Extracellular Matrix Proteins in Organ Transplantation1

Traffic of leukocytes from the bloodstream to specific sites of inflammation is regulated by signals that stimulate cellular adhesion to vascular endothelium or extracellular matrix (ECM) proteins (1, 2). To migrate to the extravascular areas, circulating leukocytes have to acquire strong adhesion interactions to the vessel wall while resisting to continuous sheer forces (3). The emerging relevance of leukocyte-ECM interactions to cell adhesion, migration, and positioning within specific tissue microenvironments emphasizes the role ECM proteins may play in the functioning of the immune system in both physiological and pathological conditions (1, 4). This review will focus on some of the best characterized ECM proteins, fibronectin (FN) in particular, one of the most studied ECM components in organ transplantation. The text is divided into three integral parts, which include a general overview of ECM proteins and their cellular receptors, a narrative of ECM proteins in organ transplantation, and a discussion of ECM-leukocyte interactions as potential targets for therapeutic intervention. ECM PROTEINS AND THEIR CELLULAR RECEPTORS FN is a large dimeric glycoprotein composed of a series of independently folding modular domains known as FN repeats I, II, III (5). These repeating units contain regions or domains that interact with a number of other matrix molecules as well as cells, and mediate functions such as cell migration, matrix assembly, differentiation, and proliferation (6, 7). Structural diversity in FN arises by regulated alternative splicing of a single gene transcript in three segments, termed EIIIA, EIIIB, and V in rats, and ED-A, ED-B, and IIICS, respectively, in humans (8, 9) (Fig. 1). The EIIIA (or ED-A) and EIIIB (or ED-B) domains are either included or excluded as intact type III homology repeats. The V (or IIICS) may be excluded, partially included, or fully included. The latter form contains the connecting segment-1 (CS1) region known to mediate leukocyte adhesion (10). The FN variants that include the EIIIA and EIIIB segments are prominent in FN produced during embryogenesis, and their expression in the adult tissues is minimal unless under certain pathological conditions (11, 12). For example, adult liver synthesizes the plasma form of FN, and this form excludes the EIIIA and EIIIB domains. However, the EIIIA and EIIIB segments are markedly increased during cutaneous wound healing (13, 14), neointimal hyperplasia (15, 16), and glomerular nephritis (17, 18). Alternative splicing of the FN gene allows for the generation of multiple isoforms, with 20 isoforms possible in humans (19). The role of FN in lymphocyte adhesion, migration, and activation has been extensively illustrated (20–24). Figure 1: FN structure and variants. A diagrammatic representation of FN showing the three regions of alternative splicing designated EIIIA, EIIIB, and V in rats. FNs are mainly composed of three types of homologous structural units called I (rectangles), II (triangles), and III (ovals), which define several domains interacting with different molecules. Note that the cell binding domain RGD that interacts with the α5β1 integrin and the cell adhesive segment CS1 that interacts with the α4β1 integrin are included in FN-III(8–11) and in V120, respectively.Tenascin (TN), another ECM oligomeric protein functionally associated with FN, consists of an amino- terminal cystein-rich region followed by a series of EGF-like domains, a series of FN type III repeats, and a fibrinogen-like region at the carboxyl terminal (25, 26). The findings that TN is present normally and exclusively in the adult human thymus and thymus-dependent areas of lymph nodes (27) and abnormally in the matrix of tumors (28) are attracting a growing interest to this ECM component. TN is considered a major ECM glycoprotein that can interfere with the activity of FN by inhibiting cell adhesion and spreading (27). TN together with thrombospondin and SPARC, form a family of ECM proteins with counter-adhesive properties that are able to provoke the loss of focal adhesions in well spread endothelial cells (29). TN inhibits both monocyte and T cell adhesion to FN (30–33). Moreover, soluble TN inhibits proliferation of human T cells and high level induction of interleukin (IL)-2R in response to anti-CD3 antibody (Ab) co-immobilized with FN (31). TN