Cell Adhesion Molecules in Human Transplantation (Medical Intelligence Unit) [2nd ed.] 1570595178, 9781570595172, 9780585408569

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MEDICAL I N T E L L I G E N C E U N I T

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Gustav Steinhoff, M.D., Ph.D.

Cell Adhesion Molecules in Organ Transplantation Second Edition

R.G. LANDES C O M P A N Y

MEDICAL INTELLIGENCE UNIT 1

Cell Adhesion Molecules in Organ Transplantation Second Edition Gustav Steinhoff, M.D., Ph.D. Medical School Hannover Hannover, Germany

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MEDICAL INTELLIGENCE UNIT 1 Cell Adhesion Molecules in Organ Transplantation, Second Edition R.G. LANDES COMPANY Austin, Texas, U.S.A. U.S. and Canada Copyright © 1998 R.G. Landes Company and Chapman & Hall All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

U.S. and Canada ISBN: 1-57059-517-8 Reasonable efforts have been made to publish reliable data. The authors, editors and publisher believe that the accuracy and interpretation of the data (including but not limited to drug selection and dosage, methods and procedures), as set forth in this book, are in accord with current recommendations and practice at the time of publication. They make no warranty or endorsement, expressed or implied, with respect to the material described in this book and are not responsible for the consequences of its use. In view of the ongoing research, product and equipment development, changes in governmental regulations and the rapid accumulation of bioscience information, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Steinhoff, Gustav, 1958– Cell adhesion molecules in human organ transplantation / Gustav Steinhoff —2nd ed. p. cm. — (Medical intelligence unit) Includes bibliographical references and index. ISBN 1-57059-517-8 (alk. paper) 1. Transplantation immunology. 2. Cell adhesion molecules. I. Steinhoff, Gustav, 1958- Cell adhesion molecules in human organ transplants. II. Title. III. Series. [DNLM: 1. Transplantation Immunology. 2. Cell Adhesion Molecules— immunology. 3. Major Histocompatibility Complex—immunology. 4. Organ Transplantation. WO 680 S822c 1998] QR188.8.S74 1998 617.9'5—dc21 DNLM/DLC for Library of Congress 98-10904 CIP

MEDICAL INTELLIGENCE UNIT 1 PUBLISHER’S NOTE

Cell Adhesion Molecules in Organ Transplantation

R. G. Landes Company produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books work directly with the physical systems they describe and contribute with avid interest to progressing knowledge about these systems. Typically, topics are fascinating pieces of important biological puzzles; often few or no similar books exist on these topics.

Second Edition

Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to match the fast pace at which information grows in bioscience. We aim to publish our books within six months or lessSchool of receipt of the manuscript. We thank Medical Hannover our readers for their continuing interest and welcome any comments or Hannover, Germany suggestions they may have for future books.

Gustav Steinhoff, M.D., Ph.D.

Judith Kemper Production Manager R. G. Landes Company

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

DEDICATION For Antje, Elsa and Tristan

CONTENTS Part 1: Cellular Adhesion Molecules, Major Histocompatibility Complex Antigens (MHC) and Immune Reactivity 1. Mechanisms of Interaction Between Immune Cells and Organ Transplants ............................................................... 3 Gustav Steinhoff Organ Specific Manifestation of the Immune Reaction ........... 6 Considerations on Somatic Differences in the Susceptibility to Allogenic Immune Processes .............................................. 8 Considerations About the Genetical Modification of Immunological Reactivity in Organ Transplantation ....... 9 2. Molecular Basis of Transplant Infiltration and Rejection ...... 11 Gustav Steinhoff Endothelial Adhesion Molecules and Leukocyte Adhesion Receptors in Transplant Inflammation ................................ 14 Initial Phase of Transplant Inflammation ............................... 15 The Intermediate Phase of Transplant Inflammation ............ 17 The Late Phase of Transplant Inflammation ........................... 18 The Infiltration Phase of Transplant Inflammation ................ 21 3. Structure and Function of MHC and Adhesion Molecules .... 25 Gustav Steinhoff Major Histocompatibility Complex Molecules (MHC) ......... 25 Intercellular Adhesion Molecules ............................................. 27 Part 2: MHC and Adhesion Molecules in Clinical Organ Transplantation 4. Major Histocompatibility Complex Antigen and Cell Adhesion Molecule Expression in Clinical Renal Transplantation ......................................................................... 37 Susan V. Fuggle Introduction .............................................................................. 37 Major Histocompatibility Antigen Expression ........................ 38 Clinical Correlations and Diagnostic Potential of Upregulated Class II Antigens .......................................... 39 Adhesion Molecules .................................................................. 41 Discussion .................................................................................. 52

5. MHC and Cell Adhesion Molecules in Clinical Heart Transplantation ......................................................................... 57 Gustav Steinhoff and Matthias Wilhelmi Expression of Major Histocompatibility Complex (MHC) Molecules in Human Heart Transplants .............................. 57 Expression of Adhesion Molecules in Human Heart Transplants ............................................................................ 64 6. Cell Adhesion Molecules in Clinical Lung Transplantation ... 77 Gustav Steinhoff Introduction .............................................................................. 77 Patients and Methods ............................................................... 77 Results ........................................................................................ 78 7. MHC and Cell Adhesion Molecules in Clinical Liver Transplantation ......................................................................... 89 Gustav Steinhoff Major Histocompatibility Complex (MHC) Molecules in Human Liver Transplants ................................................. 89 Intercellular Adhesion Molecules in Human Liver Transplants ............................................................................ 94 Part 3: Pathophysiological and Clinical Aspects 8. Role of Adhesion Molecules in Reperfusion Injury and Rejection ........................................................................... 117 Gustav Steinhoff Transplant Reperfusion Reaction ........................................... 117 Acute and Chronic Transplant Rejection .............................. 118 Longterm Adaptation of Organ Transplants ......................... 121 9. Cytomegalovirus (CMV) Infection, Expression of Cell Adhesion Molecules and Allogeneic Rejection .......... 123 Xiaomang You Introduction ............................................................................ 123 Manifestation of CMV Infection in Allografts ...................... 124 Expression of MHC Molecules with CMV Infection ............ 126 Expression of Cell Adhesion Molecules ................................. 128 Mechanisms of Regulation for Allogeneic Rejection by CMV Infection ................................................................ 130 Impact of CMV Infection on Allogeneic Grafts .................... 133

10. Therapeutic Options by Blocking Adhesion Molecules ........ 137 Michael Brandt Introduction ............................................................................ 137 Reperfusion Injury .................................................................. 137 Acute Allograft Rejection ........................................................ 139 Chronic Allograft Rejection .................................................... 140 Future Outlook ........................................................................ 141 Part 4: Adhesion Molecules in Xenotransplantation 11. Adhesion Molecules in Xenotransplantation ........................ 145 André R. Simon, Anthony N. Warrens and Megan Sykes Introduction ............................................................................ 145 The Immunosuppression Approach ...................................... 146 The Genetic Approach ............................................................ 147 The Tolerance Approach ........................................................ 147 Specific Adhesion Molecule-Ligand Interactions .................. 150 Comments ............................................................................... 162 References .......................................................................................... 165 Index .................................................................................................. 215

EDITORS Gustav Steinhoff, M.D., Ph.D. Department of Thoracic and Cardiovascular Surgery Leibniz Research Laboratories of Biotechnology and Artificial Organs (LEBAO) Medical School Hannover Hannover, Germany Chapters 1, 2, 3, 5, 6, 7, 8

CONTRIBUTORS Michael Brandt, M.D. Department of Thoracic and Cardiovascular Surgery Medical School Hannover Hannover, Germany Chapter 10 Susan V. Fuggle, D. Phil. Nuffield Department of Surgery University of Oxford John Radcliffe Hospital Headington, Oxford, United Kingdom Chapter 4 André R. Simon, M.D. Transplantation Biology Research Center Surgical Service Massachusetts General Hospital Harvard Medical School Boston, Massachusetts, U.S.A. Chapter 11 Megan Sykes, M.D. Transplantation Biology Research Center Surgical Service Massachusetts General Hospital Harvard Medical School Boston, Massachusetts, U.S.A. Chapter 11

Anthony N. Warrens, M.D., Ph.D. Transplantation Biology Research Center Surgical Service Massachusetts General Hospital Harvard Medical School Boston, Massachusetts, U.S.A. Chapter 11 Matthias Wilhelmi Department of Thoracic and Cardiovascular Surgery Medical School Hannover Hannover, Germany Chapter 5 Xiaomang You, M.D. Division of Immunoloigy and Infectious Diseases Dalhousie University Halifax, Nova Scotia, Canada Chapter 9

ABSTRACT

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dhesion molecules play a central role in the regulation of immune responses after organ transplantation. This book gives a broad survey about the current knowledge of expression and induction of various intercellular adhesion molecules in clinical kidney, liver, heart and lung transplantation. Furthermore, the relevance of cellular adhesive interactions for the developing field of discordant xenotransplantation is reviewed. This book gives a complete survey on the cellular expression patterns on endothelia, resident cells and graft infiltrating cells in human transplants. The patterns of cellular expression and inducibility in different pathological conditions of the graft are reviewed. The implications for the organ specific appearance of inflammatory reactions in human heart, kidney, liver, and lung transplants for immunosuppressive and therapeutic interventions are described. The future prospects concerning tolerance induction, specific immunosuppression, and immune escape mechanisms for allo- and xenotransplantation are discussed.

PREFACE

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he control on the immune reaction of a host to an organ transplant is of crucial importance for its function and acceptance. In recent years, it has become clear that immunological changes of the organ transplant contribute to the control of the recipient’s immune reactivity. A number of signal transducing molecules have been identified that regulate leukocyte reactivity in a receptor-ligand mode. It became clear that the presence of certain ligand molecules on endothelial cells and in the organ tissue is significantly important for the regulation of processes as leukocyte-endothelial adhesion and tissue infiltration. A central role could be attributed to the class I and class II MHC molecules by the presentation of antigen peptides to the T cell receptor. In an organ transplant, the MHC (HLA) molecules are by their genetic polymorphism additionally the main transplantation antigens. For the mediation of specific and unspecific leukocyte-endothelial adhesion, transmigration and interstitial infiltration a number of further cell-cell and cell-matrix adhesion molecules are necessary that may function in a multiple receptor binding or in certain binding sequences. In this book different aspects of the expression of immunological ligand molecules are described in human kidney, heart, lung and liver transplants. Organ specific peculiarities are of interest for the investigation of organ specific manifestations of immune processes. These concern peculiarities in the histocompatibility after transplantation and local differences in the manifestation of the rejection reaction. The analysis of cell adhesion molecules in organ transplants forms a molecular basis for the kind of immunological reactivity between host immune system and transplant cells. The molecules can be attributed to regulate the induction and course of antigen specific and unspecific inflammatory reactions in the graft. The pattern and coexpression of certain adhesion ligand molecules and cell-matrix receptor molecules may explain the diversity of immunological compatibility and susceptibility of different cell types. This knowledge forms a basis for the understanding of organ specific pathophysiology and local manifestation of rejection and inflammation processes. A main result from the investigation is the knowledge that the different cell types in a transplanted organ may possess distinct immunological reactivity. This may explain special immunological functions of organ specific blood stream compartments for the control of leukocyte reactivity. It could further explain organ specific features and functions on the background of the local expressed and soluble secreted adhesion molecules. Further investigations have to

define the intercellular steps of reactions involving different cell adhesion molecules and to evaluate effects of toxins, cytokines and drugs. It can be foreseen that this may lead to a more specific and differentiated immunological treatment of transplant recipients in the sense of the manipulation of immune reactivity by intervention in intercellular adhesion reactions. The compatibility and incompatibility of adhesive molecular interaction and their inducing signals (i.e. cytokines) between species is a main field of basic research in xenotransplantation. This investigation deals with the specific immunological problems arising by xenotransplant rejection, but also deals with the specific perspectives in genetical engineering of intercellular contacts to induce tolerance of the host or anergy of the graft. The modification of adhesive interactions between graft cells and immune cells by the tools of gene transfer or temporary blockade (i.e. drugs, ligand absorption) will be a major field of development in the improvement of allogeneic and xenogeneic organ transplantation. Gustav Steinhoff Hannover, Germany September 1997

ACKNOWLEDGMENTS The authors wish to acknowledge Stefanie Hartung and Ingo Meents for their help in the preparation of the manuscript.

Part 1 Cellular Adhesion Molecules, Major Histocompatibility Complex Antigens (MHC) and Immune Reactivity

CHAPTER 1

Mechanisms of Interaction Between Immune Cells and Organ Transplants Gustav Steinhoff

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he basic problem in the transplantation of organs or cells between species (xenotransplantation) and between individuals (allotransplantation) is the immunological reaction of the host against foreign tissue antigens. This process leads to the rejection of an organ transplant. In the beginning of the transplantation of vascularized xenografts and allografts rejection phenomena were observed that first were attributed to difference in histocompatibility between species and individuals.99,100,464 Considerations on the possible causes of transplant rejection were made by the serological knowledge about human blood group antigens.230,242,423 The identification of a genetic basis of histocompatibility was founded on the discovery of the importance of the ABO-blood group system for the rejection of skin grafts first postulated by Shawan711 and proved by kidney transplants by Simonsen and Sorensen.727 This was confirmed by the first clinical observations by Hume et al.344 Research on the transplantation of tumors led to the identification of histocompatibility antigens by Gorer.263 Apart from the recognition of genetic difference in histocompatibility Loeb479 postulated somatic differences on the basis of discrepancy in the rejection of different organs. The insight in genetically determined histoincompatibility between individuals of one species was initiated by clinical experiments with skin grafts by Gibson and Medawar255 and further experimental research by Medawar.508,509 From this, the knowledge was deducted that the rejection of organ transplants within one species is mediated by specific immunological reactions directed against genetically determined antigeneic structures of the transplanted tissue.510 Experiments done by Mitchinson524 and Billingham et al79 could prove the transferability of this immunity from one to the other individual by lymphoid cells. The immunological reaction leading to transplant rejection was recognized as a cell-mediated process. Finally, a system of histocompatibility antigens on human leukocytes (HLA-system) was discovered by Dausset174 and van Rood.870,871 This led to the description of the genetic region of the human major histocompatibility complex (MHC).68,69 In the last two decades it has become clear that the function of the MHC encoded Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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HLA-molecules is the regulation of T lymphocyte reactivity. The MHC molecules are able to present autologous and foreign peptides to the T cell antigen receptor (TCR).70,694 The progress of immunological knowledge in the last years has further enraveled the cellular and molecular processes that lead to inflammatory immune reactions and to the rejection of organ transplants. The induction of the cellular alloantigen directed immune reactions as well as the contribution of different immunological cell types to the process of rejection have become more and more clear. This immunological cell interaction, however, involves also the interaction of a number of adhesive ligand molecules transplant cells as endothelial cells, tissue macrophages and interstitial dendritic cells. The main cell types involved in the interaction between immune system and transplant are listed in Table 1.1. It is now clear that the intercellular reaction of host immune cells with transplant cells is mediated by a number of specific adhesive cell membrane receptor molecules in addition to the interaction of TCR and MHC.746 The regulation of their expression on recipient as on transplant cells is dependent on soluble mediators and cytokines.153,408,613 The interplay of different immunocompetent cell types is the precondition for the generation of an immune reaction. Apart from the specific immune reactions, specifically equipped T and B lymphocytes also “accessory” cells are necessary for the delivery of an antigen-specific reaction.855 These are of central importance for the presentation of antigen peptides and to connect T lymphocytes by adhesive interactions and cytokine stimulation. The main “accessory” cell types are interstitial dendritic cells793-796 and macrophages.793 In organ transplants HLAincompatible “accessory” cells of the donor as well as immigrating recipient type “accessory” cells play a major role in the regulation of the immune reactivity of T lymphocytes against transplantation antigens.45 A major function is the presentation of antigens and stimulation of CD4+ T-helper lymphocytes.738,835,854 In the human liver, these are mainly interstitial dendritic cells in the portal tract,625 Kupffer cells,793 but also endothelial cells.614 Similarly, the distribution of perivascular dendritic cells and tissue macrophages in heart grafts538 and lung grafts underlies an organ specific variation. It is now clear that in the course after organ transplantation not all accessory cell types of the donor are preserved. In different experimental and clinical investigations the cell migration and exchange of these cell types after organ transplantation has been described.45,751 In that especially, the reduction of immunogenicity of organ transplants by the decrease of dendritic cells as emigrating “passenger cells” has been observed.446 The exchange of Kupffer cells in the liver after bone marrow or liver transplantation has been found by different authors.247,249,265,616-618,779 The partial longterm-exchange of dendritic cells in human liver grafts and the influence of immune reactions on the exchange of Kupffer cells has been described.766,768,779,783 A main factor for the initial activation of T lymphocytes is their reactivity to endothelial cells of organ transplants. It has become clear that this interaction may decide about the further course or suppression of an immune reaction.613 The molecular interaction of TCR to MHC or alloantigeneic peptides is under intensive study at present. This analysis, however, has also made clear that the activation of alloantigen-reactive T lymphocytes is not only a single receptor-ligand interaction.408,677,746 A main prerequisite for the adhesive binding of T lymphocytes to endothelial cells is an additional interaction of intercellular adhesion molecules.

Mechanisms of Interaction Between Immune Cells and Organ Transplants

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Table 1.1. Interaction between immune system and organ transplant Cell Type

Receptors

Ligand Molecule

Target Cell

TCR,CD8 TCR,CD4 CD2 LFA-1

MHC class I MHC class II LFA-3 ICAM-1,2,3

endothelial cell dendritic cell macrophage epithelium/ parenchymal cell

VLA-4 CD28 L-selectin sialyl LewisX

VCAM-1 B7 sialyl LewisX E-/P-Selectin

Antigen Specific Reaction T lymphocyte

B lymphocyte

MHC, Kl.II FcR Mac-1 LFA-1 CD22 Nonspecific Immune Reaction

Antigen (MHC peptide) endothelial cell Ig macrophage ICAM-1 parenchymal cell ICAM-1,2 sialic acid glycans

Monocyte/macrophage

MHC, Kl.II Mac-1 p150,95 LFA-1 VLA-4 L-selectin sialoadhesin CD33

antigen (MHC peptide) ICAM-1 ? ICAM-1,2 VCAM-1 sialyl LewisX sialic acid glycans sialic acid glycans

endothelial cell parenchymal cell lymphocyte

NK-cell

NCAM LFA-1

NCAM ICAM-1

endothelial cell parenchymal cell

Polymorphonuclear leukocyte

Mac-1 (LFA-1) L-selectin (VLA-4)

ICAM-1 ICAM-1 sialyl LewisX (VCAM-1)

endothelial cell parenchymal cell

This has not only the function to arrest passing lymphocytes or leukocytes at endothelia, but also to mediate additional intracellular signals leading to cell activation or de-activation. The first main receptor-ligand pairs identified were LFA-1/ICAM-1 and CD2/LFA-3. Their interaction potentiates the lymphocyte activation and cell adhesion processes.196,488 The expression of LFA-1 is restricted to leukocytes.336 The expression of ICAM-1 (CD54) was found at first on activated lymphocytes and endothelia. Later its inducibility by cytokines492 and expression on other cell types was detected.203,909 Faull and Russ216 found in 1989 the inducibility of ICAM-1 on the tubular cells of rejecting kidney grafts. Similar observations were made on

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hepatocytes of the liver.6,760 It could be assumed that the induction of ICAM-1 ligand molecules would influence the leukocyte reactivity in transplanted tissue. On monocytes the importance of ICAM-1 for accessory cell function and clustering of dendritic cells was described.551 In contrast to ICAM-1, a second ligand to LFA-1 (CD11a), ICAM-2, showed a high basal expression on endothelial cells and absence of inducibility.181,488 CD2 receptor molecules are only expressed on lymphocytes. Its ligand molecules LFA-3 (CD58) showed a broad expression on different cell types.488 In addition, a number of endothelial-specific ligand molecules were identified: PECAM (CD31), VCAM-1, ELAM-1 (E-selectin), and CD62 (P-selectin; PADGEM, GMP140).746 Also on leukocytes a number of additional adhesion receptor molecules were found as the integrins VLA-1, VLA-4 and the selectin LECAM (L-selectin). These react to a number of cellular ligand molecules and cellmatrix molecules.346,746 By the discovery of these molecules a molecular basis for the understanding of the regulation of intercellular processes between T lymphocytes, endothelial cells, perivascular dendritic cells and interstitial cell-matrix molecules was laid.124-126,150,168,477 A schematic description of the main molecular interactions in the intravascular and interstitial phase of transplant rejection is given in Figure 1.1. The current hypothesis about the adhesion molecule interactions involved in different steps of leukocyte-endothelial interaction is depicted in Figure 1.2

Organ Specific Manifestation of the Immune Reaction The studies on immunological changes of organ transplants in this book show the foundation of molecular interactions between immune cells of the host and the cells of the organ transplant. The study of kidney, liver, heart and lung transplants show a cell type- and organ-specific expression of various immune adhesion molecules. Similar as in heart and lung the major “immunocompetent” cell types as interstitial dendritic cells and macrophages expressed a manifold of adhesion molecules in high density. The immunocompetence of other cell types in the organ tissue is most likely dependent on their respective expression of intercellular adhesion molecules. The endothelia in different parts of the vascular stream bed inside the kidney, liver, heart or the lung display such differences in MHC and vascular adhesion molecules. This refers to both the basic expression (high in the liver sinusoid and lung capillaries) as to differential inducibility (low in liver sinusoid and lung capillaries). It is conceivable that these differences in “immunocompetence” result in a fine regulation of leukocyte reactivity inside the vascular stream bed. Furthermore, by specific receptor-ligand combinations the homing of leukocyte subpopulations to certain vascular compartments may be regulated. This has to be discussed especially for the interaction of memory T lymphocytes (CD45RO+) and NK-cells that are specifically accumulated in the liver sinusoidal and lung capillaries.686,879 Similar mechanisms may, however, exist in other organs as in lymph nodes or thymus. Organ specific differences in the immunological susceptibility can be explained by differences in patterns of basal or induced expression of intercellular adhesion molecules on the different organ cell types. The expression of MHC and other adhesion molecules including cell-matrix receptors is clearly cell-type specific. Despite major differences in the patterns of ligand molecules and their inducibility by cytokines it can be generally postulated that almost all cell types can be immu-

Mechanisms of Interaction Between Immune Cells and Organ Transplants

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Fig. 1.1. Mediation of intravascular T lymphocyte-endothelium interaction and interstitial antigen presentation by dendritic cells to T lymphocytes by intercellular adhesion molecules—concept of a multireceptor interaction. The receptor-ligand binding of different cell adhesion molecules is shown. A specific antigen recognition evolves by the binding of the T cell receptor (TCR) to MHC and antigen peptide or allo-MHC. This process is supported by further binding- and activation-adhesion molecules intravascular (cell-cell) and interstitially (cell-cell and cell-matrix). APC - antigen presenting cell/ interstitial dendritic cell; X – carbohydrate molecules necessary for the rolling process of L– (LECAM-1) and E– (ELAM–1) selectins.

nologically recognized by immune cells upon induction of the main ligand molecules MHC (class I) and ICAM-1. A relative resistance against immunological recognition can exist by deficient basal expression (for instance on hepatocytes and myocytes) or by a relative resistance against cytokine mediated induction (myocytes). The composition of such differing immunocompetent cell types in an organ transplant may determine its susceptibility to immune destruction. Generally, a minor basal expression of adhesion ligand molecules or incomplete ligand pattern can be overcome by the action of local released cytokines from infiltrating leukocytes or tissue macrophages. The susceptibility to cytokine stimulation in the different cell types may also determine patterns of adhesion ligand molecules induced in a specific organ. Therefore the regulation of the anti-alloantigeneic immune response and its manifestation may depend on the one side on organ specific intravascular stimulation (in vascularized organ transplants). On the other side the localization and kind of interstitial infiltration may depend on the organ specific composition in adhesion ligand and cell-matrix molecules allowing

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Fig. 1.2. Leukocyte-endothelial interaction: Stepwise receptor-ligand interaction leading to firm leukocyte adhesion and transmigration (© Biotest Aspekte, 3/96).

differences in the stimulation and manifestation of immune reactivity. The local cellular sensitivity—mainly read by MHC and other adhesion molecule expression—may determine an organ specific difference in cytokine mediated effector mechanisms and the extent of organ destruction by the rejection response.

Considerations on Somatic Differences in the Susceptibility to Allogenic Immune Processes Deducting from specific immunological and cellular aspects of the human liver that may influence the immunogenicity of a transplanted liver the question arises, if similar peculiarities exist in other organs. The diversity of various cell types in immunological compatibility may thereby form a foundation of somatic differences in transplant rejection. Distinct susceptibility of certain intravascular regions as the liver sinusoid and lung capillaries as well as differences in adhesion interaction between arterial and venous endothelia probably exist in different organs similar to liver and lung transplants. It can be stated that the local specific vascular reactivity as in lung capillaries or in kidney glomeruli may determine the organ specific appearance of rejection activity. Surveying different organ systems the special pathology of arterioles and postcapillary venules in organ transplants with acute and chronic rejection has to be stressed as a predilectory site of lymphocyte adhesion and infiltration.457 It can be assumed that in all afferent and efferent vascular beds of different organs similar to liver and lung a special pattern of adhesion molecules exists as the particular expression of ICAM-2 and the coexpression of VCAM-1, E- and P-selectin. Further investigations are needed to analyze experimentally and by in vivo microscopy organ specific differences of the vascular stream beds regarding to their molecular interaction with passing leukocyte/lymphocyte subpopulations and thrombocytes. It can expected that common and organ specific features of the endothelial mo-

Mechanisms of Interaction Between Immune Cells and Organ Transplants

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lecular ligand structures exist that are sufficient to explain specific rejection patterns. Furthermore, a common aspect in several organs is the respective cellular baseline expression of MHC and other adhesion molecules as well as the cellular sensitivity or receptors for cytokine stimulation. This may explain organ specific aspects in rejection resistance as for example known for the myocytes in the heart muscle. For these differences probably not the expression of donor MHC on transplant cells is the determinant but only the coexpression of ICAM-1, LFA-3, and VCAM-1. A high of low immunogenicity of a transplanted organ or transplanted cells thus may be deducted from its molecular cell surface structure in immunological interaction (adhesion) molecules and their inducibility by cytokines. Advantage in the selection of single cell types in their resistance against immunological recognition and effector processes thus may involve mainly their membrane adhesion ligand structure. It is not considered for this that the host to transplant reaction is primarily a passive event from the side of transplant cells that are only activated by the alloantigeneic response. This is in contrast to other types of inflammatory immune reactions as in viral or bacterial infection or in postischemic reperfusion injury with a primary reaction of tissue cells that secondarily recruit and activate leukocytes. Especially the dynamic of changes in membrane expression of adhesion molecules on parenchymal and endothelial cells points to their active contribution in the afferent phase of immunological and inflammatory reactions. Thus it seems likely that a regulation of an immune reaction is usually exerted by tissue cells, mainly endothelia, in response to ischemic, toxic, or infectious stimulants in the organ tissue. Signal transduction by intercellular binding of adhesion molecules as between perivascular dendritic cells/macrophages and endothelial cells as well as the release of a variety of cytokines are most likely the primary mechanisms. In a transplanted solid organ such a primary reaction of transplant cells to ischemic, toxic, or infectious causes interference with the immunological rejection reaction against histoincompatible MHC antigens. Finally, it can be postulated that the function of the described system of intercellular and cell-matrix adhesion molecules is not restricted to the interaction with immune cells, but that a number of other reactions as the organotypical cell-matrix organization of cells and homotypical adhesion, cell proliferation and metabolic reactions may be influenced by this communication system.

Considerations About the Genetical Modification of Immunological Reactivity in Organ Transplantation In the current clinical practice, the immune reactivity to a transplanted organ is only controlled by immunosuppressive drug treatment. This allows a rather selective intervention in the immunocompetence of transplant recipients suppressing T lymphocyte dependent immunological reactions. Inflammatory cellular activation of transplant cells upon stimuli as postischemic reperfusion injury and viral infection, however, remain rather unchanged. The preservation of a limited immune response to the graft may help to control opportunistic infections as especially important in lung allotransplantation. Longstanding graft inflammation on the other side may lead to destruction of tissue structures as in obliterative bronchiolitis of the lung, transplant coronary artery disease in the heart and vanishing bile duct syndrome in liver transplants. Even more, this problem may cause

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Cell Adhesion Molecules in Organ Transplantation

transplant dysfunction in xenotransplantation. The question arises, if a specific genetical manipulation of the graft may allow a reduction of inflammatory upregulation of graft cells and open the possibility to downregulate immune recognition. In the last years cellular chimerism of the transplant recipient has been discussed as a means to induce tolerance to allo- or xenografts.751,817-819 On the other side transplant chimerism with the partial restitution of the transplanted organ by recipient cells as f.i. Kupffer cells265,618,774,779 may allow to induce graft acceptance by the chimerism of the transplanted organ.751,752 This limited process may be manipulated by cellular seeding techniques to reduce the antigenicity of organ transplants. Seeding techniques for endothelial cells have been introduced for vascular prostheses and heart valves256,558 and may be transferable to solid organ transplantation. The physiological genetical transformation of a transplanted graft by the ingrowth of recipient cells thus may form a tool for immunological manipulation of the graft. Ultimately, tissue engineering using autologous cells to grow tissue or organ transplants may overcome the problem of transplant rejection. Genetical manipulation of vascular adhesion molecules both in human allotransplantation and xenotransplantation will be another important option to modify the contact surface of organ transplants.347 The study of transplant immune reactions in animals deficient in or transferred with genes for adhesion molecules is of critical value in the judgment about the possibility for the modification of immune responses in clinical organ transplants.691 Moreover, genetical manipulation by using sense or anti-sense gene-transfer to up- or downregulate the expression of adhesion molecules may be applied to the modification of cell and solid organ transplants.799 The modification of leukocyte-endothelial interaction by the manipulation of graft endothelia is of principal interest for the application of gene manipulation to increase longterm graft acceptance and induce graft tolerance. This refers to the reduction in antigenicity by downregulation or masking of MHC molecules in allotransplantation as well as to the reduction of oligosaccharide ligands (sialic acids or galactosyl residues) in allo- and xenotransplantation.154,386 Downregulation of main leukocyte ligands as ICAM-1 799 or carbohydrate ligands109,602 may serve as a potent tool for specific intervention in leukocyte interaction to transplant endothelia. Furthermore, new approaches to the downregulation of intracellular signal pathways of cytokine/adhesion molecule activation show the principal direction of the genetical engineering of endothelial cell reactions in the next years.151,221,222,260 A new era of experimental and clinical investigations calling adhesion molecule interactions at the center of interest for genetical manipulation can be envisaged.

CHAPTER 2

Molecular Basis of Transplant Infiltration and Rejection Gustav Steinhoff

T

he analysis of the expression of MHC and intercellular adhesion molecules in kidney, liver, heart and lung transplants reveal major changes in basic expression and induction patterns. Strong differences were found in the reactivity—and by that the immunological compatibility—of different cell types. These differences in immunological susceptibility and compatibility of organ transplants are provoked by rejection processes and other inflammatory conditions of the transplant. Studies on clinical biopsy material revealed that a number of additional inflammatory and postischemic stimuli induce similar changes in the composition of intercellular adhesion molecules in transplants.310,435,777,765 Intragraft accumulation of cytokines as TNFα and IL-1β could be demonstrated in endothelial lining cells and macrophages in local concordance with ICAM-1 induction.333 A major aspect of the shown alterations is that they show variant states of the immunological activation and immune competence of the transplanted organ. This can be deducted from different densities in the cellular expression of incompatible MHC and other adhesion molecules (ICAM-1, LFA-3) in the transplant. It can be postulated, however, that only the intravascular coinduction of certain ligand molecules leads to an adhesive reactivity of T lymphocytes and leukocytes. Although the intravascular donor MHC expression in liver grafts is reduced in the late course after liver transplantation by the exchange of Kupffer cells,265,779 the basal expression of class I and class II donor MHC antigens on endothelial cells is a constant feature in kidney, heart and lung allografts. By that, the exposition of allogeneic MHC structures to T lymphocytes by graft endothelia is a continuous event after implantation of the organ. It can be assumed that this may lead to a constant stimulation of immune reactivity even under effective immunosuppression. The results of the expression pattern of immune adhesion molecules in clinical organ transplants show a differential inducibility on the different cell types as well as a time dependent sequence of the induction of ligand molecules. These findings suppose that the immune reaction of transplant cells involves a number of T lymphocyte dependent and independent inflammatory phases. At first, an intravascular and an interstitial phase of the immune reaction have to be distinguished. Then theoretically a rejection dependent immune reaction in a transplanted organ can be divided in sequential events. Four main steps can be discussed. Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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Cell Adhesion Molecules in Organ Transplantation

The first event, intravascular rolling of leukocytes, can be considered as a physiological process (Step 1). The primary contact between leukocytes and endothelial cells is based on the interaction of selectins (L-selectin, P-selectin, E-selectin) with carbohydrate molecules (sialyl LewisX) and similar ligand molecules in endothelia (E- and P-Selectin).125,439,747 The induction of the ligand molecules E- and P-selectin on endothelia causes a deceleration of the leukocyte passage as a rolling event. Lindbohm et al475 could show that this selectin mediated rolling process is a prerequisite for a β2 integrin mediated leukocyte-endothelial interaction. The missing inducibility of E- and P-selectin on liver sinusoidal lining cells as well as on lung capillaries suggests that the rolling of leukocytes is modified in this vessel segment or even altered in such a way that the leukocyte-endothelial interaction is influenced. Other endothelial cells in arteries and veins show the typical inducibility of selectins upon inflammatory stimulation as known from in vitro studies.611 It cannot be excluded, however, that a similar rolling interaction can be controlled by different molecular mechanisms as the interaction of CD44 molecules and hyaluronic acid. The endothelial cell activation by the alloantigeneic immune reaction (binding of HLA-antibodies, release of cytokines by reactive T lymphocytes) and by other pathomechanisms is most likely the generator of further reactions leading to tissue inflammation (Step 2). It has to be emphasized that the activation of endothelial cells may be the consequence of immunological stimulation by cytokine release by activated intravascular leukocytes as in transplant rejection. However, it can also be the consequence of tissue ischemia as in organ transplant preservation/reperfusion, drug toxicity, and infections (HBV,CMV,EBV a.o.). By that, the endothelial activation may be the result of a local amplification of an effector immune reaction as the HLA-directed alloreaction, on the other side it may also be the promoter of an immune reaction by the on-site activation of lymphocytes. In this situation the primary activation of the endothelium may be caused by a different stimulus as ischemic or toxic damage. The recruitment of adhesive leukocytes is probably mediated primarily by a process of temporary adhesion preceded by the rolling of leukocytes. The temporary adhesion probably depends on the presence of the corresponding leukocyte ligands on the endothelium, mainly ICAM-1 and 2; LFA-3; MHC class I and class II; VCAM-1. The coexpression and a critical membrane density of these ligand molecules may be necessary for the induction of leukocyte binding necessary for intercellular cytokine stimulation and antigen presentation. In this respect the sinusoidal endothelium of the liver and capillaries of the lung are of special interest, because—in the absence of selectin molecules and partially VCAM-1—they possess a broad panel of basally expressed intercellular ligand molecules, such as ICAM-1,-2, LFA-3 and MHC molecules (HLA-DR). In the liver sinusoid, in addition CD4 molecules are expressed on endothelial cells. The broad spectrum of the expressed molecules may present a highly adhesive ground for passing leukocytes. The absence of E-, P-selectin and VCAM-1 probably is necessary to prevent firm adhesion and microvascular thrombosis. The intravascular or in situ activation of leukocytes is upon the endothelial activation the precondition of a firm or definitive leukocyte-endothelial binding (Step 3). This process most likely implies intracellular activation signals that produce changes in receptor constellation and composition on the cell membrane. It can be induced by cytokines and most likely also by soluble adhesion ligand mol-

Molecular Basis of Transplant Infiltration and Rejection

13

ecules as MHC, ICAM-1, LFA-3, E- and P-selectin. Leukocytes carrying highly affine receptors (LFA-1, CD2) may more easily bind to activated endothelia. For the process of firm adhesion probably a stabilization by additional components as complement factors is necessary. The firm binding to endothelia in organ transplants then may cause a direct damage of the vascular endothelium seen as histopathological endothelialitis.735,913 This damage or induction of the retraction of endothelial cells from the basement membrane may enable the leukocyte to easily transmigrate the vessel lining cells and emigrate in the perivascular interstitial tissue. A special anatomical feature of the liver sinusoid is the perivascular space of Disse. Hereto certain leukocyte subpopulations as natural killer cells and monocytes are able to transmigrate. It is very well possible that this selective transmigration process is facilitated by special adhesion molecules. In this context the exclusive induction of NCAM adhesion molecules on sinusoidal endothelia during transplant rejection and strong inflammation could enable NK-transmigration by homotypic NCAM interaction. The lack of lymphocyte infiltration in the liver sinusoid may very well be the result of the lack of VCAM-1 and selectin expression. It is possible, that the VCAM-1 ligands are necessary for their vessel wall transmigration.570 These ligand patterns show differences in local typical endothelial differentiation that influences leukocyte subpopulation and thrombocyte reactivity. It is unclear, however, how far the reactivity of different leukocyte populations is influenced by this composition of vessel wall ligands. The clarification of this needs experimental and in vitro investigations. The interstitial phase of the inflammatory immune reaction (step 4) enables target cell interactions with parenchymatous cells (epithelia, cardiomyocytes) and interstitial dendritic cells. A main part of the interstitial phase is in addition the cell-matrix interaction of leukocytes by the use of integrin receptors. The importance of the cell-matrix interaction may be reflected by the increased and generalized expression of VLA-4 and the de novo expression of additional matrix receptors (VLA-1,5,6 and CD51) on infiltrating leukocytes.765 It can be suspected that these receptors exert functions in the interstitial migration of leukocytes and possibly in their activation. The binding of integrin receptors to matrix molecules as collagen and fibronectin may relate to the initiation of a local metalloproteinaserelease. This could already be the case at the basal membrane to enable leukocytes to leave the vessel into the interstitium. The question arises, if this process is dependent on the binding of matrix specific integrin receptors to activate leukocyte metalloproteinases and to distinguish different matrix component proteins. In vitro investigations could demonstrate a matrix-molecule dependent T lymphocyte activation mediated by integrin receptors (VLA-4 to fibronectin CS1 splice variant).559 Further studies are needed to clarify such cell-matrix interactions that may have basic relevance in cell biology. The induction of cell-cell as well as cell-matrix receptor molecules on parenchymal cells is most likely a cytokine effect of infiltrating leukocytes.630 This may render the organ cells susceptible to T lymphocyte recognition and lysis (with rejection) by the induction of mainly MHC and ICAM-1 molecules. It also leads to the direct activation of organ transplant cells that is reflected in the increased expression of cell-matrix receptor molecules.765 It can be stated that the local fibrotic reaction with the deposition of matrix proteins may result from this cell activation.86 However, it is not excluded that also metabolic changes as regeneration and proliferation processes may be influenced.30

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Cell Adhesion Molecules in Organ Transplantation

The regulation of the different steps of tissue inflammation may depend on a limited repertoire of adhesion molecules and their respective counterparts and involves a manifold of intercellular reactions as well as intracellular activation and suppression events. Thus it can be estimated that the use of a limited adhesion receptor repertoire may steer quite different intercellular processes. For the induction phase of inflammation it can be assumed that the binding of LFA-1/MAC-1 to ICAM-1 and -2 plays a central role in the generation of a firm leukocyte-endothelial binding.125,747 This molecular interaction, however, is also involved in the process of transendothelial migration.570 On a molecular basis the diversity of receptor functions could be explained by a sequential binding of different molecular isoforms with different glycosylation of ICAM-1.585 In the interstitial infiltration phase the binding of LFA-1 to ICAM-1 together with TCR to MHC is a central prerequisite in antigen presentation by interstitial dendritic cells683 as well target cell cytolysis.872 The latter reaction involves not only antigen specific cytolysis but also nonspecific cytolysis by macrophages and NK-cells.632 A similar diversity in intercellular reactions and functions has to be attributed to the CD44 molecule.306 It is known that a molecular microheteroenicity of this molecule exists that may cause the mediation of different intra- and intercellular reactions. A further example for a multiple functional binding capacity is the VLA-4 receptor molecule that shows binding sites to cellular (VCAM-1, M-addressin) and matrix (fibronectin, thrombospondin) molecules.144,627 The exact contribution of the single molecules to the different steps in tissue infiltration, cell activation, cytolysis and proliferation is not unraveled yet. Given the above hypothesis it can be assumed that the molecules are involved in different intercellular reactions. In addition for special purposes there may exist separate molecules as the selectins for intravascular rolling. The sequence of events in inflammatory reactions and with that the rejection of an organ transplant may involve a certain pattern. The knowledge of the molecular mechanisms may then open the way to find tools for intervention.

Endothelial Adhesion Molecules and Leukocyte Adhesion Receptors in Transplant Inflammation In the last years it has been understood that the endothelial cells play a central role in the regulation of leukocyte adhesion and extravasation between the intravascular and extravascular compartment. A major function probably is exerted by adhesion molecules in a differential and sequential mode of expression on endothelial cells.763-765 It is conceivable that their regulation of expression determines a certain order of events during tissue inflammation that can be described in a phase model. The assumed hypothetic phases can be deducted from the changes in sequential transplant biopsies, mainly in liver transplants. The progress to a next phase may very well depend on the stepwise completion of stipulated adhesive interactions. By this every step of the inflammatory reaction may decide on the further course. The regulation mechanisms may require constellations of adhesion molecules as cytokine effects. Four phases of intravascular and extravascular tissue inflammation in organ transplants are postulated, namely an initial phase of endothelial activation: (1) immediate interactions; (2) intermediate phase; (3) late phase of definitive or firm leukocyte adhesion to activated endothelial cells; and (4) a phase of leukocyte transmigration and interstitial infiltration with cell-matrix interactions.125,611,747

Molecular Basis of Transplant Infiltration and Rejection

15

Two models of a stepwise generation of tissue inflammation have been postulated by Pober and Cotran611 and Butcher.125 These were based on in vitro data and presumed a three or more step genesis of inflammation. Especially the Butchermodel supposing a three step interaction between leukocytes and endothelial cells may be attributed to the changes observed in vivo in the inflammation of liver, heart and lung transplants. A first step postulated would be the binding of constitutive leukocyte receptors to endothelial ligands. This process of primary adhesion could be mediated mainly by lectin-carbohydrate interactions. To these both leukocyte and endothelial selectin molecules and related oligosaccharide ligands may contribute. This initial adhesion process should be a temporary and reversible event, if not a second factor is initiated, namely the activation of leukocytes by specific chemoattractive or cell-adhesion triggered intracellular signal pathways. These may firstly induce receptor conformation and avidity changes of constitutional adhesion receptor and secondly the coinduction of secondary adhesion receptors that are dependent on cell activation. The interaction of activation-dependent adhesion receptors with endothelial ligand may result as a third step in firm adhesion that completes processes as antigen recognition of MHC peptides and may precede the transmigration processes. The best characterized activation dependent leukocyte adhesion receptors according to Butcher125 are the heterodimeric Integrins of the β2 (CD18) or β1 (CD29) classes. The model implicates that leukocyte-endothelial recognition can be controlled on each of the presumed steps. This may lead to a mechanism dependent on a combination of initiation factors and may create a specificity and diversity of leukocyte-endothelial interaction. The in vitro based model of Pober and Cotran611 distinguishes also three phases in the generation of inflammation, that are ordered in a time dependent initial phase (5-30 min), intermediate/early phase (2-6 hr), and late phase (12-48 hr). The presumed phases contain the previously described events as intravascular rolling, endothelial and leukocyte activation, and definitive/firm adhesion up to transmigration and tissue infiltration. Each phase is characterized by a special pattern of mediators that lead to discrete patterns of adhesion molecules induced. Table 2.1 gives a survey on the model of Pober and Cotran.611 The sequential fashion of induced adhesion molecules as based on in vitro findings on umbilical vein endothelia may very well fit into observations done here on expression patterns in different situations sequentially after the implantation of allogeneic organ transplants.

Initial Phase of Transplant Inflammation An initial phase as a first step of rejection, postischemic or infection related transplant inflammation presumably may depend on the initiation by intravascular rolling of leukocytes. Studies using intravital microscopy have revealed that this is induced within minutes after tissue ischemia and organ reperfusion.499,500,513,514 It involves the rolling of neutrophils on vessel walls. The process is reversible in the vessel segments involved. Intravital studies and experimental models have revealed the specific nature of this process at activated vessel segments.439,513 A major impact of the rolling process could be the deceleration of neutrophils at the site of injured or inflamed (heat, toxins, ischemia) vessel areas and to induce the possibility for the neutrophils to check the endothelial surface for activating or chemoattractive signals.125

16

Cell Adhesion Molecules in Organ Transplantation

Fig. 2.1. Immediate phase of inflammation. This event is characterized by physiological intravascular “rolling” of leukocytes on the endothelial surface. This process is mediated by lectin-carbohydrate interactions and an activation dependent selectin expression.

