The role played by adhesion molecules during allograft rejection

Transplant Immunology S u m m e r School

de prrparation du greffon pancrratique en vue de la transplantation: donnres exprrimentales et applications cliniques au cours de 9 transplantations chez 8 sujets diabetiques. Chirurgie 1978; 104: 242-58. 5 Groth CG, Lundgren G, Gunnarsson R, Arner P, Berg B, Ostman J. Segmental pancreatic transplantation with duct ligation or drainage to a jejunal Roux-en-Y loop in nonuremic diabetic patients. Diabetes 1980; 29 (suppl 1): 3-9. 6 Wahlberg JA, Love R, Landegaard L, Southard JH, Belzer FO. 72-hour preservation of the canine pancreas. Transplantation 1987; 43: 5--8. 7 Ulbig M, Kampik A, Thurau S, Landgraf R, Land W. Long-term follow-up of diabetic retinopathy for up to 71 months after combined renal and pancreatic transplantation. Graefes Arch Clin Exp Ophthalmol 1991; 229: 24245. 8 Bilous RW, Mauer SM, Sutherland DE, Najarian JS, Goetz FC, Steffes MW. The effects of pancreas transplantation on the glomerular structure of renal allografts in patients with insulindependent diabetes. N Engl J Med 1989; 321: 80-85. 9 Rosen CB, Frohnert PP, Velosa JA, Engen DE, Sterioff S. Morbidity of pancreas transplantation during cadaveric renal transplantation. Transplantation 1991; 51: 213--27.

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10 Sutherland DE. Pancreas and islet transplantation. I. Experimental studies. Diabetologia 1981;20".161-85. 11 Scharp DW, Lacy PE, Santiago JV et al. Insulin independence after islet transplantation into type I diabetic patient. Diabetes 1990; 39: 515-18. 12 Warnock GL, Kneteman NM, Ryan E, Seelis REA, Rabinovitch A, Rajotte RV. Normoglycemia after transplantation of freshly isolated and cryopreserved pancreatic islets in type 1 (insulindependent) diabetes mellitus. Diabetologia 1991; 34: 55-58. 13 Ricordi C, Tzakis AG, Carroll PB et al. Human islet isolation and allotransplantation in 22 consecutive cases. Transplantation 1992; 53: 407-14. 14 Sullivan SJ, Maki T, Borland KM et al. Biohybrid artificial pancreas: long-term implantation studies in diabetic, pancreatectomized dogs. Science 1991; 252: 718-21.

Recommendedreading Groth CG ed. Pancreatictransplantation. Philadelphia: WB Saunders, 1988. Ricordi C ed. Pancreatic islet cell transplantation. Austin, TX: RG Landes Co, 1992.

The role played by adhesion molecules during allograft rejection John A Kirby Department o f Surgery, The Medical School, University of Newcastle upon Tyne

Introduction Many parenchymal cells express surface molecules which interact with, and stabilize adhesion to, structures on adjacent cells or components of the extracellular matrix. In addition to maintaining normal body structure, these 'adhesion molecules' are fundamental to normal immune system function. In this review the nature and purpose of leukocyte adhesion to various target cells will be discussed in the context of rejection of a vascularized organ allograft. Following organ transplantation, blood-borne recipient lymphocytes have the potential to interact with endothelial cells within the allograft. Such interaction may result directly in the activation of aUoreactive lymphocytes) Alternatively, leukocytes may be stimulated to pass through the vascular endothelium to infiltrate graft tissues. Adhesion molecules are vital to both processes.

Seleetins Leukocytes do not pass unhindered through a blood vessel but tend to 'roll' across the endothelial surface. This process is made possible by cyclic formation and breakage of adhesive bonds between the two cell types (Figure 1). These relatively weak bonds are formed between a family of heavily glycosylated proteins known as the 'selectins'; blocking studies have shown that the glycosylated or lectin domain is essential for adhesive function. The predominant members of this family on endothelial cells are E-selectin (or ELAM-1) and Pselectin. Leukocytes variously express L-selectin conjugated Address for correspondence: John A Kirby, Department of Surgery, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK.

