Transplant immunobiology: a crucial role for heparan sulfate glycosaminoglycans?

After allotransplantation, a solid organ can suffer a number of injurious processes that result in a functional deficit. These include responses to stress associated with brain death of the donor; ischemia-reperfusion injury inflicted before, during, and immediately after surgery; and, potentially, local binding of preformed antidonor antibodies. These insults result in endothelial cell activation, local shedding of anticoagulant factors from the endothelial cell surface (1), and immediate release of granule-stored E-selectin molecules and chemokines such as interleukin (IL)-8 (2). This causes local immobilization of leukocytes on the surface of the graft endothelium and, if sufficiently severe, can generate a procoagulant intravascular environment, leading to thrombosis and hyperacute graft failure. Grafts that survive the perioperative period are susceptible to specific T-cell–mediated immune responses, which are orchestrated by a range of cytokines including graft- and immune-cell–generated chemokines such as interferon (IFN)-inducible protein 10, proinflammatory cytokines such as IFN-γ, and a range of T-cell growth factors including IL-2 and IL-4 (3). After the main phase of T-cell–mediated rejection, grafts often show a gradual deterioration in function associated with interstitial fibrosis and vascular occlusion (4). This process is relatively poorly understood but is likely to involve excessive activity of locally produced growth factors, such as transforming growth factor-β (5). Glycosaminoglycans (GAG) are complex polysaccharides involved in the preservation of the critical anticoagulant surface of vascular endothelial cells and in binding and regulating the function of many cytokines, including those involved in every stage of graft damage. Therefore, it is reasonable to consider GAG as central molecular components of the posttransplant response and, therefore, as excellent targets for immunomodulatory intervention. This review considers the role of GAG as regulators of the graft rejection process through their interactions with a variety of proteins. In addition, it surveys the prospect of using these molecules for specific anti-inflammatory therapy after transplantation. PROTEOGLYCANS AND GAG GAG are components of proteoglycan (PG) molecules found on the cell surface and within the extracellular matrix (6). PG consist of a range of core proteins that are covalently linked to one or more GAG chains. These chains are chemically defined as heparan sulfate (HS), chondroitin sulfate, dermatan sulfate, or keratan sulfate that range in molecular weight up to 100 kDa. HS is generally the most abundant GAG, and can reach a concentration of up to 200 μg/mL within the thin PG layer on 50% to 90% of endothelial cell surfaces. Both the core protein and the GAG component of a PG can contribute to the molecule’s function (7). There are two major families of plasma membrane-bound HS containing PG, known as the syndecans (four members) and the glypicans (six members). Both families have distinct functions even when expressed on the same cell type (for an in depth review, see Bernfield et al. (8)). Several minor membrane PG containing HS have also been described, including epican and betaglycan. Cells that produce basement membranes also secrete the HS-PG perlecan, agrin, and collagen XVIII. In addition to core protein variability, the number and type of GAG chains attached to the core can also vary considerably (9). There has been an increasing realization that specific sequences in HS-GAG chains are responsible for selective interactions with certain proteins and that these interactions result in the regulation of protein activity. Cells can also dynamically alter the structure of HS-GAG sequences in response to their environment, leading to the view that they can act as a new class of multifunctional cell regulator. In common with most GAG, HS is synthesized in association with the core protein to form a mature PG. The first step in the heparin (a highly sulfated variant of HS)-HS biosynthetic pathway occurs within the Golgi apparatus and consists of the assembly of a tetrasaccharide linkage region (xylose-galactose-galactose-glucuronic acid) on a selected serine residue of the core protein. In the case of HS, chain extension occurs with the alternate transfer of N-acetyl-d-glucosamine (GlcNAc) and d-glucuronic acid (GlcA) monomers from uridine diphosphate-sugar donors to the nonreducing terminus of the new chain. The result is an unmodified polymer consisting simply of repeating GlcNAc-GlcA units that are then acted on in a sequential manner by a series of modifying enzymes (Fig. 1). However, not all of the potential sites will be modified, and few reactions will reach completion, leading to a large degree of structural diversity. Figure 1: Model of HS-GAG structure on luminal surface of vascular endothelium: HS-GAG exists on the cell surface as protein conjugates called proteoglycans. Dark rectangles show regions of high sulfation; the linkage region depicts undersulfated regions. Also shown is the HS-GAG disaccharide repeat: a uronic acid (iduronic or glucuronic) linked to glucosamine. X, A position that can be either sulfated or free; Y, an acetate or sulfate moiety.Members of the N-deacetylase–N-sulfotransferase (NDST) (10) enzyme family replace some acetyl groups with sulfate groups to form clusters of disaccharide units containing N-sulfated