Further studies concerned the fact that the interaction of lectins with carbohydrates and the activation dependent selectin expression is the mediator of this first inflammatory reaction.440,475,884,885 The selectins as key molecules of intravascular rolling act either as receptors on circulating leukocytes and thrombocytes or on the corresponding endothelial cells. It seems that the increase of intercellular adhesion is a main aim, but not the mediation of intracellular activation. The selectins induce a “stumbling” of leukocytes and thrombocytes on branches of complex N- or O-bound sugar molecules. It could be shown that these act as ligand for the selectins. The effect is a selective reduction of the flow of leukocytes inside the blood and to increase the secondary interaction of low-affine β1 or β2 integrin adhesion receptors. A main finding is that the selectin expression on endothelial cells underlies major differences in the organ specific vessel compartments. Sinusoidal endothelia of the liver and capillaries of the lung are spared from the induction of endothelial selectins. This may implicate a major difference in leukocyte activation and contact at these vessel sites and bear consequences for the local appearance of the inflammatory response. Intravital microscopy of the liver and in part of the lung in the rat during organ reperfusion after ischemia could confirm such regional difference in intravascular rolling.412,499,514 It seems that rolling does not occur at these capillary sites upon activation, but that only a process of temporary adhesion of leukocytes and thrombocytes is possible. The more interesting is the fact that tissue infiltration of T lymphocytes and leukocytes during transplant rejection cannot be observed in the liver sinusoid and lung alveolar tissue.

Molecular Basis of Transplant Infiltration and Rejection

17

Fig. 2.2. Intermediate phase of inflammation. The event is characterized by the activation of endothelial cells and a temporary adhesion of leukocytes. It is induced by cytokines, “rolling” contact of leukocytes, tissue ischemia, toxins as lipopolysaccharides and (viral) infectious pathogens.

The Intermediate Phase of Transplant Inflammation The intermediate phase of an inflammatory reaction may involve mainly endothelial activation and temporary leukocyte adhesion at activated endothelia. The intravascular adhesion molecules as CD44, VCAM-1, LFA-3 and PECAM (CD31) may appear in this stage of inflammation. Their contribution to temporary leukocyte-endothelial adhesion and to the binding of different leukocyte subpopulations, however, is speculative at present. It can be assumed, however, that their endothelial expression is the result of activation by different stimuli as cytokines, contact activation by rolling activated leukocytes, local ischemia, toxins as lipopolysaccharides and infectious pathogens. From the observation of the patterns of in vivo expression in organ transplants probably the following leukocyte and endothelial receptors are of importance in an intermediate inflammation phase. A main function may be exerted by CD44 homing receptors that bind to homotypic ligands on activated endothelia (possibly CD44 variants) or to hyaluronic acid. A second important interaction for temporary adhesion may be mediated by CD2 binding of T lymphocytes to LFA-3 that may be able to induce cell activation. In the liver, heart and lung LFA-3 is present on capillary endothelia upon stimulation. This interaction may facilitate the binding of CD4 to MHC class II. In the liver sinusoid this reaction occurs in two ways, as sinusoidal endothelia and Kupffer cells possess both MHC class II and CD4 receptors. A reversible binding of MHC to TCR may allow a fluent contact to antigen peptides. The binding of VLA-4 to VCAM-1 and of LFA-1/MAC-1 to ICAM-1

18

Cell Adhesion Molecules in Organ Transplantation

Fig. 2.3. Late phase of inflammation. This event is characterized by the activation of leukocytes and their definitive/firm adhesion to activated endothelial cells. Three main reactions mediate this interaction: (1) adhesion receptor-ligand binding to endothelial cells leading to transmembrane signaling of CD2, CD44, LFA-1, or VLA-4; (2) conformational change of receptor affinity (LFA-1, MAC-1) upon firm binding or due to intravascular preactivation by cytokines and soluble factors (sICAM-1, sLFA-3, sMHC, sELAM-1, sCD62); (3) a two (or more) receptor (VCAM-1, ICAM-1, ELAM-1) requirement for the achievement of a steady state adhesion reaction.

and 2 ligands only may be reversible or temporary, if the leukocyte receptors run in a state of low affinity. Especially in the liver sinusoid and lung capillaries with broad expression of ICAM-1 and -2 this interaction may be facilitated.

The Late Phase of Transplant Inflammation This hypothetic phase would be characterized by the firm binding of activated leukocytes to activated endothelia and the induction of consecutive transmembrane signals. Already intravascular a preactivation of leukocytes by cytokines and soluble factors as ICAM-1, LFA-3 and MHC molecules may occur. The transmembrane signal activation may then involve certain leukocyte adhesion receptors as CD2, CD44, LFA-1 or VLA-4477 capable to induce intracellular activation pathways. These may involve increased cytosol calcium by calcium-channel influx and increase phosphatidyl inositol turnover.584 Furthermore, a receptor re-conformation may lead to a higher grade of intercellular affinity. This phenomenon has been especially described for LFA-1 and MAC-1 integrin receptors: In the absence of activating signals they are presumed to be rather inactive. Therefore, monoclonal antibodies directed to β2 integrins do not affect leukocyte rolling and

Molecular Basis of Transplant Infiltration and Rejection

19

Table 2.1. Model of a stepwise regulation of inflammation according to Pober and Cotran611 Phase

Event

Effect

Mediators

Immediate phase (5-30 min)

1. endothelial secretion of prostacyclin, EDRF 2. endothelial contraction

1. vasodilatation and increased leukocyte recruitment 2.increased permeability and local blood stasis 3. neutrophil/monocyte adhesion 4. neutrophil activation and chemokinesis 5. neutrophil infiltration

1. histamin, thrombin LTC4, a.o. 2. histamin, thrombin, LTC4, a.o. 3. histamin, thrombin, LTC4, a.o. 4. histamin, thrombin, LTC4, a.o. 5. PAF, C5a,LTB4, a.o.

1. sustained vasodilatation and increased leukocyte recruitment 2. vascular permeability and local blood stasis 3. neutrophil (monocyte) adhesion 4. neutrophil/monocyte activation and chemokinesis 5. neutrophil infiltration

1. IL-1, TNF

3. endothelial expression of CD62 4. endothelial synthesis of PAF 5. β2 integrin dependent transmigration of activated neutrophils Intermediate phase (2-6 hr)

1. increased endothelial secretion of prostacyclin (EDRF?) 2. endothelial reorganization 3. endothelial expression of ELAM-1 4. endothelial secretion of IL-8/MCP-1 5. β2 integrin-dependent transmigration of activated neutrophils

Late phase (12-48 hr)

1. increased endothelial secretion of prostacyclin 2. maintained endothelial reorganization 3. increased endothelial expression of ICAM-1, VCAM-1; decreased ELAM-1 4. endothelial secretion of IL-8/MCP-1 5. β2 integrin dependent and independent (VLA-4or CD44-dependent?) transmigration of lymphocytes/monocytes

1. preserved vasodilatation and leukocyte recruitment 2. increased vascular permeability and local blood stasis 3. lymphocyte/monocyte adhesion; decreased neutrophil adhesion 4. lymphocyte/monocyte chemokinesis 5. mononuclear cell infiltration

2. IL-1, TNF 3. IL-1, TNF 4. IL-1, TNF 5. IL-8, PAF, C5a, LTB4, a.o.

1. IL-1, TNF 2. IL-1, TNF, IFNγ

3. IL-1, TNF, IFNγ

4. IL-1, TNF, IFNγ 5. IL-8, MCP-1, others?

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Cell Adhesion Molecules in Organ Transplantation

affinity in a state before β2 integrin re-conformation. Neutrophil leukocytes, however, that already stick to endothelia are released into the circulation again by antiβ2 antibodies. Earlier studies by Pardi et al585 have allowed conclusions that the adhesive reaction between cells depends on the relative concentrations of receptor and ligand on the membrane, whereby the intrinsic affinity of the receptor for its ligand and its diffusion along the cell membrane undergoes modulation. These seem to be the most important regulative factors for the formation of a strong receptor-ligand binding. The first observation showed that the local density of a given receptorligand pair has a critical influence on the affinity of the cell interaction, when receptor and ligand are membrane-bound. It is known that a 2- to 3-fold decrease or increase of adhesion receptor density have significant functional influence. With respect to ligand affinity the molecular mechanism of an allosteric change leading to an altered receptor affinity is not clarified yet. A number of intracellular events, however, have been described that parallel the kinetics of affinity change of leukocytes or even precede them.585 In conclusion the change of intercellular affinity of leukocytes is probably a consequence of conformation changes in the extracellular domain of the LFA-1 heterodimer either by physical association with the actin based cytoskeleton or by direct biochemical modification of the cytoplasmic domains. The first measurable event is in this context the multistep Ca2+-dependent protein kinase C activation204 in combination with a temporary increase in intracellular Ca2+-concentration. In connection to this a network of proteinkinase C dependent phosphorylations on a variety of substrates in addition to the LFA-1 heterodimer may cause an affinity change of this molecule. The cytoskeleton associated isoforms of LFA- and other β2 integrins are changed on their extracellular domain conformation with the creation of neo-epitopes24,868 and the loss of other epitopes that are existent in heterodimers, but not associated with the cytoskeleton. The polarization of a cell resulting from the new arrangement of the cytoskeleton may help to direct the secretory part of the leukocyte toward the cell bound. This may allow to carry out effector functions with the secretion of soluble mediators in a very efficient paracrine form. Lastly the description of the adhesion receptor diffusion or lateral mobility of the receptors is an important factor that determines the fate of the interaction between receptor and respective ligand. The observed event is probably a mechanism caused by a high lateral mobility of glycosyl-phosphatidyl-inositol bound forms that accumulate soluble or transmembranous ligands in the area of cellular attachment.144 This is supported by the fact that adhesion to transmembranous forms of the ligands are less efficient, when they are expressed in a lesser density. According to the localization of various ligand isoforms in certain areas of the endothelial cell membrane623 the hypothesis may be allowed that the sequential leukocyte adhesion to different isoforms of these molecules are related to a mechanistic movement from the site of initial contact (apical) to areas of transmigration (basement membrane). The activation dependent interaction of leukocyte-integrin receptors is stable under physiological shear stress conditions for minutes, but seems to be a reversible event.478 The reversibility has the consequence that the extravasation is not obligatory following endothelial cell binding. The progress from temporary leukocyte-endothelial binding then may depend on a second signal that may originate

Molecular Basis of Transplant Infiltration and Rejection

21

from extravascular stimulants as chemoattractive substances. This second signal may be a requirement for the initiation of diapedesis and tissue migration. However, if the second permissive signal is derived from the endothelial cell itself, extravascular chemoattractants may then function for the initiation of extravasation. Thus in several situations, and probably organ specific sites, the kind of extravascular signals and chemoattractive substances released determine, if an endothelial bound leukocyte is able to emigrate or to be released to the circulation again.125 In the late phase of inflammation, two categories of adhesion receptor-ligand molecule interactions can be differentiated that probably play an important role in the postulated leukocyte activation and definitive/firm adhesion. The first category involves the leukocyte receptors CD2 and the integrins LFA-1, MAC-1 and p150,95. As stated above especially the receptors LFA-1 and MAC-1 can reach a state of higher affinity by several mechanisms that allow higher specificity in the late phase of inflammation. The respective ligands LFA-3 and ICAM-1 are expressed not only on endothelial cells, but on a variety of parenchymal cells as the hepatocytes of the liver and the pneumocytes of the lung. Probably, they are also present in soluble isoforms during inflammation. In the liver the interaction of these receptor-ligand pairs may be facilitated in the sinusoid, in the lung in the capillaries by the presence of high ligand concentrations on endothelia. The second category of receptor-ligand pairs contains either the receptors VLA-4 and LECAM-1 (L-selectin) in the interaction to VCAM-1 and E-selectin (ELAM-1). The distribution of the ligands shows differences to the first category with a predilection to arterioles and veins both in the liver and the lung. VCAM-1, however, can be induced at other sites during inflammation (liver sinusoid, heart capillaries). It is remarkable in this context that all vessel wall adherent leukocytes in the pathological specimens expressed either LFA-1 or MAC-1, CD44 and VLA-4. Furthermore these molecules were present on tissue infiltrating cells. The broad positivity for these receptor molecules in immunohistological analysis cannot distinguish fine differences in affinity binding or glycosylation of receptors,306,382 but points to their central role in the processes of definitive adhesion, leukocyte emigration and tissue infiltration.

The Infiltration Phase of Transplant Inflammation The last step for the completion of tissue inflammation is the phase of tissue infiltration. It involves the transendothelial migration of leukocytes with consecutive migration through the interstitial perivascular tissue. A major role for the mediation of cellular interactions play cell-cell as well as cell-matrix binding. The first is probably functional with transendothelial migration and in binding to interstitial dendritic cells or parenchymal target cells. The second interaction involves binding to basement membrane matrix components and further tissue specific matrix proteins in the interstitium. The process of transendothelial migration firstly involves cell contacts of the receptors LFA-1/MAC-1 and VLA-4 that function in firm intravascular adhesion, but then either change affinity state or interact with modified isoforms or alternative ligands to allow leukocytes to migrate on the endothelial surface. In the liver and the lung this process during rejection seems to have local predilections either for the portal tract and the perivascular/ peribronchial space. This also can be observed in different forms of liver inflammation. A possible explanation for the special infiltration patterns of leukocyte

22

Cell Adhesion Molecules in Organ Transplantation

Fig. 2.4. Infiltration phase of inflammation. The event is characterized by the transendothelial migration of leukocyte subpopulations and their consecutive tissue infiltration. The processes are mediated by cell-cell interactions (transendothelial, interstitial) and cell-matrix interactions (subendothelial, interstitial).

subpopulations could be differences in the expression of isoform ligands, mainly to be discussed for ICAM-1 or ICAM-2. The second reaction needed for extravasation is the contact and cleavage of basement membrane matrix proteins. For this process the matrix binding of VLA-4 receptors is most likely the major tool. The binding to fibronectin and possibly other matrix proteins may allow the recognition of matrix components, the release of metalloproteinases needed for cleavage. Furthermore a process of interstitial migration and cell movement may involve the use of integrin receptors to matrix components. The coexpression of the receptors VLA-1, VLA-5, and CD51 on interstitially infiltrating lymphocytes during late rejection supports this assumption.765 This points to their functional role in the subendothelial, interstitial migratory and possibly proliferative phases of the inflammation of liver grafts.713 Resulting from the described findings in organ grafts and functional data two main categories of receptor-ligand interactions for cell-cell and cell-matrix binding in the phase of tissue infiltration can be differentiated. The first involves highly affine LFA-1 and VLA-4 that bind to cellular ligands ICAM-1 (isoforms) and VCAM1. The second category involves cell-matrix receptor binding, mainly of VLA-4, secondary of VLA-1,5,6 and CD51—possibly in dependence on the kind of the surrounding matrix protein composition. The concordant function of cell and matrix contacts of the migrating leukocytes may allow the process of tissue infiltration.

Molecular Basis of Transplant Infiltration and Rejection

23

Chronic inflammatory reactions as chronic liver graft rejection result in the fibrotic transformation of the organ. In the human liver a central regulator of fibrogenesis is the activation of lipocytes.86 Perisinusoidal lipocytes (ITO cells) are present in the normal liver, where they exert function in the deposition of retinoids. Upon activation by cytokines they produce new collagen types, the proteoglycan synthesis and retinoid metabolism is changed and α actin is expressed. These activated lipocytes are probably identical to myofibroblasts found in fibrotic livers. A number of activation signals as cytokines and retinoids have been identified, but this may also be assumed for extracellular matrix proteins that may activate cells by integrin receptor binding as shown for fibronectin splice variants.559 Thus the activation by matrix compound may not only activate immigrating leukocytes, but even to a higher extent the locally present ITO-cells and tissue macrophages. This and a chronic stimulation by cytokines released from immigrating leukocytes may induce a circle of events leading to an excess production of matrix proteins resulting in organ fibrosis during chronic organ transplant rejection.

CHAPTER 3

Structure and Function of MHC and Adhesion Molecules Gustav Steinhoff

Major Histocompatibility Complex Molecules (MHC)

T

he genetic polymorphism of molecular groups causing individual differences forms the basis for the problem of histocompatibility in organ transplantation. The polymorphic epitopes of the molecules of the major histocompatibility complex (MHC) are the major transplantation antigens.285,298 Their expression at the membrane of the different types of donor cells causes recipient sensitization and the development of an alloantigen-directed immune response. In addition, it has become clear that the MHC molecules form the ligand of the T cell receptor for the presentation and recognition of antigens.694 The regulation of the expression of self-MHC molecules in tissue may therefore determine the kind and course of an immune response.69 Whereas a static expression of these molecules has been assumed in the past it is now clear that their expression is dynamic and underlies regulatory influences.285,298 Two classes of major transplantation molecules bearing polymorphic epitopes are encoded by genes of the MHC on chromosome 6. The class I molecules (HLA-A, B, and C) are glycoproteins composed of an α chain (338 amino acids) with three immunoglobulin-like domains. Out of the three α domains, the polymorphic sequence is restricted to the α1 and α2 domains that are also a binding site of the T cell receptor molecule. The entire α chain can be divided into three structural regions (beginning at the amino-terminal end): an extracellular hydrophilic region (amino acids 1 through 281), which can be further divided into the three α domains, a transmembrane hydrophobic region (amino acids 282 through 306), and an intracytoplasmatic region (amino acids 307 through 338). The noncovalent associated β chain, β2-microglobulin, completing the receptor complex of the class I MHC molecule, is not encoded by the MHC, but on chromosome 15, is not polymorphic and consists of only one extracellular domain. The α3 domain and β2microglobulin are structurally homologous to the constant region of immunoglobulins, identifying the class I molecule as a member of the immunoglobulin supergene family. Three different polymorphic class I molecules are expressed that were defined serologically by their differences in antigenicity (HLA-A, HLA-B, Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

26

Cell Adhesion Molecules in Organ Transplantation

HLA-C). The class II molecules are glycoproteins and composed of an α and an β chain that are both polymorphic (HLA-DR, HLA-DP, HLA-DQ) and both contain two domain regions. Additional HLA-D region genes have been defined (HLA-DO, HLA-DN, HLA-DM, HLA-DV, HLA-DX, HLA-DZ) that may have yet undefined different products (Fig. 3.1). The polymorphic regions of both class I and class II molecules are the binding site to the T cell receptor molecule.847 The polymorphism of this binding site between individuals allows the distinction of self and nonself.447 At the cleft of the polymorphic α1 and α2 domains of the MHC molecule the peptide-fragments of foreign antigens are bound and presented.87,88 In transplanted organs due to difference in the polymorphic sequence of the HLA-molecules a T cell sensitization to nonself antigen is induced. By similarity in sequence clones crossreactive with viral or bacterial antigens may be activated. The T cell reactivity first and most powerful is determined by the expression of donor HLA-II-molecules on the membrane of so called antigen presenting cells (APC) such as dendritic cells, mobile bone marrow derived donor cells which are present within the transplanted tissue (‘direct route’ of antigen presentation) A second, ‘indirect’ route of T cell activation is maintained by autologous accessory cells (recipient dendritic cells, macrophages) which is usually weaker than the first route. Furthermore, the binding of T lymphocytes may be determined by the additional expression of adhesion molecules.205 Studies concerning the expression of the two classes of MHC molecules: class I (HLA-A,B,C) and class II (HLA-DR,DQ,DP) on different cell types using monoclonal antibodies have shown a heterogeneous distribution in normal human tissue.169,170 Whereas class I MHC antigens were expressed on the majority of cell types, the class II MHC antigen expression was mainly restricted to B lymphocytes and accessory cells.169,170 The limited distribution of class II MHC antigen positive accessory cells (dendritic cells, macrophages) in tissue may therefore cause a local restriction of T lymphocyte reactivity and sensitization. However, a number of studies have shown that class II MHC antigens can be induced on a number of additional cell types during immune reactions and infections.183,285,298,419,521,792 These also may be capable to present antigens.480 Lymphokines have been identified as the mediators of the induction of MHC expression.277,474,754 During immune reactions in organ transplants the induction of MHC antigens on donor cell types additionally increases the target antigens for the alloreactive T lymphocytes. Studies in experimental organ transplant models have demonstrated a massive induction of donor class I and class II MHC molecules on graft cells during acute rejection. Immunosuppressive drugs such as cyclosporine A have been shown to abrogate the production of lymphokines and the induction of class II molecules.285 The regulation of the expression of donor and recipient MHC molecules in transplanted organs may therefore determine the alloreactivity and be of importance for the outcome and function of the graft. The availability of monoclonal antibodies to a variety of monomorphic and polymorphic epitopes of the different MHC molecules and the development of sensitive immunohistological methods has prompted the intensive study of the regulation of the tissue expression of MHC molecules. Not only the two classes of MHC molecules were studied in their cellular expression, but also the differential regulation of the expression of the different MHC molecules so far defined. Tissue

Structure and Function of MHC and Adhesion Molecules

27

Major histocompatibility complex (MHC)

Fig. 3.1. Physical map of the HLA-gene region (major histocompatibility complex: MHC) modified according to Trowsdale and Campbell.247 The gene region contains 3500 base pairs (bottom). Defined genes with known HLA-antigen products are black, so far undefined genes are white. Between the genes for HLA class I and class II molecules additional genes coding for 21-hydroxylase, complement factors 2, 4 and Bf as for Tumor Necrosis Factor (TNF) are located.

biopsies have been taken of liver in different pathological states and in sequence after liver transplantation. This allowed the study of changes in MHC antigen expression during rejection and other complications and under immunosuppressive drug treatment.

Intercellular Adhesion Molecules For the development, structure and function of multicellular organisms adhesive structures are necessary to maintain the cellular and/or extracellular components in temporary or continuous contact. The molecules mediating contact function between cells form the basis for processes as cellular morphology, histogenesis, cellular differentiation and migration. Moreover they form the basis for differentiated tissue reactions as wound healing, immunological reactions, hemostasis, thrombosis and malignant transformation.346 Cellular adhesion molecules (CAM) are specialized molecules functioning in adhesive interactions and are classified according to their molecular structure in different families: Cadherins, selectins, immunoglobulin supergene, integrin and complementary cellular adhesion molecules (unclassified).138,139,744-746,881 It is now known that the regulation of tissue inflammation and repair mechanisms involving cells of the immune system depends on a number of cell-surface interactions.124-126,746,747 The processes of intravascular adhesion, transmigration

Cell Adhesion Molecules in Organ Transplantation

28

Table 3.1. Classification of the molecules of the human major histocompatibility complex (MHC) Class I

Class II

Gene region

A,B,C,E,F,G,J,H

DR,DQ,DP,DN,DO,DM,DV,DX,DZ

Molecules

HLA-A,B,C

HLA-DR,DQ,DP

Structure

44,000 Dalton heavy chain

+/- 34,000 Dalton α chain

12,000 Dalton β2-microglobulin

+/- 29,000 Dalton β chain

restriction of CD-8+ zytotoxic T lymphocytes

Restriction of CD-4 positive helper T lymphocytes

antigen presentation

antigen presentation

HLA-A,B,C

HLA-DR,DQ,DP

Function

Polymorphism

and infiltration by leukocytes and platelets are mainly mediated by receptor-ligand interactions with target cells (cell-cell) and extracellular matrix proteins (cell-matrix). Three main molecular families of adhesion receptor/ligand molecules have been identified: the integrin, immunoglobulin supergene, and selectin families. Table 3.2 gives a characterization of the known adhesion molecules and their reactivity to ligand structures. The cell surface expression of intercellular and cellmatrix adhesion molecules on leukocytes, endothelia and tissue cells is in part constitutive and in part synthetized in response to a variety of cytokines. A number of cytokines and eicanosoids have been identified to modify the expression of receptor molecules on leukocytes and/or the production of their ligand structures.124,125,346,611 It can be assumed that inflammatory reactions consist of a network of intercellular reactions that organize the interplay between immune cells, endothelia and tissue cells. The use of the adhesion cell receptor repertoire in a step-by-step reaction thereby may facilitate cellular activation, intravascular adhesion, migratory and proliferative reactions, fibrogenesis and cytolysis.125,346,585,611 Although general characteristics and functions of various cellular adhesion molecules are known, the manifestation of inflammatory reactions in different tissues is most likely dependent on organ- and cell-type specific factors which, by establishing different cytokine milieus, lead to different patterns of the expression of adhesion molecules. The current classification of intercellular and cell-matrix immune adhesion molecules in man is shown in Table 3.1.

Structure and Function of MHC and Adhesion Molecules

29

Immunoglobulin Supergene Family

VCAM-1 CD2

LFA3

ICAM-1

ICAM-2

T-cell receptor/CD3 CD4

CD8

MHC class II MHC class I

Integrin Family

LFA-1

MAC-1

p150,95

VLA-5

VLA-4

LPAM-2

Selectin Family

Mel-14 LECAM-1

ELAM-1 E-selectin

CD62/PADGEM/GMP140 P-selectin

Fig. 3.2. Molecular structure of human immunoglobulin supergene-, integrin-, and selectinadhesion molecules (according to Springer TA, Nature 1990; 346:425-434).

Immunoglobulin Supergene Family The immunoglobulin supergene family of cell membrane adhesion receptors is characterized by immunoglobulin domains of 90-100 amino acids arranged in two sheets of anti-parallel β-strands, stabilized by a disulfide bond at its center.118,914 The T cell receptor (TCR) and immunoglobulins show hypervariable regions that undergo somatic diversification and function as recognition sites for antigens. Immunoglobulins, TCR and MHC molecules consist of paired immunoglobulin domains. They have to be considered as specialized molecules for the presentation and recognition of antigens and require the transient interaction of additional IgSF receptor molecules.178,881,883 In contrast, the other adhesion molecules as CD4, CD8, CD2, LFA-3, ICAM-1, ICAM-2, ICAM-3, VCAM-1, NCAM and PECAM/CD31

Cell Adhesion Molecules in Organ Transplantation

30

Table 3.2. Classification of human cell-cell and cell-matrix adhesion molecules Adhesion Molecule Immunoglobulin supergene family MHC class I MHC class II TCR1 TCR2 CD3 CD4 CD8 CD28 CD2 (LFA-2) LFA-3 (CD58) ICAM-1 (CD54) ICAM-2 (CD102) ICAM-3 (CD50) VCAM-1 (CD106) NCAM (CD56) PECAM-1 (CD31) MAdCAM-1 Sialoadhesin CD22 CD33 MAG Integrin family LFA-1 (CD11a,CD18) (αLβ2) Mac-1 (CD11b,CD18) (αMβ2) p150,95α (CD11c,CD18) (αXβ2) p150,95β (CD-,CD18) (αdβ2) VLA-1 (CD49a/CD29) (α1β1) VLA-2 (CD49b/CD29) (α2β1) VLA-3 (CD49c/CD29) (α3β1) VLA-4 (LPAM-1; CD49d,CD29) (α4β1) VLA-5 (CD49e,CD29) (α5β1) VLA-6 (CD49f/CD29) (α6β1) LPAM-2 (CD49d,CD-) (α4β7) CD103/CD- (HML-1) (αEβ7) CD49f/CD- (αEβ4) CD41/CD61,gpIIbIIIa (αIIbβ3) CD51/CD29 (αVβ1) CD51/CD- (αVβS) CD51/CD18 (αV/β2) CD51/CD61,VNR (αVβ3)

Counter Receptor/Ligand

TCR,CD8 TCR,CD4 MHC class I (HLA-A,B,C) MHC class II (HLA-DR,DP,DQ)

MHC class II MHC class I B7.1 (CD80), B7.2 (CD86) LFA-3 (CD58), CD48 CD2 LFA-1, Mac-1 LFA-1 LFA-1, αd/β2 VLA-4 NCAM, heparan sulfate? CD31, HECAM, αV/β2 α4β7, L-Selectin Neu5Acα2,3Gal Siaα2,6Gal Neu5Acα2,3Gal Neu5Acα2,3Gal ICAM-1,ICAM-2 C3bi, factor X, fibrinogen, ICAM-1 fibrinogen ICAM-3 laminin, collagen laminin, collagen fibronectin, laminin, collagen VCAM-1, fibronectin, thrombospondin fibronectin laminin MAdCAM-1 E-Cadherin laminin fibrinogen, fibronectin, vWF fibronectin vitronectin, fibronectin PECAM-1 vitronectin, fibrinogen, vWF, thrombospondin

Structure and Function of MHC and Adhesion Molecules

31

Table 3.2. (continued) Adhesion Molecule Selectin family L-Selectin, CD62l, LECAM-1, Mel-14, LAM-1 E-Selectin, ELAM-1, CD62e P-Selectin, CD62p, PADGEM,GMP140 Unclassified CD44, HCAM-1 HECA452 CD34 Cadherins

Counter Receptor/Ligand

sialyl LewisX,a; GlyCAM, CD34, MAdCAM-1 sialyl LewisX,a; ESGL-1 sialyl LewisX,a (CD15s); PSGL-1 hyaluronic acid, chondroitin keratane sulfate ? L-selectin homophilic; αE/β7

are single stranded cell membrane molecules (CD2, Meuer et al;518 LFA-3, Springer et al,743 Meuer and Resch;517 ICAM-1, Rothlein et al,658 Marlin and Springer,494 Makgoba et al;487 ICAM-2, De Fougerolles et al,181 Seth et al;705 ICAM-3, De Fougerolles et al;180 VCAM-1, Osborn et al,576 Rice et al,637-639 Elices et al;210 NCAM, Hercend et al,321 Lanier et al;431 PECAM/CD31, Simmonds et al,717 Albeda et al,15 Liao et al,469 Metzelaar et al515). These molecules exert different functions in intercellular adhesion, transendothelial migration, transmembrane signaling, cell activation processes. While most IgSF molecules exert heterologous receptor-ligand interaction, homologous interaction is known for NCAM and PECAM-1.182,623 Furthermore, the interaction of PECAM-1 with heterophilic receptors (heparin sensitive calcium independent interaction) with αV/β3 has been reported.603 Recently, a new family of immunoglobulin supergene receptor molecules for carbohydrate ligands has been defined, the I-type lectins. They react with sialic acid dependent carbohydrate ligands: CD22, CD33, MAG, Sialoadhesin.385-388,619-622,713 CD22 is a 140kDa cell membrane molecule with seven extracellular domains which is exclusively found on B cells.749 The evidence for the sialic acid dependent binding of CD22 to α2,6-linked sialic acids and CD45RO was demonstrated in the recent years.619-622,882 It was demonstrated that CD22 could mediate adhesion of B lymphocytes to activated endothelia which express high levels of α2,6 sialyltransferase.289,290 The biological function of CD33 is quite unclear at present. CD33 is expressed on myeloid cells in the bone marrow and has been related to maturation of bone marrow cells.234 Sialoadhesin is an IgSF molecule with 17 extracellular domains present on macrophages and reacting with the carbohydrate ligand Neu5Acα2,3Gal on glycoproteins and glycolipids.163-167,385-387 The function of Sialoadhesin has been attributed to the development of myeloid cells in bone marrow and leukocyte traffic in lymphatic organs. Also macrophage/T cell and macrophage/granulocyte interactions have been postulated.386

32

Cell Adhesion Molecules in Organ Transplantation

Integrins The integrin family of adhesion molecules consists of cell membrane receptors to intercellular adhesion ligand molecules and cell matrix proteins.158,394,665 They are composed of noncovalently associated α and β-subunits and classified into subfamilies according to the respective β-subunits as β1 (CD29), β2 (CD18) and β3 (CD61).336,346,746 β2 receptors form heterophilic binding to cellular ligands of the IgSF family.677-679,746 The adhesive binding of several matrix β1 receptor molecules is related to crosslinking with peptides containing the sequence ArgGly-Asp (RGD). On the α subunits several divalent cation binding sites are present that require Ca2+ or Mg2+. Additional ligand binding domains in the α chains are contained in inserted domains as it could be shown for VLA-4.144,627 By this VLA-4 has been demonstrated to function in physiological lymphocyte/leukocyte-endothelial interaction by binding to VCAM-1.22,73 The avidity and binding capacity of receptor molecules as LFA-1 to the IgSF ligand ICAM-1 and VLA-4 to VCAM-1 are thought to be influenced by conformational changes.125,362,382 These may be regulated by chemoattractants and transmembrane signaling from the intracellular domains of the molecules upon cellular activation. Van der Vieren et al865 demonstrated recently the binding of αd/β2 to ICAM-3.

Selectins The molecular family of selectins consists of three membrane glycoproteins: E-selectin (or CD62E, LECAM-2, ELAM-1),75 P-selectin (or CD62P, LECAM-3, GMP140, PADGEM)114,252,505 and L-selectin (or LAM-1, LECAM-1, CD62L, MEL14).742 These mediate adhesive interactions between leukocytes and endothelial cells.652,833 Their aminoterminal domain is the C-type lectin (calcium dependent) or the carbohydrate recognition domain (CRD).198 This explains the characteristics of selectin binding: (1) calcium dependency; and (2) carbohydrate binding capacity. Due to their pivotal role in leukocyte-endothelial interactions, the selectins became the up to now best studied mammalian lectin-like receptor.373,386 The adhesion of blood leukocytes to endothelia had been subject to intensive research that concentrated on lymphocyte homing receptors. Rosen et al652 provided evidence that sialidase-sensitive carbohydrate structures on specialized high endothelia venules (HEV) of lymph nodes are essential for this homing interaction. This could be specifically inhibited by carbohydrates like the monosaccharide mannose-6-phosphate, the polyvalent carbohydrates polyphosphomannan ester (PBME) or fucoidin, a polymer of fucose-4-sulfate.652,833 The primary sequence of all three adhesion molecules, E-selectin, P-selectin and L-selectin were described by several laboratories.76,323,389,640,720 These showed that they are related proteins with relatively high sequence similarity. Particularly, they contain the same structural elements identified by sequence data comparisons. The extracellular parts contain two to nine short consensus repeats (SCR), typically found in complement binding proteins, followed by a domain containing an epidermal-growth-factor analog sequence and then by a N-terminus domain which is the C-type lectin as a carbohydrate binding domain.916 The EGF-like domains of the selectins determine the protein interaction.374 The close genetic localization of the selectins on chromosome 1q21-24 demonstrates their close relation and that to complement regulator proteins. The three selectins bind to similar, but different glycosylated determinants. Common is their binding to terminal α2,3

Structure and Function of MHC and Adhesion Molecules

33

sialic acids derivatives, α1,3 and α1,4 fucosylated lactosaminoglycans. These are for instance sialyl LewisX (sLeX, CD 15s) and sialyl Lewisa (sLea) that are part of several glycolipids and glycoproteins and function as membrane signaling molecules in monocytes.601,615 The importance of this interaction is demonstrated by the leukocyte adhesion deficiency (LAD) syndrome type II with the inability to express SLex.602 These patients exhibit neutrophilia and recurrent infections. Recently, additional ligands for E-selectin: ESGL-1,257,587,588,755,839 P-Selectin: PSGL-1,42,483 and L-Selectin: CD34, sulfoglucoronosyl paragloboside and MAdCAM-161,72,372,433 have been reported. Selectins have a different tissue distribution: E-selectin is expressed only by activated endothelia, L-selectin by leukocytes and P-selectin by activated endothelia and thrombocytes. P-selectin is stored intracellular in the endothelial WeibelPalade bodies and in thrombocytes in α-granula up to cellular activation. This storage modus explains the in vivo and vitro observed fast translocation-mediated postactivation expression of P-selectin induced by agents as thrombin, histamin or complement proteins that cause a fusion of the granula with the cellular plasma membrane.252,300,505 It mediates the adhesion and rolling of neutrophils and monocytes.440,482 In contrast, the E-selectin expression is detectable 4-6 hr after activation of endothelial cells by cytokines, lipopolysaccharides (LPS) or other inflammatory mediators. Inducing signals are TNF, IL-1, IFNγ and LPS.76,454 It mediates neutrophil adhesion by binding to carbohydrate ligands (sialyl LewisX) and CD15.606,839 The regulation of E-selectin expression is mediated on the transcriptional levels by activation of NFκB transcription factors.152,155 L-selectin, however, is shed upon cellular activation from the membrane of leukocytes by the action of proteases.145,151 This process initiates the inflammatory binding of leukocytes to endothelia.145,151,393 The L-selectin functions as a lymphocyte recirculation receptor, but also contributes to neutrophil emigration.132,640 The mechanisms of cell-cell adhesion by this molecule is associated with the presentation of oligosaccharide ligands to ELAM-1.606 It can be assumed that the molecule is involved in lymphocyte activation and the initial phase of leukocyte-endothelial adherence, thus in the intravascular adhesion and margination process.125

Cadherins Today about 15 members of the cadherin family are known which all have in common the so called ‘Cadherin-Repeat’. The amino terminal domains show immunoglobulin like structural characteristics which point out a structural relation between cadherins and the members of the immunoglobulin supergene family. They are responsible for adhesive interactions with their homophilic ligand molecules.278,398,708 Recently, the heterophilic interaction of cadherins with integrin receptors has been demonstrated for the interaction of E-Cadherin and αE/β7 integrin on T lymphocytes. This has been proposed as a mechanism of intraepithelial retention of T lymphocytes and may regulate the tissue specific organization of T lymphocyte epithelial subpopulations.142,377,826 Unclassified adhesion molecules have been mainly attributed to functions in the regulation of lymphocyte homing and recirculation: CD44 and HECA452.71 Their contribution to the specific binding to high endothelial venules (HEV) and the homing of memory T lymphocytes has been implicated.199,201 Differences in

34

Cell Adhesion Molecules in Organ Transplantation

fine specificity have been shown for CD44.306 However, the function of these molecules seems not to be limited to lymphocyte recirculation.712 CD44 has been implicated to play an important role in metastasis formation of migrating tumor cells.613 CD44 is a group of proteins derived from a single gene in alternative splice variants.663 It is an integral membrane protein involved in the stabilization of chondroitin-keratan sulfate proteoglycan and hyaluronic acid binding. The binding avidity is modulated by glycosylation.376 The CD44 binding to extracellular matrix results in signal transduction and integrin upregulation in different cell types.460 CD34 is a sialomucin expressed on bone marrow progenitor cells and endothelial cells and is involved in selectin interaction.407,719,721

Part 2 MHC and Adhesion Molecules in Clinical Organ Transplantation

CHAPTER 4

Major Histocompatibility Complex Antigen and Cell Adhesion Molecule Expression in Clinical Renal Transplantation Susan V. Fuggle

Introduction

I

n clinical renal transplantation, matching between donor and recipient MHC (Major Histocompatibility Complex) antigens significantly improves allograft survival. Presentation of incompatible donor MHC antigens provides a major stimulus for the rejection response to a renal allograft, an inflammatory response which involves a myriad of intercellular interactions mediated by adhesion molecules. These molecules are involved at all stages of the response including: the initial contact between antigen presenting cells and responding leukocytes, the cascade of interactions between leukocytes and endothelium culminating in extravasation of leukocytes into the graft, the migration of leukocytes through the extracellular matrix and the interactions between leukocyte effector cells and their targets. Adhesion molecules are also important in maintaining the structural integrity of the kidney. The expression of the MHC antigens and the adhesion molecules has been extensively studied in the normal and transplanted kidney and is reviewed in this chapter. For clarity the distribution of MHC antigens is dealt with first, followed by the adhesion molecules, grouped according to the family of molecules. Salient points regarding the function of each molecule are included. The expression of many of the molecules is not static, but is upregulated by cytokines during an inflammatory response. The diagnostic potential of such changes are discussed, together with possible means of therapeutic intervention in renal transplantation using reagents directed at the adhesion molecules.

Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

38

Cell Adhesion Molecules in Organ Transplantation

Major Histocompatibility Antigen Expression In the mid 1980s the distribution of MHC antigens in transplanted organs became an area of intense interest because of their importance in stimulating an alloimmune response. Furthermore cells expressing allogeneic MHC antigens are potentially targets for alloreactive effector cells. MHC antigens are not uniformly expressed on cells throughout the body,169,170 and the expression is not static; upregulated and de novo expression can be demonstrated during an immune response. The upregulation is reversible and following the cessation of an immune response can return to a normal level.

HLA Class I Antigens HLA class I antigens are broadly expressed on nucleated cells and are present on all cells within a normal kidney. The endothelium of glomeruli, intertubular capillaries and large vessels is intensely positive while the renal tubules are more weakly stained.228,238 It is clear from in vitro studies that class I antigen expression may be increased following cytokine stimulation. Indeed increased expression has been demonstrated by quantitative analysis in animal models of renal transplantation.522 Nevertheless, the high constitutive level of expression present within the normal human kidney has largely precluded study of increased levels following clinical transplantation.

HLA Class II Antigens HLA class II antigens have a more limited tissue distribution. Numerous studies of HLA class II expression have been performed using antibodies against monomorphic determinants of class II antigens, incapable of distinguishing the individual HLA-DR, DQ and DP antigens. In the human kidney class II antigens are consistently detected on the glomerular mesangium and endothelium and on intertubular structures, including both capillaries and interstitial leukocytes.238 The expression of class II antigens on large vessel endothelium is variable, ranging from weakly positive to negative. The distal tubules do not express class II antigens but cytoplasmic antigen has been demonstrated within proximal tubules in some studies57,213,291,631 whereas in other studies all renal tubules were reported to be class II negative.406,549,679 In our own analyses class II antigens were detected in the proximal tubules of 56/75 (72%) of preanastomosis wedge biopsies from cadaver donors.238,241 In a recent analysis of preanastomosis biopsies from living related donors, class II antigens were consistently detected on endothelium, but all renal tubules were class II negative (Koo and Fuggle; unpublished data). Thus, it is possible that the discrepant tubular staining may be explained by the source of ‘normal’ kidney. There have been numerous studies of class II antigen expression following clinical transplantation.25,57,157,240,241,259,284,308,320,631,886 In a transplanted kidney the glomerular and intertubular capillaries still express high levels of class II antigens, but it is evident that the expression may be significantly upregulated within the cytoplasm and on the cell membranes of the renal tubules. In studies of our own patients we noted two different patterns of induced tubular staining, either a generalized induction on tubules throughout the kidney or a focal induction, either

MHC Antigen and CAM Expression in Clinical Renal Transplantation

39

perivascular or associated with focal interstitial infiltration. The endothelium of larger vessels was invariably class II positive when close to an area of positive tubules.

HLA-DR, DQ and DP Antigens HLA-DR, DQ and DP antigens (Table 4.1). Studies using locus specific antibodies have shown that the HLA-DR antigens represent the majority of the class II antigen expression in normal kidney. HLA-DR-specific antibodies give an identical pattern to that described in the early studies with the broadly reacting antibodies. In contrast, HLA-DQ and DP antigens are expressed at a much lower level, being weakly present on glomerular and intertubular structures, but totally absent from the renal tubules.212,239 In a transplanted kidney with upregulated tubular class II antigen expression, HLA-DR, DQ and DP antigens are all upregulated. Staining with polymorphic antibodies in mismatched donors and recipients has demonstrated that the antigen is of donor origin.212,239 In some kidneys where generalized, upregulated HLA-DR antigen expression was detected on all renal tubules, HLA-DP and DQ antigens were only detected on the proximal tubules. It is possible that the proximal and distal tubules have a differing susceptibility to induction.

Clinical Correlations and Diagnostic Potential of Upregulated Class II Antigens MHC antigen induction has been shown to be associated with the extent of leukocyte infiltration within a renal allograft.25,84,239,241,284,308,320 As described above, the tubules in the vicinity of focal infiltration frequently express upregulated levels of class II, while normal levels persist in other areas of the biopsy, suggesting that the upregulation is caused by cytokines, probably interferon-γ, released locally from activated leukocytes. The observation that class II antigen induction was present when the infiltration had a blastogenic component is consistent with this hypothesis.308 It was anticipated that the presence of upregulated antigen would provide a useful tool for the diagnosis of transplant rejection, but these expectations have not been fulfilled. It is clear that increased MHC antigen expression is frequently found during rejection episodes, but it is not invariably present during clinical rejection, nor is upregulated antigen specific for rejection. Upregulated antigen has been demonstrated in transplanted kidney following ischemic injury,258,707,714 in early biopsies from nonrejecting grafts,631 during chronic vascular rejection,97 in nonrejecting renal allografts where rejection has been prevented by pretransplant donor specific transfusion,37,919 during systemic CMV infection66,886 and in the autologous kidney when rejection is evident in the transplanted organ.520 Analysis of sequential transplant biopsies has shown that elevated levels of class II antigens may persist for several weeks after rejection is successfully treated, before returning to a baseline level.241 Thus, the increased expression may not pertain to the current clinical status and limit the diagnostic power of the presence of upregulated class II expression. In theory, class II antigens should not be upregulated during cyclosporine nephrotoxicity, thus allowing differentiation from rejection.

