin [Baumheuter etø1.,1993;Baumhueteretal.,1994].Incontrast to GIyCAM-1, CD34 is expressed on a diverse range of lymphoid and non- lymphoid endothelium and on immature haematopoietic cells [Simmons et ø1.,
1992; Baumheuter et al., \993; Baumhueter et al., L9941. The low levels of CD34 expression on endothelial cells at sites of cutaneous inflammation [Norton et ø1.,
19931 suggests the molecule is not essential for leukocyte migration during inflammation, but may play a significant role in normal circulation. This is supported by in aitro studies demonstrating a reciprocal down regulation in CD34 protein and mRNA levels as ICAM-L and E-selectin are upregulated on ECs stimulated with eitherlL-1', TNF-o or IFN-y [Delia et ø1.,1993].
L.2.5 Cadherins
Cadherins aÍe a family of CAMs expressed by cells forming solid tissues. These molecules are primarily involved in homotypic interactions and are thought to regulate cell polarity and tissue morphology [Takeichí, L991]. Twelve molecules have been identified, representing four main subclasses: E-(epithelial), P-(placental), N-(neural) cadherins and L-CAM (Liver cell adhesion molecule)
[Luna and Hitt, 1992). Flowever, a recent study has demonstrated that E-cadherin also binds integrin cxEP7, expressed by intraepithelial lymphocytes, suggesting that it may also participate in lymphocyte homing [Cepek et ø1.,7994]. As with many other CAMs, Cadherins also interact with the cytoskeleton suggesting that the expression of these molecules at cell-cell junctions may involve intercellular signalling [Takeichi, 1991]. 26
L.2.6 cD40
CD40 is a 50 kDa phosphorylated glycoprotein expressed by B cells, ECs, macrophages, dendritic cells and fibroblasts [reviewed in Stout and Suttles,t996).
It is a member of the TNF-receptor superfamily due to sequence homology with
TNF-a and nerve growth factor [Stamenkovic et ø1.,1989].
Cultured HUVECs constitutively express low levels of CD40 which can be significantly upregulated by stimulation for 24 hours with TNF-a,IL-1.cr or B, or
IFN-y or B [Hollenbaugh et ø1. ,1995; Karmann et al. , 1995; Yellin et al' ,1995]. CD40 has also been detected on the vascular endothelium in frozen sections of normal human spleen, skin, muscle, kidne/, lung and umbilical cord [Yellin et ø1.,1995]. Furthermore, increased endothelial CD40 was observed in sections of tissue from inflammatory skin diseases [Hollenbaugh et ø1.,1995].
The counter-receptor for cD40 is cD40L (gp39 or T-BAM), a 30-39 kDa glycoprotein transiently expressed on activated T cells, but absent from resting T lymphocytes [Armit age et ø1. , L992; Lane et ø1.,1992; Noelle et ø1.,1992].
Ligation of CD40 on cultured HUVECs with soluble trimeric CD40L,
CD40L+ Jurkat cells or CD40L+ 293 transfectant cells resulted in the induction of ICAM-1, VCAM-I and E-selectin on the ECs [Hollenbaugh et ø1.,1995; Karmann eú a\.,1995; Yellin et a\.,19951. The kinetics of induction of the CAMs following the interaction of CD40-CD40L is similar to that observed for cytokine mediated upregulation (see below). Flowever, the interaction of CD40 and its ligand did not alter the level of MHC class II molecule expression on the HUVECs. These observations suggest that endothelial CD40 may be involved in signal transduction whereby activated T cells amplify inflammatory responses through the upregulation of endothelial CAMs and possibly the induction of cytokine synthesis by both leukocytes and endothelium. 27
The involvement of CD40 in the alloimmune resPonse is supported by studies in which blockade of CD40 significantly prolonged the survival of murine cardiac allografts, while blockade of both CD40 and CD28 pathways produced
long term acceptance of murine skin and cardiac allografts [Larsen et ø1.,1996].
1..2.7 cD44
CD44 is a heterogeneous family of glycoproteins which have had various
names including Pgp-I,Ly-24, gp90 Hermes, H-Cam. It has a broad distribution
on lymphocytes , monocytes, neutrophils, epithelial cells, glial cells, fibroblasts and monocytes [Pigott and Power,1993]. Many isoforms of CD44 are generated due to alternative splicing of at least 12 exons and extensive post-translational
modifications [Scre aton et a1.,1992], giving rise to molecules with a wide range of molecular weights (85-250 kDa). The two most abundant isoforms are CD44H, associated with mesenchymal cells, and CD44E, which is present on actively
dividing epithelial cells [Underhill, 1992].
CD44 primarily mediates cell adhesion to hyaluronic acid (HA), but can also bind to fibronectin, collagen and endothelial proteoglycans [Gallatin et ø1.,
1989; Aruffo et ø1. ,1990; Miyake et ø1. , 1990; |alkanen and Jalkanen, 1992; Toyama- Sorimachi and Miyasaka, 19941. While CD44 has been reported to play an
important role in lymphocyte activation, adhesion and transmigration [falkanen eú
al., \986b; Huet et ø1., 1989; Picker et al., L989; Shimizu et øL, 19891, the precise function of CD44 in leukocyte migration to either normal or inflamed endothelium is unclear. The finding that mAb to CD44 inhibited lymphocyte homing to Peyer's patch and PLN HEV, and to activated endothelium, suggested
a role for CD44 in lymphocyte-endothelial cell adhesion [Jalkanen eú ø1., L9871. This was further supported by observations of mAb inhibition of leukocyte migration through endothelial monolayers in aitro [Oppenheimer-Marks and
Lipsky, 1994; Brezinschek et a1.,t9951. 28
However, others have reported the inability of anti-CD$[ mAbs to inhibit lymphocyte binding to HEV in aitro [Yang and Binns, 1993f , or limit the trafficking of MNCs to lymph nodes [Camp etø1.,1993; Mikecz et a1.,1995), although migration into inflamed synovial tissue was blocked [Mikecz et ø1.,
1ee5l.
Nonetheless, CD44 may function as an accessory molecule involved in lymphocyte activation. It has been demonstrated that CD44 interacts with chondroitin sulphate on the invariant chain associated with MHC class II antigens, generating class Il-dependant allogeneic and mitogenic T cell responses
[Naujokas et n\.,1993]. In addition, engagement of CD44 results in the activation of T cells [Conrad et ø1., 1992], NK cells [Galandrini et ø1., f994] and promotes the homotypic adhesion of T cells through the LFA-L pathway [Koopman et ø1.,1990]. CD44 expressed by ECs is thought to bind chemokines, such as MIP-IB, presenting them to bound leukocytes and triggering integrin activation and firm leukocyte-endothelial adhesion [Tanaka et ø1.,1993] (see below).
1..3 VASCULAR ENDOTHELIUM
Historically, ECs were perceived as a passive layer of cells, providing a protective, nonadherent surface separating the blood from the extravascular tissue. Flowever, the development of techniques enabling the establishment of continuous EC cultures []affe et aI., 1973; Gimbrone et ø1., 1974; Johnson, L980], provided a tool to study the function of these cells under different conditions. It is now well appreciated that ECs are actively involved in a variety of physiological functions, including coagulation and thrombolysis, vasotonus regulation, antigen presentation and haematopoietic regulation [Pober and Cotran, 1990b].
It has been estimated that greater than 1012 endothelial cells line the inside of blood vessels, with a surface area of more than 1000rr;.2 gaf.ie,1g87l, possibly representing the body's largest complex organ. In the embryo the cardiovascular 29 system is the first organ system to develop [Risau, 1995] and it has been suggested that ECs and haematopoietic cells may share a common development lineage
[Risau et a\.,1983]. Support for this proposal came from the observations that antibodies against immature blood cells cross-react with ECs [Pardanaud et al', lg8gl and that the mature ECs retain expression of many haematopoietic cell differentiation markers [Favaloro, 1993). Furthermore, ECs share various functional characteristics with cells of the macrophage/monocyte lineage, including antigen-induced T-cell proliferation [Hirschberg et aL,,1980; Wagner ef
al., 19851, presentation of peptides to primed T cells [Nunez et ø1., 1983] and
induction of Fc and complement receptors [Ryan et ø1.,1981].
L.3.L Morphology of Endothelial Cells
Quiescent, resting ECs in the adult represent a highly heterogeneous cell population that varies not only on different anatomical sites, but also between
large and small vessels [Risau, 1995]. Depending upon the organ or tissue, endothelial cells can be divided into different phenotypes consisting of either a tight, continuous monolayer in organs where they perform important barrier
functions, such as the brain or lungs, or they can form a discontinuous layer of
cells with intercellular gaps or intracellular fenestrae, as found in the kidney,liver or spleen. Furthermore, within any one organ it is possible to have several different types of ECs, for example fenestrated diaphragmed endothelium of the kidney peritubular capillaries and discontinuous endothelium of glomerular
capillaries [Risau, 19951. It has been proposed that endothelial phenotype is governed by the surrounding microenvironment, with the endothelial cells receiving cues from adjacent cells, basement membrane, or both [Saunders and
Damore, t992). 30
L.3.1.L High Endotheliøl Venules
In contrast to the thin flattened morphology of cells that line the majority of the body's blood vessels, the HEVs within lymph nodes and Peyer's patches display a characteristic plump high cuboidal morphology [Ager, L987]. It is through these HEVs that a high percentage of lymphocyte extravasation occurs and the presence of discontinuous junctions between the high endothelial cells probably facilitates this process [Anderson and Shaw, 1993]. The role of HEV in leukocyte migration will be discussed in greater detail later in this Chapter, in the context of leukocyte-endothelial cell interactions.
HEV are found in all secondary lymphoid organs, with the exception of the spleen, where small arterioles may terminate open ended into the parenchyma, enabling continuous unrestricted access of blood leukocytes. At sites of inflammation in non-lymphoid tissue, capillary endothelium can take on morphologic and functional features similar to HEV. This has been associated with chronically inflamed tissue, such as in rheumatoid synovium [falkanen et ø1.,
1,986a; Freemont, 1988] and acute inflammation of peritubular capillary endothelium during acute renal allograft rejection [Renkonen et ø1., 19901. Moreover, a recent study of rat lymph node autografts demonstrated the differentiation of HEV from capillaries which grew into the grafts followed by lymphocyte extravasation through these venules [Sasaki et a1.,1994].
The "high" morphology of HEV is thought to function in two important processes, protection against vascular leakage and facilitation of lymphocyte extravasation [Kraal et n1.,1987]. In contrast to the tight junctions of capillary and arterial endothelium, the presence of discontinuous "spot-welded" junctions between HEVs [Anderson and Shaw, 1993], probably allows the massive recirculation of lymphocytes associated with HEV.
Ligation of afferent lymphatics in rats or mice results in the loss of HEV phenotype, GIyCAM-L expression, and the ability to support lymphocyte 31 extravasation, with the endothelium reverting to flat-walled non-functional venules [Hendriks et ø1., L980; Hendriks and Eestermans, 1983; Drayson and Ford, 1984; Mebius et øL,1991,; Mebius et ø1., 19931. In contrast, studies of HEV in irradiated, lymphocyte depleted mice demonstrated a partial reduction in endothelial cell height, but no effect on lymphocyte binding [Duijvestijn et øl',
1990]. From these studies it has been suggested that either the presence of antigen draining from peripheral tissue into the lymph node [Hendriks and Eestermans/
1983] or the production of cytokines by monocytes and dendritic cells may play a significant role in the maintenance of HEVs [Duijvestiin et aL.,t990].
L.3.2 Study of Endothelial Cells in oitro
7.3.2.7 Large Vessel Endotheliøl Cells
Although considerable detail regarding the morphology, structure and antigenic properties of the vascular endothelium have been obtained from histological studies of tissue sections, the development of techniques for culturing
ECs enabled these cells to be studied in great detail in aitro []affe et ø1., 1973; Gimbrone et a\.,1974; johnson, 19801. Much of the understanding of vascular biology has come from the use of ECs isolated from large vessels. In particular, the supply of human umbilical cords from Maternity wards has made human umbilical vein endothelial cells (HUVECs) a readily available tool to investigate a variety of physiological functions in aitro. Animals have also represented a convenient source of ECs for research purposes, with cells being obtained from bovine and ovine aorta, carotid artery and jugular vein [Mueller et ø1., 1980; Abernethy and Hay,1989; Borron and Hay, 19941. Although ECs derived from large vessels have a limited life span, the addition of various growth supplements, such as endothelial cell growth factor (ECGF) and heparin, to culture medium improves the growth characteristics of the cells [Maciag et al., 1979; Thornton ¿f 32 al., t9831. In addition, large vessel ECs can be isolated with relative ease by collagenase digestion []affe et ø1.,1973; Gimbrone et aL.,19741.
1.3.2.2 Miuoo øsculør Endotheliøl Cells
However, while large veins and arteries represent a convenient source of ECs they are not representative of the majority of the bodies endothelium. Approximately 95% of the bodies vasculature is of the microvascular form
[Hewett and Murray,1993a] and it is at this level that many of the physiological functions, including immune responses, occur [Bishop et nl,, 1989; Murray et ø1.,
L9941. Microvascular ECs have been isolated from numerous sites including human foreskin [Davison et ø1.,1980; Swerlick et ø1., L9911synovium []ackson eú at.,1990) and lung [Hewett and Murray,1993a; Hewett and Murray,l993bf , rct glomeruli [Laulajainen et ø1., L993] and murine skin, peritoneum, mesentery and liver [Macphee et ø1., L9941. The isolation and culture of microvascular ECs has to date been associated with several major problems, including the rapid overgrowth by fibroblasts and/or smooth muscle cells, their fastidious growth requirements in aitro , and a restricted life span [Ades et al',1992].
Some of these problems have been attributed to mechanical or enzymic damage occurring during harvesting lRyan et ø1., 1982). Flowever, several modifications have recently been developed in order to eliminate these problems, including the selective enrichment of human lung microvessel ECs at the first passage using magnetic beads coated wilh Ulex europaeus agglutinin-L [Hewett and Murray, \993a] and collagenase digestion of fibroblast growth factor L- impregnated sponges [Macphee eú ø1., 19941. Chen et ø1. 11995] reported the ordered migration of ECs out of pieces of lung or muscles after erythrocytes and leukocytes, but before fibroblasts and other cell types. 33
L.3.2.3 Trønsformed Endotheliøl Cell Lines
The development of transformed EC lines may overcome some of the restrictions incurred by the limited life span for either small or large vessel- derived ECs. Several transformed EC lines have been described, including human dermal microvascular ECs [Ades et ø1.,1992) and rat glomerular ECs [Laulajainen et ø1., tggSlwhich were immortalised with the aid of transforming viral elements. Continuous HUVEC lines have also been generated by viral transformation
[Vicart et ø1., LggS], fusion with a human lung carcinoma line [Edgell et ø1.,1983] and. following a spontaneous transformation event [Cockerill et ø1.,19941. Each of these transformed cell populations retain a number of the typical characteristics of
ECs, including the expression of von Willebrand factor, cobblestone morphology when grown in monolayers, uptake of acetylated low-density lipoproteins and the expression of CAMs.
Despite the development of these continuous endothelial cell lines, HUVECs remain the most frequently used cell type for investigation of the role of
ECs in a range of patho-physiological states. Flowever, although the endothelium in most parts of the body constitutes a uniform continuous layer of cells with a thin flattened morpholog/, there is considerable evidence describing the phenotypic heterogeneity of these cells. The next section of this chapter is a brief introduction to ECs and some of the data concerning their antigenic and functional properties and the heterogeneity associated vascular endothelium from different anatomical sites.
L.3.3 Heterogeneity of Vascular Endothelium
L.3.3.1 Weib el-P aløde b o dies (WPB s)
In addition to morphological differences, ECs exhibit variability in several other aspects, both within species and between species. For instance, Weibel- 34
Palade bodies are generally considered as one of the classical phenotypic markers of ECs [Weibel and Palad e, \964] and are typically present in the endothelium of humans and rats [Weibel and Palade,1964; Trillo and Prichard, L979]. However WPBs are uncommon in the arteries of primates [Trillo and Prichard, 1979], variable in distribution in ECs from different sites in the vasculature tree of pigs
[Gebrane-Younes et ø1.,1991] and absent from bovine aorta [Schwartz,1978) and
murine ECs [Murrary, 1987].
1..3.3.2 oon Willebrønd føctor (ztWF)
WPBs are the storage site for vWF [Wagner et ø1.,1982; Bonfanti et a1.,1989;
Hattori et ø1.,19391 which is synthesised by the ECs [Jaffe et ø1., 1973]. Although
distribution of vWF has been shown to vary according to the Presence of WPB in
the pig [Gebrane-Younes et ø1., 1991.), its distribution in humans also varies, despite the uniform presence of WPBs in all ECs. For instance in the adult endothelium, immunochemical staining has revealed high levels of vWF in large
vessels and HEV of lymphatic tissue, whilst being either weakly expressed or
absent in capillary ECs [Turner et ø1.,1987).
L.3.3.3 Bio synthetic Hetero geneity
ECs also display heterogeneity in a number of metabolic functions, including the production of ECM components.In aitro irrurrran ECs cultured from the umbilical vein and vena cava secrete collagen IV into the medium and
collagen IV and V into the ECM lJaffe, L9871. ECs from cultured bovine aorta, pulmonary aftery, or mesenteric vein release collagen III into the medium and collagen III, IV, and V into the ECM lJaffe, 19871 while the production of fibronectin is lower in porcine cardiac valvular ECs than in aortic ECs [fohnson
and Fass, 19831. 35
There is also variability in the insulin-binding ability of ECs, with arterial cells binding 2.5 times more insulin than venous ECs lJaffe,1987l. There are also
differences between large and small vessel ECs in the effect of insulin on glucose
incorporation into glycogen and thymidine incorporation into DNA lJafifi.e, L9871. Finally, while most ECs secrete prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet adhesion, human foreskin capillary ECs synthesise mainly
PGEz and PGFza lJaÊfle,19871.
L.3.3.4 Antigenic Heter ogeneitY
Not only do ECs display morphological and function differences, but they
also exhibit extensive antigenic heterogeneity. Page et al. 11992] examined the
expression of a panel of antigenic markers, including CAMs and MHC molecules
on ECs from a number of different vascular sites. Their results demonstrated that
small vessel endothelium strongly express MHC class I & II antigens,ICAM-L and
CD36, whereas these molecules are weak to undetectable on large vessel ECs. However, HLA-DR and VCAM-L were expressed strongly on normal coronary arteries, while absent from all other large vessels. Several studies have also demonstrated that ECs from different compartments within the liver, lung and kidney display different profiles of surface antigens [Nagura et ø1., \986;
Yamamot o et al. , 1988; Kinjo et al. , 19591. For instance, Kinjo et al. 119891 reported peritubular capillaries in the renal medulla to be CD36 positive, whereas those in the cortex were rarely, if at all, reactive with the anti-CD36 mAb, OKMS. Glomerular capillary loops were also unreactive with OKMS. All capillary vessels
were positive for HLA-DR, which was weak or absent from large vessel ECs.
Studies of the level of ICAM-1- expression on resting and TNF-cr-treated human aortic ECs have demonstrated heterogeneity between cultures from
different donors [Everett et al., 1994]. Furthermore, the expression of ICAM-1, VCAM-1, E-selectin and CD36 have been reported to vary between large vessel 36
ECs and. microvascular endothelium, and between venous and arterial
endothelium [Swerli ck et al. , L99t; Detmar et al. , 1992; Swerlick et al. , 1992; Hauser
et a1.,1993; Petzelbauer et ø1.,L9931.
1.3.4 Endothelial Cell Functions
The ability to study ECs in aitrohas enabled investigation of the role of the endothelium in both immunological and haemostatic processes. The majority of
this thesis concerns the cell adhesion molecules involved in leukocyte-endothelial cell interactions during immune responses. However, the endothelium also actively participates in a number of other processes, including coagulation and
angiogenesis. As these functions can contribute to the survival of a vascularised
allograft, they will be reviewed briefly below.
7.3.4.L Coøguløtion
The ability of perturbed or injured ECs to initiate coagulation is a critical
aspect of their role in the maintenance of haemostasis. Through the expression of different thrombogenic or antithrombogenic properties ECs are able to participate in both procoagulant and anticoagulant mechanisms. One important modulator of
blood coagulation is thrombomodulin, a protein expressed on the surface of ECs which enhances the ability of thrombin to activate protein C [Esmon, L987; Esmon, 1989). Activated protein C functions with its cofactor, protein S, as an anti-coagulant by inactivating the coagulation factors Va and VIIIa by proteolysis
[Dahlback, 1986; Stern et nl., 1986). Protein S is synthesised by ECs fFair et ø1.,
1e861.
A second mechanism involves the expression of glycosaminoglycans, such as heparan sulphate and dermatan sulphate, on the surface of ECs. These 37 molecules catalyse the inactivation of thrombin by antithrombin III through the formation of a covalent thrombin-antithrombin complex which is then rapidly
cleared by the liver [Rosenberg and Rosenbery, \984].
The interaction of the endothelium with platelets is another important
factor in coagulation. Adhesion of platelets to the subendothelium occurs rapidly after damage to the endothelial lining. Platelets bind to either subendothelial collagen [Coller et ø1., 1989] or to vWF, which in turn anchors to the subendothelial matrix through collagen types I, III, IV and V [Meyer and Baumgartner, L983]. As mentioned above, vWF is synthesised by the ECs [affe et
ø1. ,19731 and stored in WPBs [Wagner et al., L982; Bonfanti et al., 1989; Flattori et
ø1., 19891. Thrombin has been reported to stimulate the release of vWF from HUVECs [Levine et al., 1.982]. Aggregating platelets release adenosine diphosphate (ADP), which recruits other platelets into the platelet plug, and
adenosine triphosphate (ATP), which is vasodilator. The effects of ADP and ATP are modulated by their rapid metabolism by ectosenzymes synthesised by
endothelium [Pearson et al., L980].
Flowever, ECs normally inhibit platelet activation and adhesion, and clot
formation [Rodgers and Shuman,1983; Rosenberg and Rosenberg,1984; Rodgers, 1983]. The inability of unstimulated platelets to adhere to ECs has been attributed to the presence of endothelium-derived relaxing factor on the surface of ECs
[Palmer et ø1., 19871. The inhibition of activated platelet adhesion to ECs is mediated by PGI2, produced by ECs in response to numerous factors including
thrombin and trypsin [Weksler et ø1.,19781, histamine [Baenziger et ø1., 1980; Baenziger et øl.,lgïLl,bradykinin [Hong, 1980l, immunological injury [Goldsmith and McCormick, 19841, activated neutrophils [Miller et aL.,1985] and cytokines
(II--L, TNF-cr) [Goldsmith and McCormick, L984; Jafifte, 1987). PGI2 synthesis is inhibited by aspirin [Weksler et al., L978] and other non-steroidal anti- inflammatory drugs such as indomethacin [Weksler et aL.,1977]. 38
The two components of coagulation, plasma factors and platelets, can function co-operatively in blood clotting with platelet activation accelerating thrombin formation, while fibrin deposition strengthens platelet aggregates [Roth, te92l.
The endothelium participates in fibrinolysis by the synthesis of plasminogen activators in two forms, tissue plasminogen activator (TPA) and
prourokinase [Erickson eú ø1,, !985]. TPA is produced by ECs in response to stimulation by thrombin, histamine and bradykinin [Erickson et ø1.,1985]. The plasminogen activators cleave plasminogen to form plasmin, the principle fibrinolytic enzyme. ECs also secrete plasminogen activator inhibitor, which is
partly neutralised by protein C [Esmon,1987\, enhancing fibrinolysis.
L.3.4.2 Angiogenesis
Angiogenesis refers to the formation of new microvessels and occurs during the processes of wound repair, tumour growth and inflammation
[Folkman and Shing, t992]. Capillary endothelium is normally quiescent and surrounded by an intact basement membrane. In resPonse to an angiogenic stimulus, ECs separate exposing segments of the basement membrane which are
degraded. ECs then migrate into the interstitium, followed by multiple rounds of
EC proliferation and migration, which continue until the microvascular network
is complete [Furcht, 1986].
Many factors have been identified which stimulate angiogenesis including epidermal growth factor, acidic and basic fibroblast growth factors, heparin and prostaglandins which can be derived from both normal and tumour cells
[Folkman and Klagsbrun, 1987; Fajardo, t989]. Soluble E-selectin and soluble VCAM-1 have been reported to be chemotactic for human ECs in aitro and also
exhibited angiogenic potential in rat cornea [Koch et ø1.,1995]. Furthermore, the 39 chemotactic and angiogenic activities of rheumatoid synovial fluid could be inhibited by antibodies to either soluble CAM.
Several antagonists of angiogenesis have been described including corticosteroids, IFN-1 and TGF-B [Folkman, t984; Baird and Durkin,1986; Beck ef al., 1986; Folkman and Klagsbtun,ISST; Friesel et ø1., 19871. The role of TNF-0 is unclear as reports that it stimulates angiogenesis ln aiao fFrater-Schrodet et øL,
79871conflict with observations that it inhibits the proliferation of capillary ECs in culture [Schweigerer et øt., 1987]. However, TNF-ct has been shown to be chemotactic for capillary ECs in aitro and to stimulate the formation of tube like structures [Leibovich et nl., L9871.
7.3.4.3 Cy t okine Pr o iluction by En d o theli øl C ells
Cytokines play an important role in the recruitment of leukocytes from circulation to sites of tissue damage or inflammation. Leukocytes are a major source of cytokines which may act upon ECs to promote migration, proliferation and activation [Pober and Cotran, 1990a; Cavendet, 19911. However, ECs themselves are also capable of producing soluble inflammatory mediators which are able to act in both autocrine and paracrine functions.
Interleukin 1 (IL-1) is a pleiotropic cytokine which not only activates ECs
[Carlos and Har1an,1994f ,but is also produced by ECs [Libby et a1.,1986; Miossec et al., 1986; Kurt fones et ø1.,1987; Miossec et ø1., 1988]. The production of endothelial ll--1cr and B is induced by LPS, TNF and IL-L itself [Warner and Libby,
7989; Maier et ø1.,1990). EC-derived IL-L stimulates the binding of T lymphocytes to ECs, is chemotactic for T cells and stimulates fibroblast proliferation [Miossec ef at. ,1988]. Furthermore, it has been reported that hypoxic ECs release IL-L cr which acts in an autocrine manner resulting in the induction of E-selectin and upregulation of ICAM-1 [Shreeniwas et ø1., 1992]. Enhanced expression of these CAMs enabled increased leukocyte adhesion, which was partially blocked by the 40 use of mAbs to E-selectin,ICAM-L or IL-l-u. Endothelial lL-L production was also suppressed by specific antisense oligonucleotides, resulting in decreased leukocyte adhesion.
Various cell types, including ECs, smooth muscle cells and monocytes, produce high levels of IL-6 following exposure to IL-L [Aarden et ø1.,1987; Sironi et a1.,19891 .IL-6 acts upon T and B cells [Aarden et ø1.,1987] and stimulates the production of acute phase proteins by the liver [GauIdie et ø1.,1987].
ECs produce a number of haematopoietic growth factors including granulocyte (G)-, granulocyte-macrophage (GM)- and monocyte (M)- colony stimulating factors (CSFs) which are induced by stimulation with a variety of mediators including IL-L, LPS or TNF [reviewed by Mantovani and Dejana, 19891.
The production of CSFs, as well as IL-L and IL-6 which have CSF activity, suggest ECs may participate in the regulation of haematopoiesis. In addition to synthesising CSFs, human ECs also possess receptors for GM-CSF and G-CSF
[Bussolino et ø1,, L989]. Studies in aitro and in r¡kto have also shown that the primary effect of GM-CSF and G-CSF on ECs is the stimulation of angiogenesis
[Bussolino et ø1., 1991,]. Furthermore, while GM-CSF does not modulate the expression of VCAM-1, E-selectin or MHC class II antigens on ECs [Bussolino ef ø1., 19971, it does enhance both the expression of MHC class I antigens lLeszczynski and Hayry,1990] and leukocyte adhesion, presumably through
ICAM-I [Bussolino et ø1., t991'].
Activation of ECs byIL-1,, TNF-cr and LPS results in the slmthesis of several chemotactic cytokines, including IL-8, GRO and monocyte chemotactic protein
(MCP) [Sica eú al., 1990a; Sica et aL, 1990b]. While IL-8 and GRO are potent chemoattractants for neutrophils and lymphocytes [Oppenheirn et ø1.,199L), tl,:re effect of IL-8 on neutrophil-endothelial cell interactions is less clear. It has been reported that IL-8 induces upregulation of B2-integrins, thereby increasing neutrophil adhesion to normal ECs [Carveth et ø1.,1989; Huber et aL, \991], 41, whereas EC-derived IL-8 reduces neutrophil adhesion to stimulated ECs
expressing E-selectin [Gimbron e et ø1., 19891.
In addition to inflammatory mediators, the monocyte chemoattractant MCP is induced in ECs and smooth muscle cells by minimally modified low density lipoprotein [Cushing et a\.,1990]. Human renal mesangial cells also
produce MCP lZoja et ø1.,199t1. Another active signalling molecule produced by activated ECs is PAF, which has been reported to activate granulocytes and induce the upregulation of LFA-L and Mac-L [Zimmerman et ø1., 1990; Lorant et
at.,19911. PAF is absent from resting ECs, but is rapidly expressed on the cell surface within minutes following stimulation by agonists including thrombin,
histamine and leukotriene C4 [Prescott et a1.,1984; Zimmermanet ø1.,1990].It has
been proposed that localisation of PAF at the EC surface enhances "juxtacrine" activation of granulocytes tethered to ECs at sites of inflammation by P-selectin
[Lorant et al., t991,]. PAF is rapidly degraded by ECs, thereby limiting the duration of the activation signal [Rot, 1992].
7.3.4.4 The RoIe of Endotheliøl Cells in Antigen Presentøtion
The stimulus for an allo-immune response is the recognition of foreign antigens on the transplanted tissue by the recipients immune system. In a vascularised allograft, the endothelium forms a large body of foreign cells that represent the first point of contact between the host's immune system and antigenic molecules. Recognition of foreign antigen in the context of MHC class II
antigen on the surface of antigen presenting cells (APCs) results in the activation
of CD4+ T tymphocytes, the first step in initiating an effective immune resPonse
[Unanue and Allen, 1987].
MHC class II antigens are constitutively expressed by many cell types
including macrophages, B cells, Langerhans cells and dendritic cells [Unanue and
Allen, 19871. As described earlier, ECs are also capable of expressing MHC class II 42 antigens, with many small vessels displaying basaI, low levels of class II molecules [Nagura and Ohtani, 1992].
Unstimulated cultured ECs express MHC class I constitutively in aitro,but not class II antigens. However, they become positive for class II after stimulation
with IFNI [Pober et ø1.,1983a; Pober et ø1.,1983b; Collins et a\.,1984; Geppert and Lipsky, 19851, medium conditioned by activated T lymphocytes [Pober et al., 1983b; Wagner et ø1., 1985\ and phytohaemagglutinin (PHA) [Pober and
Gimbrone ,19821. Maximum surface expression of class II is evident 4-6 days after exposure to IFN-y, and can be detected several weeks after the removal of the
cytokine lCollins et ø1., t984| IFN-1 also induces MHC class II expression on dermal fibroblasts and smooth muscle cells [Pober et ø1., L983b; Collins et ø1.,1984; Geppert and Lipsky, 1985l. Expression of class II is regulated by the activation of
protein kinase C [Mattila and Renkonen, 1991-].
Evidence for the ability of ECs to present antigen comes from bolh in rsitro
and in aiao studies. Experiments in aitro have shown ECs to function as APCs to
sensitised T cells [Hirschberget ø1.,1980; Wagner et ø1.,L984; Wagner et a\.,1985] and augment mitogen-induced T lymphocyte proliferation [Ashida et ø1.,l98t;
Hashimoto et ø1.,1989; Wilcox et ø1.,1939]. Furthermore, cultured ECs were able to
stimulate allogeneic T cells [Hirschberg et al., 1975; Pober et øL, 1983a; Geppert and Lipsky, L985; Savage et ø1., 19931, while antibodies to HLA-DR or CD4
blocked this response [Savage et ø1,, L993]. Fibroblasts and smooth muscle cells were unable to support a primary immune response, despite the induction of
MHC class II antigens by IFN-y [Pober et ø1., 1983a; Pober et nl., 1983b; Geppert and Lipsky, L985]. Fibroblasts have however, have been shown to process and
present antigen effectively in the presence of monocytes or fixed ECs [Geppert and Lipsky,1987; Hughes et ø1., 1990b1, or by the addition of exogenous IL-2
[Umetsu et a\.,1986]. 43
In contrast, ECs dramatically augment the production of IL-2 by T lymphocytes in a contact-dependant mechanism [Guinan et ø1., 1989; Hughes eú
ø1., 1990b; Adams et a\.,19921. The involvement of CD2 on the T cells and LFA-3 on ECs in this process was implicated by the partial inhibition (50-70%) of the IL-2 response by mAbs to CD2 and LFA-3 [Adams et a\.,1992].In addition, mAbs against these accessory molecules also inhibited the primary allogeneic resPonse between CD4+ T cells and IFN-T activated ECs, as did mAbs to CDL8 and CD44
[Savage et ø1.,1993]. Interestingly, anti-CDlgd (VLA-4 cr chain) and anti-CD29 (þ chain) mAbs were not inhibitory and enhanced the proliferative response. This suggests VLA-4 delivers costimulatory signals, consistent with earlier reports of its involvement in T cell activation [Bednarczyk and Mclntyre, 1990; Campanero et al., 1990; Nojima et ø1., 1990; Yamada et ø1., 1991). Recent studies indicate that VLA-4 costimulation may involve tyrosine-phosphorylation of proteins [Nojima et ø1.,1992;Sato et ø1.,19951.
The evidence presented above demonstrates co-culture of ECs with sensitised or mitogen-stimulated T cells, or allogeneic PBLs, induces a primary immune response involving the production of IFN-y by the lymphocytes and subsequently class II expression on ECs. However, it has been reported that purified allogeneic CD4+ T cells do not proliferate in response to unstimulated class II negative ECs [Savage et ø1.,1993], but rather produce an inhibitor of class
II [Doukas and Pober, t990).In the same study however, Doukas and Pober [1990] demonstrated that class II antigens could be induced on resting ECs by CD8 T cells, an observation also reported by Adams et ø1.119921.
These observations led to the hypothesis that unstimulated ECs generate a CD8 T cell primary response, independent of CD4 T cells, resulting in the activation of ECs and the subsequent initiation of a CD4 T cell proliferative response [Epperson and Pober,1994]. Experiments by these researchers indicated that it was primarily the CD4SRO+ (memory) CD8 T lymphocytes that responded to the ECs by producing IL-Z and IFN-y. The requirement lor IL-2 to drive the 44 proliferate response, presumably in an autocrine/paracrine mechanism, was demonstrated by the abrogation of the primary response in the Presence of anti-
IL-2Rcr mAb. Furthermore, anti-CD2 and anti LFA-3 mAbs were shown to inhibit the proliferation response, consistent with the earlier report concerning the involvement of CD2 and LFA-3 in IL-2 production [Adams et ø1., 1992]. In addition, anti-MHC class I mAb, but not class II mAb, blocked proliferation of
CD8 T cells by resting ECs, whereas class II mAb, but not class I mAb, blocked the response of CD4 T cells to IFN-¡treated HUVECs [Adams et aL.,1994; Epperson and Pober,1994l.
Adams et ø1. ft994] have proposed that activation of allogeneic human T cells by ECs can occur through at least three pathways: (L) resting ECs directly stimulate CD8+ T cells, but not CD4+ T lymphocytes, (2) IFN-ltreated ECs can directly activate CD4+ T cells, and (3) indirect activation of CD4+ T cells by presentation of EC alloantigens on autologous monocytes. The studies with autologous monocytes suggested that these cells function to recruit CD4+ T cells, but not CD8+ T cells, onto the immune response, resulting in T cell proliferation and cytokine production.
Epperson and Pober 179941suggest that induction of class II antigen on ECs in aitro by CD8+ T cells, followed by activation of CD4+ T cells may explain the sequential infiltration of CD8+ T cells followed by CD4+ T cells observed in human cardiac allograft rejection [Duquesnoy et a\.,19871 and virally-induced delayed-type hypersensitivity (DTH) response in mice [Moskophidis et a1.,t990].
