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Immunology and Cell Biology (2002) 80, 52–64 Special Feature Recruitment of lymphocytes to the human liver PATRICIA F LALOR, PHILIP SHIELDS, ALLISTER J GRANT and DAVID H ADAMS Liver Research Laboratories, University of Birmingham MRC Centre for Immune Regulation, Institute of Clinical Research, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom Summary This review discusses the function and localisation of lymphocytes resident within the human liver, under both physiological and pathological conditions. Through description of the mechanisms that mediate lymphocyte recruitment into tissues, this article explains how hepatic endothelial and epithelial cells regulate the recruitment of specific lymphocyte subpopulations. We illustrate that the expression of adhesion molecules and chemokines is crucial to the control of lymphocyte adhesion. Thus, in the normal liver, adhesion molecules such as vascular adhesion protein-1 (VAP-1), intercellular adhesion molecule-1 (ICAM-1) and intercellular adhesion molecule-2 (ICAM-2), and chemokines such as regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), interferon γ inducible protein-10 (IP-10), MIG and interferon inducible T-cell alpha chemoattractant (ITAC) are involved in lymphocyte binding to different endothelial compartments. However, in response to inflammation or injury, additional expression of adhesion molecules such as VCAM-1, P-selectin and E-selectin, as well as higher levels of chemokines, permits the attraction and retention of specific effector populations of lymphocytes. We also discuss the expression and function of a newly defined adhesion protein, (VAP-1), and suggest that the unique functions of this protein may provide therapeutic potential for the treatment of liver disease. Key words: adhesion molecules, chemokines, endothelium, liver, lymphocyte homing. Introduction The normal liver contains a large number of lymphocytes that include not only specialized NK and NKT cells, but also CD4 and CD8 T cells. Whereas some of these cells are terminally differentiated effector cells that are destined to die by apoptosis, many of these cells are not and include immunocompetent cells that traffic through the liver to provide continuing immune surveillance as well as epithelial-associated effector T cells. In inflammatory liver disease the number of lymphocytes in the liver increases and the type and distribution of these infiltrating cells will determine the nature of the inflammation, for instance, a predominance of parenchymal inflammation is a feature of lobular hepatitis associated with viral hepatitis while a predominantly portal infiltrate centred on bile ducts is a feature of the biliary diseases primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). In this review we discuss the molecular mechanisms that regulate the entry of lymphocytes to the normal and inflamed liver and thereby determine the patterns of inflammatory damage in liver disease. The liver vasculature The liver has a unique dual blood supply with arterial blood delivered via the hepatic arteries and venous blood from the Correspondence: Dr PF Lalor, Liver Research Laboratories, University of Birmingham MRC Centre for Immune Regulation, Institute of Clinical Research, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK. Email: [email protected] Received 16 October 2001; accepted 31 October 2001. gut via the portal veins. This complex supply is necessary to allow nutrients from the gut to be transported to the liver, which is thus constantly exposed to gut-derived antigens in the portal blood. Because portal blood also presents a route through which infectious organisms can enter the liver, specific mechanisms have evolved to allow rapid and selective immune responses within this tissue. Thus, the liver contains a large resident and migratory population of lymphocytes and macrophages that provide immune surveillance against foreign antigens (Fig. 1). This population can be rapidly expanded in response to infection or injury by recruiting leucocytes from the circulation, a process that is dependent on the ability of lymphocytes to recognize, bind to and migrate across the endothelial cells that line the hepatic vasculature. The complex nature of the liver vasculature means that there are several points at which lymphocytes can interact with endothelium and be recruited into different anatomical compartments. The hepatic microvascular system consists of portal venules, hepatic arterioles, sinusoids, central venules and lymphatics. Most blood entering the sinusoids comes from the portal venules, the terminal branches of which enter into the sinusoids at inlets that are protected by contractile sinusoidal lining cells that act as a sphincter to regulate blood entry and, hence, blood flow within the sinusoids. Arterial blood enters the sinusoids through branches of the hepatic arterioles and via occasional direct arterioportal anastomoses at the level of the terminal portal venules.1 These structures are also contractile and, therefore, contribute to the control of blood within the sinusoids. The sinusoids take the blood through the lobule from the portal