– –

– –

HLA-DQ Normal Rejection

HLA-DP Normal Rejection

– –

– –

w+ w+

+ +

w+ w+

++ ++

– +/–

– +/–

+/– ++/–

Mesangium Endothelium Proximal

– –

– –

– –

Distal

Tubules

nd, not determined; +/–, indicates differences in the level of staining

– –

HLA-DR Normal Rejection

Bowman’s Capsule

Glomerulus

w+/– +/–

w+/– +/–

+/– ++/–

Arteries

w+/– +/–

w+/– +/–

+/– ++/–

Venules

Large Vessel Endothelium

w+ +

w+ +

++ ++

Capillaries

nd

nd

++

Resident Interstitial Leukocytes

Table 4.1. Expression of major histocompatibility complex antigens in normal and rejecting kidney

w+/–

w+/–

+/–

T Cells

w+

w+

+

Macrophages

Inflammatory Infiltration during Rejection

40 Cell Adhesion Molecules in Organ Transplantation

MHC Antigen and CAM Expression in Clinical Renal Transplantation

41

The results of an initial study looked promising,57 but the association was less clear in other analyses where it appeared that previously upregulated antigen expression was a confounding factor.240,631 It is well established that cyclosporine therapy inhibits class II induction, probably as a result of inhibition of cytokine release.46,277,286,522,633 In clinical renal transplantation, we noted a significantly decreased incidence of induced class II antigens, both in stable function and rejecting grafts, in patients receiving cyclosporine A or triple therapy when compared to azathioprine/prednisolone immunosuppression.240 Despite the failure of class II antigen status to provide diagnostic information in the early postoperative phase, persistent high levels of tubular class II antigens may indicate subliminal intragraft events and identify patients with a poor longterm prognosis.

Adhesion Molecules Integrin Family (Table 4.2) β1 Integrins (VLA, Very Late Antigens, 1-6) β1 integrins include receptors for components of the extracellular matrix, collagen (VLA-1, VLA-2, VLA-3), laminin (VLA-1, VLA-2, VLA-3, VLA-6) and fibronectin (VLA-3, VLA-4, VLA-5). There are two aspects of the expression of β1 integrins which are relevant to renal transplantation; first the expression on the renal parenchyma and vascular endothelial cells, where these molecules are important in mediating adhesion to the basement membrane to maintain the structural integrity of the kidney and enable it to perform efficient filtration, and second, the expression on interstitial leukocytes. On leukocytes they are the major receptors for the extracellular matrix and are thus important in controlling the localization of leukocytes within the interstitium. Activated T lymphocytes have increased levels of certain VLA antigens which may reflect an increased capacity to bind to the extracellular matrix. The distribution of the antigens, VLA-1-6, in human kidney has been the subject of many investigations including both immunohistochemical studies of tissue sections161,217,402,725,938 and of isolated renal components.53,161,726 The distribution of each antigen in the kidney is summarized in the following paragraphs. There are some minor variations between studies in the pattern of reactivity described, but these may be the result of antibody affinity, sensitivity of detection etc. Analysis of tissue sections from normal and transplanted kidneys has been performed in our laboratory, where indicated, using antibodies from the adhesion panel from the VIth Human Leukocyte Differentiation Antigen Workshop. VLA-1. VLA-1 (CD49a) has a widespread distribution being detected on glomerular mesangium and capillary loops, intertubular capillaries, intima and media of larger vessels and occasionally reported as weakly present at the base of tubular cells. This widespread distribution was found in our own study using the monoclonal antibody, HP2B6.1.2, (van Agthoven AJ, Marseille, France). No differences in expression were detected in the transplanted kidney as previously shown by Faull and Russ,217 but many of the interstitial infiltrating cells were positively stained.

VLA-1 Normal Rejection VLA-2 Normal Rejection VLA-3 Normal Rejection VLA-4 Normal Rejection VLA-5 Normal Rejection

+++ +++

+ +

+++ +++

– –

w+ w+

– –

+++3 +++3

– –

– –

++ ++

– –

+++ +++

+ +

+++ +++

– –

– –

– –

– –

w+1 w+1

– –

– –

– –

+2 +2

– –

+2 +2

Venules

– –

+ +

– –

++ ++

Capillaries

++ ++ ++ ++/+++ ++/+++ ++/+++

– –

+2 +2

– –

+1 +1 ++ ++

+2 +2

Arteries

Large Vessel Endothelium

w+1 w+1

Distal

Tubules

Mesangium Endothelium Proximal

– –

Bowman’s Capsule

Glomerulus

Table 4.2. Expression of integrins in normal and rejecting kidney

nd

–

–

–

nd

Resident Interstitial Leukocytes

+/–

+

–

–

++/–

T Cells

+/–

+

–

–

++/–

Macrophages

Inflammatory Infiltration During Rejection

42 Cell Adhesion Molecules in Organ Transplantation

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

++ ++

– –

– –

– –

++1 ++1

– –

– –

– –

++1 ++1

1 basal localization 2 Intimal, medial and endothelial staining 3 Parietal epithelium of Bowman’s capsule and basal and lateral surface of podocytes

nd, not determined; +/–, indicates differences in the level of staining

VLA-6 Normal Rejection LFA-1 Normal Rejection MAC-1 Normal Rejection p150,95 Normal Rejection – –

– –

– –

nd nd

– –

– –

– –

nd nd

– –

– –

– –

++ ++

++

+++

+++

nd

–

–

+++

–

+++

+++

+++

–

MHC Antigen and CAM Expression in Clinical Renal Transplantation 43

44

Cell Adhesion Molecules in Organ Transplantation

VLA-2. VLA-2 (CD49b) can be detected on the mesangial and endothelial cells within the glomerulus, where it was localized by electron microscopy to the luminal and abluminal endothelial surfaces.53 Distal tubules express VLA-2 where the staining is focused around the basal surface, while the proximal tubules are consistently negative. The expression of VLA-2 remained unchanged in transplanted kidneys and the infiltrating leukocytes were negative/insignificantly stained.217 VLA-3. VLA-3 (CD49c) is present throughout the glomerulus being expressed on the mesangium, luminal and abluminal surfaces of the capillaries, parietal epithelium of Bowman’s capsule and uniquely amongst VLA antigens on the basal and lateral surfaces of the podocytes. VLA-3 is also expressed at the base of the distal tubules. There are no differences in expression in transplanted kidneys and the infiltration is unstained217 (and results from our study using antibodies ASC-1, and ASC-10, A. Skubitz, Minneapolis, USA). VLA-4. VLA-4 (CD49d) VLA-4 was not detected on vascular endothelium or renal tubules in normal kidney, nor in biopsies from acutely or chronically rejecting patients, but leukocytes within the inflammatory infiltration are positively stained26,217,555 (and results with HP2.1, 9F10, Immunotech, Marseille, France). VLA-5. VLA-5 (CD49e) is expressed on endothelium throughout the kidney and is weakly present on the mesangium. Renal tubules are consistently negative for VLA-5. VLA-5 is the only one of the VLA-1-6 antigens to have altered expression after transplantation, with a reported increase on endothelial cells during allograft rejection.217 A proportion of the infiltrating leukocytes were stained when tested with the anti-VLA-5 antibody SAM1 (Immunotech). VLA-6. VLA-6 (CD49f) is found at the basal aspect of the renal tubules, both proximal and distal, a pattern consistent with mediating extracellular matrix contacts. Glomerular and intertubular capillary endothelium has been reported to be positively stained for VLA-6. There are no reported changes in expression following transplantation nor significant staining of infiltrating leukocytes.217 The localization of the VLA antigens in the human kidney, predominantly within areas of cells adjacent to the basement membrane, is consistent with their role in maintaining structural integrity. There is a specific cellular and subcellular distribution of these antigens within the kidney, as clearly revealed by the staining patterns within the renal tubules. This distribution suggests variability in the composition of the basement membranes of different cell types. VLA-5 is the only antigen reported to have an altered level of expression during renal allograft rejection, being increased within the blood vessels. In response to inflammation blood vessels secrete increased amounts of fibronectin and thus an increased level of VLA-5 during rejection may not be unexpected.217 A significant proportion of the leukocytes within the inflammatory infiltration present during allograft rejection express VLA-1, and VLA-4.217 VLA-4, in addition to binding to fibronectin, binds to VCAM-1 expressed on activated endothelium and thus may play a role in mediating the extravasation of lymphocytes into the allograft.

MHC Antigen and CAM Expression in Clinical Renal Transplantation

45

β-2 (Leukocyte) Integrins LFA-1. LFA-1 (CD11a/CD18), mediates functional interactions between leukocytes and leukocyte adherence to endothelium at inflammatory sites by means of interactions with the ligands ICAM-1, ICAM-2 and ICAM-3. The interstitial leukocytes within the normal kidney and inflammatory infiltration in a transplanted kidney express LFA-1, while the endothelium and parenchyma are consistently negative. CR3, Mac-1 and p150/95. CR3, Mac-1 (CD11b/CD18) and p150/95 (CD11c/CD18) have a more restricted distribution than LFA-1 and are involved in monocyte and granulocyte adhesion to endothelium at inflammatory sites. Staining performed with antibodies to CR3 and p150/95 (‘44’ Pharmingen, San Diego, USA; LeuM5, Becton Dickinson, San Jose, USA) demonstrated that many of the interstitial leukocytes within a normal kidney express CR3 and p150/95, but only a subpopulation, probably macrophages, of the interstitial infiltration in a rejecting kidney were stained.

Immunoglobulin Superfamily (Table 4.3) CD2. CD2 is expressed on lymphocytes and mediates adhesion by interaction with its ligand, LFA-3. In pretransplant renal biopsies CD2 may be detected on an occasional interstitial T lymphocyte and in transplant biopsies CD2 is found on the lymphocytes within the inflammatory infiltration. The renal parenchyma does not express CD2.83 LFA-3. LFA-3 (Lymphocyte function-associated antigen-3, CD58) the counterreceptor for CD2, is widely expressed within tissue. In the human kidney LFA-3 is expressed on all cells, being strongly expressed on glomerular and intertubular capillaries and large vessel endothelium and more weakly on renal tubules. Studies of biopsies obtained during allograft rejection have demonstrated the presence of LFA-3-positive leukocytes within the inflammatory infiltrate, no increased expression on the parenchyma, but a possible reduction in LFA-3 expression on intertubular capillaries.83,85 ICAM-1. ICAM-1 (Intercellular adhesion molecule-1, CD54) is the counter receptor for the leukocyte integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) and is involved in antigen presentation and leukocyte adhesion to endothelium. Expression of ICAM-1 in normal kidney and in renal transplant biopsy material has been studied by many research groups.25,26,83,121,193,207,216,242,246,254,328,343,533,555, 810,836,887-889,935 ICAM-1 is constitutively expressed on the glomerular and intertubular capillary endothelium and the endothelium of arterioles and venules. While distal tubules are consistently ICAM-1 negative, the expression on proximal tubules is variable. In a recent study of a large number (n=65) of pretransplant biopsies from cadaver donors, ICAM-1 was detected on the proximal tubular epithelium in 86% of biopsies, but in the remaining biopsies the proximal tubules were ICAM-1 negative. The ICAM-1 is most intensely expressed apically, at the brush borders. In contrast, ICAM-1 was not detected on the tubules of pretransplant biopsies tested from living related donors (Koo and Fuggle, unpublished data). The results of in vitro analyses have shown that expression of ICAM-1 can be induced/upregulated by cytokines on endothelium and renal tubules.25,206,354,366,473 ICAM-1 expression has been shown to be upregulated after transplantation in

CD2 Normal Rejection LFA3 Normal Rejection ICAM-1 Normal Rejection ICAM-2 Normal Rejection ICAM-3 Normal Rejection

– –

w+ w+

++ ++

– –

– –

w+ w+

++ ++

– –

– –

– –

++ ++

++ ++

w+ w+

– –

– –

– –

+/– ++/+/–

w+ w+

– –

– –

– –

– –

w+ w+

– –

Distal

Tubules

Mesangium Endothelium Proximal

– –

Bowman’s Capsule

Glomerulus

– –

+ +

++ ++

+ +

– –

Arteries

– –

+ +

++ ++

+ +

– –

Venules

Large Vessel Endothelium

– –

+ +

++ ++

+ +

– –

Capillaries

Table 4.3. Expression of immunoglobulin superfamily molecules in normal and rejecting kidney

+

nd

nd

nd

–

Resident Interstitial Leukocytes

+

+

+

+

+

T Cells

+

+

+

+

–

Macrophages

Inflammatory Infiltration During Rejection

46 Cell Adhesion Molecules in Organ Transplantation

w+ w+

– –

– –

+++1 +++1

– –

– –

+++ +++

– –

– –

– –

– –

+/– ++/+/–

– –

– –

– –

+++ +++

– –

– w+/–

+++ +++

– –

– +/–

1Parietal epithelium of Bowman’s capsule only

nd, not determined; +/–, indicates differences in the level of staining; occ+, occasional positive cell

VCAM-1 Normal Rejection NCAM-1 Normal Rejection PECAM-1 Normal Rejection +++ +++

– –

– +/–

nd

–

–

–

–

–

occ+

–

+

MHC Antigen and CAM Expression in Clinical Renal Transplantation 47

48

Cell Adhesion Molecules in Organ Transplantation

association with rejection and leukocyte infiltration. It is difficult to comment on the level of expression on endothelium, as the constitutive level is so high that quantitation of any upregulation is difficult with commonly used immunohistochemical techniques. Nevertheless upregulation of tubular ICAM-1 expression may occur after transplantation. The upregulated antigen expression is frequently focal and associated with areas of interstitial infiltration. The interstitial infiltrating leukocytes, both lymphocytes and macrophages are positively stained for ICAM-1. ICAM-2. ICAM-2 (CD102), an alternative ligand for LFA-1, is widely expressed on endothelium and on a subpopulation of lymphocytes.180 In the normal kidney ICAM-2 is constitutively expressed on the endothelium.555 In our own study of normal and transplanted kidneys using antibodies BR-7 and B-S9, (Vermot Desroches C./ Widgenes J. Helsinki, Finland), endothelium throughout the kidney was positively stained for ICAM-2 with no appreciable changes after transplantation. The inflammatory infiltration was positively stained for ICAM-2. A slight decrease in intertubular capillary staining in relation to controls has been reported.555 ICAM-3. ICAM-3 (CD50) is a third ligand for LFA-1, and in contrast to ICAM-1 and ICAM-2, the expression is restricted to leukocytes.4,219 ICAM-3 expression in normal kidney is restricted to interstitial leukocytes and occasional leukocytes within the glomeruli. In renal biopsies obtained from rejecting grafts infiltrating leukocytes were positively stained for ICAM-3. Upregulated ICAM-3 was not found on the renal endothelium or parenchyma.397,555 VCAM-1. VCAM-1 (Vascular cell adhesion molecule-1, CD106) is a cytokine inducible adhesion molecule which plays a major role in the adhesion of lymphocytes to activated endothelium through interaction with the β1 integrin, VLA-4.576,639 VCAM-1 expression is not restricted to endothelium, but is also expressed on antigen presenting cells and certain epithelia.638 VCAM-1 expression in normal and transplanted kidney has been the subject of many investigations.26,93,111,113,121,242,254,328,472,555,703,836 In the pretransplant kidney VCAM-1 is constitutively expressed on the parietal epithelial cells of the Bowman’s capsule, weakly detected on mesangium and may be observed on an occasional intertubular structure (either interstitial leukocyte or capillary). Distal tubules appear to be consistently negative, but as described for MHC class II antigens and ICAM-1, there is variation in the level of VCAM-1 reported on the proximal tubules. The distribution of antigen expression is different to ICAM-1 being most dense at the basal surface of the tubule, usually surrounding the nucleus. In a recent study of pretransplant biopsies from cadaver donors 72% of kidneys expressed high levels of proximal tubular VCAM-1, whereas none of the pretransplant biopsies from living-related donors expressed high levels (Koo and Fuggle, unpublished data). VCAM-1 expression may be upregulated after transplantation in association with leukocyte infiltration and rejection. Cytokine mediated induction of VCAM-1 has been demonstrated on renal tubules in vitro.472 The upregulation observed in transplant biopsies is most apparent on proximal tubules, but VCAM-1 positive distal tubules may be present. Intertubular capillaries and larger vessels may become positive but, the glomerular endothelium remains negative. As may be anticipated, strong induction of tubular and endothelial expression is frequently a

MHC Antigen and CAM Expression in Clinical Renal Transplantation

49

Fig. 4.1. (A) and (B) illustrate the range of expression of VCAM-1 found in preanastomosis biopsies from cadaveric kidneys. VCAM-1 is invariably expressed on the parietal cells of the Bowman’s capsule, but there is variation in the level of expression on the tubules ranging from (A) an isolated basal area of an occasional proximal tubule to (B) extensive cytoplasmic proximal tubular staining (x 10). (C) Tubular induction of VCAM-1 in a post-transplant biopsy associated with an area of inflammatory infiltration (x 10). (D) A consecutive section stained for ICAM-1, showing expression within the glomerulus, induced ICAM-1 on the renal tubules and positively stained leukocytic infiltration, (x 10).

50

Cell Adhesion Molecules in Organ Transplantation

Fig. 4.2. (A) PECAM-1 staining indicating the extent of the vasculature within the normal kidney (x 10). (B) and (C) illustrate the varied levels of expression of E-Selectin in preanastomosis biopsies from cadaveric kidneys, ranging from (B) extensive staining of intertubular capillaries (glomerular capillaries are always negative), to (C) staining of occasional, isolated intertubular capillaries (arrow, x 10). (D) shows a transplant biopsy in which E-Selectin expression is induced on the endothelium of a small vein, in close proximity to an area of interstitial leukocytic infiltration, (x 25).

MHC Antigen and CAM Expression in Clinical Renal Transplantation

51

focal, rather than a generalized phenomenon and as such is usually associated with areas of interstitial infiltration. Occasional leukocytes within the infiltration are VCAM-1 positive. NCAM-1. NCAM-1 (Neural cell adhesion molecule-1, CD56) is transiently expressed in many tissues during embryogenesis and is expressed on NK cells and a subpopulation of activated T lymphocytes. Preanastomosis wedge biopsies from 20 cadaver donors and routine needle core transplant biopsies obtained from the same patients on days 7 and 28 after transplantation were stained with the antiCD56 antibody, NKH-1 (Griffin et al,273 Coulter Immunology, Hialeah, Florida, USA). A very occasional positively stained leukocyte was detected in the intertubular areas of the pretransplant biopsies. After transplantation the majority of the interstitial infiltrating cells were negative, but again a very occasional positive leukocyte was detected. The renal parenchyma and the endothelium were negative in all biopsies studied (Fuggle, unpublished data). PECAM-1. PECAM-1 (Platelet endothelial cell adhesion molecule-1, CD31) is constitutively expressed on vascular endothelial cells where it is concentrated at high levels at the intercellular junctions and appears to participate in the cascade of events leading to leukocyte transmigration into tissue at inflammatory sites. Furthermore it is expressed on the surface of circulating platelets, neutrophils, monocytes and certain subpopulations of T lymphocytes553,869 and can mediate both homotypic and heterotypic adhesion.552 PECAM-1 is strongly expressed on all endothelial cells within the normal and transplanted kidney, consequently it is a useful marker of endothelial integrity.242 Focal decreases in interstitial PECAM-1 staining in transplant biopsies may indicate areas of endothelial damage. PECAM-1 is not expressed on the renal parenchyma, but a proportion of the interstitial infiltrating leukocytes are positive.

Selectin Family (Table 4.4) E-Selectin. E-Selectin (CD62E) unlike many of the adhesion molecules discussed so far, has a very restricted tissue distribution, being expressed transiently on endothelium following cytokine stimulation in vitro. E-selectin mediates the initial capture and rolling of leukocytes, principally neutrophils, monocytes and memory T lymphocytes, at the site of an inflammatory response. The expression of E-selectin, with respect to renal transplantation has been reported by a number of groups.26,113,242,254,836,888,889 As with other cytokine inducible molecules studied, ICAM-1, VCAM-1 and HLA class II antigens, we have reported variation in a level of E-selectin expression in pretransplant biopsies.242 The expression ranges from entirely negative, to multiple foci of positive intertubular capillaries with isolated positive capillaries elsewhere. Glomerular capillaries have always been negative in all biopsies we have studied, but occasionally a large vein may be positively stained. In a recent study of pretransplant biopsies from cadaver kidneys, 64/65 pretransplant biopsies had some areas of E-selectin expression, in 29/64 (44%) of biopsies an occasional intertubular capillary was stained, but in 55% of biopsies there were extensive areas of strongly positive intertubular capillaries (Koo and Fuggle, manuscript in preparation). This pattern is in marked contrast to that found in pretransplant biopsies from living related kidneys where none of the biopsies

52

Cell Adhesion Molecules in Organ Transplantation

had this high level of expression, an occasional intertubular capillary was positive in only 2/15 (13%) of biopsies studied, and the remaining biopsies were entirely negative. In our experience, E-selectin expression may be, but is not invariably, induced on intertubular capillaries and on larger veins in rejecting kidneys. As with the other cytokine inducible adhesion molecules, the induced expression is frequently associated with areas of focal infiltration. L-Selectin (CD62L). L-Selectin (CD62L) mediates initial adherence of leukocytes at inflammatory sites and lymphocyte binding to high endothelial venules of peripheral lymph nodes. Normal and transplanted kidney biopsies were stained with the monoclonal antibody, Dreg 56, (Pharmingen, San Diego, USA) L-selectinpositive interstitial leukocytes were present in the normal kidney and, as may be anticipated, a proportion of leukocytes within the inflammatory infiltration of the transplanted kidney were stained. P-Selectin (CD62P). P-Selectin (CD62P) is stored in the α-granules of platelets and the Weibel-Palade bodies of endothelial cells. It is released and translocated to the cell surface within minutes of stimulation. P-selectin is expressed at a very low level in normal kidney, being detected on an occasional intertubular capillary and area of venous or arterial endothelium.26,237 A similar low level was detected in preanastomosis wedge biopsies from cadaver kidneys and kidneys from living related donors (Koo and Fuggle, unpublished data). In transplant biopsies increased expression of P-selectin may be detected focally within intertubular capillaries, usually in close proximity to interstitial infiltration.26,237

Unclassified Adhesion Molecules CD44. CD44 is strongly expressed on the parietal epithelium of Bowman’s capsule, glomerular endothelium and mesangium. There is extensive staining of capillary endothelium and vessel wall and endothelium of larger vessels. Renal tubules are entirely negative.226 There is no detectable change in the level of CD44 expression during rejection, but the infiltrating T lymphocytes and macrophages are strongly stained (CD44 antibody- B-F24, Wijdenes, Bescancon, France).

Discussion Clinical Correlations and Implications for Diagnosis Adhesion Molecule Expression The cytokine inducible adhesion molecules ICAM-1, VCAM-1 and E-selectin have been most widely studied as potential diagnostic markers of renal allograft rejection. The immunohistochemical studies summarized above clearly demonstrate that following transplantation, upregulated ICAM-1 and VCAM-1 may be detected on the proximal tubules and E-selectin and VCAM-1 on the endothelium. These upregulated antigens may play a role in transmigration of leukocytes from the circulation into the allograft and later in the effector stage of the response. There are many similarities in both the distribution and the clinical associations described for upregulated adhesion molecule expression and for HLA-class II antigens. Upregulated adhesion molecule expression is frequently perivascular or associated with focal areas of interstitial infiltration and there is a significant

– –

– –

– –

++ ++

– –

– –

– –

++ ++

++ ++

– –

– –

– –

– –

– –

– –

– –

Mesangium Endothelium Proximal

– –

– –

– –

– –

Distal

Tubules

++ ++

–/w+ –

– –

– –

Arteries

++ ++

–/w+ +/–

– –

–/w+ +/–

Venules

Large Vessel Endothelium

occ+, occasional cell positive; –/w+, indicates differences in the level of staining; nd, not determined

E-Selectin Normal Rejection L-Selectin Normal Rejection P-Selectin Normal Rejection CD44 Normal Rejection

Bowman’s Capsule

Glomerulus

Table 4.4. Expression of selectins and CD44 in normal and rejecting kidney

+ +

– +/–

– –

occ+ +/–

Capillaries

nd

–

occ+

–

Resident Interstitial Leukocytes

++

–

occ+

–

T Cells

++

–

occ+

–

Macrophages

Inflammatory Infiltration During Rejection

MHC Antigen and CAM Expression in Clinical Renal Transplantation 53

54

Cell Adhesion Molecules in Organ Transplantation

association between the presence of upregulated adhesion molecules and the level of leukocyte infiltration. In our study we generally found concomitant induction of ICAM-1, VCAM-1 and E-selectin,242 despite the differing kinetics of induction reported from in vitro studies.611 However, increased levels of class II antigens were frequently detected when adhesion molecule expression was at a baseline level. This phenomenon cannot be explained on the basis of the kinetics of induction,354,366 but it is possible that, once induced, class II antigens remain elevated for a longer period. The presence of class II antigens in the absence of rejection is a major factor preventing the use of class II induction as a diagnostic marker for allograft rejection. Thus it is possible that induced adhesion molecule expression may have a greater specificity than class II induction. Indeed, it has been suggested that ICAM-1 expression may be of value in differentiating allograft rejection from cyclosporine nephrotoxicity.489 Nevertheless, while many groups have reported an association between upregulated levels of adhesion molecules and clinical rejection, the upregulation is not specific for rejection, detracting from its value as a prognostic indicator.

Circulating Adhesion Molecules Soluble isoforms of cell adhesion molecules (sCAM) have been found in the circulation and the level may be raised during disease processes.250 The physiological role of these molecules is unknown, but it is conceivable that they may compete in cell-cell adhesion and trigger responses in ligand bearing cells. The presence of elevated levels of circulating cell adhesion molecules may indicate endothelial activation and therefore may have diagnostic utility. Given the upregulated expression of ICAM-1, VCAM-1 and E-selectin following renal transplantation, serum804 and urinary sCAM62 levels have been measured in renal transplant patients. Samples obtained in different clinical circumstances have been studied to examine the possibility that increased levels may assist in the differentiation of rejection from other clinical conditions. Encouraging results were obtained from initial studies of sICAM-1 levels in renal transplant patients.250,804 Markedly increased levels of sICAM-1 were detected in samples obtained during, and immediately preceding allograft rejection, when compared to samples obtained during periods of stable graft function. Increased levels were not detected in cyclosporine nephrotoxicity, permitting differentiation of this condition. However, increased levels were also detected during CMV infection, limiting the application of such a test.804 There have been conflicting conclusions from more recent studies; E-selectin, but not ICAM-1 or VCAM-1 was found to be increased during acute rejection,445 but VCAM-1 was increased in CMV infection,444 possibly permitting a differential diagnosis. Authors of a subsequent study concluded that none of these sCAM were useful indicators of rejection.16 Their results showed that patients with chronic renal failure had increased sCAM levels, possibly reflecting decreased clearance in this condition. It is possible that blood levels may not be at all meaningful, since circulating sCAMs may bind to their ligands and thus decrease potentially high blood levels. Analysis of urinary sCAMs from renal transplant patients suggested that while sE-selectin was not excreted, measurement of a combination of sICAM-1, sVCAM-1 and sC4d may prove useful in the diagnosis of steroid resistant rejection.62

MHC Antigen and CAM Expression in Clinical Renal Transplantation

55

Cellular Adhesion Molecules as Targets for Therapeutic Intervention The dual role of adhesion molecules in mediating the egress of alloreactive leukocytes from the circulation and their subsequent interaction with target cells makes them attractive targets for immune intervention. Antibodies to adhesion molecules have been used to modify the immune response in all types of vascularized organ allografts, but in renal transplantation the major interests have been in inhibiting ICAM-1 and LFA-1 interactions. Antibodies to ICAM-1 and LFA-1 used in combination have been shown to induce a state of tolerance in a mouse model of cardiac transplantation.358 Encouraging results were obtained from studies of renal transplantation in cynomolgous monkeys. A mouse monoclonal antibody to ICAM-1, administered prophylactically as the sole immunosuppressive agent was found to significantly delay allograft rejection when compared to untreated controls (24.2 ± 2.4 vs. 9.2 ± 0.6 days). Furthermore, the antibody reversed pre-existing rejection episodes resulting from the reduction in therapeutic cyclosporine levels.159 ICAM-1 has a very similar distribution in cynomolgous monkey tissue as in human141 and in the rejecting cynomolgous grafts was present on the infiltrating leukocytes and at an increased level on endothelium and tubules. In antibody-treated animals the antibody was shown to coat the endothelium, and despite the presence of an immune response to the antibody, there was no evidence of vascular injury.159 The same anti-ICAM-1 antibody was subsequently used in combination with conventional immunosuppression in a phase I clinical trial, in renal recipients at high risk of delayed graft function. Both highly sensitized recipients and recipients of grafts with extended ischemia times were included. The results suggested that in patients where an adequate level of circulating antibody was achieved, the incidence of delayed graft function and rejection was significantly decreased.301 In another clinical study, anti-LFA-1 was used in an attempt to reverse acute rejection episodes occurring in renal transplant patients receiving azathioprine and cyclosporine immunosuppression.441 Despite the presence of high levels of circulating antibody, the treatment was ineffective. The antibody was also used prophylactically in recipients of renal allografts with more encouraging results. In this phase I clinical study, patients were immunosuppressed with azathioprine and corticosteroids in addition to anti-LFA-1 and cyclosporine administration was delayed until day 9. The administration of LFA-1 during the first 10 days reduced the incidence of rejection episodes in the first month after transplantation.341 The therapeutic potential of antisense oligonucleotides to inhibit adhesion molecule expression is beginning to be explored in models of transplantation. Initial results with antisense reagents to ICAM-1 suggest that it is possible to inhibit ICAM-1 expression in stimulated endothelial cells in vitro. Furthermore when administered either in a preoperative perfusion or as induction and maintenance immunosuppression, the antisense reagents prolonged rat renal and heart allograft survival.801 Ischemia/reperfusion injury causes physiological changes which may ultimately lead to impaired function of an organ allograft.421 There are many possible therapeutic agents which may be used to reduce this damage.275 The benefit of intervention is illustrated by the improved long term survival of renal allografts treated with superoxide dismutase, an oxygen free radical scavenger.422 Adhesion

56

Cell Adhesion Molecules in Organ Transplantation

molecules become upregulated as a result of ischemia/reperfusion injury and thus targeting adhesion molecules, and inhibiting recruitment of leukocytes into the allograft, is a possible means of intervention. Antibodies to P- and E-Selectin, CD11a, CD18 and ICAM-1 have been used in experimental models to limit the damaging effects of ischemia. Furthermore it may be possible to diminish the damaging effect by targeting the selectin ligands by infusion with oligosaccharides. With respect to renal transplantation, the overall findings of the phase I clinical trial, in which ICAM-1 was administered to renal transplant patients, suggested that the antibody appeared to limit the ischemic injury to the transplant.159 An anti-ICAM-1 antibody has been shown to protect against functional impairment in an experimental model of renal ischemia, even when administered up to 2 hr after restoration of blood flow. In this model, lower doses of ICAM-1 and LFA-1 antibodies acted synergistically to protect, indicating the involvement of leukocytes in the pathophysiology of ischemic damage.383 However an anti-CD18 antibody did not appear to confer protection in a rabbit model of renal ischemia.105 It is clear that the potential to manipulate adhesion interactions with the aim of modifying ischemia/ reperfusion injury and allograft rejection has not been rigorously explored. Further studies are justified to determine the therapeutic applications of appropriate reagents.

CHAPTER 5

MHC and Cell Adhesion Molecules in Clinical Heart Transplantation Gustav Steinhoff and Matthias Wilhelmi

Expression of Major Histocompatibility Complex (MHC) Molecules in Human Heart Transplants

C

linical heart transplantation is successfully practiced across major differences in major histocompatibility complex (MHC) antigens between donor and recipient. This implies a high degree of allosensitization. The factors that determine the severity and organ specific appearance of the rejection response could very much depend on how the donor MHC antigens are expressed in the graft. Since the mid 1980s it has been known that the expression of MHC antigens is not static, but influenced by immune stimuli.285 The induction of MHC antigens has been demonstrated in allograft rejection of different organs189,241,419,521,792,777 and in autoimmune and infectious disease.33,656 Lymphokines were identified as the main mediators of MHC antigen induction and prostaglandins as inhibitors.474 Early studies in rat heart transplants had demonstrated an induction of donor class I and class II MHC antigens on myocyte membranes and endothelia in allografts that are being rejected.521,792 Similar findings were reported in studies in which human heart transplants are investigated.649,775,811 In this chapter a survey about the expression of monomorphic determinants of MHC antigens, class I (HLA-A,B,C) and class II (HLA-DR,DP,DQ), analyzed in endomyocardial biopsy specimens taken sequentially after transplantation is given. The changes in MHC antigen expression were compared with histopathologic and clinical events. In addition, the distribution of donor and recipient MHC antigens studied by the use of monoclonal antibodies directed to polymorphic epitopes of HLA-A and HLA-B is described. The expression of polymorphic determinants of HLA-A and HLA-B antigens was studied in 11 patients after transplantation. Differences between donor and recipient in HLA-A and HLA-B antigens allowed specific staining of donor or recipient cells with monoclonal antibodies directed to polymorphic epitopes of MHC antigens HLA-A of HLA-B (Table 5.1). The specificity of the monoclonal antibodies was tested in tissue sections of donor spleens. The specimens studied were obtained 1 week, 1 month and up to 1 year after transplantation. Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

58

Cell Adhesion Molecules in Organ Transplantation

Monoclonal Antibodies The monoclonal antibodies used in the study were directed against monomorphic or polymorphic determinants of class I (HLA-A, HLA-B, β2-microglobulin) or class II (HLA-DR, HLA-DQ, HLA-DP) molecules (Table 5.1). Monoclonal antibodies that detected polymorphic determinants on HLA-A and HLA-B loci were produced by Dr. J. Happrecht, Institute of Immunology, University of Kiel, Kiel, FRG297 and Dr. S. Ferrone, Department of Microbiology and Immunology, New York Medical College, NY, USA.668 The antibodies 48C1, 83A5, and CR11-351 were specific for HLA-A2 and A28, the MCAs 38D4 and KS4 bound specifically to B7 and B27, MCA 38D4 also reacted with Bw22, and the MCA 2BC4 HLA-Bw6 polymorphic determinants (Table 5.1).

Expression of MHC Antigens, Classes I and II (Monomorphic Epitopes) Normal Heart The expression of MHC antigens in normal heart tissue was studied in seven individual atrial specimens that were obtained as described. In Table 5.2 the staining pattern of class I and class II antigens found in these samples is summarized. The myocytes showed negative staining for all MHC antigens. No staining of intercalated discs was observed in the specimens studied. In addition, the smooth muscle cells of arterial vessel walls were negative for MHC antigens. On all vascular endothelia class I antigens were moderately expressed. HLA-DR antigens were only weakly expressed on some vessel endothelia, and findings were negative on capillary endothelium. MHC antigens HLA-DQ and HLA-DP were not found on endothelia of the normal heart. Interstitial cells showed positive staining for all class I and class II MHC antigens. MHC antigens HLA-DQ and HLA-DP were expressed only by a few interstitial cells.

Post-transplant Biopsy Specimens Quiescence. Fifty-nine biopsy specimens from 26 patients that were obtained on days 0 to 399 showed no signs of rejection activity. All samples were taken in periods of clinical quiescence between rejection episodes. No clinical infections were present at the time of the biopsy. Focal positive class I antigen staining on myocyte membranes was found in 11 specimens from 9 patients (days 23 to 145). Five of these biopsies were performed 1 to 3 weeks after a previous treatment for rejection, and three biopsies were performed 1 to 3 weeks before a rejection episode that required steroid treatment. Three specimens showed no signs of acute rejection but demonstrated chronic rejection activity. Compared with the normal heart muscle HLA-DR antigens were expressed in increased amounts on endothelial cells. This varied from weak to strong staining intensity, and most vessels showed staining. Moderately increased HLA-DR expression was found in biopsy specimens irrespective of the time (days 7 to 365) after transplantation. With strong HLA-DR expression in a few specimens weak HLA-DQ and HLA-DP expression was found. Rejection. In 57 of 78 rejection episodes, class I MHC antigens were induced on myocyte membranes. This induction appeared in one or more sequential biopsy specimens. The incidence of this finding is listed in Table 5.3 according to the histopathologic diagnosis. In moderate and severe rejection the majority of speci-

MHC and Cell Adhesion Molecules in Clinical Heart Transplantation

59

Table 5.1. Monoclonal antibodies directed to MHC antigens used Specificity Monomorphic determinants HLA-A,B,C HLA-A,B 0602 β2-microglobulin HLA-DR L243 HLA-DP B7/21 HLA-DQ IU22 Polymorphic determinants HLAA2,28 A2,28 A2,28 (weak) A25,32 A25,32 A2,11,10 (25,26) 28-31,33 B7,27 B7,27, w22 (54,55,56) B13 Bw6

Antibody

Reference

W6/32 Pellegrino et al596 0592 L368 Lampson et al 420 Watson et al p902 Ziegler et al944 Leu10

Barnstable et al56 Pellegrino et al596 Brodsky et al116

Chen et al146

CR11/351 48C1 83A5 1BA2 9B6 66C4

Russo et al668 Harpprecht et al297 " " " "

KS4 38D4

Russo et al668 Harpprecht et al297

40A2 2BC4

" "

Table 5.2. Expression of class I and class II MHC antigens in normal human heart

Cell Type Cardiomyocyte Vascular endothelium Interstitial cell

Class I HLA-A,B,C

Class II HLA-DR

HLA-DP

HLA-DQ

0/7 7/7 7/7

0/7 7/7 7/7

0/7 0/7 7/7

0/7 0/7 7/7

mens were found to contain class I positive myocytes. Usually the induction was weak and focal on some myocyte membranes and intercalated disks in and around areas of lymphocyte infiltration and myocytolysis. With prolonged and severe rejection the majority of myocytes in the biopsy specimen were moderately or strongly stained for class I MHC antigens. This generalized strong class I antigen induction was found in 16 (10 patients) of 78 rejection episodes. Four of these 10 patients also had infectious complications at the time of biopsy.

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Reversal of class I MHC antigen induction within 1 to 3 weeks after effective steroid treatment for rejection was observed in 22 of 78 rejection episodes of informative sequential biopsies. A prolonged or late class I MHC antigen induction on myocyte membranes in the course of a rejection episode was also seen in 20 of 51 rejection specimens with resolving rejection without myocytolysis. The biopsy specimens of six patients diagnosed as demonstrating moderate rejection contained no class I positive myocytes, but strong induction was present in the following specimens taken 1 week later with resolving rejection. In three patients with episodes of rejection persisting for several weeks class I positive myocyte membranes were found in all biopsy specimens obtained during this period (heart transplant patients 81, 82, and 86). In four patients only weak class I staining of myocyte membranes, or none at all, was detected on myocyte membranes during rejection. Class II antigen induction was not found on myocytes during rejection. Endothelia HLA-DR was strongly expressed during rejection episodes, and most of the capillary endothelia also were stained. Weak HLA-DQ and HLA-DP expression was found on some endothelia in specimens with strong HLA-DR expression. Class II antigen positive (HLA-DR and in part HLA-DQ and HLA-DQ) lymphocytes appeared in the graft with acute rejection. Class II positive (HLA-DR, -DP,-DQ) interstitial cells increased in number and staining intensity in particular for HLA-DQ and HLA-DP. Infections. Class I antigens were strongly induced on myocyte membranes at the time of clinically manifest CMV infection. In four of five cases there was a generalized induction of class I antigens on all myocyte membranes. In each case, however, a moderate rejection response was also present. In six of nine rejection episodes that occurred shortly after the manifestation of the CMV infection, the class I induction on myocyte membranes was generalized. HLA-DR antigens were expressed strongly by all endothelial cells, HLA-DP and HLA-DQ antigens were expressed weakly on a few vessel endothelia. In one patient (patient 99) in whom sequential biopsy specimens were informative early in the postoperative period and after the development of CMV infection, the HLA-DR antigen expression on endothelia increased from moderate focal to strongly generalized expression. In biopsy specimens obtained during bacterial infection with rejection activity focal induction of class I antigens on myocyte membranes was detected in five of seven specimens. In four biopsies taken during periods of bacterial infection without rejection no class I antigen induction on myocyte membranes was present. No distinct pattern of class II antigen expression was found during episodes of bacterial and fungal infections. Strong HLA-DR antigen expression on most vessel endothelia was in some cases associated with a weak expression of HLA-DQ and HLA-DP antigens on a few vessels.

Expression of Polymorphic Epitopes of MHC Antigens HLA-A and HLA-B Donor class I MHC antigens were stained in biopsy specimens of nine patients after transplantation. In specimens with class I positive myocyte membranes HLA-A and HLA-B antigens of donor origin could be demonstrated on the intercalated disks of only a few cells, compared with the total expression analyzed with antibodies to monomorphic determinants. Endothelia and interstitial cells strongly

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Table 5.3. Induction of class I MHC on cardiac myocytes after transplantation

Diagnosis (%)

Class I Positive Myocyte Membranes* No. of Biopsies Focal Generalized Patients

Clinical quiescence Mild rejection Moderate rejection Resolving rejection With myocytolysis With old myocytolysis

Total

27 21 27

64 35 58

12+ 14 29

0 1 4

19 43 57

18 23

25 51

12 12

3 8

60 39

* Expression of class I MHC antigens on myocyte membranes in cardiac allograft biopsies classified according to the histopathologic diagnosis + Positive staining relates to the number of biopsy specimens that contain positive myocyte membranes. Focal or generalized staining of the membranes was differentiated. Specimens obtained during rejection treatment of 1 week after treatment demonstrated mild rejection (3/35), moderate rejection (4/58), resolving rejection (with myocytolysis, 20/25; with old myocytolysis, 26/51).

expressed both donor HLA-A and HLA-B antigens in comparable staining intensity. One year after transplantation most of the interstitial cells between muscle fibrils showed a positive reaction to donor MHC antigens (HLA-A and HLA-B). Recipient class I MHC antigen expression was studied in six patients. Infiltrating lymphocytes in the biopsy specimen reacted to staining, whereas endothelial cells and myocyte membranes did not. One week to 1 month after transplantation infiltrations of dendritic interstitial cells of recipient type were found around vessels and between muscle fibrils. At 6 months and at 1 year after transplantation the amount of interstitial cells of recipient type was not further increased. All patients studied had several moderate rejection episodes that responded well to steroid treatment.

Discussion In the normal human heart myocytes do not express class I MHC transplantation antigens. The absence of staining with anti-class I monoclonal antibodies by means of immunohistologic techniques does not provide final proof for the total absence of the antigens on myocyte membranes. It can be assumed, however, that class I antigen expression is very low or negative in normal heart tissue. Early studies have agreed about the negative staining of outer myocyte membranes.33,521,649,775,792,811 For intercalated disks, by contrast, a weak positive staining has been reported in humans.33 In heart transplantation the lack of MHC antigens on the main functional cell type of the heart could be an important aspect with respect to the rejection response. The finding that in almost all biopsy specimens obtained during clinical quiescence after transplantation myocyte membranes remained negative for class I antigens implies that in this state these cells are probably resistant to T cell recognition and lysis. The presence of MHC alone and not costimulatory adhesion molecules determines the alloantigeneic reactivity.813

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The induction of class I antigens on myocyte membranes was found during different states of rejection activity. In the majority of the rejection episodes an induction, either focal or generalized, could be found. The staining of class I positive myocyte membranes and the extent of staining foci were usually related to lymphocytic infiltration and myocyte necrosis. Thus it is very likely that class I induction on myocytes forms part of the rejection process. It is probably caused by the local release of lymphokines from infiltrating lymphocytes. Additional potential alloantigens are generated by this induction and exposed to the host immune response. Effective rejection treatment enabled reversal of this induction process, which also has been demonstrated by other investigators.811 This decrease in MHC antigen expression in the graft probably results in a reduced allostimulatory capacity whereas a prolonged MHC induction after rejection treatment could result in repeated rejection episodes. In long-standing and severe rejection episodes myocyte class I induction was generalized and lasted several weeks. Studies on the kinetics of the MHC induction in rat and mouse have shown that class I antigens are induced very early (day 3) in unmodified allograft rejection.521 In this clinical study, however, acute rejection developed under potent immunosuppression. It has been shown that immunosuppressive drugs have an abrogative effect on lymphokine production and thus probably on MHC induction.580 Therefore, it can be expected that class I induction on myocytes of human cardiac allograft rejection is rather slow and delayed. This could explain why many specimens that showed mild and moderate rejection activity contained no myocytes positive for class I antigens. Furthermore, a full induction was mainly found when the process of rejection was established in a generalized fashion or was even resolving under treatment. As all of the rejection episodes identified by histopathologic examination were treated early, full class I induction on myocytes may not develop or may be found only in the state of resolving rejection after treatment. This is supported by the finding that class I induction on myocyte membranes was not found in six cases with moderate rejection until after 1 week following treatment. Another possibility is that successful treatment of rejection has a delayed effect on the termination of activated lymphocytes in the graft. Also, it might take some time before the induced class I antigens on myocytes are modulated. An easy inducibility of HLA-DR antigens on endothelia and a constantly elevated expression were observed after transplantation. The HLA-DR antigen expression was elevated irrespective of the time after transplantation. During rejection episodes, however, and in patients with repeated rejection and infection the expression was particularly strong; further the expression of MHC antigens HLA-DP and HLA-DQ was found. Interestingly, the capillary endothelium that is only weakly focally positive for HLA-DR antigens in the normal heart became strongly positive after transplantation. This enhanced class II expression could be of major importance and could be alluding to a pivotal role of capillary endothelial cells during immunoreactions. Endothelial cells are the major contact site to the host immune response, and the strong expression of class I and class II alloantigens may attract and stimulate alloreactive T lymphocytes. This could be a major determinant for the long-term outcome with respect to chronic rejection and graft atherosclerosis. In addition the recognition and sensitization to non-MHC molecules on endothelia143 could be mediated and facilitated by an increased expression of MHC class II molecules.