The observation that the CD4SRA+ (naive) subpopulation of CD8+ T cells did not respond as well as the CD4SRO+ (memory) subpopulation to ECs may reflect both the lower signal threshold required to activate memory cells and the differences in recirculation pathways used by naive and memory cells [Mackay ef øL, 1990; Mackay, 1991.; Mackay et ø1., 1992b; Butcher and Picker, 1996] (see below). While ECs are considered as "semi-professional" APCs and therefore may 45 be able to activate circulating memory cells, naive cells preferentially recirculate through lymph nodes where they will encounter professional APCs, such as dendritic cells, which are fully capable of activating naive cells. Furthermore, mAbs against CD28 (important in the activation of T cells by dendritic cells,
activated monocytes and activated B cells [Linsley and Ledbetter, \993)) failed to inhibit the proliferative response of allogeneic CD8+ T cells to ECs [Epperson and
Pober,19941.
L.3.5 Endothelial Cells and Allograft Reiection
The role of ECs as functional APCs in oiao came from the study of allograft
rejection. Sensitisation to a non-vascularised allograft aPPears to occur following the presentation of donor antigen to host T cells by APCs in the draining lymphoid tissue. In contrast, rapid rejection of kidneys in an ovine model of renal transplantation in which renal lymph is drained from the body, demonstrates that
sensitisation to a vascularised allograft occurs within the graft itself [Pedersen and
Morris, 1970; Grooby et al., L992bl, although migration of dendritic cells from allografts to the spleen may also be a critical event in graft rejection [Larsen et al.,
19901. The prolongation of allograft survival in transplanted organs depleted of "passenger leukocytes" [Lafferty et al., 1975; Lechler and Batchelor, 1982] suggested that ECs did not play a major role in antigen presentation in aiao. However, subsequent studies involving the removal of class II expressing leukocytes from rat and human allografts showed ECs to be capable of initiating
allograft rejection [Ferry et ø1.,1987; Stegall et a\.,1990]. Moreover, the detection of notable microvascular abnormalities during hyperacute, acute and chronic rejection is indicative of the significant involvement of ECs in allograft rejection
[Sedmak and Orosz, 1991]. 46
7.3.5.L Hyper øcute Rej ection
The hyperacute rejection reaction occurs within the first few hours after implantation of the transplanted organ. The endothelium of an allograft represents the primary target in hyperacute rejection where preformed antibodies in the recipient bind to antigens expressed on the donor endothelium, resulting in destruction of the endothelial layer. Antibody targets expressed by the endothelium include the ABO lPaul et ø1.,1981] and HLA [Kissmeyer-Nielsen ¿f al., 1,9681 blood group systems. Two less well characterised polymorphic alloantigen systems, involving endothelial-monocyte (EM) [Moraes and Stastny,
19771and endothelial specific [Jordan et ø1., L988; Miltenburg et ø1.,19891antigens, have been described. Although these antigen systems are thought to play only a minor role in clinical transplantation, they are possibly the basis of hyperacute rejection in HLA and ABO compatible individuals [Cerillí et ø1., 1985; Yard et aI., 1ee3l.
Destruction of the endothelium in hyperacute rejection is the result of complement activation by the EC-antibody complexes and aggregation of neutrophils and platelets, and subsequent activation of the coagulation system.
The endothelium may play an active role in these steps by the rapid expression of
CAMs, such as P-selectin and E-selectin, following stimulation with thrombin.
L.3.5.2 Acute Rej ection
Acute rejection reactions generally occur between 6-90 days after transplantation and are mediated by two mechanisms. Mononuclear cells mediate one mechanism which characterised by progressive graft dysfunction and cellular infiltration. In the case of renal allografts, destruction of peritubular capillaries and venules is an early feature of acute cellular rejection [Bishop et ø1.,1989] . The ECs initially develop a characteristic plump, hypertropiod appearance and separate from each other [Pedersen and Morris, 1970]. The major site of 47 extravasation of cells into a renal allograft is the peritubular capillaries [Bender ef a\.,1989; Renkonen et ø1.,19891.
The other form of acute rejection is mediated by a humoral response. Antibodies newly produced by the recipient result in endovasculitis involving vascular occlusion, fibrin deposition, interstitial haemorrhages and tubular
necrosls.
By functioning as APCs, ECs may actively participate in the development
of acute rejection reactions. As described earlier, ECs are capable of activating T cells and the subsequent production of cytokines by these lymphocytes can induce expression of MHC antigens and CAMs on the surface of the ECs, which
enhance lymphocyte activation, adhesion and infiltration.
1..3.5.3 Chronic Rei ection
Chronic allograft rejection may not appear for months or years after engraftment. It remains the most important cause of graft failure, with progressive functional deterioration associated with vascular obliteration and
fibrosis [Sedmak and Orosz, 1991.; Azuma and Tilney, 1994]. Although the molecular mechanism of chronic rejection is unknown, the changes are thought to
be due to chronic immunologic inflammation, directed toward the endothelium and involving the participation of cytokines, growth factors and eicosanoids
[Sedmak and Orosz, t991,; Azuma and Tilney, 1994].In renal transplantation, chronic rejection is associated with clinical symptoms of hypertension, proteinuria and declining function [Hayry et al., 1993]. Morphological features include glomerulopathy, arteriopathy, tubular atrophy and interstitial fibrosis [Hayry eú
a\.,19931.
Thus in allograft rejection the endothelium can serve multiple roles by
acting as a stimulator, amplifier and target of allo-immune responses [Sedmak 48 and Oros z, 19911. While the role of leukocytes in chronic allograft rejection remains to be defined, leukocyte-endothelial cell interactions are essential to the development of inflammatory reactions, such as acute and hyperacute rejection. Similar processes are also involved in normal leukocyte recirculation and these
mechanisms are examined in the next section.
1..4 LEUKOCYTE-ENDOTHELIAL CELL INTERACTIONS
7.4.1 Differentiøl Migrøtion of Naiae ønd Memory T Cells
The provision of immunological surveillance by lymphocytes requires extensive movement throughout the body, involving emigration into tissue
followed by a return to circulation, via the lymphatics. The extravasation of T cells
occurs through three basic pathways, with naive and memory cells using distinct
trafficking patterns [Mackay, 1991.; Butcher and Picker,7996; Mackay et a\.,1996]. The two pathways principally used by memory T cells are (L) through flat endothelium of normal tissue, and (2) through endothelium of inflamed tissue, while the third pathway (3), through HEV within a lymph node, is primarily used by naive T ceIls. Consequently, the majority of T cells found in nonlymphoid
tissues such as skin [Foster et ø1., 1990f, gut lamina propria and lung epithelial surfaces [Saltini et al., 1990] are of the memory phenotype. In addition to migrating across the flattened endothelium of non-flamed tissue, T cells also cross through the inflamed endothelium and represent the predominant cell type
within sites of inflammation [Pitzalis et a\.,1988; Damle and Doyle, L990]. In contrast, naive T cells constitute the vast majority of cells within the efferent lymph, having left the blood via HEVs in lymph nodes [Mackay et al., 1990;
Butcher and Picker,L996;Mackay et ø1.,19961.
The different migratory routes of naive and memory T cells is attributed to the differential expression of cell adhesion molecules. Memory T cells express 49 higher levels of cell adhesion molecules such as LFA-1, VLA-A, CD2 and LFA-3 compared to naive cells [Shimizu et nl., 1990c; Springer, 1994]. In addition, memory cells displayed tissue-specific homing pathways based on the pattern of
expression of the adhesion molecules [Mackay et a\.,7992b], and the type of tissue in which they initially encountered antigen [Mackay et al., t992a; Picker and
Butcher, 1992; Butcher and Picket,1996l.
Flowever, an alternative model has recently been proposed to explain the
tissue-specific migration of lymphocytes, based on the functional differentiation
of T cells into Th1 or Th2 subtypes following activation [Meeusen et ø1.,1996]. The
authors argue that preferential migration of T cells to peripheral lymphoid tissue or mucosal tissue is associated with the ThL or Th2 phenotypes, rather than the
expression of organ-specific homing receptors. Moreover, Meeusen et ø1. propose that the microenvironment of the peripheral or mucosal lymphoid tissues may induce the differentiation of the cells into functionally distinct ThL and Th2 subpopulations.
The authors discuss the evidence for the production of Th1 and Th2-type
cytokines in the mucosal and peripheral lymph nodes, respectively. In addition,
the presence of the steroid hormones dehydroepiandrosterone (DHEA) and 1,25- dihydroxyvitamin D3 (1,25(OH)ZDg) in the peripheral and mucosal lymph nodes, respectively, also influences the localisation of the ThL and Th2 lymphocyte
subsets.
Furthermore, these same microenvironmental factors may also induce the
vascular endothelium in the lymphoid tissue to express receptors complementary to those induced on the ThL and Th2 subpopulations. Thus irrespective of their origins, the ThL and Th2 cells would preferentially migrate back to sites that favour the appropriate ThL or Th2-inducing microenvironments. 50
1.4.2 Lymphocyte-Endothelial Cell Adhesion øt HEVs
Since the description by Stamper and Woodruff lL976l of their elegant technique for studying the binding of viable leukocytes to HEVs found within frozen tissue sections, many groups have applied this methodology to the study of leukocyte-endothelial interactions.
Similar to the "adhesion cascade" model thought to be involved in leukocyte adhesion and migration at sites of inflammation [Butcher, 1991';
Shimizu et ø1., 1992; Springer, 19941, (discussed below), a multistep process of lymphocyte emigration at lymph node HEVs has been described [Picker, 1994; Springer, 1994; Girard and Springer, 19951. The four steps involved are (1) lymphocyte attachment and rolling, (2) adhesion triggering, (3) sticking and arrest and (4) transendothelial migration.
The involvement of L-selectin in the initial lymphocyte attachment and rolling on peripheral lymph node HEVs was identified by the use of anti-L- selectin antibodies [Gallatin et ø1., L983; Tedder et ø1., I990a; Tedder et a\.,19931 and in studies of lymphocyte migration in L-selectin deficient mice [Arbones et al.,
19941. This is particularly the case for homing of unactivated/naíve T cells [Mackay et ø1., 1990; Bradley et nI., 1994]. These observations undoubtedly demonstrate an important role for L-selectin in controlling lymphocyte binding to
HEV and subsequent migration into lymph nodes. As described earlier L-selectin binds to it's HEV ligands, MAdCAM-1, GIyCAM-L and CD34 [Lasky et ø1.,1992;
Baumheuter et a1.,1993;Berget a\.,1993; Baumhueter et ø1,,19941.
The specific mechanism regulating the second step of adhesion triggering remains unclear. However it is thought that G-protein coupled receptors in the plasma membrane of leukocytes mediate the activation of leukocyte adhesion to
HEVs [Springer, 1994]. Activation of lymphocytes (and neutrophils) results in the shedding of L-selectin, a mechanism which may be important in allowing leukocytes to migrate through the endothelium [Iung et al., L988; Kishimoto et ø1., 5L
1989a1. Furthermore, activation of leukocytes has been reported to result in a rapid and transient increase in L-selectin activity prior to it being shed [Sperttni et aI.,t991a].
Following activation, firm attachment (step 3) of the lymphocyte occurs through two integrins crLB2 and u4þ7. The role for uLþ2 was demonstrated by inhibition of lymphocyte migration through PLN HEV by 80% and through
Peyer's patch HEV by 50% using monoclonal antibodies against cLþz [Hamann ef aL,\994j. Similarly, monoclonal antibodies againsto.4þ7, or it's ligand MAdCAM-
1, showed inhibition of up to 75% of lymphocyte migration in Peyer's patch HEV
[Hamann et ø1., 7994]. Evidence also exists for ECM proteins to play a role in lymphocyte adhesion and migration at HEVs. Immunohistochemical studies have identified the presence of laminin and fibronectin on both the basal and luminal surfaces of HEVs [van den Berg et ø1., L993]. Furthermore, antibodies against laminin have been shown to limit lymphocyte migration into PLNs in aiao
[Kupiec Weglinski and De Sousa, l99tl1, while anti-fibronectin or anti-c5B1 (fibronectin receptor) antibodies blocked adhesion to HEVs in frozen sections of
human lymph nodes [Szekanecz et a1.,1992].
It has been suggested that CD3L may play an important role in the final step, that of transendothelial migration lMuller et al., 1993]. This proposal is supported by the observations that anti-CD3L mAbs blocked transendothelial migration of monocytes and neutrophils, without affecting the firm adhesion of leukocytes to endothelium [Muller et ø1., 1993; Muller, 1995], and inhibited
neutrophil migration into inflamed peritoneum in uiao [Bogen et aL.,1994].
1,.4.3 L euko cy t e -En d o theli øl C ell Int er acti o n s D uring lnfl ømm øti o n
While the early concepts of leukocyte-endothelial adhesion were based upon the interaction of a single adhesion molecule and its ligand, it is now clear
this process is much more complex. 52
The triggering of an immune response decreases the specificity that regulates normal trafficking and facilitates the influx of T cells into the inflamed tissue. The principle change that occurs at sites of inflammation is an increase in endothelial cell adhesion molecule expression involving upregulation of ICAM-1 and de noao exptession of VCAM-1-, E-selectin and P-selectin [Carlos and Harlan,
1994). In the case of leukocytes, rapid modulation of integrin avidity from a non- adhesive, low affinity state to a transient high affinity state is an important adhesion mechanism [Hynes, 1992).
As described earlier, the presence of different inflammatory mediators, either individually or in various combinations, can influence the pattern of expression of adhesion molecules by ECs. This is further influenced by the heterogeneity of cell adhesion molecule expression by ECs from different vascular beds. In addition, the differences in the kinetics of expression of endothelial adhesion molecules may play a major role in defining the types of leukocytes recruited into sites of inflammation (Figure L.4).
P-selectin is the first activation CAM to be expressed on the endothelial cell surface. It appears within minutes of EC activation by stimuli including cytokines, LPS, thrombin and histamine, and returns to baseline within 20-30 minutes [Hattori et al., 1989]. However, subsequent studies have reported P-selectin expression persisting for between 4 and 24 hours following EC stimulation with
LPS [Sand ers et al., 1992] or phorbol esters [Vadas et ø1., L992] .
While E-selectin, ICAM-I and VCAM-I- are also induced by many of the mediators of P-selectin induction (i.e., TNF-c, IL-l, LPS, thrombin) [Carlos and
Harlan, 1994], their expression on the EC surface occurs many hours later. ln aitro, maximum E-selectin expression occurs 4 hours after stimulation and returns to basal studies levels within 24 hours [Bevilacqua et ø1., L987]. However studies in aiao have demonstrated the presence of E-selectin on ECs 24 hours after cytokine- activation [Munro et al., 19891. Maximum ICAM-1 and VCAM-1- expression on 53
ECs occurs approximately 8-1.2 hours after stimulation and remain elevated for at
IeastT2hours [Dustin et a\.,1986; Osborn et ø1.,1989].
tcAM-1 c P-selectin E-selectin .o at, U, o) VCAM-1 o-x LrJ o ¿ oC) o tcAM-2 g .9 U' Eo E
24 0 4 I 12 16 20 Time (hr)
Eigure 7.4 Temporal expression profiles of endothelial cell adhesion molecules induced by inflammatory mediators. Adapted from Westphal and de Waal 119921.
Whilst the adhesion of lymphocytes to unstimulated ECs occurs primarily through the interaction of LFA-L/ ICAM-1 [Haskard et ø1., 1986; Arnaout et ø1.,
1988; Haskard et ø1.,L999;Oppenheimer-Marks et ø1.,1990; Oppenheimer-Marks ef
ø1. , L99L; Shimizu et al. , 199'J,a; Meerschaert and Furie, 1995], other inducible cell surface proteins such as VCAM-I and E-selectin have been shown to provide alternative pathways of adhesion and transmigration for leukocytes, both in aitro
and in aiao lBevilacqua et al., 1987; Bevilacqua et ø1., \989; Haskard et ø1., 1989;
Osborn et ø1. , 1989; Carlos et al. , L990a; Munro et al., 1991'; Oppenheimer-Marks ef
ø1., L99'J.; Shimizu et ø1., t991.a; Meerschaert and Furie, 1994; Meerschaert and
Furie, L9951. 54
7.4.4 Adhesion Cøscøde
The evidence described above demonstrates that multiple CAMs are involved in leukocyte-endothelial cell adhesion and a simple model of one receptor-one ligand is inadequate. The inhibition of neutrophil rolling and subsequent firm adhesion to post capillary venules by anti-L-selectin and anti- CD18 mAbs, respectively, led to the proposal of a two-step model of adhesion, involving selectins and integrins [von Andrian et ø1., 1991]. This model, initially formulated for neutrophils, has since been modified to include three (or more) steps to account for the process of extravasation by all leukocytes and is known as the "adhesion cascade" [Butcher,l99L; Shimizu et ø1.,1992).
The first step, rolling, involves low-strength, activation-independent, reversible adhesion of leukocyte to endothelium via selectin-mediated interactions. The second step involves the activation of leukocytes to upregulate integrin function which mediate the third step, firm adhesion to the endothelium, followed by extravasation. Two further refinements have been proposed for the three-step model [Springer, 1994; Mackay, 1995]. The first involves segregation of the selectin-dependent step into two components, namely the initial tenuous tethering of leukocytes to the endothelium and subsequent rolling. The second modification suggests that rather than the adhesion cascade representing a series of distinct sequential steps, the process actually involves considerable overlap at each of the stages (Figure 1.5).
1-.4.4.1 Step 1-- Tetheringl Rolling
Much of the initial work identifying the role of selectins in the first step of extravasation involved studies of neutrophil-endothelial cell interactions. Studies in aioo using anti-CDL8 mAbs demonstrated inhibition of leukocyte extravasation at sites of inflammation while having no effect on neutrophil rolling [Arfors et ø1.,
1e871. 55
Select lns Chemoattractants + <- lntegrins
Eood vessel lumen
\\ + + \ dot helium
ECM
la. Tethering lb. Rolling 2. Activation 3. Firm Adhesion 4. Transendothelial migration
Eigure 1.5 The multistep "adhesion cascade" model of leukocyte-endothelial cell adhesion and transendothelial migration.
Furthermore, the observation that L-selectin and Mac-l are inversely regulated irn activated neutrophils suggested that L-selectin plays a role in the initial adhesion events under physiological sheer stress conditions [Jutila et al.,
L989; Kishimoto et a\.,1989a1.
Subsequent studies using anti-L-selectin monoclonal and polyclonal antibodies in uitro and in aiuo demonstrated a dramatic reduction in the number of neutrophils rolling on stimulated endothelium [Ley et al., 799t; Smith et al.,
1991; von Andrian et a\.,79911. Using intravital video microscop/, von Andrian eú aI. [1991) further showed that while anti-CD18 mAbs inhibited firm adhesion of leukocytes, it did not effect neutrophil rolling. 56
The contribution of P-selectin in leukocyte rolling and tethering has been examined in elegant studies in aitro using a modified parallel flow chamber with phosphotipid bilayers containing purified P-selectin and ICAM-I [Lawrence and
Springer, 19911. Under physiological flow conditions, neutrophils rolled on the bilayers in the presence of P-selectin, but did not attach to layers containing ICAM-1 only. Moreover, the addition of a chemoattractant resulted in neutrophil arrest, spreading and firm adhesion to the bilayer" Conversely, in the absence of ICAM-I, and despite the presence of a chemoattractant, activated neutrophils did not become arrested. In addition, studies with P-selectin-deficient mice revealed the absence of leukocyte rolling in mesenteric venules [Mayadas et aL.,1993]. This work demonstrates the co-operative nature of CAM families and chemoattractant molecules in leukocyte extravasation.
It has been suggested that the increased numbers of intravascular leukocytes in patients with LAD II (leukocyte adhesion deficiency type II) syndrome may be due to a marked decrease in the ability of these leukocytes to roll [Etzioni, 1994]. This condition is characterised by an absence of sl-ex carbohydrates on leukocytes.
The observation that anti-E-selectin mAbs completely inhibit neutrophil rolling on E-selectin transfectants [Abbassi et nl., 1993] supports in oitro studies demonstrating the involvement of E-selectin in neutrophil rolling and adhesion
[Lawrence and Springer,7993). In addition, neutrophil rolling on E-selectin was significantly inhibited by anti-L-selectin mAb [Abbassi et ø1.,1993), indicating that L-selectin may partially mediate E-selectin dependant adhesion. Furthermore, neutrophils were able to roll on E-selectin, in the absence of L-selectin, but required L-selectin for tethering under flow conditions [Lawrence et al., 1994].
These observations support the suggestion by Springer 11994) that the initial rolling step actually consists of two processes, tethering and rolling. 57
E-selectin and P-selectin have also been shown to mediate T lymphocyte tethering and rolling under physiological flow conditions [Alon et ø1.,1994b]. While VLA- /VCAM-1 interactions are generally associated with firm adhesion (step 3), a recent study in aitro demonstrated that both peripheral blood lymphocytes and VLA-4 transfectants were capable of tethering and rolling on TNF-stimulated endothelium and in a flow chamber containing purified VCAM-1
[Alon et a\.,1995].
Finally, the fast reaction rate of carbohydrate-lectin interactions, characterised by the selectins, provides these molecules with the ability to mediate rapid on-off adhesion necessary to initiate leukocyte rolling along endothelium lWilliam s, 1991].
L.4.4.2 Step 2- Actia ationlTriggering
Selectin mediated adhesion is transient and reversible in the absence of an appropriate chemotactic signal. The role of chemoattractants in mediating leukocyte activation and firm adhesion was elegantly demonstrated by the in uitro flow chamber studies of Lawrence and Springer 119941, described above.
At least 12 different leukocyte chemoattractants have been identified including the family of a-chemokines (e.g., IL-8, ENA-78, gro), the B-chemokines
(e.g., MIP-1u,p, RANTES, MCP-1.) and the classical chemoattractants (e.g., FMLP,
CSa, PAF), each of which act on diverse cell populations [Springer, 1994; del Pozo et ø1., 1995). The mechanism of the chemokine-mediated integrin activation involves binding to transmembrane, rhodopsin-like receptors which signal through coupled trimeric G proteins, as well as via second-messenger-generating enzymes, for instance adenylate cyclase [Simon et al., 1991; Wu et ø1., 7993; Baggiolini,l994l. Chemokines also participate in the induction of lymphocyte uropods and the redistribution of adhesion molecules such as ICAM-1, ICAM-3, CD43 and CD44 into these structures [del Pozo et ø1., 19951. This process is 58 thought to be important for lymphocyte motility, adhesion and recruitment of additional cells [del Pozo et ø1.,1995].
The critical role of chemoattractants and G-proteins in signal transduction and leukocyte extravasation was demonstrated by the observation that pertussis toxin-treated lymphocytes were unable to migrate into PLNs and PPs, although the cells were still capable of rolling along PP HEVs in oiao [Spangrude et ø1.,1984;
Bargatze and Butcher,1993l. Pertussis toxin irreversibly inactivates the G protein a-chain [Simon et ø1.,1991.;Wuet n\.,1993; del Pozo et ø1.,L995).
Chemokines possess a heparin-binding domain which enables these factors to be immobilised on endothelial surface proteoglycans, such as CD44, lor presentation to circulating leukocytes [Tanaka et al., 1993; Webb et ø1., 19931. Macrophage inflammatory protein-1a (MIP-1cr) has been shown to bind to CD44 on ECs and trigger the VlA-4-dependent adhesion of T-cells to VCAM-L [Tanaka et ø1., 19931. Similarly, TNF-a has been reported to bind to fibronectin and augment the Bl-integrin-mediated adhesion of CD4+ T cells to fibronectin [Alon et al.,t994al.
Monocyte chemoattractant protein-L (MCP-1) has also recently been shown to function as a chemoattractant for lymphocytes [Carr et al., 1996]. This study demonstrated that MCP-1 activation of both VLA-4 and VLA-S resulted in increased adhesion to fibronectin, but not to purified ICAM-1 or VCAM-1. The authors suggest that MCP-1 (and RANTES and MIP-LÞ) may be more important in the process of lymphocyte transmigration (step 4, discussed below), rather than the initiation of integrin-mediated firm adhesion.
The interaction of leukocytes with CAMs expressed on the surface ECs may also function to activate rolling cells. Studies of CD31, expressed by all endothelia, monocytes, neutrophils and some T cells, demonstrated that ligation of the molecule on T cells resulted in signal transduction and activation of B1 integrins [Tanaka et a1.,1992]. Furthermore, activation of T cells by adhesion to co- 59 immobilised T cell antigen receptor and ICAM-1, ICAM-2 or VCAM-1 has been
demonstrated [van Seventer et ø1., 1990; Burkly et aI., L991'; Damle and Aruffo, 1991,; van Seventer et ø1.,I99La; Damle et ø1.,1992a; Damle et al.,L992bl. In addition, mAb blocking of CDLS, the E- and P-selectin ligand, resulted in
neutrophil activation and upregulation of B2 integrins, LFA-1 and Mac-l- [Forsyth
et ø1.,L999;Lund-Johansen ef a\.,1992; Stockl et ø1.,19931.
A number of additional mechanisms regulating integrin binding affinity
have been described. Cabanas and Hogg 119931demonstrated the need for LFA-1 interaction with ICAM-I and prior to stimulation by an agonist in order to achieve full activation of LFA-1, a mechanism referred to as the "ligand induced binding" (LIB) interaction. A second method of integrin regulation involves the production of an integrin modulating factor-L (IMF-l) by PMNs which may
function in an autocrine or paracrine fashion [Hermanowski-Vosatka et aL.,1992]. Addition of IMF-1 to lymphocytes resulted in modulation of LFA-I binding activity, while addition to resting PMNs increased the ability of the PMNs to bind
C3bi-coated erythrocytes.
1,.4.4.3 Step 3- Stuong Adhesion to Endothelium
Integrins, which are inactive on resting cells but rapidly activated by various stimuli, undoubtedly mediate the third step in the adhesion cascade.
The contribution of CD18 integrins in providing strong adhesion to endothelium and subsequent extravasation, was highlighted in patients with LAD type I syndrome [Harlan, 1993; Etzioni, 1994]. LAD-1, an inheritable
immunodeficiency disease, is due to mutations in the CDL8 B subunit which
blocks expression of all three CD18 integrins [Kishimoto et a\.,1987; Etzioni, 1994]. This disease is characterised by the absence of stable adhesion between leukocytes and endothelium, coupled with inability of neutrophils from LAD patients to
undergo transendothelial migration lSmith et a\.,1988; Smith et øL,1989]. 60
In addition, while neutrophils did not adhere to ICAM-L contained in lipid bilayers under physiological shear stress conditions, static incubation of stimulated neutrophils resulted in adhesion to ICAM-I that was 100-fold more
resistant to shear stress than adhesion to P-selectin [Lawrence and Springer,l99L]. Besides LFA-I/ICAM-I and Mac-1/ICAM-l, other pathways mediating strong
leukocyte-endothelial adhesion are VLA- /VCAM-1, LFA-L /ICAll4-z
[Oppenheimer-Marks et ø1., t990; Oppenheimer-Marks et ø1., t991'; Shimizu et ø1.,
1991,a1, while a4þ7 /MAdCAM-1 contribute in adhesion to Peyer's patch HEVs
[Nakache et ø1.,1989; Strauch et n\.,I994;Mackay et a1.,1996].
The role of soluble CAMs in regulating leukocyte adhesion remains
unclear. Release or shedding of molecules such as L- and P-selectin may serve to limit an inflammatory response and provide a protective mechanism that prevents the interaction of activated neutrophils, which inadvertently find themselves back in circulation. Such an anti-inflammatory role has been suggested for P-selectin following the observation that a soluble form of the
molecule prevented TNF-o stimulated PMN adhesion to ECs [Gamble et ø1.,L9901,
1..4.4.4 Step -Leuko cyte Tr ønsmigr øtion
The process of transmigration involves the movement of adherent
leukocytes across the luminal endothelial surface, followed by passage between
endothelial cell junctions and then through the basement membrane.
While rapid modulation of integrin avidity from low to high-avidity state enables leukocytes to adhere firmly at sites of inflammation, it is equally important for cells to "reverse" integrin avidity, from high to low, to enable
migration over surfaces [Kuijperc et ø1.,1993]. Shedding of L-selectin by activated
leukocytes may also facilitate diapedesis [Jung et ø1,,1988]. 61.
A spatial gradient of high-affinity receptors at the leading edge and inactivated low affinity receptors at the trailing edge, is thought to generate a treadmill like movement of adhesion receptors responsible for leukocyte migration [Dustin and Sprin ger, \99L].
studies with mAbs directed against LFA-1, Mac-1, ICAM-1, VLA-4 and
CD44 have shown these CAMs to be involved in leukocyte transmigration in response to a chemotactic gradient [Furie et ø1., ].99; Smith et ø1.,7988; Smith et ø1., |9}9;Chuluyan et ø1.,1995a; Chuluyan et ø1.,1995b; Issekutz et ø1.,1995; Issekutz,
19951. Ex aiao monoclonal antibody blocking studies on frozen tissue sectíons from rat cardiac allografts showed anti-VLA- and anti-LFA-L mAbs inhibited the binding of lymphocytes to capillary endothelium [Turunen et al., 1992]. Thre involvement of VLA-4 in cell motility was demonstrated in aitro by the ability of a VlA-4-transfected Chinese hamster ovary (CHO) cell line to undergo cell adhesion, spreading and motility on a fibronectin substrate [Wu et a\.,t995].
In addition, VLA-4 may be actively involved in lymphocyte transmigration
[Hourfüan et ø1.,1993]. Triggering of VLA-4 by VCAM-1 induces the expression of a 72 kDa gelatinase on the surface of T cells that can digest collagen, possibly facilitating lymphocyte migration into perivascular tissue [Romanic and Madri,
1994). Anti-CD31 mAb inhibit monocyte and neutrophil migration into inflamed peritoneum [Muller et a1.,7993; Bogen et ø1.,1994; Muller, I995J.
7,4.5 "Trøffic signøls" or "Areø codes"
As mentioned earlier, many of the initial studies on CAMs were based on the concepts of a single receptor/ligand pair with expression of specific molecules by particular leukocyte subsets controlling migration pathways in an organ- specific manner. Flowever, as the number of CAMs and ligands identified rapidly increased it quickly became apparent that this simple model could not account for the observed specificity in leukocyte-endothelial interactions, in particular the 62 often selective recruitment of a leukocyte subtype in inflammatory or immune reactions.
There are several factors which may contribute to this selectively, including the heterogeneity of CAM expression by ECs in different vascular beds and the differences in the kinetics of expression of these molecules, as discussed earlier. The alternative splicing of many CAMs and the differential glycosylation of selectins, depending upon cell type, all provide for different combinations of CAMs.
Development of the three-step model has been extremely important in understanding the process of leukocyte extravasation as it has shown the requirement for cooperation of different molecules at the various steps in the cascade [Butcher, 199L; Shimizu et al., 1992). Springer 11994] has extended the concept by suggesting that the selectivity of leukocyte emigration is best explained by an "area code" or "traffic signal" model in which each of the three steps identify a code that facilitates extravasation at a particular site. This multi- step model allows combinations of different adhesion molecules, chemokines, and their receptors which generate extensive diversity and specificity in leukocyte- endothelial interactions.
For example, following antigen challenge there is an accumulation of eosinophils in the lungs of asthmatic patients [Bousquet et a\.,1990]. While part of this specificity may be explained by the presence of VLA-4 on eosinophils and not neutrophils [Bochner et nl., L991.; Dobrina et a1.,199I], the expression of VLA-4 by all other leukocytes [Hemler et ø1., 1,990] cannot account for the selective recruitment of eosinophils. Furthermore, a recent study of L-selectin-mediated adhesion of eosinophils and neutrophils to activated ECs (under flow conditions) indicates that different functional epitopes on L-selectin may be used by these two leukocyte populations [Knol et a1.,1994]. 63
The ability of ECs to vary their array of signals provides further understanding for the co-ordinated and sequential migration of leukocytes observed during immune responses, such as allograft rejection [Duquesnoy et al., 1987) and DTH reactions [Moskophidis eú ø1., 1990]. Furthermore, although proposed as a model for leukocyte emigration, it may be possible to extend the area code model to explain how activated cells are selectively retained within the tissue [Picker, 1994].
Finally, the presence of circulating or soluble forms of CAMs (sCAMs) has increased the level of complexity with regard to leukocyte-endothelial cell interactions. As discussed earlier, although soluble forms of selectins are found in human plasma the functional significance of these sCAMs remains unclear. Shedding of L-selectin may facilitate firm leukocyte adhesion at sites of inflammation. However, all soluble selectin molecules are biologically active and may possibly serve to regulate leukocyte adhesion and modulate inflammatory reactions by competing in cell-cell adhesion [Kishimoto et a\.,1989a; Gamble et ø1. , ree}l.
Similarly, functionally active forms of soluble ICAM-L and VCAM-L
[Rothlein et nl., 1991; Gearing et al,, 1992; Pigott et al., 1992] have been reported, but again their specific role is not clear. Several groups have investigated the clinical significance of changes in sCAM levels in sera of patients with respect to the diagnosis and therapeutic monitoring of a range of disease states. The results from these studies are discussed in the following section. 64
1.5 ADHESION MOLECULES IN DISEASE
The inappropriate localisation of leukocytes at specific sites is a central factor in the development of a range of immune diseases and inflammatory disorders. There is a vast wealth of literature reporting the expression of CAMs in a variety of disease states, both in humans and in experimental animal models, some of which has been summarised in Table 1.2.
CAMs have subsequently become the target of extensive investigation with respect to development of therapeutic agents designed to block both leukocyte- endothelial interactions and the progression of inflammatory resPonses.
L.5.L Anti-adhesion Therapy: Potential Targets and Strategies
The increasing understanding of the molecular basis of leukocyte adhesion and extravasation has expanded the number of potential targets and strategies designed to regulate the adhesion cascade.
These approaches include (L) competitive binding of endothelial-leukocyte adhesion molecules by monoclonal antibodies or soluble receptor antagonists
(e.g., peptides, oligosaccharides), (2) inhibition of signalling pathways involved in integrin modulation or induction of endothelial cell adhesion molecules, (3) prevention of activation dependent upregulation of adhesion molecule surface expression by blocking translocation of receptor from cytoplasm to cell surface
(e.g., P-selectin, Mac-L) and (4) use of antisense oligonucleotides to interfere with gene expression of CAMs.
1..5.7.1- Mono clonøl Antib o dy Ther øpy
The application of mAb therapy with respect to the control of cell-cell adhesion and signal transduction may be grouped into four general clinical 65
Tøbte 7.2 Involvement of cell adhesion molecules in human disease.
Disease ICAM-1 VCAM-1 E-selectin
Kidney Renal allograft rejection Yes Yes Yes Glomerulonephritis Yes Yes Heart Cardiac allograft rejection Yes Yes Yes Atherosclerotic Yes Yes
Liver Liver allograft rejection Yes Yes No Viral hepatitis Yes Yes Yes Alcoholic hepatitis Yes Yes Weak Cholangitis Yes
Bone Rheumatoid arthritis Yes Yes Yes
Thyroid Grave's disease Yes No No Hashimoto's thyroiditis Yes
GI Tract Inflammatory bowel disease No Yes
Eye Uveitis Yes Yes, no Herpes keratitis Yes
Skin Psoriasis Yes Yes Yes Scleroderma Yes Yes Graft vs host disease Yes Contact dermatitis Yes Yes Yes Lichen planus Yes Mycosis fungoides Yes Alopecia areata Yes Yes Yes
Central Nervous System Multiple sclerosis Yes Yes
Cancer metastasis melanoma Yes (shed) Yes osteosarcoma Yes carclnoma Yes adapted from Bennett and Crooke ll994l 66 settings: (1) immunosuPPression (e.g., organ transplantation, autoimmune diseases), (2) infectious diseases (bacterial and viral), (3) cancer (diagnostic and cytotoxic mAbs) and (4) detoxification (e.g., poisoning by animal or bacterial toxins, ischaemia-reperfusion syndromes) [Back. et ø1.,1993]. As the focus of this thesis concerns the role played by CAMs in organ transplantation, particular attention will be paid to the first category with regard to the efficacy of mAbs in prolonging allograft survival.