may also support the tethering and rolling of peripheral blood lymphocytes under flow conditions, with several more binding sites, compared with the rolling on E selectin (34). Laminin (LN), one of the first ECM protein detected during embryogenesis, is found predominantly in basement membranes (35, 36). LN is composed of three subunits, designated α, β, and γ, where the association of different isoforms of those subunits into heterotrimers gives rise to the seven known LNs (37). The expression of specific LN isoforms is tissue specific, and it may be altered in disease, suggesting that the interactions of leukocytes with different LN isoforms can elicit differences in leukocyte functions (38). Indeed, LN has been found to play a role in T cell migration (39, 40), and T cell differentiation (41). Cellular adhesion to ECM proteins is mediated primarily by a superfamily of heterodimeric molecules, the integrins (42). The leukocytes have been shown to interact with sequences within the type III repeats of FN primarily through two different receptors of the β1 integrin family, the α4β1 and α5β1 (43, 44) (Table [tbc]). Thus, α4β1 integrin, interacts with the CS1, which is located within the V region (44). In addition to the CS1 binding domain, activated α4β1 integrins are able to recognize the KLDAPT (45) and RGD domains on FN. For example, adhesion of B lymphocytes to FN is exclusively mediated by the interaction between α4β1 and the CS1 domain (46). However, antigen activation can induce α4β1 integrin expression on B cells to recognize the RGD domain (47). The flexibility of the α4β1 integrins in recognizing other domains in the FN molecule supports the important role this ECM protein may play in an array of lymphocyte biological functions. The other main receptor for FN is the α5β1 integrin that interacts through the RGD sequence, and it is known to be present on a subpopulation of resting CD45RA dim memory T cells (48). Although, the RGD loop is the critical recognition site for the α5β1 integrin, it is thought that in humans the PHSRN sequence located in FN-III9 is required for high affinity binding (49). Other integrins have also been identified as alternative receptors for FN, such as α4β7 (50), α3β1 (6), α8β1 (51), αvβ1 (52), αIIβ3 (53), αvβ3 (54), αvβ6 (55), and α?β8 (56) (Table). Adhesion to TN has been attributed to several receptors, such as α2β1, α9β1, and FN, although the mediators of leukocyte adhesion to this protein need to be identified (34). Recently, it has been suggested that α9β1 is critical for neutrophil migration on both vascular cell adhesion molecule-1 (VCAM-1) and TN (55). Finally, α6β1 integrin represents the principal leukocyte LN receptor on macrophages, neutrophils, and T cells (57–59). The adhesive capacity of integrins expressed on circulating leukocytes is highly regulated. For example, adhesion of T cells to FN has been shown to be regulated by the engagement of antigen-specific T-cell reactivity (TCR)/CD3 complex, as well as CD2, CD7, and CD28 (60–62). The fact that TCR engagement up-regulates adhesion of T lymphocytes to FN highlights the importance of this protein in host immune responses. Integrin-dependent biding of lymphocytes to FN is also regulated by a number of chemokines, including the C-C chemokines, such as RANTES, monocyte chemotactic protein 1 (MCP-1) and macrophage inhibitory protein 1 (MIP-1) (63–65). Modifications in ECM expression may also have a regulatory role in the integrin-mediated leukocyte adhesion. Indeed, there is good evidence that ECM protein binding can modulate integrin expression (66, 67).Table 1: Fibronectin and its integrin receptorsECM PROTEINS AND ORGAN TRANSPLANTATION There is a growing number of reports describing changes in ECM composition associated with reperfusion injury or acute and chronic rejection in human organ transplantation (68–71). Because much of mechanistic studies on the immune modulation by ECM proteins derive from experimental setting, this review emphasizes work done primarily in animal transplantation models. In our own immuno-histological and in situ hybridization studies of rat cardiac allografts, a markedly increased expression of FN and LN, mostly vascular, in the early posttransplantation period preceded cellular infiltration (72, 73). This initial up-regulation of vascular FN and LN is a common step in both allotransplantation and isotransplantation and may