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with the sialyl Lewis* (sLe*) ligand. Activated and memory T cells carry a ligand which binds specifically to E-selectin. During periods of inflammation the expression of endothelial cell-associated selectins is upregulated by the presence of cytokines2 such as interleukin 1 (IL-1) and tumour necrosis factor (TNF-a). There is also evidence that the affinity of individual selectin molecules can increase after appropriate stimulation. Together these processes effectively reduce the speed with which a leukocyte passes through a blood vessel. Leukocytes are arrested and bound tightly to the endothelium after activation of a second family of adhesion molecules, the integrins.

Integrins These glycoproteins are heterodimeric consisting of an a- and a, generally smaller, I~-subunit. The major integrins involved in leukocyte adhesion are lymphocyte function-associated antigen-1 (LFA-I; dimer of CD11a and CD18 subunits) and very late antigen-4 (VLA-4; dimer of CD49d and CD29 subunits). Both LFA-1 and VLA-4 are expressed constitutively by leukocytes including lymphocytes, monocytes and polymorphonuclear cells. Additional members of the family such as Mac-1 ( C D l l b and CD18) and leukocyte adhesion receptor P150,95 (CD11c and CD18) play an important part in binding neutrophils and monocytes to endothelial cells during inflammatory responses. Some integrins bind to a specific arginine-glycine-aspartic acid sequence on extracellular matrix proteins such as collagen, fibronectin, laminin and yon WiIlebrand factor; for example VLA-4 binds to fibronectin. Integrins also bind to ceil-surface members of the immunoglobulin superfamily of proteins.

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Transplant Immunology Summer School Direction of Movement

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lmmunoglobulin superfamily The members of this family are thought to have evolved from a single structural motif based on two parallel 15-pleated sheets which are generally stabilized by disulphide bridges. However, the family, which contains over 70 members, shows an enormous diversity of primary sequence with variation in the number of structural domains. During evolution it is likely that the simplest form of intercellular adhesion occurred between identical structures expressed on adjacent cells. A critical adhesive interaction can occur between T cell associated CD2 and lymphocyte function-associated antigen-3 (CD58) which is widely distributed within the body on cells including those of the endothelium. These immunoglobulin superfamily members show a high degree of structural homology but share only a limited sequence identity. Adhesion molecules within the immunoglobulin superfamily are also able to bind to ceil-surface molecules that are not members of the immunoglobulin superfamily. The two most important immune cell interactions in this category occur between intercellular adhesion molecule-1 (ICAM-1) and the integrin LFA-1, and vascular cell adhesion molecule1 (VCAM-1) and the integrin VLA-4. The major adhesion molecule interactions responsible for the binding of T lymphocytes to vascular endothelium are shown in Figure 2.

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pression result in relatively large changes in intercellular avidity. For example, work with the neural cell adhesion molecule has shown that a twofold increase in expression can cause a 30-fold increase in adhesion. 3 During periods of inflammation a variety of 'proinflammatory' cytokines are produced. These include IL-1, tumour necrosis factor (TNF)-c~ and -13 and interferon-gamma (IFN~/). In addition to their effect on the selectins, these agents also stimulate increased endothelial cell expression of the VCAM-1, ICAM-1 and LFA-3 adhesion molecules. There is evidence that cytokines are synergistic in their effects. For instance, it has been reported that TNF-ct synergizes with IFN-'y by upregulating the expression of IFN--/ receptors. 4 Cytokines are also thought to regulate the level of adhesion counter-receptors expressed by leukocytes. It has recently been suggested that the cytokine macrophage inflammatory protein-ll3 (MIP-113) may become fixed to glycosyl residues on the endothelial cell surface, s In this immobilized state the protein induces the binding between VCAM-1 and the T cell ligand VLA-4. It is also known that activation of T lymphocytes results in upregulated expression of molecules such as CD2, LFA-1 and VLA-4. 6"7 These processes all result in greatly enhanced intercellular adhesion. In addition to regulation of the number of adhesion molecules, it is known that the function of individual adhesive integxins is regulated by specific signals. For example, T cell activation results in rapid and transient activation of LFA-1. 8 Inhibition of G-protein linked signal transduction causes a corresponding inhibition of integrin-related intercellular adhesion. It is thought that phosphorylation of a cytoplasmic domain of the 13-chain of the heterodimer causes a conformational change which is responsible for the regulation of integrin function. The integrin VLA-4 is thought to occur in three states. One of these fails to adhere to either VCAM-1 or fibronectin, one adheres to both of these ligands and one binds only to VCAM-1. In addition to reflecting intracellular activation events, both integrins and adhesive members of the immunoglobulin superfamily are capable of transducing extracellular signals through the cell membrane to the cytoplasm. Signal transduction It is known that stimulation of CD2 with monoclonal antibodies directed simultaneously at two epitopes causes T cell activation; the place of one of these antibodies can be taken by the natural LFA-3 ligand. It has been suggested that the