The use of mAbs as therapeutic agents has largely stemmed from the production of mAbs for the purposes of identification and characterisation of cell surface molecules. The anti-CD3 mAb, OKT3, is one such example where the mAb demonstrated significant immunosuppressive effects on T-cell function lø
zrlfro [Reinherz et ø1.,1980], and was subsequently evaluated in clinical trials soon
after [Cosimi et a\.,1981]. The immunosuppressive properties of OKT3, its role in transplantation, and the problems associated with murine mAb therapy have been studied extensively [reviewed in Burlingham, L992] and will not be considered in anymore detail here. More relevant to the work presented in the
thesis are the studies describing the effects of mAbs targeting the B I and B2 integrins, the Ig family and the selectins, both in animal models and human
clinical settings [reviewed in Soulillou, 1994].
Monoclonal antibodies against LFA-L (CD11a) have been used with
varying success in human and animal organ transplantation settings. The mAbs
have been shown to prolong survival of human bone marrow allografts [Fischer eú
ø1., L986; Fischer et ø1.,1991], heterotopic cardiac allografts in rabbits (S'adfåiro43Ì
[Sadahiro et a\.,1993] and allogeneic tumours in mice [Heagy et al.,1984]. An anti- LFA-I mAb has also been used successfully as prophylaxis in human renal
allograft recipients [Hourmant et ø1.,1994; Le Mauff et ø1,,1995].In addition, anti- LFA-L or CDLS mAbs have been shown to protect against reperfusion injury
[Horgan et ø1.,1990; Vedder et ø1., L990;Ma et a\.,1991; Lefer et a\.,1993;Tamiya et al., L995f , uveitis [Rosenbaum and Boney, 1993), invasion and metastasis of 67 lymphomas [Harning et a\.,t993], allergic airway responses [Rabb et a\.,1994]and be partially effective in the treatment of lupus [connolly et ø1,,1994].
In contrast, others have reported the failure of such mAbs to prevent
rejection in humans of renal [Le Mauff et a\.,1991] and bone marrow transplants
[Baume et ø1., 1989], nor prevent the development of adjuvant arthritis in a rat model [van dellangerijt et ø1.,1994; Issekutz and Issekutz,1995). Furthermore/ an anti-LFA-L mAb was unable to prolong renal allograft survival in a sheep
transplantation model [Grooby et al., 1992a], while a combination of anti-LFA-L and anti-ICAM-1 mAbs not only failed to prolong rat liver allografts, but resulted
in accelerated graft loss [Omura et aL,1992; Morikawa et ø1.,\994].
Flowever, studies of anti-ICAM-l mAbs in allograft transplantation have proved very encouraging. Cosimi et nl.11990] demonstrated that a 12 day course of anti-ICAM-L mAb prolonged renal allograft survival in a cynomolgus monkey model. Therapeutic administration of the mAb also reversed acute allograft rejection episodes in monkeys receiving suboptimal doses of cyclosporine A. Subsequent studies with this mAb in a Phase L clinical trial showed it to be beneficial in patients who received renal allografts which were at high risk of delayed graft function (either due to prolonged preservation time or a highly- sensitised recipient) [Haug et al., 1,993]. Using a murine heterotopic transplantation model, Guymer and Mandel ll992l demonstrated prolonged survival of corneal, pancreas and skin grafts by an anti-ICAM-L mAb. Similar to LFA-L mAbs, anti-ICAM-L mAb have been effective in blocking experimental
uveitis lUchio et ø1., 1994] and ameliorating reperfusion injury fi|i4a et ø1.,1992; Rabb et ø1., t995; Tamiya et ø1., 19951. The latter result is supported by the observations that IcAM-l-deficient mice are protected from ischaemic injury to
the kidney [Kelly et a1.,1996i.
Consistent with the in aitro studies described earlier demonstrating the involvement of multiple adhesion pathways in leukocyte-endothelial adhesion, 68 combinations of mAbs have also proved therapeutically efficacious. In particular,
Isobe et ø1. [1992] observed that although mAbs to ICAM-L or LFA-1 alone resulted in significant prolongation of murine heterotopic cardiac allografts, a combination of the two mAbs led to long-term specific tolerance of the allografts. Other groups have shown combined LFA-1 and ICAM-L mAbs to be effective treatment against murine corneal allograft rejection lIJe et ø1., t9941, rat nerve allograft rejection [Nakao et a\.,1995], glomerulonephritis [Nishikawa et øL,1993; Kawasaki et al.,1995l,murine acute graft-versus-host disease [Harning et ø1.,t991] and reperfusion injury to the lung [Horgan et ø1., 1990]. Combinations of LFA-1 and VLA-4 mAbs have proved effective in prolonging survival of rat cardiac allografts [Paul et ø1., 1,9931, rat islets [Yang et nl., 1995], blocking leukocyte migration into the inflamed peritoneum of rabbits [Winn and Harlan,1993) and adjuvant arthritic joints of rats [Issekutz and Issekutz,l995l and inhibiting DTH- induced inflammation in rats [Issekutz,1993].
Furthermore, there is recent evidence that specific targeting of the VLA- 4/VCAM-L pathway alone may be efficacious. Antibodies against VLA-4 have been reported to inhibit contact hypersensitivity in mice [Chisholm et al., 1993], experimental autoimmune encephalitis in mice [Yednock et øL, 1992], inhibit insulitis and prevent diabetes in NOD mice [Tsukamoto et al., 19951, decrease allergic airway responses in rats [Rabb et ø1.,1994] and prolonged murine cardiac allograft survival [Isobe et ø1,,1994b].
In contrast, anti-VLA-4 mAbs have exhibited little or no efficacy in the treatment of cardiac allografts in rats [Paul et ø1.,19931 or rabbits [Sadahiro et ø1. ,
19931, nor were they able to block the migration of leukocytes in a rat model of adjuvant arthritis [Issekutz and Issekutz, L995].
Studies with anti-VCAM-l mAbs have shown these reagents to be capable of preventing acute graft-versus-host disease [Schlegel et al., 1,995] and cardiac allograftrejection[Orosz etal.,L993; Pelletier etø1., 1993a; Isobe etal.,1994a]in 69 mice. Furthermore, combined anti-VLA-4 and anti-VCAM-L mAb treatment resulted in both long term cardiac graft survival and acceptance of donor-type skin grafts in murine cardiac allograft recipients [Isobe et ø1,, 1994b], and inhibition of insulitis and prevention of diabetes in NOD mice [Tsukamoto et ø1.,
1ee5l.
Inhibition of selectin function has been studied with regard to ischaemia- reperfusion injury. Anti-P-selectin mAbs have been shown to attenuate reperfusion injury to the rabbit ear following ischaemia [Winn and Harlan, !9931, as have anti-L-selectin mAb in the rabbit ear [Mihelcic et ø1., 1994] and cat myocardium [Ma et ø1.,1993]. An anti-E-selectin mAb has been reported to inhibit damage to vascular endothelium in a rat model of adult respiratory distress syndrome [Mulligan et ø1.,t99t].
Studies using mAbs directed against other surface molecules include the reduction of rejection episodes in human renal allograft recipients [Brewer et al.,
1989] and the prevention of renal allograft rejection and reversal of acute rejection in mice by anti-CD45 mAb [Lazarovits et al., 1996), prolongation of murine cardiac allografts by anti-CD2 and anti-CD3 mAbs, either alone or in combination
[Chavin et nl., 1993a; Chavin et ø1., 1993bJ and long term survival of murine cardiac and skin allografts following blockade of CD40 and CD28 pathways
[Larsen et a\.,1996].
Finally, the application of mAbs as "magic bullets" for the delivery of drugs to specific sites has been a long term goal [Davies, 198'],; Brodsky, 1988; Clark,
19921. Recent studies examining this mechanism demonstrated that an anti- ICAM-I mAb coupled to a ricin A-chain was cytotoxic to human myeloma cell lines [Huang et al., 19931 and that ICAM-L mAbs could be incorporated into liposomes which function as drug carriers [Bloemen et a\.,1995].
The examples of mAb therapy described above represent only some of the studies reported in this area, with particular reference in those involved in organ 70
transplantation. Further discussion on the role of anti-adhesion therapy, including summaries of animal disease models inhibited by anti-CAM mAbs, has been presented in several recent reviews
93) [Waldmann and Cobb old, t993; Carlos and Harlan, 1994; Talbott et ø1., 1994;
Elices, 19951.
Flowever, despite the success of mAb therapy in a variety of disease states,
there are still a number of obstacles to overcome. The most serious disadvantage of rodent mAb therapy is the development of an immune resPonse against the mAb by recipients. For instance, patients treated with murine anti-CD3 mAb all develop human anti-mouse antibodies (HAMA) after the second week of
treatment [Chatenoud et ø1., L982]. HAMA can result in accelerated clearance of mAb, thereby reducing its therapeutic effect and pose a risk of anaphylactic shock
in response to subsequent treatment with the mAb.
In order to overcome this problem humanised antibodies are being engineered which are reported to retain functional characteristics, possess
improved pharmacokinetics and reduced immunogenicity [Sims et ø1., 1993;
Winter and Harris,1993; Poul ef a1.,19951.
Other problems involve the "cytokine release syndrome" associated with
the injection of mAb [Cosimi et ø1., I98l; Chatenoud et ø1., 1991] and the need to establish a balance between the immunosuppressive benefits of anti-adhesion therapy, while not causing undue risk to the patient through inhibition of normal
immune functions [Bach et ø1.,1993),
1..5.L.2 S oluble Receptor Antøgonists
Other approaches to blocking leukocyte adhesion to endothelium have involved soluble receptor antagonists targeted toward selectin and integrin- dependent adhesion mechanisms. A slex-oligosaccharide was shown to reduce 71, lung permeability and neutrophil accumulation following cobra venom factor- induced [Mulligan et ø1.,1993b] or IgG-induced [Mulligan et al.,t993a] lung injury in the rat and to reduce feline myocardium reperfusion injury [Buerke et ø1.,I994J. A cyclic RDG pentapeptide has been reported to block MNC adhesion to both cultured ECs and CS-1-coated plates, by interfering with oc4p1 and cr5Bl interactions [Nowlin et al. ,7993). Similarly, a cyclic peptide has been reported to inhibit the adhesion of a4Bl-expressing cells to immobilised VCAM-1' lWang et al., 19951. An L-selectin-Ig chimera has been reported to reduce neutrophil migration into inflamed peritoneum [Watson eú nl., 199t]. Inositol polyanions, carbon ring structures with ester-linked phosphate or sulphate groups, also inhibit adhesion of cells to immobilised L- and P-selectin, but not E-selectin lCecconi et ø1,,1994].
7.5.1..3 Antisense Oligonucleotide Ther øpy
Another therapeutic strategy for preventing leukocyte-endothelial cell interactions is the use of antisense oligonucleotides, short synthetic sequences (L0-
25 bases in tength) designed to hybridise to the mRNA which codes for a specific protein [Wagner, 1994].
The effectiveness of the original generation of oligonucleotides was limited by their rapid degradation by serum and cellular nucleases, due to their phosphodiester backbones [Shaw et ø1., 1991]. Modified oligonucleotides containing phosphorothioate backbones offer greater resistance to nucleases.
These molecules have been demonstrated to reduce the expression of ICAM-I
[Chiang et ø1.,1991.; Bennett et ø1.,1994; Stepkowski et ø1.,1994], VCAM-1 and E- selectin [Bennett et ø1., t994] by TNF-a-stimulated ECs. ICAM-I antisense oligonucleotides were also shown to be effective in prolonging murine cardiac allografts [Stepkowski et ø1.,1994]. Moreover, a combination of ICAM-1 antisense oligonucleotides and anti-LFA-l mAb treatment resulted in both long term 72 cardiac graft survival and acceptance of donor-type skin grafts in murine cardiac
allograft recipients [Stepkowskí et a\.,L994]. Liposomal delivery systems have also been used in several studies to enhance cellular uptake and biological activity of
oligonucleotides [Bennett et al., 1992].
1..5.7.4 lnhibition of Surføce Expression of Cell Adhesion Molecules
An alternative strategy involves the use of anti-inflammatory compounds which block the expression of CAMs at the cell surface. One such group of agents, the leumedins, consist of a group of N-(fluorenyl-9-methoxycarbonyl) amino acids which display anti-inflammatory activity in aioo and in aitro lBurch et al., Ieell.
Although the mechanism of action has yet to be fully defined, one possible pathway may involve the inhibition of upregulation of surface expression of Mac- 1 (CD11b /CD1,8), with subsequent inhibition of neutrophil adhesion to
endothelium [Bator et al,, L992]. In addition, leumedins inhibit PAF-mediated leukocyte adhesion in aiao lMcCaffefty et ø1., 1993] and the LDl-induced production of MCP-L mRNA and protein by human aortic ECs [Navab et ø1,, 1ee3l.
Leumedins have been shown to reduce inflammatory tissue damage in models of adjuvant arthritis in rats and rabbits and the Arthus reaction in rats
[Burch et al., 1991] and inhibit leukocyte migration in models of sepsis in rats
[Noronha-Blob et a\.,1993] and mice [Noronha-BLob et a\.,1991; Burch et ø1.,19931. In addition, leumedins inhibited the accumulation of neutrophils in a guinea pig-
to-rat heterotopic cardiac xenotransplant model lZehr et al.,I994l and in a porcine
model of ischaemia-reperfusion injury [Curtis et al.,1993].
Thus, several different approaches (i.e., mAb, oligosaccharides, antisense
oligonucleotides) are actively being pursued to develop new therapeutic reagents. 73
As described above, some of these substances have demonstrated significant efficacy in both animal models and clinical settings, and in aitro. However, disruption of the adhesion cascade may interfere with normal leukocyte-mediated defence, as seen in patients suffering from the LAD syndromes, and it will therefore be necessary to establish a balance between the immuno-suPPressive of anti-adhesion therapy, while not causing undue risk to the patient [Bach et ø1.,
1ee3l.
Finally, reports of increased levels of circulating CAMs in patients suffering from various diseases [Gearing et ø1.,1992; Mason et ø1, ,1993; Wellicome et nl., 1993; John et ø1., 19941 suggests these molecules may be useful in the diagnosis and therapeutic monitoring of the disease states.
However, there is conflicting evidence and opinion as to the significance of these results. The levels of sICAM-1 and sVCAM-L have been reported to increase by 3-6 fold during renal allograft rejection, with the rise in sVCAM-L occurring in parallel with, or slightly earlier than, the increase in serum creatinine levels
[Gearing and Newman,1993].In contrast, although John et ø1. 119941 observed elevated sICAM-L levels in renal allograft recipients compared to normal controls, they did not observe any correlation between levels of sICAM-L and serum creatinine. In addition, sICAM-L levels did not decrease following drug induced clinical remission, prompting the authors to suggest that the measurement of sCAMs may not be clinically informative of disease activity.
The reasons for these differences may be associated with the use of different ELISA kits and consequently differences in antibody specificity and standards. Similarly, differences in timing of the initial sample collection may account for some of the variations in observations [John et a\.,1994]. Finally, very littte is known about the mechanism of clearance or the half-life of sCAMs, especially in patients with abnormal kidney function or under immunosuppressive therapy. 74
1..6 AIMS OF THE THESIS
The literature reviewed in this chapter clearly demonstrates that the vascular endothelium and CAMs are involved in both normal haemostatic and immunological functions. In recent years there has been considerable progress made towards understanding the role played by CAMs in leukocyte-endothelial cell interactions and in the pathogenesis of many inflammatory diseases.
In particular, the elucidation of the molecular basis of leukocyte adhesion has led to the evolution of the current paradigm of leukocyte-endothelial cell interaction, the "adhesion cascade". Subsequent refinements to the model, including the "area code" or "traffic signal" concept, are helping to explain preferential migration pathways of leukocyte populations both in normal and inflamed tissue. The increased understanding of the co-operation between adhesion molecules and chemoattractants will also assist in the development of new therapeutic reagents capable of regulating leukocyte-endothelial adhesion.
Many of these new reagents have been shown to function effectively by blocking adhesion in aitro and in aiao, using small animal (often rodent) models.
While such in aiao models are invaluable in the development and characterisation of new therapeutic reagents, it raises questions concerning the relevance of such models to human clinical settings, and the need to test these reagents in larger, clinically relevant animal models in order to confirm the initial observations.
In summary, this thesis examines the development of an ovine renal transplantation model and the isolation and characterisation of both ShUVECs and sheep specific mAbs. The availability of these tools enabled the simultaneous study of ECs and CAMs in oitro and in aiao.In particular, the ovine model was used to investigate the therapeutic potential of anti-LFA-1 and anti-VCAM-l mAbs to prolong allograft survival in this clinically relevant,large animal model. 75
Ch"pter 2
Materials and Methods
2.1. Materials
2.1-.1 Monoclonal øntibodies
25-32: anti-ovineCD44 monoclonal antibody (mAb) (murine IgGL); The hybridoma cell line was a generous gift from Dr. M. Brandon, Centre for Animal
Biotechnology, University of Melbourne, Parkville, Australia.
72-87: anti-bovine LFA-L mAb (murine IgGl) which cross-reacts with ovine
LFA-I. The hybridoma cell line was also a gift from Dr. M Brandon.
HAE2-1,: anti-human VCAM-L mAb, murine IgGl; a kind gift from Dr. T.
Tedder, Dukes University Medical Center, Durham, NC, USA. Previously shown to cross-react with ovine VCAM-I.[Mackay et a1.,1992b1.
HAEIa: anti-human E-selectin mAb, murine IgGl; also a kind gift from Dr.
T. Tedder, Dukes University Medical Center, Durham, NC, USA.
P-selectin: polyclonal antibody; a gift from Dr. M. Berndt, Baker Medical Research Institute, Prahran, Australia. 76
QE.2E5: murine IgGZb mAb; recognises CD29, the Þt integrin subunit. The mAb was produced in the host laboratory.
KM5.L1.2: murine IgG2a mAb directed against human HLA class II. The hybridoma cell line was obtained from Dr. A. d'Apice, St. Vincent's Hospital,
Fítzroy, Australia.
SBU-I: (clone 41,-19) murine IgGl mAb recognising ovine MHC Class I.
This mAb and the following panel of anti-ovine mAbs were generous gifts from
Dr. M. Brandon, Centre for Animal Biotechnology, University of Melbourne,
Parkville, Australia.
SBU-II: (clone 28-1) murine IgGl mAb recognising ovine MHC Class II.
SBU-TL: (clone 25-91) murine IgGl mAb recognising ovine CDs.
SBU-T : (clone 44-38) murine IgG2a mAb recognising ovine CD4
SBU-78: (clone 38-65) murine IgG2a mAb recognising ovine CD8
TIB-191,: murine IgGl mAb raised against TNP-KLH (trinitrophenyl-
Keyhole Limpet Hemocyanin) that does not bind to ovine or human tissues was used as negative control. The cell line was obtained from the American Type
Culture Collection (ATCC), USA.
W6/gZ: murine IgG2amAb directed against human HLA class I. The hybridoma cell line was obtained from the ATCC.
aWF antibody: rabblt anti-human, purchased from DAKO (Dako A082),
Carpinteria, USA 77
2.1.2 Cytokines ønd cDNAtemplates
Recombinant humøn (rh)-IL-Lþ purchased from Genzyme Diagnostics, USA.
Re c o mb in nnt humøn -T N F - a p urcha se d f rom Sigma, USA
Re c o mb in nnt humøn -/FN- 7 p urchase d from Genente ch Inc, USA
Recombinønt oaine (ro)-TNF-a andro-IL-lBwerc a generous gift from Dr. A.
Nash, Centre for Animal Biotechnology, University of Melbourne, Parkville, Australia.
Humøn-ICAM-l cDNA template was a kind gift from Dr. A. Boyd, WEHI,
Melbourne, Australia.
Human-VCAM-L cDNA template was generously supplied by Dr. Y.
Takada, Scripps Research Institute, La Jolla, CA, USA.
Conditioned Medium (CM), used for the stimulation of ShUVECs, was obtained by treating sheep MNCs (7 x106 /ml RPMI + L0% FCS) with Con A (10 pg/ml) for 24hr. The MNCs were removed from the culture by centrifugation at
25009 for 10 min and 10% (v/v) CM was added to the VEC culture medium.
ShUVECs were stimulated with this combined CM-VEC medium for periods of overnight up to 3 days.
2.7.3 Cell lines
IMR-90, a human fibroblast cell line was obtained from the ATCC.
Sheep fibroblasts were obtained from sheep nuchal ligament explants
SP2/0-Ag14, a murine myeloma cell line was obtained from the ATCC.
ELAM-I-CHO cell transfectanú and non-transfected CHO cells; a gift from
Dr.J. Gamble, Hanson Centre for Cancer Research, Adelaide, Australia. 78
VCAM-I,-CHO cell trønsfectønt,was also a gift from Dr.l.Gamble.
WEHI-ICAM-I L-cell transfectant and non-transfected L-cells were a generous gift from Dr. A. Boyd, WEHI, Melbourne, Australia.
2.7.4 Buffers ønd culture mediø
2.1.4.L Ce\| culture bffirs
Hank's buffer:5.6mM D-glucose, 5.4mM KCl, 0.4mM KHZPO4, 1.37mM
NaCl, 0.3mM NaZHPO4,4.2mM NaHCO3
RPMI 1.640: supplemented with 50 units/ml of penicillin/streptomycin,
100mM sodium pyruvate, 0.2"/" sodium bicarbonate, 10mM HEPES and 2mM L- glutamine. pH was adjusted to7.3 by bubbling through COZ.
Trønsfectønt ceII line culture medium: RPMI-1640 supplemented with L0%
FCS and 400 pg/ml Geneticin (G418).
VEC culture medium: RPMI-L640 additionally supplemented with 20'/.FCS,
20 ¡tg/ml endothelial cell growth factor (ECGF) and 30 Units/ml porcine mucous heparin sodium.
2.L. 4.2 Immunopr ecip it øtion ønd S D S -P AGE buffer s
Coomassie blue støining solution:0.259 Coomassie Brilliant Blue R250 , 45'/o ethanol and9"/o Glacial acetic acid.
Destain Solution: Staining solution without Coomassie Blue.
High buffer PBS:132mM NaCl, 16mM Na2FIPO4, 4mM KHZPO¿
Lysis Bffir1/' Triton X-100 in PBS ¡
SAC Btffir: PBS plus 0.5% Triton X-100, SmM KI, 0.02% sodium azide 79
SDS PAGE Løemmli Sample Bffir: 62rr.MTrízrna Base, 10% glycerol,0.2o/o
SDS, + 50mM dithiothreitol, pH 6.8
Stncking gel bffir:O.sM Trizma base, 0.4'/. SDS, dHZO. Adjust to pH6.8 with LM HCl. Make up to L00 ml and store at 4C
Resolaing gelbffir:1.5M Trizmabase, 0.4% SDS, dHZO. Adjust to pH8.8 with 1M HCl. Make up to L00 ml and store al4C
Running buffr, (L0X stock): 25mM Trizma base, 190mM glycine, 0.1% SDS
Do not titrate. Store at RT
Støphlycoccus øureus (Pønsorbin) for use in immunoprecipitøtions: A 1- gm quantity S. øureus (pansorbin) was reconsituted and centrifuged at 17,0009 for L0 min at 4'C. The pellet was resuspended in cold SAC buffer and washed twice as above. Finaiiy the S. øureus pellet was resuspended in a volume of SAC buffer equal to the original reconstitution volume. The final addition of SAC buffer may be supplemented with 1 mg/ml ovalbumin. The cell suspension was then stored in 200 pl aliquots at -70'C. Each aliquot of S. aureøs without the ovalbumin supplement was centrifuged at 13,5009 for 60 sec, and the supernatant removed before use. Aliquots of S. aureus supplemented with ovalbumin were used without any further manipulation.
2.1..4.3 Bøcteriological culture mediø, øntibiotics ønd buffers
LB (Luriø Bertøni) broth:Sg yeast extract, 10g tryptone , 109 sodium chloride,
1L water, pH7.4
LB agør:1..59 agar (Oxoid, UK) per 100 ml of LB broth.
Ampicillin: 35 mg/ml solution in water
XGAL (5-Bromo-4-Chloro-3-Indolyl-B-D-Galactopyranoside) : 20 mglml in dimethyl formamide. 80
IPT G (Isopropyl- B-D-thiolalactopyranoside) z 24 mg / rnl in water.
Tetracycline:35 rng/ ml in ethanol/water (50"/' v /v)
Miniprep Sol. 1:50mM glucose, 25mM Tris-HCl pH 8.0, L0mM EDTA.
Miniprep Sol.2:0.2M NaOH, 1% SDS
Miniprep Sol. 3:3M potassium acetate,2M glacial acetic acid pH 4.8
TE buffer: L0mM Tris-HCl, LmM EDTA, pH 8.0
Buffer H (for Restriction Enzyme digestions): 50mM Tris-HCl, 100mM NaCl,
LOmM MgCI2, LmM dithioerythritol, pF{7.5
2.1.44 RNA prepørøtion bffirs
Chloroform: AII chloroform for RNA isolation was prepared as a 49:1 solution with isoamyl alcohol. All chlorofom for DNA isolation was prepared as a
24:1 solution with isoamyl alcohol.
Diethylpyrocørbonøte (DEPC)-treøted water: Redistilled water was stirred with 0.L% v/v DEPC for 30 min at room temperature, then autoclaved.
Stock denaturing solution: 4M guanidine thiocyanate salt, 25mM sodium citrate ptJ7.0, 0.5% n-lauroylsarcosine, dissolve at 65'C.
Solution D:72¡t"I of B-mercapthoethanol added to L0 ml of stock denaturing solution.
Phenol (water-eqrtilibrøted) for RN,4 extrøction'. Hydroxyquinoline was added to colour the phenol yellow, prevent oxidation and inhibit RNases. The phenol was equilibrated by adding an equal volume of sterile distilled water, and centrifuging at 8509 for 5 min to separate the two phases. The aqueous phase was 81 removed, and the equilibration repeated. The second volume of water was removed and replaced with fresh water.
2,1.4.5 Northern trønrtr buffers
Hybridisøtionbffir:60% deionised formamide, L x SSPE, 0.5% nonfat milk
powder, 10% dextran sulphate,0.5 mg/ml salmon sPerm DNA, 1% SDS
MOPS buffer:O.2M MOPS, 0.05M Na acetate, 0.01M EDTA
RN,4 loadingbffir:150 pi 10X MOPS, 100 pl glycerol, 240 ¡t'l formaldehyde
solution, 750 ¡tlformamide (deionised), 20 pl bromophenol blue (saturated), L60 pl dH2O. Stored as 1 ml aliquots at -20"C.
SSCbuffer (20X):3M NaCl,0.3M Na3 Citrate.ZH2O, adjust pH to 7.0 with
1M HCI
SSPE brffn (20X):3M NaCl, 0.2M NaHZPO4.?Í12O,0.02M EDTA-Na2,
adjust Io p}{7.4 with 10M NaOH
2.L.4.6 Miscellaneous bffirs
Bicarbonøte bffir pH 9.5 (FITC conjugation):8.69 NaCO3 and 17.2gNaHCOe
to 1L
Diethønolømine buffer:0.25M MgCI2.6H2O, 1.54M NaN3. Add d i-
ethanolamine (97 mi/L) slowly and iast. Adjust pH to 9.8 and store at 4C in the
dark for up to 3 months.
ELISA coøting buffer:1.5M Na2CO3,3.49M NaHCO3, p}{9.6
FACS wøshing buffer: l- x PBS, 0.1% sodium azíde,2% FCS
Phosphøte Buffered Saline (PBS):4mM NaHZPO¿.2H2O,120mM NaCl, 16mM
Na2FIPO4 82
2.1.5 Reøgents ønd supplements for culture media
BM-Condimed HL Boehringer Mannheim, Germany
Collagenase Type II Worthington Biochemical, USA
Endothelial cell growth factor (ECGF) Sigma, USA
Foetal Calf Serum (FCS) CSL, Australia
Geneticin (G418) GibcoBRL, USA Heparin (Porcine mucus) Fisons, NSW, Australia HAT-Media Supplement Boehringer Mannheim, Germany
HT - Media Supplement Boehringer Mannheim, Germany
HEPES Sigma, USA L-glutamine Cytosystems, Australia
Penicillin / Streptomycin Cytosystems, Australia
RPMI-1640 Sigma, USA
Sodium bicarbonate M&8, Australia
Sodium pyruvate ICN Biomedicals, USA
2.L.6 Other chemicøls ønd reøgents
8-Hydroxyquinoline Sigma, USA ABC staining kit Vector Laboratories, USA Acetone Ajax, Australia Ac-LDL (1.1'-dioctadecyl-1-3,3,3'3, Biomedical Technologies, USA -tetramethyl-indocarbocyanine perchlorate; Dil-Ac-LDL)
Acrylamide Ø0%) 37.5:1. Acrylamide : Bis Bio-Rad, USA Agar Oxoid, UK Agarose Progen, Australia Ammonium persulphate Bio-Rad, USA Ammonium sulphate BDH, Australia Ampicillin Boehringer Manlheim,Germany B-mercapthoethanol Sigma, USA B3
Biotin blocking kit Vector Laboratories, USA
Biotinylated horse anti-mouse IgG Vector Laboratories, USA
Bromophenol blue Bio-Rad, USA
Calcium chloride Ajax, Australia Calf intestinal alkaline phosphatase Boehringer Mannheim,Germany
Chloramphenicol Boehringer Mannheim,Germany Chloroform Ajax, Australia
Collagenase Type II Worthington Biochemical, USA
Concanavalin A type IV (Con A) Sigma, USA
Coomassie Brilliant Blue R250 Bio-Rad, USA
D-glucose BDH, Australia
Dextran sulphate T-500 Pharmacia, Sweden
Diaminobenzidine tetrahydrochloride (DAB) Sigma, USA Diethanolamine Ajax, Australia
Diethylpyrocarb onate (DEPC ) Sigma, USA Dimethyl formamide BDH, Australia
Dimethyl sulphoxide (DMSO) Ajax, Australia Disodium hydrogen orthopohosphate Ajax, Australia Dithiothreitol Bio-Rad, USA
EDTA (ethylenediamine tetraacetic acid) Sigma, USA
Eosin Y Sigma, USA
Ethanol BDH, Australia
Ethidium Bromide Sigma, USA
Ethidium monoazide (EMA) Molecular Probes, Inc., USA
FACS Lysing solution Becton Dickinson, USA
FlTC-conjugated sheep anti-mouse Silenus Labs, Australia secondary antibody (DDAF)
Fluorescein isothiocyanate (FITC) Sigma, USA
Fluothane Zeneca, UK
Formaldehyde solution Ajax, Australia 84
Formalin (buffered L0%) Orion, Australia
Formamide BDH, Australia
Frusemide Alphapharm, Australia
Gelatine (porcine skin) Sigma, USA
Gene-Clean Bresatec, Australia
Glacial acetic acid BDH, Australia
Glycerol Ajax, Australia
Glycine Sigma, USA
Guanidine thiocyanate salt Sigma, USA Flarris' modified haematoxylin Sigma, USA Hybond N* membrane Amersham, UK
IPTG (Isopropyl- B -D-thiolalactopyranoside) Boehringer Mannheim,Germany Isoamyl alcohol Sigma, USA
Isopropanol, anhydrous Sigma, USA
Lactoperoxidase Calbiochem, USA
Lipopolysaccharide (LPS) Sigma, USA Lymphoprep Nycomed, Norway
Magnesium chloride Sigma, USA Mannitol Baxter, Australia
Message Maker Kit (MMK-1) Bresatec, Australia
Methanol Ajax, Australia
N-butanol Sigma, USA
N-lauroylsarcosine Sigma, USA
Nembutal Boehringer Ingelheim, Australia
Normal rabbit serum ICN Biomedicals, USA
Normal saline (0.9% sodium chloride) Baxter, Australia
Ovalbumin Sigma, USA
Penstrep (sheep) Troy Labs, Australia
Phenol Progen, Australia
Phorbol myristate acetate (PMA) Sigma, USA 85
Phosphatase substrate tablets (104-105) Sigma, USA
Potyethylene glycol 1500 (PEG) Boehringer Mannheim,Germany
Potassium acetate BDH, Australia
Potassium chloride Ajax, Australia
Potassium dihydrogen orthophosphate Ajax, Australia
Potassium iodide Ajax, Australia
Pristane (2,6,1.0, L 4-tetramethylp enta dec ane) Aldrich Chemical Co., USA
Rabbit anti-mouse IgG DAKO, USA
Rabbit complement Pel Freez, USA
Reaction buffer H Boehringer Mannheim,Germany
Ross kidney perfusion solution Orion Labs, Australia
Salmon sperm DNA Sigma, USA
Serotec MMT RCL isotyping kit Serotec, England
Sheep anti-mouse alkaline phosphatase Sigma, USA
Sodium acetate Ajax, Australia
Sodium carbonate, anhydrous Ajax, Australia
Sodium chloride M&8, Australia
Sodium citrate BDH, Australia
Sodium di-hydrogen orthophosphate .Ãjax, Australia Sodium dodecyl sulphate (SDS) BDH, Australia
Sodium hydrogen carbonate Ajax, Australia
Sodium hydrogen orthophosphate Ajax, Australia
Sph 1 restriction endonuclease New England Biolabs, USA
S t aphly co c cus aur eus (p ansorbin) CalBiochem, USA
T4 DNA ligase Promega, USA TEMED Bio-Rad, USA
Tetracycline Sigma, USA
Tissue Tek II mounting medium Miles Scientific, USA
Triton X-100 Bio-Rad, USA
Trizrna Base (Tris [hydroxymethyl] Sigma, USA 86 aminomethane) Trypsin Sigma, USA Tryptone Oxoid, UK
Tween-20 Bio-Rad, USA
Water for irrigation (pyrogen-free) Baxter, Australia
Yeast extract Oxoid, UK
Xba restriction endonuclease New England Biolabs, USA XGAL (5-Bromo-4-Chloro-3-Indolyl- Diagnostic Chemicals, Canada B-D-Galactopyranoside)
2.7.7 Rødiochemicøls
MethlY-39-thYmidine Amersham, UK
1251ut sodium iodide DuPont, Australia lo(-32p1-UTp Bresatec, Australia
2.2 Methods
All micro-centrifugation steps were conducted in an Eppendorf microfuge or a Phoenix Orbital 100 microfuge. Centrifugation of volumes greater than 2 ml was conducted in a Beckman TJ-6 centrifuge using the TH-4 swing-out rotor unless otherwise stated. All optical density measurements were determined using a Beckman DU650 spectrophotometer.
2.2.7 Cell prepørøtions
The cell lines used in this thesis were cultured in RPMI 1640 medium plus supplements. Foetal bovine serum was added to the above medium at a final concentration of 1.0% (v /v), except for hybridoma lines which used 75o/. and endothelial cells required 20%. Medium for transfected cell lines also contained
Geneticin (400 ¡tg/mt). All cells were cultured at37"Cin1%COz. 87
2.2.1.1. Isoløtion of oaine mononucleør cells (MNC)
Sheep blood was collected into heparinised tubes and centrifuged at 1-40g for L0 min, followed by removal of the platelet rich plasma. The remaining blood was diluted L in 4 ín 0.9% saline and distributed into V-bottomed l-0 ml plastic tubes (Johns Biolab Scientific, Australia). The diluted blood was then underlayed with 2 ml of Lymphoprep and centrifuged at 11009 for 20 min. The mononuclear cells at the Lymphoprep-plasma interface were removed by pipetting into V- bottomed 10 mt plastic tubes, diluted L in 3 in 0.9o/' saline, and pelleted at 13009 for 5 min. The pellet was resuspended in 10 ml of 0.9% saline and the celI numbers determined with a Neubauer haemacytometer.
2.2.1.2 Isolation of sheep umbilical cord aein endotheliøl cells
ShUVECs were obtained from sheep umbilical cord veins essentially by the method of Jaffe Í19731, with some modifications [Grooby et ø1., 7997]. Umbilical cords were obtained from pregnant ewes at the time of slaughter and stored in sterile Hank's buffer at 4C until processed later the same day.
At the time of harvesting, the cords were warmed to 37"C and clamp marks from each end of the cord were removed. The umbilical veins were then cannulated with sheathed 21G winged infusion sets (Terumo, Australia) which were gently clamped in place. The presence of multiple vessels (3 veins and 2 arteries) often allowed simultaneous cannulation of 2 veins. The veins were flushed gently but thoroughly with 40 ml warm sterile Hank's and the free end of the cord clamped. Three ml of warmed 0.1% collagenase Typ" II were injected through the infusion set and the filled cord was incubated at 37"C for L7 min. After incubation the collagenase and detached cells were washed into a tube containing 5 mI FCS (to inactivate the collagenase) by perfusing the veins with 30 ml of warmed sterile Hank's. Cells were pelleted by centrifugation at 5009 lor 7 min and resuspended in 3 ml of VEC culture medium (2.1,.4.1) and placed in a 88
0.5% gelatin-coated 25 cm2 tissue culture flask (Sarstedt, USA). After overnight incubation, any non-adherent cells were washed out with pre-warmed sterile
Hank's and 3 ml of fresh medium added to the adherent ShUVECs. As a general rule, cells from individual cords were cultured separately.