Fig. 5.1. MHC expression in human heart transplants. (a) Staining of class I antigens (HLA-A,B,C) in a heart transplant undergoing acute rejection. Induction of class I on myocyte membranes and intercalated discs is noted. Endothelial and interstitial dendritic cells are strongly class I positive. (b) Staining for donor type HLA-antigens in a cardiac transplant 10 months after transplantation: Strong positivity of vascular endothelia for donor class I MHC and of persisting interstitial cells (HT 90, day 298, MCA 2BC4: HLA-B35(w6), polymorphic epitope). (c) In parallel section expression of recipient type MHC: positivity of perivascular infiltrating cells and few interstitial cells (HT 90, day 298, MCA 48C1: HLA-A28, polymorphic epitope). (d) Coinduction of HLA-DP class II MHC molecules on vascular endothelia during rejection (HT 86, day 24, MCA B7/21).

b

d

a

c

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In contrast to the differential expression and induction of the class II MHC antigens, polymorphic class I HLA-A and HLA-B antigens were expressed on endothelia and interstitial cells in comparable density. On myocytes, however, donor HLA-A and HLA-B antigens could be detected only on a few cells. This could be caused either by a decreased sensitivity of the monoclonal antibodies reacting with polymorphic epitopes of HLA-A of HLA-B or by the differential expression of the separate alleles on different cell types. Furthermore, it cannot be excluded that HLA-C of nonclassic class I antigens are expressed. The infiltration of recipient interstitial cells of dendritic shape occurred within the first weeks after transplantation. These immigrating cells seen around vessels most likely are tissue macrophages. A detailed analysis of their phenotype, however, has not been performed. The origin of the interstitial cells sited between myofibrils remains unclear. The majority of these cells remained of donor type up to 1 year after transplantation. They could be either fibroblasts that are not replaced by bone marrow-derived cells or persisting dendritic cells of the donor. Further studies are needed to analyze the phenotype of donor and recipient-derived interstitial cell types by doublestaining techniques. These results indicate that the pattern of donor antigen expression in the long-term and that only a limited number of bone marrow-dependent cells (macrophages and dendritic cells) of recipient origin appear in the graft in the first weeks and during episodes of rejection in the first year after transplantation. This is in contrast to the extensive exchange of Kupffer’s cells by recipient macrophages in human liver grafts, which reduces the allogenic MHC expression.265,774 Thus it is likely that differences in rejection complications between organ grafts are dependent on the patterns of cell exchange and distribution of donor MHC antigens. Especially the donor MHC class I/II expression of endothelial cells may stimulate the recipients rejection response and maintain grafts susceptibility to lymphocyte recognition.245

Expression of Adhesion Molecules in Human Heart Transplants In recent years the expression of adhesion molecules in human heart transplant rejection has been thoroughly investigated. However, the overall knowledge about the relevance of adhesion molecules in different types of inflammatory heart disease is still limited, especially with regard to the role of different molecules in the regulation of immunological reactions. The current knowledge from results of various investigators and own investigations in endomyocardial biopsies and larger tissue preparations is reviewed in this chapter. The induction patterns during rejection, membrane expression, soluble release of these molecules in human heart allografts and the implications regarding diagnostic and therapeutic interventions are discussed. As data in humans are still incomplete single paragraphs are supported by in vitro and animal data. Whenever possible, results are described together with findings of other investigators.

Patients and Methods In the present study 417 biopsies of 53 patients following orthotopic heart transplantation and 12 larger right ventricular tissue preparations of 7 heart grafts were studied. In 53 cases it was the first heart transplantation for the recipient, two underwent retransplantations, carried out between 1982 and 1996. Five hearts were not transplanted. The postoperative maintenance immunosuppression consisted

Fig. 5.2. Vascular adhesion molecules in chronic heart transplant rejection. (a) Pselectin induction on small arterioles in a chronic rejected heart at the time of retransplantation (7 years). (b) In the same specimen induction of Sialyl Lewis X (SLex) was found on arterioles or venules. (c) CD34 ligand molecules were found strongly induced on microvessels of the myocardium in chronic heart transplant rejection. (d) On coronary arteries with intimal proliferation an endothelial upregulation of CD44 was found.

b

d

a

c

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of cyclosporine A, prednisolone and azathioprine. Antithymocyte globulin (ATG) was used as induction therapy for 3 or 4 days after transplantation. Rejection episodes were treated with 500 mg methylprednisolone on three consecutive days. Clinical diagnosis of acute rejection as performed by standard histology according to the criteria of the International Society for Heart and Lung Transplantation 1990.78 Eighty-four biopsies showed no signs of rejection (level ‘0’ according to ISHLT classification), 122 level ‘1A’ and ‘1B’, 72 level ‘2’, 14 level ‘3A’, and 123 recovering rejection (46 with fresh myocytolysis, 77 with old myocytolysis). Five of the larger tissue preparations were of donor origin and were used for heart valve banking only. The grafts not transplanted and those which showed no rejection were seen as representatives for the physiological situation. Immunohistology was performed in standard technique.

Results The results for this study are summarized in small paragraphs according to the different families of adhesion molecules. Findings from the literature are brought together with our own results to give an overview to the current knowledge. Tables 5.4 and 5.5 summarize the results.

Immunoglobulin Supergene Family CD2. CD2 is the receptor molecule for LFA-3.516 It is expressed on lymphocytes and functions as an intercellular adhesion molecule in T lymphocyte activation and cytolysis and as transmembrane activation molecule synergistic with the T cell receptor.516,517,710 In human heart grafts CD2 is expressed on lymphocytes only. In biopsies of rejecting cardiac allografts the number of CD2+ lymphocytes correlates with the degree of lymphocyte infiltration. No endothelial staining was observed.758 LFA-3. LFA-3 or CD58 is the ligand molecule to CD2 and may specifically facilitate the interaction of CD2+ T lymphocytes. Two isoforms exist that show differences in the transmembrane part of the molecule.488,743 Up to now there are only a few papers dealing with LFA-3 expression in heart tissue. The data show weak baseline expression758 up to strong baseline expression on nearly all tissues, with strong expression on intercalated discs of the cardiac muscle.733 One possible explanation for this difference may be the use of different monoclonal antibodies which recognize distinct epitopes on the LFA-3 molecule. In heart biopsies after transplantation a clear relationship between LFA-3 expression and rejection episodes was found.758 ICAM-1. ICAM-1 is by far the most intensively studied adhesion molecule in normal tissue and its inducibility under various pathological and experimental situations has been reported by many independent investigators. Although there are still some differences in the baseline expression reported by different groups, expression in the normal heart is now clear. Small differences may be due to different monoclonal antibodies which may recognize slightly different epitopes on the immunoglobulin domains. In the fetal human heart the capillaries are ICAM-1+, whereas the endocardium is ICAM-1–. In the adult human heart capillaries, venules, arterioles, coronary arteries, aorta, pulmonary artery and endocardium express ICAM-1 in

Fig. 5.3. Upregulation of integrin receptors in chronic heart transplant rejection. (a) VLA-1 was found upregulated on venous endothelia and interstitial myocardial cells (chronic rejection 7 years after HTX). (b) The same pattern of expression was found for VLA-2 and VLA-6 (c). (d) A strong induction of CD51 on endothelia and positivity of cardiomyocyte membranes was found in chronic rejection.

b

d

a

c

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decreasing intensity. In heart transplants, in the absence of rejection, this pattern remains basically unchanged. Possibly the baseline expression in heart transplants is slightly increased.65 With allograft rejection marked induction of ICAM-1 on all endothelia cells is observed. The capillaries and the endocardium show by far the strongest inducibility. Some investigators report ICAM-1 expression on intercalated disks650 and on the myocardial membrane.829 With resolving rejection ICAM-1 expression is downregulated to baseline. Our own results show a slower fall in ICAM-1 expression in the capillaries than in infiltrating lymphocytes.65 Viral myocarditis can lead to a similar induction pattern as rejection.702,758 A large number of studies have demonstrated the possibility of therapeutic intervention to prevent or treat allograft rejections by moderating the ICAM-1/LFA-1 interaction. Moreover ICAM-1 plays a critical role in the mediation of reperfusion injury after circulatory interruptions. ICAM-2. ICAM-2 is a ligand molecule for LFA-1. Some other receptor molecules are under discussion. Normal human heart capillaries express ICAM-2 strong. With rejection this expression is decreased a little. A small population of infiltrating cells is ICAM-2+. VCAM-1. VCAM-1 (INCAM-110) has recently attracted attention as cell adhesion molecule induced on vascular endothelial cells at sites of inflammation.638 VCAM is one of the ligands for the integrin VLA-4210 which is functionally expressed on activated lymphocytes, monocytes and NK cells.14,138,713 In vitro data suggest that certain cytokines are responsible for the expression of VCAM-1 on endothelial cells.501,838 The data for baseline expression and inducibility of VCAM-1 are more unique than for other adhesion molecules. In a murine cardiac allograft model no baseline expression was found. There was inducibility on microvascular and venous endothelia cells by allograft rejection and viral myocarditis.597 In the fetal human heart there was no VCAM-1 expression on capillaries or endothelial cells.578 In the adult human heart most investigators report staining on capillaries, aorta or pulmonary artery.110 Arterioles, venules and endocardium are sometimes reported to be weakly positive. Coronary arteries seem to have a little stronger baseline expression. With rejection there is de novo induction of VCAM-1 on arteriolar endothelial cells, capillaries and postcapillaries venules as on endocardium.136,220,829 With resolving rejection the expression of VCAM-1 even precedes the histological findings of rejection for 1 or 2 weeks.220 Apart from endothelial staining, some monocytes and macrophages showed VCAM-1 positively. NCAM. NCAM or CD-56, the neural cell adhesion molecule, plays a critical role during cardiac morphogenesis and innervation.123,262 In the embryonic heart NCAM is expressed by mesenchymal cells and by myocytes.29,418 It is also found on endocardial cells and on nerve fibers. The overall intensity of NCAM staining in rat heart seems to increase until birth and declines thereafter. The expression pattern in human fetal and adult heart seems to be similar.13 In the adult human heart NCAM staining is mainly restricted to cardiac innervation. There it is localized to neuronal cell bodies, nerve fascicles and fibers. The sacrolemnal immunoreactivity is weak.261 In animal models of myocardial hypertrophy an increase of NCAM immunoreactivity in sacrolemnal and intercalated disks was found.261 This confirmed by extensive sacrolemnal and intercalated disk immunostaining in biopsies after human heart transplantation. With rejection there is NCAM expression

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on myocyte membranes and capillaries. Infiltrating cells are NCAM negative. In human cardiac diseases a range of NCAM induction showed the inducibility by hypertrophic and hypoxic stimuli. PECAM. PECAM, the platelet endothelial cell adhesion molecule also known as CD31, is found on all endothelial cells and platelets.829 In fetal and adult human hearts capillaries and endocardium stain PECAM+.578 In rejecting cardiac allografts there is an induction of PECAM on capillaries. The staining intensity on endocardium, arterioles and postcapillary venules is normal.829 There is no staining of allograft infiltrating cells.

Integrin Family LFA-1, CR-3 or Mac-1. LFA-1 (CD11a/CD18), CR-3 or Mac-1 (CD11b/CD18) and p150.95 (CD11c/CD18) are leukocyte receptors found on majority of leukocyte subpopulations. Mac-1 and p150,95 are more restricted to monocytes/macrophages than LFA-1. As they are not found on capillaries endothelia, myocytes and intercalated disks, their expression in heart transplants is reported together. In the normal human heart just a few interstitial cells and intravascular leukocytes express these molecules. With allograft rejection there is an infiltration mainly of lymphocytes but also of macrophages and granulocytes. Most of these graft infiltrating cells are LFA-1 positive. Few are Mac-1 and p150,95 positive. LFA-1+ cells are mainly located around ICAM-1+ endothelia. VLA-1. VLA-1 (CD49a/CD29) is a cell-matrix receptor for collagen and laminin. In the normal adult heart it is found on capillaries, coronary arteries, the endocardium and on some interstitial cells. With acute rejection its expression on capillaries and endothelial cells of arterioles and venules seem to be a little decreased and totally absent on the endocardium. During episodes of chronic rejection we found an increasing expression on small myocardial vessels, the endocardium and coronary arteries. Most of the infiltrating lymphocytes are VLA-1–, but nevertheless a small population of lymphocytes expresses VLA-1. VLA-2. VLA-2 (C49b/CD29) is a cell-matrix receptor or collagen and laminin, just as is VLA-1. The expression of VLA-2 in the normal heart is very weak. Some endothelial cells of arteries and venules seem to have low, coronary arteries no VLA-2 expression. With rejection there is no remarkable induction of VLA-2, but some of the infiltrating leukocytes weakly express VLA-2. VLA-3. VLA-3 (CD49c/CD29) is a cell-matrix receptor for fibronectin, laminin and collagen. In the normal human heart there is clear VLA-3 expression on capillaries, the endocardium and coronary arteries. Interstitial cells and myocytes are VLA-3 negative. With acute rejection there is no remarkable VLA-3 induction, but a decrease of expression on endocardium. During episodes of chronic rejection small myocardial vessels showed an expression comparable to that of normal heart tissue. Graft infiltrating cells are VLA-3 negative. VLA-4. VLA-4 (CD49d/CD29; LPAM-1) is a receptor molecule for VCAM-1, fibronectin, thrombospondin and M-addressin. It is basally expressed on resting lymphocytes and monocytes. In the normal human heart the endothelial cells lining small vessels, arterioles, venules, the endocardium and coronary arteries show a VLA-4 expression. With chronic but not acute rejection this expression increased. Additionally some capillaries show VLA-4 induction. Graft infiltrating lymphocytes are VLA-4 positive.

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VLA-5. VLA-5 (CD49e/CD29) is a cell-matrix receptor for fibronectin. In the normal human heart small myocardial vessels and coronary arteries show a strong VLA-5 expression. Some interstitial macrophages are VLA-5 positive. With chronic but not acute rejection there is an induction of VLA-5 on small myocardial vessels and the endocardium. Myocytes remain VLA-5 negative. Monocytes and macrophages within lymphocyte infiltrates are also VLA-5+, whereas most lymphocytes are VLA-5 negative. VLA-6. VLA-6 (CD49f/CD29) is a receptor molecule for laminin. In human heart it is found on small myocardial vessels, coronary arteries, the endocardium, and interstitial cells. With acute rejection it is downregulated on small myocardial vessels and the endocardium, but shows an upregulation during episodes of chronic rejection. Monocytes and macrophages strongly express VLA-6. Lymphocytes in the infiltrates are VLA-6–. CD51. CD51 is also called VNR-α chain and is presented on platelets and nonleukocytes. The 125 + 25 kDa molecule associates with the VNR-β chain (CD61) to build up a vitronectin receptor. CD51 shows a clear expression on small myocardial vessels and coronary arteries, but only a weak expression on the endocardium of the normal adult heart. In acute rejection the endocardium is totally negative, but shows an upregulation during episodes of chronic rejection. Furthermore, myocytes were found positively stained for CD51 in chronic rejection. CD61. CD61 is also called gpIIIa and presents a VNR-β chain with an molecular weight of 110 kDa. It is presented on platelets, and megakaryocytes and, when associates with gpIIb or VNR-α (CD51) to form a receptor for fibrinogen.CD61 shows little decrease in endomyocardial and microvascular expression in acute rejection. Normal adult hearts and those which showed chronic rejection episodes show a weak CD61 expression on small myocardial and a clear and strong expression on coronary arteries and the endocardium respectively. CD29. CD29 is a VLA-β chain with a molecular weight of 130kDa which is presented on T cells and many other cell types. It belongs to the integrin subgroup of β1 integrins and pairs at least with 6 different integrin α chains. CD29 shows an expression which is comparable to that of CD51.

Selectin Family E-selectin. E-selectin, endothelial leukocyte-adhesion molecule-1 (ELAM-1), or leukocyte endothelial-cell-adhesion molecule-2, is a cell-surface glycoprotein that mediates the adhesion of blood neutrophils, memory T cells and some monocytes.76,744 It contains an N-terminal lectin-like domain and is absent from resting endothelial cells. Its expression is mainly induced by IL-1 and TNF but also by direct contact with primed CD-4 cells.172,612 ELAM-1 mediates neutrophil adhesion to the endothelium an is only transiently expressed (a few hours) following induction in vitro. Its ligand molecules in addition to sialyl LewisX are ESGL-1 and sLea.257,453,601,755,839,899 In fetal human heart no E-selectin expression was found.578 In adult human heart (donor heart before transplantation) it was detected only weak on small vessel endothelial cells, but on larger vessel endothelium, including coronary arteries. Endocardium showed no E-selectin staining. Venules express E-selectin weakly. Other investigators report that capillary endothelium expresses little, if any

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E-selectin.829 During rejection the overall amount of E-selectin expressed on capillaries increases, whereas staining on endocardium is still absent in acute, but clearly to be seen in chronic rejection. In Quilty lesions there is no remarkable Eselectin staining.629 In a prospective longitudinal study Ferran showed E-selectin expression on capillaries and postcapillary venules preceding the histological characteristics of acute rejection. In these cases ELAM-1 expression was not detectable on myocardial biopsies taken a week later.220 Interestingly animal studies comparing the tissue distribution in IL-1-stimulated animals, show by far the highest ELAM-1 concentration in heart.63 P-selectin. P-selectin also called PADGEM, CD62p or GMP-140, is an integral α-granule membrane glycoprotein and has a molecular mass of about 140 kDa. It is stored in α granules (Weibel Palade bodies) of platelets and endothelial cells. After cellular activation by stimulatory agents like histamine, thrombin or lipopolysaccharides it shows a translocation on the cellular surface of these cells within minutes and can be restored in Weibel-Palade bodies for reuse. It mediates the adhesion and rolling of neutrophils and monocytes.439,482 In heart tissue it was found to be basally expressed on myocardial arterioles within normal hearts.763 In the present analysis we found a weak expression on small myocardial vessels, but not on coronary arteries. The endocardium was found to be negative in all cases. However, negative observations under activated conditions as in episodes of acute rejection could be the result of a ‘too late’ investigation, because P-selectin expression is known to be present only a few hours after activation.

Unclassified Adhesion Molecules CD44. CD44 is a receptor molecule for hyaluronic acid. Other receptors are under discussion. In normal human heart interstitial cells like monocytes, macrophages and lymphocytes show CD44 expression. Additionally some intravascular cells positively stain CD44. Microvascular endothelial cells, coronary arteries and endocardium of normal hearts are weakly CD44 positive. With rejection most infiltrating cells show CD44 expression. There is also a weak induction of CD44 on capillaries and endothelia. In chronic transplant coronary arteriopathy CD44 is induced on coronary and microvascular endothelia. Myocytes are CD44 negative. Sialyl LewisX. Sialyl LewisX Sialo-2-3, sialysated lacto-N-fucopentaose III, which is presented on granulocytes and monocytes and interacts with ELAM-1 and PSelectin. SLex was negative in all right heart endomyocardial biopsies with acute rejection. In two patients with chronic vascular rejection SLex was found positively stained on the venules of right and left ventricular specimens. CD34. CD34 the 105 to 120 kDa molecule is known to be presented on hematopoietic progenitor cells and endothelial cells. It has been demonstrated to function as a ligand for L-Selectin. In the normal heart CD34 is expressed on endothelia of myocardial small vessels and coronary arteries. Endocardium is weakly positive. In acute rejecting heart biopsies CD34 was reduced expressed in microvascular vessels. In chronic rejection, however, CD34 was found strongly induced on microvascular vessels of the myocardium. Von Willebrand Factor. Von Willebrand factor, formerly known as Factor-VIII related antigen or FVIIIrAg, is the second, larger unit of Factor VIII. Factor VIII is generally believed to circulate in blood as a multimeric complex of two glycoproteins which are physiologically and immunologically distinct. One component of

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the factor VIII complex is factor VIII procoagulant activity (FVIII:C), which is associated with factor VIII/procoagulant antigen (FVIII:Ag, formerly FVIII/Cag). The second, larger unit is factor VIII/von Willebrand factor (vWF:Ag), which is investigated in this study. FVIII:C has anti-hemophilic activity and is defective or deficient in patients with classical hemophilia; and vWF:Ag, which is important in the activation of the coagulation cascade, is absent in patients with von Willebrand disease. Von Willebrand factor, like fibronectin and P-selectin, is localized in intracellular granules (Weibel-Palade bodies) of endothelial cells300 and in the extracellular fibrils. Both fibronectin and vWF are codistributed. They are coaligned with each other to be components of the extracellular matrix and also show a codistribution in intracellular granules. In normal hearts vWF could not be found in any of the specimens studied. In acute rejecting hearts, however, small myocardial vessels showed clear expression. Endocardium revealed strong vWF positivity in acute rejection. Biopsies taken during chronic rejection and at the time of retransplantation showed no vWF in any region of the heart.

Discussion For the understanding of heart allograft rejection the endothelial cell and its surface molecules are of major interest. The initiation or regulation of allograft rejection is likely to be influenced by recipient endothelial cells.758 The first step of graft infiltration takes place there. So there is increasing interest in the distribution and regulation of endothelial immune adhesion molecules.

Implications for Transplant Pathology The present study gives nearly complete survey of the baseline expression and inducibility of adhesion molecules in human heart allografts. The discussion attempts to explain why adhesion molecule expression in cardiac allograft is important for scientific, diagnostic and therapeutic purposes. In particular, some implications for the future are discussed. Finally, interesting opportunities to use monoclonal antibodies against adhesion molecules outside transplant medicine are evaluated. The study of the regulation of adhesion molecules may aid in understanding the pathophysiology of allograft rejection.322,827 Moreover, the inflammatory reaction in transplanted organs can serve as a model for immune reactions. But more than this, increasing knowledge about distribution and induction of immune adhesion molecules gives insight regarding the regulation of leukocyte trafficking. One of the most interesting points about endothelial cell adhesion molecule expression is the baseline expression of these molecules. It shows that the microenvironment of endothelial cells differs in various sites.787 The microscopic finding of a specialized morphology of different endothelial cells is confirmed by molecular studies. Some factors from the extracellular matrix from the subendothelial tissue must lead to this local-specific differentiation.771 Otherwise, this proves that the endothelial cell is not just the lining for the blood vessel but is a local-specific regulator of leukocyte trafficking.313,609,851,852 Signals are passed on between the blood cells and subendothelial cells and matrix.787

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Adhesion molecule expression in human cardiac allograft varies considerably between the endothelial cells of the heart.457,578,830 First, the adhesion molecule equipment of capillaries differs from all other endothelia. But interestingly, venules and arterioles differ also in their microenvironment. It is still unclear whether these differences are due to metabolic, hemodynamic or extracellular stimuli, or if the endothelial cells differ from each other intrinsically. This would mean that the distinct precursor cells must exist or that a precursor endothelial cell undergoes local and specific differentiations to fit into its area of destination. But in spite of this lack of knowledge about endothelial differentiation, the variety of adhesion molecule expression on endothelial cells in different vascular beds indicates that this may be a main regulative factor for leukocyte traffic in circulation. Thus, the various vascular sites, by expressing in part different kinds and quantities of cellular adhesion molecules, seem to have different importance for immunological reactions. Special attention has been given to the role of adhesion molecules in transplant coronary artery disease. Here the induction of ICAM-1 together with HLA-DR has been associated with the chronic allograft vasculopathy.416 The contribution of different adhesion molecules to coronary artery pathology has been thoroughly discussed.471 The antigen presenting capacity of endothelial cells to T lymphocytes is a potential factor of local immunogenicity of coronary endothelium.650 However, nonspecific endothelial activation, for instance, by antibody binding may lead to the expression of adhesion ligand molecules and transplant arteriopathy.651 This may be accompanied by the induction of cytokine expression and the consecutive release of growth factors as PDGF and FGF.659

Implications on Clinical Diagnosis Although an increasing knowledge about adhesion molecule expression in allograft rejection has been collected in the last years, the implications for clinical diagnosis are very limited. A number of applications, however, can be envisaged. First, the results of immunohistology evaluations may serve as confirmation of standard histology. This may be true even in the immediate future when the initial studies regarding adhesion molecule expression are repeated and confirmed by an increasing number of independent groups. But more than this, some initial encouraging studies showed an increased expression of E-selectin preceding the histological findings of cellular rejection by one or two weeks.20,112,220 Although it is unclear whether this is an early sign of rejection or feature of the etiology of rejection, e.g., viral induction, it has promising implications for postoperative monitoring. The possibility of diagnosing rejection before cellular infiltration and myocytolysis is indeed fascinating. Also standard histology might be combined with adhesion molecule immunohistology as a means of following treatment. Perhaps monitoring of adhesion molecule expression will demonstrate chronic rejection not apparent by conventional histology techniques—such as lymphocyte infiltration (as shown by Allen for VCAM-1; Allen et al18 ). Eventually immunohistology will help distinguish between rejection and infectious complications.18 The last, but not least, promising feature of adhesion molecule immunohistology may be the gradation of rejection. It is indeed conceivable that one may distinguish between steroid and nonsteroid sensitive rejection episodes. In this way studying immune adhesion molecule expression could lead to individualized treatment of

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Cell Adhesion Molecules in Organ Transplantation

rejection. Methods for quantitative diagnosis of adhesion molecule induction in organs are still provisional. Although scintigraphic evaluations of ICAM-1 induction have been propagated,356 this may be of limited clinical value for rejection diagnosis.564,565 A considerable number of studies have concerned the value of circulating soluble adhesion molecules and cytokine serum levels as a means of diagnosing heart transplant rejection.28,54,268 These parameters, however, are not suitable for noninvasive rejection diagnosis by lack of specificity.

Therapeutic Options A great deal of preliminary data are published about trials with monoclonal antibodies against different adhesion molecules for the prevention or treatment of graft rejection. These studies can be divided into those for induction therapy (tolerance induction), maintenance immunosuppression and treatment of acute rejection. Most studies have been conducted in animals, but a few human trials have been done. The results are not conclusive. A prophylactic administration of anti-VCAM-1 in Cynomolgus monkeys with renal allografts without other immunosuppression showed a remarkable increase in graft survival.159 In the same setting treatment of acute rejection was also possible.908 Interestingly, the intensity of lymphocytic infiltration was not altered, but the lymphocytes seem to be somewhat inactivated. In a Cynomolgus monkey heart transplant model similar results prolonging graft survival were achieved.227 In rodents various combinations of interventions involving adhesion molecules have been attempted. The results indicate that prolonging of graft function is possible with monoclonal antibodies against ICAM-1, ∝LFA-1,(CD11a), CD18, CD4, VCAM and VLA-4.227,365,370,371,400,545,589,591,592,598,821 Combination of two or three monoclonals are additionally effective and may have an effect on transplant atherosclerosis.357,400,589,667 The addition of low dose cyclosporine A in some models supported the effect remarkably. Nevertheless there are strong indications that the effectiveness of antibody treatment is closely related to the strength of the genetic background.370,371 The prevention of acute rejection with monoclonal antibodies against adhesion molecules is far better in weak than in strong histoincompatible rat strain combinations. The results of the first application of an antibody against ∝LFA-1 as rejection treatment in human kidney transplantation was not as promising as the results in animal models.455 One interesting aspect of the use of monoclonals for prevention of allograft rejection is the theoretical possibility of inducing some kind of donor-specific tolerance in animal models.355,358 In some models skin grafts from the same donor are tolerated, whereas third party grafts are strongly rejected.400 This effect on immunological tolerance in mouse minor histoincompatible models is still under discussion. A similar observation has been made by Stepkowski et al799,800 in a rat model using anti-sense oligonucleotides to ICAM-1 and a monoclonal antibody to LFA-1. In contradiction Morikawa et al535 found an accelerated graft loss after the application of a combination of ICAM-1 and LFA-1 monoclonal antibodies in rat heart graft recipients. Orosz et al found a long-lasting allograft acceptance in rats after the administration of anti-VCAM-1 antibodies.572,574,575 This field of immunosuppressive strategies in the induction phase to induce longterm alloantigeneic tolerance is still in its infancy.

MHC and Cell Adhesion Molecules in Clinical Heart Transplantation

75

The effect of some of the newer immunosuppressive drugs on adhesion molecule expression has been not definitely examined. Initial results point to modulation of adhesion molecule expression for RS 61443 and AF&F 105685687 as well as the cyclosporine adjuvant ricinoleic acid.179 New aspects are given by data of adhesion inhibition by new drugs as NPC15669,860,940 antioxidant pyrrolidine dithiocarbamate,221 castanospermine,325 apigenin253 and pentoxiphilline.115 The use of these drugs may open fascinating possibilities for drug treatment conceivable with yet unexpected features. The knowledge gained in transplant research regarding expression and induction of immune adhesion molecules has profound implications outside transplantation medicine. From biopsies before explanation and before and after reperfusion, we know that adhesion molecule expression is upregulated in the donor. The early phase of reperfusion injury, which is clearly not in immunology phenomenon, guides the interest in the role of adhesion molecules in reperfusion injuries after cardiac surgery in general. It is now clear that adhesion molecules are the principal mediators of reperfusion injury after ischemia or circulatory arrest. Several investigators have explores different systems of injury and studied the changes in adhesion.338,378,729 The next question was whether intervention on the adhesion molecule system could reduce the reperfusion injury or myocardial stunning. Experiments so far show a remarkable influence of monoclonal antibodies on ICAM-1/CD18 with regard to the extent of tissue change and myocardial stunning.32,211,326,337,484,519,732 It is likely that pharmacological interventions in the immune adhesion molecule system will become routine within the next few years.

CHAPTER 6

Cell Adhesion Molecules in Clinical Lung Transplantation Gustav Steinhoff

Introduction

I

n recent years the clinical transplantation of heart-lung, single- and double-lung has become an established treatment option for terminal lung disease.305 Problems in transplant preservation, operation technique,303 rejection diagnosis274,937 and postoperative management of opportunistic infections767 have experienced major improvements in recent times. In addition to the organ specific exposition to opportunistic infections, especially cytomegalovirus infection and fungal infections, a major threat to transplant recipients comes from chronic rejection with obliterative bronchiolitis.381 An interaction of inflammatory events caused by infection of the graft or CMV-related leukocyte infection and the upregulation of chronic rejection activity has to be discussed.269,923,924 Inflammatory changes of graft cells and especially lung endothelia have to be assumed as causative in the mediation of inflammatory lung disease after transplantation. In this context the analysis of intercellular adhesion molecules is of particular interest for the interpretation of the organ specific appearance and extent of inflammatory events in transplanted lungs. Their analysis, however, has been restricted by the low availability of lung biopsy material. Only the analysis of the expression of MHC antigens has been described in human lung allografts by Taylor et al831 and in the rat by Romaniuk et al.646 The need for lung retransplantation either for acute or chronic rejection304 allowed to study lung transplant tissue for the question of tissue distribution of cell adhesion molecules.771

Patients and Methods Lung transplant biopsy material was studied in patients requiring retransplantation for acute or chronic rejection. The four grafts studied were resected at day 11a, 66, 68, and 463 post LuTx because of acutea (n = 1) or chronic rejection with bronchiolitis obliterans (n = 3). Normal lung specimens from organ donors (not used for transplantation) were studied for comparison (n = 4). The postoperative maintenance immunosuppression consisted of Cyclosporine A, prednisolone and azathioprine. Acute rejection was primarily treated by steroid bolus (3 days 500 mg methylprednisolone) and secondarily upon treatment resistance Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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with ATG or OKT3. The biopsies were all frozen immediately and stored in liquid nitrogen. The expression of intercellular immune adhesion molecules was studied on cryostat sections using standard immune peroxidase and alkaline phosphatase immunohistological techniques. The monoclonal antibodies used for detection of cell adhesion molecules are listed in Table 6.1.

Results The presented results give an overview on expression patterns of leukocyte adhesion receptor and ligand molecules in normal human lungs and rejected lung allografts. The adhesion molecules studied and monoclonal antibodies employed are specified in Table 6.1. Included are immunohistological data of explanted grafts of four patients with irreversible acute (n = 1) or chronic (n = 3) rejection. The expression patterns are described for immunoglobulin superfamily (Table 6.2), integrin (Table 6.3), selectin and other cell adhesion molecules (Table 6.4). The expression patterns of the ligand molecules MHC class I and class II in human lung grafts have been previously described.831 Adhesion molecules in clinical lung transplantation have been studied by Shreeniwas et al715 and Steinhoff et al.762,763,771 Experimental data on the expression of MHC and adhesion molecules in rat lung allografts were obtained by Romaniuk et al,646 Han et al,288 You et al936 and Steinhoff et al.786

Immunoglobulin Superfamily LFA-3. LFA-3 (CD58) was expressed in normal lung on all vessel endothelia, bronchial epithelia and alveolar macrophages. The pattern of expression underwent no major change during acute and chronic rejection, the intensity of expression was found upregulated on alveolar macrophages. ICAM-1. ICAM-1 (CD54) showed a different expression pattern from LFA-3 in normal lung. Although ICAM-1 was found on almost all endothelial cell types— with less staining intensity of larger arteries—it was absent on bronchial epithelia, alveolar macrophages and lymphocytes found. However, ICAM-1 was in general strongly positive on all pneumocytes. In rejecting grafts the staining intensity on larger arteries and veins was upregulated. Bronchial epithelia were negative in the grafts studied, whereas alveolar macrophages and lymphocytes were found generally positive with varying staining intensity. ICAM-2. ICAM-2 was expressed on arteriolar, capillary and venous endothelia and a few interstitial cells in the normal lung, but absent on larger arteries and other cell types. In the rejecting grafts ICAM-2 was found generally induced on larger arteries. Interstitial dendritic cells were positive in part. Other cells remained negative. VCAM-1. VCAM-1 (INCAM110), was expressed only on large arteries and a few lymphocytes in the normal lungs studied. The rejecting grafts showed an induction of VCAM-1 on arterioles and venous endothelia, but in general not on capillary endothelia. In some areas pneumocytes were also found to be induced to express VCAM-1 during severe rejection. Infiltrating lymphocytes and interstitial dendritic cells were VCAM-1 positive to a large part.

Cell Adhesion Molecules in Clinical Lung Transplantation

79

Table 6.1. Adhesion molecules studied and monoclonal antibodies used Adhesion Molecule

Monoclonal Antibody

Producer/Source

Immunoglobulin Superfamily CD2 LFA-3 LFA-3 (CD58) CD2 ICAM-1 (CD54) CD11a/b

6F10.3;8E6B3 G26.1;TS2/9 84H10;RR1/1;6.5B5

ICAM-2 VCAM-1 NCAM (CD56) PECAM-1 (CD31)

CD11a VLA-4 NCAM NCAM

CBR-IC2/1 1.4C3 T199 5.E.6

a a; T. Springer, Boston a; Rothlein, Boston; Haskard, London b a a a

ICAM-1,ICAM-2 ICAM-1,C3bi,Factor X fibrinogen, ICAM-3

25.3.1 Bear1 Bu15 BL5 TS2/7 Gi9 P1B5 HP 2/1 SAM-1 GoH3 AMF/7

a a a a c a a a a a a

Integrin Family LFA-1 (CD11a) CR3 (CD11b) p150,95 (CD11c) CD18 VLA-1 VLA-2 VLA-3 VLA-4 VLA-5 VLA-6 CD51

Ligand Structure

laminin, collagen laminin, collagen fibronectin, lam.,coll. VCAM-1, fibronectin fibronectin laminin vitronectin, fibrinogen vWF, thrombospondin, fibronectin

Selectin Family LECAM (Lam-1,Leu8) ELAM-1 CD62

addressins, ELAM-1,CD62 CD15,sialyl LewisX sialyl LewisX

Leu8,Dreg56 1.2B6; HP18 CBL.thromb6

d; a a; d a

Unclassified CD44

hyaluronic acid

SBU24-32,F10-44-2

HECA452

?

Mem-85 HECA-452

McKenzie, Melbourne; Dalchau, London Horeysi, Prague Duivestijn, Maastricht

Source of monoclonal antibodies: a DIANOVA, Hamburg, FRG; b BENDER MED/SERVA, Heidelberg, FRG; c BIERMANN/T-CELL SCIENCES, Bad Nauheim, FRG; d BECTON-DICKINSON, Heidelberg, FRG.

NCAM. NCAM (CD56) was expressed on nerve cells in normal lung and a few mononuclear cells, presumably NK-cells. In the rejecting grafts a few arteries were NCAM positive as well as a few pneumocytes. The number of NCAM positive NKcells in the graft was increased. CD31. CD31 (PECAM, CD13) was expressed on all endothelia in the normal lung, as well as on alveolar macrophages and some lymphocytes. This expression pattern underwent no change in the rejected grafts studied, only the staining intensity on alveolar macrophages and the number of CD31+ lymphocytes were found increased.

++ ++

+ ++

– +++

+++ +++

– (++)

+++ +++

LFA-3 normal rejection

ICAM-1 normal rejection

ICAM-2 normal rejection

VCAM-1 normal rejection

NCAM (CD56) normal rejection

PECAM (CD31) normal rejection +++ +++

– –

– +++

+++ +++

+++ +++

++ ++

+++ +++

– –

– –

+++ +++

+++ +++

+++ +++

+++ +++

– –

– ++

++ +++

++ +++

++ ++

Arterial Arteriolar Capillary Venous Endothelia Endothelia Endothelia Endothelia

– –

– –

– –

– –

– –

+/++ +

– –

– (++)

– ++

– –

+++ +++

– –

++ +++

– –

– –

– –

– ++

++ +++

++ ++

(+) +

++ –

– –

– ++

++ +++

Bronchial Pneumocytes Alveolar LymphoEpithelia Macrophages Cytes

Table 6.2. Expression of immunoglobulin superfamily adhesion molecules in human lung transplants

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81

Integrin Family LFA-1. LFA-1 (CD11a) was expressed on alveolar macrophages and lymphocytes in normal lungs. This pattern of expression remained unchanged in the rejected grafts studied, however, the number of LFA-1 positive vascular adherent and interstitially infiltrated leukocytes was strongly increased. CR3, Mac-1 and p150/95. CR3, Mac-1 (CD11b/18) and p150/95 (CD11c/18) were partially positive on alveolar macrophages and leukocytes. In the four rejected transplants, the expression on alveolar macrophages was upregulated. Lymphocytes or leukocytes were generally positive for CD18 when vascular adherent and located in perivascular lymphocyte clusters. VLA-1. VLA-1, a cell-matrix receptor for collagen and laminin, was found strongly positive on all normal lung endothelia. This pattern remained unchanged in the rejected grafts studied. VLA-2. VLA-2, a cell-matrix receptor for collagen, was only found positive on capillary endothelia, while negative on arteries and veins in the normal lung. Further expression was found on bronchial epithelia. This pattern was changed in the rejected lungs: de novo VLA-2 expression was observed on arterial and venous endothelia, pneumocytes and some alveolar macrophages. Some interstitially infiltrating leukocytes were found VLA-2 positive. VLA-3. VLA-3 (ECMRI, CD49c/CD29), a cell-matrix receptor for fibronectin, laminin and collagen, was expressed on part of the vascular endothelia, all bronchial epithelia and pneumocytes (alveolar luminal expression) in normal lungs. The same pattern of expression was found in the four transplants with advanced rejection and inflammation. The endothelial positivity (arterial) varied in the pathological specimens. VLA-4. VLA-4 (LPAM-1, CD49d/CD29) was expressed only on a few lymphocytes/leukocytes in the normal lungs studied. In the rejecting grafts almost all vascular adherent and interstitially infiltrating leukocytes were VLA-4 positive. In addition, alveolar macrophages expressed VLA-4 in part. No other cells were found VLA-4 positive. VLA-5. VLA-5, a cell-matrix receptor molecule binding to fibronectin, was positive on all endothelia in normal lung, as on alveolar macrophages and a few leukocytes. In the rejected transplants the general strong VLA-5 expression on all endothelia and alveolar macrophages remained unchanged, whereas in acute rejection bronchial epithelia and part of the pneumocytes expressed VLA-5 de novo. VLA-6. VLA-6, a cell-matrix receptor molecule binding to laminin, was found on capillary, venous and arteriolar endothelial cells, but not in larger arteries. In normal lungs bronchial epithelia were VLA-6 positive at the basal membrane site. This pattern of expression was also found in rejecting transplants, the expression on endothelial cells, however, was upregulated and generally positive, including larger arteries. A few VLA-6 positive alveolar macrophages and infiltrating leukocytes were found. CD51. CD51, the α chain of receptors to fibronectin, vitronectin, von Willebrand factor, and thrombospondin, was found on all resident cell types in normal lung, as well on a few alveolar macrophages and leukocytes. The pattern of expression in rejecting grafts was mainly the same, whereas alveolar macrophages and interstitial leukocytes were positive to a larger extent.

– –

– –

+++ +++

– +

++ ++

– –

+++ +++

LFA–1 normal rejection

Mac–1 normal rejection

VLA–1 normal rejection

VLA–2 normal rejection

VLA–3 normal rejection

VLA–4 normal rejection

VLA–5 normal rejection +++ +++

– –

++ ++

– +/++

+++ +++

– –

– –

+++ +++

– –

+++ ++

++ ++

+++ +++

– –

– –

+++ +++

– –

+ ++

– (+)

+++ +++

– –

– –

Arterial Arteriolar Capillary Venous Endothelia Endothelia Endothelia Endothelia

– +++

– –

+++ ++

++ ++

– –

– –

– –

– +/++

– –

+++ +++

– ++

– –

– –

– –

+++ +++

– ++

– –

– ++

– –

+++ +++

+++ +++

+++ +++

+++ +++

– –

– +

– –

+++ +++

+++ +++

Bronchial Pneumocytes Alveolar Lympho– Epithelia Macrophages Cytes

Table 6.3. Expression of integrin adhesion molecules in human lung transplants

82 Cell Adhesion Molecules in Organ Transplantation

+++ +++

CD51 normal rejection ++ +++

++ +++ ++ ++

+++ +++ ++ ++

++ +++ +++ n.d.

++ + ++ ++

– – ++ ++

– –

++ – – +++ +++ +++

E–selectin (ELAM–1) normal (+) rejection –

P–selectin (CD62, GMP140) normal ++ rejection +++

+ +

– –

CD44 normal rejection

HECA452 normal rejection – –

– –

L–selectin (LECAM–1) normal – rejection –

– –

+++ +++

– –

– –

– –

– ++

+++ +++

++ ++

– –

– –

Arterial Arteriolar Capillary Venous Endothelia Endothelia Endothelia Endothelia

++ ++

– –

– –

+++ +++

– –

– –

– –

– +++

+++ +++

– –

– –

– –

– +++

+++ +++

– –

– –

– –

+++ +++

+++ +++

– –

(+) (+)

++ ++

Bronchial Pneumocytes Alveolar Lympho– Epithelia Macrophages Cytes

Table 6.4. Expression of selectin and unclassified adhesion molecules in human lung transplants

– +++

VLA–6 normal rejection

Cell Adhesion Molecules in Clinical Lung Transplantation 83

Fig. 6.1. (a) Expression of ICAM–1 in lung rejection with alveolar fibrosis. Positivity of pneumocytes and endothelial cells (day 68 post double lung transplantation; ICAM–1, orig. magn. 200x). (b) Expression of ICAM–2 in the same graft: Strong positivity of arterial endothelia and staining of capillaries (ICAM–2, orig. magn. 325x). (c) VCAM–1 induction was only found on arterial and venous endothelia, but not on capillaries in lung rejection (day 66 post transplant; VCAM–1, orig. magn. 325x). (d) E–selectin (CD62e) induction was found only on arterial and venous endothelia during lung rejection (day 11 post lung transplant; orig. magn. 325x).

b

d

a

c

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85

Selectin Family LECAM-1. LECAM-1 (Lam-1,Leu8,Mel-14), the L-selectin, was only expressed in the normal human lung on a few intravascular lymphocytes. With acute and chronic rejection a few LECAM-1+ lymphocytes were present intravascular adherent and in perivascular infiltrates. No other LECAM-1 positive cells were found. ELAM-1. ELAM-1, the E-selectin, was found only on a few perivascular lymphocytes and some arteries and arterioles (not in all specimens). In rejecting grafts not ELAM-1 positive endothelia were found in acute or chronic rejection. A few lymphocytes displayed ELAM-1 expression. CD62. CD62 (GMP140, PADGEM), the P-selectin, was found positive on the endothelium of large arteries and veins in the normal lung. In graft rejection CD62, in addition was induced on part of the arterioles and found on large arteries and vein as well. No positive staining could be found on lung capillaries.

