Lymphocyte Homing to Allografts1

A complex system of lymphocyte trafficking has evolved to provide immune surveillance and recognition of foreign antigens (1). When the regulation of this process is disrupted leukocyte recruitment continues inappropriately resulting in pathological inflammation (2, 3). During graft rejection activated lymphocytes are recruited to and retained in the graft (4) and therefore the factors that regulate lymphocyte recruitment are crucial in determining the outcome of allorecognition. In this article we review the current understanding of how lymphocytes recognize and bind to endothelial cells and how these interactions regulate T cell recruitment into tissue. We discuss the implications of leukocyte recruitment in the context of allograft rejection. LYMPHOCYTE RECIRCULATION UNDER PHYSIOLOGICAL CONDITIONS Lymphocyte recruitment from the circulation into tissue is dependent on the ability of lymphocytes to bind to molecules on endothelial cells that promote adhesion and transendothelial migration into tissue. A multi-step model of leukocyte adhesion to vascular endothelium has been described and is broadly applicable, although the details of the signals involved will differ depending on the setting. In the generally accepted model (1, 2, 5) tethering or rolling receptors expressed on endothelial cells, capture free flowing leukocytes. These receptors may be members of either the selectin family of adhesion proteins (6) or the immunoglobulin superfamily (7–9). Once captured, the leukocyte can receive activating messages presented by endothelial cells in the form of chemotactic cytokines or “chemokines,” which bind to specific G protein coupled receptors on the leukocyte surface (10–12). Under some circumstances cell surface receptors, including the L-selectin ligand glycosylation dependent cell adhesion molecule 1 (GLYCAM-1), CD31 and CD73, can also trigger rapid integrin activation (13, 14). Occupancy of these receptors triggers a cascade of intracellular signals that result in presentation of high affinity integrin receptors on the leukocyte surface. These activated integrins then bind competently to their immunoglobulin family receptors expressed on the endothelium to promote arrest and firm adhesion of the leukocyte to the vessel wall (15–17). In the presence of the appropriate migratory signals the leukocyte will then migrate across the endothelium into tissue where it follows a hierarchy of chemotactic signals towards the focus of inflammation (see Fig. 1) (18). Figure 1: Adhesion of lymphocytes to endothelial cells under conditions of flow. Free-flowing lymphocytes in the circulation are captured by tethering receptors (usually carbohydrate dependent selectins) expressed on endothelial cells that induced the cell to roll on the vessel wall bringing it into contact with chemokines immobilized in the endothelial glycocalyx. Chemokine activate specific, G-protein-linked receptors on the lymphocyte that results in a conformational activation of lymphocyte integrins to a high affinity state, permitting firm adhesion to endothelial-expressed immunoglobulin adhesion molecules. Chemokine recognition also results in cytoskeletal reorganization within the adherent lymphocyte, which facilitates migration across the endothelial monolayer and into the tissue. Once within the tissue the leukocyte follows a hierarchy of chemotactic gradients toward the site of inflammation. The shaded triangles represent the relative contribution of the different classes of adhesion molecule to each step of the cascade (adapted from Adams and Shaw, Lancet 1994 and Picker and Butcher, Science 1996).HOMING PATTERNS OF NAÏVE AND MEMORY/EFFECTOR LYMPHOCYTES Adhesion of lymphocytes to endothelial cells within different tissues appears to follow this paradigm, under both physiological and pathological conditions. However, subsets of lymphocytes display different receptors that will alter their propensity to be recruited to different sites. This is best illustrated by the marked differences between naïve and memory T lymphocytes (Fig. 2). Figure 2: Adhesion molecules involved in lymphocyte endothelial interactions. Details are given in the text. PNAd, Peripheral node addressin a complex that contains CD34, podocalyxin, GlyCAM-1, and Sgp 200. Adapted from Salmi, Adams, and Jalkanen, Am J Physiology 1998.Naïve lymphocyte migration. These cells migrate almost exclusively between the circulation and secondary lymphoid tissues whereas memory cells are largely excluded from lymph nodes and instead migrate into tissue, returning to the circulation via lymphatics (19). The recruitment of naïve T cells to secondary lymphoid tissue is regulated by their ability to recognise specific molecules on lymph node high endothelial venules (HEV) (20). These molecules include endothelial adhesion molecules, such as the peripheral node addressin (PNAd) which binds to L-selectin on naive T cells and the chemokine SLC (secondary lymphoid tissue