When the monolayers attained confluenc/, the ShUVECs (and HUVECs, below) were passaged up to 75 cm2 tissue culture flasks by brief incubation with either 0.725% Trypsin/O.1% EDTA in sterile Hank's or 3 min incubation with 0.1% EDTA in sterile Hank's. The detached cells were washed with sterile Hank's containing 1% FCS (to inactivate the trypsin), resuspended in fresh VEC medium and placed in new flasks.
2.2.L.3 Isoløtion of human umbilicnl cord aein endotheliøl cells
HUVECs were isolated from umbilical cords obtained from the labour ward of The Queen Elizabeth Hospital. The cords were stored in sterile Hank's buffer at 4"C until use. The HUVECs were harvested essentially as described above for ShUVECs. In the case of human umbilical cords, incubation time with collagenase was 15 min at 37"C. As with ShUVECs, cells from individual cords were cultured separately.
2.2.2 Leukocyte-endotheliøl cell ødhesion øssøy
Leukocyte-endothelial cell adhesion assays were performed essentially as described by Bevilacqua [1987], using an inverted centrifugation assay [Charo ef ø1.,1985). ShUVECs were plated at 2x 104 cells/well onto gelatin-coated 96-well fiat-bottomed microtiter trays (Corning) and grown to confluence. Stimulated
ShUVECs were obtained by addition of CM (see 2.L.2) to the appropriate wells, followed by incubation for L8 hr. The trays were washed twice with RPMI1.640 containingl% FCS before the addition of 1x 105 sheep MNCs/well in 50 pl aliquots and an equal volume of mAb (diluted ascites or neat tissue culture 89 supernatant) were added to the wells (in replicates of 4). In the case of activated MNCs, the cells were incubated with PMA (15 nglml) for 30 min prior to addition to the microtiter plates. The cells were incubated for 60 min at 37"C,5/o
CO2 and the non-adherent MNCs removed by inverted centrifugation (1509 for 5 min). The remaining adherent cells were quantified by scoring the number of adherent MNCs in a random low-power field in each 4 well replicate and expressed as the mean (t SD).
2.2.3 Proliferøtion øssøys
2.2. 3.1- Mixe d lympho cy te-endothelial cell r eøction ( MLER)
MLER assays were performed by the method of Hirschberg et ø1. 119751.
ShUVECs were plated onto gelatin-coated flat-bottomed96 well microtitre plates
(Corning) and sheep MNCs and mAbs were added (in replicates of 6) as described for the adhesion assays. Plates were cultured at 37"C and the wells pulsed on day 4 with 1 pCi of 3H-thymidine. After a further 20 hr of culture, the cells were harvested with a Skatron cell harvester (Skatron, Norway) and the incorporated counts measured in a Beckman LS 2800 beta counter (Beckman,
USA). Control wells which contained ShUVECs or MNCs alone showed that these cells in isolation were minimally proliferative. The results are expressed as an average of the 6 replicates + SD.
2.2.3.2 Mixed lymphocyte culture (MLC)
MNCs from the donor sheep were prepared aseptically and resuspended at a concentration of 2x 106 ce[s/ml in MLC culture medium (RPMI ]-640 medium plus 20'/. heat-inactivated FCS). Aliquots of 50 pl of each of the donor MNCs were mixed together in the wells of a round-bottomed 96 well microtitre plate
(Corning). Dilutions of monoclonal antibody ascites were also added (replicates of
6), and the cells were cultured at 37"C. The wells were each pulsed with 1 pCi of 90
3-H thymidine on day 4 andharvested 20 hr later, as described for the MLER. The results are expressed as an average of the 6 replicates + SD.
2.2.4 Protein cøpping ønd interuølisation øssøys
VCAM-1 capping studies were performed by a modification of the method of Burn [1988]. Either ShUVECs stimulated with CM for L8 hr, or the VCAM-L transfectant CHO cells, were eluted from flasks, washed and resuspended in
PBS /I% FCS. The cell samples were incubated with L00 pl of mAb (1/ 250 dilution of ascites or neat tissue culture supernatants) on ice for 20 min. The cells were again washed, then incubated with rabbit anti-mouse IgG on ice for 20 min, and after a further wash, transferre d to 37"C. Aliquots were removed at various time points, immediately washed in ice cold PBS/\%FCS/0.02 M NaN3 and incubated with QE4G9-FITC in the dark on ice for 30 min. The samples were fixed with FACS Lysing solution for l2min at room temperature. After two washes, the cells were resuspended in 500 pl of filtered saline and stored in the dark at 4'C until analysed on a FACScan.
2.2.5 Metøbolism of Acetyløted-low density lipoprotein (Ac-LDL)
Endothelial cells and fibroblasts were assessed for their ability to metabolise Ac-LDL labelled with 1,L'-dioctadecyl-1-3,3,3',3',-tetramethyl- indocarbocyanine perchlorate (Dil) as described by Voyta et al. 119841. Cells were incubated at 37"C for 4 hr in the presence of 10 pglml Dil-Ac-LDL, then eluted from the flasks by 0.125% Trypsin/0.1% EDTA digestion, washed twice in FACS wash and analysed on a FACScan flow cytometer.
2.2.6 Antib o dy-dep endønt cellulør cytotoxicity
Cytotoxicity assays were performed by a modification of the dye exclusion method of. ZoIa [1987]. EssentialIy,25 ¡rl of Hank's was added to each well of a 91. round bottom 96-well microtiter plate followed by the addition of 25 pl of mAb to the first column (in quadruplicate) and diluted,by doubling dilutions, across the plate. Twenty five microliter aliquots of sheep MNCs at4xtO6/mI in Hank's were then added to each well and incubated at 37"C for 30 min. Rabbit complement (25 pl) was then added to the wells and the plate incubated for a further 30 min at 37"C. Five microliters of filtered 5% aqueous Eosin Y were added to each well and plated incubated for 3 min at room temperature. Aliquots
(25 pl) of 10% buffered formalin (pH7.2) were then added to the wells and the plate read using an inverted phase microscope (Diaphot, Nikon, Japan). Control wells contained mAb but no complement or complement in the absence of mAb.
Reactions were scored as follows : 0=0-19'/' cell death ,2=20-39o/o, 4=40-59o/o, 6=60-
79o/o,8=80-1,00fo.
2.2.7 Production of monoclonøI øntibodies [Kohler, 1981]
2.2.7.1. Animals
Female BaIb / c mice were used for immunisation and production of immune ascites.
2.2.7.2 Immunisøtion
Mice were immunised by intraperitoneal injection of approximately 3 - 5 x
106 stimulated-ShUVECs or WEHI-ICAM-1 transfectant ceils suspended in 0.5 ml of plain RPMI 1640 at 28 and 3 days before the fusion. ShUVECs were stimulated overnight with either ro-TNF-o (100¡rg/m1) or conditioned medium (70% v /v) and eluted with 0.1% EDTA in sterile Hank's. All cells were washed 4 times in plain RPMI L640 prior to injection. 92
2.2.7.3 Fusion partner cell line
The murine cell line SP2/0-A91,4, which was originally produced as a sub- clone of a hybrid between aBalb / c spleen cell and the myeloma cell line X63-4g8, was used for fusions. SP2/0-4g14 cells are resistant to 20 þB/ml9-azaguanine but die in HAT-supplemented medium and do not synthesise immunoglobulin chains
[Shulman et a1.,1978].
2.2.7.4 Cellfusion
The immunised mouse was killed by cervical dislocation and the spleen aseptically removed. A single cell suspension was produced by teasing the spleen through a sterile sieve in plain RPMI 1,640, and was washed 3 times. A total of L x
108 spleen cells were mixed with 1 x1.07 SPz/0-Ag14 cells and centrifuged at 600g for 7 min. The supernatant was completely removed by aspiration, and the cell pellet disrupted by "flicking". The tube was placed in a water bath at 37"C and 0.7 ml of prewarmed 50% polyethylene glycol 1500 was added over 1 min with gentle stirring. The mixture was stirred for a further minute and 10 ml of plain RPMI
1.640 at 37'C added over the next 10 min with continual stirring. A small sample of the mixture was observed microscopically to determine if fusion had taken place, and the remainder was centrifuged at 6009 for 7 min. The pellet was resuspended in 30 ml of RPMI L640 plus 15% FCS and 70o/o BM-Condimed HL
(hybridoma cloning supplement), and 100 pl aliquots were placed in the wells of flat-bottomedg6 well microtitre trays (Corning, USA). Plain medium was used to fill the outside wells of the plates, and they were incubated overnight at 37'C. The next day 100 pl of 2xHAT medium was added to each well, and from then on the wells were observed daily for growth of hybridoma colonies. 93
2.2.7.5 Screening of fusions
At 7 to L4 days after fusion, supernatant was taken from the weils containing hybridoma colonies and assayed for the presence of specific antibody.
The assay method used was indirect immunofluorescence staining and FACS analysis (2.2.II.1) of cultured ShUVECs or appropriate cell adhesion molecule
expressing transfectant. The supernatants were screened in parallel against both unstimulated and CM-stimulated ShUVECs to detect antibody against antigens more highly expressed by activated ShUVECs. Hybridomas of interest were then
expanded, cloned, and the cells frozen and supernatant kept for further testing.
The culture medium was changed to HT (hypoxanthine, thymidine) supplement
after L0 to 14 days, and several days later the HT was omitted.
2.2.7.6 Cloning of hybridomas
Hybridomas were cloned by limiting dilution in flat-bottomed 96 well microtitre trays (Corning) in the presence of BM-Condimed HL medium supplement (10% v /v). Thirty six wells were seeded with viable hybridoma cells at a concentration of 5 cells/well, 36 wells at L cell/well, and 24 wells at 0.5 cells/well. After 3 to 4 days of culture, the number of colonies in each well were counted, and when they were of sufficient size (day 7 to I0 usually), the supernatants were assayed again for antibody against ShUVECs by fiow cytometry. A colony from a single colony well was grown up and recloned, and the hybridoma was considered to be monoclonal when all wells containing
hybridoma cells produced supernatants containing antibody of the same activity. The cells of a positive well containing a single colony were then expanded for
crypreservation and production of tissue culture supernatant and immune ascites. 94
2.2.7.7 Cryopreseraation of cells
Approximately L0 ml of well-grown culture was centrifuged at 6009 for 7 min and the pellet resuspended in L ml of RPMI L640 plus 20% FCS. An equal volume of 20% DMSO in the same medium was added to the cell suspension at 4'C while the tube was shaken. The mixture was placed in cryotubes (Nunc, Denmark) and fuozen at a controlled rate in a Handi-Freeze fueezing tray (Union
Carbide). The ampoules were stored in the liquid phase of liquid nitrogen in a Union Carbide 35VHC liquid nitrogen tank until used.
2.2.7.8 Prepørøtion of immune øscites
Balb / c mice were injected twice, one week apart, with 0.5m1 of Pristane.
Three days after the second injection, they were injected intraperitoneally with 2 x
106 washed hybridoma cells suspended in plain RPMI. The mouse was inspected daily and when it developed marked abdominal distension (usually after 6 to L0 days), the mouse was sacrificed and the ascites aspirated by incising the peritoneal membrane. Ascites produced for in aiao studies was harvested using de-pyrogenated pasteur pipettes and collected into pyrogen-free, sterile tubes to minimise introduction of endotoxin (see 2.2.8). The sample was centrifuged and the ascites aspirated and stored at -70"C until required.
2.2.7.9 lsotyping of øntibodies
The isotypes of the monoclonal antibodies produced as a part of this thesis were determined using a Serotec MMT RC1 isotyping kit.
2.2.8 Ammonium sulphøte precipitøtion of monoclonøl øntibodies
The monoclonal antibodies intended for both general laboratory use and ln oiao studies were purified by the following technique, as described by Goding
[1986]. In addition, the mAbs destined for in uiao use were produced under 95 stringent endotoxin-free conditions. This primarily involved the preparation of all reagents in pyrogen-free water (i.e., water for irrigation), using baked glassware and working under aseptic conditions.
Antibody was purified from ascites by the precipitation of immuno-
globulin in a 50% solution of ammonium sulphate. An equal volume of cold (4'C)
saturated solution of ammonium sulphate, adjusted to pH 7.2 with NaOFI, was added dropwise to the protein preparation while being agitated with a vortex
mixer. The mixture was left standing on ice for L hr. The resulting precipitate was washed three times with a 50% solution of ammonium sulphate. After washing, the precipitate was dissolved in an appropriate volume of PBS and the solution was dialyzed against approximately 200 volumes of PBS (t 0.02% sodium azide)
to remove remaining ammonium sulphate.
2.2.9 FlTC-conjugøtion of monoclonøl antibodies
Monoclonal antibodies were directly conjugated with fluorescein
isothiocyanate (FITC) by the method of Goding 119861, using ammonium sulphate precipitated murine ascites. The mAbs were dialysed overnight against pH 9.5
carbonate/bicarbonate buffer. FITC was prepared as a 1,0 mg/ ml solution in dimethyl sulphoxide (DMSO) and L0 llg/mg antibody was added to the dialysed mAb dropwise whilst maintaining gentle stirring. The solution was then incubated at room temperature for 2 hr with continued gentle mixing, followed by separation of conjugated mAb from unbound FITC on a disposable PD-10 column (Pharmacia, Sweden). Confirmation of optimal conjugation was obtained by measuring the absorbance at 280 nm and 495 nm and determining the
fluorochrome:protein ratio using the following formula :
2.87 xOD¿ss F /P ratio = (Goding 1986p257) ODzao-0.35xODae5 96
2.2.70 Monoclonøl øntibody concentrøtion determination - ELISA
Monoclonal antibody concentrations were determined using the method of
Lems Van Kan et ø1. 119831. The wells of a flat-bottomed 96 well microtitre plate
(Corning) were coated with sheep anti-mouse Ig (2 ¡t"g/mI) in coating buffer by incubating at37"C Íor 2hr, followed by overnight incubation at 4'C. Next day the plate was washed 3x with PBS /0.05% Tween-2O, which was used in all subsequent wash steps. A standard curve was constructed by serially diluting at 1:3 mouse Ig (2 ltg/ml in PBS). The mAb was also serially diluted L:3 commencing at L:100 or L:1000 dilutions. The plate was wrapped in cling wrap and incubated for 2hr at 37"C in a humid chamber. Following incubation, wells were washed 3 times and 100 pl of a 1/1000 dilution of sheep anti-mouse alkaline phosphatase conjugate added to each well, followed by a further 2l:rr incubation at 37"C in humidified chamber. Wells were then washed 3 times and 100 pl of phosphatase substrate (2 tablets into L0 ml diethanolamine buffer) was added. After a l-5 min incubation at room temperature the OD+OS was then obtained using a Titertek plate reader.
2.2.1,L 5t øining techniques
2.2.11.1- Indirect immunofluorescence for flow cytometric nnølysis (FACS)
Adherent endothelial and transfected cells were eluted with 0.1-% EDTA or
0.725% Trypsin/O.1% EDTA. Ali cell samples (i.e. adherent, non-adherent and
MNCs) were counted and sufficient taken to give approximately 0.5 x 106 cells per antibody being tested. They were washed twice in cold FACS wash buffer (2.1,.4.6) by centrifugation at 2009 for 5 min at 4C. All subsequent washes in the procedure were performed in the same manner, and the cells were resuspended in between by gentle flicking of the tube. All manipulations were performed on ice unless otherwise stated. 97
After washing, the cell pellet was resuspended in cold FACS washing buffer. Aliquots of L00 pl were distributed into separate tubes, and heat inactivated rabbit serum was added to a final concentration of 1.0"/. (v /v) to block non-specific antibody binding to Fc receptors. Ethidium monoazide (EMA) was also added to a final concentration of 1,0% (v /v) for discrimination between live and dead cells. Samples were then incubated in the dark for L0 min and kept in the dark as much as possible for all remaining manipulations.
Following the L0 min incubation, primary antibody was added (50 pl of tissue culture supernatant or L0 pl of a7/500 dilution of ascites) and the samples were incubated in the dark for 30 min. The samples were then placed under bright fluorescent light for 20-30 min to photoactívate the EMA. All tubes were filled vigourously with FACS washing buffer and centrifuged at 2009 for 5 min. The supernatant was removed from each tube and the pellet resuspended by flicking, prior to the addition of the FlTC-conjugated sheep anti-mouse IgG secondary antibody at a final concentration of 1/500 in FACS washing buffer. All samples were incubated in the dark for 20 rnin, followed by a 5 min incubation at room temperature. A 1 ml aliquot of freshly prepared 1,0"/" FACS lysing solution was added to each tube, followed by a 20 min incubation at room temperature. The cells were then washed twice by filling the tube vigourously with FACS washing buffer and centrifuging at 2009 for 5 min. Finally, the cells were resuspended in 350 pl of filtered saline and stored in the dark at 4"C until
analysed on a Becton Dickinson FACScan flow cytometer.
2.2.1,1,.2 Indir ect immunop er o xi døs e
Cell preparations or frozen tissue sections were stained with antibodies using an indirect immunoperoxidase technique. The isolated cells were cytospun onto glass slides, allowed to dry, and fixed by immersion in cold (4"C) acetone for
10 min. 98
Tissue samples were embedded in Tissue Tek II embedding medium and snap frozen in liquid nitrogen. Cryostat sections 5 Lrm thick were mounted onto gelatinised glass slides, air dried overnight and fixed in cold acetone for 10 min. Nonspecific staining was blocked by incubation with 3% normal horse serum
(NHS) in PBS for 10 min. This and all subsequent incubations were performed at room temperature in a humidified chamber. Endogenous biotin was blocked by a
L5 min incubation with 2-3 drops (neat) of avidin D solution (Biotin Blocking kit,
Vector). Sections were then washed briefly in PBS and2-3 drops of biotin solution were added. After a L5 min incubation, sections were washed in PBS for 5 min. The specimens were then incubated for 45 min with 100 pl of mAb (titred dilutions of ascites or neat tissue culture supernatants). Sections were then washed thoroughly in PBS and incubated for 30 min with L00 pl of biotinylated horse anti-mouse IgG (1/200 dilution ín 3% NHS). After washing, endogenous peroxidase was quenched by a 30 min incubation with 0.3% HZOZ in methanol, followed by several washes in PBS. Sections were then incubated with a 1:1 preparation of avidin DH and biotinylated horseradish peroxidase H (ABC kiÐ for 60 min. After further washing, the sections were exposed to 0.5 rng/mI diaminobenzidine tetrahydrochloride (DAB) and 0.0L% hydrogen peroxide in
0.05 M Tris-HCl buffer (pH7.2) for 7 min. A positive reaction gives a brown stain.
The specimens were then counterstained with Harris' haematoxylin and mounted under a glass cover-slip.
2.2.1,2 Immun op re cþ it øti on of surf øc e øntigens
2.2.L2.L Lactoperoxidøse catølysed cell ntrføce iodinøtion
All manipulations with unconjugated 1251ursyg performed in a fume hood in a dedic¿1s¿ 1251 laboratory. Ali remaining manipulations were performed behind lead-lined perspex shielding. 99
Cells were harvested by centrifugation at 5009 for 7 min, aliowing 2 x 106 cells for each antibody being tested. The supernatant was discarded, and the cells washed twice in High Buffer PBS (2.1.4.2), as above. After the final wash, all of the supernatant was removed with a drawn-out pasteur pipette, and the cells were resuspended in 200 pl of cold High Buffer PBS. A volume of 75 p I of lactoperoxidase (O.2mg/ml) was added, followed by 0.5mCi 1251. At L min intervals, 10 pl aliquots of increasing concentrations of H2O2 were successively added: i.e. 1. / 27000, 7 / 9000, 1 / 3000, 1/ 1000. One minute after the addition of the 1/1000 dilution of H2O2, the tube was filled with cold PBS plus 0.02% NaAzide and L mM KI. The cells were washed three times in the same solution, by centrifugation at 5009 at 4'C.
2.2.1-2.2 Detergent solubilisation of løbelled cell surface molecules
After the final wash the supernatant was removed, and the pellet was resuspended in 1ml of cold Lysis buffer (2.L4.2) for each aliquot of up to 107 cells.
The sample was incubated on ice for 30-45 min, with occasional mixing to allow the cells to lyse. The solution was then centrifuged at 2,5009 at 4C, and the supernatant transferred to a new tube.
2.2.L2. 3 Immunopr ecipit øtion us ing S t aphylo co c cus øur eus c ells
Pre-clearing of the supernatant involved the addition of 50 pl of normal rabbit serum for each l- ml of cell lysate, followed by incubation on ice for 30 min.
A pellet of S. øureus cells from a200 pl aliquot (2.1,.4.2) was resuspended with the cell lysate and incubated on ice for 10 min. The cell lysate was then centrifuged at
2,5009 for 5 min at 4C, and the supernatant used to resuspend a second pellet of
S. aureus cells. The samples were incubated on ice and centrifuged as above. The cell lysate was then distributed equally into a number of individual tubes sufficient for each antibody tested. The relevant antibody was added to each tube 100
(200 pl culture supernatant or 5 pl ascites), and the samples were incubated overnight by rotating at 4'C.
The following day,S pl of rabbit anti-mouse immunoglobulins was added to each tube and incubated for 60 min on ice. A volume of 100 pl of S. øureus supplemented with L mg/ml ovalbumin (2.1..4.2) was added to each tube, and the incubation was continued for a further 60 min al 4C on a rotating table. Each tube was then filled with SAC buffer (2.1,.4.2), and centrifuged at2,5009 at 4"C for
15 min. The supernatant was discarded, and the pellet was washed twice, as above. The pellet was then resuspended in 100 pl of SDS Sample Buffer (2.1.4.2) (+ dithiothreitol), and boiled for 4 min. Finally, the sample was centrifuged at
2,5009 for 5 min, and an aliquot o11,8-25 pl was loaded on to a polyacrylamide gel for protein gel electrophoresis (see below).
2.2.L3 SD S-P oly øcryl ømide G el Electr ophorcsis (SD S -P AGE)
Proteins were separated by electrophoresis on polyacrylamide microslab gels consisting of either a 5o/",7 .5'/" or a 70% resolving gel and a 4.5"/' stacking gel, as described by Matsudaira and Burgess 119781. The concentration of acrylamide in the resolving gel was determined by the molecular weight of the antigens to be examined. The proportion oÍ 40"/" acrylamide stock was therefore adjusted accordingly and added to a mixture of resolving gel buffer (2.1,.4.2) and distilled water, foliowed by 0.05% ammonium persulphate, 0.I3'/o TEMED, and was poured to allow for a 1 cm stacking gel. The resolving gel was overlaid immediately with N-butanol to avoid aeration while setting, typically 45-60 min, and to ensure a level interface between the two gel types.
The stacking gel contained 4.5'/. acrylamide in stacking gel buffer (2.1..4.2) and dH2O, 0.05"/. ammonium persulphate, and 0.2'/o TEMED. Protein samples were loaded in L x SDS polyacrylamide gel loading buffer (2.1.4.2) and electrophoresed at200 volts in a vertical mini-gel apparatus for app min (Bio-Rad Mini-Protean II).
At the completion of electrophoresis, gels were stained for 30 min in Coomassie blue staining solution. Destaining consisted of a 10 min wash, followed by a 30 min wash in destaining solution. Gels were then dried onto
Whatman paper using a vacum operated gel drier (Bio-Rad model 11258).
The dried gels were placed against Kodak X-OMAT AR film inside a
Kodak X-OMATIC film cassette. The films were exposed to the gels for varying lengths of time at -70"C, and developed in a Kodak X-OMAT developer at the
Department of Radiology, The Queen Elizabeth Hospital.
2.2.1-4 cDNA insert subcloning and lineørisøtion
The ICAM-1 cDNA was supplied in the pCDM8 expression vector (Invitrogen) and required subcloning into a riboprobe vector to facilitate the transcription of cRNA probes for Northern analysis experiments. The plasmid pGEM-7Zf(-) (Promega, USA) was used as the vector. It contains the ampr gene allowing for selection of transformed bacteriaby growth on Luria Bertani (LB) agar plates supplemented with ampicillin at l-00 þB/m1rThe Escherichiø coli strain BB4 was used as the host bacteria for the transformation. Cultures of BB4 were supplemented with 10 pg/ml of tetracycline and grown al 37"C in a shaking water bath for liquid cultures, or a warm air incubator for agar cultures.
The VCAM-L cDNA was supplied as a L Kb PCR product insert in the PCR
1000 vector (Invitrogen, USA) which contains a T7 promotor at the 3' end. The vector was linearised with the Xba restriction endonuclease (RE) (see 2.2.1.4.8) in preparationlor inuifro RNA run-off transcription (see 2.2J,5.4). 1.02
2.2.1.4.1 Digestion of Vector ønd Insert
The ICAM-I- insert was excised from pCDM8 by digestion with Xba L in reaction buffer H (2.1,.4.3), according to the manufacturers directions. The riboprobe pGEM-7Z was linearised with the same RE and reaction buffer. A quantity of 10 pg of both ICAM-1/pCDM8 and pGEM-7Z werc digested with Xba i- by overnight incubation at37'C.
2.2.L4.2 Recoaery of ICAM-I Insert
The ICAM-L insert was isolated from remaining RE digest products by separation on a 1o/" agarose gel, poured with a toothless preparatory comb. The ICAM-L insert band was cut from the gel and residual agarose removed with
Gene-Clean, according to the manufacturers directions.
2.2.L4.3 Dephosphorylation of pGEM-7Z Vector
Dephosphorylation of the linear plasmid was conducted following the method of Sambrook et aI.,1989, for the removal of phosphate residues from 5' termini. The Xba l-digested vector (see 2.2.1.4.L) was dephosphorylated by the addition of 0.5 units of calf intestinal alkaline phosphatase and 1 x reaction buffer supplied by the manufacturer. The reaction mix was then incubated at 37"C for 45 min, followed by the addition of a further 0.5 units of alkaline phosphatase, and the continuation of the incubation at37"C for 45 min. The reaction was terminated by the addition of 5 mM EDTA pH 8.0, and a L0 min incubation at75'C.
2.2.L4.4 Ligation of Vector ønd Insert
A ligation reaction at a vector:insert ratio of 1:3 was prepared using 100 ng of digested vector DNA and 200 ng of the 2 Kb ICAM-1 insert. In addition to the vector and insert DNA the ligation reaction also contained 1.5 Weiss units of T4 103
DNA ligase, 2¡tI of 10x ligase buffer, and water to a final reaction volume of 20pi.
The ligation reactions were incubated overnight at L4'C. A "no insert" control ligation reaction, containing vector only, was included in each cloning experiment to check for incomplete cutting or re-ligatation of vector. Additional controls included uncut vector and cut-dephosphorylated vector
2.2.L4.5 Prepørøtion of Competent Cells
Competent BB4 cells were prepared as needed and used within 24 hr. On day one a stock culture of BB4 was streaked on to a LB agar plate to obtain fresh single colonies, and allowed to grow overnight. The following day a 5 ml LB broth culture was innoculated with a single colony from the agar plate and allowed to grow overnight. On day three, 1 ml of the overnight broth culture was added to 25 ml of fresh LB broth and allowed to continue growing until log phase growth was reached, and the optical density of the culture was 0.4-0.6 at 600 nm
(approximately 1,1/ z -2 hr). The culture was then incubated on ice for l-0 min, followed by centrifugation at 1,3009 for L0 min at 4C. The pellet was resuspended in 2 ml of 100 mM CaCl2,20 mM MgCl2 and incubated on ice for a minimum ol2hr prior to transformation.
2.2.14. 6 B øcterial Tr ansþrmation
An aliquot of L0 pl of each ligation reaction was added to a 200 pl aliquot of competent cells, mixed briefly and gentl/, and incubated at 42"C for 2 min. The samples wete then incubated on ice for 10 min, and allowed to stand at room temperature for a further L0 min. A volume of 0.5 ml of LB broth, containing 35 Itg/m\ tetracycline, was then added to each sample and the bacteria were incubated at 37"C for 45 min to allow the antibiotic resistance gene to be expressed, before culturing onto selective agar plates. The bacteria were then spread on to selective LB agar plates containing 100 pglml ampicillin, L0 ltg/mI L04 tetracycline,40 pg/ml XGAL, and 48 þ1/mIIPTG (2.1..4.3), in L0 pl, 100 ¡tl and 500 pl aliquots, and allowed to grow overnight.
In addition to the control samples prepared in section 2.2.1,4.3 above, competent cells only (no DNA) were also included to monitor the success of transformation and the ampicillin sensitivity of the cells.
The following day all agar plates were analysed for the presence of growth.
Well-isolated white colonies were chosen and the presence or absence of an insert was confirmed by analysis of small-scale bacterial cultures .
2.2.14.7 Smøll-scøle BacteriøI Cultures
Small scale cultures of isolated clones were grown to determine if successful transformation of the ligated plasmid had occurred. These "minipreps" were prepared by alkaline precipitation following the method of Sambrook et al.,
1989. The contents of miniprep solutions L,2, and 3 are listed in section 2.1,.4.3.
Positive colonies identified by XGaI/IPTG white selection were sub- cultured into 5 ml of LB broth containing 100 ItB/mI ampicillin and L0 þB/mI tetracycline, and grown overnight at37"C. A 1-.5 ml aliquot of well-grown culture was transferred to a microfuge tube and spun at 15,000g for 1 min. Cells were resuspended in 100 pl of solution L, followed by addition of 200 pl of solution 2 and a 5 min incubation on ice. A 150 pl aliquot of solution 3 was added, followed by a further 5 min incubation on ice. The solution was microfuged at 15,0009 lor 2 min and the supernatant (approx. a50 pl) transferred to a clean microfuge tube. The nucleic acid content was recovered by phenol/chloroform extraction, followed by ethanol precipitation. The dried nucleic acid pellet was then resuspended in 50 pl of TE buffer containing 20 pg/mlRNAse. 105
An aliquot of 1 pl of miniprep sample was digested with Sph-I, followed by
analysis on a 1o/o agarose gel to ascertain whether the plasmid contained an insert of the expected size.
2.2.L4.8 Lørge-scøle B acterial Cultur es
Following confirmation of the correct síze insert by miniprep analysis, large
scale preparative cultures were prepared. All the following centrifugation steps were conducted in a Sorvall RC-58 centrifuge using the SS-34 fixed angle rotor. A L25 ml LB broth containing 100 ItB/mI ampicillin and L0pg/ml tetracycline was innoculated with a 2.5 ml starter culture from the miniprep and grown for 6-8 hr at37"C. Chloramphenicol (150 pglml) was then added and the culture continued growing overnight. Next moming the culture was harvested by centrifugation at
4,0009 for 20 min at 4'C. Purified plasmid DNA was then prepared using gravity- flow columns, following the procedure recommended by the manufacturer (Qiagen, Germany). The alkaline lysis purification protocol, and all buffers and
reagents were supplied by the manufacturer (Qiagen, Germany).
The concentration of the purified plasmid DNA was measured at 260 nn-, with an ODunit of l- corresponding to 50 þg/ml of double-stranded DNA. An aliquot of approximately 1 pg of plasmid DNA was then digested with the
appropriate restriction enzyme and run on a1-o/" agarose gel to once again confirm
the presence of the expected size insert.
2.2.14.9 Lineørisøtion of ICAM-L øndVCAM-IcDNA aectors
Stocks of linearised cDNA were prepared by bulk RE digestion overnight at 37"C. A 50 pl aliquot of ICAM-1/pGEM-72 was cut with Sph-I and 50 pl of
VCAM-1/PCR 1000 digested with Xba-1. After overnight incubation, the nucleic acid content was recovered by phenol/chloroform extraction, ethanol washes and
resuspended in 50 pl sterile dHZO. Stocks were quantitated and stored at -20C. 106
2.2.75 Nothern blot ønøly sis
2.2.1.5.1 RNA preparntion
Total RNA was isolated from stimulated and unstimulated ShUVECs by the acid guanidinium phenol chloroform (AGPC) method of Chomczynski and
Sacchi l1g87| Confluent monolayer¡of cells ín a75 cm2 culture flask (approx. 3 x
L06 cells) were washed with sterile PBS. A volume of 1 ml of solutionD (2.1..4.a) was added directly to the flask and mixed briefly to lyse the cells. The suspension was transferred to a microcentrifuge tube and the following components were added sequentially, mixing briefly between additions: 100 pl of 2M sodium acetate pH 4.0, 1ml of water-saturated phenol (2.1.4.4), and 200 ¡rl of chloroform:isoamyl alcohol (49:1) (2.1.4.4). The sample was incubated on ice for
1-5 min, followed by centrifugation at 13,5009 for 30 min at 4'C. The RNA in the upper aqueous phase was removed and transferred to a clean tube, and 1 ml of isopropanol was added, followed by an incubation at -20"C for L hour or -70"C for 15 min. The tube was then centrifuged at 13,5009 for 30 min at 4"C, and the supernatant discarded. The pellet was dissolved in 300 pl of solution D, to which L x volume of isopropanol was added, followed by an incubation at -20"C or
-70"C as above. Finally, the sample was centrifuged at 1-3,5009 for l-5 min and washed twice in L ml o1 75% ethanol. The purified RNA pellet was air dried, resuspended in 50 pl of DEPC-water (2.1,.4.4) and heated at 65'C for L0 min to dissolve.
The concentration of RNA was measured by preparing a I/50 dilution in
DEPC-water and measuring the absorbance al 260 nm, with an ODunit of 1 corresponds to 40 þg/mI RNA.
2.2.1.5.2 RN,4 gel electrophoresis
RNA was resolved by electrophoresis in formaldehyde/I% agarose gels (containing ethidium bromide; 0.01% of 10 mg/mI stock) prepared in MOPS 1.07 buffer (2.7.4.5) and electrophoresed in a horizontal submarine gel apparatus
(Pharmacia, Sweden). Ten Fg of total RNA (5pl aliquots) were mixed with 25pl of RNA loading buffer, and electrophoresed at 100 volts in MOPS buffer.
2.2.15.3 20X SSC trønrtr
Following electrophoresis, RNA was transfered to Hybond N+ nylon membrane by overnight transfer in 20X SSC by the method of Sambrook [1989]. The gels were washed 2 x 20 min in 10X SSC at room temperature to remove formaldehyde and placed on Whattman 3MM filter paper wick, followed by the nylon membrane, sheets of filter paper, absorbent towel and a weight (-500 gm). After overnight transfer the nylon membrane was exposed to UV light to cross- link the RNA to the membrane.
2.2.15.4 32P -tabetting of cRN A probes
cRNA probes were prepared by RNA run-off transcription using a
"Message Maker in aitro Transcription Kit". [o(-32P]-UTP was incorporated into antisense RNA (cRNA) with T7 RNA polymerase by mixing 1 pg of linearised
DNA with the kit reagents (according to manufacturers directions) plus 50 mCi
[o¿-32P]-dUTP and incubating for 60 min at 37"C. The DNA template was removed by addition of DNAse and incubation for L0 min at 37'C. Labelled cRNA was then isolated with Tris-saturated phenol:chlorof orrr. (24:1) by centrifugation at
13,5009 for 5 min at 4C. The RNA in the upper aqueous phase was removed and transferred to a clean tube. A volume (1,/1,0) of Na acetate pH5.5 was added followed by a 2.5 volume of cold absoiute ethanol and incubation at -20"C for I hour or -70'C for 30 min. The tube was then centrifuged at 13,5009 for 30 min at
4C, and washed twice in 1 ml of cold 70'/o ethanol. The labelled cRNA pellet was air dried, resuspended in 100 pl of DEPC-water (2.1-.4.4) and heated at 55'C for L5 min to dissolve. 108
2.2.L5.5 Prehybridisøtion
The membrane was rinsed briefly in 2X SSPE and placed into a sealed plastic bag containing prehybridisation buffer (L00 ptlcm2¡. fhe membrane was
then prehybridised at 65'C for a least 4 hr in a shaking water bath.