glucosamine (GlcNS) residues. This crucial modification creates a target for further modification (11). A glucuronosyl C5 epimerase can act on some GlcA monosaccharides to produce iduronic acid (IdoA). Regions rich in GlcNS and IdoA are termed S domains, whereas those where the precursor saccharide backbone has undergone few modifications are called N-acetylated (NA) domains. The domain structure and sulfation pattern of HS has important functional implications (12). For example, the relatively undersulfated NA sequences that intersperse the heavily sulfated S domains are flexible and can enhance the formation of dimers between HS-bound chemokine molecules such as IL-8 (13). Several sulfotransferase enzymes further modify the S domains, including 2-O-sulfotransferase (2OST), which adds a 2-O-sulfate to IdoA (14); 6-O-sulfotransferases (6OSTs) (15), which adds 6-O-sulfates to GlcNAc and GlcNS residues; and 3-O-sulfotransferases (3OSTs) (16), which act infrequently to add 3-O-sulfates to GlcNS residues. Like the NDST, the sulfotransferase and epimerase enzymes do not act on all potential sites and thus create regions of hypervariable structure and sulfation (17). There are at least 32 possible unique disaccharide units, so the potential variation, even within short sequences, is enormous; this places HS as one of the most information-rich molecules currently identified. HS-GAG IN TRANSPLANTATION In xenograft transplantation models, binding of natural antibodies and activation of the complement cascade leads to rapid loss of HS from the endothelial surface after reperfusion (18). Size fractionation studies indicated the loss of intact HS-PG, implicating proteases in the release, including elastase produced by neutrophils (19). Loss of HS from the cell surface may compromise the endothelial barrier and result in a loss of anticoagulant function. Indeed, loss of antithrombin III from the basement membrane may lead to the development of fibrin deposits commonly seen in allograft rejection. Shed HS has the ability to activate a variety of antigen-presenting cells (APC), such as macrophages, dendritic cells, and B cells, which in turn affect allogeneic T cells, increasing proliferation and cytolytic activity (20–23). In macrophages, HS stimulation rapidly leads to the production of IL-1 and IL-6 and translocation of nuclear factor-κB into the nucleus, an essential transcriptional regulator for a number of cytokines. Maturation of dendritic cells by shed HS also leads to the production of cytokines including tumor necrosis factor (TNF)-α, and increased allostimulatory capacity. Soluble HS and heparin, through their effects on APC, may promote the development of the T-helper (Th) 1 type response seen in allograft rejection in addition to providing the cytokines necessary for the initiation of the early chemokine cascade in acute rejection (24). Cytokines produced by activated APC will also lead to the eventual increase of HS synthesis by the endothelium. Ongoing work in the authors’ group indicates that both IFN-γ and TNF-α increase the expression of NDST-1 and NDST-2, leading to an increase of heavily sulfated HS on the cell surface. In support of these data, sections from normal and rejecting renal tissue showed staining for heparan sulfate that was largely restricted to the basolateral surface of renal tubules (Fig. 2) (Au et al., personal communication, 2002). A significant increase in the levels of HS-GAG were observed in acutely rejecting renal biopsy tissue when compared with normal controls. Such an increase in cell surface HS would lead to an increased capacity for the binding of soluble factors involved in the inflammatory response. Figure 2: Expression of HS in rejecting and nonrejecting kidney biopsy tissue. Semiquantitative immunofluorescence confocal microscopy was carried out on biopsy sections from acutely rejecting and non–heart-beating donor kidneys. (A) HS staining in control nonrejecting tissue detected with an antibody for N-sulfated HS moieties (Seikagaku America, Ijamsville, MD). (B) Staining with the same antibody in rejecting tissue. (C) Average pixel intensity of the fluorescence signal (n=12 for both rejecting and control sections) showing a statistically significant difference in staining intensity (P =0.03, Student t test) indicating an increase of HS expression in rejecting tissue.PROTEIN INTERACTION WITH GAG Many cytokines have affinity for anionic HS-GAG molecules (Table 1) (7,13,25–46); indeed, heparin-affinity chromatography is commonly used to purify many of these proteins (43). One of the first proteins recognized with natural affinity for HS was antithrombin, which prevents intravascular blood coagulation. Although the interaction between antithrombin and HS is stabilized by hydrogen bonding, mapping studies have defined a precise amino acid sequence in the protein with a charge distribution complementary to a specific, highly sulfated pentasaccharide sequence in HS. When amino acid residues, including Arg129, Lys125, Arg46, and Arg47, in antithrombin bind to HS, a conformational change occurs in the protein that increases the anticoagulatory activity of the molecule 300-fold (47). Therefore, loss of HS by damaged or activated endothelium can result in the localized failure of this important interaction, leading to thrombus formation.Table 1: Table 1. Representative heparin-binding cytokines and growth factorsTable 1A: Table 1. ContinuedTable 1C: Table 1. ContinuedA further, well-characterized