chemokine) that binds to a receptor, CCR7, preferentially expressed on naive T cells (1, 21–24). Antigen presenting cells (APCs) in lymph nodes have the capability to activate naïve T cells and these interactions are also regulated by chemokines. For example, another CCR7 ligand called, EBV-induced molecule-1 ligand chemokine (ELC) , is secreted by dendritic cells (DCs) in the T cell compartment of the lymph node and acts to bring T cells and activated DCs (which also express CCR7) together thereby promoting optimal T-APC interactions (22, 25, 26). Memory/effector T cell migration. After activation in secondary lymphoid tissue, naïve T cells differentiate into activated effector cells and long-lived memory T cells. These cells exhibit different migratory pathways dictated by changes in their cell surface expression of adhesion receptors (27). For example, activated T cells lose expression of L-selectin and CCR7 (which prevents them from binding efficiently to HEV in lymph nodes) but up-regulate molecules such as integrins and the chemokine receptors CCR2, CXCR3, and CCR5 that promote adhesion to activated endothelium in inflamed tissue (28–31). If the lymphocyte is activated in tissue it down-regulates the expression of chemokine receptors such as CCR2 and CCR5 while activating integrin-mediated adhesion to cellular and extracellular matrix ligands resulting in immobilization of the lymphocyte at the site of antigen exposure (32, 33). Thus in the main, memory T cells respond to tissue derived inflammatory signals but not to the physiological signals that drive recruitment to lymph node. However, recent studies suggest that a subset of Th1 memory T cells maintain CCR7 expression allowing them to recirculate through lymph nodes but excluding them from B cell areas and preventing them from providing B cell help (34, 35). Differences in adhesion molecules determine the migration patterns of Th1 and Th2 cells. An even more subtle regulation of adhesion molecule expression has been reported on Th1 and Th2 lymphocytes. Because these functional subsets are delineated by distinct patterns of cytokine secretion differences in Th1 versus Th2 cell recruitment could effect the outcome of alloactivation. Th1 cells secrete interleukin- (IL) 2, interferon-γ (IFN-γ) and tumor necrosis factor (TNFβ) whereas Th2 cells secrete IL-4, IL-5, IL-10, tumor growth factorβ (TGFβ), and IL-13 and have been associated with suppression of allograft rejection in some models (36). There are marked differences in the expression of chemokine receptors and adhesion molecules on Th1 and Th2 cells reflecting their different requirements for recruitment to tissue (37). Although both Th1 and Th2 lymphocyte subsets express the P-selectin ligand PSGL-1, only Th1 cells bind both E- and P-selectin (38), a property determined by their ability to glycosylate PSGL-1. The fucosyl transferases (39, 40) responsible for this glycosylation show differential expression between Th1 and Th2 cells (41) and determine whether the cell will bind P-selectin at sites of inflammation (42). The expression of these enzymes is influenced by the local cytokine milieu. For example, IL-4 down-regulates α3 fucosyltransferases in Th2 cells thereby altering their ability to home to inflamed sites. However, although the differences between Th1 and Th2 cells are clear-cut in vitro, current evidence suggests that the expression of chemokine receptors and the ability of T cells to bind selectins may be more strongly associated with tissue specificity rather than in determining Th1 versus Th2 responses in vivo (43, 44). TISSUE SPECIFIC EXPRESSION OF ADHESION RECEPTORS AND CHEMOKINES In addition to acquiring molecules which promote their recruitment to inflamed tissue there is evidence that memory cells display tissue-specific homing receptors that allow them to recognize endothelium in the tissue draining into the lymph node where they were originally activated (1, 18, 27). Hence, a T cell activated in an axillary lymph node will subsequently recirculate preferentially to the skin, whereas one activated in a mesenteric node will be programed to home to the gut (19). This tissue tropism is facilitated by organ-specific expression of endothelial molecules; particularly those involved in lymphocyte capture. For example, recruitment of lymphocytes to the gut is mediated by an endothelial molecule mucosal addressin cell adhesion molecule-1 (MAdCAM-1) which is largely restricted to mucosal vessels and its ligand α4β7 integrin, which is preferentially expressed on T cells that display gut tropism (45). In contrast memory T cells that infiltrate the skin express a unique skin-homing receptor called the cutaneous lymphocyte-associated antigen (CLA) which results from fucosyltransferase VII-mediated glycosylation of the cell-surface receptor P-selectin glycoprotein ligand-1 (PSGL-1) (46). Although PSGL-1 is expressed constitutively on most human T cells, CLA is found on memory T cells in the skin but not on T