tract to the central (terminal vein). Close to the Recruitment of lymphocytes to the human liver 53 Figure 1 The normal human liver contains functional T cells. (a) Staining of normal donor liver for CD8 reveals CD8 T cells both in the parenchyma and portal tracts. (b) Freshly isolated T cells from normal or hepatitis C virus infected human liver were stimulated to secrete cytokines detected by intracellular cytokine staining. A significant proportion of T cells from normal liver secreted either IFN- γ or IL-10, suggesting that these cells are functionally active. (c) The phenotype of freshly isolated intrahepatic T cells reveals them to be activated as demonstrated by the expression of CD69 and CD38, but with low numbers of proliferating/cycling cells as shown by CD25 and CD71 expression. (d) The majority of intrahepatic T cells are CD45RO +, suggesting that they are primed or memory T cells. portal tract the sinusoids form an interconnecting network that becomes organized into parallel vessels connected via short intersinusoidal sinusoids. Before leaving the sinusoids and entering the central vein, blood flows through outlet sphincters that control the rate of blood flow entering the central veins. Matsumoto renamed hepatic lobules as hepatic microvascular subunits because each lobule is supplied by one inlet venule and its associated hepatic arteriole. 2 Thus, not only is the vasculature within the liver lobule complex, but there are also several points where blood flow can be regulated by the myofibroblast sinusoidal lining cells. This means that blood flow through the sinusoids can vary greatly both between different compartments and with time or injury. Intravital studies suggest that transient plugging of some sinusoids, particularly those in the periportal areas, by flowing leucocytes is a normal feature of liver blood flow. 3,4 Heterogeneity of endothelium within the liver The endothelial cells that line the different vascular beds within the liver show marked heterogeneity. The most obvious difference is between the vascular endothelium that 54 PF Lalor et al. lines the portal vessels and terminal veins and the specialized fenestrated endothelium that lines the sinusoids. However, there is also evidence for differences between portal and hepatic venous endothelium and differences within the sinusoids.5–9 The sinusoidal endothelial cells (SEC) are morphologically unique and are characterized by an absence of tight junctions between cells, a lack of basal lamina and by the presence of open fenestrations that are arranged into characteristic sieve plates.4,10 These ultrastructural features have evolved to facilitate solute transport between the blood and the underlying hepatocytes, which are separated from the endothelial cells by the space of Disse. Sinusoidal endothelial cells express the scavenger receptor for oxidized low-density lipoprotein (LDL), CD36, which is absent from vascular endothelium in the portal tracts and they lack factor VIII related antigen and Wiebel–Palade (WP) bodies, two characteristic features of vascular endothelium. In contrast, vascular endothelial cells lining the hepatic arterial branches, portal and central veins are continuous with an underlying basement membrane, express factor VIII related antigen and contain WP bodies.5–7,11,12 There are also important differences in the expression of adhesion molecules and the secretion of chemokines between vascular and sinusoidal endothelium. Consistent with the lack of WP bodies, SEC fail to express P-selectin in vivo and in vitro and in addition show markedly reduced or absent expression of E-selectin.7 This absence of selectins on sinusoidal endothelium extends to inflammatory conditions such as PBC, in which E- and P-selectin are strongly induced on portal vascular endothelium.13 This may reflect a lack of requirement for selectin-mediated tethering in the low-flow environment of the sinusoid, where leucocyte recruitment in vivo has been shown to occur in the absence of selectins. 14 Sinusoidal endothelial cells express low levels of an antigen (CD31) that is strongly expressed at cell–cell junctions in vascular endothelium and the lack of such junctions in sinusoidal endothelium might explain this difference. In contrast to CD31, intercellular adhesion molecule-1 (ICAM-1) is constitutively expressed at high levels on sinusoidal endothelium compared with the low basal expression on non-inflamed vascular endothelium.6 Chemokine expression also varies between the vascular beds in the liver. In the non-inflamed human liver, several chemokines, including regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-8, can be detected on vascular endothelium in portal tracts and this expression increases markedly with inflammation.15,16 However, little chemokine is detected on non-inflamed sinusoidal endothelium and the pattern seen with inflammation differs from that seen on portal vascular endothelium.17 The interferon-inducible chemokines, interferon γ inducible protein-10 (IP-10), interferon inducible T-cell alpha chemoattractant (ITAC) and human monokine induced by interferon γ (HuMig) are detected on sinusoids in association with necroinflammatory activity, whereas