2.2.L5.6 Hybrídisøtion
The labelled cRNA probe was mixed with 100 pl of hybridisation buffer
(2.1.4.5) and heated at 95'C for 10 min, pulse centrifuged and placed at 65'C. The probe was then injected into a corner of the plastic bag, resealed and mixed
evenly throughout the bag. Hybridisation was carried out overnight at 65'C in a shaking water bath. Posthybridisation washes were performed in 2X SSC/0.1%
SDS at room temperature for 2x75 min followed by 2x30 min in 0.2XSSC/7%
SDS at 68'C.
2.2.1-5.7 Autor adiogr øphy
The Hybond N+ membranes were blotted briefly and exposed to Kodak
XAR film for 1 to L0 days at -70"C.
2.2.16 Døtø Anølysis
Data are expressed as means + SD (n = number of subjects). Differences between means were assessed for statistical significance using paired Student's t- test. The Wilcoxon rank sum test was used to compare survival data. Ali calculations were carried out with the Statview program (Abacus Concepts,
Berkeley, CA, USA) using a Macintosh computer. t09
Ch apter 3
Establishment of a sheep renal all ograft model: Immunophenotypic comparison of cells in the ly*phatic drainage and peripheral blood
INTRODUCTION
Animal models ranging in size from small rodents up to primates, dogs
and the small ruminants, such as sheep and goats, have been used to study a diverse range of physiological and pathological conditions. Sheep have increased in usage and importance as a large laboratory animal for a number of reasons. Firstly, with the size and weight of sheep being comparable to that of humans, these animals can provide data which is of clinical relevance. Secondly, once sheep have acclimatised to the laboratory for one to two weeks, they generally become accustomed to the presence of handlers which enables the investigation of physiological function in health/, unstressed conscious animals [Hecker, 1983]. In addition, the size of sheep also makes them idealiy suitable for solid organ transplantation, eliminating the obvious requirements for more difficult and time consuming microsurgical techniques necessary lor 110
such procedures in rodents. After surgery sheep recover well from anaesthesia and given correct care, they are generally not affected by post-operative
infection [Hecker, 1974]. Finally, the size and weight of the animal enables cannulae and catheters to be readily introduced at a number of different sites
around the body. This increases the ease and frequency with which blood and body fluids can be collected with minimal effects on cardiovascular function, and in volumes that would often not be possible in small animal models. This is particularly useful in models of solid organ transplantation where biopsies can be collected daily to enable the temporal study of histological and physiological changes.
The ability to collect large volumes of lymph from sheep was demonstrated by two seminal studies reported in the early L970s.In the first,
Mclntosh and Morris 119711showed that the ovine renal lymphatic drainage is unique in that there are no capsular lymphatics and that all lymph drainage in the sheep kidney occurs entirely via 2 to 5 hilar lymphatics, which in turn
flow to the renal lymph node. This study was also the first to provide a detailed description on the origin and composition of renal lymph obtained from a kidney functioning in situ under physiological conditions.
In the second study, Pedersen and Morris [1970] utilized the unique anatomy of the ovine renal lymphatics to undertake a detailed study of the role of the lymphatic network in the rejection of renal allografts in sheep. In particular, their study revealed a dramatic increase in the volume and cellular content of lymph from rejecting allografts compared to little or no change in the lymph from autografts.
The work presented in this chapter concerns the establishment of an ovine model of renal transplantation, based on that described by Pedersen and Morris ll970l. The initial aim was to confirm that the observations from the model were consistent with those reported by Pedersen and Morris 119701, and 1L1 to then extend their observations with regard to the cellular content of lymph draining from rejecting renal allografts. With the passage of time, we now have the benefit of several technological advances not available in the early
1970s, such as monoclonal antibody production and flow cytometry. These tools enabled a more detailed examination of the immuno-phenotypic characteristics of the cellular content of lymph during allograft rejection than was possible in the earlier studies of Pedersen and Morris 119701.
In addition to the "standard" T-lymphocyte subset markers such as CD4 and CD8, it was also of interest to examine the expression of CD44, a heterogeneous family of transmembrane glycoproteins expressed on many cell types. At the time of commencing these studies CD44 was considered to be one of the essential molecules required for lymphocyte homing and adhesion and had been reported to play an important role in the activation, adhesion and transmigration of lymphocytes [Jalkanen et ø1., 1986b; Huet et al., 1989; Picker et øL, 1989; Shimizu et al., 19891. Furthermore, there is increased expression of CD44 by lymphocytes following the activation of T and B cells and differentiation from naive cells to antigen-primed memory cells [Budd et al., 7987; Shimizu et øL, 1.990b; Springer, 7990; Camp et ø1., 1.99L; Hathcock et al.,
7ee3l.
Thus the availability of a mAb (25-32) recognising the ovine and bovine equivalent of CD44 [Mackay et ø1., 1988b; Hein and Mackay, 1997; MacHugh ef ø1., 1993) provided an opportunity to study simultaneously the expression of this activation marker by leukocytes in the peripheral blood and afferent lymph compartments during ovine renal allograft rejection. 112
DEVELOPMENT OF THE MODET
Sheep Ren øl Tr ønsplantøtion Pro ce dure
Merino sheep, approximately 2 yrs of age and weighing between 38 and 50 kg, were used for all transplantation studies described in this and subsequent chapters. All experiments were conducted with approval of The
Queen Elizabeth Hospital Institutional Animal Ethics Committee.
The animals were fasted Íor 12 hr prior to transplantation but had free access to water. Anaesthesia was induced with Nembutal (2ml/Skg) and maintained with a Fluothane-oxygen gas mixture delivered via an endotracheal tube. At the commencement of surgery, a 20 cm central venous catheter (CVC) (Arrow Int, USA) was inserted into the right jugular vein to allow systemic infusion of solutions during the transplant procedure and the collection of peripheral blood samples during the course of the experiments. During surgery the sheep were hydrated by intravenous infusion of l- L of normal saline (0.9% sodium chloride), followed by u further L L during fecovery.
The left kidney was used for all transplantations as the renal vessels are longer than those of the right kidney, allowing greater flexibility for positioning of the kidney during and after anastomosis. Allograft transplantation involved removal of the left kidney from the donor followed by placement into the left side of the recipient's neck (Figure 3.1). In the case of an autograft, the kidney was removed from the donor and placed into the neck of the same donor animal.
The transplant procedure involved careful dissection and removal of the left kidney, which was immediately placed into a slurry of sterile frozen saline and perfused intra-arterially with cold Ross kidney perfusion solution 113
Eigure 3.L Transplanted kidneys were placed under the skin flap on the neck of sheep. Lymphatic and ureteric cannulas were brought to the surface through small incisions in the skin below the graft. 774 containing 5000U heparin, whilst the final preparations were made to the site on the left lateral aspect of the recipients neck. The kidney was transplanted with an end-to-end arterial anastomosis between the renal and carotid arteries and an end-to-side anastomosis of the renal vein to the jugular vein.
]ust prior to revascularisation, 20 mg of Frusemide and 100 ml of 20% Mannitol were given through the CVC to assist in the promotion of diuresis, minimisation of potential damage caused by oxygen free radicals and to prevent oliguria associated with acute renal failure [Bernier and Hearse, ].988; Richards et al., 7989; Gupta and Marmor, 1,9931. Immediately following revascularisation, 1000U heparin were also given to reduce the risk of thrombosis occurring within the graft. At the completion of vascularisation, one afferent lymph duct was cannulated with a polyethylene tube (0.8 mm ID, 0.96 mm OD) (Critchley Electrical, Australia) which was brought out to the surface, near the base of the kidne/, through a small incision in the skin flap covering the graft. The remaining non-cannulated lymphatics were ligated and lymph was then collected into a tube containing heparin. Collection of lymph at timed intervals enabled the calculation of hourly flow rates. The cellular content of lymph was determined using a Neubauer haemacytometer.
Urine was collected by cannulation of the graft ureter using an FGS paediatric feeding tube (Indoplas, Australia) which was also brought out to the surface through a separate small incision in the skin flap. The urine drained into a small bottle strapped under the animals neck and collection at timed intervals enabled the calculation of hourly flow rates. After surgery, the animals received daily intramuscular injections of antibiotícs (penicillin and streptomycin).
During the development of this model, the remaining native kidney was left in place and the onset of rejection in the allograft was assessed by measurement of urine output and percutaneous biopsy of the graft 115
approximately every three days using a Monoptyt"tbiopsy needle (Bard Inc., USA). In experiments described in later chapters, renal function in allograft recipients was provided by the transplanted kidney alone, as bi-lateral nephrectomy of the native kidneys was performed. This enabled graft function to be monitored by measurement of serum creatinine concentration, in addition to urine output and histological assessment. These modifications will be discussed in detail in the next chapter.
RESULTS AND DISCUSSION
3.1, Grøft Function
Whilst establishing this model, a total of 9 transplants (5 allografts and 4 autografts) were performed. To increase the likelihood of achieving fully- functioning grafts, without the added complication of acute rejection, the first
3 procedures attempted were autografts. However, the first 2 out of these 3 autografts failed due to cortical necrosis, secondary to thrombosis of the renal artery. Therefore to minimise potential damage to the kidneys perfusion of the remaining 2 autografts and 5 allografts with Ross solution was improved and increased through the use of a Hamilton Bailey needle. This modification enabled better cannulation of the renal artery and infusion of the perfusate which resulted in 7 functional grafts that produced urine within 5 min of revascularisation. Flowever, one allograft developed a major infection around the kidney and despite continued blood flow through the graft, the production
of urine stopped at day 4 and resulted in the animal being sacrificed at day 6. Thus of the 9 transplants performed, 4 allografts and 2 autografts functioned continuously until the time of either allograft failure or euthanasia of the
autografted animals (Figure 3.2). 1,1,6
MI o isìÈswqt Renal artery thrombosis (! ñ and cortical necrosis L 2 ol W o a 3
4
5 lnfection
a¡, 6 õ cD 9 7 a 8
9
0123 4 5 6 7 8 I 10 11 No. Days of urine output
Figure 3.2 Comparison of renal allograft and autograft function in the series of animals used to develop the ovine model of renal transplantation.
3.2 CeIIulør lnfiltrøtion ønd Grøft Histopøthology
The onset of rejection was determined by histological assessment of percutaneous biopsies taken from the graft every three days. In the allografts there was evidence of focal mononuclear cell (MNC) infiltrates in biopsy samples taken at day L post-transplant which progressively increased in association with the onset of severe rejection from day 4 onwards. In contrast, MNC infiltration was not evident in the autografts and graft function remained unaffected till the animals were euthanased (Figure 3.3).
Histological analysis of the allografts between days 6 and 9 showed tissue infarction and subsequent autopsy revealed extensive thrombosis and oedema of the cortex, medulla and collecting system, resulting in significant enlargement of the allografts. These changes were not evident in the autografts (Figure 3.4). rt7
AUTOGRAFT ALLOGRAFT
CT
+Ì t:-
b e
c f
, ¡L.
Figure 3.3 H & E staining of tissue samples from a sheep renal autograft and an allograft. A ple-transplant wedge biopsy was collected at day 0 (a t d) and percutaneous core biopsy sampies were obtained on day 7 (b €i e) and day 6 post-transplantation (c t fl. (x50). 118
Figure 3.4 Autograft kidney (top) and allograft kidney (bottom) were examined at autopsy for evidence of rejection, including thrombosis, infarction and enlargement due to oedema. 119
3.3 Lymph ønd Urine Outpttt
With the exception of the one animal with an infection surrounding the graft, urine flow from the remaining 4 allografts continued for at least 6 days, despite the development of severe rejection evident on biopsy at day 4. In addition, lymphatic data from one of the allografts was unreliable due to difficulties in retaining the lymphatic cannula in the lymph vessel and maintaining patency of the cannula. Therefore, data on lymphatic drainage was available for 3 allografts and 2 autografts. The results presented below were obtained from one allograft and one autograft, and are representative of the animals in their respective grouPs.
The flow rate of iymph in the sheep allografts on day 0 was <5 mllhr and increased to a mean hourly rate of 45 ml/hr by day 2. The fiow of lymph continued at this increased level for several days after cessation of urine output, decreasing only as complete rejection and failure of the allograft occurred (Figure 3.5). It has been suggested that the effects of anaesthesia and surgery are responsible for the low flow rate of lymph in the early stages after cannulation, which stabilises and attains maximum flow by approximately 2
days post-surgery [Hecker, 1974].
The output of cells immediately after allograft transplantation was approximately 1 x 106 ceils/hr and this value increased to a peak of up to 8 x 108 ceils/hr between days 2 and 4. This was followed by a rapid decrease in lymph cell output, concurrent with the fall in urine production (Figure 3.6).
In comparison to the allografts, the lymph flow, cellular content and urine flow for autografts were generally stable throughout the period of sample collection. Furthermore, the lymph flow and celi output from the
autografts were approximately 10-100 fold less than the allografts (Table 3.1). 120
ALLOGRAFT
Bx Bx Bx Bx
25 125 -.r Lymph Ur ne w 00 100
I 7sS E 75 E 3 3 o o LL LL o- s0g'= E 50 ì l
25 25
ô 0 0123 456 7 I I 10
Day (post-operative)
Eigure 3.5 Measurement of allograft lymph and urine flow rates. Samples were collected on an hourly basis to enable calculation of flow rates. Kidneys were biopsied on days 1, 4, 6 and L0 to determine the onset of rejection and confirm renal failure. 121.
ALLOGRAFT
Bx Bx Bx Bx
125 100 # Lymph
@ urine 100
F* 75 o x 7ss -c g =o = o 50 II-9 o) s0g o f o- E J 25 25
0 0 0123 456 7 8 I 10 Day (post-operative)
Figure 3,6 Measurement of the cellular output in lymph and urine flow from a renal allograft. Kidneys were biopsied on days 7, 4, 6 and L0 to determine the onset of rejection and confirm renal failure. 122
Tøble 3.1 Comparison of lymph drainage from ovine renal autografts and allografts
Lymph Measurements # Autograft (n=2) Allograft (n=3)
Lymph flow rate (ml/hr) 0.7 - 1,.5 30-60
Cell ouþut (cells/hr x1.07) 0.1 - 0.63 3-80
Cell concentration (cells/ml x 1'07) 0.2 - 0.9 3 - L6.5
# Data shown represents the range of values for samples collected at day 2 post-transplant.
On day 2 the autografts demonstrated peak flow rates of l-.5 ml/hr
(Figure 3.7) and, cell output of only 6.3 x 106 cells/hr on day 2 (Figure 3.8). These observations are similar to the observations reported previously for lymph from ovine renal allografts and autografts [Pedersen and Morris, 1.970] and the afferent lymph of the ovine popliteal lymph node [Hail and Morris,
1963; Mackay et ø1.,1988a1.
Although the onset of severe rejection of the allografts was confirmed on biopsy at days 4 and 6, it is likely that the decreasing cellular output in lymph was associated with the specific accumulation of MNCs in the graft. It has been proposed that the expression of signals (e.g., cytokines, cell and ECM receptors or antigens) by the local microenvironment may act as "traffic signals" [Springer, I994f , directing whether the cells remain at the site of inflammation or continue to recirculate [Picker, 7994]. The histology of the sheep allografts demonstrated an extensive infiltration of MNCs from day 3 onwards, suggesting that many of these cells remain at the inflammatory site and fail to pass into the lymphatics. 123
AUTOGRAFT
Bx Bx Bx Bx
+ + 70 10 Lymph
Urine I
-c -c 6 E E ì 3 a _9 LL LL -co o E 4 l -J 0
2 0
0 0 0 2 34s 6 7 I Day (post-operative)
Eigure 3.7 Measurement of autograft lymph and urine flow rates. Samples were collected on an hourly basis to enable calculation of flow rates. Kidneys were biopsied on days 1,, 4, 6 and 8 to confirm the absence of rejection. r24
AUTOGRAFT
Bx Bx Bx Bx
+ + 10 10 Lymph 60 U¡ine I 50 F-o X 40 -c 6 f E o- 3 = 30 _9 o LL c) c) C) 4 c _c. l o- 20 E I 2 10
0 0 0 2 345 o 7 I
Day (post-operative)
Figure 3.8 Measurement of the cellular output in lymph and urine flow from a renal autograft. Kidneys were biopsied on days 1, 4, 6 and 8 to confirm the absence of rejection. I25
3.4 lmmunophenotypic Anølysis of Lymphøtic Drøinøge
The cell populations present in samples collected simultaneously from lymph and peripheral blood were examined by flow cytometry following staining by indirect immunofluorescence (2.2.1.1.1) using the following panel of mouse anti-sheep mAbs; SBU-TI (CDs), SBU-T4 (CD4), SBU-T8 (CD8), SBU-
II (MHC class II) and25-32 (CD44). T18-L91 was used as negative control mAb.
3.4.L CD5+ T Lymphocytes in Drøining Lymph ønd Peripheral Blood
The proportion of T lymphocytes within draining lymph was determined by reactivity with the anti-ovine CDS mAb, SBU-TI, which has previously been demonstrated to recognise an antigen present on all sheep T lymphocytes, but absent from sheep B cells [Mackay et ø1.,1985].
Flow cytometric (FACS) analysis of allograft lymph immediately following transplantation (day 0), showed 57% of cells present were CDs+ T lymphocytes. This percentage increased rapidly and by day 2 had risen to 95o/o, where it remained for the duration of the study. This compares with 37% CDs+ T lymphocytes in peripheral blood of both the recipient and donor at day 0 and this proportion showed little variation through to duy 8 post- transplantation (Figure 3.9).
3.4.2 CD4:CDS ratio ønd MHC cløss II Expressionby Cells in Drøining
Lymph ønd Peripherøl Blood
Having confirmed the earlier studies of Pedersen and Morris [1,970] regarding lymphocyte concentration and output in renal lymph, it was then possible to extend these observations by examining the distribution of CD4 and
CD8 lymphocyte subpopulations and the expression of MHC class II by cells in the lymph and peripheral blood of the allograft recipient. 126
100
90
80 -# Lymph u, * PBL 970 + rf)
860\o
50
40
30
0 1 2 3 4 5 6 7 I
Day (post-operative)
Eigure 3.9 Comparison of the proportion of CD5+ T lymphocytes in the lymph and peripheral blood of an allograft recipient. Samples of blood and lymph were collected simultaneously and the MNC population analysed. Results are representative of those seen in the allograft recipients in this study. 127
The CD4:CD8 ratio in lymph showed an initial increase from 2.2:1 on day 0 to 2.6:1, on day L, followed by a fall at day 5 to 1:1 and at day 8 a reversed value of 0.53:1 was obtained. This compares to a constant ratio of 1.3:L in the peripheral blood of the recipient (Figure 3.10)'
In addition, the proportion of MHC class II positive cells in the lymph increased from 65% to 85'/" at day L and a peak value of 95'/o on day 6 was observed. This increase in MHC class II bearing cells was not seen in peripheral blood until day 8 when it rose to 64"/o, compared to approximately
50% for days 0 to 7 (Figure 3.L1-). A similar rapid increase in expression of MHC class II has been observed on sheep MNCs stimulated in aitro with mitogen
resulting in an upregulation of class II antigens within 1 day [Mackay et ø1., 1e88bl.
Although the significance of the changes in the phenotypes of the cells within the lymph is unclear, the involvement of T lymphocytes in the rejection of allografts is well documented and has been discussed in several recent reviews [Mason and Morús, 1,986; Colvin, 7990; Hutchinson, 1991.; Kirkpatrick and Rowlands, 7992; GLII, 1993; Suthanthiran and Strom, 1994]. In
the case of acute rejection, as seen in the sheep used in this study, the response is mediated by two mechanisms, an antibody-dependant cellular cytotoxic (ADCC) response and a cell-mediated response. ADCC involves the production of new antibodies by plasma cells after exposure to alloantigen and has been shown to play a major role in renal allograft rejection in a rat model
[Gracie et a1.,1990). The second mechanism is mediated by T lymphocytes with CD4+ cells responding to MHC class II alloantigens and CD8+ lymphocytes reacting to disparate MHC class I antigens of the allograft lcili, 1993], although some functional overlap does occur between these iymphocyte subsets. 128
4.0
# Lymph
* PBL 3.0
ôo o 2.0 o\f O
1.0
0.0 0 1 2 345 6 7 I Day (posÈoperative)
Figwe 3.70 The CD4:CD8 ratios for cells collected from the lymph and peripheral btood of an allograft recipient. Samples of blood and lymph were collected simultaneously and the MNC population analysed. Results are representative of those seen in the allograft recipients in this study. 129
100
90
Lymph ah 80 # zO ! -+ PBL
8to(ú () ïo = -60o\
50
40 0 2 345 6 7 I
Day (post-operative)
Figure 3.77 Expression of MHC class II by MNCs in the lymph and peripheral blood of an allograft recipient. Samples of blood and lymph were collected simultaneously and the MNC population analysed. Results are representative of those seen in the allograft recipients in this study. 130
CD4+ T cells have traditionally been attributed the role of amplifying the inflammatory response by proliferating and secreting cytokines, resulting in the activation of both endothelium and CD8+ cytotoxic T lymphocytes (CTL). These CTL, along with ADCC, are subsequently responsible for the tissue injury and graft dysfunction associated with rejection. The lysis of cultured rat kidney parenchymal cells by CTLs demonstrated the cytolytic potential of CTLs [Nietbsvaara et a1.,79901.
Furthermore, Bishop et al. 179921 studied the movement and functions of CTL and helper T lymphocytes (HTL) following allogeneic heterotopic cardiac transplantation in mice. Their studies demonstrated a number of similarities to the changes observed in the CD4 and CD8 subpopulations in lymphoid compartments of our sheep which received renal allografts. Firstly, they noted a large increase in the number of CTL within mesenteric lymph nodes (MLN) and spleen following transplantation, which was not reflected by the HTL population. Secondly, the number of CTL in the MLN returned to normal levels by day 9 after transplantation, at which time the allograft had been rejected. This pattern of migration to lymphoid organs is consistent with the increase in CD8+ lymphocytes seen in the sheep allograft lymph associated with the onset of severe rejection and the plateauing of the CD4:CD8 ratio at the time of graft failure and rejection (see Figure 3.10).
Bishop et al. 11992] also reported that the early cardiac infiltrate was essentially all donor specific HTL, followed by a large increase in the number of CTL infiltrating at the time of peak rejection. In addition, depletion of CD4+ HTL inhibited the infiltration of CTL into the allograft and MLN. These results argue the case for CD4+ HTL amplifying the inflammatory response, through the production of cytokines such as IL-2 and IFN-y, thereby facilitating the activation of CD8+ cytotoxic lymphocytes and subsequent destruction of the graft. This was further supported by Cobbold et al. 119841who showed that 131 the depletion of CD4+ lymphocytes, but not CD8+ lymphocytes, prolonged the survival of skin allografts in mice.
The observations in the mouse are consistent with the sheep renal allograft studies presented both here and by Pedersen and Morris [1970] which demonstrated that the removal of lymph-borne CD8+ cells from circulation by cannulation of the renal lymphatics did not diminish the rejection process. Therefore, although CD8+ lymphocytes can play a major role in the
destruction of an allograft [Nietbsvaara et a1.,7990], they are clearly not solely responsible for allograft rejection and failure.
The results presented in this section provide additional information on the changes occurring to the phenotypic profile of cells in the lymph draining from a rejecting ovine renal allograft. Furthermore, the results are consistent with the proportions of CDs+ T lymphocytes, CD4 and CD8 subpopulations and the MHC class II+ cells found within the afferent lymph from the popliteal and intestinal lymph nodes of sheep [Mackay et al., 7988a; Abernethy et ø1.,1991; Mackay et nL.,1992b1.
3.5 CD44 Expression by Cells in Drøining Lymph ønd Peripheral Blood
FACS analysis of lymph and peripheral blood for the putative lymphocyte homing receptor and activation marker, CD44, revealed that all cells in the lymph were positive, whereas only 85% of cells in the peripheral blood were strongly positive (Figure 3.I2). Cells in the lymph showed a single
çp44high phenotype, whereas in the peripheral blood, MNC demonstrated a much broader profile of negative through to high CD44 expression. The CD44 phenotypes of cells from both compartments showed little variation during the period of observation (Figure 3.13). The CD44high phenotype in lymph from a rejecting allograft suggests that (L) these cells may acquire this 132
100
90
zO + t\f ()ô òe 70
# CD44 Lymph 60 * CD44 PBL
50 0 1 2 345 6 7 8 Day (post-operative)
Figwe 3.72 Expression of CD44 by MNCs in samples of lymph and peripheral blood, collected simultaneously from an allograft recipient. 133
Day I Day 5
E Ê lD att Lymph rl E E
(v¡ E o st -o E E ¿ z_ o õ o l0 10 I
E E l! E Peripheral Ð tIÞ ,ö o Blood E¡ tfT r) E o T T € e(.î f., (\¡af <\¡ É o 4 t0 1 t0
Fluorescence lntensity (Log)
Figwe 3.73 Flow cytometric analysis of CD44 expression by MNCs in samples of lymph and peripheral blood collected simultaneously from an allograft recipient. Cells were collected on day 1 and day 5 post-transplantation. Negative control mAb, TIB-191, is shown by the orange overlay. 734 phenotype through an activation-associated event as a consequence of their contact with foreign antigen, or (2) only the CD44high ."11t are able to migrate into tissue or lymph as a consequence of the interaction with endothelial ligands.
Previous studies in aitro on transendothelial migration by lymphocytes have demonstrated that the endothelium plays an important role in the
migration of CD44high çp4+ cells [Brezinschek et ø1., 7995].In the absence of ECs, fewer lymphocytes were found to migrate into the basement collagen gel layer. Furthermore, ligation of CD44 on monocytes by immobilised anti-CD44 mAb induced the production of the inflammatory cytokines, TNF-cx and IFN-y
[Webb et al., 7990]. The release of these mediators at sites of inflammation may stimulate both endothelial cells and peripheral blood lymphocytes resulting in an upregulation of cell adhesion molecules, including CD44 variant isoforms, thereby enabling the lymphocytes to bind and migrate through the endothelium.
Mobley and Dailey 1L992) reported the isolation of çp44high CD8+ cytotoxic T lymphocytes from the draining lymph node of a murine skin allograft and also a sponge matrix allograft. They propose that these cells have differentiated into effector CTL, with the ability to be highly efficient killers of allogeneic target cells and that the differentiation involved a change in the expression of several other CAMs, including increased CD44, LFA-1, ICAM-1 expression and decreased L-selectin expression. The authors postulated that the changes in CAM expression enabled the redirection of these cells to sites of inflammation, away from the normal recirculation route involving lymphoid tissue. These observations are supported by the preferential migration of çp44high CDB+ lymphocytes through endothelial monolayers during in aitro
studies of lymphocyte transmigration [Pietschmann et ø1.,1992]. 135
Based on the evidence above, it is possible to speculate that the çp44high phenotype of cells in the ovine renal allograft lymph is the result of lymphocyte activation following interaction with foreign antigen, in this case the endothelium of the allograft. CD44 has been shown to deliver co- stimulatory signals, in association with CD2 or CD3 [Shimizu et a\.,1989], and this may result in activation or upregulation of other CAMs, thereby enhancing adhesion and transmigration.
In addition, the çp44high population may also reflect the infiltration of CD4SRO+ cells and the differentiation of lymphocytes from naive to memory phenotype. Increased numbers of CD4SRO+ cells, plus cells in a transition state of CD4SRA+/CD45RO+, have been observed in rejecting human renal allografts [Akbar et a\.,1990]. Furthermore, Li et al. [1993] observed an increase in the çp44high phenotype in the peritoneal exudate of mice immunised i.p. with Listeria-monocytogenes. Despite eliminated of viable L. monocytogenes from the peritoneal cavity by day 7, the çp44high phenotype was evident for
up to 1-20 days after immunisation. This suggested the high level ol CD44 expression was associated lymphocyte activation followed by the acquisition of
a memory phenotype as a consequence of contact with foreign antigen. Similar high levels of CD44 expression have been reported for antigen-primed B cells
[Camp et a\.,1991].
The role of CD44 in homing and migration was suggested following the observations that mAbs to CD44 inhibited human lymphocyte binding to HEV
[Jalkanen et ø1., t9871 and the migration of leukocytes through endothelial monolayers in aitro [Oppenheimer-Marks and Lipsky, 1994; Brezinschek et ø1., 19951.In contrast, anti-CD44 mAbs were neither able to inhibit the binding of porcine lymphocytes to rat high endothelial cells in aitro [Yang and Binns, 1,993), nor limit the accumulation of MNCs in the draining lymph node during a cutaneous delayed-type hypersensitivity (DTH) reaction in mice [Camp et al., 1993]. A similar result was noted by Mikecz et ø1. U9951where 136 anti-CD44 mAb did not alter the ability of cells to migrate to the lymph node of arthritic mice, although the mAb inhibited migration into the inflamed synovial tissue.
The CD44 molecule has multiple isoforms due to a combination of post- translational modifications and alternative mRNA splicing of at least L0 variant exons inserted in the extracellular portion [Screaton et ø1., 7992]. Although the specific function of these isoforms is still being determined it has been suggested that the expression of particular variant isoforms may represent an "area postcode", providing specific directions for the emigration
of cells [Jackson et a|.,1992].
Thus, although CD44 does not appear to be necessary for normal leukocyte recirculation, there is some evidence that it may be involved in the
trafficking of cells to sites of inflammation [Mobley and Dailey,1992; Camp et ø1., 1993; Li et al., 1,993; Mikecz et al., 19951. Consequently, the precise role played by CD44 in homing and extravasation remains unclear. Furthermore, there is now an increasing volume of evidence demonstrating the involvement of selectins and integrins in leukocyte adhesion and transendothelial migration [Butcher, 1'991'; Springer, 1994; Alon et ø1., 7995; Girard and Springer,1995; Mackay, 1995; Tedder et øL,19951.
SUMMARY
This chapter describes the establishment of an ovine model of renal transplantation and the analysis of the lymphatic drainage collected from both allografts and autografts. In the case of the allografts, two events occurred within the first 24 to 48 hr following transplantation which clearly distinguished them from the autografts. The first is the massive influx of MNCs into the allografts which easily differentiated them from the autografts. 737
The second aspect was the significantly larger flow of lymph and cells from the allografts, which was up to 100 fold greater than the autografts'
Flow cytometric analysis of lymph revealed that cells are predominantly T lymphocytes and that the ratio of CD4 and CD8 subpopulations in lymph from rejecting grafts showed an initial increase, followed by a substantial fall, consistent with the observed increase in the proportion of CD8 positive cells. Neither the reversal of the CD4:CD8 ratio nor the increase in expression of
MHC class II antigen by cells in lymph were reflected in the peripheral blood of the recipient animal. In addition, the lymph-borne cells exhibited a single gp44high phenotype, whereas peripheral blood lymphocytes demonstrated a broader CD44 phenotype, ranging from negative to high.
Within this group of unmodified animals, the timing of the onset of renal allograft rejection was found to be consistent, with evidence of rejection detected as early as day 3, followed by complete graft failure at day 8 post- transplantation. This information was subsequently used in assessing the efficacy of an anti-LFA-1 mAb to prolong survival of sheep renal allografts, which is the subject of discussion in the next chapter. 138
Chupter 4
Studies of an anti-ovine LFA-L monoclonal antibody in aitro and a xn TxTo
INTRODUCTION
Lymphocyte function-associated antigen (LFA-1 or cxl-) is a member of the
B2 integrin family of cell adhesion molecules [Hynes, 1989], along with Mac-L (crM) and p150,95 (o¿X) and the recently described aDB2 molecule [Danilenko ef aL, 1,9951. All of these moiecules are heterodimeric structures composed of different cr-subunits non-covalently associated with a common B-subunit (CD18). For LFA-1-, the u and p subunits consist of a l-80 kDa (CD1La) and a 95 kDa
(CD18) chain, respectively [Kürzinger et al.,19BI], as discussed in Chapter 1.
The early studies of LFA-1 demonstrated this molecule to be involved in a wide range of cellular functions including T-cell mediated cytotoxicity, T and B cell activation, NK cell mediated killing and leukocyte-endothelial cell adhesion
[Davignon et ø1., 1981,; Krensky et al., 7983; Springer et ø1., 1987; Arnaout, 1990]. LFA-1 participates in this range of activities through its capacity to bind to multiple ligands, including intercellular adhesion molecule-1 (ICAM-1) [Rothlein 739 et ø1,,1986; Marlin and Springer,l987l,ICAM-2 [Staunton et ø1. ,1989a] or ICAM-3
[de Fougerolles and Springer,1992; Fawcett et ø1.,1992; Vazeux et a1.,1992].
Consequently, LFA-1, has been studied in a number of experimental and clinical settings to examine the ability of LFA-L blocking antibodies to prolong allograft survival. In addition, mAbs against one of its counter-receptors, ICAM-1, have been studied. The expression of LFA-1 on lymphocytes is particularly relevant to allograft transplantation as alloreactive T-cells are considered the primary mediators of acute rejection [Bishop et nl., 1992; Kirkpatrick and Rowlands,1992; Suthanthiran and Strom, L994; Kahan, 19961.
Flowever, whilst in oitro studies conducted by others and reported in this Chapter clearly demonstrate an inhibitory capacity for anti-LFA-l mAbs during T-cell interactions, there is conflicting evidence for the in uiao efficacy of such antibodies in allograft rejection. For example, several groups have reported that anti-LFA-l [Heagy et ø1.,1984; Fischer et ø1.,1986; Fischer et a\.,1991,; Isobe et ø1.,
1992;He et ø1.,1994; Hourmant et ø1.,1994; Le Mauff et ø1.,1'9951and anti-ICAM-1 mAbs [Cosimi et ø1.,1990; Guymer and Mandel,1992; Isobe et ø1.,1992; Haug et ø1.,
19931prolong allograft survival. Of particular interest are the studies by Isobe ef
ø1. 119921who not only demonstrated the ability of individual anti-LFA-1 or anti- ICAM-1 mAbs to significantly prolong murine cardiac allograft survival, but also the induction of long-term donor-specific tolerance by the use of a combination of these mAbs.
In contrast, others have reported no enhancement of graft survival using the same or similar mAbs [Baume et ø1., L989; Le Mauff et aL.,199L]. Moreover, there have been reports of accelerated loss of cardiac and liver allografts in rats following the use of anti-ICAM-1 mAb [Omura et al., L992] or a combination of anti-ICAM-L and anti-LFA-1 mAbs [Morikawa et ø1.,1994].
The availability of both the ovine renal allograft model and an anti-ovine
LFA-1 mAb provided the opportunity to study the effects of such a mAb in a r40 clinically relevant large animal model in order to provide further evidence for the use of anti-LFA-1 mAbs in the treatment of acute allograft rejection.
The studies presented in this chapter examine the functional effects of an anti-LFA-1 mAb, designated 72-87, which was shown to recognise the ovine equivalent of LFA-1 at the First International Workshop on Ruminant Leukocyte
Differentiation Antigens [Haig et ø1.,1991.; O'Reilly et al., 1991.; Gupta et al., 7993;
Hopkins et a\.,1993; Pépin et al. ,1993). The chapter consists of three main sections; in the first section the characterisation of in aitro effects of 72-87 are studied, while the second section investigates the use of 72-87 as a prophylactic immuno- suppressive agent in an ovine renal aliograft model. In the third section, the side effects of 72-87 therapy are discussed, including the potential effect of endotoxins and cytokine release. This work confirms lhat 72-87 recognises a functional epitope in the ovine LFA-1 molecule but fails to show improved renal allograft survival.
MATERIALS AND METHODS
O a ine Ren øl Tr ønspl ønt øti on
Five pairs of sheep underwent cross-transplantation, each receiving a renal graft from its partner, as described in Chapter 3. In these studies the remaining native kidney was also removed such that the recipient was dependent on the function of the allograft for survival. In addition, two autografts were transplanted, giving a total of 72 animals for this study. One sheep of each pair received 72-87 and the other TIB-191 commencing on day 7 post-transplant, with the exception of the last pair of allografts which both receivedT2-87 that had been produced under stringent endotoxin-free conditions (discussed below) (i.e., 6 allografts and 1 autograft received 72-87;4 allografts and 1- autograft received TIB-
191). The mAbs were diluted in a 100 ml pack of normal saline and infused daily over a period of 30 min. I4I
Graft function was monitored by measurement of serum creatinine. Graft failure was defined as occurring when both the creatinine had risen to >600 pmol/L and histological assessment of the graf.t confirmed the presence of rejection.