cells infiltrating other inflammatory sites (47, 48). Expression of CLA by T cells facilitates their homing to inflamed skin by promoting adhesion to E-selectin on dermal vessels (49). Tissue-specific endothelial adhesion molecules have also been proposed for the liver (50–52) and lung (53–55). There is now evidence that chemokine receptor expression also determines where lymphocytes are recruited. Thus, skin homing T cells express high levels of CCR4, the receptor for TARC, a chemokine that is expressed at inflammatory sites in the skin (56). In contrast gut and liver homing T cells express very little CCR4 but instead high levels of CCR5 the receptor for the chemokines regulated with activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein- (MIP) 1α and β (30) and gut-derived cells express CCR9 (18, 57). Two factors, therefore, determine where a lymphocyte is recruited; the adhesion molecules and chemokine receptors expressed on the lymphocyte (which will be determined by the microenvironment in which the cell was activated) (58) and the combination of chemokines and adhesion counter-receptors expressed on the endothelium in the target tissue. REGULATION OF ADHESION MOLECULE AND CHEMOKINE EXPRESSION BY ENDOTHELIUM Under basal conditions the expression of endothelial adhesion molecules in a particular tissue will be determined by signals from the microenvironment. For example lymphotoxin (TNFβ) is essential for the development of mucosal lymphoid tissue and the expression of MAdCAM-1 (59). The nature of many of these signals is currently poorly understood but will be crucial to understanding tissue specificity of homing (60). Adhesion molecules expressed by activated endothelium. The signals responsible for activating endothelium in the presence of inflammation are understood. Molecules such as intercellular adhesion molecule-2 (ICAM-2) and CD31 are constitutively expressed on endothelial cells and change little with inflammation. Other molecules such as ICAM-1 are normally expressed at low levels but increase after activation, whereas others such as E-selectin and vascular cell adhesion molecule-1 (VCAM-1) only appear after activation (61). The local presence of proinflammatory cytokines such as IL-1, TNFα, or IFNγ, causes increased expression of many endothelial adhesion receptors, thereby extending the range of leukocyte subsets that can be recruited (62). The precise phenotype of activated endothelium depends on the nature of the local inflammatory response because some cytokines have differential effects on adhesion molecules. For example, a combination of IL-4 and TNFα selectively up-regulates VCAM-1 on endothelial cells in culture (63) (64), and IFNγ can inhibit activation-induced selectin expression (65). The time course of endothelial adhesion molecule expression differs. For example P-selectin is stored as intact protein in endothelial Weibel-Palade bodies and can be rapidly mobilised to the cell surface in response to agonists such as hydrogen peroxide and histamine (66). In contrast, E-selectin, in common with most endothelial adhesion receptors, requires de novo protein synthesis and takes several hours to be expressed (61). The leukocytes recruited to tissue can themselves amplify endothelial activation by secreting cytokines and via poorly understood adhesion-dependent mechanisms. For example, engagement of endothelial ICAM-1 by lymphocyte leukocyte function associated molecule-1 (LFA-1) results in increased secretion of chemokines and expression of adhesion molecules such as VCAM-1 that promote transendothelial migration (67–69). In addition endothelial cells express CD40, a member of the TNF receptor superfamily that can interact with its ligand, CD154 expressed on activated lymphocytes. Expression of CD40 increases with cytokine treatment of endothelial cells in vitro and engagement of CD40 with trimeric CD154 increases expression of the adhesion molecules, E-selectin, VCAM-1, and ICAM-1. CD40 can be detected immunocytochemically on endothelium in rejecting human allografts (70) and may thus acts as a signaling receptor to promote lymphocyte infiltration to the graft (71). Thus as the local inflammatory response develops the phenotype of the endothelium will change over time accounting for the sequential recruitment of different subsets of leukocytes. Endothelial secretion and presentation of chemokines. The nature of an inflammatory stimulus will also determine the local secretion and presentation of chemokines by the endothelium. Their expression can be rapidly induced in most cell types following stimulation by a variety of agents including bacterial products, viruses, cytokines and activation of cell surface receptors (11, 72). Activated T cells express a range of chemokines at both the mRNA and protein levels and non-hematopoeitic cells including endothelial and epithelial cells are potent sources of many chemokines. The chemokines secreted in response to particular stimuli show differences between