reflect a response to injury or ischemia occurring during the interval of about 45 min of cardiac engraftment (73). The initiation of vascular FN and LN synthesis so early may represent an important signal that triggers lymphocyte recruitment at the graft site. Indeed, administration of anti-LN Ab to T-cell deficient (B) rat recipients of cardiac allografts significantly decreased the accumulation of adoptively transferred lymphocytes selectively in the peripheral lymph nodes and in the cardiac grafts (39). At later posttransplantation periods, simultaneous detection of FN and LN in cardiac allografts by laser scanning confocal microscopic analysis, revealed a preferential accumulation of FN in the interstitial and perivascular areas where infiltrating mononuclear cells (MNC) localize. Sensitized lymphocytes adoptively transferred to test recipients also localized in FN-rich areas of both cardiac transplants and lymph nodes (72). Furthermore, treatment of rats with a neutralizing anti-TNF-α serum significantly prolonged cardiac allograft survival, downregulated local production of FN and reduced intragraft MNC infiltration (74). This supports a key role of FN as an in vivo adhesive factor for lymphocytes to home to specific tissue microenvironments, including an organ transplant (75). Our combined immunohistological and in situ hybridization analyses have demonstrated that the prime sources of FN in rejecting cardiac allografts are macrophages in the myocardium and smooth muscle and endothelial cells in the vessels (73, 74). It remains to be determined how different cell types regulate FN expression in vivo. Although transforming growth factor (TGF)-β1 is known to increase FN expression by fibroblasts, its role in modulating FN expression in other cell types remains unclear (76, 77). Our data suggests a potential role for TNF-α in modulating macrophage appearance, and potentially FN expression, during cardiac allograft rejection (74). Other data also indicate that, in coronary arteriopathy after transplantation, endothelial and smooth muscle cells produced increased FN under the regulation of IL-1β and TNF-α (78, 79). Moreover, soluble TNF-α receptor reduces the expression of FN and leukocyte infiltration within areas of intimal thickening (80). In cardiac allografts, FN exists in multiple isoforms with a distinct temporal and spatial expression patterns (73). Newly synthesized FN in cardiac grafts includes EIIIA+, EIIIB+, and CS1 variants that are generated by alternative splicing of FN premRNA (Fig. 1). Local synthesis of FN by cells in the transplanted organ is not likely the only FN present. The extravasation of plasma FN along with other plasma proteins, such as fibrinogen, seems to be a ubiquitous feature of all inflammatory sites examined (81). Extravasated plasma proteins constitute a provisional matrix that is a prominent feature of early healing wounds and cutaneous delayed hypersensitivity reactions (82, 83). An important consequence of FN alternative splicing that occurs during inflammation is the gradual replacement of plasma FN, which largely lacks the EIIIA and EIIIB segments, with locally produced FNs that contain these segments. Very little is known about the function of the EIIIA and EIIIB domains of FN, although, by comparing the patterns of EIIIA and EIIIB expression in cardiac allografts and isografts, our data support the hypothesis that these FN variants are involved in lymphocyte adhesion, migration, or differentiation in cardiac allografts. Both EIIIA and EIIIB domains are highly expressed in the myocardium of cardiac allografts associated with increased numbers of infiltrating T cells and appearance of infarcts. In contrast, the FN splicing variants are not detected in the myocardium of cardiac isografts, neither are a similar increase in T cells, nor are infarcts. Because the EIIIA segment mediates adhesion of fibroblasts and promotes the differentiation of lipocytes in the liver to become myofibroblasts (84), one may envision similar processes in lymphocyte biological functions in organ allografts. For example, CD8+ T-lymphocyte-mediated lysis of target cells requires the formation of tight conjugates between T cytotoxic cells and the targeted cell. Whereas the cellular receptors for EIIIA and EIIIB domains are not yet fully characterized, the CS1 domain recognized by the α4β1 integrin is known to mediate lymphocyte adhesion (10, 43). The CS1 subunit of FN is the first alternatively spliced domain found to be increased in cardiac allografts, it is up-regulated as early as 3 hr after transplantation, and it is preferentially expressed after day 4 (73). The presence of specific FN variants may provide a spatial address at which appropriate co-stimulatory signals could initiate or augment critical T cell signaling events. Supporting this assumption, are our recent findings in rat recipients rendered tolerant to MHC-incompatible cardiac grafts by exogenous immunosuppressive therapy. In long-term tolerant recipients, FN is up-regulated in the vessels in the absence of other vascular adhesion molecules, such as VCAM-1 and intercellular adhesion molecule-1 (ICAM-1), highlighting the putative role of FN in the recruitment of leukocytes at the graft site (85). Moreover, despite the high number of MNC infiltrating well-functioning grafts, intramyocardial infiltrating macrophages failed to express FN. This contrasted with long-term control recipients undergoing chronic rejection, in which FN expression was readily detectable in the myocardium (85). The observation that MNC do accumulate in the myocardium in the absence of cellular FN in tolerant hosts, indicates another role for this ECM protein that is unrelated to cell migration or tissue positioning. Furthermore, FN may regulate IL-2 and IFN-γ expression, which indicates an active role of this ECM protein in cellular activation (Coito et al., April, 1999, unpublished data). Our results are consistent with the concept that FN exerts synergistic effects on T cell activation by acting as a co-stimulator for both CD4+ and CD8+ T cells through TCR (23, 86) and cytokine release. For example, the density of immobilized CD3 or TCR monoclonal antibody (mAb) required to induce degranulation and tyrosine phosphorylation of cellular proteins by CD8+ T cells is about 10 times lower in the presence of FN (23). Several studies have also shown that adhesion to FN activates tyrosine phosphorylation of several T cell proteins (23, 87, 88). The production of IL-2, IFN-γ, and TNF are also stimulated in vitro by interactions between CD4+ cells and FN Moreover, it has been that the binding of CD4+ cells to ECM proteins, most likely through changes in to their may in cytokine activity For a cytokine with yet in organ transplantation, is by most cells in an form and its active or form is highly on the in vivo association to thrombospondin In addition to cell migration and differentiation, there is evidence that ECM may also function as a leukocyte factor in cardiac allografts. also called cell has been in organ transplants undergoing chronic rejection Moreover, recent studies have a role of mediated by activated MNC in the of allografts and in the vascular of chronic rejection In it has also been illustrated that cells that are from to ECM proteins may Indeed, the interactions support of cell α5β1 or cells these findings support the that ECM proteins are active in the immune to graft The role of adhesive interactions has been in both and transplantation animal models. MNC infiltration occurs in followed by and In cells from increased adhesion to FN in an increased adhesion in has been after of cells with FN that interact with integrins cell It is that of FN leukocyte tissue and the of and the for this The of FN has been also in the rat of only FN inhibitory to acute and chronic also found to and local leukocyte and the of Figure T cell interactions in the of FN, which provide the for possible therapeutic intervention. Figure of T cell interactions in the of FN. Cellular interactions with FN are mediated primarily by the α4β1 and α5β1 The interactions of T lymphocytes with FN may have distinct during allograft T cell interactions with FN expressed by endothelial cells may the up-regulation of other vascular adhesion molecules, such as and the recruitment of leukocytes at the graft T cell interactions with FN expressed mainly through macrophages may as a co-stimulatory signal on T cell activation and cytokine release. indicate FN by in a rejecting rat cardiac own data are consistent with the in which in vivo interactions between the α4β1 integrin receptor and the CS1 of FN are critical in the acute allograft rejection Indeed, treatment of rat recipients of cardiac allografts with a of CS1 the of acute rejection, and the transplant of vascular expression of