there is relatively little induction of RANTES, MIP-1α or IL-8 on sinusoidal endothelium. The reasons for these differences are unclear. One possibility is that they reflect fundamental differences in the response of sinusoidal versus vascular endothelium to inflammation. Some evidence supports this because sinusoidal endothelial cells secrete IP-10 in response to IFN-γ in vitro.17 However, it is also possible that the differences are explained by an effect of the local microenvironment. Other cell types influence gene transcription in sinusoidal cells as part of a complex process of autocrine cross-talk. For example, hepatocytes support SEC differentiation by secreting vascular endothelial growth factor (VEGF).18 Thus, cytokines from stellate cells, hepatocytes or other local cell types may determine the chemokines secreted by sinusoidal endothelium. Alternatively, the chemokines detected on sinusoidal endothelium might be secreted by other cell types, such as fibroblasts or inflammatory cells, in the immediate local environment, which are subsequently transported across the SEC to be presented by binding to endothelial surface proteoglycans. 19 This model in which chemokines secreted elsewhere are ‘posted’ on the endothelium has been demonstrated in other systems and is regulated by active processes of chemokine uptake, transcytosis and presentation by endothelial cells. 20–23 Thus, the unique phenotype of sinusoidal endothelium is probably due to the local microenvironment in the sinusoids as well as differences in differentiation of the endothelium. Lymphocyte adhesion to endothelial cells The nature of the lymphocytes recruited to the liver will be determined by the ability of specific leucocyte subsets to recognize and bind to hepatic endothelial cells under different conditions. The molecular basis of lymphocyte endothelial interactions has been studied in many systems, resulting in the acceptance of a ‘generic’ model that is broadly applicable to most tissues and situations in which leucocytes bind endothelium. However, it is emerging that the fine details of the molecules involved differ between tissues depending on the local requirements. In the generally accepted model,24–26 rolling receptors expressed on endothelial cells capture fast moving lymphocytes from the bloodstream. These receptors may be members of either the selectin family of adhesion proteins27 or the immunoglobulin superfamily.28–30 Once captured, the adherent cell detects activating messages presented in the form of chemokines presented on proteoglycans in the endothelial glycocalyx.31 These chemokines activate leucocytes via specific cell surface G-protein-coupled receptors, resulting in a cascade of intracellular signals involving the small GTPases Rho and Rac and PI3Kinase.32,33 These signals trigger the conformational activation of leucocyte integrins, cytoskeletal reorganization and cellular motility,34 bringing the leucocyte to a halt and allowing it to respond to migratory signals 31,35 that direct it across the endothelium and into tissue in which it follows a hierarchy of chemokines to the focus of inflammation (see Fig. 2). Thus, it is the combination of molecules on the endothelium and receptors on lymphocytes that act together to determine whether a lymphocyte is recruited. This provides several points at which diversity can be introduced to permit tissuespecific recruitment of different leucocyte populations. Subsets of lymphocytes with tropism for specific tissues can be defined by their expression of particular adhesion molecules and chemokine receptors, which allow them to respond to a combination of molecules on the endothelium that confer Recruitment of lymphocytes to the human liver 55 Figure 2 Lymphocyte adhesion to endothelial cells occurs in a series of stages. Free flowing lymphocyte subpopulations in the bloodstream (A + B) express different profiles of adhesion and chemokine receptors. Cells expressing appropriate counter receptors are captured by tethering or rolling receptors expressed on endothelial cells (1). Chemokine proteins localized on the glycocalyx of the endothelial cell are detected by specific, G-protein-linked receptors expressed on the leucocyte (2). This results in a conformational change in lymphocyte integrins, which permits firm adhesion of the lymphocyte to endothelial-expressed immunoglobulin adhesion molecules (3). Chemokine recognition also results in cytoskeletal reorganization within the adherent lymphocyte, which facilitates migration across the endothelial monolayer and into tissue (4). Once within the tissue the leucocyte follows a chemotactic gradient of chemokine signal towards the site of inflammation. Other cells in the microenvironment can affect the molecules expressed by the endothelium either by altering the differentiation in a tissue-specific manner (e.g. hepatocyte-derived growth factors drive the differentiation of sinusoidal endothelium), by secreting activationg factors, such as cytokines, that activate endothelium or by secreting chemokines that are then transported through the endothelium and presented within the glycocalyx. a molecular address for that particular tissue. 