cell types. For instance epithelial cells secrete large amounts of the CXC chemokines ENA-78 and IL-8 in response to LPS whereas they fail to respond to IL-10 or IFNγ (73). However at sites of chronic inflammation local IFN-γ secretion causes expression of the CXCR3 ligands IP-10, MIG and I-Tac by endothelium thereby promoting the recruitment of effector lymphocytes and monocytes (30, 74). Infiltrating monocytes and activated lymphocytes are a major source of the chemokines that will determine the subsequent composition and duration of the inflammatory response. For example, CD8+ CTL, which are antigen specific for myelin proteolipid protein peptides, a putative antigen in multiple sclerosis, secrete the chemokines MIP-1α, MIP-1β, IL-16, and IP-10 that act to recruit CD4+ T cells of the same TCR specificity. Thus CD8+ cytotoxic T cells can promote and maintain inflammatory responses by recruiting specific CD4 subsets (75). If they are to trigger adhesion and migration at the endothelial surface chemokines be retained at the vessel wall to with leukocytes. This is by chemokines on in the endothelial or by their binding to receptors such as the antigen on cells and receptors show differential binding to and because from tissue to tissue and with activation, this a by which tissues can selectively express a particular of chemokines The of chemokine presentation by endothelium is because endothelial cells can both chemokines secreted by cells by a process of and also capture chemokines in the circulation ADHESION AND CHEMOKINES OF in adhesion molecule and chemokine expression by endothelial cells and of adhesion molecules and chemokine receptors by leukocytes determine and where leukocytes to the vessel wall and tissue. thus follows that such will regulate the recruitment of leukocytes to allografts and determine the outcome of allorecognition. endothelial cells within can be activated to express a range of adhesion molecules and chemokines. The of endothelial activation in allograft tissue follows that in other inflammatory and with T cell infiltration into the graft are on several graft including the and endothelium and local secretion is then by infiltrating lymphocytes There is now and evidence that endothelial activation as a of to the graft the process of and and that this the recruitment of leukocytes graft also the by providing for lymphocyte activation as as activating endothelium thereby the subsequent recruitment of lymphocytes rejection and studies have that adhesion molecules, including VCAM-1, and E-selectin and chemokines such as IL-8 and are in graft tissue this activation has been associated with subsequent rejection and may the between and graft rejection The of selectins in lymphocyte trafficking to Endothelial E- and P-selectin are particularly in recruitment This is by the ability of selectin ligand to and the of are also in lymphocyte However, it is that selectins will different in different tissues or Thus Th1 responses in the skin are associated with lymphocyte binding to endothelial selectins whereas T cells at Th1 sites of inflammation in the gut not express E- or P-selectin receptors This lymphocytes in rejecting liver allografts not bind E-selectin In contrast there is in vivo evidence that P-selectin is for lymphocyte recruitment to lung When a was to P-selectin function in a model of lung there was a in the of graft rejection expressed by other cell types such as and lymphocytes may also be of allograft rejection. adhesion to graft endothelium very and the high levels of P-selectin expressed by such adherent can as a focus to capture lymphocytes There is also evidence for of lymphocyte expressed L-selectin in lymphocyte recruitment to rejecting allografts fail to migrate into skin in and the of rejection is with L-selectin ligands have been detected on the endothelium of allografts rejection in and in human and reported increased expression of L-selectin ligands on the endothelium of human which with lymphocyte infiltration and the of graft rejection. this that the expression of L-selectin ligands was an in rejection associated with the as as the of lymphocyte infiltration The ability to inhibit adhesion with them for in The of other adhesion molecules in lymphocyte trafficking to In some circumstances molecules other than selectins may the capture of lymphocyte Two are the gut addressin which can promote lymphocyte rolling via interactions with either L-selectin or the integrin α4β7 and vascular adhesion which dependent capture of lymphocytes to lymph node high endothelial venules and liver these molecules display expression in particular tissues but whether they maintain this tissue specificity in the context of allograft is not the of MAdCAM-1 in lymphocyte recruitment to the gut it that it will be involved in lymphocyte trafficking to and studies this is another adhesion molecule that can function both as a molecule and to lymphocyte adhesion to endothelium. of to lymphocyte infiltration in