adhesion molecules such as VCAM-1 and of intragraft infiltration by CD4+ and CD8+ cells, and of activation at the graft as by decreased infiltration by cells, and expression of T cell 1 and T cell type These immunosuppressive effects could be and acute rejection after treatment with IL-2, suggesting that CS1 may induce a of T cell in vivo. of the adhesion functions mediated by α4β1 integrin are attributed to interactions with its two known the CS1 in the FN and VCAM-1 expressed on endothelial cells (Table and Figure 2). Although it is possible that CS1 may interfere with α4β1 VCAM-1 α4β1 has been shown to have distinct sites for VCAM-1 and FN suggesting that the CS1 that α4β1 integrin not likely the binding by Moreover, although VCAM-1 and may biding sites on the of FN that interfere with the α4β1 VCAM-1 binding are several times those required for the α4β1 CS1 it seems that the effects of CS1 on the and local cell recruitment associated acute graft rejection, could be attributed to the of α4β1 VCAM-1 Supporting this are findings from cardiac transplant of T cell infiltration in allografts, despite and T cell in isografts, which are of VCAM-1 expression These a role for that mediate leukocyte during the of acute It has been shown that the of leukocyte interactions by treatment with with to the of rat allografts Moreover, combined of α4β1 and VCAM-1 prolonged cardiac allograft and in some In the of α4β1 with only in a of the graft However, in a more recent administration of CS1 to recipients of allografts not only graft rejection, also graft suggesting a role for α4β1 FN interactions in leukocyte to the graft site In to the of interactions by CS1 the effects by the α4β1 integrin by and on mononuclear and endothelial cell to graft rejection in transplant have also the of CS1 in rat recipients in which with rejection at hr not the cardiac allograft loss at These long-term grafts of a of chronic rejection Indeed, an of CS1 in the early posttransplantation in the of the inflammatory and smooth muscle cells from the of long-term or large in rats undergoing those CS1 and of neointimal that CS1 the of chronic Moreover, treatment with CS1 reduced gene transcript and for T and and which are known to to the of allograft A similar was in a in which treatment with CS1 vascular and markedly coronary arteriopathy with FN intragraft infiltration by macrophages and T cells, the role of FN in the of inflammatory cells in of cardiac However, in our rat CS1 not the of cardiac rejection in the as by the cell and interactions are critical for T cell adhesion, and local to chronic inflammatory of cellular and recruitment by FN an important for modulating the adhesion and inflammatory the of FN to neointimal thickening in coronary this may interfere with the migration of smooth muscle cells from the into the with of neointimal consistent with the expression of α4β1 integrin by smooth muscle cells The of chronic rejection may from a more effects on long-term in vivo interactions between endothelial vascular and the ECM with rejection in by and T cell cytotoxic and At this interactions are to the cellular with ECM and the endothelial administration of CS1 during this early posttransplantation may the engagement of T cells with FN as of the transplantation structural At the CS1 may intragraft by its for host the the in intragraft MNC and positioning are by the macrophage The of smooth muscle neointimal and to be Other findings the of cell to and microenvironments to as by was also in cardiac recipients with In this of chronic recipients are through a CD4+ T although treatment with an antibody the LN receptor the of CD4+ cells to migrate into the graft and rejection Moreover, the of decreased of activated lymphocytes and prolonged allograft in of and transplantation. is a cell receptor that to ECM components such as and a major of the extracellular can mediate activated T cell extravasation into sites of inflammation by the adhesion to vascular endothelium under conditions of However, a is a monocyte Indeed, highly up-regulated may be readily detected in cardiac allografts in with increased macrophage infiltration unpublished these data the hypothesis that local synthesis of ECM components is an feature and adhesive are critical for the acute and chronic rejection in transplant of interactions may potential sites for in the control of transplant rejection, and may to the of of host