36,37 This paradigm has been used to explain why naive T cells are selectively recruited to lymph node and why mucosal lymphocytes are selectively recruited to the gut (see Fig. 3). T cells circulate continuously between the blood and the tissues and can be divided into naive or virgin cells that have not been exposed to antigen and primed cells that have been activated by specific antigen presented in lymphoid tissue. Primed cells consist of ‘effector’ cells, which home to inflammatory sites to mediate immune responses, and memory cells, which are long-lived cells that have reverted to a lessactivated phenotype and provide both immunological memory and the ability to respond rapidly to subsequent encounters with an antigen.38 Naive T cells migrate exclusively to lymph nodes, which they enter using combinations of the adhesion molecule L-selectin and the chemokine receptor CCR7. This allows the T cells to interact with the endothelial receptor peripheral node addressin and the chemokine SLC, which are largely restricted to lymph node high endothelial venules.39,40 After activation by antigen, primed and memory lymphocytes acquire a different complement of adhesion receptors that promote their recruitment into tissue. Some of these receptors, including the chemokine receptors CXCR3 and CCR5, appear to play a role in recruitment into inflammatory sites, whereas others promote tissue-specific recruitment. For example, CCR9 and the integrin α4β1 are selectively expressed on mucosal lymphocytes and allow them to bind to vessels in the gut that express the chemokine TECK and the α4β1 ligand MAdCAM-1.37,41 The absence of these endothelial ligands from most other sites means that these lymphocytes are selectively recruited to the gut, whereas lymphocytes that express CCR4 and CLA bind to dermal vessels and are recruited to the skin. 42–44 These processes insure that primed lymphocytes recirculate through the organ from which its stimulatory antigen originated, thereby increasing the likelihood that it will re-encounter its cognate antigen. Lymphocyte recruitment to the liver The normal liver contains resident large granular lymphocytes (pit cells) in the sinusoids. These cells probably enter the liver from the circulation via the sinusoidal endothelium and are retained in the sinusoids where they provide protection against viral infections and tumour cells. 45–48 In addition, the normal human liver contains significant numbers of T lymphocytes in the portal tracts and scattered through the 56 PF Lalor et al. Figure 3 Recirculation of lymphocyte subpopulations is regulated by differential expression of adhesion molecules and chemokine receptors. After leaving the thymus naive lymphocytes, which have not yet encountered antigen, recirculate continuously between the lymphoid system and the bloodstream in the absence of antigen. This is facilitated by their expression of L-Selectin, CCR7 and low levels of LFA-1 integrin, which can bind to PNAd, the chemokine SLC and ICAM-1, respectively, expressed on lymphoid high endothelial venules (HEV). When naive cells encounter antigen within the lymphoid environment they differentiate into memory/effector cells. These cells lose the molecules that permit entry into tissue draining lymph nodes and acquire new molecules that promote recruitment to tissue and specifically in the case of the gut and skin to the organ from which their stimulatory antigen was derived. This process is facilitated by the expression of specific homing receptors on subsets of lymphocytes and tissue-specific adhesion molecules and chemokines on endothelium in target tissue. Thus, skin homing lymphocytes express CLA and CCR4, which bind to E-selectin and TARC expressed on skin vessels, whereas gut homing lymphocytes express α4β7 integrin and CCR9, which recognize MAdCAM-1 and the chemokine TECK, which are largely restricted to mucosal vessels. To date no specific liver homing lymphocyte populations have been identified, but it is possible that such cells would express high levels of the receptor for vascular adhesion protein-1 (VAP-1), and may express CXCR3 for binding IP-10, Mig and ITAC. parenchyma (Fig. 1). Animal models suggest that terminally differentiated effector T cells are removed by the liver, where they die by apoptosis,49–51 and there is evidence from studies using radiolabelled lymphocytes that a similar phenomenon occurs in man.52–54 However, in normal human liver relatively few liver-infiltrating lymphocytes express apoptotic markers and, in addition to terminally differentiated T cells, other subsets including CD45RA+ cells can be detected, suggesting that many of the T cells in the liver are involved in providing local immune surveillance.38 Infiltration of the liver by lymphocytes occurs in response to many insults, including not only the hepatitis viruses, but Recruitment of lymphocytes to the human liver 57 Figure 4 This figure shows molecules that may be involved in lymphocyte recruitment to different compartments of the human liver. The establishment of inflammation requires both recruitment via the endothelium and retention in tissue. Some of the molecules that have been reported to be involved under normal and inflamed conditions in