Effector Mechanisms in Transplant Rejection


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Effector Mechanisms in Transplant Rejection

  1. 1. Paulo N. Rocha Effector mechanisms in transplant Troy J. Plumb Steven D. Crowley rejection Thomas M. Coffman Authors’ addresses Summary: Antigens, provided by the allograft, trigger the activation Paulo N. Rocha, Troy J. Plumb, Steven D. Crowley, and proliferation of allospecific T cells. As a consequence of this Thomas M. Coffman, response, effector elements are generated that mediate graft injury Duke University and Durham VA Medical and are responsible for the clinical manifestations of allograft rejection. Centers, Durham, NC, USA. Donor-specific CD8þ cytotoxic T lymphocytes play a major role in this process. Likewise, CD4þ T cells mediate delayed-type hypersensitivity Correspondence to: responses via the production of soluble mediators that function to Thomas M. Coffman, MD further activate and guide immune cells to the site of injury. In Chief, Division of Nephrology addition, these mediators may directly alter graft function by modulat- Box 3014, Duke University Medical Center ing vascular tone and permeability or by promoting platelet aggrega- Durham, NC 27705 tion. Allospecific CD4þ T cells also promote B-cell maturation and USA differentiation into antibody-secreting plasma cells via CD40–CD40 Tel.: þ1 919 286 6947 ligand interactions. Alloantibodies that are produced by these B cells Fax: þ1 919 286 6879 exert most of their detrimental effects on the graft by activating the E-mail: coffm002@mc.duke.edu complement cascade. Alternatively, antibodies can bind Fc receptors on natural killer cells or macrophages and cause target cell lysis via antibody- dependent cell-mediated cytotoxicity. In this review, we discuss these major effector pathways, focusing on their role in the pathogenesis of allograft rejection. Introduction Injury and destruction of an organ transplant during rejection is carried out by effector elements generated as part of the immune response to alloantigens on the graft. These effector responses are redundant and can cause precise, antigen-specific cell injury or can affect the physiological functions of the graft through the non-specific actions of inflammatory medi- ators. Immune effector pathways are shaped by the differen- tiation and maturation processes of alloantigen-specific T and B lymphocytes that are described elsewhere. In this article, we review three major effector elements that are important in Immunological Reviews 2003 the pathogenesis of allograft rejection: the cytotoxic T-cell Vol. 196: 51–64 Printed in Denmark. All rights reserved response, delayed-type hypersensitivity, and antibodies and complement. We focus on recent advances in understanding Copyright ß Blackwell Munksgaard 2003 Immunological Reviews the role of these effector mechanisms in organ transplant 0105-2896 rejection. 51
  2. 2. Rocha et al Á Effector mechanisms in transplant rejection Lymphocyte-medicated cytotoxicity receptor expressed on T lymphocytes], a newly recognized member of the tumor necrosis factor (TNF) family expressed Generation of antigen-specific cytotoxic T lymphocytes (CTLs) on activated T cells, can also influence CTL activation in the is a major immunological effector mechanism in allograft alloimmune response (13–15). These alternative pathways rejection. Much of the current understanding of CTLs derives may assume a more important role after blockade of CD40/ from in vitro studies exploring the cellular immune response to CD40L and/or CD28/B7. alloantigens, infectious agents, and tumors. This work has In the 1–3 days following activation of CTL precursors, form- been summarized in several recent reviews (1–4). The role ation of cytotoxic granules containing perforin and granzymes of CTLs in rejection has long been a popular theme in trans- can be detected. When the target cell is identified and engaged plantation research. For example, Strom and associates (5) through specific interactions between the T-cell receptor (TCR) showed that donor-specific CTLs could be eluted from reject- and CD8 on the CTL and MHC class I on the target cell, these ing human renal allografts. Later, Rosenberg et al. (6) showed granules fuse with the effector cell membrane and extrude the that adoptive transfer of CD8þ effector T cells was sufficient to granule contents into the immunological synapse (Fig. 1) (4). induce rejection of major histocompatibility complex (MHC) Along with perforin and granzymes, the cytotoxic granules class I-mismatched skin grafts in mice. Additional complexity also contain serglycin, calreticulin, Fas ligand (FasL), and of the role of CTLs in rejection was suggested by studies of granulysin. In the presence of calcium, perforins assemble Wood and Morris (7), showing that donor-specific