Transplant Immunology Summer School

stimulation of CD2 by endothelial cell-associated LFA-3 plays a vital role during T cell activation by immunogenic endothelial cells. L9 Crosslinking of LFA-1 on T lymphocytes by anti-a-chain antibodies causes a rise in intercellular Ca 2÷ levels together with enhanced production of cytokines and cell proliferation. It has been suggested that adhesion molecules on T cells can supply an accessory signal which augments that transduced by the T cell receptor-CD3 complex after recognition of a donor specific antigen. For example, ligation of VLA-4 with fibronectin enhances the proliferation of CD4+ lymphocytes.

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Extravasation After adhesion to the endothelium, leukocytes can pass between the endothelial cells to infiltrate the subendothelial matrix and the graft tissues. In many cases the infiltration is vectorial with leukocytes migrating between parenchymal components as they climb the concentration gradient of a chemotactic factor. Many adhesive interactions can immobilize leukocytes on the endothelial surface but the binding between ICAM-1 and the integrins LFA-1 and Mac-1 is thought to play a predominant role in leukocyte extravasation. In this respect it is of significance that the distribution of VCAM-1 is limited to the apical surface of endothelial cells whilst ICAM-1 can be detected on the apical and basal surfaces and around the tight junctions that occur between endothelial cells. It is known that antibody blockade of ICAM-1 can inhibit the extravasation of leukocytes; similar blockade of VCAM-1 has no effect on the rate of extravasation. L y m p h o c y t e activation a n d e f f e c t o r f u n c t i o n The interaction between donor major histocompatibility antigens (MHC) and allospecific recipient T lymphocytes provides the basis for alloreactivity and graft rejection. In most cases this interaction is relatively weak despite some stabilization through binding between CD4 or CD8 and the MHC antigen. It is now recognized that ligation between the immunoglobulin superfamily molecule CD28 on T lymphocytes and B7 on professional antigen-presenting cells, such as dendritic cells or activated B lymphocytes, provides the major costimulatory signal resulting in productive T cell activation. Recent data have shown that adhesion molecule stimulation of both the LFA-1 and VLA-4 receptors enhances the response of T lymphocytes to costimulatory signals delivered by CD28. w This underlines the point that stable adhesion must occur between the T lymphocyte and a donor antigenpresenting cell prior to lymphocyte activation. A number of experimental studies have illustrated this by demonstration that blockade of lymphocyte adhesion molecules by treatment with monoclonal antibodies can abrogate mixed leukocyte activity in vitro, n Patients with a defective 132-integrin fail to express the adhesion molecules LFA-1, Mac-1 and p150,95. This results in impaired lymphoproliferative responses to mitogens, allogeneic cells and antigens; furthermore, cytotoxic T cell killing is reduced to about 20% of the normal result. Adhesion molecules play an important role in immune effector function. For example, it is known that both cytotoxic T lymphocytes and natural killer cells must adhere to their cellular targets before initiation of the cytolytic process. Inhibition of this adhesion by antibody-mediated blockade of LFA-1 produces a significant reduction in subsequent cytol-