different liver compartments are shown. These include molecules involved in recruitment via endothelium and those involved in attracting lymphocytes to and retaining them at the epithelial surface of the bile ducts. Under inflammatory conditions broader profiles of adhesion molecules and chemokines permit recruitment of a wider range of lymphocyte subsets. also autoimmune and toxic damage such as that mediated by alcohol. The distribution of hepatic infiltration by lymphocytes varies depending on the inflammatory stimulus. In acute allograft rejection and biliary diseases such as PBC, lymphocytic infiltration is localized predominantly to the portal tracts, whereas lobular inflammation and infiltration of the parenchyma characterize acute viral hepatitis and autoimmune hepatitis. The factors that determine the distribution of hepatic infiltration in any given situation are poorly understood, but are likely to be determined by the site of entry of lymphocytes into the liver and factors that retain lymphocytes at a particular location.55 The recognition that the expression of adhesion molecules and chemokines is not uniform throughout the liver vasculature begins to provide a molecular explanation for the distinct patterns of lymphocytic infiltration in liver disease (Fig. 4). 58 PF Lalor et al. Lymphocytic infiltration of the portal tracts Lymphocytes are seen in portal tracts in the normal liver, and infiltration increases in many inflammatory diseases. In normal liver both CD4 and CD8 T cells are found in portal tracts albeit at low numbers and a population of cells with the characteristics of intraepithelial lymphocytes are found in association with biliary epithelium.56 Furthermore, cells with a dendritic morphology can be detected in portal tracts and animal studies suggest that in response to infection dendritic cells (DC) migrate via the sinusoids to the portal tracts, where they stimulate the development of portal-associated lymphoid tissue.57,58 We have recently reported similar findings in human inflammatory disease. These observations suggest that the portal tracts serve at least two immunological functions: (i) as a site of mucosal protection for antigens entering via the biliary epithelium; and (ii) as a form of lymphoid tissue to coordinate and orchestrate antigen-driven responses in the liver. This may explain why even in diseases such as viral hepatitis, in which the antigen is predominantly localized in hepatocytes, a portal infiltrate is a universal finding. Thus, lymphocyte recruitment into portal tracts will be important for normal immune surveillance as well as a factor in pathological inflammation. The exact route of entry for lymphocytes into portal tracts is unclear. The portal vessels include hepatic arteries and their portal tract capillaries and portal veins, which do not have classic postcapillary venules, but instead empty into the sinusoids. During chronic inflammation new microvessels develop in portal tracts that have morphological and phenotypic similarities with the secondary high endothelial venules and their development during inflammation is likely to facilitate the recruitment of lymphocytes. 59 Under non-inflamed conditions portal vessels express vascular adhesion protein-1 (VAP-1) and ICAM-2 and low levels of several chemokines, including RANTES, MIP-1α and MIP-1β.55 The ability of VAP-1 to support lymphocyte capture under some conditions suggests that appropriate signals for lymphocyte recruitment are present even in normal liver, although the range of lymphocyte subsets recruited may be restricted.60 In inflammatory conditions, the portal vessels express P-selectin, E-selectin, and vascular cell adhesion molecule-1 (VCAM-1), none of which is detected on noninflamed portal endothelium, and high levels of ICAM-1. 13 In addition, strong staining with the chemokines MIP-1α, MIP-1β, RANTES and MCP-1 can be detected. 16 Thus, the selectins induced on inflamed portal vessels could promote primary adhesion and tethering of lymphocytes with subsequent secondary adhesion occurring via integrins binding to endothelial VCAM-1 and ICAM-1. In support of this, increased expression of both very late antigen-4 (VLA-4) and leukocyte function related antigen-1 (LFA-1) have been reported on lymphocytes infiltrating inflamed liver. 61 Few lymphocytes in inflamed liver express either L-selectin or E-selectin ligands,62 suggesting that these pathways are less important, but they do express the P-selectin ligand, PSGL-1, and P-selectin is strongly induced on portal endothelium. P-selectin binding is a characteristic feature of Th1 cells, suggesting that it might be involved in recruiting specific functional subsets to the portal tracts. 