Sømple Collection from Trønsplønted Sheep
PeripherølBlood
Samples of peripheral blood were collected daily prior to infusion to confirm mAb binding to PBLs and for the measurement of serum creatinine concentrations, electrolyte levels and blood formed elements (i.e., WBC, RBC & platelets), which were performed by the Clinical Chemistry and Haematology laboratories at The Queen Eiizabeth Hospital. Blood samples were also coliected at 2, 4 and 6 hr after infusion of mAb to allow monitoring of effects on blood formed elements.
Tissue Sømples
For histological analysis, wedge biopsy samples were collected from kidneys both at the time of transplantation and autopsy, and daily percutaneous core biopsies were also performed.
RESULTS AND DISCUSSION
4.L Chørøcterisation of 72-87 in aitro
4.L.7 mAb 72-87 Recognises Otsine LFA-L
Lysates from sheep MNCs labelled with [1251] were immunoprecipitated with 72-87 and then separated by SDS-PAGE on a 5% gel. Under reducing and non-reducing conditions two bands were identified with molecular weights of 180 742 and 95 kDa (Figure 4.L), corresponding to the LFA-L o¿ and B subunits, respectively.
The reactivity of 72-87 with ovine PBLs was examined by indirect immuno- fluorescence staining of sheep whole blood samples, followed by flow cytometric analysis (2.2.11.1). These studies demonstrated strong staining of all cells from the peripheral blood compartment by mAb 72-87 (Figure 4.1). These results are consistent with the original descriptions of LFA-L expression on murine cells [Kürzinger et ø1.,1981), and identical to that previously reported for sheep leukocytes [Haig et al., 1991'; O'Reilly et al. ,1991'; Gupta et ø1., 1993].
4.1.2 Inhibition of Proliferøtionby 72-87
To study the ability of 72-87 to inhibít allogeneic cell proliferation, a2-way ovine mixed tymphocyte culture (MLC) was performed in the presence of increasing dilutions of mAb (2.2.3.1). Addition of 72-87 to the MLC resulted in a dose-dependent inhibition of proliferation, ranging from 63"/. lo 95'/" for ascites dilutions of L/20,000 to 1/500, respectively (Figure 4.2), which is similar to the dose-related responses reported in the early descriptions of anti-LFA-1 mAb functions [Davignon et a1.,198i].
4.1.3 Inhibition of Leukocyte-Endotheliøl Cell Adhesionby 72-87
The mAb was tested for inhibition of binding in leukocyte-endothelial cell adhesion assays (2.2.2).Inhibition of adhesion by dilutions of 72-87 ascites of
1/5000 to 1,/200 ranged from 75"/' to 39o/o, rcspectively, for unstimulated ovine
MNCs and unstimulated ShUVECs, compared to 1,0% to 37o/. for phorbol ester
(PMA)-stimulated MNCs and unstimulated ShUVECs (Table 4.1). The addition of the PMA to the MNCs resulted in a dramatic increase in binding to unstimulated 143
ø
Mr 200 > rDI|; 116 > 97 -> 66 + 42 >
æ z B o. TJ J o
b
g ranulocyt es o) -9 o co o o U' oL o monocyt es tE lymphocyt es
cell size
Fìgure 4.7 Chancterisation and distribution of ovine LFA-1 on sheep PBLs. 12sl-labelled sheep MNC surface proteins were immunoprecipitated wíth72-87 or the negative control mAb TIB-1-91 and then separated under reducing and non- reducing conditions on a 5% polyacrylamide gel (a). The LFA-L cr-subunit corresponds to a band at 180 kDa and the B-subunit to a band at 95 kDa. Pnnel b, flow cytometric analysis of LFA-1 expression by sheep PBL. The distribution of LFA-I on sheep PBLs was examined by indirect immunofluorescence staining of whole blood samples with72-87. 1,M
100
75
cr) o
x -50 *** * * *
o_ o
25
0 Ø Ø o 5 t o N) ('l oì- f\) o (D o o o o- = o o o o o o E E õ' o o o o o o o (p o o o = f 6 72-87 dilutions
Eígure 4.2 Dose-dependant inhibition of proliferation in an ovine MLC. Dilutions of 72-87 ascites were added to wells containing allogeneic sheep MNCs (6 well replicates) and the level of proliferation determined by the uptake of 3H- thymidine. * p< 0.001, relative to cultures treated with control mAb, TIB-191. 145
ShUVECs (Table 4.1), consistent with the described change in conformation and increase in avidity of LFA-1 binding following MNC activation [Dustin and Springer, t989l.In spite of PMA stimulation, T2-87 was still able to significantly inhibit adhesion (from 1.5"/. to 21%; Table 4.\), consistent with the major role played by LFA-1 in MNC binding to unstimulated VECs [Haskard et al., 1986;
Amaout et ø1.,1988; Oppenheimer-Marks et ø1.,1991,; Meerschaert and Furie, 1994;
Issekutz and Issekutz,1995; Meerschaert and Furie, 19951.
Adhesion assays were also performed with PMA-stimulated MNCs and ro-
TNF-cr-stimulated ShUVECs. Under these conditionsT2-87 demonstrated minimal inhibition of binding (Table 4.L). This is in accordance with previous reports that other LFA-l-independent pathways come into play following activation of endothelium [Haskard et al., 1989; Carlos et a|.,1990b]. Consequently, blocking only one adhesion pathway is likely to have little effect on the ability of leukocytes to bind to activated endothelial cells.
4.1.4 Cytotoxicity of 72-87 in aitro
The cytolytic effect of 72-87 on ovine MNCs in aitro was studied using a complement-mediated cell lysis assay (2.2.6).In the presence of exogenous rabbit complement,T2-87 displayed a dose-dependent cytotoxic response which was not evident in wells containing either 72-87 without complement, or the negative control mAb with complement (Figure 4.3). Therefore it would appear that72-87 is capable of triggering complement fixation, resulting in the initiation of the complement cascade and ultimately the lysis of Ab-coated cells. Tøble 4.1 Comparison of the adhesion of MNCs to endothelial monolayers under different stimulation conditions in aitro#
UnstimMNC & unstimVEC PMA-stim MNC & unstim VEC PMA-stim MNC & TNF-stim VEC ol ol ol mAb No. cells /o significance No. cells /o significance No. cells /o significance dilution 4 bound b inhibition c bor:nd b inhibition c Q)d bound b inhibition c (P)d r/200 52r8 37.3 0.003 243 t12 21.1 0.0004 255 + 52 7.9 NS
1/500 56+1L 32.5 0.012 231, + 17 25.0 0.0022 370 + I02 0
1/1000 63+20 24.1, NS 262+ 34 74.9 NS 289 + 67 0 t/2000 73 +'J,4 t2.t NS 327 + 32 0 281, + 73 0
1/s000 75+5 9.6 NS 337 + 1,6 0 279 + 81, 0
1/5000 75 t5 9.6 NS 337 + 16 0 279 + 81 0 TIB-191 83+L0 308 r 14 227 + 45
# dutu from quadruplicate wells in one experiment and representative of 3 similar assays a dilutions of ascites of anti-LFA-1 mAb, 72-87 or 1/500 dilution of negative control mAb IB-191 b M"urr + SD of No. of cells bound per high power field from quadruplicate wells c percentage (%) inhibition compared to No. of cells bound in wells containing negative control mAb, T18-19L d statistical significance of No. of cells bound at each dilution of 72-87 compared to TIB-l9L calculated using paired Student's t-test Ê rÞ o\ L47
100
..# 72-87 + C' 80 o 72-87 - c' o\\o q) o TIB-191 õ C)OU o
40
20 ooooooooo oooooooooc\tsco(oNt@(o cÐ (o Õl ro C\I
mAb dilution
Figure 4.3 Cytotoxicity of 72-87 in uitro. Dilutions of 72-87 or TIB-l9L ascites were added to wells containing sheep MNC in the presence or absence of complement. The viability of cells was assessed by the exclusion of eosin dye. 1.48
4.2 In aizto Studies
4.2.1- mAb Dosage ønd SøfetY
Prior to the transplantation experiments, safety and dosage regimens for these mAbs were evaluated in two non-transplanted sheep. FACS analysis of peripheral blood MNCs was used to determine the daily dose of 72-87 which would provide pre-dosin g 24 hr trough blood levels of mAb sufficient to ensure MNCs remained coated wíth 72-87. Tlne dose of mAb used in the non- transplanted sheep was estimated from similar earlier studies in humans [Fischer et ø1.,1986).
Blood samples were taken from the two non-transplanted sheep immediately prior to infusion and then at L hr and 24hr post mAb injection. The pre-injection samples were stained in aitro by indirect immunofluorescence staining methods (2.2.11,.1) with either 72-87 or TIB-L9L to determine the maximum level of staining under saturating concentrations. Samples collected after mAb injection were stained with only the FlTC-conjugated sheep anti-mouse secondary antibody to detect mAb which bound to the MNCs whilst in circulation. Using this method, a daily dosage of 0.1'25 mg/kg/day (approximately 5 mg/ day) was established which was administered i.v. for up to
10 days.
FACS analysis of PBL following injection of 72-87 showed near saturation of LFA-1 on circulating lymphocytes up to 6 hr after injection, with significant binding still detectable at24hr, compared to the level of staining achieved in aitro using saturating concentrations of 72-87 (Figure 4.4). The two non-transplanted animals showed no evidence of adverse effects following infusion of the antibodies for up to 1-0 days. There were no changes in temperature, respiration or eating patterns, nor were there any variations in haematological or biochemical parameters. 149
a l¡ E rE ÚIo ro rrl (\t
o It 1
b ñJÊ
É bE -()Uc ZE=Ë ()(l)fl E _-¡----_-".-* tü 10
c
E co
,E' rE E
r.J 4 I 10 10 It Fluorescence lntensity (log)
Figure 4.4 FIow cytometric- analysis of 72-87 and TIB-191 binding in aiao and in aitro to sheep MNC. Panel a shows maximum level of binding achieved in aitro under saturating mAb conditions. Panel b indicates the level of saturation on circulating MNCs L hr after injection of. 72-87, and panel c shows the level of binding of 72-87 to MNC in aiao at24l;rr after injection, immediately prior to the next dose. TIB-191 staining of MNC samples is shown by the orange overlays. 150
4.2.2 Effect of 72-87 on Renal Allogrøft Suraiztøl
In spite of the strong in aitro inhibitory actions of 72-87, there was no prolongation of allograft survival in the animals receiving anti-LFA-l mAb. Graft failure occurred significantly earlier (p<0.05; Wilcoxon rank sum) in the animals receiving 72-87 (n=6), at a median of day 7.0 (10.5), compared with day 9.3 (t 0.5) for the control group (n=4). There was no loss of renal autografts (Figure 4.5). In tine 72-87-treated animals, creatinine and urea levels showed early increases compared to the control animals, typically rising rapidly from day 4 onwards (Figure 4.6). Notwithstanding the accelerated allograft failure in the animals receiving 72-87, the autografted animals showed no signs of graft dysfunction and creatinine levels remained stable for the duration of treatment (Figure 4.6).
Daily assessment of creatinine, urea and electrolyte levels and liver function tests revealed that with the exception of creatinine and urea, there was no significant difference in the concentration of several clinically relevant parameters (e.g., ALP, bicarb. & phosphate) in the plasma of unmodified allografts, autografts and non-transplanted sheep (Appendix 1).
4.2.3 Effects of 72-87 on Graft Infiltrøte
Histology of the allografts at autopsy revealed intense interstitial MNC infiltrates consistent with acute cellular rejection. There was no difference in the level of MNC infiltrate observed in the kidneys of the animals treated with72-87 compared to animals receiving TIB-1,91 (Figure 4.7), indicating 72-87 was unable to reduce MNC adhesion and migration into the allografts.
At autopsy, histopathology of kidneys from both treatment groups revealed evidence of significant oedema throughout the cortex, medulla and collecting system, identical to that seen in the kidneys from the unmodified animals
(Chapter 3; Figure 3.4). 151
100
80 r ¿ J 60 U) g (5 (n=6) \o 40 # 72-87 Allo
TIB-191 Ano (n=4)
20 -# 72-87 Auto (n=1) # TIB-191 Auto (n=1) 0 0 1 2 3 4 5 6 7 I I 10 11 12
Days (Post-Tx)
Eigure 4.5 Survival of ovine renal allografts and autografts in sheep treated with either 72-87 or TIB-L9L. The percentage of functioning grafts is shown for each day of the observation period. 152
1 000 a 4- Allo72-87 800 -J Allo TIB-191 =\o * E ¿ * Aulo 72-87 o 600 .c .c # Auto TIB-191 (ú 0) o 400 E o U) 200
0 0 1 2 3 4 s 6 7 I I 10 11
Day (post-operative)
70 þ .# Allo72-87 60 * Allo TIB-191 50 * Aulo 72-87 J o 40 Auto TIB-19'l E + E (ú 30 lI 20
10
0 01234 567 I I 10 11
Day (post-operative)
Figure 4.6 Creatinine concentrations in ovine renal allografts and autografts of sheep treated with either 72-87 or TIB-191. Creatinine (panel ø) and urea levels (panel b) were measured on a daily basis and graft failure was assessed as creatinine > 600 pmol/L. The data shown are from one pair of allografts and one pair of autografts and are representative of the data obtained from each group of animals in the study. 153
tl
l.',.
j a
t
I é ie' t ¡ çr t.. f, '1i ¡ t r.¡ \ Ít t;.. o I I t I !¡ ì \
I +
N ?-
f .-'
','. .t: ¡ l..¡¡-. ,t ,¡. 1.,' t l¡2 "- '
a tat..
lr
Figu'e 4.7 Htstokrgv of ther ren¿rl alklgrafts at aLìtopsy. H & E staining rlf kidnev sectic)r-rs from an animal treated vtitl¡,72-87 (¡tnrrcl ¿), and a cotìtrol a11irììal treatecj with TIB-19I (¡tutrcl b). (x50). 1.54
4.2.4 Effect of 72-87 on Peripherøl Blood Leukocytes
4.2.4.L White Cell Counts
Samples of peripheral blood were collected dally, and in some instances at 2 hourly intervals, after mAb infusion for analysis of WCC and lymphocyte subsets. The daily monitoring of WCC revealed no depletion in circulating cell numbers over the course of treatment. However, the WCC of samples taken atZ,4
and 6 hr post mAb injection revealed a 3-fold decrease in cell numbers, which
subsequently returned to normal or slightly higher by 24 hr (Table 4.2).
The depletion of leukocytes immediately after administration of 72-87 may be associated with complement-mediated lysis of cells in the peripheral blood or opsonisation and phagocytosis of Ab-coated ceils, similar to the mode of action
reported for the anti-T cell mAb, OKT3 lCosimi et al., 1981,; Abramowicz et aL, 1989; Debets et ø1., 19891. The dyspnoeic and tremulous state of the sheep (discussed below) is consistent with the reports of rapid TNF release by human peripheral blood monocytes during opsonization and phagocytosis of Ab-coated
cells [Nathan et a1.,1980; Debets et ø1.,1988].
4.2.4.2 CD ønd CD8 subpopuløtions
FACS analysis of the CD4+ and CD8+ lymphocyte subpopulations in the peripheral blood of 72-87 treated animals revealed the CDB+ population decreased from 75"/" to 4% ln the 6 hr after mAb infusion. Unlike the WCC
decrease, this reduction in CD8+ cells was not transient and remained low for the duration of the study (Table 4.2), suggesting the preferential lysis of this sub-
populationby 72-87.
There was less effect on the CD4+ population which, like the CD8+ cells,
initially showed an increase in numbers from approximately 20"/" pre-transplant L55 to 25% immediately after surgery. This was followed by a decrease to a plateau of approximately 15"/' lor the remainder of the study (Table 4.2).
Tøble 4.2 White cell counts and % T cell subsets for sheep treated with anti-LFA-1 mAb and control mAb #
72-87 treated-Sheep a TIB-191 treated-Sh eep b
Duy WCC x 109 CDA%C CDg% WCC x 109 CD % CD8 %
0 (pre-Tx) 6.3 20.6 13.6 7.0 20.0 t2.3
1, Ts hr 6.6 25.7 1,5.9 9.2 23.1 14.6
1T2hr 1,.9 1,4.6 4.2 9.4 21.0 11.3
L T¿hr 2.1. 15.6 3.9 7.1 21.1. 11.8
lTohr 2.9 15.0 4.0 7.1. 20.6 L1,.2
2 8.8 12.2 3.8 6.0 21,.2 72.3
õ J 7.3 14.0 4.L 5.9 19.8 17.9
6 7.5 1,4.1, 4.r 7.2 21.3 1.t.2
7 10.0 21,.0 12.2
8 7.2 20.8 11.0
9 9.2 21,.2 11.1
# th" dutu shown are from one pair of animals and are representative of all sheep studied a data representative of 6 sheep treatedwirhT2-87 b duturepresentative of 4 sheep treated with TIB-191 c figures are percentage of MNCs
There was no significant variations in either CD4 or CD8 populations in the animals receiving TIB-191., with the exception of a slight increase in cell numbers the day after transplantation as seen with the 72-87-Ireated animals, presumably due to surgical trauma.
A transient leukopenia induced by mAb has also been observed by others whilst studying the side effects of two anti-CD3 mAbs used prophylactically to 1.56 treat renal transplant patients [Parlevliet et a\.,L994]. An alternative hypothesis to cell lysis has subsequently been proposed which suggests that the lymphocytopenia observed following infusion of mAb is the result of T cell activation and transient adhesion of lymphocytes to the vascular endothelium
[Buysmann et a\.,1994]. Furthermore, Buysmarvt et aI 11994) demonstrated in aitro that maximal activation of CD3+ cells occurred within 3 min of the addition of
OKT3 to human MNCs, followed by a gradual decrease in activation status over
the next 4.5 hr. This response could account for variations seen in WCCs observed
in the sheep after infusion of 72-87 as deactivated MNC progressively detach and
return to circulation. SimilaÃy, Raasveld et ø1, 11992] reported sequestration of activated granulocytes within the pulmonary vasculature of renal allograft
recipients treated with OKT3.
In addition, the FACS analysis of 72-87 stained peripheral blood leukocytes
(see Figure 4.1) demonstrates that the mAb is highly reactive with PMNs. While
the effects of 72-87 binding to neutrophils is unknown, it has been reported that mAbs against LFA-1 (and p150,95) activate human neutrophils resulting in the triggering of a respiratory burst involving the release of H2O2and O¡ from
neutrophils [Berton et ø1., 1,992]. Furthermore, von Asmuth et al. 11991] demonstrated that in the presence of TNF-ct or PMA, activated neutrophils mediated endothelial cell damage.
4.3 Side Effects of 72-87 Therapy
Although the two non-transplanted animals demonstrated no adverse effects from the mAb, several of the allograft recipients and the autografted
animal showed signs of dyspnoea and became tremulous during each infusion.
The possible mechanisms which may account for these side effects and the
transient leukopenia include (1) cytokine release syndrome, (2) endotoxin in the 157 mAb preparations, (3) sequestration of PBL into organs such as the lungs and, (4) widespread cell lysis and/or phagocytosis.
It is possible to speculate that the most likely causes of the side effects of 72-87 treatment are due to activation and sequestration of PBLs following cytokine release. The dyspnoeic response of the sheep to 72-87 is consistent with the accumulation of cells within the lungs. This could be due to the presence of large amounts of cytokines in circulation, similar to the cytokine-release syndrome seen during the treatment of acute allograft rejection in humans following the infusion of OKT3 [Abramowicz et ø1., 1989; Debets et ø1.,1989]. Although the release of cytokines after OKT3 therapy has generally been associated only with a "first-dose response", there have been several reports describing patients experiencing adverse reactions and cytokine reiease during second and subsequent doses of OKT3 [Norman et a1.,1987; Vasquez et ø1.,1.995], similar to that observed in the sheep throughout the 10 day course of therapy.
Therefore the binding of 72-87 to sheep PBL may have resulted in the activation of leukocytes with two possible inter-related consequences. Firstly, activated PBLs may release large amounts of inflammatory mediators, as suggested by increased expression of TNF-cr mRNA by human T-cells following the binding of an anti-LFA-L mAb in aitro lEan et ø1,,7993]. Similarly, human NK cells are reported to produce TNF-cx in the presence of soluble anti-LFA-1 and
anti-CDl-6 mAbs [Melero et ø1,, 79931. Secondl], activation of leukocytes, in conjunction with the production of cytokines, may result in an increase in the avidity of adhesion of cells to the endothelium, thereby facilitating their transient
sequestration from circulation as described earlier.
4.3.7 Endotoxin Leaels in Monoclonal Antibody Preparøtions
An alternative explanation for the laboured breathing and tremors observed in the sheep could have been the presence of the small amounts of 158 endotoxin in the mAb preparations, which themselves may have generated the pyrogenic effects. There was the additional concern that this may have promoted a systemic inflammatory response resulting in the induction of cell adhesion molecules, thereby promoting the infiltration of MNC into the allograft and exacerbating the rejection process. This response may have contributed to the accelerated loss of grafts in animals receiving 72-87.
Flowever, the original batches of 72-87 contained endotoxin levels of 6
EtJ /mI, which equates to 0.27 Eulkg in an average 45 kg sheep. As this is well below the FDA approved standard of 5 EUlkg for injectible grade human pharmaceuticals [Munson, 1985; Munson, 19871, these levels of endotoxin should not have any effect on the animal's physiology.
Unfortunately at the time of conducting these experiments it was not possible to measure the levels of ovine cytokines in plasma or urine samples, which may have helped determine whether 72-87 was responsible for the systemic release of inflammatory mediators.
Nevertheless, to exclude this possibility antibody stocks were produced under stringent endotoxin-free conditions (2.2.8) resulting in a 15-fold decrease in pyrogen levels (down from 6EU /mI to 0.38 EUlml).When this latter batch of 72- 87 was administered to a pair of allograft recipients, both animals displayed evidence of dyspnoea and tremors. These observations suggest that the side effects elicited by 72-87 were not endotoxin related and probably represented a cytokine-release phenomenon.
The release of such mediators may contribute to the upregulation and de noao production of cell adhesion molecules, including ICAM-I and VCAM-L, as observed during allograft rejection [Bishop and Hall, 1989; Faull and Russ, 1989;
Rice ef ø1.,L991.; Andersen et ø1.,1992; Wuthrich,1992; Fuggle et aL.,L993;HJIL et al.,
19951. The availability of other adhesion pathways (e.g., VCAM-1/VLA-4), and the multiplicity of ICAM-L ligands (LFA-1-, Mac-1, CD43), provides alternative 159 mechanisms for leukocytes to adhere to and migrate through the endothelium
and initiate the rejection process.
Ishikura et ø1.119911 demonstrated the induction of ICAM-1 on cultured human renal tubular epithelial cells after incubation with either recombinant cytokines or supernatant from a mixed lymphocyte reaction. The authors speculate that the upregulation of ICAM-1 after the release of cytokines (IFN-1 and TNF-cr) by T cells and APCs may result in increased adhesion of graft- infiltrating T cells to the renal tubular cells, via the LFA-I/ICAM-1 pathway. Although not examined by the authors, increased T celi attachment presumably
also involves the VLA- /VCAM-1 pathway, as VCAM-1 is also induced by TNF-
cr and has been detected on inflamed tissue, as described above.
This redundancy in cell adhesion molecule pathways has been well highlighted by the ability of T cells from leukocyte adhesion deficiency (LAD) patients to migrate through endothelial monolayers/ despite the T cells being
LFA-1 negative [Haskard et aL,1989; Vennegoor et ø1.,1992].
Therefore, both activation and cytokine release could potentially account
for the adverse side effects and transient leukopenia observed following each dose of 72-87. In addition, studies in oitro have also demonstrated that different adhesion molecules are utilized during lymphocyte-endothelial cell adhesion, depending upon both the differentiation and activation status of the T-
lymphocytes [Oppenheimer-Marks et ø1., \990]. Thus the inability of anti-LFA-1 mAbs to completely inhibit MNC transendothelial migration is not only a result of the redundancy within the cell adhesion molecule pathways, but may also
depend on the maturation state of the lymphocytes.
Thus blocking multiple adhesion pathways with mixtures of mAbs may be more efficacious than targeting only one CAM in a highly redundant system [Hutchinson,7991]. Therefore, to examine the efficacy of blocking multiple adhesion pathways in aiao, a number of sheep were given a combination of anti- 1.60
LFA-1 and anti-VCAM-L mAbs. The results from those studies are discussed in detail in Chapter 7.
SUMMARY
The studies presented in this chapter examined the functional capabilities of an anti-ovine LFA-I- mAb in aitro and in aiao. The capacity of 72-87 to inhibit MNC proliferation and adhesion in aitro demonstrater its recognition of a functional epitope within the ovine LFA-I- molecule.
There are numerous conflicting reports in the literature concerning the efficacy of anti-LFA-L mAbs when used as therapy in transplantation. Thus it was of interest to study a functionally active anti-LFA-1 mAb in the clinically relevant large animal model of ovine renal transplantation in an attempt to provide further evidence for the use of anti-LFA-L therapy.
Flowever, when used as an immunosuppressive prophylaxis in the ovine allograft model, 72-87 was unable to either prevent graft rejection or inhibit MNC infiltration into the allografts, compared to animals treated with a control mAb.
The reasons for this are not clear, although it is likely the apparent adverse systemic effects evident after 72-87 infusion may have contributed to rapid rejection of the grafts. In particular, the fact that side effects were generated after each infusion and not just the initial dose, may have exacerbated the rejection process by continually inducing expression of cell adhesion molecules, thereby enhancing adhesion and migration of MNCs into the graft.
This response is consistent with a recent hypothesis suggesting that treatment with some mAbs, such as OKT3, results in a transient lymphocytopenia due to adhesion of lymphocytes to the endothelium following activation of LFA-1.
Unfortunately it was not possible to determine if similar events were occurring in this model due to an inability to study the activation status of the ovine MNCs. 167
Furthermore, during allograft rejection cell adhesion molecules are induced and/or upregulated, thereby providing multiple pathways for MNC adhesion and migration through the endothelium. Thus, blocking only the LFA- 1/ICAM-L complex is unlikely to provide the level of inhibition of adhesion necessary to prolong graft survival. This was evident in uitro where varying the activation status of both the MNC and endothelial cells dramatically affected the ability of 72-87 to inhibit MNC adhesion to ShUVECs.
In view of the in aitro data presented here and by others demonstrating the important role played by adhesion molecules expressed by activated endothelial cells in the adhesion of MNC, it was of interest to extend both the in aitro and in oiao studies to include combination mAb therapy.
However, there are few, if any, mAbs directed against endothelial cell adhesion molecules, such as ICAM-L or VCAM-1, which recognise the equivalent ovine molecules and it therefore became necessary to produce these mAbs before being able to proceed to the mAb combination studies. In order to generate such antibodies it was also necessary to establish a supply of ovine endothelial cells.
The sheep umbilical cord vein was chosen as the source of ovine endothelial cells and the isolation and characterisation of these cells are described in the following chapter. 1.62
Cnapter 5
Characterisation of ovine umbilical vein endothelial cells and their expression of cell adhesion molecules: comparative study with human endothelial cells
INTRODUCTION
The ability to isolate and culture vascular endothelial cells (ECs) fJaffe et ø1.,1973; Gimbrone þ., 1.976; Johnson, 1980] has enabled their extensive study in aitro. Much of the understanding of vascular biology has come from the use of ECs isolated from large vessels, such as human umbilical vein endothelial cells (HUVECs).
As discussed in Chapter L, it is now well accepted that ECs do not merely
serve as a passive lining of the vascular system, but are actively involved in a range of immunological and haemostatic functions, including antigen presentation, cytokine production and modulation of vascular tone and thrombogenesis [Pober and Cotran, 1,990b]. HUVECs have been used extensively, both in the host laboratory and by others, to examine both the 1.63 expression of cell adhesion molecules (CAMs) on ECs and the role these molecules play in the interaction with leukocytes.
Activation of human endothelial cells in aitro by the addition of cytokines or LPS has been shown to induce multiple adhesion pathways and enhance MNC adhesion lYu et ø1., 1986; Arnaout et al., 1.988; Oppenheimer- Marks et ø1., 1990; Oppenheimer-Marks et ø1., 1991,; Meerschaert and Furie, 1994; Issekutz, 7995; Meerschaert and Furie, 79951. Similarly, in oiuo, endothelial cells at sites of inflammation have also demonstrated de noao and upregulated expression of CAMs [Bishop and Hall, L989; Faull and Russ, 7989; Rice ef al., 1991,; Andersen et ø1., 1992; Wuthrich,7992; Fuggle et ø1., 19931. Consequently, the inability of the anti-LFA-1- mAb, 72-87, to prolong allograft
survival, as described in Chapter 4, could in part be attributed to the induction of CAMs on the sheep renal vascular endothelium. It was therefore important to investigate the expression, induction and receptor-ligand interactions of ovine endothelial CAMs to determine if they function in a manner which we would predict from studies with human endothelial cells.
Thus in order to study ovine leukocyte-endothelial cell interactions and ovine EC adhesion molecule expression, it was first necessary to establish an adequate supply of ovine endothelial cells.
Whilst the aorta is a well established source of bovine endothelial cells [Mueller et ø1., 1980], sheep endothelial cells have previously been isolated from large vessels such as the carotid artery or jugular vein [Abernethy and
Hay, 1989; Borron and Hay, 19941. However, as discussed in Chapter 1, there is considerable heterogeneity between ECs derived from different anatomical sites [Fajardo, 1989; Kinjo et ø1., 1,989; Swerlick et al., 1991-; Page et nl., 7992]. Consequently, the decision was made to culture cells from the sheep umbilical vein to allow direct comparison with cells derived from the equivalent human vessel. 164
The work to be presented in this chapter describes the isolation and characterisation of sheep umbilical vein endothelial cells (ShUVECs) and examines the kinetics of expression of intercellular cell adhesion molecule-l (ICAM-l) and vascular cell adhesion molecule-l (VCAM-1) by these cells. Each of these aspects was studied concurrently in HUVECs to provide a comparison.
MATERIALS AND METHODS
Isoløtion ønd Culture of Endotheliøl Cells
Endothelial cells were obtained from sheep or human umbilical cord veins essentially by the method of Jaffe 119731, with some modifications [Grooby et ø1,, 1997], as described in Chapter 2.2.1..2. As a general rule, cells from individual cords were cultured separately.
RESULTS AND DISCUSSION
5.7 Isoløtion of ShUWCs
The anatomy of the sheep umbilical cord differs from that of the human cord in three major areas. Firstly, there are 3 veins and 2 arteries traversing the length of the umbilical cord, compared to a single vein (and 2 arteries) in human cords. Secondly, the cords are relatively short, generally only 10 cm in length. Thirdly, the vessels are encased in a soft, semi- transparent jelly-like matrix as opposed to the solid, coiled tissue of human cords (Figure 5.1). The presence of 3 veins in the sheep umbilical cord aliowed multiple cannulations. 165
Figre 5.I Comparison of a full-length sheep umbilical cord (Ieft) and a partial length of human umbilical coñ (right). 1.66
5.2 Phenotypic characterisøtion of ShUWCs
In general, sheep umbilical cords provided fewer ECs compared to the human cords. Consequently it took approximately 7 days for the ShUVEC cultures to become confluent in a 25 crn2 flask as opposed to 4-5 days for HUVECs. When grown to confluence ShUVECs displayed the characteristic "cobblestone" monolayer of cultured ECs when examined under inverted phase contrast microscopy (Nikon, Japan) (Figure 5.2). In comparison, sheep and human fibroblasts, which may contaminate isolated EC cultures, are elongated in shape and grow in clearly recognisable parallel swiriing patterns with multiple overlapping layers [Jaffe et ø1., 1973]. After reaching confluence the ShUVECs were then subcultured at the same rate as HUVECs, approximately every 3-5 days with a split ratio of 1.:3, without observable
change in morphology or over growth by contaminating cells up to passage 10.
5.3 Metøbolism of Acetylated-Low Density Lipoprotein (Ac-LDL)
Another distinguishing marker of EC phenotype is the ability to metabolise Ac-LDL. ShUVECs, HUVECs and fibroblasts from both species were
evaluated for their ability to metabolise Ac-LDL (Chapter 2.2.5). Both groups of ECs demonstrated strong uptake and metabolism of the fluorescent-labelled Ac-LDL, while sheep fibroblasts, obtained from sheep nuchal ligament explants, and the human fibroblast cell line, IMR-90, showed minimal binding of the molecule (Figure 5.3). It has been shown previously that ovine smooth muscle cells are also unable to metabolise Ac-LDL [Abernethy and Hay,1,989]. These data confirm the endothelial nature of the umbilical vein isolates and provide a marker to assess the purity of the cultures following isolation from collagenase digested sheep veins. The data presented here demonstrate that the ShUVEC cultures were >97"/. pure, confirming minimal contamination by fibroblasts or smooth muscle ceils. t67
OVINE HUMAN
Figure 5.2 Phase contrast photomicrograph of ovine and human endothelial cell monolayers. The typical "cobblestone" morphology is exhibited by
ShUVECs and HUVECs at fourth Passage (x10). 1.68
5.4 Endotheliøl CeII Phenotypic Møtkers
5.4.1- Expression of oon Willebrønd Føctor
The expression of vWF was examined on ShUVECs and HUVECs by indirect immunofluorescent staining (2.2.1,1,.1) and immunoperoxidase staining of cytospun cells (2.2.7I.2).
FACS analysis of ShUVECs demonstrated levels of expression comparable with HUVECs (Figure 5.3). Immunoperoxidase staining of cytospun ShUVECs revealed strong granular cytoplasmic staining which was also evident in HUVECs (Figure 5.4).
5.4.2 Expression of P-selectin
The expression of P-selectin was demonstrated in both ShUVECs and HUVECs by strong staining with an anti-human P-selectin polyclonal antibody (Figure 5.3). In humans, the fact that the tissue distribution of P-selectin is restricted to vascular endothelium, platelets and megakaryocytes [McEver ef ø1.,79871further supports the endothelial phenotype of the ovine cell isolates.
5.4.3 Weib el-P øløde b o dies
Electron microscopic examination of cultured ShUVECs and sections of aorta and jugular vein failed to reveal any Weibel-Palade bodies (WPB), which are generally considered as one of the classical phenotypic markers of ECs
[Weibel and Palade, 1964j. However, the absence of WPBs from ShUVECs is not unusual as there are several reports describing the heterogeneity of WPB distribution in ECs, including their absence from bovine aortic ECs [Schwartz, 1.978), rarely detected in monkey arteries [Trillo and Prichard, 1,9791 169
OVINE HUMAN
F- rE'Ð
Ac.LDL
o t0 tfl4r 1û
|.- oo E z vWF oo
tû Itl
o E IJT flÌ Tõ '= sl fî P-selectin (\¡
1û lfl
Fluorescence I ntensity (Log)
Figure 5.3 Flow cytometric analysis of the expression of endothelial cell markers by ovine and human ECs. Uptake of Dil-Ac-LDL by ShUVECs and HUVECs (blue histograms) was compared to species-specific fibroblasts (green overlay) stained under the same conditions. The expression of vWF and P- selectin (blue histogram) was examined by indirect immunofluorescence staining with polyclonal Abs directed against human vWF, P-selectin or a control mAb, TIB-191 (orange overlay). 170
4
b
Figure 5.4 vWF expression by cultured ovine and human endothelial cells. Samples of ShUVECs (a) and HUVECs (b) were cytospun onto slides and the expression of vWF assessed by indirect immunoperoxidase staining. (x50)' 177 and variable expression in cells from different sites of the porcine vascular tree
[Fajardo, 1989; Reidy et ø1., 1.989; Gebrane-Younes et al., 1991].In comparison, these rod-shaped tubulated organelles were confirmed to be present in HUVECs.
5.5 Stimuløtion of Endotheliøl Cells - Expression of Cell Surføce Molecules
Due to the unavailability of ovine IFN-1 and the lack of responsiveness of ovine cells to IFN-y from other species (personal communication, Dr. A. Nash), a crude source of ovine cytokines was prepared by conditioning sheep MNC with Con A (Chapter 2.1,.2). This followed the observation that co- cuiture of sheep MNC with ShUVECs for 67 hr resulted in the induction of VCAM-1 expression on the surface of the ShUVECs (Figure 5.5), similar to the induction of endothelial MHC class II observed during co-culture with allogeneic human MNCs [Pober et ø1., 1983b]. Several groups have previously reported the use of medium conditioned by activated T-lymphocytes to study the expression of MHC antigens on cultured human endothelial cells and shown it to be effective in stimulating HUVECs lPober et ø1., 1983b; Wagner ef
ø1., L9851.