Transplant Immunology 1994; 2:129-132

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Inhibition of T Cell Adhesion Figure 3 Graph showing the correlation observed between antibodymediated inhibition of the adhesion of allospecific cytotoxic T cells to donor renal epithelial cells and the inhibition of target cell lysis produced by the same treatment

ysis. The results in Figure 3 show that inhibition of T cell adhesion correlates closely with inhibition of target cell lysis. During rejection many of the parenchymal cells within a graft respond to high levels of cytokines within the local environment and express significant levels of adhesion molecules. For example, it is known that cardiac myocytes,]2 renal tubular epithelial cells 13a4 and biliary epithelial cells 15 express molecules such as ICAM-1, VCAM-1 and LFA-3 during episodes, respectively, of cardiac, renal and hepatic allograft rejection. As these cells also express both class I and class II MHC antigens, they are vulnerable to cytolysis mediated by donor specific cytotoxic T lymphocytes.

Therapeutic intervention Monoclonal antibodies specific for adhesion molecules have been used in an effort to modulate the immune system by inhibiting leukocyte adhesion. It has been reported that allograft survival in animal models is increased by treatment with antibodies specific for either LFA-1 ]6 or ICAM-1.17 Treatment of human renal allograft recipients with antibodies specific for the C D l l a component of LFA-1 has produced more equivocal results. TM Polyclonal antilymphocyte antibody reagents have been known to possess immunosuppressive properties for many years. ]9 It has generally been presumed that such reagents function by causing the destruction of recirculating lymphocytes. However, it is now clear that these antibody preparations are capable of binding to and blocking the function of adhesion molecules expressed on the surface of lymphocytes. 2° Experiments performed in vitro have shown that polyclonal antilymphocyte antibody preparations produce extremely effective blockade of lymphocyte adhesion to target cells and protect donor cells from lysis by activated cytotoxic T cells. 21 It is possible that some of the immunosuppressive properties of polyclonal antibody drugs can be ascribed to these functions.

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Transplant I m m u n o l o g y S u m m e r School

Conclusion It can be seen that adhesion molecules and their ligands play an important part in every aspect of allograft rejection. The molecules immobilize recirculating leukocytes and direct their extravasation and subsequent passage through the subendothelial matrix into graft tissues. Adhesion molecules are responsible for holding allospecific T cells in contact with donor antigen-presenting cells in order that specific signal transduction and lymphocyte activation can occur. It is also necessary for adhesion molecules to hold specific immune effector cells in contact with their targets prior to initiation of the cytolytic process. Adhesion molecules would appear to be a prime target for therapeutic intervention during the allograft rejection process. However, care must be exercised. The term adhesion molecule may be viewed as a misnomer as most of these molecules have subtle regulatory functions that are more sophisticated than those required for simple cell--cell binding. Interference with any single interaction may produce effects which differ from those predicted for simple adhesion blockade. Nevertheless, continued research will certainly improve our ability to manipulate this system in order to ameliorate the effects of allograft rejection.

References 1 Pober JS, Cotran RS. Immunologic interactions of T lymphocytes with vascular endothelium. Adv lmmunol 1991; 50: 261-302. 2 Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone MA. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci USA 1987;84: 9238--42. 3 Hoffman S, Edelman GM. Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule. Proc Natl Acad Sci USA 1983;80: 5762~6. 4 Aggarwal BB, Eessalu TE, Hass PE. Characterisation of receptors for human tumour necrosis factor and their regulation by gamma-interferon. Nature 1985;318: 665--67. 5 Tanaka Y, Adams DH, Hubscher S, Hirano H, Siebenlist U, Shaw S. T-cell adhesion induced by proteoglycan-immobilizedcytokine MIP-1 beta. Nature 1993;361: 79-82. 6 Shimizu Y, van Seventer GA, Horgan KJ, Shaw S. Roles of adhesion molecules in T-cell recognition: fundamental similarities between four integrins on resting human T-cells (LFA-1, VLA-4. VLA-5, VLA-6) in expression, binding and costimulation, lmmunol Rev 1990; 114: 109-43. 7 Springer TA, Dustin ML, Kishimoto TK, Marlin SD. The lympho-