interaction occurs between soluble HS and basic (b) fibroblast growth factor (FGF). In this case, binding of bFGF to HS promotes oligomerization of the protein and is required for specific receptor ligation (41). Therefore, the HS-GAG chain constitutes an essential component of the signaling complex. The proinflammatory cytokine IFN-γ also binds HS through a basic Lys-Arg-Lys-Arg sequence toward the carboxyl terminus of the molecule. However, in this case, interaction with soluble HS inhibits the signaling potential of the cytokine (33), possibly by steric hindrance of specific receptor ligation or by the induction of a regulatory structural change in the protein (32,34). It is speculated that this interaction protects the protein from degradation and allows the development of a local reservoir for rapid release during inflammation. The chemokine family plays a central role in the initiation of graft inflammation after organ transplantation (2). Members of this family are known to bind HS and, typically with lower affinity, other GAG. This interaction occurs between sulfated domains on HS and characteristic basic sequence motifs on the chemokine molecule (29). In common with HS-binding domains on other proteins, the motif on chemokines frequently takes the form BBXB, where B is a basic residue; some chemokines also contain a BBXXB motif. In general, these GAG-binding motifs are located at a site distant from the specific receptor-binding domain, typically within the carboxyl terminal region of the molecule. The significance of the BBXB motif in the CC chemokine RANTES (regulated on activation normal T-cell expressed and secreted) has been defined recently by a site-directed mutagenesis study that produced a functional protein with reduced HS-binding potential (25). Significantly, this mutant variant of RANTES supports reduced transendothelial chemotaxis (30), presumably as a consequence of a reduced potential for apical presentation on the endothelial cells or for the formation of a stable concentration gradient. IMMUNOBIOLOGY OF HS-PG In view of the highly sulfated nature of HS-GAG, it is not surprising that they interact with a wide variety of proteins. This led to the perception that most interactions were relatively nonspecific. However, this has now changed with increasing evidence for interactions between consensus structural motifs both in HS and in many HS-binding proteins (31). There are several important functional consequences of these proposed interactions, including the following. Stabilization from Proteolysis Proteolytic enzymes are present in abundance at sites of inflammation. Enzymes such as plasmin, elastases, and cathepsin G, released by activated neutrophils, are able to modulate or destroy the activity of several proteins such as bFGF and IFN-γ (35). This process has been studied extensively with IFN-γ; many enzymes appear to act on the carboxyl region of IFN-γ, in particular, the basic domains referred to as the consensus sequence for HS-heparin recognition (36). Because the carboxylic terminal of IFN-γ is critical for its biologic activity, proteolytic cleavage may represent an important mechanism for regulating the activity of both this and other cytokines. Binding to HS-GAG, which is predominantly mediated through the C-terminus of proteins, may prolong the half-life of proteins and protect them from this targeted degradation. Sequestration-Concentration Chemokines often form in and this process may be after interaction with HS-GAG on the cell surface or within the extracellular matrix In a study using an which that promote oligomerization of the have shown that is necessary for the biologic activity of However, the of the interaction between either or and cell surface HS-GAG for was by the of chemotaxis of mutant cells of chemokines a role in and local chemokine at the cell surface. The affinity for GAG families and their capacity to will increase the local of chemokine at a as for RANTES in the formation of a complex with HS-GAG the potential to other molecules such as on T cells in cell Such a be of significance at sites of inflammation. of of its or both with HS-GAG can result in conformational leading to the of or the of a required protein The interaction of and its receptor has been studied most extensively in this (41). is the interaction of IFN-γ with HS (36). The C-terminus of IFN-γ the of the protein of the C-terminus in a protein that is and to bind to its to HS, IFN-γ its charge but is able to bind to its receptor and functional it has been shown that IL-2 by HS in promotes T-cell proliferation and cell death The of IL-2 is also critical in the of T cells after IL-2 is not by HS in the extracellular membrane to be critical even for T-cell Chemokines produce a signal to leukocytes to sites of and, in addition, increase molecules such as to the cell et al. showed that chemokines to be produced at inflammatory and for chemokines would be in by the blood from inflammatory HS-GAG expression at the endothelial cell surface allows for chemokine production by providing an in for and Chemokines may to GAG such as HS and be in form to The that heparin is able to to chemokines in in transendothelial and that the heparin-binding RANTES mutant has a the of GAG such as HS in transendothelial A has that apical chemokines to cell surface HS-GAG on endothelium can blood the cells in the of In the of apical and chemokines to the process of will likely vary with the endothelial chemokine and and the of