Unclassified Adhesion Molecules CD44. CD44 (Pgp-1;EDM-III;p80;Hermes1,2,3;Hutch-1) was expressed on almost all cell types in normal lung including leukocytes and alveolar macrophages. Only larger arteries were partially positive. This pattern of expression was also found in rejecting grafts. All interstitially infiltrating leukocytes and vascular adherent lymphocytes/leukocytes were CD44 positive. HECA452. HECA452 was found only on lymphocytes in normal lungs. In lung rejection additional expression was found on a few venous endothelia, pneumocytes and alveolar macrophages.

Discussion The patterns of distribution found for cell adhesion molecules in rejecting lung allografts reveal a number of cell-type and organ-specific aspects. These may have implications for lung transplant physiology and specific aspects in the manifestation of transplant rejection. Three main points can be remarked: (1) The broad basal expression of ICAM-1 on pneumocytes; (2) regional differences in arterial, venous and capillary cell-cell and cell-matrix adhesion molecule expression; (3) inflammatory changes of adhesion molecule expression on alveolar macrophages. 1. It was found that the cells in normal lung already express a number of leukocyte adhesion ligand molecules. It was described previously by Taylor et al831 that the expression of MHC class I and class II antigens was broadly found in normal lung pneumocytes. Major induction was found for bronchial epithelium and endothelium MHC class II in transplanted lungs with bronchiolitis obliterans. In this context it is remarkable that lung pneumocytes coexpress a variety of adhesion ligand molecules as MHC class I and II, ICAM-1, and CD44 in the normal lung. During rejection this expression was enhanced and an additional expression of VCAM-1, HECA 452 and NCAM (few cells) could be observed. It seems that the pneumocytes, either type I and type II possess a broad panel of ligand molecules enabling intensive interaction with leukocyte receptors. It is possible that this is especially required for the intense interaction with alveolar macrophages in the defense of infectious pathogens. It is not excluded, however, that interaction to other leukocytes is possible. This is contradicted by the fact that acute lung transplant rejection finds only minor manifestation in the lung alveoli, but instead at

Fig. 6.2. (a) PECAM–1 (CD31) was expressed on all endothelia and induced on alveolar macrophages during lung rejection (day 68; CD31, orig. magn. 200x). (b) CD44 was expressed on almost all resident in infiltrating cell types in lung rejection (day 68 post transplant; CD44, orig. magn. 200x). (c) VLA–6 was not expressed on endothelia of large veins and arteries in normal lung (orig. magn. 325x). (d) VLA–6 was found induced on endothelial cells of lung arteries during rejection (day 66; VLA–6, orig. magn. 200x).

b

d

a

c

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87

bronchial epithelium and perivascular at arterioli. Thus a presumed high susceptibility of pneumocytes to alloantigeneic lymphocyte interaction—even in the presence of a full panel of ligand molecules—seems not to exist. In contrast, bronchial epithelia that express a lesser panel of ligand molecules (MHC class I, LFA-3, CD44) and are only induced to express class II MHC during rejection831,646 seem to be a major target of the acute and chronic immune response leading to obliterative bronchiolitis. 2. The second observation of basal expression of ICAM-1, LFA-3, ICAM-2, PECAM on capillary, venous and arterial endothelia of the lung may also point to a physiological high reactivity and susceptibility of lung endothelia to circulating immune cells. In rejecting grafts this expression was increased, however main changes could be observed on the endothelia of larger arteries. These have differences in the normal state with a basal expression of VCAM-1 and P-selectin and a lesser expression of ICAM-1 (few) and ICAM-2 (negative). Only in the rejecting grafts a complete pattern was found to be induced, whereas arteriolar, capillary, and venous endothelia failed to express VCAM-1. Capillary endothelia failed to express VCAM-1, E- and P-selectin. This resembles the pattern found in liver graft sinusoidal lining cells.765 It seems that similar differences exist in the lung between the arterial, capillary and venous vessel compartments in the pattern of vascular adhesion ligand molecules both in the basal state of expression and in induced state with transplant inflammation. A true absence of inducibility for the early adhesion molecules VCAM-1, E- and P-selectin seems to exist in lung capillaries. Although it is not excluded, that especially a temporary or focal induction of E-selectin is possible220 and not present anymore in the explanted grafts with longstanding rejection, this possibility seems to be excluded for VCAM-1 and P-selectin, as these molecules are very well induced on arteries or veins (partly). Thus a differential pattern of ligand molecule expression and differences in susceptibility to inflammatory induction clearly exist for lung vascular endothelia. The lack of inducibility of the early adhesion molecules E-, P-selectin needed for rolling of leukocytes/thrombocytes and VCAM-1 necessary for definitive adhesion and extravasation in the capillary stream bed could explain the almost complete absence of lymphocyte infiltration in acute or chronic rejection. This mechanism could very well compensate for the high expression of other ligands as ICAM-1,-2 found on endothelia and pneumocytes and possibly facilitating leukocyte interaction in a temporary way. The lack of the initiation step, the rolling of leukocytes by selectins for leukocyte adhesion, may effect a different mode of leukocyte-endothelial interaction and prohibit the induction of further steps leading to tissue infiltration.475,787 A similar difference in adhesion receptor patterns was found for cell-matrix integrin receptors between the vascular endothelia of the lung. Whereas VLA-1, VLA-5, and CD51 were found on all endothelial cell types, the receptor VLA-2 (receptor to collagen and laminin) was basally expressed only on capillary endothelia, and the receptor VLA-6 (to laminin) only negative on the endothelia of larger arteries. Thus differences in cell-matrix interaction or possibly in the composition of the subendothelial matrix may exist between arterial, venous, and capillary endothelia. It is very well possible, that such differences either reflect cell differentiation or the local microenvironment composed of cell-matrix produced by perivascular cells. Such differences may have function in the special interaction of

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endothelial cells with the surrounding tissue cells and in their site specific anchoring to basal membrane matrix proteins as it has been demonstrated in vitro for fibroblasts.171 A second function is probably the regulation of leukocyte reactivity to basal membrane matrix components upon retraction of endothelial cells. Thus differences in the organ specific composition of cell-types possessing different properties of adhesion ligand molecule expression as well as the special composition of the perivascular and interstitial cell-matrix may influence the manifestation of the immune response by offering distinct cell- or matrix-adhesion targets. A molecular microheterogeneity of cellular ligands as ICAM-1 isoforms or CD44 may exist in addition,306,704 so that the regulation of immune responses inside an organ may underly various special restrictions as dictated by the presence of respective activating or deactivating ligand structures. 3. A further indication for inflammatory changes of the resident lung transplant cells during long-standing rejection comes from an adhesion receptor change on alveolar macrophages. Although this cell type does not seem to be affected by the rejection reaction and is physiologically replaced by recipients bone-marrow cells, inflammatory changes leading to lung pathology can be suspected, especially since the release of cytokines upon infections or rejection of the graft may be a major promoter of interstitial fibrotic change in the lung alveoli. A broad induction of adhesion molecules was found for ICAM-1, LFA-3, PECAM, VLA-2, VLA-4, and HECA-452 during lung transplant rejection. Both cell-cell as cell-matrix molecules are induced. This could be a general effect of cytokines released by lymphocytes or endothelial cells, but points to an activated state of the tissue macrophages. The chronic inflammatory upregulation of alveolar macrophages itself could be a major threat to the integrity of the lung transplant as it could maintain interstitial inflammation even in the absence of lymphocyte infiltration. This ultimately could cause increased cytokine levels leading to T lymphocyte activation and the increased processing and presentation of alloantigeneic-MHC antigens that may induce the rejection activity.

CHAPTER 7

MHC and Cell Adhesion Molecules in Clinical Liver Transplantation Gustav Steinhoff

Major Histocompatibility Complex (MHC) Molecules in Human Liver Transplants

L

iver transplantation is performed successfully across major HLA-difference between donor and recipient.605,753 This may be influenced by the organ specific expression of MHC molecules. It could determine the local immune reactivity and rejection response. The tissue expression of MHC molecules on parenchymal and infiltrating cells has been studied in transplanted human liver using monoclonal antibodies and immunohistological methods. A strong induction of class I (HLA-A,B,C; β2-microglobulin) and class II (HLA-DR,DQ,DP) MHC antigens was demonstrated on hepatocytes, bile duct epithelium and endothelial cells during rejection episodes, viral and bacterial infections. The massive induction of donor antigens on hepatocytes, bile ducts and endothelia forms part of and may also augment the rejection response. During quiescent states without infection or rejection after transplantation, a rather restricted expression of class I and class II donor MHC antigens is present. In addition, the donor Kupffer cells and interstitial dendritic cells are gradually replaced by recipient accessory cells expressing self-MHC molecules. These changes in the antigen density and distribution of donor MHC alloantigens as the replacement of accessory cells capable to present antigens to T lymphocytes may influence the course of immune reactivity and the rejection response in the liver.

Expression of Class I MHC molecules Early studies have shown that MHC molecules are not uniformly expressed on the different parenchymal components of organs.176,177,549,586 Hepatocytes of the liver were found to express no or low amounts of MHC molecules.55,434 Increased expression of α chain and β2-microglobulin class I MHC antigens was found on hepatocyte membranes in a number of pathological states and during rejection after liver transplantation.544,736,777,780,781 No major difference in the density and pattern of expression was noted between the α chain of HLA-A,B,C and β2-microglobulin.777,780 Studies employing sequential follow-up biopsies during various Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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complications after liver transplantation could show that the induction of class I MHC antigens on hepatocyte membranes occurred not only during rejection but also during various inflammatory and infectious processes.777 The induction of class I MHC antigens was reversible after cessation of the inflammation and after successful rejection treatment.780 In graft biopsies without any complication the pattern of expression resembled that of normal liver, but a part of the hepatocytes remained class I MHC antigen positive. In an experimental model of bile duct ligation in the rat Innes et al could show that not only immunological stimuli, but cholestasis alone, was able to increase the class I expression on hepatocytes.352 In addition, in a rat liver transplant model it could be demonstrated by Settaf et al706 that class I MHC antigens were induced on hepatocyte membranes not only in acute rejection but also in nonrejecting isografts. In human transplants, interestingly in the majority of biopsies taken before and during the transplant operation an induction of class I MHC antigens was found on hepatocyte membranes.706,707 This may very well be caused by toxic damage or inflammatory reactions in the donor and during hepatectomy. The de novo class I MHC antigen induction on hepatocyte membranes has been further differentiated using HLA-A and HLA-B specific antibodies directed to polymorphic epitopes.778 In patients with appropriate difference in HLA-type it could be shown that the induced antigens were donor derived.265,774 The expression and induction phenomena for both donor HLA-A and HLA-B were the same as previously found with antisera to monomorphic epitopes. Moreover, an equal induction of HLA-A and HLA-B antigens during rejection could be demonstrated in sequential biopsies of a limited number of patients.778 However, it is not known if HLA-C molecules are induced and expressed in a similar way as HLA-A and HLA-B. The production and expression of the different class I MHC molecules may very well underlie genetically determined individual differences. Furthermore, it is unclear if the regulation of the production of β2-microglobulin is regulated by the same stimuli as the class I α chain in hepatocytes or may be produced in response to other factors. Although class I MHC antigens are expressed on bile duct epithelia, endothelia, Kupffer cells and interstitial cell types in the normal human liver, an increased expression of HLA-A and HLA-B antigens can be demonstrated on bile duct epithelia during rejection and infections as cholangitis.777 Although induction phenomena can be qualitatively demonstrated on the normally negative hepatocytes, these may also quantitatively occur on other cell types as bile duct epithelia and endothelia. However, this cannot be quantified by the conventional immunohistological methods.

Expression of Class II MHC Molecules MHC class II molecules are induced during rejection, infection and inflammatory complications on parenchymal liver cells in a way similar to class I MHC molecules, but their expression is more restricted. In the normal liver HLA-DR antigens are only expressed by Kupffer cells, portal interstitial cells and a few endothelia.170,434,549,777 HLA-DQ and HLA-DP are only present on a subpopulation of Kupffer cells and portal interstitial cells.777 Bile duct epithelia and hepatocytes are negative for class II molecules as detected by immunohistological methods.170 De novo expression of HLA-DR on bile duct epithelia in rejected liver grafts has first been reported by Takacs et al820 and Demetris et al.189 The detailed analysis of the

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Table 7.1. Expression of class I and class II MHC molecules on normal liver cells Class I HLA-A,B β2-Microgl. Hepatocyte Bile duct epithelium Portal endothelium Sinusoidal endothelium Kupffer cell Portal dendritic cell

(+) ++ ++ ++ ++ ++

(+) ++ ++ ++ ++ ++

DR

Class II DP

DQ

– – – +/– ++ ++

– – – – + +

– – – – +/– +

Table 7.2. Patterns of MHC antigen expression on liver cells during different complications after transplantation Hepatocyte HLANormal state Acute Rejection Viral hepatitis Bacterial cholangitis Cholestasis

Biliary Epithelium

Sinusoidal Endothelium

A,B

DR

DP

DQ

A,B

DR

DP

DQ

A,B

DR

DP

DQ

+/– ++ ++ ++

– + + –

– – – –

– – – –

+ ++ + ++

– ++ + ++

– +/– +/– +

– – – +/–

+ ++ ++ ++

+ ++ ++ ++

– + + +/–

– +/– +/– –

+

–

–

–

++

–

–

–

++

++

–

–

HLA-DR induction on biliary epithelium in human liver grafts revealed that an induction takes place both with rejection and infectious complications (cholangitis, CMV-hepatitis)777 and that already during transplantation a few bile ducts display HLA-DR antigens.265 With strong induction of HLA-DR also HLA-DP antigens were coexpressed.777 Interestingly, in patients developing a rejection with a vanishing bile duct syndrome a strong expression of HLA-DR and HLA-DP was found on the bile duct remnants. The de novo expression of HLA-DR antigens on hepatocyte membranes was observed in liver grafts during acute rejection and CMV-hepatitis.777,781 This was found focally around infiltrates in acute rejection and at sites of inflammation in CMV-hepatitis. HLA-DP and HLA-DQ were not found to be coexpressed on the positive cells. In experimental studies in rat and rhesus monkey also the weak induction of class II MHC antigens on a limited number of hepatocyte membranes could be demonstrated.706,772 In these models the induction was related to the rejection process and reversible within 5-10 days after effective rejection treatment.772 Other authors studying isolated hepatocytes in transplant aspiration biopsies found variable numbers of HLA-DR positive hepatocytes during rejection that decreased after effective treatment.875,939 However, it is not clearly distinguishable whether the induction of HLA-DR on hepatocytes is a rejection related process initiated by

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the local release of lymphokines, or due to local viral reactivation with viral hepatitis.231,776,864 In many patients a combination of stimuli can be postulated. Studies of nontransplanted livers with Hepatitis B infection and Primary Biliary Cirrhosis were able to show a similar focal induction of HLA-DR antigens on hepatocyte membranes as in liver grafts during rejection.231,741,864 In vitro IFNγ was able to induce HLA-DR on cultured hepatocytes.231 Moreover, in liver grafts with CMVhepatitis without rejection an induction of both class I and HLA-DR antigens was observed on hepatocyte membranes.777 Thus, a clear discrimination of the immune processes causing the induction of class I and class II MHC molecules thus is not possible. Moreover, in many situations after transplantation different stimuli may exist, that cannot be distinguished by clinical data, histological and immunohistological morphology. MHC antigens are not only induced on parenchymal liver cells, but also on vascular endothelial cells of the portal vessels and the sinusoids.777 These cells are the major contact site to circulating lymphocytes and may play an important role in the elicitation and regulation of local immune responses. HLA-A,B and DR are expressed on endothelial cells in quiescent state after transplantation. HLA-DP and on a few endothelial cells HLA-DQ can be additionally found during rejection and severe inflammation.777 In liver transplants the endothelial cells remained of donor type and expressed donor class I and class II MHC antigens as detected by antibodies directed to polymorphic epitopes of donor or recipient HLA.774 In contrast, sinusoidal Kupffer cells of the donor disappeared gradually in normal postoperative courses and rapidly within a few weeks with severe rejection or infection.265,774 Thus in the patients with mismatched HLA-type a mixed cell population of endothelial cells expressing donor MHC molecules and recipient Kupffer cells exists. It is not known so far, how this expression of self and nonself MHC molecules in the liver sinusoids may interfere with allogenic and nonallogenic immune reactions. This might very well be of importance for the development and clinical course of viral hepatitis and Hepatitis B reinfection in liver transplants.

Diagnostic Aspects A diagnostic use of the assessment of MHC antigen expression in liver tissue after transplantation has been postulated by some authors. The detection of induction phenomena as the HLA-DR expression on bile ducts and hepatocytes indicates severe complications.776,777 The pattern of induction and localization may indicate the kind of the immune process (rejection vs. cholangitis/viral hepatitis) or by the lack of immune stimulation other processes as ischemia and toxic damage.776,777 However, the analysis of patterns of MHC antigens is a nonspecific parameter found during different types of immune reactions and tissue inflammation. Although the induction of HLA-DR antigens on hepatocytes in early acute rejection may correlate with the clinical and histological diagnosis,875,939 at later term it also may indicate a viral hepatitis.777 It can be used, however, to evaluate the effectiveness of immunosuppression as rejection treatment and the donor alloantigen exposure of the graft as a guide for the immunosuppression. Especially an increase of donor class II antigens on parenchymal and endothelial cells may represent a risk situation for the transplant even when histological signs of rejection are not apparent.

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The tissue interaction of the T cell receptor (TCR) and different MHC molecules in vivo during the process of antigen presentation and alloreactivity has to be investigated in more detail. The availability of appropriate reagents and more advanced immunohistological and immunogold-electronmicroscopic methods may allow to study the receptor density, T cell receptor/MHC interaction and the propagation by adhesion molecules as CD2, LFA1, ICAM-1, ICAM-2 and LFA3.205 The question arises, if a different interaction of T lymphocyte subpopulations with accessory cells of donor type or alloantigen-presenting recipient accessory cells (Kupffer cells/interstitial dendritic cells) may occur. The tissue distribution of the different accessory cell subpopulations could then determine the localization and kind of T lymphocyte reactivity. Moreover, their expression of different MHC products and adhesion molecules may determine T lymphocyte reactivity. The expression of the alloantigeneic sites of the different MHC molecules on the liver cell types may differ depending on the HLA-type and cell membrane related factors. This could influence the T lymphocyte reactivity and the alloantigenicity of the different MHC molecules. The tissue immune reactions may be characterized more clearly and effects of intervention by immunosuppressants, monoclonal antibodies and cytokines can be described. A more detailed knowledge about the induction of donor antigens and their molecular interaction with recipient T lymphocytes may then lead to a more specific immunosuppressive treatment. The binding characteristics of alloantigen specific T lymphocytes and mapping of the reactive T cell receptors by anti-idiotypic antibodies are further fields of interest. A major field of development will be in-situ DNA/RNA hybridization techniques to study the translation of the different MHC molecules during cellular induction. In vitro studies have to discriminate the effects of the different cytokines on the MHC gene transcription and translation. Also secondary and direct effects of immunosuppressive and other drugs may be characterized with a more advanced methodology. The production of β2-microglobulin as a soluble protein and of possible additional nonclassical class I and class II MHC gene products that may be produced by liver cells740 may be studied using molecular genetic methods.

HLA-Matching With recent progress in organ procurement and prolongation of preservation time, the discussion about the beneficial effect of prospective HLA-matching in the major MHC loci between donor and recipient has become actualized.788 The longterm outcome of renal and bone marrow-transplants has been widely improved by HLA-matching.562,569 Recently, with the availability of a large number of patients who received a heart or a liver transplant, retrospective analysis concerning the effect of HLA-mismatches on rejection and graft outcome has been possible. In a study of 2000 heart transplants a beneficial effect of matching of HLA-B and HLA-DR MHC antigens on graft outcome has been demonstrated.567,922 These results resemble the effects seen in renal transplants.569 In a series of 500 liver transplants, however, a different outcome of MHC class I (HLA-A,B) and MHC class II (HLA-DR) matched grafts was reported.493 The outcome of grafts matched for class II (HLA-DR) loci was inferior to those without match. This surprising effect has been intensively discussed, but a similar negative effect of HLA-DR matching as a potential cause of the vanishing bile duct syndrome has been demonstrated in

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another paper.59,193 Both enhancement of an antiviral and autoimmune or the T cell response to minor histocompatibility antigens in grafts with full HLA-DR compatibility are possible mechanisms of graft injury. This may then lead to recurrent disease, but also augment the rejection activity by the induction of donor HLA and secondary sensitization to alloantigens. Also a sensitization to noncompatible class I molecules can be mediated by compatible HLA-DR. These mechanisms can be discussed in a variety of patients with liver diseases as PBC, PSC, viral and autoimmune hepatitis. At present, the limited analysis of immunological events, patient data and the small and heterogeneous patient population studied cannot justify definite conclusions about a differential or dualistic effect of matching in class I or class II MHC loci in human liver transplants.791 Moreover, similar effects of HLA-DR matching should occur in patients with autoimmune or viral disease receiving heart, pancreas, and renal allografts. The benefit of HLA-matching and the analysis of postoperative immune events in liver grafts first have to be characterized in large scale prospective clinical studies and specific laboratory investigation.

Intercellular Adhesion Molecules in Human Liver Transplants In recent years a complete analysis of the expression patterns of cell adhesion molecules in human liver transplants in different clinical situations has become available. The tissue expression and inflammatory changes of a number of immune adhesion molecules as ICAM-1, LFA-3, VCAM-1 and ELAM-1 have been studied before, in part, in human liver disease and after liver transplantation.6,65,696,759-761,769,770,876,877,879,880 Endothelial cells play a central role in the regulation of leukocyte adhesion and extravasation. In liver allografts endothelial cells remain of donor-type in the long-term course after transplantation. Thus they remain the main target and regulatory cell of the allogeneic immune response. A stream bed specific difference in adhesion ligand expression has been described on different endothelial cell types in liver allografts.764,773 Moreover, cell type specific changes in integrin receptor and selectin ligand expression may have relevance to both immune reactivity and cellular functions in the transplanted liver. An overview on expression patterns of leukocyte adhesion receptor and ligand molecules is given in human liver grafts. Included are immunohistological studies of follow-up liver transplant biopsies and explanted grafts of 36 patients with normal courses or different rejection and nonrejection related pathological conditions. The expression of T cell receptor related molecules for antigen recognition as TCR, CD3, CD4 and CD8 is not included. The expression patterns of the ligand molecules, MHC class I and class II,777,780,781,790 and that of ICAM-16,760,761 and LFA365,765 in human liver grafts have been previously described in part. A summary of the pattern of basic expression of the different adhesion molecules in normal human liver and their inducibility on the liver cell types as the presence on infiltrating leukocytes is given in Table 7.3. Table 7.4 gives a summary of characteristic patterns for different acute and chronic types of liver transplant inflammation on resident liver cells. The receptor pattern on intravascular adherent or tissue infiltrating leukocytes is depicted in Table 7.5.

Fig. 7.1. Expression of donorand recipient-MHC in human liver transplants—exchange of Kupffer cells. (a) Expression of class I MHC molecules (HLA-A,B,C) during severe transplant rejection. MHC class I is induced on all cell types (LT 140, day 55, MCA W6/32: monomorphic epitope). (b) Expression of donor type class I MHC molecules in the same biopsy. a Recipient cell infiltrates in the b portal field are not stained. Endothelia, bile duct epithelium and hepatocytes are positive; also a few dendritic cells in the portal tract are of donor type (LT 140, day 55, MCA 2BC4: HLA-B7 polymorphic epitope). (c) Parallel section stained for recipient MHC class I molecules: Portal infiltrating cells are stained for recipient HLA as well as the majority of Kupffer cells, thus exchanged d c by recipient macrophages (LT 140, day 55; MCA 48C1:HLAA28: polymorphic epitope). (d) Expression of MHC class II in liver transplant rejection: Massive positivity of graft infiltrating cells in the portal tract. Coinduction of HLA-DR on bile duct epithelia (LT 140, day 55; MCA L243: monomorphic epitope of HLA-DR).

MHC and Cell Adhesion Molecules in Clinical Liver Transplantation 95

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Immunoglobulin Supergene Family CD2. In human liver grafts CD2 was expressed on intrasinusoidal vessel adherent and portal infiltrating lymphocytes. In addition, a few portal interstitial cells were found CD2 positive in rejection and chronic inflammatory liver disease. LFA-3. LFA-3 (CD58) is the ligand molecule to CD2 and may specifically facilitate the interaction of CD2+ T lymphocytes. Two isoforms exist that show differences in the transmembrane part of the molecule.746 In normal liver LFA-3 is expressed on sinusoidal endothelia and Kupffer cells. With acute rejection the expression is upregulated on sinusoidal endothelia and induced on portal endothelia, bile ducts and hepatocytes. De novo expression of LFA-3 on hepatocytes and bile duct epithelia was found focally in Rappaport zones I and III in also in chronic rejection and viral hepatitis. Kupffer cells, interstitial dendritic cells and infiltrating lymphocytes were strongly LFA-3 positive in all inflammatory complications. ICAM-1. ICAM-1 (CD54) is the ligand molecule to the LFA-1 receptor on different types of leukocytes facilitating various types of leukocyte adhesion including T lymphocyte antigen recognition. In contrast to other organs ICAM-1 is already basally expressed in the liver sinusoids on endothelia and a part of Kupffer cells. With transplant rejection the expression of ICAM-1 is strongly upregulated and additional cell types as hepatocytes, bile ducts, portal and central vein endothelia become positive for ICAM-1. However, also infiltrating lymphocytes and interstitial dendritic cells in the portal tract display strong ICAM-1 membrane expression. In and around infiltrates the induction of ICAM-1 is more pronounced both on infiltrating and resident cells; the inflammatory upregulation on sinusoidal endothelia and hepatocytes, however, is generalized and independent from local infiltrates in cases of prolonged acute and chronic inflammation. ICAM-2. ICAM-2 is the second ligand molecule for the LFA-1 receptor. Its expression is more or less restricted to endothelial cells and present on most endothelial cell types.181 In the human liver a strong basal expression is present on portal arterioles and—more weakly—on sinusoidal endothelia. During transplant rejection and other types of liver inflammation this expression undergoes no major changes. Additional expression on portal vein endothelia, portal interstitial cells and a few Kupffer cells was, however, found in chronic rejection and prolonged inflammation. VCAM-1. VCAM-1 (INCAM110), vascular-cell-adhesion-molecule 1, was in normal liver basally expressed on a part of the Kupffer cells, portal interstitial dendritic cells and very few portal vein endothelia. Inflammatory induced expression of this molecule was found mainly on portal (arterial and venous) and central vein endothelia. This was found in early acute rejection and infections as cholangitis, sepsis, viral hepatitis. The VCAM-1 expression on Kupffer cells and portal interstitial dendritic cells also was found increased under these conditions. In advanced inflammation as irreversible and chronic rejection the sinusoidal endothelia became in part (Rappaport zone I) VCAM-1 positive. NCAM. NCAM (CD56), the neural-cell-adhesion molecule, was basally expressed on nerve fibers in the portal tract and weakly on a few bile duct epithelia. Natural killer cells in normal liver parenchyma and in rejection or nonrejection related infiltrates expressed NCAM, known as HNK-1 antigen on NK-cells. In normal human liver NCAM was only present on a few NK-cells and nerve tissue. With acute and chronic rejection as with cholangitis NCAM was induced on bile duct

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epithelia and regenerating bile ducts. This was not observed with early inflammation as in organ reperfusion. A weak induction of NCAM was observed on sinusoidal endothelia in several types of transplant inflammation. CD31. CD31 (PECAM, CD13), is a platelet-endothelial-cell adhesion molecule found on the surface of platelets, some leukocytes and at endothelial intercellular junctions in cell culture and is involved in thrombocyte/leukocyte adhesion to endothelia. In normal liver it was basally present on all endothelial cell types (PA,PV,SE,CV), on Kupffer cells, some intravascular lymphocytes and interstitial cells. During transplant rejection the endothelial expression of CD31 was upregulated. CD31+ lymphocytes were found intravascular and in portal infiltrates together with CD31+ portal interstitial cells. Hepatocytes were found focally CD31 positive in two patients with advanced chronic rejection.

Integrin Family LFA-1. LFA-1 (CD11a) was expressed in normal liver on adhering leukocytes in the sinusoids, a few portal interstitial cells and Kupffer cells. Shortly after transplant reperfusion the number of LFA-1-positive leukocytes (neutrophils and Kupffer cells) in the sinusoids was strongly increased. Increased numbers of LFA-1 positive leukocytes were found in moderate acute rejection and infectious complications after transplantation. They were mainly located in portal tract and central vein infiltrates. In these areas ICAM-1 was also induced on parenchymal cells and lymphocytes. In severe acute and chronic rejection as in bacterial infections or sepsis also the LFA-1 expression on Kupffer cells was strongly increased. In portal tracts not only the majority of the infiltrating leukocytes expressed LFA-1, but this molecule was also strongly expressed by portal interstitial dendritic cells. CR3, Mac-1 and p150/95. CR3, Mac-1 (CD11b/18) and p150/95 (CD11c/18), macrophage/monocyte adhesion molecules, both showed a similar expression on macrophages and Kupffer cells in liver grafts. They were basally expressed on Kupffer cells. The expression of both molecules was upregulated on Kupffer cells with acute rejection, viral hepatitis, and bacterial infection. Interstitial dendritic cells and infiltrating monocytes/macrophages in portal tracts and central vein areas displayed the expression of both adhesion receptor molecules. Vascular adherent and portal infiltrating polymorphonuclear neutrophils expressed Mac-1 (CD11b/18). VLA-1. VLA-1, a cell-matrix receptor for collagen and laminin, was in normal human liver broadly expressed by all endothelia, portal interstitial cells, Kupffer cells and hepatocytes. Induced expression on these cell types was found in rejection and other types of liver transplant inflammation. VLA-1 positive portal infiltrating lymphocytes were only found in advanced or chronic rejection. VLA-2. VLA-2, a cell-matrix receptor for collagen, was in normal and inflamed liver expressed strongly present on bile duct epithelia, weakly on portal artery/ vein and a few Kupffer cells. Focal expression on hepatocytes was found only in acute rejection, however no expression on infiltrating and vessel adherent leukocytes. VLA-3. VLA-3 (ECMRI, CD49c/CD29), a cell-matrix receptor for fibronectin, laminin and collagen, was in normal liver expressed on portal artery and vein endothelia as well as bile duct epithelia. Weak expression was present on sinusoidal endothelium and interstitial dendritic cells. The expression on bile ducts, portal arterial and venous endothelium was strongly upregulated during rejection and

PA,PV,SE,CV

PA,PV,SE,CV (PA,PV) PA,PV,(SE) (SE) PA,PV,SE,CV PA,PV PA,PV,SE

PV PV, (PA)

LFA-1 Mac-1,CR3 p150/95 VLA-1 VLA-2 VLA-3 VLA-4 VLA-5 VLA-6 CD51

LECAM-1,Leu8 ELAM-1 CD62

SE SE PA,SE (PV)

CD2 LFA-3 ICAM-1 ICAM-2 VCAM-1 NCAM CD31(PECAM)

Normal State Endothelia

Hep BD (BD)

Hep BD BD

NT

Epithelia ao

(Ly1)

(IDC) Ly,IDC,KC, IDC,KC

Ly,PMN,IDC,KC IDC,KC,PMN IDC,KC (PMN) IDC

Ly KC KC IDC KC Ly/NK KC,Ly

Leukocytes

PV,PA,CV PV,PA,CV

PA,PV,SE,CV PA,PV,CV PA,PV,SE SE PA,PV,SE,CV PA,PV,SE PA,PV,SE,CV

PA,PV,CV,SE PA,PV,CV,SE PA,PV,SE PA,PV,SE,CV SE PA,PV,SE,CV

Hep BD,Hep Hep,BD

Hep BD,(Hep) BD,(Hep)

Ly,PMN,IDC,KC

(Hep) BD,NT (Hep)

Hep,BD Hep,BD

Transplant Inflammation/Rejection Endothelia Epithelia ao

Table 7.3. Expression of adhesion ligand molecules in normal and inflamed human liver transplants

Ly (Ly) (Thr)

IDC,KC/Mo,PMN IDC,KC/Mo,(PMN) IDC,KC (KC) (IDC,KC) Ly,IDC,KC IDC,KC,Ly/PMN (IDC,KC,CV) IDC,KC,Ly/PMN

Ly,(IDC) Ly,KC,IDC Ly,KC IDC,KC KC,IDC Ly/NK KC,IDC,Ly

Leukocytes

98 Cell Adhesion Molecules in Organ Transplantation

Hep,(BD)

Ly,Mo PMN, KC,IDC PV,SE

(PV,SE,CV) KC,IDC Hep,BD Ly

Ly,Mo,PMN,

Portal vein

Endothelial Cells Portal artery

Cell Type

CD62, ELAM-1

VLA-1,2,3,5,6 CD51

VLA-1,2,3,5,6 CD51

VLA-1,2,3,5,6

VLA-1,3,5,6

(ELAM-1)

ICAM-2,CD62 ELAM-1

Organ Reperfusion

ICAM-2

Normal Liver

CD62,ELAM-1 ICAM-1,VCAM-1 LFA-3 VLA-1,2,3,5,6 CD51

ICAM-2,CD62, VCAM-1,ELAM-1 ICAM-1,LFA-3 VLA-1,2,3,5,6

Acute Rejection

CD62,ELAM-1 ICAM-1,VCAM-1 LFA-3 VLA-1,2,3,5,6 CD51

ICAM-2,CD62 VCAM-1,ELAM-1 ICAM-1,LFA-3 VLA-1,2,3,5,6

Irreversible and Chronic Rejection

Table continues on next page.

CD62,ELAM-1 ICAM-1,VCAM-1 LFA-3 VLA-1,2,3,5,6 CD51

ICAM-2,CD62 VCAM-1,ELAM-1 ICAM-1,LFA-3 VLA-1,2,3,5,6

Sepsis, Cholangitis, Viral Infection

Table 7.4. Patterns of adhesion molecule expression on resident liver cells in acute and chronic transplant inflammation

Abbreviations: Liver cell types: PA, portal arterial endothelium; PV, portal vein endothelium, SE, sinusoidal endothelium, CV, central vein endothelium; IDC, portal interstitial dendritic cells; KC, Kupffer cells; BD, bile duct epithelium; Hep, Hepatocytes; NT, nerve tissue. Infiltrating cells: Ly, lymphocytes; Mo, infiltrating monocytes/macrophages; NK, natural killer cells; Thr, thrombocytes; PMN, polymorphonuclear leukocytes. Explanations: The patterns of rejection and nonrejection (cholangitis, viral infection: CMV,EBV, bacterial sepsis) related types of transplant inflammation are comprehended when giving a uniform pattern. The induced pattern is shown in the section “Transplant inflammation/rejection”. 1 only found in organ reperfusion.

HECA452

CD44

MHC and Cell Adhesion Molecules in Clinical Liver Transplantation 99

VLA-1,5

VLA-2,3,6

Central vein

Epithelial Cells Bile duct

VLA-1,5 (HECA452)

ICAM-1,ICAM-2 LFA-3 CD-4,CD51 VLA-1,(3,4),5

Sinusoidal endothelium

Hepatocytes

Normal Liver

Cell Type

Table 7.4. (Continued)

VLA-1,5 ICAM-1 (HECA452)

VLA-2,3,6

VLA-1,5 (CD62)

ICAM-1,ICAM-2 LFA-3 CD-4,CD51 VLA-1,(3,4),5

Organ Reperfusion

VLA-1,2,3,5,6 ICAM-1,LFA-3 CD-51,HECA452

VLA-2,3,6 ICAM-1,NCAM LFA-3,CD51

VLA-1,5 CD62,ELAM-1 (VCAM-1,LFA-3)

ICAM-1,ICAM-2 LFA-3,(VCAM-1) CD-4,CD51,CD44 VLA-1,3,4,5,6

Acute Rejection

VLA-1,5,6;HECA452 ICAM-1,LFA-3 (CD-51,CD31,VCAM-1)

VLA-2,3,6 ICAM-1,NCAM LFA-3,CD51

VLA-1,2,5 CD62,ELAM-1 VCAM-1,LFA-3 ICAM-1

ICAM-1,ICAM-2 LFA-3,VCAM-1,NCAM CD-4,CD51,CD44 VLA-1,3,4,5,6

Irreversible and Chronic Rejection

VLA-1,5,(6) ICAM-1;HECA452 (CD-51)

VLA-2,3,6 ICAM-1,NCAM LFA-3,CD51

VLA-1,5 CD62,ELAM-1 VCAM-1,LFA-3 ICAM-1

ICAM-1,ICAM-2 LFA-3,VCAM-1 CD-4,CD51,CD44 VLA-1,3,4,5,6

Sepsis, Cholangitis, Viral Infection

100 Cell Adhesion Molecules in Organ Transplantation

Fig. 7.2. (a) Induction of ICAM-1 on hepatocyte membranes after liver transplantation (hepatitis B reinfection, chronic transplant rejection). Sinusoidal endothelia are generalized ICAM-1 positive, whereas portal endothelia display no or weak expression (OLT 172, day 403 post transplantation; ICAM-1; orig. magn. 200x). (b) In the same biopsy ICAM-2 molecules are expressed on all endothelia and Kupffer cells. The expression on portal artery is particularly strong (OLT 172, day 403; ICAM-2; orig. magn. 200x). (c) ELAM-1 molecules are induced only on portal endothelia during chronic transplant rejection (OLT 296, day 110; ELAM-1;orig. magn. 325x). (d) CD44 molecules are expressed on almost all endothelia and infiltrating leukocytes/Kupffer cells in acute allograft rejection (OLT140, d.55; orig. magn. 200x).

b

d

a

c

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other types of inflammation. In biopsies taken during acute rejection also hepatocytes become VLA-3 positive in part. No VLA-3 positive intravascular and portal infiltrating leukocytes could be detected. VLA-4. VLA-4 (LPAM-1, CD49d/CD29): In normal human liver VLA-4 was basally expressed on Kupffer cells and few lymphocytes. During rejection and other types of liver transplant inflammation vessel adherent and almost all portal infiltrating leukocytes expressed the VLA-4 molecule. The VLA-4 expression on Kupffer cells was upregulated in all types of liver inflammation. In severe inflammation a few sinusoidal endothelia became VLA-4 positive. VLA-5. VLA-5, a cell-matrix receptor molecule binding to fibronectin, was in normal human liver expressed on all endothelia, some hepatocytes, Kupffer cells/ interstitial cells, but not on bile duct epithelia. In liver graft rejection VLA-5 was induced mainly on endothelia and Kupffer cells and additionally on some bile duct epithelia. In portal infiltrates a part of the lymphocytes also was found VLA-5 positive. Only few intravascular VLA-5 positive leukocytes were found in acute rejection. In portal infiltrates interstitial dendritic cells were in part positive for VLA-5 in all types of liver inflammation. VLA-6. VLA-6, a cell-matrix receptor molecule binding to laminin, was in normal human liver only positive at portal endothelia (PA and PV) and the basal membrane of bile ducts. In liver rejection and other types of inflammation the molecule was induced on hepatocytes, bile duct epithelia, Kupffer cells/sinusoidal endothelia. No expression was present on intravascular leukocytes and graft infiltrating cells. CD51. CD51, the α chain of receptors to fibronectin, vitronectin, von Willebrand factor, and thrombospondin, was in normal liver only expressed on portal (PA and PV) and sinusoidal endothelia. A broad induction on liver cells and graft infiltrating cells was found during graft rejection and other types of transplant inflammation.

Selectin Family LECAM-1. LECAM-1 (Lam-1,Leu8,Mel-14), the L-selectin, was in normal human liver only expressed on a few intravascular lymphocytes. With acute and chronic rejection a few LECAM-1+ lymphocytes were present intravascularly mainly in portal vessels and very few in the sinusoids. A large number of LECAM-1 positive lymphocytes were present in interstitial infiltrates of the portal tract. In grafts with cholangitis or initial nonfunction only a few LECAM-1 positive lymphocytes or PMN were present intravascularly or in portal infiltrates. ELAM-1. ELAM-1, the E-selectin, was in normal liver only weakly expressed on a few portal vein endothelia. The molecule was found induced during graft reperfusion and in early acute rejection on portal arterial, venous and central venous endothelia. Sinusoidal cells were not found ELAM-1 positive in acute or chronic rejection, cholangitis, viral infection and sepsis. Additionally, in acute rejection a few ELAM-1+ lymphocytes were detected in portal infiltrates. CD62. CD62 (GMP140, PADGEM), the P-selectin, is basally only weakly expressed in portal vein endothelia. The expression was upregulated already during organ reperfusion on portal vein and arterial endothelia. In these biopsies showing early portal endothelial activation intravascular CD62 positive thrombocytes were found intravascularly both in the portal and sinusoidal vessels. In all types of

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rejection and nonrejection related liver inflammation CD62 was induced only on portal venous and arterial endothelium and on central veins. Sinusoidal lining cells remained negative under all acute and chronic inflammatory conditions studied in liver transplants.

Unclassified Adhesion Molecules CD44 (Pgp-1; EDM-III; p80; Hermes1,2,3; Hutch-1) was in normal liver expressed on all leukocytes (IDC, KC, Ly, Mo) and a few sinusoidal endothelia. All infiltrating and vessel adherent leukocytes (Ly, PMN, Mo) expressed strongly CD44 in the different types of liver inflammation and rejection. In acute and chronic rejection portal vein, central vein, and sinusoidal endothelia and a few bile duct epithelia were induced to express CD44. No difference in staining pattern of the three antibodies used was found.

HECA452 In normal liver HECA452 was expressed weakly on hepatocyte membranes and a few bile ducts. With acute and chronic rejection as well as cholangitis/viral infection the expression on hepatocytes was upregulated in hepatocytes. Bile duct positivity remains unchanged. In addition a few HECA452 positive portal vein endothelia were found in acute rejection and cholangitis. A few intravascular and tissue infiltrating lymphocytes expressed HECA452 in acute rejection.

Discussion The inflammatory response exerted by immune cells and liver cells to various pathological stimuli is regulated by a network of cytokines and intercellular interactions. Currently it can be assumed that the generation and regulation of the intercellular reactions is regulated by the specific interaction of a family of cell membrane adhesion molecules that have been identified and classified in the recent time.746 Immunohistological studies on normal and pathological liver tissue have revealed, in part, information about the tissue specific expression of these molecules on resident liver cells and infiltrating immune cells.6,65,760,769,877,880 The present study gives a complete survey about the distribution of the currently known adhesion molecules in human liver transplants. The following discussion will consider organ specific factors in the regulation of adhesion molecule expression and relevant factors for the specific pathology of liver transplants. These have implications for intravascular leukocyte homing and selection, lymphocyte antigen recognition and target cell cytolysis.