63 In addition to the selectins, VAP-1 expression is maintained on inflamed portal endothelium where it can also support lymphocyte binding. In some chronic inflammatory liver diseases, including PBC, chronic hepatitis C infection and PSC, portal tract infiltrates organize into lymphoid follicles containing B and T lymphocytes, DC and new CD34+ vessels with the morphology of high endothelial venules.64,65 The development of new lymphoid tissue in inflammatory sites at times when normal lymph node development is complete is termed lymphoid neogenesis,66 and provides a microenvironment for the recruitment and retention of lymphocytes at sites of chronic inflammation.67,68 Recent evidence suggests that these follicles express chemokines such as SLC, which are usually restricted to lymph nodes, and may therefore provide a specialized microenvironment for the continuing recruitment and differentiation of lymphocytes within the chronically inflamed liver. In addition to lymphoid follicles, the fibrous septa, which are a characteristic feature of cirrhosis, also contain newly formed blood vessels and have been referred to as ‘fibrovascular membranes’.69 These vessels express high levels of adhesion molecules and chemokines and provide an additional pathway for lymphocyte recruitment. Because they are located at the periphery of portal tracts at the interface with the parenchyma they may be critical for the development of the lesion of interface hepatitis and the spreading of inflammatory damage from the portal tract to the parenchyma. 70 Lymphocyte recruitment via the sinusoids Intravital microscopy has revealed that leucocyte recruitment to the hepatic parenchyma can occur through the sinusoids in a process that involves direct adhesive interactions with sinusoidal endothelium.14,51,71,72 Moreover, the heterogeneity within the sinusoidal bed suggests that recruitment through the perivenular and periportal sinusoidal endothelium might be differentially regulated.73 Several features of the sinusoidal bed are profoundly different from other vascular beds and will affect how lymphocytes interact with sinusoidal endothelium. The sinusoid is a low blood flow environment in which leucocytes come into contact with the sinusoidal lining cells as they make their way through the liver microvasculature. These conditions are likely to reduce the need for molecules, such as selectins, that mediate capture of fast-flowing lymphocytes in other vascular beds and might explain why selectins are largely absent from the sinusoids. 14,62 Several other adhesion molecules are constitutively expressed on hepatic sinusoidal endothelium including ICAM-1 and VAP-1, both of which support lymphocyte adhesion. 60,61 VAP-1 can mediate lymphocyte capture under some conditions, although recent evidence suggests that it is also required for transendothelial migration.74 This latter function may be particularly important in the sinusoids that lack a basal lamina, tight junctions and junctional adhesion molecules such as CD31 that have been shown to regulate transmigration across other vascular endothelial cells.75–77 Increased expression of adhesion molecules and chemokines is detected on sinusoidal endothelium during inflammation when ICAM-1 is increased, and VCAM-1 and CD31 induced. In addition, the development of chronic inflammation can stimulate the development of neovessels at areas of interface hepatitis in which the sinusoidal endothelium undergoes neocapillarization with the development of a rudimentary basement membrane and expression of microvascular Recruitment of lymphocytes to the human liver markers, such as CD34.78,79 In inflammatory conditions, the expression of VAP-1 remains constant, even in diseases in which portal vessels express few selectins or no E-selectin or P-selectin expression is detected on the sinusoids. In these inflammatory conditions, primary adhesion to sinusoidal endothelium could also be mediated by VAP-1 or VCAM-1, which are induced and can support the capture of lymphocytes via α4-integrins. Subsequent firm adhesion appears to be mediated predominantly by β2 integrins and ICAM-1. Role of chemokines in regulating lymphocyte recruitment to liver compartments The detection of chemokines on sinusoidal endothelium also changes with inflammation and there is evidence that specific chemokine/chemokine receptor interactions regulate the recruitment of activated T cells to different compartments of the inflamed liver.17 Furthermore, the pattern of chemokine secretion in the liver may determine the distribution and severity of the T-cell inflammatory infiltrate. CC chemokines with activity for T cells such as MIP-1α and MIP-1β are found predominantly on vascular endothelium within portal tracts, whereas the CXC chemokines, Mig and IP-10, which bind to CXCR3, a receptor that is upregulated on activated lymphocytes, are preferentially expressed by sinusoidal endothelium. Furthermore, the expression of IP-10 is associated with localized lymphocyte lobular infiltrates in areas of necroinflammatory activity in hepatitis C and parenchymal damage in chronic allograft rejection. 19 Hepatic sinusoidal endothelial cells secrete IP-10 and Mig in vitro in response to IFN-γ and increased IP-10 mRNA has been detected in inflamed livers in association with local inflammation. In addition, hepatocytes stimulated with cytokines 17,80,81 and activated Kupffer cells19 also secrete CXCR3 ligands, suggesting that chemokines sequestered in the sinusoidal glycocalyx are likely to reflect the production by several cells types within the local microenvironment (Fig. 2). Further evidence for the importance of IP-10 and Mig is provided by the high levels of their receptor CXCR3 on liver-infiltrating T cells compared with autologous peripheral blood T cells. 