a skin through different depending on which of was the homing of activated lymphocytes into the whereas the of T cells The of integrins in lymphocyte adhesion to graft endothelium. There is evidence from and studies for the of both and integrins in lymphocyte adhesion to graft endothelium. The endothelial expression of VCAM-1 and ICAM-1 increases rejection and both molecules can lymphocyte adhesion as by binding studies In several models of treatment with to either or lymphocyte infiltration into the graft and graft The relative of each between models with effects both pathways are Thus, although it is accepted that both and integrin dependent can promote lymphocyte endothelial adhesion the precise of each will differ between tissues and in response to different inflammatory the of studies in which and are in vivo is because in addition to lymphocyte recruitment both pathways are also involved in Because the studies molecule has been in several as a the of the human molecule was to of rejection. Although the was in most it was in rejection In a of in reported rejection and in levels the reported the results of given either or a for together with There was differences in the of graft or graft The of chemokines in lymphocyte trafficking to The for integrin activation to rolling adhesion into adhesion lymphocyte recruitment suggests that chemokines expressed by graft endothelium will be crucial of lymphocyte from and human studies increased expression of several chemokines that act on lymphocytes in allograft rejection. studies but have increased expression of MIP-1α, MIP-1β, and IP-10 graft rejection studies allow the and function of chemokine expression to be in more a skin allograft model that the expression of MIP-1α, MIP-1β, and IP-10 in the graft was increased in and rejection and that this expression was dependent on the presence of T cells. studies have the functional of chemokines in allograft rejection. The of a of to lymphocyte recruitment and graft as a of lymphocyte capture by graft endothelium and chronic rejection of allografts has been reported in that the receptor The of that recognise specific chemokine receptors lymphocyte subsets of to specific chemokines to be The MIP-1α, receptor CCR5 has been to be increased on infiltrating lymphocytes in both and liver allograft rejection the for CCR5 ligands in allograft rejection. chemokine receptor which appears to be for effector cell into inflamed tissue is CXCR3 (30, The ligands for CXCR3, IP-10, and are by and evidence suggests that they are in graft rejection in with infiltration by CXCR3 expressed T cells In the it will be to determine which chemokines and chemokine receptors are involved in determining the outcome of allorecognition. For instance graft is by a in local chemokine to the recruitment of Th2 over Th1 cells, of chemokines or chemokine receptor function may allow responses to be toward the recruitment of lymphocytes that promote graft The development of models with chemokines and chemokine receptors will determine the specific of particular more and will be in a subsequent LYMPHOCYTE Although that regulate lymphocyte endothelial adhesion are recent suggests that recognition of on endothelial cells by T cells can effect not only the of lymphocyte activation but also their ability to transendothelial migration Because endothelial cells in allografts express molecules they have the to activate T cells via the a stimulus for the of the high affinity integrin resulting in lymphocyte A recent has more evidence for an in lymphocyte recruitment to and that presentation of antigens by molecules on endothelium to T cell the of transendothelial migration that antigen presentation by endothelium can the recruitment of T cells into tissues The have major in understanding the regulation of lymphocyte endothelial interactions. is that the endothelium within allografts will other inflammatory tissue and express molecules that promote lymphocyte recruitment graft rejection. the nature and function of these molecules has for The endothelium in the graft may be by to expression of molecules such as ICAM-1 and VCAM-1 which appear to be for lymphocyte The development of or agents that inhibit adhesion molecule function may to the of with the is that expressed adhesion molecules will also inhibit lymphocyte to If specific molecules regulate lymphocyte recruitment to particular tissues the of these molecules tissue-specific and lymphocyte the most is to the nature of the lymphocyte subsets recruited to the graft that cells are excluded and subsets preferentially recruited. For example, the understanding of the particularly that the recruitment of lymphocyte subsets to tissue the of in which the recruitment of cells is However, in vivo to inhibit lymphocyte endothelial interactions have been reflecting the of the interactions involved and the large of chemokines by the studies in chemokine receptors have results that the outcome of particular receptor interactions will be