Intravascular/Sinusoidal Adhesion Molecule Expression Sinusoidal endothelial cells display a specific pattern of adhesion molecule expression both in a normal and inflammatory state of human liver transplants. A large number of adhesion ligand molecules is basally expressed. The expression of certain molecules such as E-selectin, P-selectin, and VCAM-1, however, is neither basally present nor inducible in most transplant pathologies. The question arises of whether this specific pattern of adhesion molecule expression in the liver sinusoid may have functional consequences for the leukocyte-endothelial cell interaction. In transplant rejection the liver sinusoid is mainly spared from lymphocyte infiltrates. Its huge intravascular surface of donor endothelial cells and—before

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exchange—donor Kupffer cells should be regarded a prime target for reactive lymphocytes and antibodies by the expression of incompatible MHC antigens.774 It is likely that the lack of expression or inducibility of the early adhesion molecules Eand P-selectin as well as VCAM-1 may influence the reactivity of sinusoidal endothelia with recipient lymphocytes. This may eliminate an early cascade step required for the initiation of definitive leukocyte adhesion.125,126 Moreover, this may reflect a specific constellation of the hepatic sinusoid caused by the cell differentiation of sinusoidal lining cells and a specific local cytokine milieu. Not only the transplant alloreaction but also other types of antigen-specific T lymphocyte dependent immune reactions may underlie specific restrictions in the liver sinusoid. In vitro studies have suggested a low antigen presenting capacity of sinusoidal lining cells.661 This may be caused by the specific adhesion molecule constellation or other suppressive factors. As a consequence it can be postulated that even the persistence of hepatic viruses or tumor cells in this organ compartment can be facilitated by specific local mechanisms.

Intercellular and Cell-Matrix Adhesion Receptor Molecules on Graft Infiltrating Cells The distribution of the different leukocyte adhesion receptors on intravascular adherent cells and in tissue infiltrates allows to discriminate their sequential expression and to classify their possible contribution to different steps of inflammation. Regional differences between portal tract and sinusoid were noted for the intravascular expression of the early leukocyte adhesion molecule L-selectin (LECAM-1) similar to the E- and P-selectin. Even with strong inflammation few Lselectin positive lymphocytes were present in the sinusoid, whereas an intravascular and perivascular accumulation of positive lymphocytes was found in the portal tract. A possible explanation may be the early positivity of portal vessels for the E- and P-selectin ligand structures in inflammation as organ reperfusion may induce the rolling process of L-selectin leukocytes already in the afferent vessel compartment of the liver. This process may cause their activation and by that the shedding of the L-selectin.393 Intrasinusoidal lymphocytes could therefore be L-selectin negative, but proceed to a state of intravascular activation with increased adhesive potential. This could include conformational changes in LFA-1 or MAC-1 (CD11b)

Fig. 7.3. (opposite) (a) Tissue staining of VLA-1 in an irreversibly rejected liver graft (OLT 211, day 27; cryostat section, MCA TS2/7, POD-staining, orig. magn. 240x). All hepatocytes, sinusoidal and portal endothelia (PA,PV) are VLA-1 positive, bile ducts (BD) negative. A part of the periportally infiltrating lymphocytes displays VLA-1 expression. (b) In the same graft VLA-2 (MCA Gi9, POD, orig. magn. 240x)) is strongly positive on all bile duct epithelia and a few portal interstitial cells. Kupffer cells, hepatocytes, and endothelia are not induced. (c) VLA-4 is expressed on endothelial adherent and marginating leukocytes (L), interstitial and periportally infiltrating mononuclear leukocytes (arrow) and sinusoidal lining cells (OLT 211, day 27; MCA HP2/1, POD, orig. magn. 325x). (d) CD44 expression in a chronic rejecting graft (OLT 380, day 86; SBU 24-32, AP, orig. magn. 240x). CD44 is expressed on intravascular adherent (arrow), periportal infiltrating lymphocytes and monocytes, Kupffer cells, and pericentralvenous mononuclear infiltrates.

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a

b

c

d

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Abbreviations: IDC, portal interstitial dendritic cells; KC, Kupffer cells; Ly, lymphocytes; Mo, infiltrating monocytes/macrophages; NK, natural killer cells; Thr, thrombocytes; PMN, polymorphonuclear leukocytes. Explanations: “intravascular” refers to endothelial adherent leukocytes. “interstitial” refers to portal infiltrating leukocytes. 1 bone marrow derived leukocytes including Kupffer cells and portal interstitial dendritic cells; 2 cholangitis, sepsis, viral infection; 3 no expression found; 4 only found in late/advanced rejection.

Normal Ly KC KC KC KC Ly/NK KC,(Ly) Ly,KC KC KC –3 – – Ly – – – (Ly) – – KC –

Molecule

CD2 LFA-3 ICAM-1 ICAM-2 VCAM-1 NCAM CD-31(PECAM)

LFA-1 CD11b CD11c VLA-1 VLA–2 VLA-3 VLA-4 VLA-5 VLA-6 CD51

LECAM/Leu8 ELAM-1 CD62

CD44

HECA452

Ly,Mo PMN,KC (Ly)

Ly – Thr,(Mo)–

Ly,KC,PMN KC,Mo KC,Mo – – – Ly,Mo,KC – – KC

Ly Ly,KC Ly,KC KC KC Ly/NK KC,Ly

Rejection

Intravascular

Adhesion

Ly,Mo PMN,KC (Ly)

(Ly) – –

Ly,KC,PMN KC,Mo KC,Mo – – – Ly,Mo,KC – – KC

Ly Ly,KC Ly,KC KC KC Ly/NK KC,Ly

Others2

–

KC

– – –

Ly,IDC IDC IDC – – – Ly,(IDC) – – –

Ly IDC IDC IDC IDC Ly/NK –

Normal

Ly,Mo PMN,KC Ly

Ly (Ly) (Mo)

Ly,PMN,IDC IDC,Mo IDC,Mo Ly4 – – Ly,IDC Ly,(IDC) – Ly,IDC

Ly,(IDC) Ly,IDC Ly,IDC IDC IDC Ly/NK IDC,(Ly)

Rejection

Tissue Infiltrate

Ly,Mo PMN,KC

(Ly) – –

Ly,PMN,IDC IDC,Mo IDC,Mo – – – Ly,IDC (Ly,IDC) – IDC

Ly Ly,IDC Ly,IDC IDC IDC Ly/NK IDC

Others

Table 7.5. Distribution of leukocyte adhesion receptor expression in human liver grafts on leukocytes1

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receptors585 leading to affinity binding to ICAM-1 and ICAM-2 ligands that are continuously present on sinusoidal endothelia. The definitive binding of leukocytes to endothelial cells, however, may depend on a two (or more) receptor interaction. It can be postulated that despite the presence of LFA-1/ICAM-1 the lack of additional ligand structures as E-selectin, P-selectin, and VCAM-1 in the sinusoid may prohibit the definitive firm adhesion of leukocytes (except Kupffer cells) at sinusoidal endothelia, but favor a process a reversible adhesion. The induction of E-selectin and VCAM-1 as reported to occur during viral Hepatitis B infection on sinusoidal endothelia880 may therefore break through this specific constellation and induce sinusoidal inflammation and infiltration. It is possible that the inducibility of E-selectin and VCAM-1 on sinusoidal endothelia may depend either on the presence of high cytokine concentrations or may be induced by the virus itself. This was not found for transplant pathology related to rejection or bacterial infection. It is not excluded, however, that the post-transplant immunosuppression given may decrease the full pattern of inducible adhesion molecules in the graft. The partial inducibility of VCAM-1 on sinusoidal endothelia in Rappaport zone I with advanced rejection may point in this direction. It is remarkable that all intravascular adherent leukocytes expressed LFA-1/ MAC-1, CD44 and VLA-4. Furthermore, these molecules were also expressed on all portal tissue infiltrating cells. The broad positivity of these molecules cannot distinguish fine differences in affinity by receptor re-conformation or glycosylation,306,382 but points to their central role in the processes of irreversible firm adhesion at endothelial cells, transmigration and tissue infiltration. Especially the VLA-4 receptor molecule with different functional binding sites to matrix and cellmembrane molecules can be suspected to play a key role in the process of transendothelial and tissue migration.359,693 In this context it is remarkable that additional integrin cell-matrix receptors to VLA-4 were expressed in portal infiltrates. VLA-1, VLA-5 and CD51 were present on a part of portal infiltrating lymphocytes in advanced inflammation as late rejection. This finding points to functional role of these receptor molecules on leukocytes.713 The integrin receptor expression on leukocyte subpopulations can give a molecular explanation for special infiltration patterns of the portal tract. In portal infiltrates of acute liver transplant rejection an accumulation of eosinophils, but not neutrophils, with mononuclear infiltrates has been an unexplained observation. The expression of VLA-4 receptors on eosinophils, basophils, monocytes and lymphocytes, but not on neutrophil leukocyte subpopulations94 may explain the specific composition of portal interstitial infiltrates. Firstly, the lack of considerable numbers of neutrophils in rejection infiltrates points to the special importance of the VLA-4 receptor molecule for the process of perivascular extravasation and tissue infiltration. This has already been demonstrated in blocking studies using an anti VLA-4 monoclonal antibody.359 A second argument for the special importance of VLA-4 for T-lymphocyte, monocyte, and eosinophilic tissue infiltration in the portal tract is the preferential endothelial induction of its ligand molecule VCAM-1 on portal arterial/venous endothelia in contrast to the sinusoidal endothelia. In vitro studies have demonstrated the importance of the VLA-4/ VCAM-1 pathway for intravascular T lymphocyte adhesion to activated endothelia, but point to a main importance of LFA-1/ICAM-1 for the process of transendothelial migration.570 It is likely that the VLA-4 molecule is functioning

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sequentially as a cell matrix receptor in binding to subendothelial matrix fibronectin and other interstitial matrix proteins. The central role of the LFA-1/Mac-1 and VLA-4 receptors, however, for the margination and infiltration process of leukocytes is supported by their immunohistological expression both in portal rejection infiltrates as intravascular adherent to portal vessel endothelia.

Cytokines and Adhesion Molecule Induction The sequence of adhesion molecule induction during the stepwise inflammation of the liver graft is most likely dictated by the presence of cytokines.492 The initializing “rolling” process in the interaction of leukocytes to endothelial cells is selectin dependent. The induction of P-selectin is regulated by the cytokines histamin, thrombin, and leukotriene C4 (LTC4). Within a few hours the expression of E-selectin and ICAM-1 are strongly upregulated by the cytokines TNFα and IL-1β, which are produced by Kupffer cells, monocytes, and ITO-cells.611,612 In the late phase of adhesion (12-48 hr) ICAM-1 may also be induced by IFNγ. The VCAM-1 expression is upregulated by interleukin 4 and IFNγ. IL4 and IFNγ are derived from T lymphocytes. The stimulation of adhesion molecule expression on endothelial cells attracts circulating leukocytes to the liver. In the extravascular compartment cytokines activate cytotoxic effector cells resulting in tissue destruction. During all phases of inflammation the leukocyte extravasation and transmigration is regulated by the induction of PECAM-1 (CD51) and VCAM-1 by IL-8 and PAF. The comparative analysis of the distribution of cytokines and adhesion molecules in human liver grafts could demonstrate that local differences in ICAM-1 expression on endothelial cells relate to the local concentration of cytokines. The increased TNFα and IL-1 levels and their known fibrogenetic potential suggest a causal relationship to the progressive hepatic fibrosis in chronic graft dysfunction. In liver biopsies during an uncomplicated course after liver transplantation only a few cytokine positive cells can be observed. During and even 1-2 days prior to acute rejection episodes, TNFα, ILA-1 and IFNγ have been detected by increased plasma levels. Therefore a number of pro-inflammatory cytokines may be involved in the rejection reaction.333 Integrin receptors are upregulated by cytokines. By this mechanism cytokines take part in the regulation of tissue inflammation, cellular activation and fibrogenesis.

Fig. 7.4. (a) LECAM-1 (L-selectin) positive leukocytes accumulate in the portal area during rejection. Only few LECAM-1+ cells are present in the sinusoids (OLT 211, day 27; MCA Leu8, POD, orig. magn. 240x). (b) The P-selectin (CD62) ligand expression in the same graft is also restricted to portal endothelia. In the sinusoids only thrombocytes are CD62+ (OLT 211, day 27, POD, MCA CBL thromb.6, orig. magn. 325x). (c) NCAM (CD56) was found to be de novo inducible on sinusoidal endothelia in late rejection (OLT 211, day 27, POD, MCA T199, orig. magn. 240x). (d) HECA452-antigen expression on hepatocytes, bile ducts and few portal vein endothelia during rejection (OLT 211, day 27, POD, MCA HECA452, orig. magn. 240x).

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b

c

d

109

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Liver Cell Adhesion Molecule Expression The distribution of cell-matrix adhesion receptor molecules in the subendothelial and interstitial matrix as on parenchymal liver cells is of particular interest for the pathology of liver transplants and of other types of inflammatory liver disease. The basal composition and upregulated synthesis of cell matrix molecules produced by myofibroblasts (Ito-cells) and other liver cell types as hepatocytes in response to cytokines has been intensively studied in vitro.86,235 The present analysis demonstrates a differential expression and induction of the cell-matrix receptor molecules VLA-1 through 6 and CD51 on resident liver cells reflecting cell type specific cell-matrix interactions and inflammatory changes. Portal interstitial cells, portal and sinusoidal endothelia as well as Kupffer cells show a broad positivity and inducibility for most integrin cell-matrix receptor molecules. Bile duct epithelia and hepatocytes, however, show a distinct basal expression of VLA-2,3,6 and VLA-1,5 respectively. With transplant rejection and also infectious inflammation the expression of VLA-1 and VLA-5 on hepatocytes is strongly upregulated and the additional receptor molecules VLA-2,6 and CD51 are partially induced. It is presently unclear which role the differential induction of cell-matrix receptor molecules on epithelial cells may play for the inflammatory process. It can be supposed that these cell types may exert bystander reactions in response to local inflammatory cytokine stimulants, but on the other hand they may also actively take part in processes as fibrogenesis and cell proliferation. The different pattern and inducibility of integrin receptor expression on bile ducts and hepatocytes may reflect their cell differentiation and local adherence to specific matrix-molecules, but also different cytokine milieus. The question arises, if fibrogenetic inflammatory processes as the portal fibrosis during chronic rejection are related to the induced expression of cell-matrix receptor molecules. The broad expression of integrin receptors on portal interstitial cells (including myofibroblasts), portal and sinusoidal endothelia as Kupffer cells may point to an activated state of these cell types. Further studies have to reveal, if the pattern of induced integrin receptors relates to the local produced matrix proteins as collagen, fibronectin, laminin, and vitronectin. It seems likely that the effects of cytokines on the expression of integrin receptors, the activation of interstitial cells and Ito-cells as well as on the production of matrix molecules form a circuit of inflammatory fibrogenesis. Not only the induction of cell-matrix interactions on graft infiltrating cells and the local reaction of resident macrophages and Ito-cells seems to be of relevance for transplant pathology, but also the major changes in cell-cell adhesion ligand molecule expression on parenchymal liver cells. Bile duct epithelia are of particular interest because of a wide pattern of basally expressed and inducible cell-cell adhesion molecules. This may reflect general immune-interactive functions of gut epithelial cells, but on the other hand may also relate to specific properties of this cell type. The expression of NCAM (CD56) on bile duct epithelia and especially on regenerating bile ducts in acute inflammation is particularly remarkable. It is possible that it facilitates the homotypic ligand-interaction of immune cells as NK-cells bearing this receptor molecule. A similar mechanism may also apply to sinusoidal endothelia that become NCAM positive with advanced rejection. The strong expression of a wide spectrum of immune interactive ligand molecules may render the bile duct epithelia especially susceptible to the immune re-

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sponse. This may involve not only alloantigeneic T lymphocyte reactions to MHC, but also the nonspecific inflammatory response of other locally infiltrated leukocyte subpopulations.685 Hepatocytes, in contrast, show a more restricted expression of intercellular adhesion ligand molecules. Here the expression and inducible synthesis of ICAM-1 is probably of major importance. As known for class I MHC molecules175 ICAM-1 may also be released in a soluble form and effect systemic immunomodulatory functions as an acute phase protein in the blood.7,704,705 The full inducibility of T lymphocyte ligand molecules MHC class I, ICAM-1, LFA-3 and even HECA452 on hepatocytes supposes that this cell type may be very susceptible to T lymphocyte interaction with alloantigen recognition and cytolysis. The histological appearance of the rejection response sparing the liver parenchyma, however, does not support this mechanism. In vitro studies on cultured mouse hepatocytes have shown the susceptibility of this cell type to the alloreactive T lymphocyte response.92,93 The lack of T lymphocyte stimulation in the absence of antigen presenting cells have been attributed to the lack of basic MHC class II expression in the mouse and minor inducibility of these molecules in humans780 necessary for antigen presentation on hepatocytes. It is not excluded, however, that hepatocytes may be less susceptible to T-helper lymphocyte interaction by active suppression. In this context the production of soluble class I MHC molecules175 and of HECA452201 by hepatocytes may be mentioned. These mechanisms may be able to block T lymphocyte-hepatocyte interaction by T cell receptor or homing receptor binding.

Clinical and Therapeutic Aspects In clinical transplantation it is often difficult to differentiate between rejection and other causes of graft dysfunction. The analysis of adhesion molecules could be of diagnostic interest. The induction phenomena in different inflammatory processes, however, are quite similar and allow no distinction of rejection or infectious origin. Only the inflammatory state of the graft can be determined. In this respect the measurement of soluble ICAM-1 and cytokines in the bile may be a useful marker for the assessment of graft rejection and inflammation.5,7 Furthermore, the inflammatory release of VCAM-1 in serum and ICAM-1 in bile may enable a differential diagnosis of infectious and rejection related liver inflammation.427,428 Inflammatory release of circulating adhesion molecules may aid in the diagnosis of complications after multivisceral organ transplantation. 681,682 Lautenschläger et al435,437 reported that inflammatory upregulation of adhesion ligand molecules on hepatocytes preceded lymphocyte infiltration in rejection and cytomegalovirus infection. A selective immunosuppressive treatment by blockade intercellular adhesion pathways is a prospect for clinical treatment of liver transplant inflammation. Experimental studies using anti-ICAM-1159,227 or anti CD11/CD18338,729 antibodies are available. From these data it can be suspected that the further use of anti-inflammatory reagents affecting mechanisms of intercellular adhesive reactions is a prospect for clinical use. Conflicting reports about the effect of anti-ICAM-1 antibodies, however, demonstrate the need to analyze organ specific features.184,568 Not only blockade of adhesion receptors by monoclonal antibodies can be pursued, but also possibilities to modify immune reactivity by specific designed adhesion peptide molecules may develop. Here the biological model of soluble forms of

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adhesion molecules as ICAM-1, LFA-3, ELAM-1, CD62 and MHC may guide to possible immunological effects. It can be postulated that these molecules similar to cytokines effect leukocyte activation or de-activation by membrane receptor binding. The intravascular binding to leukocyte receptors as LFA-1 and CD2 may induce a state of preactivation and receptor conformational change facilitating a highly adhesive state. On the other side, it is also possible that adhesion receptors of circulating leukocytes are blocked by soluble ligand molecules prohibiting endothelial binding as a downregulatory mechanism. The possible implications and effects of different molecular isoforms have to be investigated in more detail. The production of these molecules or pharmacological derivatives, however, might become a field of major interest in clinical research. Not only the modification of liver transplant inflammation may be important, but also possibilities for the treatment of other types of inflammatory liver disease. For clinical hepatology there might be special importance, since the liver may be a major production site of soluble adhesion molecules as ICAM-1, LFA-3 and class I MHC/β2-microglobulin in the peripheral blood. These may form part of the homeostatic and acute phase functions of the liver sinusoid as known for the production of coagulation factors and complement in the blood.641 A major point of interest in liver transplantation is the possibility to modify organ damage caused by the initial reperfusion injury.148 The presented results of intraoperative changes in the expression of the early leukocyte adhesion ligand molecules CD62 and ELAM-1 point to their potential importance in the regulation of the early reactivity and influx of recipient leukocytes. However, not only the induction of early adhesion molecules, but also the extent of endothelial inflammation leading to a cascade reaction with cytokine release and expression of additional adhesion ligand molecules as VCAM-1 and ICAM-1 may effect late intravascular changes of the reperfusion damage after hours to days. The pattern of a preferential ELAM-1, VCAM-1 and ICAM-1/2 induction in the afferent portal vessels may render this vascular stream bed particular sensitive to the inflammatory reaction. Secondary leukocyte stasis, coagulation and hypoxemic effects may also affect the liver sinusoidal cell and hepatocyte function. Beneficial effects on organ reperfusion damage as the treatment with prostaglandin E1730 and abrogation of leukocyte adhesion by anti-ICAM-1 or anti-CD11/CD18338,729 point to the importance of these molecular mechanisms. In this context it is noteworthy to mention that the induction of the rejection response may be upregulated by the extent of initial reperfusion induced nonspecific organ inflammation. Clinical data comparing the rejection rate in initial good and bad functioning grafts point in this direction.342 The present analysis shows a wide pattern of inflammatory changes in the expression of adhesion molecules in the human liver. The changes are most likely caused by cytokines released from inflammatory leukocytes, endothelia and resident liver cells.30 It can be assumed that the different inflammatory cytokines as TNFα, interleukin-1, interleukin-6, and IFNγ induce specific patterns of adhesion molecules. The pattern of cytokines released by inflammatory leukocytes may therefore open their way into the liver tissue and facilitate cell-interactions. The inducibility and time sequence of adhesion molecule expression, however, may be the definitive regulator of the inflammatory reaction. It can be assumed that a few key molecules are necessary for the initiation and generation of these reactions. These

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and the presence of their stimulatory cytokines may determine the sequence and a stepwise progress of the inflammatory reaction. The manifold of other adhesion receptors, however, is involved in the fine regulation of the different intra- and intercellular reactions. Future studies have to clarify the stepwise regulation of the molecular reactions and interactions of different leukocyte populations. Resident liver cells, however, are of equal interest as the broad changes in adhesion molecule expression on all endothelial and nonendothelial liver cell types point to their active involvement in the regulation of liver inflammation.

Part 3 Pathophysiological and Clinical Aspects

CHAPTER 8

Role of Adhesion Molecules in Reperfusion Injury and Rejection Gustav Steinhoff

Transplant Reperfusion Reaction

A

n important mechanism of immediate transplant dysfunction is ischemia/ reperfusion injury caused by a nonspecific inflammatory implantation response affecting graft function in the first days after transplantation.148 This mechanism is dependent of ischemia related damage to graft endothelia.806 The endothelial upregulation of adhesion ligand molecules (P/E-Selectin, ICAM-1, VCAM-1) then leads to the binding and infiltration of leukocytes. Possibilities to modify this inflammatory response that is mediated by donor endothelia and passing recipient leukocytes are of main interest. Moreover, the immunological changes that already lead to intraoperative inductions of adhesion molecules and MHC in the transplant probably have major influence on the immune reaction against transplantation antigens.310,572,840 The early inducibility of E-/P-selectin and VCAM-1 in different organ transplants demonstrates that the endothelial activation precedes leukocyte adhesion. Further steps of firm leukocyte adhesion and infiltration may then be initiated in dependence on the extent of the postischemic reaction and the generation of a cascade reaction by cytokines and expression of additional adhesion ligand molecules on the injured and activated endothelial cells.536,789 The extent of adhesion molecule induction may very well relate to the metabolic impairment of the transplant. The endothelial activation and injury that results in a clinical disturbance of coagulation factor synthesis—especially in liver transplantation— on the other hand may induce thrombogenesis in the transplant by direct adhesion ligand induction. Furthermore, inhibitory mechanisms of leukocyte and thrombocyte adhesion similar to the synthesis of antithrombin III3 may be disturbed. Firstly, it can be stated that an arteriolar or capillary leukocyte stasis induced by the endothelial reperfusion reaction may cause regional microcirculatory perfusion defects. 513,499,500 Their additional ischemic consequence may potentiate the tissue damage even in a later course after one or two days, when additional ligand molecules and soluble molecules as ICAM-1 and MHC are Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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induced.695 This concept is depicted in Figure 8.1. Secondly, the first passage sensitization of alloreactive T lymphocytes and the induction extent of rejection activity may very well be influenced by the initial postischemic inflammatory reaction. This has been stated for the early induction of class I and class II MHC molecules707 and may also relate to the coinduction of other adhesion ligand molecules.534 Clinical observations of an increased rejection rate in initial bad functioning grafts point in this direction.536,840 In experimental research the postischemic reperfusion damage could be successfully inhibited by anti-CD11/CD18 monoclonal antibodies;130,229,338,729 L/E/P selectin,757 Slex/Fucoidin carbohydrates107,109 and prostaglandin E1.730 This may open possibilities to clinically modify the reaction by the use of such tools and may include new pharmacological agents as NPC15669,860,940 antioxidant pyrrolidine dithiocarbamate,221 castanospermine,325 apigenin253 and pentoxiphilline.115 It can be assumed, however, that a number of alternative pathways have importance in the induction phase of inflammation (Sialoadhesin, CD31, VLA-6/fibronectin, vWF).

Acute and Chronic Transplant Rejection The understanding of the pathophysiology of transplant rejection has undergone major change by the analysis of molecular changes in the transplant. Until a few years ago the molecular mechanisms of transplant infiltration were rather unclear and the morphological and functional knowledge on the contributing effector cell populations remained in the center of interest.266,267,307 It was not known that on transplant cells—especially on endothelial and parenchymatous cell types— immunological reactions may occur in an extent as known at present. Starting with the observations of Lampert et al419 and De Waal et al,183 changes in the MHC expression on parenchymatous transplant cells were found. Although this seemed to explain the effector mechanism of reactive T lymphocytes at MHC incompatible transplant cells on the first sight, the basal and normal expression of class I and class II MHC molecules especially on endothelial cells in a transplant without major rejection activity referred to the existence of additional binding molecules.408 Furthermore, the nonspecific coinfiltration of other leukocyte subpopulations with transplant rejection was an unexplained event on a molecular level. By the identification of molecular families of accessory adhesion molecules and their functions in specific and nonspecific lymphocyte/leukocyte reactivity, various mechanisms in the recognition of cells and the genesis of tissue infiltration have been unraveled. At the present time the main receptor and ligand molecules needed for the intercellular and immune reactions, and many aspects of their cellular function and interactions, are clarified and allowed to draw conclusions on their functional order by their sequential distribution patterns during transplant rejection.47,150,168,802,822 In addition, a manifold of functional in vitro data exist that may help to interpret in vivo events. A central event in the antigen specific reaction of CD4+ T-helper lymphocytes is the counterexpression of class II (HLA-DR) MHC molecules. These are present or may be induced by cytokines on many cell types of liver, heart or lung transplants. The stimulation of T lymphocytes by the various cell types, however, may have different effectivity. This could have a basis in the fact that additional costimulatory ligand molecules as B7 (BB7 ligand to CD28/CTLA4), LFA-3, ICAM 1 and 2, as VCAM-1 find a variable expression on different cell types and are

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Fig. 8.1. Sequential pathomechanisms of ischemia/reperfusion inflammation in leukocyteendothelial interaction.

coexpressed in high density mainly on the “immunocompetent” considered interstitial dendritic cells and monocytes/macrophages. Therefore, the order of reactivity of different cell types to T lymphocytes may depend on the presence of a panel of adhesion ligand molecules.173 This is underlined by in vivo data on coblockade of LFA/ICAM and CD28/B7 pathways in allogeneic bone marrow transplantation.90 In addition, accessory cell types are capable of producing stimulatory cytokines such as interleukin 1 and IFNγ upon binding of T lymphocytes. Not at last intracellular functions as the cleavage and presentation of antigen-peptides with MHC molecules determine their immunological interactivity. For the target cell interaction and cytolytic process of CD8+ “cytotoxic” T lymphocytes also the presence of the target antigen, alloantigeneic MHC molecules, is a prerequisite.31 However, for the regulation of cytolytic cellular interaction, the additional binding of costimulatory molecules as ICAM-1, ICAM-2, and LFA-3 may also be necessary to induce the cascade reaction leading to cytolysis. Therefore, the presence, or rather the inducibility, of certain immune ligand molecules in an organ transplant is the prerequisite for the genesis of an alloantigen directed T lymphocyte sensibilization551 and the manifestation of an effector reaction. For the specific and nonspecific immunosuppression of the anti-allogenic immune response after transplantation, a number of new aspects are arising. The clinical immunosuppression after liver, heart and lung transplantation consists of two main components: T cell specific inhibition by abrogation of interleukin 2 mediated stimulation (cyclosporine A, FK506/tacrolimus) or by a short term elimination of lymphocytes by anti-lymphocyte sera or monoclonal antibodies (antiCD3, anti-IL2rec). A secondary effect of the abrogation of T lymphocyte stimulation is a suppression of the B lymphocyte antibody response. This is additionally immunosuppressed by azathioprine, brequinar, rapamycin (sirolimus) or RS61443

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(Cellcept). Secondly, nonspecific suppression of leukocyte activation and adhesion by corticosteroids, which are likely to interact with adhesion molecule interaction. Further specific abrogation of leukocyte-endothelial adhesion by other compounds has been studied experimentally and clinically in the last years. Experimental studies using anti-ICAM-1,159,227 anti-CD11/CD18 monoclonal antibodies311,338,378,729 and anti-VLA-4359 support the central role of these molecular interactions for the infiltration process. For clinical purposes in modification of immunosuppression, however, not only the usage of such monoclonal antibodies340 may be a major prospect, but the identification of anti-adhesive capacities of other drug substances as discussed above (apigenin, castanospermin a.o.). Of particular relevance for the outcome of chronic transplant rejection is the approach of anti-inflammatory treatment possibilities.590 The results in chronic liver,765 heart758 and lung transplant rejection show an endothelial activation with the increased expression of adhesion ligand molecules as ICAM-1, VCAM-1, and MHC class II. Similar findings have been done in renal transplants.113,242 The trigger mechanisms are probably antibody binding to endothelial alloantigens and local cytokine release of reactive memory T lymphocytes.172,313,314 Furthermore, general effects of altered organ homeostasis as vascular effects of flow related shear stress on endothelial cells in a denervated organ (heart) and opportunistic viral infections have to be considered as triggering events. It can be stated that even with absent T lymphocyte stimulation the endothelial activation in the transplant alone can recruit leukocytes and therefore maintain a chronic inflammatory reaction. The increased expression of adhesion molecules with endothelial activation and the induced intravascular adhesiveness in the transplant may play a central role in this pathomechanism.200 Although closer analysis of the pathomechanisms has to be awaited, it can be stated that the main target in the treatment of chronic rejection is the inhibition of endothelial activation and increased adhesiveness. A number of possibilities are under experimental and clinical evaluation.590 Recently, gene transfer for factors abrogating intracellular activation pathways have gained major consideration.220-222,260 A special aspect for new approaches to immunosuppression and immunomodulation arise from the biological model of soluble forms of adhesion molecules as ICAM-1, LFA-3, E- and P-selectin and MHC.7,175,704,740 It can be estimated that these molecules have a major function in the intravascular regulation of the immune response in addition to the known cytokines and growth factors. Processes of intravascular leukocyte activation and depression may be influenced by the release and interaction of these solubilized ligand molecules. It is possible to imagine that the intravascular prebinding of ICAM-1 or LFA-3 to their respective receptors on leukocytes leads to a change in expression density or receptor conformation either to increase or decrease the state of intercellular affinity (leukocytethrombocyte, leukocyte-leukocyte, leukocyte-endothelium). It is just as possible as the intravascular or interstitial blockade of interactive adhesion receptors by soluble adhesion ligands as a downregulation to target cell binding. In this context the identification of various molecular isoforms306,704 may point to a differentiated usage of these for the regulation of intercellular contacts. Similar effects have been postulated for the binding of soluble alloantigeneic MHC to the T cell receptor. For the alloantigeneic situation of organ transplants, effects of soluble MHC in previous blood transfusions or simultaneous organ transplants (liver-kidney, heart-

Role of Adhesion Molecules in Reperfusion Injury and Rejection

121

Fig. 8.2. Multireceptor interaction and cytokine stimulation in leukocyte-endothelial interaction to define specificity of immune reaction.

lung) may also modify the T lymphocyte response. The usage of soluble adhesion molecules or specifically designed peptides may open new avenues for the manipulation of the immune response. This may involve not only organ transplant related questions as the induction of tolerance or modification of chronic rejection, but has relevance for the treatment of all inflammatory reactions in general. The human liver in that respect may be of special interest as a main producer of soluble adhesion molecules in the blood stream (ICAM-1, MHC/β2-microglobulin). These may exert physiological function in the acute phase reaction similar to complement and coagulation factor synthesis.641

Long-Term Adaptation of Organ Transplants The main aspects of the immunological changes found in liver transplants regarding to a longterm adaptation are, first, the fact that regardless of the time after transplantation systemic and infectious complications lead to immunological activation of transplant cells. These may influence the immune reactivity of the host and form a major risk situation of the transplant against rejection reactivity. It can be assumed that a constant endothelial activation as observed in heart transplants during clinical quiet courses758 may lead to chronic transplant vasculopathic changes. Although this may not apply in a similar extent to liver transplants, these changes are also found on arterioles of the liver. This includes endothelial damage and smooth muscle proliferation in chronic transplant rejection. Secondly, the organ specific immunological changes—especially observed in liver transplants with a major change in donor/recipient cell composition—may influence the longterm behavior of organ transplants in the host. As found experimentally in the rat446 clinical transplants of the liver and the heart774,782 show an almost complete exchange of immunocompetent donor cells as macrophages and dendritic cells. The results, however, also show that interstitial donor dendritic cells may persist for longer than 1 year in liver and heart transplants. This is also supported by experimental data in rat heart transplants.122 Particular intravascular changes are occurring in liver transplants with the exchange of the intravascular located Kupffer cells and possibly some endothelial cells. Whilst the exchange of accessory cells alone may not explain a lower immunogenicity of a transplant, because other cell

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types produce membrane bound and soluble MHC molecules and immigrated autologous accessory cells are presumed capable to present alloantigens, it may be assumed that a major reduction of highly immunocompetent donor cells may reduce the chance of T lymphocyte stimulation. It is not excluded that further mechanisms lead to a specific tolerance induction after organ transplantation. Experimental observations of tolerance after liver transplantation exist.36,131 According to recent suggestions a stable cellular chimerism of donor and recipient cells may exist, not only in the transplant, but in other host organs.686,751,752 This may be the result of the state of tolerance or the emigration of immunocompetent cells from the transplant, an event inducing graft acceptance. It is unclear so far whether or not the transplant’s chimeric state itself may induce MHC specific T lymphocyte suppression by altered antigen presentation, by a certain composition of deregulating cofactors as other adhesion molecules or by the production of soluble donor MHC molecules. It is likely that immunological adaptation of the transplant, as well as regulatory mechanisms inside the host immune system, may influence the alloantigeneic reaction. The composition of MHC and adhesion molecules on the cells of an organ transplant therefore determines the local appearance and systemic upregulation of the host immune response.

CHAPTER 9

Cytomegalovirus (CMV) Infection, Expression of Cell Adhesion Molecules and Allogeneic Rejection Xiaomang You

Introduction

C

ytomegalovirus (CMV) infection is still one of the most important infectious complications after organ transplantation associated with a considerable mortality in transplant patients.660 Symptomatic CMV infection occurs in 8%, 29%, 25%, and 39% of kidney, liver, heart, and heart-lung transplant recipients, respectively.331 CMV infection commonly results from the activation of latent infected recipients,223 it can also be transmitted to transplant recipients by the donor organ and to a lesser degree by blood products.147 In the solid organ transplant recipients, CMV is related to: (1) infectious disease syndromes and organ involvement (lung, liver, gastro-intestinal, retinitis, among others); (2) increased immunosuppression (hence the frequent association with other opportunistic infection, e.g., fungal and pneumocystis)647 and (3) although controversial, acute and chronic allograft injury.39,660 After primary infection, CMV remains latent in various cells including bone marrow-derived white blood cell.302,647 The mechanism leading to the reaction of latent virus are not well known. After transplantation, T cell activation may induce viral reactivation,17,660 This view is supported by experimental data from the murine system.409,660 In clinical transplantation, it is difficult to determine whether infection with CMV activates allograft rejection or rejection activates CMV infection. No single event is responsible for the high incidence of CMV infections in transplanted patients, but the overall state of immunosuppression is the most important single agent.223,767 There is a definite and mutual relationship between CMV infection and rejection. The sequence in which infection and rejection occur is

Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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still a matter of debate. Two hypotheses that rejection favor CMV infection and that CMV infection is an adjutant triggering graft rejection, are not mutually exclusive.17,151,660 In spite of the hypothesis, it has been understood that HCMV plays an important role in acute and chronic rejection of solid organ transplants.635,660,893 Additionally, the viral infection may even lead to fatal complications in immunosuppressed patients. Although the underlying mechanisms by which CMV infection is associated with rejection have not been elucidated, a major role is attributed to a virus-linked vascular wall inflammation resulting in a secondary alloantigeneic immune injury toward allograft endothelia and vascular wall structures.404 Viral infection can induce or upregulate the expression of MHC class I and class II on donor tissues. CMV-infected endothelial cells have the ability to activate CD4+ T lymphocytes in the absence of HLA DR and induce the expression of cell adhesion molecules.867,886,894 A major factor for the induction of immune responses to alloMHC in the transplanted organ by CMV-infection is the inflammatory upregulation of adhesion ligand molecules on transplant endothelia by viral infection with subsequent leukocyte activation.700,701,739,894 This may form a major pathomechanism for the acceleration of chronic transplant rejection and allograft vasculopathy in transplant recipients.269,456,458

Manifestation of CMV Infection in Allografts Abrustini et al2 reported a total number of 143 transbronchial biopsies in 32 cases after lung transplantation. Histopathological study showed overt infection in 18 of 143 biopsies (12.6%); immunohistochemical staining with anti HCMV immediate-early (IE) antigen showed 21.76% positive infection. However, HCMV infection was determined in 73 of 143 (51%) lavage fluids by PCR. Double immunostaining showed that in human transplanted lungs, HCMV early infection cell populations seem to be represented exclusively by epithelial cells and alveolar cells that were found to be positive in any of the specimens. In these cases, 27 of 32 (84.3%) developed post-transplant CMV infection in first two months of operation. In 23 cases symptomatic systematic CMV infection developed. To study the manifestation of viral disease in transplanted organ and recipients, a rat model of experimental lung transplantation with cytomegalovirus infection was established to investigate its interaction in allografts.923 RCMV-mismatched infection combinations of donor (D) and recipient (R) were included as: D-/R+, D+/R- and D+/R+. Enhanced RCMV infection in lung allografts of D-/R+, D+/R- and D+/R+ was documented from day 3 to 21 as compared to RCMV-infected nontransplanted controls. Similar observation was obtained in further study of a rat lung transplantation model with RCMV infection by Steinhoff et al.785,786 After initial immunosuppression an increased virus manifestation was present in allogeneic lung transplants. Moreover, a more severe systemic infection developed after allogeneic lung transplantation as read by salivary gland virus titers. Interestingly, viral infection of the allogeneic lung transplant preceded the detection of infected cells both in the nontransplanted contralateral lung and the salivary gland. Accelerated organ manifestation of RCMV in the allogeneic lung transplant as well as enhancement of systemic disease may be influenced by two distinct immunological mechanisms.

CMV Infection, Expression of Cell Adhesion Molecules and Allogeneic Rejection 125

First, the viral infection may escape the self-MHC restricted immunological control in the environment of the transplant initially containing only allogeneic MHC incompatible antigen presenting cells (including endothelia) not able to effectively present RCMV antigen to reactive recipient T lymphocytes. This mechanism may enable the manifestation of RCMV and infiltration of RCMV positive cells in the allogeneic transplant. This may only be operative until more immunocompetent autologous antigen-presenting cells (APC) have migrated into the graft later after the transplant especially with ongoing graft rejection. This mechanism may then allow for a higher rate of clearance of infected (mononuclear) cells by recipient T lymphocytes in the graft. Interestingly, in this model the RCMV infection of the lung transplant ceased with the development of acute rejection. It is very well possible that the rejection process did limit viral replication and RCMV positive cells allowing for a more effective antiviral response in the graft. The endothelial cells as a prime target cell of the CMV-infection,117,894 however, are not replaced by recipient endothelium. This may allow for persistence of the virus. This experimental setting of high dose immunosuppression in the absence of higher numbers of immigrating immunocompetent cells early postoperatively may constitute a favorable environment for viral persistence and replication in the allogeneic lung graft as a result of an almost complete lack of T cell control. Thus, a combination of immunosuppression reducing T cell reactivity and a low rate of antigen recognition by T-effector cells to viral antigens on MHC mismatched endothelial cells and APC may enable graft infection. A comparable situation may occur in the first weeks after clinical lung transplantation in HCMV-seronegative (heart-) lung transplant recipients of CMV-positive donor organs, This scenario renders the lung transplant at high risk for viral (HCMV) reactivation and intragraft replication. The dismal clinical experience345 with critical viral disease and pneumonitis in cases without antiviral treatment using virostatic drugs and hyperimmunoglobulin767 may reflect such a special risk situation, at least in the early phase after transplantation. A second potential mechanism enhancing systemic viral disease and organ manifestation would be a temporary generalized decrease in the recipients T cell response during the initial phase after lung transplantation. Besides the effect of immunosuppressive drugs, postoperative T cell elimination and bone marrow suppression may be initiated by a limited graft-versus-host disease (GVH) mediated by immunocompetent donor cells.642,752,818 This mechanism is quite likely to occur after single lung transplantation in the rat since the graft contains a large number of lymphoid cells in the bronchus associated lymphoid tissue (BALT).912 The BALT may actually represent a primary site of HVG and GVH as well as a source of migrating donor T lymphocytes and dendritic cells.626,861 Enhanced viral replication after lung transplantation as compared to renal or heart grafts, where such an induction of viral disease after allogeneic transplantation has not been observed,119 could very well be explained by such a mechanism. In 355 cases of clinical liver transplantation, however, reported by Manez et al490 that the frequency of CMV hepatitis was 44% for HLA-DR-matched livers but 14% for HLA-DR-unmatched livers. In seropositive recipients (n = 187), these frequencies were 12% and 2% for HLA-DR-matched and unmatched liver grafts. These findings suggest that an HLA-DR match between donor and recipient increases the incidence of CMV hepatitis in both primary and secondary CMV infections.