5.5.L Expression of MHC molecules
After stimulation with Con A conditioned medium (CM) lor 72 hr, ShUVECs showed upregulation of MHC Class I above constitutive levels and de noao expression of MHC Class II (Figure 5.6). Similar profiles were observed for HUVECs stimulated for the same period of time with rh-IFN-y
(Figure 5.6).
The absence of MHC Class II expression on unstimulated ShUVECs, followed by its strong induction by CM, is consistent with previous reports on 172
E a u1 rE
fr)Ë
(\¡e o
E
o -o E Zп b:]L EE Þtn rÉ ||¡ E ß¡ = E 4
+ Fluorescence lntensity (Log)
Figwe 5.5 Flow cytometric analysis of VCAM-1 expression on stimulated sheep endothelial cells. ShUVECs were co-cultured with allogeneic sheep MNCs for 67 hr (ø) or incubated overnight with conditioned medium from Con A stimulated sheep MNCs (b) and then stained with HAE2-1. VCAM-1 expression on unstimulated cells (green overlays) and negative control mAb
TIB-191 (orange overlay) are also shown. 773 the functional behaviour of HUVECs and other large vessel endothelial celis
[Pober et ø1.,1983b; Collins et aL,1984; Geppert and Lipsky,l9B5; Hughes et øL, 1990a; Page et ø1., 19921. Similarly, the high level of constitutive expression of MHC Class I on resting ShUVECs, and the degree of upregulation of this molecule on stimulated ShUVECs, ate comparable to the profiles seen on
HUVECs cultured under similar conditions [Pober et ø1., 1983b; Collins et øL,
1984; Geppert and Lipsky,1985; Favaloro et ø1.,1990; Favaloro, 19931.
Based on the induction profiles for the ShUVEC surface molecules described above it would appear that the CM obtained from sheep MNC contains a mixture of cytokines. MHC class II expression on HUVECs can only be elicited by IFN-y and not TNF-cr, although they both have the ability to increase MHC class I expression [Pober et ø1.,1983b; Collins et ø1., 1.984; Pober et al., 1986b1. IL-1P is unable to induce expression of either MHC class I or II antigens [Collins et al., 1,984). Therefore the increased expression of these molecules on CM-treated ShUVECs indicates the presence of IFN-1in the CM. However, the induction of VCAM-Iby either the allogeneic sheep MNCs, or overnight treatment with CM alone (see Figure 5.5), indicates the presence of TNF-cx and/ or IL-18 in the CM as these cytokines (and LPS) have been shown to induce VCAM-1 on HUVECs, whereas IFN-y does not lOsborn et a\.,1'989;
Carlos et øL, 1.990b; Thornhill and Haskard, 1990; Thornhill et aL, 1991).
5.5.2 Expression of 81. integrin molecules
While resting ShUVECs demonstrated a high level of Þr integrin expression, as demonstrated using the anti-CD29 mAb QE2E5, only a modest increase was observed after stimulation with CM (Figure 5.6). The profiles of CD29 expression are consistent with those observed for HUVECs cultured under similar conditions, both in this study (Figure 5.6) and in previous reports [Favaloro et ø1.,7990; Favaloro, 1.993]. 174
OVINE HUMAN
E Er $l clr
MNC Class I
Ð tû 1tl
J= .lr L \Ê o -o lJì E E f T E z f., MHC Class ll q) É t\¡ o {= e 4 4 til It 10 1ü
f,r E l¡l E T Ê f.¡ cD29 (\l
4 l0 10 10
Fluorescence I ntensity (Log)
Eigure 5.6 Flow cytometric analysis of cell surface antigens expressed on resting and stimulated ovine and human ECs. Cultured ShUVECs and HUVECs were incubated for 72 hr in the presence of either CM or rh-IFN-y, respectively, and then stained with species-specific mAbs directed against MHC Class I & II, and a cross-reactive anti-human CD29 mAb (blue histograms). Expression on unstimulated ECs (green overlay) and control
mAb (TIB-191; orange overlay) stained in parallel, are also shown for each sample. 175
5.5.3 Cell Surføce Expression of VCAM-I
TNF-cr stimulated ShUVECs and HUVECs were analysed by flow cytometry for the expression of VCAM-1 using the mAb, HAE2-I. The induction of ovine VCAM-1 on endothelial cells revealed peak expression occurring between 6Io 1.2 hr after stimulation, followedby a slight decrease to a plateau extending beyond 48 hr (Figure 5.7). This profile is similar to that seen on HUVECs both during this study and that previously reported for human endothelial cells [Osborn et ø1.,1989; Carlos et ø1.,1990b1.
It was noted that some populations of unstimulated ShUVECs displayed a low level of VCAM-L protein expression, as seen in Figure 5.5. This was generally associated with ShUVEC cultures grown to higher densities than HUVECs, demonstrating tightly packed cobblestone monolayers. The basal expression of VCAM-1 by these cultures may reflect the adaptation of ShUVECs to culture conditions primarily designed for HUVECs. Whilst this low level VCAM-1 expression was not evident in the HUVEC cultures used in this study, similar observations have previously been reported for HUVECs [Carlos et al., 1,990b; Schwartz et ø1., 7990; Meerschaert and Furie, 1'994]. Although these groups were unable to detect VCAM-L expression on unstimulated HUVECs by ELISA or FACS, the ability of an anti-VCAM-L mAb
to inhibit lymphocyte [Carlos et ø1., 1,990b1 or [Meerschaert and Furie, 1.994) monocyte adhesion indicated the presence of low, but significant levels of
VCAM-L. Schwartz et ø1. 119901 and Carlos et ø1. 11.9901 postulated that the expression of VCAM-I on unstimulated HUVECs may have resulted from the presence of cytokines produced by contaminating leukocytes in the initial passage. As this low level of VCAM-1 expression was observed in ShUVEC
cultures at passages 3-5, any residual cytokines or leukocytes would have been
removed during earlier passages and the ShUVECs would have reverted to a resting state. Therefore this is unlikely to be the explanation and subsequent 176
HUMAN
o -o E z o O
48hr 48hr 24hr 24hr 6hr 2hr Unst. Unst.
Figwe 5.7 Flow rytometric analysis of the kinetics of VCAM-I expression by ovine and human endothelial cells in aítro. ShUVECs and HUVECs were stimulated for periods of 0-48 hr with species-specific r-TNF-cr and then stained with the anti-VCAM-l mAb, HAE2-1. 777 modifications to the culture conditions, primarily involving the maintenance of ShUVECs at lower densities largely resolved this basal expression.
5.5.4 Cell Surføce Expression of ICAM-7
Unfortunately, the unavailability of either a cross-reactive or a sheep specific anti-ICAM-L mAb did not allow comparison of the protein expression. However, the detection of ICAM-L mRNA in ShUVECs, as discussed below, suggests it is likely that surface expression of the molecule also occurs.
5.6 Northern Blot Anølysis of VCAM-L ønd ICAM-L øRNA Induction
For the mRNA induction studies, ShUVEC and HUVEC cultures were stimulated by incubation with LPS L00 ng/mI or species-specific TNF-o¿ or IL- LB for periods between 2to 48 hr. ShUVECs were stimulated with 100 ng/mI recombinant ovine-TNF-cr or l-0 ng/mI ro-Il-1p and HUVECs were stimulated
with either 1.0 ng/ml recombinant human-TNF-c¡¿ or 1 nglml rh-IL-18.
Total RNA was extracted from the cultured endothelial cells, transferred onto nylon membrane and probed with [cr-32p]-Ufp-labelled cRNA probes synthesised from human-ICAM-1 and -VCAM-1 cDNA templates (2.2.15).
5.6.L ICAM-L øRNA Expression
Northern analysis of ICAM-L mRNA in ECs of both species identified a 3.3 Kb transcript, verifying the cross-reactivity of the human ICAM-1 cDNA probe with ovine mRNA and confirming the basal constitutive expression of ICAM-1 in HUVECs. Stimulation with either LPS or species-specific r-TNF-cr or r-IL-1p induced rapid expression of ICAM-1 mRNA in both human and 778 ovine cells, peaking at between 2 to 6 hr. This was followed by a decrease to a level which persisted beyond 48 hr (Figure 5.8), as previously reported for HUVECs [Springer, 1990]. Although not evident in the RNA obtained from stimulated ShUVECs, two minor transcripts o12.4 and 1.9 Kb were detected in samples from stimulated HUVECs, similar to those reported by others
[Simmons et ø1., 1988; Staunton et ø1.,1988].
5.6.2 VCAM-7 øRNA Expression
Northern blot analysis for the presence of VCAM-1 mRNA showed the expression in both species of a3.2 Kb band which was absent in unstimulated ECs but became evident between 2 to 6 hr after stimulation (Figure 5.9). This profile is similar to previous reports for human endothelial cells [Osborn ef
ø1.,1989; Carlos et ø1., 1990b; Wellicome et ø1.,19901.
SUMMARY
The studies reported in this Chapter describe the isolation and characterisation of sheep endothelial cells derived from sheep umbilical cord veins. Although sheep endothelial cells have been isolated from a number of different ovine sites, the work presented here represents the first detailed report of ECs derived from sheep umbilical veins. The endothelial origin of these cells was confirmed by comparison with many of the phenotypic and functional markers displayed by the "gold standard" of cultured endothelial cells, HUVECs.
In aitro, ShUVECs display several of the classic markers associated ECs, including the typical cobblestone morphology of EC monolayers and the ability to metabolise Ac-LDL. 179
UNSTIM LPS TNFO rL-1p hrs 2 6 24 48 262448262448 262448 Kb { ees g.g ) z.q* r.g ) 1 8S
8S
HUMAN UNSTIM LPS TNFa rL-1p hrs 262448 2 62448 2 6 24 48 262448 Kb 28S s.e ) 2.4> r.s ) <18S
+ q¡# l, ìi '6^t rr'l <1 8S
Figure 5.8 Northern blot analysis of ICAM-I mRNA kinetics of induction in stimulated ECs. Total mRNA was prepared from cultures of ovine or human ECs stimulated with either LPS or species-specific r-TNF-cr or r-IL-1B for periods of 0-48 hr. 10 pg of the extracted RNA was loaded in each lane for electrophoresis (equal lane loading shown in 18s bands from agarose gel) followed by overnight transfer to nylon membrane and probing with a [a-32f1- labelled cRNA probe synthesised from a human-IcAM-l cDNA template. 180
OVINE rL-1Ê UN LPS 4$1Fcr="^':Þ hrs 2 26 6 26 Kb < 28S 3.2> < 18S
< 18S
HUMAN UN LPS TNFa rL-1p hrs 2 26 2 6 26 Kb < 28S 3.2> < 18S
< 18S
Figure 5.9 Northern blot analysis of VCAM-1 mRNA exPression in stimulated ECs. Total mRNA was prepared from cultures of ovine or human ECs stimulated with either LPS or species-specific r-TNF-cr or r-IL-1B for periods of 0-48 hr. 10 pg of the extracted RNA was loaded in each lane for electrophoresis followed by overnight transfer to nylon membrane and
probing with a ¡¡¿-32f1-labelled cRNA probe synthesised from a human- VCAM-I cDNA template. 181
FACS analysis of vWF exPression on ShUVECs and HUVECs demonstrated comparable levels on both cell populations. This was supported by immunoperoxidase staining with both populations displaying a strong cytoplasmic staining pattern. The restricted tissue distribution of P-selectin to vascular endothelium, platelets and megakaryocytes further supports the endothelial phenotype of the ovine cell isolates, as did the expression profiles for the Br integrin subunit and the MHC class I and II antigens.
Stimulation of the ECs with species-specific cytokines or LPS demonstrated that the kinetics of ICAM-1 and VCAM-1 mRNA expression were similar to that previously reported for human endothelial cells. While VCAM-1 cell surface expression was also consistent with previous reports for HUVECs, the absence of either a cross-reactive or a sheep specific anti-ICAM-1 mAb did not allow comparison of ICAM-L protein expression. However, the Northern blot data for ICAM-L mRNA in ShUVECs suggests that surface expression of the molecule does occur.
The availability of ShUVECs provides a tool to enable studies of the expression of CAMs on ovine endothelial cells and the interactions between ovine MNC and ECs, both in r:iao and in uitro. Flowever, while limited stocks of an ovine reactive anti-VCAM-1 mAb were available, they were insufficient to allow in aiao studies. Furthermore, mAbs recognising ovine ICAM-L could not be located, despite extensive enquiries.
Therefore, in order to undertake studies of sheep CAMs, and to be able to fully utilise the ovine transplantation model, it was first necessary to raise mAbs against ovine molecules. The production and characterisation of mAbs directed against sheep endothelial cell adhesion molecules are described in the next chapter. 782
Chapter 6
Production and characterisation of monoclonal antibodies against ovine VCAM-I-
INTRODUCTION
As described in Chapter L, VCAM-I is a member of the immunoglobulin gene superfamily and is an inducible monomeric cell surface glycoprotein of 110 kDa [Osb orn et al., 7989; Polte et al., 1990; Polte et ø1., 1991]. While VCAM-L is generally not detectable on normal resting endothelial cells, it is induced on ECs by TNF-cr,IL-LB and LPS [Osborn et n\.,1989; Carlos et aL.,1990a; Wellicome et ø1.,
19901. In addition, although IL-4 selectively induces the expression of VCAM-1 (but not ICAM-1 or E-selectin) on HUVECs, it is also capable of acting synergistically to enhance the level of VCAM-I- surface expression produced by
TNF-cr or IL-1p [Masinovsky et ø1.,1990; Thornhill and Haskard,1990; Thornhill ef
ø1., 1990; Thornhill et ø1.,1.9911. Similarly,IL-1.3 acts to selectively stimulate the expression of VCAM-L on HUVECs [Sironi et ø1.,1994; Bochner et a1.,7995]. 183
The data in the previous chapter demonstrates that ShUVECs display similar kinetics and levels of expression of EC adhesion molecules to that of HUVECs. It was therefore of interest to study the role of VCAM-L in ovine leukocyte-endothelial cell interactions, both in aitro and in uiuo. However/ as stated previousl/, there are few, if any, mAbs specific for ovine endothelial cell adhesion molecules. Although several aliquots of a cross-reactive anti-human VCAM-1 mAb, IHAE2-L, were obtained, the mAb was not available in sufficient quantíties to conduct in uioo studies in sheep. It therefore became apparent that in order to study ovine endothelial CAMs it would first be necessary to produce the mAbs.
This chapter describes the production and characterisation of a panel of
anti-ovine VCAM-L mAbs. FACS analysis, protein capping studies and immuno- peroxidase staining were used to confirm the identity of the antibodies. Adhesion and proliferation assays were performed to determine whether the mAbs
recognise functional epitopes on the ovine VCAM-L molecule.
RESULTS AND DISCUSSION
6.1- Production ønd Chørøcterisøtion of Oztine VCAM-L Monoclonøl Antibodies
6.L.L lmmunisøtion ønd Screening Strøtegy
In order to produced mAbs that recognise ovine VCAM-1, stimulated ShUVECs were used as the immunogen. BaIb/c mice were immunised intra- peritoneally with CM-stimulated ShUVECs and then splenocytes were fused with the murine myeloma cell line Sp2/0-4g14 (2.2.7.4).It was anticipated that an antibody against VCAM-1 (and ELAM-L) would be reactive with stimulated
ShUVECs but not unstimulated cells, whereas ICAM-1 would be weakly positive
on unstimulated ShUVECs and more strongly reactive with stimulated cells. 1.84
Of nine hundred supernatants screened by flow cytometry, three mAbs
(eE1F3, QE2G4 & QE4G9) were identified by their reactivity with CM-stimulated ShUVECs, whilst being unreactive with unstimulated ShUVECs. The flow cytometric profiles of the three mAbs were also compared against that of HAE2-L on stimulated ShUVECs and found to be similar (Figure 6.1). Furthermore, one of these mAbs, QE4G9, displayed reactivity with HUVECs that had been stimulated overnight with rTNF-ø (Figure 6.2). The specificity of QE4G9 for VCAM-I was established by reactivity with transfected CHO cells stably exPressing human
VCAM-1. QE4G9 showed strong staining on the VCAM-1 transfectant and no reactivity with non-transfected CHO cells (Figure 6.2). This was identical to the profiles seen with HAE2-1 on the transfected and non-transfected CHO cells
populations. Neither QELF3 nor QE2G4 showed any reaction with either the stimulated HUVECs nor the VCAM-1 CHO cell transfectant'
Despite being cross-reactive with human VCAM-I, neither QE4G9 nor HAE2-1 recognise porcine VCAM-1 (personal communication, ]. Malliaros, St Vinceni's Hospital, Fitzroy, Australia) [Mackay et ø1.,1992b] and at this stage
QE4G9 has not been tested for cross-reactivity with other species. In addition, none of the mAbs were reactive with ovine or human fibroblasts or peripheral blood leukocytes.
6.7.2 Protein Cøpping ønd Internølisøtion with mAbs to VCAM-I
Further confirmation of the VCAM-1 specificity of QE4G9 was obtained with in aitro protein capping and internalisation studies using either CM- stimulated ShUVECs or the VCAM-1 transfected CHO cells (Chaptet 2.2.4).
Ligation of VCAM-I on the CHO cell transfectants by HAE2-7 progressively decreased the fluorescent signal obtained with FlTC-conjugated
QE4G9. FACS analysis revealed that the capping and internalisation Process 185
É E lt¡ tft QE2G4 E TÉ T E E tî ('¡ t= ftl ñl E -obé )EE E z. t0 t0 1 oo
È rf)É tft To fllo +io e o ct I 10 1 1 t0 + Fluorescence lntensity (Log)
Figure 6.7 Flow cytometric staining profile of anti-ovine VCAM-1 mAbs on ShUVECs. Stimulated (blue histograms) and unstimulated (green overlays)
ShUVECs were stained with anti-sheep VCAM-1 mAbs, QELF3, QE2G4, QE4G9 and HAE2-1. T18-19L (orange overlay) was used as a negative control mAb. 186
HUVECS
rÐ QE4G9 r¡D HAE2-1
LE o J 4 _o t0 It Iu t0 I E zf q) o
VCAM-1 transfectant
QE4G9 HAE2.1
o 3 t lfl tû
Fluorescence lntensity (Log)
Eigure 6.2 Flow cytometric analysis of QE4G9 cross-reactivity with human VCAM-1. TNF-cr,-stimulated HUVECs (blue histograms) and unstimulated
HUVECs (green overlays) were stained with QE4G9. Specificity of QE4G9 for VCAM-I and cross-reactivity with human VCAM-1 were examined using CHO cells expressing human VCAM-1 (blue histograms) and non-transfected CHO
cells (green overlays). HAE2-1 was used as a positive control for the expression of
VCAM-1 and T18-19L (orange overlay) was used as a negative control mAb. 787 commenced within 5 min of returning to 37"C and was essentially complete within 15 min (Figure 6.3).
The specificity of mAbs QE1F3 and QE2G4 fot ovine VCAM-1 was confirmed using protein capping studies on stimulated ShUVECs. Removal of VCAM-I from the surface of stimulated ShUVECs by incubation with these mAbs (and HAE2-L) was demonstrated by the decreased staining exhibited by QE4G9-
FITC and was complete within L5 min of incubation at 37"C (Figure 6.3).
Taken together, the capping studies and the absence of reactivity of QE1F3 and QE2G4 with either the HUVECs or the VCAM-1 transfectant demonstrate that these mAbs recognise different epitopes on the VCAM-1 molecule to that of QE Gg,in a region which is not homologous with the human form.
6.1.3 VCAM-L Expression in Normøl ønd Inflømed Kidney
Further confirmation of the identity of the mAbs came from indirect
immunoperoxidase staining (2.2.1,1,.2) of frozen sheep kidney tissue sections.
Staining of biopsy specimens obtained from normal sheep kidney and rejecting ovine renal allografts demonstrated that the structures recognised by the
three sheep VCAM-1 mAbs and HAE2-1 are identical. Samples obtained from a rejecting sheep renal allograft revealed intense staining of the endothelium of peritubular capillaries and arterioles (Figure 6.4). There was also strong expression of VCAM-1 by glomerular parietal epithelium and mesangial cells. Tubular expression of VCAM-I- was minimal, being present only on a very small
number of tubules and focal in appearance.
In addition, sections of inflamed renal tissue from sheep receiving i.v. administration of LPS in a separate study of septic shock were also stained with
the panel of mAbs. As with the allograft sections, the mAbs recognised identical structures in these tissue samples (Figure 6.5). There was intense staining of 188
UJ HAE2-1 T VCAM.l t ransfect ant t\l
'= 0 1 4 0 t0 lrl
l! QEl F3
o -o 1 10 I E z= oo U OE2 G4 u, TË St imulated f+ ShUVECS +tÞ E E t0
HAE2.1
0 1t ü + Fluorescence lntensity (Log)
Figure 6.3 Flow cytometric analysis of capping and internalisation of VCAM-I on CM-stimulated ShUVECs and the VCAM-1 transfectant. Antibody-induced capping of VCAM-I was initiated by incubation with either IIAEz-l., QE1F3 or QE2G4 and a crosslinking anti-murine Ab. The loss of VCAM-1 from the cell surface was then assessed by staining with the directly conjugated QE4G9-FITC. VCAM-1 transfectant sampies were removed from 37"C incubation at To (blue histogram), Ts (red overlay) and Tts (green). ShUVEC samples were removed at To (blue), Trs (green) and Tas (orange). , f,qr " Jt, ., .jlt l;¡i;¡
'.;,,í, -,1 \*'Á Þ 'r .', '(' !'l I .l:tr..- r .^'t, t- .!z:, ,ir. , ';. ,, :: + ::iå' ':'tt .''; :.1": ;r.ì ,.., 't
', r.i I /,
T ì '(Oçx) '(a 1auut1) t6I-8IJ'qvru Io.TlLIor artlu8au pttu (p ¡auud) 7DZAÕ 'Q Tatrutl) €CIEÕ '(q Tauutl) 6Ð?80 '(u Tauud) I-ZAVH qllM PðLttEls a.rÐM daar,¡s poluðli-Sd-I ulo.IJ sr{atrpr>1 ¡o saldruus lsdorg 'lapolu ìroLIS rrldas ãLrrÄo Lru LuoJl salnlJrlJ+s IuLraJ Lro slr.raJlucl 8r-tturuls ¡o ttostrucìuo3 g'g a.nßt7
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t; 5.{ ,i{.'r.'. I'.t ( 'l*G' .I.r, 't' 1'1... :''r \.- q Lt
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06r 191. glomerular parietal epithelium, mesangial cells and capillary and arterial endothelium. Tubular staining was again minimal. A similar pattern of VCAM-1 distribution on rejecting and normal human renal allografts has been reported by several groups [Rice et øL,1991'; Alpers et ø1. ,1993; Hill eú ø1.,!9951.
The absence of staining by any of the VCAM-I mAbs on sections of normal ovine kidney indicates VCAM-1 is not expressed on uninflamed sheep kidney, although previous studies of normal human kidney have demonstrated its presence on glomerular parietal epithelium [Rice ef nI., 1991'; Alpers et al., 1993;
IJtIl et øL,19951 . This may be due to differences in the anti-VCAM-1 mAbs used in these studies or it may reflect differences in VCAM-L expression between species.
6.2 Kinetics of Oztine VCAM-L Induction
The induction of VCAM-1 expression on stimulated ShUVECs was studied
using the panel of anti-ovine VCAM-L mAbs in parallel with HAE2-1. VCAM-I is
generally undetectable on resting ShUVECs and peak expression occurs between
6 to 12 hr after stimulation with CM, followed by a slight decrease to a plateau continuing for 48 to72lnr (Figure 6.6).
The profiles observed on sheep ECs were basically the same for each of the mAbs and are consistent with the comparative studies of VCAM-I- mRNA and protein expression by ShUVECs and HUVECs described in Chapter 5. These results are also consistent with previous reports of VCAM-1 expression by
HUVECs [Osborn et al. ,1989; Carlos et ø1. ,1990b; Wellicomè et al. ,1990; Gtooby et
ø1. , 19971. 792
HAE2.1
48hr 24hr 6hr 2hr Unst.
QE4G9
48hr o 24hr _o 6hr E 2hr z= Unst oo) QE1F3
48hr 24hr 6hr 2hr Unst
QE2G4
48hr 24hr 6hr 2hr Unst. (Los1
Figure 6.6 Kinetics of induction of ovine VCAM-I. ShUVECs stimulated with CM for periods of 0-48 hr were stained with mAbs QELF3, QE2G4, QE4G9 or HAE2-1 and the profiles of VCAM-1 expression detected by each mAb compared by FACS analysis. 793
6.3 Functionøl studies of the Three Anti-ozsine vcAM-L mAbs
6.3.7 Endotheli øl Cell-Leuko cyte Adhe sion As s øy s
The ability of the three anti-ovine VCAM-I mAbs to inhibit the adhesion of pMA-stimulated sheep MNC to CM-stimulated ShUVECs was assessed in aitro. The mAb , QE G} produced a significant dose-dependent inhibition of adhesion ranging from 1.3"/" to 40% lor ascites dilutions of 1/1000 to 1./250, respectively, whilst tissue culture supernatant of QELF3, QE2G4 and QE4G9 resulted in inhibition o131"/o,36'/" and 47o/o, tespectively (Figure 6.7).
Treatment of unstimulated ShUVECs with the anti-VCAM-L mAbs did not affect the numbers of MNCs bound to the monolayers. Although this is not unexpected in view of the absence of VCAM-I- from resting ShUVECs, other
groups have reported significant inhibition of leukocyte adhesion to unstimulated HUVECs by anti-VCAM-1 mAbs, despite being unable to detect VCAM-1 by
FACS or ELISA [Carlos et aL,1990b; Meerschaert and Furie, 1994]. The reason for the inability of the ovine mAbs to inhibit adhesion of sheep MNC to unstimulated
ShUVECs compared to that seen for HUVECs and human MNC is unclear.It may relate to the particular epitope recognised by each mAb or possibly reflect the heterogeneity associated with ECs, both within and between species [Fajardo,
1989 ; Kinjo et ø1., 1989 ; Swerlick et aI., 1991'; Page et ø1., 19921 .
Alternatively, as discussed in the previous chapter, it has been suggested
that the low level expression of VCAM-L on HUVECs may have resulted from the
presence of cytokines released by residual leukocytes in the early Passages [Carlos
et ø1., 7990b; Schwartz et ø1., 7990). As the ShUVECs used in the adhesion assays were all between passage 3-5, any contaminating MNCs and cytokines would have been removed during earlier passages and hence these ShUVECs would
have reverted to a resting state. 794
Several adhesion assays were also performed to examine the effects of a combination of anti-VCAM-L and anti-LFA-L mAbs on the adhesion of sheep MNC to CM-stimulated ShUVECs. Pretreatment of stimulated ShUVECs with
QE4G9 significantly inhibited the adhesion of sheep MNCs (41/.). Preincubation of MNCs with 72-87 resulted in a significant reduction in cell adhesion (48%) to the stimulated endothelial monolayers. When both QE4G9 and 72-87 were added to wells containing sheep MNCs and CM-stimulated ShUVECs, an additive effect on inhibition of adhesion (64"/.) was observed (Figure 6.7).
6.3.2 Inhibition of Oaine MNC Proliferation in the MLER
Previous studies have demonstrated that VCAM-L is capable of delivering costimulatory signals which act in concert with the T cell antigen receptor resulting in the activation and proliferation of T cells [Burkly et ø1., 199I; Damle and Aruff o,199L; van Sevenler et a1.,1991b1. Therefore, in order to examine the
ability of QE4G9 to inhibit ovine MNC proliferation, the mAb was studied in the MLER (2.2.3.1).
The addition of QE4G9 ascites or neat tissue culture supernatant to the MLER inhibited proliferation of sheep MNC by 52% and56o/o, respectively (Figure 6.8), similar to previous reports of anti-VCAM-l mAb inhibition of MNC
proliferation [Sava ge et al., 19931. Neither QE1F3 nor QE4G9 supernatant were able to inhibit proliferation in the MLER.
6.3.3 Inhibition of Oztine MNC Proliferøtion in the MLC
A recent report described both the inhibition of proliferation by an anti- VCAM-1 mAb in a human MLC and the detection of VCAM-1 on the adherent
sub population of cells recovered from the MLC [Lukacs et a\.,1994]. Therefore, the potential role of VCAM-1 during ovine MNC proliferation was assessed in a 195
! 125 .o LL q) 100 o o-=
=o J 75 3 ()q) c o 50 o þE zo 25
0 J J o T o o o 1\){ ø o t\) (Jl J l\) (¡ m m m I m (tl (tl m 5 J l\) @ 5 = o oO o o o o o f\)I ll { - o CÐ o J o o o o o J (o Þ (o Ø a (tlIU + f\) e (tl z z z o t\){ QE4G9 dilutions I o {@
Eigure 6.7 Inhibition of adhesion between sheep MNCs and ShUVECs. Hybridoma culture supernatants from QELF3, QE2G4, QE4G9 and dilutions of QE4G9 and HAE2-1- ascites were used to assess the inhibition of adhesion of sheep MNCs to CM-stimulated ShUVECs. Assays were also performed with the anti-ovine LFA-1 mAb, 72-87, alone or in combination with QE4G9 (both mAb ascites 1/250 dilution). Control mAb was T18-19L (ascites L/250). Results are mean + SD of 4 well replicates. * p<0.01., ** p<0.005 with respect to TIB-191. 196
20000
1 5000
o- 1 0000 o
5000
0 U) Ø o o o o ø o J c o m m m m o c Þ Þ J l\) o c f o o ll o E m o õ (o (o C¡) 5 C) J U) ø, 3 U) Ø z o J o o- z o z z c' u O 3 O
Figwe 6.8 Inhibition of sheep MNC proliferation in aitro. QE1-F3, QE2G4 and QE4G9 were used to examined the effect of anti-VCAM-l mAbs on allogeneic MNC proliferation in the MLER. Control mAb is T18-L91, ascites 1,/250. Results are mean t SD from 6 well replicates and are representative of three similar ** assays. " p<0.0L, p<0.005 with respect to TIB-191. 197
2-way MLC (2.2.3.2) in the presence of the three anti-ovine VCAM-1 mAbs. In contrast to the previous report, no inhibition of proliferation was observed, nor was the expression of VCAM-I- detected on any cells extracted from the MLC.
6.4 Structurøl Stuilies of Oztine VCAM-I,tlsing the PøneI of Anti-VCAM-l mAbs
The reasons for the discrepancy described above are not clear, however it is plausible that sheep monocytes may express a different isoform of VCAM-1 not recognised by the anti-vCAM-L mAbs (e.g., 7-Domain vs 6-Domain)' It has not been possible to successfully immunoprecipitate the VCAM-1 molecule from ShUVECs with these mAbs, nor detect it by Western blotting, and therefore structural information is not available. Consequently, little is known about the structure of the ovine VCAM-I molecule and it remains to be determined whether sheep cells are capable of expressing multiple isoforms of VCAM-L, as reported
for other species [Polte et ø1.,1990; Cybulsky et al,, 1991'; Hession et øl', 1991';
Cybulsky et ø1.,19931.
SUMMARY
In this chapter the production and characterisation of three mAbs recognising ovine VCAM-1 is reported. The FACS profiles of inductíon of VCAM-
L on stimulated ShUVECs demonstrated by the three mAbs are identical to that observed with the cross-reactive anti-human VCAM-1 mAb HAE2-1', and are consistent with previous studies of VCAM-1 mRNA and protein expression by
ShUVECs and HUVECs. FACS analysis also demonstrated that one mAb, QE4G9,
was cross-reactive with human VCAM-1- expressed on transfected CHO cells and stimulated HUVECs. Protein capping and internalisation studies on VCAM-1 transfected CHO cells and ShUVECs, in conjunction with immunoPeroxidase 198 staining of inflamed renal tissue, were used to confirm the identity of the anti- ovine VCAM-I mAbs.
All three mAbs were shown to recognise functional epitopes in the ovine VCAM-I molecule by virtue of their ability to significantly inhibit sheep MNC adhesion to ShUVECs, while QE4G9 inhibited MNC proliferation in the MLER.
Thus, the work presented in this chapter describes both the production of a panel of anti-ovine VCAM-I mAbs and data indicating that these three mAbs recognise at least two functionally different epitopes on the sheep VCAM-1 molecule. Having demonstrated the ability of one of these mAb (QEaG9) to inhibit adhesion and proliferation between sheep MNC and sheep endothelial
cells in aitro , it was then selected to study its efficacy in prolonging renal allograft survival in the sheep transplantation model. 199
Chupter 7
Studies of an anti-VCAM-I- monoclonal antibody, or combined anti-VCAM-L & anti-LFA-L monoclonal antibodies, in acute reiection in the ovine model of renal transplantation
INTRODUCTION
The interaction of peripheral blood leukocytes with vascular endothelium represents a critical step in the development of a cell-mediated immune resPonse and the subsequent infiltration and accumulation of alloreactive T lymphocytes initiates acute cell-mediated allograft rejection [Mason and Morris,!986].
Several lines of evidence, from studies performed both in uitro and in oiao, indicate that VCAM-1 plays an important role in this process. Firstly, in uitro
VCAM-1 has been shown to provide costimulatory signals that act in concert with the T cell antigen receptor resulting in activation and proliferation of T cells [Burkly et ø1., 1991,; Damle and Aruff.o, 1,991.; van Seventer et ø1.,799Ia). In addition, adhesion and transmigration studies have shown that although adhesion of lymphocytes to unstimulated ECs occurs primarily through the 200 interaction of LFA-1 and ICAM-I [Haskard et ø1., 1'986; Arnaout et al',1988;
Haskard et ø1., 1989; Oppenheimer-Marks et ø1., 1990; Oppenheimer-Marks et ø1.,
1991,; Shimizu et ø1., 1991,a; Meerschaert and Furie, 19951, other inducible cell surface proteins such as VCAM-1 provide alternative pathways [Haskard et øl',
1989; Osborn et ø1., 1989; Carlos et ø1., 1990b; Oppenheimer-Marks et ø1., 1991';
Shimizu et ø1.,199ta; Meerschaert and Furie, 1994; Meerschaert and Furie, 1995)' Secondly, while generally absent from normal tissue samples, induction of VCAM-1 on inflamed tissue has been demonstrated by many groups [Briscoe eú
ø1., 1.992; Alpers et ø1., 1993; Pelletier et al., 1993b; Briscoe et ø1., 1995; }{III et ø1. ,
19951. Finally, recent studies have demonstrated that anti-VCAM-1, mAbs, either alone or in combination with anti-VLA-4 mAbs, are able to prolong allograft survival in murine models [Orosz et al., 1993; Pelletier et ø1., 1993a; Isobe et ø1.,
1994b; Gorcyznski et ø1., 19951.
In view of the success of the anti-VCAM-1 therapy in small rodent models,
we wanted to establish if such results were reproducible in the clinically relevant sheep model of renal transplantation. The work described in this chapter is
divided into 4 sections. The first section studies the profile of VCAM-1- expression during renal allograft rejection. The second section investigates the efficacy of an anti-VCAM-L mAb in prolonging renal allograft survival in the ovine model and the third section examines the effect of combined anti-VCAM-1 and anti-LFA-1 mAb therapy. The final section examines a number of the in aiao studies which
successfully blocked the VCAM-1,/VLA- pathway and discusses possible causes for the inability of an anti-VCAM-1 mAb to prolong renal allograft survival nor block MNC infiltration in the ovine model.
MATERIALS AND METHODS
Five pairs of sheep underwent cross-transplantation, each receiving a renal
graft from its partner, as described in the Chapter 3, followed by removal of the 20r remaining native kidney. Graft function was monitored by estimation of serum creatinine. Graft failure was defined as occurring when the creatinine had risen to
>600 pmol/L andhistological assessment confirmed the Presence of rejection.
Six sheep received QE4G9 only and the remaining 4 animals received a combination of QE4G9 and 72-87 commencing on day 0, immediately after completion of surgery. Unfortunately, one animal receiving combination therapy died during post operative recovery and therefore data is only available for 3 allograft recipients treated with the combined mAbs.
RESULTS AND DISCUSSION
7.L VCAM-7 Expression in Reiecting Renøl Allogrøfts
The induction of VCAM-L expression in rejecting renal allografts was examined by indirect immunoperoxidase staining of fuozen sections (2,2.1'1'.2)
from core biopsy samples collected daily.