17,19,82,83 The requirement for IFN-γ to induce CXCR3 ligands, and the finding that high levels of CXCR3 are often associated with Th1 responses,84,85 all suggest that this pathway is tightly regulated and a critical factor in the progression of Th1 responses in the hepatic parenchyma. The different patterns of chemokine expression between portal vessels and sinusoids suggest that IP-10 and Mig are predominantly involved in recruitment via the sinusoids, whereas interactions between the CC chemokines MIP-1α and MIP1β and CCR5 may be involved in the recruitment of T cells to portal areas. Because these CCR5 ligands can be detected on portal vessels in normal human liver (albeit at lower levels than in inflammatory disease) they provide a mechanism for the recruitment of CCR5high memory T cells into portal areas in normal liver as well as for recruitment in inflammatory liver diseases.15 Chronic hepatitis confined to portal areas (‘chronic persistent hepatitis’) is generally associated with a favourable outcome, whereas extension of the inflammatory process into the adjacent liver parenchyma in the form of interface hepatitis (‘piecemeal necrosis’) is associated with destruction of hepatocytes and progressive periportal fibrosis 59 (‘chronic active hepatitis’).86–88 The induction of the interferondependent chemokines IP-10, Mig and ITAC on sinusoidal endothelium may be an important mechanism for the direct recruitment of CXCR3high T cells to the liver parenchyma and the development of progressive liver damage. Role of non-endothelial cell types in lymphocyte recruitment to the liver Endothelial chemokines regulate the recruitment of lymphocytes into tissue, but the development of chronic inflammation requires that cells are retained at sites of inflammation. 89 Chemokines on stromal or epithelial cells not only attract infiltrating cells, but also retain them in tissue by activating integrin-mediated adhesion, thereby promoting the development of chronic inflammation.89 Many chemokines are detected on inflamed bile ducts in vivo and secreted by cytokine-stimulated biliary epithelium in vitro, including IL-8, MCP-1 and RANTES. These chemokines may be important in localizing infiltrating lymphocytes to the portal tracts and retaining them in close proximity to bile ducts 90,91 in biliary diseases, such as PBC, or allograft rejection. 19 Non-inflamed biliary epithelium expresses high constitutive levels of SDF, 92 a chemokine that has been shown to be a potent inducer of sustained integrin activity in lymphocytes. 67 This chemokine has been implicated in B lymphopoesis in the liver during fetal development,92 but it may also be important in the normal adult liver. Liver-infiltrating lymphocytes express the SDF receptor CXCR4 and cells entering the non-inflamed liver may be attracted and retained at the biliary epithelium, where they can provide immune surveillance against pathogens entering via the biliary tract.19 Activated stellate cells that drive the development of fibrosis and cirrhosis in the inflamed liver 93 are a potent source of several chemokines including IP-10 and MCP1.94–96 The ability of fibroblasts to recruit, retain and promote the survival of infiltrating lymphocytes has been proposed to be an important step in the evolution from acute to resolving inflammation in the joints,89 and stellate cells may play a similar role in the liver.89,93 In addition to stellate cells, other inflammatory cells, including DC, infiltrating leucocytes and Kupffer cells, will all contribute to the chemokine milieu within the liver. One particularly important chemokine in this context is MIP-3α, which recruits activated CD4+ T cells by binding to the receptor CCR6. CCR6 is expressed on CD4 T cells infiltrating the human liver and MIP-3α is detected in periportal areas associated with DC and macrophages. Furthermore, MIP-3α secretion by DC can be triggered by culturing them with apoptotic cells, suggesting that local tissue damage will lead to DC secretion of MIP-3α and recruitment and retention of activated CD4 T cells at sites of injury. The fact that CCR6 is coexpressed with CCR5 on activated CD4 T cells provides them with a mechanism for recruitment via endothelium as well as localization within inflamed tissue.97 Role of vascular adhesion protein-1 in lymphocyte recruitment to the human liver One of the most obvious differences between hepatic sinusoidal endothelium7 and endothelium in other tissues is the lack 60 PF Lalor et al. of a role for selectins in the liver vasculature. 