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Expression of MHC Molecules with CMV Infection The MHC molecules play an important role in antigen recognition and subsequent cellular reactions in the immune response. Incompatibilities between donor and recipient class II antigens provide a powerful stimulus of the rejection response. The underlying mechanism by which CMV infections are associated with rejection has not been fully elucidated. A major pathomechanism discussed is the upregulation of MHC transplantation antigens on graft cells by inflammation of viral infection and/or by CMV itself. The RCMV-linked MHC induction is believed to be involved in acute rejection mechanisms and chronic vasculopathy after transplantation.404 Although most virus-specific cytolytic T cells are MHC class I-related CD8+ lymphocytes,192 recent data support the hypothesis that the MHC class II mediated CD4+ responses (the same pathway as that of allogenic rejection) play a role in HCMV infection.101,102 Both as direct, virus-specific cytolytic activity and cytokine mediated, virus-specific CD8 recruitment. A MHC class II-mediated response to HCMV infection likely depends on whether or not HCMV-infected cells express MHC class II antigens. It needs to be clarified if CMV is able to regulate the expression of MHC molecules on donor cells. A few studies in vitro and in vivo have been done by several research groups (Table 9.1). However, there were contradictory results in vivo and vitro. MHC class I and II expression on cultured human umbilical vein endothelial cells (HUVECs) with HCMV infection were investigated by many authors. Their results have demonstrated that (1) CMV does not directly induce HLA class II expression on human endothelial cells339,689,698,866,895 and, furthermore, that infected endothelial cells are refractory to class II induction by IFNγ,689,895 (2) upon infection, endothelial HLA class I expression progressively diminished as the cell proceeds to cytomegaly.48,701 The expression of MHC antigens on the cultured human proximal tubular epithelial cells (PTEC) was investigated by van Dorp et al.867 Normal PTEC express low amount of MHC class I and an increase in MHC class I expression was seen by CMV infection, starting 24 hr after infection, reaching a maximum after 72 hr. MHC class II is not induced by CMV infection. Incubation of CMV-infected PTEC with IFNγ for 72 hr results in an enhancement expression of MHC class II compared to noninfected PTEC incubating with IFNγ and IFNα. This finding suggest that expression of MHC I on epithelial cells can be directly induced by CMV but not class II. However, CMV-infected epithelial cells display a normal increase of class II expression after stimulation of IFNγ. A similar upregulation of MHC class I expression on cultured human smooth muscle cell by CMV infection was reported.339 To better understand MHC class II expression in vivo, a rat lung transplantation model with CMV infection (Maastricht strain) was used.936 RCMV induced expression of MHC class II on vascular endothelial cells was seen in the nontransplanted lungs and allografts of RCMV-infected rats, in contrast to normal lungs which were negative for MHC class II. RCMV infection was also found to enhance MHC class II expression on pneumocytes and leukocytes. RCMV-antigens were detected on a few leukocytes and pneumocytes, but not endothelial cells. The enhanced MHC class II is consistent with the increase of allograft rejection. Ustinov et al859 determined the expression of MHC class II in a RCMV-infected rat

CMV Infection, Expression of Cell Adhesion Molecules and Allogeneic Rejection 127

Table 9.1. MHC Class I

MHC Class II

CMV Infected Cell

CMV Infected Cell with Cytokine Stimulation

CMV Infected Cell

CMV Infected Cell with Cytokine Stimulation

In vitro Endothelial Epithelial

normal or decrease increase

increase increase

decrease no response

decrease (no response) increase (no difference from noninfected cells)

In vivo Endothelial Epithelial

increase (unclear) increase

increase increase

increase (unclear) increase

increase increase

model. Their finding suggested that RCMV induces class II expression in parenchymal structures of several organs. An intense increase in class II molecules on the surface of most endothelial cells was recorded. Also, in the epithelial cells of kidney tubuli as well as in glomeruli, class II induction appeared. Liver parenchymal cells, hepatocytes, and bile duct epithelial cells also become strongly positive for class II antigen expression. In the heart, induced expression of MHC class II was recorded in the vascular endothelium only. RCMV antigens were detected in exactly the same structures as those in which the induction of class II molecules was seen. The clinical investigation has been done by Abrustini et al2 to detect CMV infection and expression of MHC class II in 143 transbronchial biopsies from 32 cases. CMV infection was determined by immunohistochemical staining with anti HCMV immediate-early (IE) antigen and IE viral gene in lavage fluids by PCR. The occult and overt CMV infection were determined in 51% of samples in human transplanted lungs. Enhanced expression of HLA-DR was found, but not all HCMV antigen positive cell express HLA-DR in lung biopsies detected by double staining. The detected HLA-DR and HCMV-IE antigen coexpress in early-infected, noncytopathic cells of biopsy samples with inflammatory infiltrates that are consistent with acute rejection of any grade. This may strongly support the hypothesis of the indirect induction. Furthermore, an association has been found between acute allograft rejection, parenchymal cell MHC class II expression and CMV infection in kidney transplantation.886 In liver transplantation, hepatocytes and other parenchymal cells in the grafts demonstrate a strong MHC class II during CMV infection.436,777,781 This was found focally around infiltrates in acute rejection and at sites of inflammation in CMV hepatitis. Although there is no causal association with acute liver rejection, clear evidence of an association with chronic rejection exists.39,561

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Expression of Cell Adhesion Molecules The expression of endothelial cell adhesion molecules for leukocytes is an important component of the inflammatory process that underlies allograft rejection.242,699 One target of CMV infection in vivo is the endothelial cell.844 These cells form the interface between an allograft and host, and have been shown to take part in allograft rejection, the latter a result of their distinct array of constitutive and inducible immunological properties.699 Besides upregulation of HLA class I and class II, these properties include the induction of adhesion molecules for leukocytes and the presentation of associated antigens. Endothelial cell adhesion molecules such as ICAM-1, 2, VCAM-1 and ELAM-1 facilitate many cell-cell interactions. The role of cell adhesion molecule and the relationship with CMV infection have been investigated in vitro and in vivo. The current knowledge is summarized in Table 9.2. The effect of CMV (VHL/E strain) infection on the constitutive and TNFαinducible expression of ICAM-1, VCAM-1 and ELAM-1 on human umbilical vein endothelial cells (HUVECs) was detected by Sedmak et al700 in vitro experiment. TNFα treatment of CMV-infected HUVECs result in a marked induction of ICAM1 on the cell membrane as compared with uninfected, untreated or only CMVinfected cultures. Neither VCAM-1 nor ELAM-1 was induced in CMV-infected cultures in contrast to induction of ICAM-1. Scholz et al690 studied the expression of ICAM-1, VCAM-1 and ELAM-1 on human umbilical vein endothelial cell (HUVEC) incubated and infected with CMV. CMV alone enhanced the basal expression of ICAM-1 but not of VCAM-1 and ELAM-1. Adequate cytokine stimulation (IL-1, IL1/TNFα) of HUVEC led to enhanced the expression of ICAM-1, VCAM-1 and ELAM1. Moreover, ICAM-1 expression on epithelial cells at 24 hr after infection with CMV is significantly more enhanced compared to epithelial cells stimulated with IFNγ for 24 hr. Significant increase in ICAM-1 expression are documented on CMV infected PTEC stimulated by IFNγ at 72 hr.867 These studies clarify that CMV can directly induce the expression of ICAM-1 on human endothelial and epithelial cells. In order to investigate the induction of endothelial activation and rejection by CMV infection, a rat lung transplantation model of rat-cytomegalovirus (RCMV) infection and acute allograft rejection was established.785,786,923,924 Postoperatively, triple drug (CiA, Aza, Pred) immunosuppression was given from day 1-10 to induce systemic RCMV-infection and acute rejection developed from postoperative day (POD) 15-25 in allogeneic transplants. In RCMV positive animals the rejection grade was gradually increased significantly at POD 15-20. In the absence of rejection infiltration a maximal induction of ICAM-1 adhesion molecules was found on lung endothelia in RCMV+ allogeneic animals as compared to noninfected controls. This induction was found to lesser degree for VCAM-1 adhesion ligand molecules. Enhanced expression of ICAM-1 and VCAM-1 was accompanied by a significant CD11a+ and CD49d+ leukocyte infiltration into the alveolar interstitium on day 11 and 15 in infected transplants. The results show an enhancement of RCMV infection after allogeneic lung transplantation leading to endothelial activation and recruitment of CD11a/CD49d+ leukocytes. This mechanism may strongly influence transplant inflammation and the long-term course of lung transplant rejection.

CMV Infection, Expression of Cell Adhesion Molecules and Allogeneic Rejection 129

Table 9.2. ICAM-1

In vitro Endothelial Epithelial In vivo Endothelial Epithelial

VCAM-1

ELAM-1

CMV Infected Cell

CMV Infected Cell with Cytokine Stimulation

CMV Infected Cell

CMV Infected Cell with Cytokine Stimulation

CMV Infected Cell

CMV Infected Cell with Cytokine Stimulation

increase

significant increase increase

normal

significant increase increase

normal

increase

increase

increase increase (unclear) increase increase

normal

increase increase (unclear) increase increase

increase

unclear

increase

unclear

increase

Using immunohistochemical technique, human endothelial expression of ICAM-1, VCAM-1, ELAM-1, and their receptor ligands LFA-1, Mac-1 and VLA-4 were investigated by Koskinen et al403 in 105 endomyocardial biopsies from 21 heart allografts. Nine of those patients suffered from CMV infection. An induction of VCAM-1 occurred in relation to onset of CMV antigenemia, in contrast to little or no VCAM-1 found during mild rejections or in control biopsies. The expression of VCAM-1 remained elevated for several weeks declining slowly to control levels. Associated with CMV infection, capillary expression of VCAM-1 and ELAM-1 was significantly increased when compared with control biopsies. ICAM-1 expression was always seen in capillaries as detected in controls. A striking difference in the expression of VCAM-1 during rejection and CMV infection was observed: in most rejecting biopsies only a few capillaries stained faintly for VCAM-1, whereas during CMV infection multifocal intense staining was found. Induction of ELAM-1 was associated with acute rejection. In general, the expression of ligand counterparts was at high level during rejection compared with CMV infection. However, a short-term induction of VLA-4 occurred after the onset of CMV antigenemia. Thus, the VCAM-1/VLA-4 ligand pair may play an important role in adhesion of lymphocytes and monocytes to capillary endothelium during active CMV infection and may also contribute to the pathogenesis of increased vasculopathic changes reported in CMV-infected heart transplant recipients.269 The soluble form of VCAM1 is a reflection of endothelial activation. It can be released by endothelial cell. An associated increase of soluble VCAM-1 level can be detected in the renal allograft recipients with CMV infection and the level of soluble VCAM-1 can be normalized after treatment with ganciclovir.444

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Mechanisms of Regulation for Allogeneic Rejection by CMV Infection CMV has been associated with allograft rejection and transplantation-associated arteriosclerosis. Graft endothelial cells play an important role in allorejection due to the interface between allograft and host. Furthermore, epithelial cells play an important role in maintaining organ function in the lung, liver, and kidney. Interaction between cytokines and endothelial cells and/or epithelial cells is considered to be of crucial important for amplification of the inflammatory response during allograft rejection. Two distinct effects can be observed: (1) the cytokines upregulate MHC antigen expression on graft cells (endothelial cells and target cells), upregulation of MHC antigens will serve to enhance recognition by T cells; (2) they induce expression of cell adhesion molecules on endothelial cells, induction of adhesion molecules will initiate leukocyte cell adhesion and migration into tissue. It is still argued whether increased expression of MHC class and cell adhesion molecules are upregulated by CMV infection directly or as a consequence associated with an increase in inflammatory mediators such as IFNγ, TNFα, IL-1β, IL-1α and IL-6 induced by the viral infection. An enhanced expression of MHC class II antigen was found on endothelial cells of heart, kidney and liver with CMV infection in a rat model, indicating a direct induction.859 There is, however, in humans an increasing evidence that CMV is not able to enhance the MHC class II689,896 as well as class I896 in CMV-infected cultured human epithelial (Iblahim et al 1993) and endothelial cells (Waldman et al 1994 and see refs. 690, 896), even in the CMVinfected endothelial cells cultured with IFNγ. However, an upregulation of class I and II expression was seen on cultured human lung epithelial cells,599 human proximal tubular epithelial cells866 and endothelial cells690 by IFNγ, TNFα and IL-1. These studies show in vitro that CMV-infection does not directly upregulate MHC expression by infecting target cells, at least much less than the influence of inflammatory mediators cytokines—induced by CMV infection. A rat lung transplantation model with CMV infection936 showed that an induction of MHC class II expression on endothelial cells was seen as compared to normal lungs which were negative for MHC class II. RCMV-antigens were not detected on endothelial cells. The detected HLA-DR and HCMV-IE antigen coexpression of human lung biopsies in early-infected, noncytopathic cells of biopsy samples with inflammatory infiltrates that are consistent with acute rejection of any grade.2 This may strongly support the hypothesis of the indirect induction. Similarly, the studies in vitro did not find that enhanced expression of VCAM-1 and ELAM-1 on CMV-infected endothelial cells.690,700 In contrast to MHC class molecules, however, VCAM-1 and ELAM-1, ICAM-1 can be significantly induced on endothelial cells689,700 and epithelial cells867 with CMV infection and stimulated by cytokines in vitro. These adhesion molecule ligands on infected cells still have the ability to respond in cytokine induction. The upregulation mechanisms of induced expression for MHC class antigens and cell adhesion molecules by CMV are still not well understood. One mechanism may be secondary to host T cell activation by CMV infection.894 The cause of inflammatory upregulation of MHC and other adhesion molecules on the surface of endothelia may play an important role and be mediated by cytokines, namely IFNγ, IL-1, IL-6 and TNFα (Fig. 9.1). Infected and activated leukocytes release these

CMV Infection, Expression of Cell Adhesion Molecules and Allogeneic Rejection 131

cytokines to induce endothelial MHC class antigen expressions, as observed in cultured human endothelial cells.895 IFNγ was detected in the supernatant of cultured peripheral blood mononuclear cells with CMV infection.360 Cytokines may either be released locally by CMV activated leukocytes binding to lung endothelia and/or systematically lead to a generalized induction of endothelial adhesive ligand structures. The capillary network of lung allografts has a basic MHC class I and ICAM-1 ligand molecule expression and may serve as a predilective site for the binding of activated leukocytes.771 Cytokines play an important role in amplifying local modifications brought by CMV infection.571,797 Furthermore, PCR studies have demonstrated the presence of the inflammatory cytokines IFNγ, TNFα, IL-1β and IL-α in human cardiac160 and renal396 allograft biopsies during the episode of rejection. Increased levels of cytokines in circulation and/or in tissue are considered to be a key role to cell-cell and cell-matrix reaction during allograft rejection. Cytokine-mediated enhanced expression of endothelial adhesion molecules and adhesion receptors on leukocytes may lead to adherence to endothelium and migration to inflammatory sites. Another mechanism leading to increased vascular inflammation may result from the infection of endothelial cells by RCMV leading to cellular gene activation and upregulation of the expression of adhesion ligand molecules. The direct effect of CMV on MHC class and cell adhesion molecule translation may likely be based on the virus’ own molecules, which modify the host’s genome products during infection. This mechanism has been postulated for the in vitro induction of MHC class II858 on CMV infected endothelial cells and induction of ICAM-1 on infected endothelial cells689,700 and epithelial cells.867 In the rat CMV-infected endothelial cells could not be demonstrated employing the detection of RCMV antigens in vivo.936 Nevertheless, a viral infection of these cells may be present at a degree below the detection level of viral antigen using immunohistology. This has been demonstrated for the broad infection of lung endothelia in mouse MCMV infection using in-situ PCR demonstration of viral DNA.399 In addition, studies of MCMV infection have elucidated that the lung is a major organ of viral manifestation and most likely a major source of viral replication399 By this pathomechanism, the transplanted organs would be an important source of hibernating and replicating virus. The cytokine-mediated expression for MHC class I and II, VCAM-1 and ELAM1 may be a main pathway to inflammatory reactions by CMV infection in comparison with direct induction of adhesion molecule expression on CMV-infected cells. However, ICAM-1 expression may be upregulated by CMV infection directly, as well as by cytokines associated with viral infection. Two mechanisms of activation of vascular endothelial cells and/or circulating leukocytes initiate a multistep inflammatory reaction. The rolling of marginated leukocytes along the vascular wall is mediated by the molecular interaction of members of the selectin family, Eselectin, P-selectin and L-selectin with their principle carbohydrate ligand being Sialyl LewisX (SLX, CD15s) or other sialylated, fucosylated structures.125,139 After this initial “tethering”, firm leukocyte adherence is established by the banding of leukocyte integrins, such as LFA-1, Mac-1 and VLA-4, to the members of the immunoglobulin supergene family such as ICAM-1, ICAM-2 and VCAM-1, which are expressed on endothelial cells, and, in the case of ICAM-1 and -2, on leukocytes.125 Cell adhesion molecule interaction between the leukocyte and endothelial cell is a

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Fig. 9.1. Leukocyte-endothelial interaction in cytomegalovirus infection—current concept of virus-host interaction in organ transplants.

prerequisite for the migration of white blood cells into areas of inflammation. By this mechanism, CMV infection triggers and promotes acute and chronic allograft rejection. The inhibitory effect of HCMV on endothelial cells expression of MHC class II (and I) and refractory to IFNγ suggest that HCMV may result in a general inability of HCMV-infected cells to respond to cytokines.701 Furthermore, the decrease of MHC class II mRNA level and normal class I mRNA level indicates the possibility that general lack of responsiveness may represent the results of multiple inhibitory pathways.48,701 Further studies have to clarify this question, if a decrease in MHC class expression may influence allograft rejection. The relationship between CMV infection and allorejection has been widely investigated. In kidney transplantation, an association has been recorded among acute allogeneic rejection and HCMV infection.886,890 In liver allografts, hepatocytes and other parenchymal cells demonstrate a strong MHC class II expression during HCMV infection.436 In the absence of a causal association with acute liver rejection, clear evidence of an association with chronic rejection exists,39,561 albeit, some controversial results have been published.593 CMV-DNA has been detected in the hepatocytes of livers exhibiting chronic rejection.39 Correlation between HCMV infection and chronic rejection has also been reported after heart transplantation.269,910 Chronic heart allograft rejection manifests itself as accelerated arteriosclerosis and primarily affects vascular structures.456,458 Accordingly, HCMV nucleic acids have also been found in the vascular walls of arteriosclerotic patients.319

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Although class I antigens were initially thought to be expressed on all nucleated cells, more evidence indicates that the distribution of class I is not ubiquitous.169 Thus, the upregulation of class I antigen and detection by T cells may be relevant to the progression of disease processes. Furthermore, the increased expression of specific class II may trigger immunologic events, resulting in diseases. Taylor et al831 investigated the expression of MHC agents in lungs removed from recipients with OB and reported that MHC class II positivity of the vascular endothelium was largely due to expression of expression of DR with patchy expression of DP antigens. HLA-DQ was never expressed on normal donor lung and was only weakly expressed in some cases of OB. Rensmoen et al635 observed that primed lymphocyte test activity which correlated with MHC class II or combined class I and class II antigens of donor. It was found that the rejection grade of allogeneic lung transplants and expression of class II on endothelia and pneumocytes were increased in the presence of RCMV. The majority of infected grafts showed a significantly higher grade of rejection on postoperative day 15-20. It must therefore be assumed that either the increased interstitial inflammation or the increased MHC class II antigen expression prior to the development of acute rejection had an additive influence on the rejection response. This may explain the ultimate detection of RCMV positive cells in inflammatory lesions as demonstrated previously in a model of obliterative bronchiolitis.923,924 A similar induction of bronchiolar inflammation has been found in allogeneic rat lung allografts infected with Sendai-virus.994 This may indicate a similar pathomechanism as in pulmonary RCMV infection. It is thus not unlikely, that the effect of RCMV on allograft rejection mainly relies on the inflammatory induction of MHC class II molecules on endothelial cells of the lung. By this pathway, allogeneic endothelial cells may become susceptible to T cell recognition, subsequently stimulating the allogeneic immune response.

Impact of CMV Infection on Allogeneic Grafts Lung CMV infectious interstitial pneumonitis plays an important role in acute lung graft rejection. With PCR and immunohistochemistry, CMV infection could be detected in 51% of lung biopsy tissue.2 In lung transplantation, enhancement expression of MHC class I and II, as well as ICAM-1, VCAM-1 P-, and E-selectin on bronchial epithelial by CMV infection, may lead to greater susceptibility to attack by CTL and as a target for epithelial injury ultimately obliterative bronchiolitis. An animal model of rat lung transplantation with RCMV-infection showed that RCMV infection was able to trigger and promote acute rejection in allografts by way of upregulating the endothelial adhesion molecules for adherence and immigration of leukocytes, which characterized especially as acute airway damage, alveolar space hemorrhage and pneumocytic necrosis (You et al, data not published). Several studies have reported increased expression of MHC molecules on the donor organ cells during both of these processes. Enhanced expression of MHC class I and class II on bronchial epithelium during rejection of lung allografts,846 and an increased expression of MHC class II on both bronchiolar epithelium and vessel endothelium in OB831 have been reported. However, the detection of both

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rejection and infection within a single given biopsy sample raises major interpretation problems for histological analysis.2 In samples with morphologically overt cytopathy, surrounding inflammatory infiltrates can be easily attributed to the infection. In certain biopsies, some samples showed viral pneumonia whereas other samples, devoid of cytopathic cells, showed infiltrates consistent with acute rejection; in the latter case, attributing the infiltrates to infection may be questionable. However, some samples show no inflammatory reaction. The absence of inflammatory response to late cytopathic cells suggests that, in immunosuppressive hosts, even viruses that completed their life cycle may be ignored by competent T cells, possibly due to immunosuppression-related leukopenia or to infective antigen presentation.

Liver Chronic irreversible rejection with CMV hepatitis is a cause of graft loss and retransplantation after orthotopic liver allotransplantation. Manez et al490 studied CMV hepatitis in 25 of 399 patients after liver transplantation. The frequency of CMV hepatitis was 44% for HLA-DR-matched livers but 14% for HLA-DR-unmatched livers. These findings suggest that an HLA-DR match between donor and recipient increases the incidence of CMV hepatitis in both primary and secondary CMV infections (44% vs. 14%). Although HLA compatibility leads to less acute cellular rejection, chronic rejection incidence was higher in the CMV hepatitis group as compared to without CMV hepatitis group (24% vs. 6%). It is suggested that DR matching may accelerate chronic rejection of liver transplants, perhaps through HLA-DR-restricted immunological mechanisms toward viral antigens, including CMV. A consecutive analysis reported by Candinas et al135 in 423 adult primary liver allograft recipients showed that IgG positive donor to an IgG negative recipient were identified as risk factors for chronic rejection.437,438

Heart There is an especially significant relationship between CMV and chronic heart allograft rejection.269,910 The rejection manifests itself as accelerated arteriosclerosis and affects the vascular structure. CMV nucleic acid have been found in the vascular walls of arteriosclerotic patients.319 CMV is an exacerbating agent in the development of transplantation-associated arteriosclerosis (TxAA),214,269 an accelerated form of arteriosclerosis that frequently compromises coronary circulation with in cardiac allografts. TxAA, known as graft vascular disease, has emerged as the major limiting factor in long-term survival of cardiac grafts.381 This lesion is characterized in the early stages by subendothelial accumulation of host mononuclear leukocytes, primarily of monocyte/macrophage and T lymphocyte lineages.688 Presumably, the consequent release of cytokines, chemotactic factors and growth factors by infiltrating mononuclear cells, and possibly proximal endothelia, initiates a sequence of events leading to a migration and neoinitial proliferation of vascular smooth muscle cells. Subsequent differentiation of these smooth muscle cells to a more synthetic, secretory phenotype results in the accumulation of relatively large deposits of extracellular matrix.840 The end result is a progressive concentric neointimal hyperplasia that can severely compromise the circulation through large epicardial coronary arteries as well as anastomotic and penetrating intramyocardial branches.840 Although this lesion appears to develop with

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higher frequency and with severe consequences in the cardiac grafts in CMV-infected recipients, similar lesions have been observed in the transplanted lung274 and kidney.909 In addition, histological changes in other luminal graft structures terminal bronchioles and bile ducts suggest that mechanisms responsible for their stenosis (i.e., obliterative bronchiolitis and vanishing bile duct syndrome) may resemble those of TxAA or may be secondary to sclerosis of supporting vasculature.274,455 Thus all of these lesions have been collectively categorized as a form of CMV-infected associated chronic allograft rejection.

Kidney The impact of CMV infection on renal allograft rejection is still unclear. Nadasdy et al543 reported that 11 renal biopsies and 13 nephrectomies from 24 patients, all showing obliterative transplant arteriopathy, were collected for this study. Of these patients, six were seropositive for CMV before transplantation, three were identified as seropositive following renal transplantation, nine had no evidence of CMV infection. Paraffin-embedded renal sections were examined for the presence of CMV by immunohistochemistry in situ hybridization and polymerase chain reaction. By these methods, only one case (1/24) was demonstrated to have CMV infected cells in the renal interstitium, tubules, and glomeruli, but none (0/24) showed CMV to be located in any of the renal arteries or arterioles. This result suggests that obliterative transplant arteriopathy can occur in the absence of demonstrable CMV and is probably unrelated to direct CMV infection of the graft.

CHAPTER 10

Therapeutic Options by Blocking Adhesion Molecules Michael Brandt

Introduction

A

cute rejection and reperfusion injury in the early postoperative period as well as chronic rejection during the longterm course are the major causes of graft failure and death after solid organ transplantation. Infiltrating leukocytes have been repeatedly shown to participate in the development of tissue injury during allograft rejection and reperfusion. Both the ischemia during organ transportation and the immunologic process of rejection results in endothelial cell activation in transplant tissue. These pathomechanisms involve an increase in adhesion ligand molecule expression.771,787 Four steps of the infiltration process have been described: (1) rolling of the cells on the activated endothelium; (2) activation of leukocytes; (3) firm adhesion of leukocytes to the endothelium; (4) migration of leukocytes into the surrounding tissue.125 Adhesion molecules, which are expressed on both endothelial cells and leukocytes, are regulating this process.463 As such, rolling of leukocytes is mediated by selectins. E- and P-selectins are found on the endothelium, while L-selectin is expressed on leukocytes.439 Firm adhesion and migration of leukocytes are regulated by β2 integrins (CD11a/CD18, CD11b/CD18) interacting with endothelial ICAM-1 and integrin VLA-4 (CD49d/CD29) interacting with endothelial VCAM-1.885

Reperfusion Injury The ischemic damage during organ preservation and the postreperfusion inflammatory response is a major problem affecting allograft function in heart, liver and lung transplantation and patient survival during the first days after transplantation.148 The endothelial activation leading to leukocyte adhesion and graft inflammation is the major pathomechanism of reperfusion injury.148 Induction of E-selectin, P-selectin, ICAM-1 and MHC expression have been observed intraoperatively and immediately after liver transplantation.764,765 Recently it has been reported that during cold storage of cardiac allografts in University of Wisconsin solution and reperfusion the expression of MHC antigens and vascular adhesion molecules (ICAM-1, VCAM-1) remains unchanged.36 Immediate upregulation of Cell Adhesion Molecules in Organ Transplantation, Second Edition, edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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P-selectin has been described by our group within 60 minutes after lung transplantation.106 This corresponds well to clinical observations that reperfusion injury is a much smaller problem in heart than in lung and liver transplantation. In experimental research, the postischemic reperfusion injury could be inhibited by intraportal injection of anti-ICAM-1 antibodies after rat liver transplantation. This treatment improves survival as well as liver enzymes, and suppressed liver cell injury histologically.548,815 Infiltration of leukocytes and area of infarction could be reduced by pretreatment with anti-CD11a, CD11bc, CD18, and anti-ICAM-1 monoclonal antibodies in a rat heart ischemia/reperfusion model. Simultaneous administration of anti-CD11bc and CD18 antibodies tended to be most effective in this study.925 Simpson et al reported that treatment with an antibody to Mo1 (CD11b/CD18) reduced tissue injury and neutrophil activation in a dog model of myocardial infarction.729 Also, administration of anti-L-selectin monoclonal antibody before reperfusion has a significant cardioprotective effect by diminishing neutrophil infiltration, preserving endothelial function and attenuating reperfusion induced myocardial injury in a cat heart model.483 Anti-CD11a and anti-ICAM-1 monoclonal antibodies inhibited reperfusion injury in a rat model of lung ischemia and reperfusion. Increase of myeloperoxidase activity and wet/dry-ratio could be significantly inhibited by these antibodies.351 Kapelanski et al reported that administration of an anti-CD18 monoclonal antibody before reperfusion attenuates the development of both shunt and abnormal respiratory gas exchange in a dog lung transplantation model.375 An antibody blocking L-selectin and E-selectin was tested in model of sheep lung ischemia/ reperfusion. This antibody preferentially inhibited the delayed phase of the reperfusion injury. The initial phase of reperfusion injury was not significantly altered because this antibody does not recognize P-selectin which is rapidly (within minutes) expressed while the maximum of E-selectin expression requires up to 4 hr.757 Uthoff et al tested a leukomedin which prevented CD11b/CD18 expression in a rabbit lung transplant model. This agent does not compete as an antagonist like antibodies. It can inhibit adherence in activated leukocytes and can reverse adherence. Pulmonary injury was reduced by decreased pulmonary edema, decreased pulmonary vascular resistance, decreased loss of compliance and reduced airway pressure.860 Another treatment option has evolved from studies using soluble sialyl LewisX for blocking selectin dependent lung inflammation in different types of lung injury.542 Oligosaccharides as synthetic analogs to sialyl-LewisX are able to reduce allograft reperfusion injury after rat lung transplantation.109 Further studies will have to show the relevance of these treatment options for reperfusion injury in the clinical setting after long ischemic times. Especially, the optimal time point for prophylaxis of reperfusion injury (graft preservation, before reperfusion, after reperfusion) have to be defined. Also, it remains unclear which adhesion molecule in which organ is the best target for therapy. Further investigation is necessary to evaluate whether monoclonal antibodies or other blocking agents, as oligosaccharides, are more effective.

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Acute Allograft Rejection Adhesion molecules play a central role in regulating the infiltration of leukocytes into the graft during rejection. Blocking adhesion molecule interaction seems to be a promising strategy for prophylaxis and treatment of rejection, because of the specificity of this therapy compared to conventional immunosuppressive agents. The development of effective and specific immunologic tolerance in adult animals and humans would solve the problems of managing acute and chronic rejection. The temporal blockade of cell adhesion by monoclonal antibodies leads to specific tolerance in some mouse models. Isobe et al detected donor specific tolerance after a 3 day administration of anti-ICAM-1 and anti-LFA-1 mAbs in a murine heart transplant model.358 Administration of anti-VCAM-1 and anti-VLA-4 mAbs also induces tolerance in the same animal model.357 Combination of anti-ICAM-1 and anti-LFA-1 could also prolong allograft survival after rat heart transplantation in a weak rat strain combination.370,371 However, it was not effective between fully incompatible rat strains as shown by our group.108 Although therapy with antiVCAM-1 and anti-VLA-4 is effective in a mouse strain and a weak rat strain combination, it fails in a strong combination.357,575,589,591,592 A combination of monoclonal antibodies with conventional immunosuppressive drugs seems to be more efficient. In a rat cardiac transplant model prolonged cardiac allograft survival using the combination of low-dose cyclosporine and anti-ICAM-1 therapy was observed.299,400 Combined treatment of monoclonal antibodies to ICAM-1 and LFA1 with donor specific transfusion and FK506 consistently led to persistence graft acceptance even in fully incompatible rat strains.58 Tolerance induction by monoclonal anti-adhesion molecule antibodies seems to be easier in a mouse model than in other animal models. However, anti-ICAM-1 monoclonal antibodies have been shown to prolong heterotopic cardiac allograft survival in cynomolgus monkeys.227 Sadahiro et al showed that administration of anti-CD18 is more effective than anti-ICAM-1 therapy in a rabbit cardiac transplantation model.674 B7/CD28 are another costimulatory factor for T cell activation. CTLA4 is closely related to CD28 and transplantation tolerance can be induced by CTLA4-Ig.52,595 Another therapeutic option, pretreatment of the recipient combined with a short term post-transplantation treatment course, seems to be additional effective.546 In an orthotopic rat lung transplantation model blocking of selectins by a synthetic analog of sialyl-LewisX or by fucoidin, another oligosaccharide, have been shown to prolong allograft survival.107,109 The use of short acting oligosaccharides, substances with potential low toxicity and side effects, seem to be a promising strategy for abrogation of severe acute allograft rejection. Recently, Gerritsen et al showed that flavonoids inhibit cytokine-induced adhesion molecule expression on endothelial cells in vitro and in two mouse models of inflammation.253 Therefore, flavonoids may also be effective for protection of inflammation of the allograft after organ transplantation. The mechanism of action whereby monoclonal antibodies prolong allograft survival is still not precisely understood. The absence of surface expression of ICAM-1 in the donor allograft or the recipient is insufficient to prolong cardiac allograft survival in a mouse model as shown by using an ICAM-1 deficient mouse

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donor pretreatment?

perfusion solution?

recipient pretreatment? short term treatment? long term treatment? Fig. 10.1. Possible therapeutic options for blocking adhesion molecule interaction.

strain for heart transplantation.691 Therefore, administration of anti-ICAM-1 antibodies seems to work not only by inhibiting leukocyte extravasation but also by other mechanisms that remain unclear at the moment. Another option for influencing adhesion molecule expression is on the mRNA level. Stepkowski et al showed that synthetic antisense phosphorothionate oligonucleotides complementary to rat ICAM-1 mRNA inhibit ICAM-1 protein expression in vitro, and block allograft rejection in vivo, when used either for preoperative graft perfusion or as induction or maintenance immunosuppression in a rat heart transplantation model.801 The role of adhesion molecules in regulating allograft response may not only differ between species but also depending on the transplanted organs involved. Immunosuppressive effects of monoclonal antibodies to ICAM-1 and LFA-1 were significantly greater on hepatic than cardiac allografts in the same strain combination in the rat according to allograft survival.295,296

Chronic Allograft Rejection Another problem in the late follow-up of clinical heart transplantation is the chronic allograft rejection known as graft vasculopathy after heart transplantation and bronchiolitis obliterans after lung transplantation. There are only few data about the mechanism of chronic allograft rejection. Repetitive endothelial injury with intimal proliferation, hypertrophy and repair may cause the vasculopathy of the graft.310 The endothelial damage caused by ischemia, host antibodies, antigenantibody complexes and complement leads to accumulation of platelets, fibrin, and activation of the clotting cascade. Injured endothelial cells release thromboxane, platelet derived growth factor (PDGF), leukotriens, platelet-activating factors, and

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express adhesion molecules.287,310 Suzuki et al reported that treatment with antiLFA-1 and anti-ICAM-1 prevented graft arteriosclerosis after cardiac transplantation in mice (Suzuki et al, oral presentation, American Heart Association, 1995). In contrast, Orosz showed that long-term allograft survival is not a result of graft acceptance after anti-VCAM-1 treatment in the same model. Although, this therapy interrupts the destructive progression of acute allograft rejection, it permits chronic rejection..573,574

Future Outlook Today it is well-known that adhesion molecules play an important role for the infiltration of leukocytes into the allograft during reperfusion injury, acute and chronic allograft rejection. A variety of anti-adhesion molecule antibodies and other substances blocking adhesion molecules or inhibiting adhesion molecule expression have been tested in different animal models and organs (Table 10.1). Some of these treatment modalities seem to be a promising strategy for clinical organ transplantation. Further studies are necessary to define optimal agents, optimal route of administration, optimal time point, and duration of treatment (Fig. 10.1).

Part 4 Adhesion Molecules in Xenotransplantation

CHAPTER 11

Adhesion Molecules in Xenotransplantation André R. Simon, Anthony N. Warrens and Megan Sykes

Introduction

D

ue to the recent success of organ and tissue transplantation, the number of potential recipients for allogeneic organs and tissues has outgrown the number of available donors by more than two-fold, with almost 50,000 people on waiting lists throughout the USA. Furthermore, this disparity between available donor organs and recipients is increasing at an annual rate of approximately 10-15%. These facts have sparked a renewed interest in alternatives to allogeneic organ transplantation. One possible alternative would be the use of xenogeneic cells and organs. The swine has been identified as the most suitable possible xenograft donor to humans because of its size, excellent breeding characteristics, immunologic and physiological similarities to humans,154,156,671,672 and because of ethical considerations. While current immunosuppressive protocols give quite satisfying mediumterm results, long-term outcomes after allotransplantation are still not satisfactory. This is due to recurring rejection crises, the currently intractable chronic rejection process, and the deleterious side effects of the immunosuppressive regimens themselves. Furthermore, these regimens are not successful in preventing rejection of discordant xenogeneic organs and tissues.154 Preliminary studies indicate that the level of immunosuppression required after xenotransplantation may be exceedingly high compared to that required after allotransplantation. However, since the severity of side effects is mainly determined by the dose of immunosuppressive drugs administered, a mere increase in the amount of nonspecific immunosuppression is not a feasible option. In contrast to allogeneic organs, xenogeneic transplants in discordant species combinations (such as pig→human) undergo a rejection process termed “hyperacute rejection” which is initiated by preformed, naturally occurring antibodies (XNA) and dependent upon complement activation. In the case of the pig→human combination, these XNA are mainly directed against Gal(α1-3)Gal epitopes, which are widely expressed on pig tissues. Hyperacute vascular rejection destroys the xenograft within minutes to hours after the Cell Adhesion Molecules in Organ Transplantation, Second Edition. edited by Gustav Steinhoff. © 1998 R.G. Landes Company.

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circulation is re-established (for extensive review see ref. 608). Other forms of rejection, which can only be seen if hyperacute rejection is prevented, include delayed (vascular) xenograft rejection,49 which seems to occur mainly in discordant xenogeneic combinations and not in allografts. In addition, both allo- and xenografts are susceptible to antibody-mediated and T cell-dependent cellular rejection. In allogeneic graft rejection, a role for many adhesion molecule-ligand pairs and costimulatory pathways has been identified. However, in xenogeneic transplantation, the importance of many of these interactions might be either decreased or increased, depending on the efficacy with which each receptor-ligand pair interacts in the particular xenogeneic combination being studied. In addition, because of the complex manner in which adhesive interactions are regulated, some receptor-ligand interactions, which play a lesser role in allogeneic settings, might play a greater role in xenotransplantation. The importance of understanding these interactions in the pig-human combination is readily apparent from a consideration of the possible effects of a failure of some interactions to occur. For instance, in the extreme (and unlikely) situation in which host (human) lymphoid cells failed to adhere to xenogeneic vascular endothelium by the selectin-integrin cascade (Fig. 11.1) and thus could neither attack the endothelial cells nor transmigrate across the vascular wall and infiltrate the graft, cellular rejection could not occur. Failure of adhesive interactions in the opposite direction, however (pig leukocyte→human endothelium) would impose severe limitations on the ability to use xenogeneic bone marrow transplantation for the induction of xenograft tolerance (Fig. 11.3), since hematopoietic cell homing and function depends on the efficacy of such interactions. In this article, we shall review those xenogeneic adhesion molecule-ligand interactions and pathways which have been examined so far, and will speculate upon their possible roles in the hypothetical xenogeneic pig→human organ transplantation setting. In order to do this, we must briefly review the different approaches used to facilitate xenotransplantation, as they define the different roles which these interactions might play. For the purpose of simplification, we shall ignore the likelihood that approaches may ultimately have to be combined in order to achieve successful discordant xenografting in man. Instead, only the three basic underlying principles are presented.

The Immunosuppression Approach The xenogeneic organ would be transplanted as allogeneic organs are today, and the patient would receive chronic immunosuppressive treatment to prevent rejection. Even though newly-developed approaches such as the selective blocking of integrin-ligand interactions can greatly prolong graft survival in certain animal models,355,358 it is unlikely that such approaches could be used as sole substitutes for chronic immunosuppressive therapy. Rather, such manipulations might be used as adjuncts to chronic immunosuppression, which, if given at tolerable doses, is likely to be insufficient to permit porcine xenograft survival in humans. An additional limitation of this approach could be the development of anti-antibody responses.

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Fig. 11.1. Ideal xenograft: complete molecular incompatibility. (1) Human leukocyte enters xenograft via bloodstream. (2) Initial contact with endothelial cell surface molecule. (3) Unsuccessful initiation of adhesion cascade due to incompatibility of adhesion molecules and ligands. (4) Failure to adhere: no transmigration, no infiltration, no rejection.

The Genetic Approach Donor pigs would be genetically modified to: (1) express protective molecules (e.g., FAS ligand or DAF67,128,129,526,527,614); (2) not express stimulatory molecules (e.g., αGal, MHC, VCAM-1 or ICAM-1 knock out282,384); or (3) both. While this approach holds great potential, it is very probable that organs derived from such animals would still generate immune responses in the host. In view of the redundancy of available pathways of antigen presentation, cell adhesion and graft rejection, it seems reasonable to expect, at best, a downmodulation of xenograft rejection with such approaches. In addition, this approach may be limited by a lack of viability of pigs bearing certain combinations of alterations.

The Tolerance Approach The immune system of the recipient would be made nonreactive towards the donor by “donor-specific tolerance induction”, thereby abrogating the need for any further treatment, so that the host would be left fully immunocompetent towards other antigens.819 This represents the most desirable situation, as it ensures freedom from rejection and excludes the requirement for chronic immunosuppressive therapy. One approach to induction of such donor-specific tolerance would involve a pretransplantation tolerance-inducing regimen that includes a bone

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marrow transplant to induce stable, mixed chimerism in the recipient’s hematopoietic system, i.e., coexistence of mutually tolerant donor and host lymphohematopoietic cells. While this induction of hematopoietic chimerism has been used successfully in animal models to induce allotolerance,283,673,734,817 and the tolerance-inducing effect of allogeneic hematopoietic cell transplantation has also been described in humans,271,845,846 xenogeneic tolerance induction through hematopoietic chimerism has only been achieved in concordant species combinations.12,348,349,350,450-452,541,581,814,819,842 In addition, while lifelong tolerance can be achieved in both allogeneic and concordant xenogeneic settings, the chimerism achieved in the concordant xenogeneic model was not stable,348,709 reflecting a competitive advantage of host hematopoiesis over xenogeneic hematopoiesis.276,451 One of the elements contributing to this host advantage, in addition to speciesspecificity of some important cytokines,932,933 may be a greater efficacy of homologous than xenogeneic adhesive interactions which are important for hematopoiesis. In this model, tolerance to organs,349,451 and in vitro tolerance in MLR, CML and humoral tolerance persist,556 even as chimerism declines. Skin grafts were eventually rejected (>100 days) in these animals, presumably because of the existence of skin specific antigens, to which hematopoietic cells cannot induce tolerance. In order to successfully and reliably use bone marrow transplantation, porcine hematopoietic progenitors would have to home to the human bone marrow and the thymus. Porcine progenitors would be administered to recipients whose existing T cell repertoire had been depleted with specific antibodies, and who had perhaps received local thymic or a small amount of whole body irradiation. In allogeneic and concordant xenogeneic recipients of similar mild, nonmyeloablative conditioning, tolerance of T cells that develop at subsequent times in the life of the animal occurs within the thymus via a deletional mechanism, resulting from the continuous presence of donor hematopoietic cells in the thymus.843 Several different types of hematopoietic cells have been shown to possess this capacity to induce deletional tolerance in the thymus,334,503,560 but it is unclear which of these is most important in the bone marrow transplantation (BMT) models described above. In this context, it is important to note that thymic dendritic cells and T cells arise from a common lymphoid progenitor.35 Thus, if engraftment of xenogeneic hematopoietic stem cells in the bone marrow can be achieved, a permanent supply of lymphoid progenitor cells with tolerance-inducing capacity would exist. To induce tolerance, it would be essential for these cells to be able to differentiate and migrate to the host thymus, two abilities which both require specific adhesive interactions. While successful engraftment of rat hematopoietic cells and deletional tolerance has been achieved in mice,556,842 success has been more difficult to achieve in the more disparate species combinations such as pig to mouse and pig to monkey. Successful engraftment of porcine progenitors has been achieved in the marrow of both species276,933 (D.H. Sachs, personal communication) and both marrow and peripheral blood chimerism can be enhanced by administration of porcine cytokines.932 Migration of porcine progenitors to the recipient’s thymus, however, has not been achieved in either model. Porcine progenitors would need to be capable of engrafting in both compartments and of differentiating into the appropriate cell types, such as thymic dendritic cells, in order to induce lasting chimerism and intrathymic deletional tolerance.

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The use of the approaches mentioned above (or combinations thereof) to facilitate the transplantation of xenogeneic organs into man leads to different considerations with respect to the roles that adhesion molecules might play, making interactions either desirable or not, sometimes even for the same adhesion molecule-ligand interaction. One of the major advantages of the pig as a xenograft donor results from its excellent breeding characteristics, which make genetic engineering feasible. Indeed, several genetically modified pigs have already been produced.391,614,807 Thus, it is likely that several different genetically engineered swine could be used, for example, as marrow and organ donors to humans, depending on the particular adhesion interactions desired for the engraftment of each tissue. These superb breeding characteristics also allow for extensive inbreeding (D.H. Sachs and S.J. Arn, personal communication),670-672,824 so that the use of various genetically altered pigs need not entail the introduction of different xenoantigens to a recipient with each porcine donor used. The basic cascade leading to infiltration and rejection of organ and tissue grafts (Fig. 11.2) has been well described and is the topic of this book. For xenogeneic organ rejection to occur, human effector cells would have to interact with xenogeneic molecules involved in this process. Of particular importance in this process are the adhesion molecules expressed by vascular endothelial cells, representing the anchor points used by lymphocytes, granulocytes, monocytes, macrophages and NK cells in their adhesion to and arrest on the vascular wall and their subsequent migration into the tissue (Fig. 11.1 and 11.2). While crossreactivity of human effector molecules with their porcine ligands may not be desirable if they contribute to rejection (Fig. 11.1), crossreactivity in the opposite direction might be of considerable importance in allowing porcine hematopoietic progenitor cells to engraft successfully and induce tolerance (Fig. 11.3 and 11.4). In such a setting, porcine bone marrow cells would have to home to and engraft in different microenvironments (e.g., bone marrow or thymus) in the host, a process that is, again, controlled by specific combinations of adhesion molecules and ligands and other molecules expressed by the vascular endothelium, the stroma and the extracellular matrix environment. (Fig. 11.3). As indicated above, the possible roles of adhesion molecules in xenogeneic transplantation are manifold. Depending on the approach used to induce graft acceptance, they might be diametrically opposed, making successful transplantation of porcine organs into man a complex procedure involving several different genetically engineered donors. In the next section, we shall examine some of the specific adhesion molecules, their ligands, and the possible significance of their interactions in xenogeneic transplantation. We will supply detailed information on porcine adhesion molecules and porcine ligands whenever possible. However, many of the porcine analogs to human molecules have not been identified or analyzed in detail, and most data on xenogeneic organ transplantation involve small animal models that can only be of limited relevance to pig→human transplantation.

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Fig. 11.2. Rejection of allograft. (1) Recipient leukocyte enters the allograft via bloodstream. (2) Initial contact with endothelial cell surface molecule. (3) Initiation of adhesion/activation cascade (rolling). (4) Firm arrest on vascular endothelium and activation. (5) Transmigration through endothelial wall and infiltration of organ graft. (6) Rejection.