cyte function-associated LFA-1, CD2 and LFA-3 molecules cell-adhesion receptors of the immune system. Annu Rev lmrnunol 1987; 5: 223-52. 8 Dustin ML, Springer TA. T cell receptor cross-linkingtransiently stimulates adhesiveness through LFA-1. Nature 1989; 341: 619-24. 9 Savage COS, Hughes CCW, Mclntyre BW, Picard JK, Pober JS. Human CD4+ve T cells proliferate to HLA-Dr+ve allogeneic vascular endothelium. Transplantation 1993;56: 128-34. 10 Damle NK, Klussman K, Leytze G e t al. Costimulation via VCAM-1 induces in T cells increased responsiveness to the CD28 counter-receptor B7. Cell lmmunol 1993; 148: 144-56. ll Hildreth JEK, August JT. The human lymphocyte function-associated (HLFA) antigen and related macrophage differentiation antigen (HMac-1): functional effects of subunit-specific monoclonal antibodies. J lmmunol 1985; 134: 3272-80. 12 Taylor PM, Rose ML, Yacoub MH, Pigott R. Induction of vascular adhesion molecules during rejection of human cardiac allografts. Transplantation 1992; 54: 451-57. 13 Faull R, Russ G. Tubular expression of intercellular adhesion molecule-1 during renal allograft rejection. Transplantation 1989; 48: 226-30. 14 Lin, Y, Kirby JA, Browell DA et al. Renal allograft rejection: expression and function of VCAM-1 on tubular epithelial cells. Clin Exp lmmunol 1993; 92: 145-51. 15 Steinhoff G, Behrend M, Schrader B, Pichlmayr R. Intercellular immune adhesion molecules in human liver transplants: overview on expression patterns of leukocyte receptor and ligand molecules. Hepatology 1993; 18: 440-53. 16 Heagy W, Waltenbaugh C, Martz E. Potent ability of anti-LFA-1 monoclonal antibody to prolong allograft survival. Transplantation 1984;37: 520--23. 17 Cosimi AB, Conti D, Delmonico FL et al. In vivo effects of monoclonal antibody to ICAM-1 (CD54) in nonhuman primates with renal allografts. J lmmunol 1990; 144: 4604-12. 18 Mauff BL, Hourmant M, Rougier JP et al. Effect of anti-LFA-1 (CDlla) monoclonal antibodies in acute rejection in human kidney transplantation. Transplantation 1991; 52: 291-96. 19 Lange EM, Medawar PB, Taub RN. Antilymphocyte serum. Adv lmmunol 1973; 17: 2-93. 20 Bonnefoy-Berard N, Vincent C, Revillard JP. Antibodies against functional leukocyte surface molecules in polyclonal antilymphocyte and antithymocyte globulins. Transplantation 1991; 51: 669-73. 21 Kirby JA, Lin Y, Browell DA et al. Renal allograft rejection: examination of adhesion blockade by antilymphocyte antibody drugs. Nephrol Dialysis Transplant 1993; 8: 544-50.

Transplantation of the aUoimmunized patient Erna MOiler Department o f Clinical Immunology, Huddinge Hospital, Huddinge

Transplant rejection is a T cell dependent process. Each individual has a unique set of peripheral immunocompetent T cells, adjusted to that individual's HLA phenotype. Positive and negative selection takes place in the thymus and forms the basis for the HLA-dependent individual T cell repertoire. Since the immune system is adjusted to the 'self' HLA phenotype, it is strange that T cells can recognize allogeneic

Address for correspondence: Erna Mrller, Department of Clinical Immunology, F79 Hnddinge Hospital, S-141 86 Huddinge, Sweden. Transplant I m m u n o l o g y 1994; 2:132-134

HLA antigens at all. However, the immune response against allogeneic tissues is exceedingly strong and involves a large set of different T cell clones. AUoreactivity is thought to depend on cross-reactivity between a person's own altered or infected cells (which should be efficiently recognized by the T cells) and unaltered foreign cells, i.e. foreign cells expressing their 'self' or autopeptides. Because of cross-reactivity, immunosuppression to avoid rejection unavoidably also causes increased susceptibility to infection. Immunocompetent T lymphocytes carry receptors that react specifically against antigenic fragment (peptides) in