However, in either case, the for binding to cell surface HS-GAG is Therefore, it is reasonable to by which a chemokine may be to its specific and interaction with HS, or in a or in with HS (Fig. Figure and presentation of chemokines to the chemokine presentation is defined as the ability of HS-bound chemokine to present to its receptor on the same a complex of the form for the presentation of presentation is defined as the presentation of a chemokine to GAG on one cell or within the extracellular matrix to a specific receptor on an It is also possible for soluble chemokine to present to its this is to be of in the high affinity of chemokines for HS-GAG OF GAG There is a between the pattern of proinflammatory cytokine expression and allograft rejection Although such as A can modulate expression of T-cell cytokines such as these do not the production of most proinflammatory cytokines. For this development of a to modulate the critical interaction between these cytokines and HS-GAG may of the activity of several important cytokines. include the following. by Soluble GAG has been shown to with including and of enzymes GAG chains of HS-PG have a charge that allows them to bind cytokines such as IFN-γ, and IL-8 by of the high of on these proteins. The have shown that exists for to μg/mL of heparin and HS the binding of RANTES to its whereas of the anionic GAG not Therefore, the charge distribution on the GAG to be of critical for protein interaction The mechanism by which soluble heparin It is possible that HS-GAG binding in will change a chemokine in a failure of normal binding to its The have shown that heparin is able to both protein and major complex class induction in endothelial cells, that heparin to the binding of IFN-γ with its receptor and its ability to IFN-γ from the endothelial cell surface has also been shown to prolong allograft in and in xenograft Because this activity is not on anticoagulant activity, it has been possible to heparin that immunomodulatory activity if used after has some potential as an anti-inflammatory but a of and is essential its potential can be is a that chemically and GAG, is and anti-inflammatory including the ability to the complement It has also been used to prolong allograft in a of transplantation In addition, other have also been shown to prolong xenograft with specific interactions may be for the of inflammatory This may to be a complex even family such as the CC chemokines and appear to for HS-GAG It has been observed that soluble HS-GAG can function as of whereas or HS-GAG are able to have the of or receptor HS-GAG molecules may be able to with HS-GAG to on the domains of proteins may also a mechanism for the interactions critical in rejection. Both the authors’ group and that of have shown that from the HS-binding domain of IFN-γ binding of protein to HS-GAG and, of in binding motif also binding of other critical such as and of The ability to bind HS-GAG is to a crucial role in chemokine formation and, therefore, function. The on HS-GAG binding a for the development of targeted these domains as a for immune with to specific anti-inflammatory therapy after transplantation. It has been shown in in that a RANTES mutant with reduced capacity to bind HS-GAG is to leukocytes when compared to the RANTES these mutant proteins will have a potential proteins they will have a half-life within the of their to bind endothelial HS-GAG, and be to form The mutant proteins are sufficiently to proteins that it is they will a significant immune response. This is further by a of the of in which showed inflammatory effects the The that responses associated with IFN-γ produce the associated with acute allograft rejection, whereas selective activation of IL-4 cells this Indeed, et al. have IL-4 and and IL-2 and IFN-γ in in by with Significantly, and cells chemokine and to cells and whereas and and, to a are expressed by The of cells has been in allograft rejection. chemokine are a for T cells and also be used as possible targets for the of an immune response by the of specific of T For example, RANTES be used to the of graft This may be by of anti-inflammatory AND It is that HS-GAG act as multifunctional regulators of protein activity through a range of that are on specific on the and synthesis of HS-GAG sequences indicates that a when of these molecules is For will to be including the are the specific sequence motifs involved in interaction are involved in biosynthetic and regulation of is the molecular mechanism of of the HS-GAG of these can now be biosynthetic The of enzyme with and at least in expression of these enzymes a for the production of specific HS-GAG the in functions of specific HS-GAG be by and molecular using These will with specific modifications to be and the to be would they in the functional role of specific but they may also in potential targets for This review supports the that GAG such as HS have an important and role in inflammation. with HS-GAG, cytokines such as IFN-γ are able to increase endothelial The have the of interactions with endothelial cell surface HS-GAG on class induction and, in addition, that IFN-γ is of the production of chemokines such as RANTES and from endothelial cells HS-GAG also appear to be necessary for HS-GAG or defined with anticoagulant activity have potential in the of allograft rejection. of these molecules to enhance the of interaction is this increases the of work required to the necessary GAG-binding for but this also increases the of intervention.