Specific Adhesion Molecule-Ligand Interactions VCAM-1(CD106)Ligand Interactions In humans, VCAM-1, an Immunoglobulin (Ig) superfamily member, is expressed predominantly on vascular endothelium in a variety of tissues, on bone marrow stromal cells, and on dendritic cells.140,293,361,401,582,583,834 The molecule exists in two alternatively spliced forms, one with 6 and one with 7 extracellular Iglike domains. The 7-domain form of VCAM-1 carries two homologous binding sites for VLA-4 in domains 1 and 4 which contains the IDSP amino acid sequence,149 whereas the 6 domain form, lacking domain 4, only carries one such site.576,577,891 VCAM-1 cell surface density is dependent on the state of activation of the cell. Expression is low on resting cells and can be upregulated by a variety of stimuli, including TNFα, IFNγ and IL-2.77 Porcine VCAM-1 has been analyzed extensively. It shows a similar pattern of expression to its human counterpart (our unpublished results). As in humans, the cell surface density is low on resting cells, and upregulation of expression is inducible by a variety of cytokines (our unpublished results). Cloning of porcine VCAM-1 by Tsang et al849 and Mueller et al537 yielded almost identical nucleotide sequences (seven nucleotide differences, which result in four amino acid changes), thus raising the interesting possibility of polymorphism within the porcine molecule. CD106

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Recipient Cell

Nontolerant Recipient Cell

Recipient Thymus Environment

Allogeneic Cell

Porcine Cell

Chimeric Thymus Environment

Recipient Bone Marrow Chimeric Bone Marrow

Fig. 11.3. Failure to induce central tolerance due to incompatibility of xenogeneic adhesion molecule-ligand interactions. Porcine hematopoietic progenitor cells are transplanted. Two possible scenarios are presented: (a) The xenogeneic cells engraft in the bone marrow of the recipient, thus creating a chimeric bone marrow compartment and supply of thymic lymphoid progenitor cells. However, the xenogeneic lymphoid progenitors fail to home or engraft in the recipient’s thymus due to incompatibility of xenogeneic adhesion molecules and ligands. (b) The xenogeneic hematopoietic cells fail to engraft into the recipient’s bone marrow due to incompatibility of xenogeneic adhesion molecules and ligands. Thus, no supply of thymic lymphoid progenitor cells is created. Either way, no colonization of the recipient’s thymus with xenogeneic cells occurs. Therefore, a chimeric thymic microenvironment is not created and intrathymic tolerance is not induced. No deletional tolerance is induced, and xenogeneic tissue is rejected.

is conserved between species, with a homology of 77% between porcine VCAM-1 and the human analog. The porcine 5 Ig-domain molecule, although one or two Iglike domains shorter than its human 6 or 7-domain counterpart, includes a conserved IDSP amino acid sequence in its domain 1. A variety of MoAbs against human (h) or (p) porcine VCAM-1 have been raised and tested.537,849 Anti-VCAM-1 MoAbs are able to block the adhesion of porcine and human cells to one another in both directions, indicating that porcine ligands can interact with hVCAM-1 and vice versa.194,537,724 The only specific ligand identified for VCAM-1 is CD49d, the α chain of VLA-4, a β1 integrin expressed on all leukocytes except neutrophils.210

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The involvement and importance of VCAM-1VLA-4 interactions in inflammatory responses and in the rejection of allogeneic organs has been well described (Molossi et al 1995 and refs. 220, 328, 530, 537, 599). In fact, firm tethering of circulating lymphocytes on vascular endothelium is mainly mediated by the VLA-4CD106 interaction,22 and blocking of this arrest by antibodies significantly enhances graft survival time574,575 in allogeneic organ transplantation. In a xenogeneic organ transplantation setting, hVLA-4 would have to interact with porcine CD106, which would be expressed and probably upregulated on the vascular endothelial surface of the organ. It has been elegantly shown by Mueller et al537 that this interaction does indeed function across the human lymphocyte→pig EC species barrier, thereby rendering the graft endothelium susceptible to arrest-dependent attack by circulating recipient leukocytes. These authors indicate that pVCAM-1, like the 7-domain isoform of hVCAM-1, has at least two distinctive, nonoverlapping binding sites involved in the attachment of human lymphocytes. They do not show, however, whether these are the same epitopes as those involved in pVLA-4→pVCAM-1 adhesion. Also, it is not clear whether porcine CD106 also exists in a 6- and alternatively spliced 5-domain form, and whether any domains of the porcine molecule other than the first carry the attachment epitopes for human and porcine VLA-4. Nonetheless, the fact that human leukocytes adhere to porcine EC via the hVLA-4pVCAM-1 interaction suggests a potentially promising target for the treatment and prevention of cellular rejection. Dorling et al suggest that the cell surface density of pVCAM-1 on unstimulated porcine endothelial cells can be downregulated in vitro by incubation with human polyclonal globulin.195 This observation was accompanied by a lower level of expression of SLA class I and resistance to complement-mediated cell lysis. The two phenomena do not seem to be functionally connected, however, and it remains unclear whether downregulation of VCAM-1 on the endothelium would have any effect on leukocyte adhesion in vivo or in vitro, in the setting of stimulation with TNFα and other cytokines.

β1 (CD29) IntegrinLigand Interactions with Respect to the β1 Chain The β1 integrin family contains six members (VLA-1-6). Their respective α chains are noncovalently associated with the β1-subunit. Other systems of designation refer to this fact and classify the VLA integrins as α1-6/β1 or CD49a-f/CD29 respectively. Porcine CD29 has not been successfully cloned and sequenced. However, immunoprecipitation has shown that it has a weight of 130 kDa, identical to the human β1 chain. Also, crossreactive anti-human monoclonal antibodies have been identified.141,413,669 Of the VLA integrins, VLA-4, VLA-5 and VLA-6 have been identified as being important in rejection and hematopoiesis. Thus, no discussion of VLA-1, 2 and 3 is presented.

VLA-4(CD49d/CD29, α4β1)Ligand Interactions

VLA-4 is a well-characterized member of the β1 integrin family.318 Like other integrins, VLA-4 exists in several different conformation-dependent states of avidity on the cellular surface. Adhesiveness can be activated by a variety of stimuli, such as phorbol myristate and others.40,362,462 Whereas VLA-4 is the only ligand for

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Recipient Cell

Nontolerant Recipient Cell

Recipient Thymus Environment

Allogeneic Cell

Porcine Cell

Chimeric Thymus Environment

Recipient Bone Marrow Chimeric Bone Marrow

Fig. 11.4. Induction of central tolerance. Allogeneic hematopoietic progenitor cells are transplanted and engraft in the recipient’s bone marrow. Thus, a chimeric bone marrow and a lasting supply of lymphoid progenitor cells is created. Recipient AND donor thymic lymphoid progenitor cells colonize the recipient’s thymus, creating a chimeric thymic microenvironment. In the thymus, donor and host cells differentiate into appropriate APCs and induce central tolerance to donor and host via deletion of host- and donor-reactive thymocytes. Donor-specific tolerance is induces, and subsequently, transplanted organs from the same donor are accepted. No rejection occurs.

VCAM-1, VLA-4 also binds to a ten-amino acid sequence (GPEILDVPST) in the alternatively spliced CS-1 region of the fibronectin A chain.279,907 In its high avidity form, VLA-4 recognizes an even smaller five- to six-amino acid epitope on fibronectin, also containing the LDV amino acid sequence.905 Using a crossreactive anti-human anti-CD49d antibody (HP2/1),413 the porcine VLA-4 α chain has been extensively analyzed, showing a similar pattern of expression and molecular weight to the human molecule (150 kDa human/160 kDa porcine under nonreducing and 130/145 under reducing conditions, respectively). Both Mueller and Dorling showed that HP2/1 was able to inhibit VLA-4-mediated adhesion of hPBL to cytokine-stimulated porcine vascular endothelial cells (PVEC).194,537 In addition, Dorling et al demonstrated inhibition of human T cell proliferation by HP2/1, using PHA and immortalized PVEC as stimulators. These data suggest that the hVLA-4-pVCAM-interaction might not only mediate adhesion, but may also play a role in stimulating the host’s immune response to xenogeneic organs in vivo.

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VLA-4 molecules are expressed on hematopoietic progenitor cells and mediate their adhesion within the bone marrow compartment.722 Indeed, blocking this interaction in long-term bone marrow cultures (LTBMC) profoundly inhibits in vitro hematopoiesis.529 Significant inhibition of CD34+ progenitor cell adhesion to bone marrow stroma and induction of apoptosis by incubation with anti-VLA-4 MoAbs has also been demonstrated.901 There is no conclusive evidence to date, however, that demonstrates that VLA-4-VCAM-1 interactions are involved in thymic homing. In fact, based on their reconstitution experiments using chimeric mice with α4-/- lymphocyte progenitors, Arroyo et al suggest that VLA-4 is not involved in thymic homing.41 Instead, they propose that VLA-4 integrin is crucial to the homing and engraftment of progenitors in the bone marrow. This finding is suggested by the fact that recipient thymi became atrophic after a short period of T cell development and that no B cell development, which is stroma-dependent in its early phase, was seen in recipients of α4-/- marrow. The defect seems to be lymphoid specific, as no defective monocyte or NK cell development was observed. Nonetheless, once progenitors have engrafted into the thymic compartment, they show distinct upregulation and activation of VLA-4 integrins at specific stages of maturation, suggesting the involvement of CD49d/CD29 in the migration process through the thymic extracellular matrix from the cortex to the medulla.162 It is possible, therefore, that porcine tolerance-inducing progenitors may need to interact via their VLA-4 molecules with either or both hVCAM-1 and the CS-1 spliced region of human fibronectin in order to engraft in the bone marrow and the thymus and induce deletional tolerance. Using different porcine and human endothelial monolayers prior to and following cytokine stimulation, we have demonstrated that pVLA-4 does indeed mediate adhesion to hVCAM-1 molecules (our unpublished results). Also, employing different inhibitory peptides and antibodies, we have established the interaction of pVLA-4 to the CS-1 sequence of human fibronectin (our unpublished results).

VLA-5(CD49e/CD29, α5β1)Ligand Interactions VLA-5, a member of the β1 integrin family, is expressed on the majority of leukocytes and on other cell types.746,906 It mediates adhesion to a binding domain in the 120 kDa fragment of fibronectin that is spatially distinct from the VLA-4 binding site, and that contains the amino acid sequence RGD.531,532,628 Like other integrins, it exists in different states of adhesiveness and can be activated by a variety of stimuli.462,498 The molecule has not been cloned or sequenced in the pig, but MoAbs to human VLA-5 have been identified which crossreact with a porcine cell surface molecule.413 While no involvement of VLA-5 in rejection of allogeneic tissues has been shown to date, and the crossreaction or blocking of VLA-5 adhesion to its xenogeneic ligand may not have a major impact on rejection of organs, the molecule has been implicated in the intrathymic development of T cells. Crisa et al have demonstrated, using in vitro adhesion and migration assays, that VLA-5 integrin is present in an active, high avidity state on developing thymocytes at a distinct stage of their maturation.162 Therefore, it is possible that the interaction of pVLA-5 with human ligands might play a significant role in the induction of donor-specific tolerance in the human thymus. However, in an in vitro allogeneic mouse T cell differentiation model, expression of functional VLA-5 was found not

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to be obligatory for the development of thymocytes into T cells, as demonstrated by successful in vitro differentiation of β1 integrin deficient progenitors,330 derived from yolk sacs of 10.5-day-old chimeric embryos.

VLA-6(CD49f/CD29, α6β1)Ligand Interactions VLA-6 expression has been demonstrated on cells in the hematopoietic lineage, as well as on endothelial cells and other cell types.329,506,654,662,737,874 The molecule has been extensively analyzed in humans188,335,823 and mice,327 but it has not been cloned or completely sequenced in pigs. However, Fujiwara et al have identified POG-2 as a putative porcine homologue to human VLA-6.243 We have identified an anti-hVLA-6 MoAb which shows crossreactivity to porcine tissues (our unpublished observations). Using this antibody, we have demonstrated similar staining patterns on porcine hematopoietic and endothelial cells compared to human cells. VLA-6 molecules have not been implicated in the rejection of allogeneic organs and tissues, so there is no indication that they would influence the rejection process in a xenogeneic transplantation setting, even if they should be shown to be fully crossreactive across species barriers. However, xenogeneic VLA-6-ligand interactions may be of greater relevance to the approach of using hematopoietic cells for the induction of tolerance. Using a mouse BMT model, Ruiz et al demonstrated that administration of anti-CD49f MoAbs reduced the number of re-colonizing thymocytes in recipient thymi by 80%, while proliferation of thymocytes in in vitro thymic organ cultures was not affected.664 Based on the observation that VLA-6 integrin was expressed on the luminal side of the thymic microvascular endothelium, they suggested that this molecule might play a role as an organ-specific “addressin” in the homing of hematopoietic progenitors. However, the expression of the CD49f, and therefore the VLA-6, molecule on the endothelium is not necessarily essential for thymic progenitor homing. This was demonstrated by Hirsch et al, who showed that bone marrow cells from hematopoietic CD29-deficient, chimeric donors, were not able to home and engraft into wild-type recipient thymi.330 Differentiation of β1-deficient cells was unaffected, however, as demonstrated by normal differentiation of these cells in in vitro cultures. Therefore, in a tolerance-inducing xenotransplantation model, porcine VLA-6 on swine hematopoietic progenitors may have to interact with human ligands on thymic endothelial cells. We are currently conducting studies in order to determine whether pVLA-6 can successfully interact with human laminin and human thymic vascular endothelium.

ICAM-1(CD54)Ligand Interactions ICAM-1 molecules (for excellent review see ref. 863) are found on a variety of cells, including hematopoietic progenitors, leukocytes, endothelial linings and stromal cells.104,203,470,723 The molecule is expressed constitutively on some cell populations, and expression can be induced by cytokine stimulation on others.863 ICAM1 is a transmembrane glycoprotein belonging to the Ig supergene family that consists of five Ig-like domains, a membrane spanning domain and a cytoplasmic tail. ICAM1 has been cloned in humans, rats, mice and dogs and a limited sequence homology of ~60% of animal to human cDNA has been shown.395,494,731,892 Although LFA1 (CD11a/CD18) is the dominant ligand for ICAM-1,496 Mac-1 (CD11b/CD18) has

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been shown to interact with another, distinct binding site on the molecule.190,191 In addition, CD43 has been identified as a third ligand to ICAM-1.655 The role of ICAM1 in leukocyte to EC adhesion and in the rejection of allogeneic organs has been well established in different animal models159,358 and in vitro.190 There has also been a number of studies suggesting a role of ICAM-1 in rejection in concordant xenogeneic transplantation models.264,292,525,563 Based on one-way MLR experiments using irradiated pPBL as stimulator cells and adding either anti-hICAM-1 or antihLFA-1 α chain antibodies to the primary cultures, Herrlinger et al suggested that hICAM-1→pLFA-1 and hLFA-1→pICAM-1 interactions have a costimulatory effect in vitro.324 A similar costimulatory effect of ICAM-1 has been described by Damle et al.173 Therefore, blocking of ICAM-1 on the endothelial surface of a transplanted organ might improve the outcome of xenogeneic pig→human organ transplantation. It remains unclear, however, whether human effector cells can actually adhere to pICAM-1 molecules, and what the effect of blocking this interaction in vivo might be. No antibody has been described which recognizes porcine ICAM-1 molecules. We are currently further elucidating the interactions of porcine and human ICAM1 with their respective xenogeneic ligands. We have shown that porcine lymphocyte adhesion to hICAM-1 can be blocked by anti-hICAM-1 MoAbs in a static adhesion assay (our unpublished data). CD43, the third ligand of ICAM-1, is expressed on hematopoietic progenitor cells655 suggesting that ICAM-1 might also play a role in hematopoiesis. However, no such involvement of ICAM-1-CD43 interactions has been demonstrated so far. Fine et al, based on an in vitro thymic organ culture model, suggest that LFA-1 is a required ICAM-1-ligand for normal T cell maturation..225 In view of the findings of Dean et al185 in ICAM-1 knockout mice, however, an essential role of ICAM-1 for in vivo hematopoiesis is unlikely. Further studies are underway to examine h/pICAM-1-xenogeneic ligand interactions in more detail.

ICAM-2(CD102) and ICAM-3(CD50)Ligand Interactions The interactions of ICAM-2 and -3 in xenogeneic organ transplantation have not been addressed so far. However, both molecules do interact with LFA-1 and therefore might play a role in rejection.127,218,675,704 Presently, ICAM-2LFA-1 interactions are thought to play a role in activation-induced cell death and NK cell activation.316 It is well established that murine LFA-1 interacts with hICAM-2 and -3, while no such interaction was demonstrable for ICAM-1.197 Since the pig is phylogenetically more closely related than mouse is to man, conservation of cross-species reactivity of ICAM-2 and -3 in the human→pig xenoreaction seems probable. Cross-species reactive antibodies against ICAM-2, but not against ICAM-3, have been identified.413

β2 (CD18) IntegrinLigand Interactions with Respect to the β2 Chain Three β2 integrins have been identified: LFA-1, Mac-1 and LeuCAMc. Their respective α chains, αL, αM and αX are noncovalently associated with the 95 kDa β2 chain, a highly conserved molecule, which has been cloned in humans as well as a variety of animals, including pigs (>80% amino acid homology between humans and animals).80,394,449,716,917 A role for the β2 integrins has been implicated in many areas connected to allo- and xenogeneic transplantation, including cell activation

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and differentiation, adhesion, extravasation and transendothelial migration, as well as effector mechanisms. While many functions of β2 integrins are independent of other leukocyte surface molecules or are themselves regulatory, firm adhesion involving CD18 integrins is L-selectin-dependent.884 There is good evidence for direct involvement of the β chain in at least some of the functions of β2 integrins.50 Notably, leukocyte adhesion molecule deficiency in humans is caused by abnormal splicing of the β2 chain.38,550 A number of antibodies have been identified which react to porcine CD18.141,413,669 As LFA-1 constitutes the member of the β2-family which has been shown to be most heavily involved in rejection processes in transplantation, we shall concentrate the remainder of the discussion of β2 integrins on this molecule.

LFA-1(CD11a/CD18)Ligand Interactions The CD11a/CD18 member of the β2 integrin family is expressed on the surface of most leukocytes.414,415 LFA-1 binds to different epitopes on ICAM-1 (CD54), ICAM2 (CD102) and ICAM-3 (CD50).81,127,180,181,191,208,494,675,750 Both the α chain (CD11a) and the β chain (CD18) participate in the binding.50 Also, LFA-1 has been shown to mediate binding of neutrophils to type I collagen.248 The molecule is expressed in different states of avidity and affinity on cellular surfaces and can be activated on leukocytes through inside-out signaling, following treatment of cells with PMA, cross-linking of T cell receptor/CD3, or a variety of other stimuli (Driessens et al 1997). Meerschaert et al demonstrated the involvement of CD11a/CD18 in leukocyte attachment and migration through endothelium.511,512 In transplantation, LFA-1 molecules are involved in the attack on donor tissues; the attack itself subsequently results in further upregulation of the target ligands ICAM-1, 2 and 3. Blocking antibodies to LFA-1 have been shown to significantly increase organ graft survival time in allogeneic and concordant xenogeneic transplantation models296,358,380,525,547,557,589,591,807,931 and have also been implicated in the in vitro stimulation of hPBL proliferation.324 Pleass et al have demonstrated LFA-1-mediated adhesion of human PBL to cultured porcine renal epithelium in vitro.610 Together with the data obtained in ICAM-1 blocking experiments, the data suggest that blocking of LFA-1 in graft recipients might be superior to blocking of ICAM-1 molecules, as the former would interfere with adhesive interactions of LFA1 with all its potential ligands. Isobe et al obtained the best results in their murine cardiac allograft model with a combination of both LFA-1 and ICAM-1 blocking MoAbs. As mentioned above, Fine et al suggested a possible role for LFA-1 in thymocyte maturation.225 Although they only show a reduction of in vitro T cell development in the presence of anti-ICAM MoAbs, the authors mention that blocking of LFA-1 has a comparable inhibitory effect on T cell development. However, the results of Dean et al185 suggest that LFA-1-ICAM-1 interactions are not essential to intrathymic T cell development. Overall, the possible role of this interaction in thymic progenitor homing and tolerance induction remains unclear. Pig-specific antibodies against CD11a have been described.448

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CD44Ligand Interactions CD44 is a ubiquitous cell surface glycoprotein that is expressed in at least ten different isoforms generated by alternative splicing. It is essentially comprised of four domains, including a highly conserved cytoplasmic domain, a transmembrane domain, a membrane-proximal domain, which contains the spliced exons, and the highly conserved amino-terminal domain.945 It binds to three repeating disaccharide residues on hyaluronic (HA) polymers,857 and the binding region seems to be contained within the amino-terminal two-thirds of the molecule, as shown by successful binding to HA of mouse T cell lymphoma AKR1 transfectants lacking the membrane-proximal domain.309 In addition, there are data suggesting that CD44 may interact with other ECM proteins such as collagen I and VI, as well as fibronectin and laminin.215,364,748 Some of these interactions seem to be dependent on the presence of covalently attached chondroitin sulfate side chains.748 CD44 is expressed at different levels by virtually all cells of the hematopoietic lineage and by a variety of nonhematopoietic cells.460 It has been suggested that it plays a role in the homing of prothymocytes to the thymus461,920 and in the migratory processes of differentiating thymocytes through the “thymic anlage”. On human peripheral lymphocytes, CD44 acts as a homing receptor to high endothelial venules,363 apparently playing a similar role to murine L-selectin. Interestingly, there is evidence that the binding site for this interaction is contained in the membraneproximal region of CD44, and no dependence on HA for this adhesion has been demonstrated. Camp et al133 have shown that blocking of CD44 molecules on infiltrating lymphocytes resulted in inhibition of a cutaneous delayed type hypersensitivity reaction, and have suggested that CD44 may be required for extravasation into inflammatory lesions. Porcine CD44 has not been studied extensively. It has been immunoprecipitated, showing a weight of 80 kDa and crossreactive and pig-specific MoAbs have been identified.669,930 With respect to xenogeneic organ transplantation, the role of CD44 and its crossreactivity have not been addressed. However, given the fact that HA is the main ligand, it seems reasonable to expect effective cross-species interactions, provided that the appropriate signals that upregulate the adhesiveness of CD44 to HA are given.460 The molecular interactions that induce this increased adhesiveness, which seems to be dependent on altered glycosylation of CD44, are as yet undetermined. There are data suggesting that interaction of donor prothymocyte CD44 with its ligands might be necessary to allow engraftment into the recipient’s thymus.461 It has also been demonstrated in LTBMC’s that antiCD44 MoAbs inhibit hematopoiesis.528 Not surprising, our own studies have demonstrated a similar effect of anti-CD44 MoAbs in xenogeneic LTBMC culture systems involving porcine hematopoietic progenitors and human stromal layers (our unpublished observations). Anti-human CD44 treatment, on the other hand, might enhance engraftment of hematopoietic progenitor cells into the recipient, as suggested by Rossbach et al, who demonstrated enhanced engraftment of donor bone marrow in a canine BMT model if anti-CD44 antibodies were administered to the recipient prior to BMT.657 Based on their in vitro findings, the authors suggest a role for CD44 in inducing engraftment-facilitating changes in the bone marrow microenvironment of the recipients. In addition, blocking of CD44 might protect

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xenogeneic organs from effects such as postischemic inflammation, which might initiate downstream rejection processes. Finally, humoral responses to porcine tissues might be downregulated by CD44 blocking, as suggested by the observation that B cell lymphopoiesis in vitro could be inhibited by blocking of the interaction of progenitor cell CD44 with stromal HA.528

Selectin (CD62)Ligand Interactions Three selectins (CD62L, E and P) have been identified thus far (for excellent review see ref. 373). L-selectin (CD62L) is expressed on most cells of the hematopoietic lineage. P-selectin (CD62-P) can be found on platelets as well as endothelium, whereas E-selectin (CD62E) expression is restricted to endothelial cells, principally in response to inflammatory stimulation. All selectins are type I proteins, composed of a NH2 terminal C-type lectin domain, an epidermal growth factor (EGF)-like domain, a variable number of short consensus repeat (SCR) domains, a transmembrane domain, and a cytoplasmic tail. The C-type lectin domain and the EGF domain are highly conserved between species, as shown by a ~72% and ~60% amino acid homology of each lectin between species. The SCR domain shows less homology (~35-40%). Also, while L-selectin contains two SCR domains in all species described, the number of SCR domains in E- and P-selectin varies between four and nine. The membrane spanning and cytoplasmic domains are also conserved between species, but no homology can be seen between individual selectins. The conserved nature of the selectin molecules and the observed crossreactivity (see below) suggest that interference with any selectin-ligand interaction might inhibit graft rejection, even in xenogeneic combinations. E-selectin is involved in primary, loose attachment (rolling) of lymphocytes on vascular endothelium at sites of inflammation and rejection.1,440,566 It has been demonstrated by Yan et al,926,928 using human skin and leukocyte transplantation to SCID mice, to be essential for homing of leukocytes to the skin. However, no suppression of leukocyte recruitment to other compartments is seen unless Pselectin is blocked simultaneously.417 Blocking of both E-selectin and P-selectin interactions might protect implanted organs from leukocyte infiltration, as suggested by the demonstrated high level of CD62E expression in allogeneic organs undergoing rejection.20,113,829 Porcine E-selectin has been extensively analyzed and cloned by Tsang et al, who demonstrated 71% homology between the human and porcine cDNA.848 The porcine molecule contains two fewer SCR domains than the human CD62E. The porcine molecule supported adhesion of human neutrophils, suggesting that crossspecies reactivity is retained. Upregulation of porcine E-selectin was observed on porcine endothelial cells after stimulation with IL-1α and TNFα.848 In addition, using a chimeric fusion molecule consisting of the porcine lectin domain and the human EGF domain linked to the constant region of human IgG, pCD62E was shown to recognize granulocytes and a subset of lymphocytes.848 Allen et al demonstrated binding to human cells in vitro and in situ of a second chimeric fusion protein comprised of the porcine lectin and EGF domains linked to human IgG, thus corroborating the data on the observed crossreactivity of CD62E.19 Finally, Rollins et al showed increased expression of E-selectin on PVEC coupled to an increased adhesion of human neutrophils after stimulation of PVEC with hTNFα.645

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Porcine L-selectin has not been analyzed. The molecule is known to be involved in physiologic lymphocyte recirculation as well as in rolling of leukocytes on endothelium. Blocking of L-selectin-ligand interactions has been shown to significantly inhibit this attachment in vivo in mice (~80% inhibition).465-468,884 In transplantation settings, L-selectin has been shown to play a role in reperfusion injury.411,485,518 These data suggest that blocking of L-selectin-ligand interactions might protect in the earliest phases of organ engraftment. Whether such blocking would have any protective effect on xenogeneic organs, however, remains to be seen. Although L-selectin can be found on hematopoietic progenitor cells, the molecule is not essential for lymphocyte differentiation.34 In the use of BMT for tolerance induction, therefore, no major role for CD62L may be expected. P-selectin has recently been cloned in the pig.644 The molecule showed 81% amino acid homology to human P-selectin and supported binding of human neutrophils. P-selectin is mainly involved in the earliest rolling of leukocytes on vascular endothelium.465 Blocking or absence504 of P-selectin does not inhibit leukocyte extravasation, but only leads to delayed recruitment of leukocytes to sites of inflammation. Furthermore, it has been implicated in playing a role in hemostasis and wound healing.579,809 As mentioned above, the simultaneous blocking of Eand P-417 or L- and P-selectin103 has profound effects on leukocyte infiltration. These data suggest that in xenogeneic transplantation, a combination of pig and human L-, E, and P-selectin blocking might have a protective effect on the xenograft. Cross-species-reactive antibodies against all selectins have been identified.19,196,413

Additional Molecules: SCF, CD34, B7 Other adhesion and costimulatory molecules (e.g., B7) may also contribute to the regulation of an immune response towards xenogeneic tissues, and may significantly influence xenograft survival. Furthermore, in the induction of donorspecific tolerance, molecules such as CD34 and SCF, as well as cytokines, may be of great importance, as they ensure organ-specific homing of and cell line-specific differentiation of progenitor cells. Therefore, we shall discuss some of the identified receptor-ligand interactions in these categories, as their efficacy may be essential to the induction of donor specific tolerance.

Stem Cell Factor (SCF, c-kit ligand)Ligand Interactions Stem Cell Factor is the identified ligand of the c-kit receptor,251 a molecule expressed on hematopoietic progenitors947 and thymic lymphoid progenitors.921 SCF is expressed in a soluble and a membrane-bound form. The latter is predominately found on bone marrow stromal and endothelial cells.315,429 SCF has been successfully cloned in humans and a variety of animals including pigs, and a high degree of amino acid homology has been observed between pigs and the other species.202,270,430,497,600,941-943,946). However, interactions between human or porcine SCF and the ligands of the other species are much less effective than the interactions between the homologous receptor ligand pairs (D. Emery, Massachusetts General Hospital, Charlestown, MA; personal communication). Ligation of c-kit-ligand to the c-kit receptor is essential for the normal differentiation and proliferation of hematopoietic cells.27,332,643,841,947 In addition, a number of studies have suggested a role for SCF in the intrathymic differentiation of lymphoid progenitors.643

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Although c-kit-ligand has not been implicated in rejection processes, it has recently been identified as a potential addressin, regulating the avidity state of VLA-4 and 5 on surfaces of progenitor cells.405 As such it may facilitate organspecific entry of cells into the bone marrow and thymic compartment. Therefore, interactions between porcine SCF and human c-kit may be relevant for the tolerance induction approach. We are currently undertaking studies to examine the role of these interactions in homing of porcine hematopoietic progenitors to human thymi.

CD34Ligand Interactions CD34, a sialomucin, is expressed on hematopoietic progenitor cells and on vascular endothelium.61,224,721,798 It has been successfully sequenced and cloned in humans, pigs, mice and dogs407,507,718 (R. Hawley, BioTransplant Inc., Charlestown, MA; personal communication). The molecule has been implicated in the homing of hematopoietic progenitors to bone marrow stroma,312 and L-selectin has been identified as a ligand for CD34.60,61 No crossreactivity of the porcine molecule to human ligands or vice versa has been demonstrated. Also, to date, no crossreactive MoAbs have been identified.

B7(CD80, CD86)Ligand Interactions The B7 molecules B7.1 (CD80) and B7.2 (CD86) are important costimulators for T cell activation. They are expressed on a variety of antigen presenting cells (APC). The molecules belong to the immunoglobulin superfamily and contain one variable and one constant extracellular Ig-like domain.594 B7.1 is expressed late after APC activation, whereas B7.2 is expressed constitutively on some APC, showing upregulation of expression early following APC stimulation. B7.1 has been cloned in the human, rat and mouse,368 and shows a moderate amino acid homology between species. Thus far, CD86 (B7.2) has been cloned in humans mice and pigs.232,233,486 The interactions of B7 with its ligand CTLA-4 have been shown to play a significant role in the regulation of T cell activation in allo- and xenogeneic systems.89,272,294,486,680,692 While the predominant effect of B7-CD28 interactions appears to be costimulatory with T cell receptor ligation, B7-CTLA-4 interactions downregulate T cell activation.410,425,666,803,898 In addition, using B7.1 transfected melanoma cells Turcovski-Corr et al demonstrated a role for CD28CD80 interactions in the regulation of T cell adhesiveness. The interaction of human T cells with porcine B7 has been studied by Restifo et al and Dorling et al who demonstrated the interaction is functional across the species barrier and plays a role in primary and secondary human-anti-pig MLR.194,636 Thus, blocking of porcine cell surface B7 molecules might have a protective effect on implanted porcine tissues. In addition, B7ligand interactions have been shown to play an important role in graft rejection in a number of animal models, including human islet→mouse xenografting.11,91,634,666,803,850 Relevant to the approach of tolerance induction, Jones et al have demonstrated that B7CD28 interactions are not required for intrathymic deletion.367 However, a role for B7-ligand interactions in the induction of tolerance has been suggested by others.426,459 In xenogeneic pig→human transplantation, successful interaction of pB7 with human ligands might therefore, be necessary for induction of donorspecific tolerance.

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PECAM-1 (CD31)Ligand Interactions PECAM-1 (CD31) is a typical member of the Ig-superfamily. The molecule is expressed on cells of multiple lineages, including platelets, leukocyte subsets, hematopoietic progenitors and endothelial cells.15,98,392,476,515,553 The molecule has been extensively analyzed and cloned in humans and mice,21,553,717,805,927,929,934 and contains an extracellular domain comprised of six Ig-like domains, a short membranespanning region and a complex, alternatively spliced intracellular domain.51,927 Intraspecies polymorphism of CD31 has been described.64,554 While much is known about the PECAM-1 molecule, definitive information about the functional pathways and ligands of CD31 interactions is still lacking. Heterotypic ligands for CD31 have been identified,82,120,186,540,624 but there is good evidence that the molecule’s main ligand is CD31 itself and that it is mostly involved in regulating the affinity of integrins and permitting transendothelial migration at intercellular junctions,74,96,187,218,603,604,684,812,824 (for models of PECAM-1 function see ref. 552). Thus, it has been suggested that CD31 plays an important role in leukocyte extravasation at sites of inflammation,95,539 and several groups have suggested a possible role for PECAM-1 in inflammation and rejection of allogeneic tissues.96,653,873 Blocking of PECAM-1 interactions has been shown to significantly reduce the recruitment of leukocytes to the peritoneal cavity in an experimental peritonitis model.95 In addition, Gumina et al demonstrated a reduction of reperfusion injury in rat and cat models of myocardial infarction by blocking of CD31 interactions.280,281 Since PECAM-1 is expressed on hematopoietic progenitor cells,43,209,443,476,903 a possible function in hematopoiesis and progenitor cell homing has been suggested. Also, endothelial CD31 might be involved in homing of lymphoid progenitors, as suggested by Vainio et al, who demonstrated increased binding of T cell progenitors to PECAM-1-expressing endothelial cells after cross-linking of HEMCAM, another adhesion molecule found on murine hematopoietic progenitors.862 If hCD31 interacts with pCD31 and the other putative porcine ligands, blocking of CD31 interactions might inhibit rejection of xenogeneic organs. Xenogeneic hematopoiesis, on the other hand, might be decreased in the absence of successful cross species CD31-ligand interactions. Porcine CD31 has not been cloned. Also, no fully crossreactive or pig-specific MoAbs have been identified.

Comments It is apparent that the efficacy of many adhesive interactions relevant to xenogeneic organ transplantation still remain to be addressed. One of the main problems in this emerging area of research is the limited availability of well-defined pig-specific or human/pig crossreactive reagents. Consequently, while a number of molecules have been implicated in the differentiation of hematopoietic progenitor cells in homologous systems, little or nothing is known of the interactions across species barriers. Molecules in this category include vanin,44 hemonectin134 and thrombospondin.481 In addition, many of the known xenogeneic interactions have been established in discordant or concordant rodent models or arise from studies in human→mouse tumor transplantation. Since each species expresses a

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different form of any given adhesion molecule, such data can only be interpreted as being relevant to the particular species combination studied, and cannot necessarily be extended to the pig-to-human xenotransplantation combination. We have also attempted to emphasize that the particular approach (e.g., tolerance induction) taken towards facilitating successful transplantation of viable porcine tissues and organs into humans greatly influences the desirability of the effectiveness of specific xenogeneic adhesion interactions in pig→human transplantation. An adhesion molecule interaction may not only play a role in adhesion, but may actively transmit signals to leukocytes and endothelial cells, thus modulating immune and inflammatory responses as well as homing and development. A deeper understanding of these phenomena in the pig→human context will be critical for the development of genetically engineered porcine donors appropriate for xenografting to humans. Because of the species specificity of many reagents, selective intervention with the interactions on either the human or the porcine side may be possible, allowing for greater specificity and efficacy than is possible in allotransplantation. For instance, blocking of porcine but not hVCAM-1 molecules may protect an organ xenograft, while the interaction of pVLA-4 with hVCAM-1, which may participate in the induction of xenogeneic, donor-specific tolerance following BMT, would be left unaffected. Finally, it is well established that there are marked differences in glycosylation between proteins of humans and swine. Indeed, this constitutes the basis of hyperacute rejection. Yet sugar moieties are also important in cellular adhesion, as demonstrated by Tavassoli et al,9,10,379,828 who have identified galactosyl and mannosyl moieties in the bone marrow stroma as “receptors” for progenitor cells. It is not clear whether such interactions are disrupted by species-specific differences in glycosylation patterns or whether there are relevant differences between pigs and humans in glycosylation in addition to the ones that have been identified to-date. The induction of tolerance via stable hematopoietic chimerism undoubtedly relies on more than crossreactivity of adhesion molecules. This approach will probably require many additional interactions, such as those between cytokines and their receptors. It seems likely that not all of the necessary links in the hematopoietic chain will be functional across any species barrier. However, once the critical missing links have been identified, genetic engineering should allow the development of pigs that can be used for donor-specific tolerance induction and subsequent organ donation to humans. Xenotransplantation holds enormous potential. However, before successful transplantation of organs and tissues from pigs to humans can be achieved, a much more detailed understanding of the critical molecular interactions involved in xenogeneic graft rejection and tolerance induction is needed. Such knowledge could then be exploited to make pig-to-human xenografting a clinical reality.

Acknowledgments We thank Drs. David Cooper and John Iacomini for critical review of the manuscript, and Ms. Diane Plemenos for expert secretarial assistance.

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Index A Accelerated arteriosclerosis 132, 134 Accessory cell 4, 6, 26, 89, 93, 119, 121, 122 Acute allograft rejection 101, 127, 128, 139, 141 AF&F 105685 75 Antibody 41, 44, 51, 52, 55, 56, 59, 73, 74, 79, 107, 119, 120, 138, 140, 146, 153, 155, 156 Antioxidant pyrrolidine dithiocarbamate 75, 118 Antisense phosphorothionate oligonucleotides 140 Antithrombin III 117

B B7 5, 30, 58, 59, 63, 95, 118, 119, 139, 160, 161 Bile 9, 89-93, 95-97, 99, 100, 102-104, 108, 110, 111, 127, 135 Bronchus associated lymphoid tissue (BALT) 125

C C-kit-ligand 160, 161 Castanospermine 75, 118 CD2 5, 6, 13, 17, 18, 21, 29-31, 45, 46, 66, 79, 93, 96, 98, 106, 112 CD4 4, 5, 12, 17, 29, 30, 74, 94, 118, 124, 126 CD11/CD18 111, 112, 118, 120 CD11b/CD18 45, 69, 137, 138, 155 CD11bc 138 CD18 15, 30, 32, 45, 56, 69, 74, 75, 79, 81, 118, 120, 137-139, 155-157 CD28 5, 30, 118, 119, 139, 161 CD31 6, 17, 29, 30, 31, 51, 69, 79, 80, 86, 97, 98, 100, 118, 162 CD34 31, 33, 34, 65, 71, 154, 160, 161 CD44 12, 14, 17-19, 21, 31, 33, 34, 52, 53, 65, 71, 79, 83, 85-88, 99-101, 103, 104, 106, 107, 158, 159 CD51 13, 22, 30, 67, 70, 79, 81, 83, 87, 98, 99, 100, 102, 106-108, 110 CD62 6, 19, 79, 83, 85, 98-100, 102, 103, 106, 108, 112, 159 Cell migration 4 Chimerism 10, 122, 148, 163 Cholangitis 90-92, 96, 99, 100, 102, 103, 106

CMV 12, 39, 54, 60, 77, 91, 92, 99, 123135 CR3 45, 79, 81, 97, 98 CTLA4 118, 139

E E-selectin 6, 12, 21, 31-33, 50-54, 56, 70, 71, 73, 85, 87, 102-104, 107, 108, 117, 131, 133, 137, 138, 159 ELAM-1 6, 18, 19, 21, 31-33, 79, 85, 94, 98-102, 106, 112, 128-131 ESGL-1 31, 33, 70

F FK506 119, 139 Flavonoids 139 Fucoidin 32, 118, 139

G Gal(α1-3)Gal epitopes 145 Graft arteriosclerosis 141

H HECA452 31, 33, 79, 83, 85, 99, 100, 103, 106, 108, 111 Hepatitis B 92, 101, 107 HLA-A 25, 26, 28, 30, 57-61, 63, 64, 8993, 95 HLA-B 25, 26, 57, 58, 60, 61, 64, 90, 93 HLA-C 26, 64, 90 HLA-D 26 HLA-DM 26 HLA-DN 26 HLA-DO 26 HLA-DP 26, 39, 40, 58-60, 62, 63, 90-92 HLA-DR 12, 26, 28, 30, 38-40, 57-60, 62, 73, 89-95, 118, 125, 127, 130, 134 HLA-DV 26 HLA-DX 26 HLA-DZ 26 Hyperacute rejection 145, 146, 163

I IFNγ 19, 33, 92, 108, 112, 119, 126, 128, 130-132, 150 IL-1α 130, 159 IL-1β 11, 108, 130, 131 IL-6 130 Immunoglobulin supergene 25, 27-31, 33, 66, 96, 131

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Integrin 6, 12, 13, 15, 16, 18-23, 27-30, 32-34, 41, 42, 45, 48, 67-70, 78, 79, 81, 82, 87, 94, 97, 107, 108, 110, 131, 137, 146, 151, 152, 154-157, 162 Interstitial pneumonitis 133 Intravascular rolling 12, 14-16 Ischemia/reperfusion injury 55, 56 ITO-cells 23, 108, 110

K Kupffer cell 4, 10, 11, 17, 89-93, 95-97, 99, 101, 102, 104, 106-108, 110, 121

P P150/95 45, 81, 97, 98 Passenger cells 4 PECAM 6, 17, 29-31, 47, 50, 51, 69, 79, 80, 86-88, 97, 98, 106, 108, 162 Platelet derived growth factor (PDGF) 73, 140 Primary biliary cirrhosis 92 Pro-inflammatory cytokines 108 Prostaglandin E1 112, 118 PSGL-1 31, 33

R L

RS 61443 75, 119

L-selectin 5, 6, 12, 21, 30-33, 52, 53, 71, 85, 102, 104, 108, 131, 137, 138, 157161 LECAM-1 7, 21, 31, 32, 85, 98, 102, 104, 108 Leukocyte activation 15, 16, 21, 112, 120, 124 Leukocyte adhesion deficiency (LAD) syndrome 33 Leukotriene C4 (LTC4) 19, 108 LFA-1 5, 6, 13, 14, 17, 18, 20-22, 30, 32, 43, 45, 48, 55, 56, 68, 69, 74, 79, 81, 96-98, 104, 106-108, 112, 129, 131, 139-141, 156, 157 LFA-1 (CD11α/CD18) 45, 69, 137, 155, 157 LFA-3 5, 6, 9, 11-13, 17, 18, 21, 29-31, 45, 66, 78-80, 87, 88, 94, 96, 98-100, 106, 111, 112, 118-120 Lymphoid progenitor cell 148, 150, 153

T cell antigen receptor (TCR) 4, 5, 7, 14, 17, 29, 30, 93, 94 Thrombin 19, 33, 71, 108 TNFα 11, 108, 112, 128, 130, 131, 150, 152, 159 Tolerance 10, 55, 74, 121, 122, 139, 146150, 153-155, 157, 160, 161, 163 Transplant coronary artery disease 9, 73 Transplantation-associated arteriosclerosis 130, 134

M

U

Mac-1 5, 14, 17, 18, 21, 30, 43, 45, 69, 81, 97, 98, 104, 107, 108, 129, 131, 155, 156 MAdCAM-1 30, 31 MHC class I 5, 12, 30, 64, 78, 85, 87, 9395, 111, 124, 126, 127, 131, 133 MHC class II 5, 17, 30, 48, 62, 85, 90, 93, 95, 111, 120, 126, 127, 130, 132, 133

Urinary sCAMs 54

N NCAM-1 47, 51 NK cell 5, 6, 14, 51, 68, 79, 96, 110, 149, 154, 156 NPC15669 75, 118

S Selectin (CD62) 108, 159 Sialyl Lewisx 5, 12, 31, 33, 65, 70, 71, 79, 131, 138 Soluble adhesion molecule 74, 112, 121 Stem cell factor (SCF) 160, 161

T

V VCAM-1(CD106) 30, 150 VLA-1 6, 13, 22, 30, 41, 42, 44, 67, 69, 79, 81, 87, 97-100, 104, 106, 107, 110, 152 VLA-2 30, 41, 42, 44, 67, 69, 79, 81, 87, 88, 97, 98, 100, 104, 110, 152 VLA-3 30, 41, 42, 44, 69, 79, 81, 97, 98, 102, 106, 110, 152 VLA-4 5, 6, 13, 14, 17-19, 21, 22, 29, 30, 32, 41, 42, 44, 48, 68, 69, 74, 79, 81, 88, 98, 102, 104, 106-108, 120, 129, 131, 137, 139, 150-154, 161, 163 VLA-5 110 VLA-6 30, 41, 43, 44, 67, 70, 79, 81, 87, 98, 102, 106, 110, 118, 152, 155