14,62 In the absence of selectin interactions, other tethering molecules are required to capture lymphocytes on hepatic endothelium and we have proposed that VAP-1 is a candidate for this role. VAP-1 is a homodimeric, class II transmembrane protein 98,99 that supports lymphocyte adhesion to high endothelial venules in lymph nodes.100 Outside the lymph node, in the absence of inflammation, endothelial expression of VAP-1 is largely confined to hepatic vessels, where it supports lymphocyte adhesion.60,61 This adhesion is sialic acid dependent and operates under shear stress in vitro,61 suggesting that VAP-1 could act as a capture receptor to support the tethering interaction between lymphocytes and hepatic sinusoidal endothelium in the absence of selectins. Further evidence for VAP-1 as a capture receptor comes from structural modelling of the D4 domain of human VAP-1, which reveals six sites for glycosylation, on the top of the molecule, from which carbohydrate chains would extend from the surface of the molecule into the vascular lumen.101 VAP-1 is not as effective as the selectins in promoting leucocyte rolling, but the architecture of the liver and the low blood flow within the liver microcirculation may circumvent the requirement for rolling receptors operating in the classical sense (i.e. permitting rapid capture of a fast moving cell). Thus, a lymphocyte moving slowly through the narrow, irregular sinusoids might be more readily captured via VAP-1. Our recent data suggest that VAP-1 plays a more complicated role in lymphocyte adhesion to hepatic endothelium. Once captured by, and firmly adherent to hepatic endothelium, a lymphocyte must breach the endothelial barrier and migrate into the tissue. In most circumstances this involves passage through interendothelial tight junctions,102 a process that can be facilitated by molecules, such as CD31, which are concentrated at these sites. 77,103 However, as outlined above hepatic sinusoidal endothelial cells form a discontinuous barrier without tight junctions and they express low levels of CD31 compared with vascular endothelium, suggesting that other factors are involved. Using a flow-based adhesion system and hepatic endothelial cells we have shown that antibodies to VAP-1 inhibit not only adhesion, but also transendothelial migration. The regulation of VAP-1 expression is poorly understood and like MAdCAM-1 it is not readily inducible by cytokines in vitro, although it is expressed in vivo in inflamed skin, joints and gut. Expression on the cell surface appears to involve mobilization of protein from the cytoplasm as well as de novo expression.104 VAP-1 is shed from the endothelial cell surface and can be detected in serum in a circulating form. In vivo the great majority of sVAP-1 is derived from the hepatic vascular bed and the highest levels of circulating VAP-1 are found in chronic inflammatory liver diseases, further supporting its unique role in the liver.105,106 Neither the mechanisms of VAP-1 cleavage nor the function of soluble VAP-1 are known. The recent molecular characterization and cloning of VAP-1 revealed that it has close sequence homology to the copper-dependent semicarbizide-sensitive amine oxidases (SSAO).107,108 It has been known for over 20 years that chronic liver disease is associated with increased monoamine oxidase (MAO) activity in serum109–111 and we have now shown that this enzymatic activity is in fact SSAO activity derived from soluble VAP-1 protein because SSAO activity in human serum is abolished by immunodepletion of sVAP-1.106 Thus, soluble VAP-1 is responsible for the vast majority of SSAO activity in human serum both in health and in inflammatory liver disease. These studies provoke several questions. Why does an adhesion protein have enzymatic activity? Are these functions related or evolutionary distinct? Other molecules have been reported to share adhesive and enzymatic properties including CD26, CD73 and the Adamalysins, but VAP-1 is the only adhesion molecule with amine oxidase activity. Salmi and colleagues have recently proposed that the active site of SSAO in the VAP-1 molecule is directly involved in the adhesion.112,113 They suggest that VAP-1 binds lymphocytes via a sialic acid dependent epitope and subsequently utilizes the catalytic reaction between the SSAO and an amine on the lymphocyte surface to link the endothelial cell and the lymphocyte by a transient covalent bond. In addition, protein-bound aldehydes that are formed on the lymphocyte as a result of the enzymatic reaction could modulate further interactions with endothelial cells. Whatever the precise mechanism the ability to block VAP-1 function with small molecule enzyme inhibitors has enormous therapeutic potential. Conclusion Lymphocyte recruitment to the human liver is mediated by distinct combinations of molecules depending on whether recruitment occurs via the portal vascular endothelium or the hepatic sinusoids. Under non-inflamed conditions the molecules involved are relatively restricted and result in selective recruitment of lymphocytes, whereas with inflammation the repertoire of molecules available for recruiting lymphocytes increases. The hepatic sinusoids are a distinct vascular bed with unique morphological, rheological and phenotypic characteristics and in this environment VAP-1 appears to play an important role both in lymphocyte capture in the absence of selectins and in transmigration. The interferon-inducible chemokines that bind the lymphocyte receptor CXCR3 are strongly expressed on inflamed sinusoids, where they are associated with necroinflammatory activity. These chemokines may be critical for the development of progressive liver injury in diseases such as chronic hepatitis C. 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