VCAM-I expression occurred first in peritubular capillaries, arterioles and
arteries, as early as day 1- post-transplantation, and the intensity of this staining was maximal at day 5 (Figure 7.1). Glomerular VCAM-1 expression became evident at day 3 showing staining of the Bowman's capsule and mesangium and
also peaked at day 5. Although VCAM-1 expression on tubules was focal and not
detected in all segments, tubular reactivity was generally seen at the basolateral
surfaces. A small number of interstitial cells expressing VCAM-1 were detected at
day 3 post-transplantation and were still present at day 7.It is unclear whether these VCAM-1 positive cells are allogeneic DCs, or represent infiltrating host
leukocytes or DCs, as seen by others [Alpers et ø1., 1993; Brockmeyer et ø1.,1993;
Fuggle et ø1.,19931. 202
c ø
d b
|ê ¡-'
Figure 7,7 Temporal profile of VCAM-1, expression in rejecting ovine renal allografts. Sections from a pre-transplant wedge biopsy (pønel a) and core biopsy samples collected at day 1, (panel b), day 3 (panel c) and day 7 (panel d) were stained by indirect immunoperoxidase with QE4G9. (x50) 203
The timing of increased VCAM-1 expression, which parallelled the increasing level of MNC infiltration, is similar to that seen in the murine cardiac allograft recipients [Pelletier et ø1., 1,993b1. Furthermore, the distribution of VCAM-1 in the rejecting sheep kidney is consistent with other immuno- histological reports [Rice et a\.,1991; Seron et ø1.,1997; Briscoe et ø1. ,1992; Alpers eú
ø1.,1993; Fuggle et ø1.,1993;HiIl et ø1.,19951.
7.2 Anti-VCAM-L mAb ThetaPY
7.2.7 Dosøge Regimen for QE4G9 Treøted-Sheep
The dose of QE4G9 used in this study was based upon previous reports by groups using anti-ICAM-l mAbs in monkey allograft recipients [Cosimi et øl',
1990] or anti-VCAM-1 mAbs in murine cardiac allograft recipients [Orosz et ø1.,
1993; Pelletier et ø1., 1993a; Isobe et øl', I994bl.
However, while the anti-VCAM-l mAbs were successful in prolonging allograft survival in the murine model, the protocols involved i.p. administration
of very large doses of mAb, which were not applicable to the sheep model. In the
studies by Orosz and colleagues [L993], the mAb was administered i.p. to the mice at 400 ltg/ day over a period of 20 consecutive days. Earlier studies by this Sroup demonstrated that smaller i.p. doses of anti-VCAM-1 mAb, 100 pglevery second
day, did not effect graft survival [Pelletier et ø1., 1.992], while 200 ¡t"g/day
prolonged allograft survival by only 5 to 6 days [Pelletúer et ø1., 1992; Orosz et ø1. ,
t993; Pelletier et ø1., t993al.
Extrapolation of this regimen to account for the size difference between mice and sheep would equate to a daily dose of approximately 640 mg. Duplication of this extremely high dosage in the sheep model was not possible in
the host laboratory. 204
In contrast, Cosimi et ø1.119901 successfully applied an anti-ICAM-1 mAb in a Cynomolgus monkey renal allograft model using a single daily dosage range of 0.01, and 2.0 mg/kg for t2 days. In comparison to VCAM-1, ICAM-1 is constitutively expressed on a wide range of cells with significant upregulation upon immune stimulation [Bevilac qua, 1993]. It was therefore surmised that for the sheep studies, a daily dose of QE4G9 similar to the dosage regimens used for the anti-ICAM- mAb therapy by Cosimi et ø1. (1990) would be sufficient to coat both constitutive and induced VCAM-1 molecules.
Based on the above reports it was decided that a daily dosage of 0.5 to 1
mg/kg (approx. 20-40 mg/ day) of QE4G9 was to be administered i.v. for up to 10 days. As described below, daily flow cytometric anaiysis of serum samples was
performed to confirm the presence of free mAb in circulation.
7.2.2 Monitoring of mAb Leztel in QE4G9 Treøted-Sheep
To demonstrate that adequate amounts of QE4G9 were being administered, serum samples were analysed on a daily basis by indirect immunofluorescent staining (2.2.1.1..1) using the VCAM-1 transfectant (see Chapter 6). In addition, the deposition of QE4G9 within the allografts was evaluated by indirect immunoperoxidase staining (2.2.1'7.2) of daily biopsy
samples.
The presence of free QE4G9 in circulation was confirmed by FACS analysis
of the VCAM-1 transfectant stained with serum samples collected daily 24hr after
infusion of the mAb, immediately before the next dose. Trough levels of QE4G9 in daily serum samples clearly demonstrated the presence of excess QE4G9 in circulation (Figure 7.2). The specificity of the staining was confirmed by the
absence of any reaction between pre-transplant serum samples and the VCAM-1
transfectant, and the daily serum samples and the non-transfected CHO cells. 205
ot- cf Day 0 ,ôo Day 5 to E Éf, V' tfl E çE T fîo hÐ o õ $.r îìl EE E zÉ o o t0 I 10 1
Ð È 1{ Day 1 f{ Day 7 E o ,ö ,Õ E e uì trì T E f'¡ tî $T (\¡ o E E E
t0 1 t0' 1 tfl
Fluorescence lntensity (Log)
Figure 7.2 Detection of excess QE4G9 in peripheral blood of treated animals. VCAM-I CHO cell transfectants (blue histograms) and non-transfected CHO cells (orange overlay) were stained with serum collected pre-transplantation (day 0), and then daily, 24 hr after mAb infusion, immediately before the next dose. Results shown are for trough level serum samples collected on days 1,, 5 and 7.
The VCAM-1 transfectant was also incubated with saturating amounts of QE4G9 (green overlays) to demonstrate the maximum level of staining on the transfectant. 206
Tissue sections from daily biopsy samples were stained with an anti-mouse
Ig to d.etect QE4G9 localised within the allograft. QE4G9 was identified on the endothelium of arterioles, arteries, peritubular capillaries and glomeruli, although notably absent from tubules. The pre-transplantation wedge biopsies were negative for staining with the anti-mouse Ig secondary Ab (Figure7.3).
7.2.3 Effect of QEaGg onRenøl Allogrøfi Suroiztal
Despite both the presence of excess QE4G9 in circulation for the duration of the studies and the deposition of the mAb on the endothelium of the allograft, there was no significant difference (p=0'32, Wilcoxon rank sum test) in graft
survival in the QE4G9-treated animals compared to the control group which received T18-L91 (see Chapter 3). Sheep receiving QE4G9 demonstrated graft
failure at a mean of 8.4 (t 0.7) days post-transplantation, compared with 9.3 (t 0.5)
days for the control group (Figute7.4).
With the exception of some early fluctuations due to ureteric catheter obstructions, serum creatinine and urea levels remained stable for 4 to 6 days after transplantation (Figure 7.5), followed by the same rapid increase observed in the
previous sheep transplantation studies. Although the allograft in the first animal of this group, V1-, began producing urine immediately after revascularisation, urine output decreased to zero over the following two days with the kidney becoming increasingly swollen and firm. The animal was subsequently sacrificed at day 4 and autopsy revealed extensive arterial and venous thrombosis and infarction within the cortex and medulla. 207
ø
b
Figure 2.3 Detection of QE4G9 mAb in ovine renal allografts was determined by immunoperoxidase staining of frozen sections from daily biopsy samples using a biotinylated anti-mouse secondary antibody only, followed by the ABC complex.
Deposition of QE4G9 in the allograft at day 1 (pønel a) was examined and a pre- transplant biopsy sample (panel b) was also stained by the same method to confirm the specificity of the process for bound murine mAb. (x50) 208
100
80 õ ¿ 60 CN
E CI 8 40
# QE4G9 (n=5) 20 * TIB-191 (n=4)
0 01234 5 6 7 I I 10 11 12
Days (Post-Tx)
Eigure 7,4 Suwival of ovine renal allografts in sheep treated with an anti-ovine VCAM-1 mAb. QE4G9-treated sheep received the mAb daily for up to L0 days, commencing on the day of transplantation. The control group received an irrelevant mAb, TIB-191, for the same period. 209
a looo
I 800 o E¿ v 600 o .c .Ec (ú 400 oE E J E 2oo
0 0 1 2 3 4 5 6 7 I I 10 11
Days Post-op
# V1 +V4 * V6 + v2 #V5 4: Control * V3
b80 70
60 3so Ê E40 ã E30 l 20
10
0 01234 5 6 7 I I 10 11 Days Post-op
Figure 7.5 Serum creatinine and urea levels of sheep receiving either QE4G9 or TIB-191. Creatinine levels (pønel a) were measured on a daily basis and graft failure was assessed as creatinine > 600 pmol/L. Urea levels (panel b) were measured for the same time points. The data for one animal treated with T18-L91 are shown and are representative of the observations for each of the control sheep. 21,0
7.2.4 Histopøthology of Allogrøfts from QE$G9-treøted Sheep
Histological examination of the allografts demonstrated an extensive MNC infiltrate associated with the onset of severe rejection which parallelled the progressive increase in VCAM-1 expression post transplantation. There was no difference in the level of MNC infiltrate observed in the kidneys of the animals treated with QE4G9 compared to animals receiving TIB-191 (Figure 7.6),
Autopsy of kidneys from animals receiving QE4G9 revealed evidence of significant interstitial oedema throughout the cortex, medulla and collecting
system, identical to that seen in the kidneys from unmodified animals (see Figure
3.4). There were also signs of haemorrhage and vascular rejection with frequent
examples of MNC adhesion to luminal surfaces.
7.2.5 Effects of QE4G9 on Peripherøl Blood Leukocytes
Samples of blood collected daily, and in some instances at 2 hourly intervals after mAb infusion, demonstrated no effect on WCC by QEaG9. The mAb was not found on any of the PBL populations following staining with anti-
mouse Ig secondary Ab. This result is consistent with the data from the previous
chapter showing QE4G9 does not recognise peripheral blood leukocytes.
7.3 Combined Anti-VCAM-7 and Anti-LFA-1- mAb Therøpy
7.3.1- Dosøge Regimen for Combined QE4G9 ønd 72-87 in Sheep
Four allograft recipients were treated with a combination of anti-VCAM-L
and anti-LFA-L mAbs. A daily dosage of 0.5 to 1. mglkg/day of QE4G9 and 0.125
mg/kg/ day o172-87 was administered i.v. for up to L0 days. Estimation of daily
trough levels of 72-87 was identical to that described in Chapter 4. 217 tl
D b
G ,
ol
Figure 7,6 MNC infiltration of rejecting sheep renal allografts fronr mAb-treated
sheep. The level of MNC infiltration in the QE4G9-treated animals (pnnel a) was
assessed by H & E staining of a day 5 post-transplantation biopsy sample. Pmtel b
shows staining of a day 5 biopsy sample from a TIB-191-treated arrimal. (x50) 212
7.3.2 Effect of combined QE4G9 ønd 72-87 on Renøl Allogrøft sutztiztøl
T¡¡e in aitro adhesion assay results described in the previous chapter demonstrated that a combination of QE4G9 and72-87 provided an additive effect on inhibition of adhesion between sheep MNCs and stimulated ShUVECs, compared to that seen with the individual mAbs (see Figure 6.7). Therefore, it was anticipated that such a combination could be more efficacious with respect to prolongation of allograft survival than has so far been the case when these mAbs are used alone.
Despite the combination of QE4G9 and 72-87, it was not efficacious in prolonging allograft survival beyond that obtained for the single mAb treatment groups or the control group. Sheep receiving QE4G9 and 72-87 demonstrated
graft failure at a median o17.7 (t 0.3) days post-transplantation compared with 9.3
(t 0.5) days for the control group (Figure 7.7).There was no significant difference between survival times for the two groups (Wilcoxon rank sum test).
In addition, the creatinine and urea levels began to increase at day 4 to 5 post-transplantation, which was sooner than that observed for either QE4G9 or control groups (Figure 7.8). This is consistent with the anti-LFA-1 (72-87) treatment of sheep undergoing renal transplantation (as described in Chapter 4) in which administration of the anti-LFA-L mAb appeared to exacerbate deterioration of graft function.
7.3.3 Histopathotogy of Allogrøfts from Combinøtion mAb'tteøted Sheep
The histology of the allografts, both at autopsy and during the course of
treatment were identical to that described above for QE4G9 (see Figure 7.6) and the unmodified allografts. There was evidence of extensive MNC infiltrate which parallelled the onset of severe rejection. Extensive interstitial oedema was evident 213
100
80
(ú
ct)=60 E CIs40
QE4G9 +72-87 (n=3) 20 # * TIB-191 (n=4)
0 01234 56789101112
Days (Post-Tx)
Eigure 7.7 Suwival of ovine renal allografts in sheep treated with a combination of anti-ovine VCAM-1 and anti-LFA-L mAb. QE4G9 artd72-87 were injected daily for up to L0 days, commencing on the day of transplantation. The control group received an irrelevant mAb, TIB-191, for the same period. 21.4
1000 a J 800 o E
c) '- 600 '-(ú o) L O 400 E o U) 200
0 0 23 4 5678 I
Days Post-op
# w1 .-.#w3 4 \L2 -+- Control
b 80
70
60 3so co Ë40 ; S30 20
10
0 0 1 2 3 45 b 7 8 Days Post-op
Figure Z8 Serum creatinine and urea levels of sheep receiving combined QE4G9
&.72-87 or control mAb, T18-19L. Creatinine (pønel a) and urea levels (pønel b) were measured on a daily basis. The data for one animal treated with TIB-19L are
shown and are representative of the observations for each of the control sheep. 215 throughout the cortex, medulla and collecting system, as was evidence of haemorrhage and vascular rejection.
7.3.4 Effects of QE4G9 ønd 72-87 on P eriphet øl Blo o d Leuko cytes
The combination of mAbs showed no effect on the WCC over the L0 day period of observations. Peripheral blood samples taken at 2 hourly intervals after mAb infusion also showed minimal variation.
7.4 Føilure of Either Single or Combined mAb Therøpy to Inhibit Celluløt Infiltrøtion or Prolong Allograft Surztioøl in the Sheep Model
The inability of QE4G9 to inhibit the cellular infiltration may in part be due to the upregulation of multiple adhesion pathways on the allograft endothelium, as discussed in previous Chapters. This explanation is supported by recent reports which suggest endothelial VCAM-L is not essential for the allograft
rejection process [Bergese et ø1. , 7995a; Bergese et ø1. , 1995b].In these reports the role of VCAM-L in murine cardiac allograft rejection was studied by pharmacological modification of its expression (i.e. pentoxifylline, anti-Il-4 mAb, soluble receptors for IL-4 or TNF). Despite the absence of VCAM-1 on the allograft endothelium, grafts were rejected at the same rate as untreated allograft recipients and although reduced, interstitial infiltration was still evident.
Furthermore, the presence of an extensive cellular infiltrate was observed
by Pelletier et al. l1992l in murine cardiac allograft recipients treated with low
doses (i.e. 200pglml) of anti-VCAM-l mAb. Moreover, high dose (i.e.,400p9/ml)
treated mice demonstrated a persistent, though less intense cellular infiltrate in the allografts at 60 days after transplantation, similar to that seen by Isobe et ø1.
ll994bl at 8 and 65 days after transplantation' 216
An alternative explanation for the lack of efficacy by QEaGg may relate to the epitope binding site of the mAb. Several studies have demonstrated that VCAM-1 has two functional binding sites for its ligand o4B1 (VLA-4), expressed by lymphocytes and monocytes [Osborn et a\.,1992; Vonderheide and Springer,
1.992; Chuluyan et nl., 1995a1, and that blocking of both sites is required to inactivate the VCAM-1./VLA- adhesion pathway [Vonderheide and Springer, t992;Chuluyan et ø1., 1995a1. There is also evidence that VLA-4 is capable of binding to endothelium via either ICAM-2 [Seth ef nl., 1'9911 or an inducible ligand, distinct from vcAM-1 [Vonderheide and springer, 1992].
As stated in the previous chapter, there is no structural information available regarding ovine VCAM-L to determine whether the molecule is expressed in the standard 7-domain or 6-domain forms. Furthermore, the epitope binding site of QE4G9 is unknown and it remains to be determined whether
QE4G9 blocks all VLA-4 binding sites.
The importance of VLA-4 in leukocyte adhesion at sites of inflammation has been d,emonstrated in a number of studies where anti-VLA- rr.\b, either
alone or in conjunction with other anti-CAM mAbs, have ameliorated the effects of inflammatory states [reviewed in Elices, 19951. Some of these experiments include the prolongation of rat islet allograft survival [Yang et ø1.,19951, inhibition
of MNC migration into inflamed peritoneum of rabbits [Winn and Harlan,7993l,
red.uced adhesion of both resting and activated T cells to rheumatoid synovial
endothelium [van Dinther-Janssen et al., 1991,] and significant inhibition of the contact hypersensitivity response in hapten-sensitised mice [Chisholm et ø1.,
1ee3l.
In addition, the integrin a4þ7, normally associated with leukocyte
adhesion to HEV in Peyer's patches via MAdCAM-1 [Berlin et ø1,,1993], has been
shown to interact with VCAM-I in aitro [Ruegg et ø1.,1992; Crowe et n1.,7994], 217
This interaction involves similar but nonidentical amino acid residues on VCAM-
L which are also required for binding of vLA-4 [Chiu et a\.,1995].
Therefore, while mAbs such as QE4G9 demonstrate significant inhibitory capacity in aitro, they wilt only be at best partially efficacious in aiao unless they btock all ligand binding sites on VCAM-1. Not only can MNCs still adhere to the allograft, but they may receive co-stimulatory signals from VCAM-I resulting in MNC activation and proliferation and enhancement of the inflammatory response. This was demonstrated by Orosz et ø1. 11993] where T cells, isolated from cardiac allografts of mice treated with anti-VCAM-1 mAb, were negative for the IL-2R. In addition, mRNA for IL-2,IL-4 and IFN-y could not be detected by
PCR, whereas untreated allografts were positive for the cytokine mRNAs and IL-
2R.
Based on these observations, and in spite of this group's success in obtaining long term survival of murine cardiac allografts with prophylactic anti- VCAM-1 mAb, they now propose that the mechanism of action of the mAb does not depend on inhibition of MNC adhesion, but rather involves the masking of graft-borne allogeneic dendritic cells (DCs) [Bergese et ø1., 1995a). This allows sufficient time for the DCs, which constitutively express VCAM-1 [Rice et ø1., Iggll and are acknowledged as potent APCs [Stingl and Bergstresser, 1995], to migrate out of the allograft, thereby possibly avoiding acute rejection. Interestingly, as mentioned earlier, although their origin remains unclear, a small number of interstitial cells expressing VCAM-L were detected at day 3 post- transplantation in the sheep renal allografts and were still present at day 7.
Finally, whilst there appear to be no reports in the literature concerning the prophylactic use of anti-VCAM-1 mAb in combination with anti-LFA-l mAb, there are numerous reports describing the use of other anti-CAM antibody combinations in the treatment of immune responses, including allograft rejection.
Examples include anti-LFA-L & anti-ICAM-1 [Isobe et ø1.,1992; Uchio et ø1.,7994; 218
Nakao et ø1.,1995; Tamiya et ø1.,19951, anti-LFA-l- & anti-vlA-4 [Issekutz, 7993; paul et ø1., 1993; Winn and Harlan, 7993; Issekutz and Issekutz, 1995; Issekutz,
1995;Yang et øL,lgg5l,anti-vCAM-l- & antiVLA- [Isobe et ø1.,1994a; Isobe et al',
Iggfibl, anti-ICAM-1 & anti-VLA-4 [Sadahiro et ø1.,1993)"
Thus targeting two independent CAM pathways in the sheep transplantation model was expected to synergistically inhibit the activation and proliferation of MNCs, and their infiltration into the allograft. The inability of the combined anti-ovine mAbs to alter the onset of acute rejection may be due to the factors discussed above regarding the requirement for QE4G9 to block all ligand binding sites on the ovine VCAM-I molecule and the apparent side effects elicited by T2-87. The use of mAb therapy does not guarantee extended graft survival, as demonstrated by earlier reports [Baume et ø1., 1989; Le Mauff et n1.,1991']. Furthermore, monoclonal antibody prophylaxis can exacerbate allograft failure, as
shown by the accelerate d graft loss in rat liver and cardiac recipients treated with anti-ICAM-L and anti-LFA-1 mAbs [Omura et øl',t992; Morikawa et ø1.,1994]
SUMMARY
Despite íne in aitro experimental results from the previous chapter
demonstrating QE4G9 effectively inhibited adhesion and proliferation of ovine MNCs when co-cultured with ShUVECs, the mAb was unable to prolong allograft survival in the sheep model. Flowever, this result is not altogether unexpected
due to the availability of multiple adhesion pathways on stimulated endothelium providing MNCs with alternative adhesion and migration routes.
Flowever, the inability of the mAb combination to effect allograft survival
was both disappointing and unexpected. There are a number of reports in the literature describing the efficacy of combined mAb therapy, whereas individually the mAbs were ineffective. Therefore while QE4G9 and 72-87 fall into the latter 219 category, additive inhibitory results from in aitro adhesion assay experiments led to the anticipation of prolonged graft survival.
potential explanations for the failure of QE4G9, either alone or in combination with 72-87, may involve its inability to block all ligand binding sites in the VCAM-1 domains. In addition, several pieces of experimental evidence, including the presence of MNC infiltrates in allografts of recipients treated with anti-VCAM-L mAb and the development of acute allograft rejection in the absence of endothelial VCAM-L expression, raise questions concerning the significance of VCAM-1 in the rejection process. Although again, this may only reflect the vast redundancy in the adhesion molecule pathways.
Finally however, most of the successful anti-VCAM-L therapy results were obtained in small rodent heterotopic allograft models, using exceptionally high doses of mAb. This in itself raises the issue of the clinical significance of results obtained from small animal models compared to the results from experiments performed in clinically relevant large animal models. 220
Chapter I
Attempted production of monoclonal antibodies against ovine ICAM-I
INTRODUCTION
The work presented in this chapter concerns the attempts to produce monoclonal antibodies directed towards ovine ICAM-1. Initially, fusions were performed using splenocytes from mice immunised with CM-stimulated ShUVECs only. Flowever, the availability of a human ICAM-1 transfectant cell line enabled adoption of several immunisation strategies designed enhance the likelihood of obtaining the desired mAb. Different combinations of ShUVECs and ICAM-1 transfectant were used for the primary immunisation and subsequent
boosts. The results obtained from these immunisation combinations are discussed below.
MATERIALS AND METHODS
Balb-c mice were immunised intraperitoneally with either CM-stimulated ShUVECs, ot a mouse L-cell transfectant stably expressing human ICAM-L 221.
(WEHI-ICAM-L), or a combination of both. Splenocytes were then fused with the murine myeloma cell line Sp2/O-Ag14 using 50% polyethylene glycol 1500 (PEG
1500) and grown in selective medium, as described in Chaptet 2.2.7.
Screening for mAbs was performed by FACS analysis of hybridoma supernatants on CM-stimulated and unstimulated ShUVECs, or WEHI-ICAM-I and nontransfected L-cells (Chapter 2.2.11,.1).
RESULTS AND DISCUSSION
In an attempt to raise anti-ovine ICAM-1 mAbs, a total of 9 fusions were performed, based on the 4 different immunisation protocols described below.
1) CM-stimuløted SbUVECs onlY
Five fusions were performed using splenocytes from mice immunised with stimulated ShUVECs. The screening process involved FACS analysis of reactivity of hybridoma supernatants with stimulated and unstimulated ShUVECs. It was anticipated that an antibody against ICAM-1 would be weakly positive on unstimulated ShUVECs and more strongly reactive on stimulated cells, whereas VCAM-1 and ELAM-1 would be negative on the unstimulated ShUVECs. It was from this immunisation regimen that the three anti-VCAM-l mAbs described in
Chapter 5 were generated.
This protocol produced 5 antibodies which displayed the FACS profile
expected for ICAM-1 on ShUVECs. Immunoprecipitation of iodinated cell surface lysates from stimulated ShUVECs revealed one of these antibodies to be recognising a structure with a molecular weight of approximately 50 kDa. The remaining antibodies did not precipitate any structures from the ShUVEC lysates. 222
While the molecular weight of the mature ICAM-L is within the range of
90-110 kDa, depending upon the level of gylcosylation in different cells [Dustin ef
ø1.,1986), the deglycosylated polypeptide form of ICAM-1 is reported to be 55 kDa [Dustin et ø1.,1986;Boyd et ø1.,1988]. Further studies involving glycosylation and protein stripping may help to determine whether the molecule recognised by this antibod.y represents the mature, fully glycosylated ovine ICAM-I- structure or the immature precursor form.
Alternatively, structural studies of ICAM-2 have identified this molecule to have a molecular weight of between 46 to 55 kDa [Staunton ef ø1., 7989a; Gahmberg et ø1., 1991.; Nortamo et ø1., t9911. However, FACS analysis of CM- stimulated ShUVECs showed the antibody to recognise an inducible molecule, whereas ICAM-2 is constitutively expressed on HUVECs and is not upregulated by inflammatory mediators [Staunton et al., I989a]. Therefore, sequencing of the molecule precipitated by this antibody, and analysis of homology with ICAMs from other species, may help determine its identity.
o e b tt øn sf e ct ønt 2) CM- stimul øt e d ShtIW C s f o ll w d y INEHI-I CAM-I
One fusion was performed using splenocytes from a mouse immunised twice with stimulated ShUVECs, followed 3 weeks later by a final boost with the ICAM-L transfectant. The strategy behind this protocol was to enhance the
response of the mouse to ICAM-1, based on the expectation that there would be
some conserved, homologous epitopes in the human and ovine ICAM-1 molecule. The use of the ICAM-I transfectant for the final boost, rather than stimulated ShUVECs, would also hopefully avoid further response to other unwanted proteins expressed on the surface of activated ShUVECs. Unfortunately this protocol did not generate any hybridomas producing suPernatants with the appropriate reactivity profiles on ShUVECs. However, L0 supernatants were 223 reactive with WEHI-ICAM-1, while being unreactive with the non-transfected L cells (and presumably recognise human ICAM-1)'
3) WEHI-ICAM-7 tuønsfectønt followed by CM-stimuløted ShUWCs
One fusion was performed using splenocytes from a mouse immunised twice with the WEHI-ICAM-L transfectant, followed a further 3 weeks later by a final boost with stimulated ShUVECs. The strategy behind this protocol was essentially the same as the one above, only priming the mouse with a human ICAM-1 expressing cell line, followed by ShUVECs with the aim of boosting the response to cross reactive epitopes. Supernatants were screened initially on the ICAM-1 transfectant and the non-transfected cells. Positive samples were then tested on stimulated and unstimulated ShUVECs. This Process revealed 4 supernatants specifically reactive with the WEHI-ICAM-I- transfectant, however none of these were reactive with ShUVECs.
4) WEHI-ICAM-I only
The final 2 fusions were performed using splenocytes from mice immunised twice with the WEHI-ICAM-1 transfectant. The supernatants were initially screened on the transfected and non-transfected cells. Only the supernatants positive on the ICAM-L transfectant were selected for testing on
stimulated and unstimulated ShUVECs. This Process yielded 30 supernatants specifically recognising the ICAM-L transfectant, again presumably human ICAM-1. Flowever, none of these supernatants were reactive with ShUVECs. 224
SUMMARY
Despite having completed 9 fusions and screened a total of approximately 2500 supernatants it had not been posssible to obtain an antibody which recognised a molecule on ShUVECs with the cell surface expression profile and molecular weight expected for ICAM-1.
Nevertheless, the Northern blot data for stimulated ShUVECs presented in
Chapter 5 clearly demonstrates that the size and induction profile of ovine ICAM- j. mRNA are consistent with that seen in HUVECs. Furthermore, the presence of the mRNA within the ShUVECs suggests that protein expression would occur on the cell surface. Flowever, the only antibody reactive with ShUVECs which partially fulfilled the induction and molecular weight requirements precipitated a surface protein of an unexpecte d size, raising doubts concerning both the identity of the structure recognised by the antibody and the nature of the ICAM-1 molecule expressed on the surface of ovine endothelial cells.
The availability of the human ICAM-1 transfectant provided a tool to specifically enhance the immunisation process in favour of ICAM-L and subsequently enabled rapid and accurate screening of supernatants, in conjunction with the non-transfected cells, resulting in the identification of 44 potential anti-human ICAM-L mAbs.
Therefore, to improve the specificity of the immunisation process in favour of ovine ICAM-I, it would be advantageous to immunise mice with a cell line transfected with ovine ICAM-I. In order to achieve this it would first require the cloning of ovine ICAM-1, followed by the production of a transfected cell line. Flowever time did not permit this aspect to be undertaken.
Finally, although it was not possible to obtain an anti-ovine ICAM-1 mAb from these fusions, a number of supernatants did demonstrate an expression profile on unstimulated and stimulated ShUVECs consistent with that expected 225 for either VCAM-I or E-selectin. Again, while time did not permit the cloning and investigation of these antibodies, the hybridomas have been expanded and stored in liquid nitrogen, hopefully for identification at a later point in time' 226
Chapter 9
Concluding Remarks
The historical perception of endothelial cells as merely an inert barrier has given way to recognition of the endothelium as a dynamic participant in a range of haemostatic and immunological functions, as described in Chapter L. Much of this understanding has come from tine in aitro study of ECs derived from large vessels, such as HUVECs. The extensive characterisation of HUVEC phenotype and function reported in the literature was a significant factor in the decision to use the ovine equivalent cells, ShUVECs, for studies in this thesis. The ShUVECs have been shown to be identical to HUVECs in almost every aspect studied and will therefore enable concurrent studies of EC function in uitro, and in aiao usíng the ovine renal transplantation model.
There has also been a wealth of knowledge accumulate over the past L0 to
L5 years regarding the distribution and expression of CAMs. The CAM families continue to grow as new molecules, such as cx,DB2 and ICAM-3, are identified and characterised. In addition to defining the kinetics of cell surface expression of CAMs, significant advances are being made toward understanding the intracellular activation events which control the upregulation or de notto 227 expression of CAMs. This research may provide valuable information to guide the development of new immunosuppressive agents aimed at down regulating the expression of CAMs.
Flowever, despite extensive knowledge of the distribution and expression of CAMs, the specific functional roles of many of the molecules remains unclear. This is highlighted by the conflicting mAb therapy studies reported both in this thesis and in the literature, and is possibly exemplified by the studies of VCAM-I expression and anti-VCAM-l mAb therapy in murine cardiac recipients lOtosz et al., 1993; Pelletier et al., 1993b; Bergese et ø1.,1995a1. In this SrouP of studies, an anti-VCAM-1 mAb induced long term tolerance to cardiac allografts, while specific inhibition of VCAM-1. expression did not block the onset of allograft rejection. These results raise doubts about the role of individual CAMs in the rejection process.
Moreover, the prediction of successful outcome of immunotherapy in large animal models or humans by the use of mAbs has relied on in ttitro and small
animal models. However, three major areas of concern with regard to this Process
are highlighted by the studies presented in this thesis;
(L) extrapolation of results fuom in aitro models of allogeneic resPonses (e.g., MLC
or MLER) are not necessarily predictive of the outcome during inaiao studies,
(2) rodent models of allograft immunosuppressive therapy do not predict the
outcome of similar studies in clinical relevant large animal models, and
(3) blocking two CAM pathways (i.e., VCAM-1/VLA-4 and LFA-1/ICAM-1) simultaneously did not prolong allograft survival, also contrary to the expected
outcome based uPon in rtitro observations.
Although the initial results from Phase I clinical trials of anti-LFA-1 and anti-ICAM-1. mAbs are encouraging, the unsuccessful results obtained in these ovine studies and by others (see Chapters 4 and 7) suggest that targeting other 228
"second signal" molecules, such as CD28, CTLA-4 or CD40, may be an alternative strategy for future mAb therapy. Several recent reports describing the successful use of mAbs against these molecules are also encouraging [Turka et a\.,1992;Lin et ø1.,1993; Larsen et øL, tgg6l, although again results from in aiuo studies in rodents will require further investigation and confirmation.
With regard to mAb therapy in general, another difficulty highlighted by t¡¡e in aiao studies reported in this thesis was the generation of adverse side effects following i.v. administration. A number of strategies have been developed to overcome some of the side effects elicited by murine mAb therapy. The construction of "humanised" mAb has been one apProach used to avoid the induction of an anti-murine Ab response in patients, potentially increasing the efficacy of second and subsequent courses of mAb therapy. However, construction of these modified mAbs can be difficult and often results in Abs with reduced affinity for their antigen.
An alternative strategy for the production of human antibodies involves the use of phage-antibody-display libraries. As discussed by de Kruif et ø1. 119961,
this technology will not only be useful in the production of human mAbs, but can
also be applied to the production of mAbs against prokaryotic and eukaryotic cell
surface molecules.
This technology could potentially be employed to extend the studies of ovine CAMs described in this thesis. In particular, in view of the difficulties in producing an anti-ovine ICAM-L mAb by traditional hybridoma techniques, the
construction of a phage-display library of sheep antibody fragments may increase
the likelyhood of identifying the desired mAb.
Further areas of investigation arising from the studies reported in this
thesis include the structural characterisation of ovine VCAM-1, sequencing of the molecule and possibly epitope mapping to determine the binding site of QE4G9. These studies will determine the number of Ig domains in the ovine VCAM-I 229 molecule and enable comparison with VCAM-L of other species. The QE4G9 epitope mapping may help to explain the lack of efficacy of the mAb in the inaiao studies reported in ChaPter 7.
Similarly, cloning and sequencing of ovine ICAM-L to determine the number of Ig domains in the molecule may also help to explain the unexpected immunoprecipitation results described in Chapter 8 obtained with a potential anti-ovine ICAM-I Ab.
A detailed knowledge of the structure and expression of these, and other ovine CAMs, will be of great value in understanding and interpreting observations from future studies in the ovine renal transplantation model. 230
Appendix L
Comparison of physiological parameters in the plasma and serum of sheep with a renal allograft or an autograft compared to non-transplanted sheep.
Allograft (n=4) Autograft (n=2) Non-transpl# -264 Creatinine Ltmol/L 79 - 78t 69 -13r 6L Urea mmol/L 7.8 - 43 t0.2 - 12.9 5.8 Hb g/dL 5.6 - 8.5 8.8 - 1.0.6 9 -15 Glucose mmol/L 2.6 - 4.2 3.1- 4.2 3.0 Sodium mmol/L 138 -]r'4 LAL -749 1.42- 148 Potassium mmol/L 3.6 - 4.9 4.0 - 4.7 4.0 - 5.2 Calcium mmol/L 2.10 -256 2.43 -2.68 2.3 -2.7 Chloride mmol/L 96 - L09 105 - 109 104-11.2 Bicarbonate mmol/L 27 -33 27 -30 27 Phosphate mmol/L 0.78 -2.22 1..57 -2.45 L.50 ALP U /L 46 -L76 48 -203 21 -276 wCC (x 709 /L) 5.8 - 10.9 5.4 - 8.8 4-12
# r"t g"r for normal, non-transplanted sheep. Some values were adapted from Hecker [1983], Gross [199a] and Adams & McKinley [1995]. 231. Bibliography
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ZojaC, Waog ]M, Bettoni s, sironi M, Renzi D, Chiaffarino F, Abboud HE, van Damme J, Mantováni A, Remuzz!G, et al.: Interleukin-l beta and tumor necrosis factor-alpha induce gene expression and production of leukocyte chemotactic factors, colony-stimulating factors, ãnd interleukin-6 in human mesangial celIs. Am. J. Pnthol. 7991,; L38: 991'. Zola1n. Monoclonøl Antibodies: A marutnl of techniques, Roco Baton: CRC Press, 1,987. UNI OCIç) 25015 () Ct t:,"1L C c.1_ ERRATUM Page iii: "dependant" should be "dependent". This error is repeated on: Page 29,line9, Page 43,Iine 2, Page 56,Iine 23, Page 90, heading 2.2.6, Page 127,Iine 19, Page 1.44,line L. Page 48, line 23, "is" should be "ate" Page 50, line 3, "methodology" should be "methods". Page 'J.07, Iine 4, "transfered" should be "transferred". Page 135, line L4, "eliminated" should be "elimination".