CTLs could into polyperforins and insert into the target cell membrane also be isolated from long-surviving, ‘accepted’ rat renal allo- to facilitate the uptake of granzyme B (GB) by an uncertain grafts. Today, it seems clear that CD8þ CTLs play a key role in mechanism into the target cell cytoplasm, where GB mediates rejection of organ transplants, but this role may be accentuated apoptosis (16). This chain of events leading to the insertion of in certain circumstances. perforin and the delivery of GB into the target cell is known as The CD8þ CTL is primed and activated by recognition of the granzyme exocytosis (GE) pathway. The final common donor MHC class I antigens. These antigens also serve as the pathway for these cytolytic processes is triggering of apoptosis targets for the mature cytolytic effector. In the prevailing view, in the target cell. recipient CD8þ T cells are primed by direct presentation of Upon entry into the target cell, GB can trigger apoptosis donor antigens by ‘passenger’ antigen-presenting cells (APCs) through several pathways, including direct cleavage of procas- from the graft (1). Although donor APCs are the major path- pase-3 and indirect activation of procaspase-9 through a ways for CTL induction, Kreisel et al. (8) reported a novel complex pathway (17, 18). In this pathway, GB acting through mechanism of direct antigen presentation by activated donor vascular endothelial cells. This pathway can induce responder CD8þ cells with an effector phenotype that are sufficient to Caspases cause acute rejection (AR). Although the role of class I MHC antigens is unequivocal, the requirement for costimulatory Target cell molecules in the allogeneic priming of CTLs is controversial. After cardiac transplantation, blockade of CD40–CD40 ligand Fas MHC I Ca++ (CD40L) interactions has been variously reported to impair (9) or to have no effect (10) on activation of allospecific CD8þ Fas pathway GE pathway CD8 TCR CD3 T cells. Moreover, the resistance to tolerance induction by FasL costimulatory blockade has been attributed to the development of CD8þ CTL effectors (11). Some of the variability in results Cytotoxic T cell in this area might reflect differences in experimental condi- tions, contributions of other pathways to provide help in the Perforin form of cytokines, or genetic background of animals used in Granzyme B Cytotoxic granule Serglycin the studies (12). Moreover, less traditional costimulatory Calreticulin Fas ligand Polyperforins molecules, such as leukocyte function-associated antigen-1 Granulysin (LFA-1), membrane lymphotoxin, or LIGHT [homologous to Fig. 1. Mechanisms of cytotoxic T lymphocyte-induced graft damage. lymphotoxins, inducible expression, competes with herpes sim- FasL, Fas ligand; GE, granzyme exocytosis pathway; MHC, major plex virus (HSV) glycoprotein D for HSV entry mediator, a histocompatibility complex; TCR, T-cell receptor. 52 Immunological Reviews 196/2003
  3. 3. Rocha et al Á Effector mechanisms in transplant rejection BCL-2 interacting domain (BID) and BCL-2 homologous Conversely, some studies show that rejection can proceed in antagonist/killer (BAK) induces the release from the mito- the absence of perforin. Diamond and Gill (31) employed a chondrion of cytochrome C and second mitochondria-derived strategy of adoptively transferring primed CD8þ cells from activator of caspase/direct inhibitor of apoptosis protein perforin- or FasL-deficient mice into severe combined immuno- (IAP)-binding protein with low isoelectric point to form an deficiency disease (SCID) murine recipients of pancreatic ‘apoptosome’ with procaspase-9. In the apoptosome, procas- islet allografts to show that in vivo allograft rejection did not pase-9 is cleaved to caspase-9, which then cleaves procaspase-3. depend on perforin or FasL. The ability of graft rejection to Caspase-3 inactivates the inhibitor of caspase-activated DNAse proceed in the absence of perforin highlights the importance of (ICAD) leading to DNA fragmentation (19). Recent studies the cation-independent mannose 6-phosphate receptor (CI- support the relevance of caspase-3 and -9 to transplantation MPR) that mediates the uptake of GB by target cells and allows physiology. For example, caspase-3 staining correlated with apoptosis in a perforin-independent manner. Blockade of GB rejection in human cardiac allograft specimens (20). In addition, interactions with CI-MPR prevents GB uptake and apoptosis in zinc chloride, an inhibitor of caspase-3, reduced apoptosis in rat target cells. Donor H-2k MPR– cells injected under the kidney cardiac allografts and prolonged allograft survival (21). Finally, a capsule of BALB/c H-2d recipients are not rejected. Thus, T-cell-specific tyrosine–kinase inhibitor, tautomycetin induced expression of the CI-MPR and its interaction with GB are recipient T-cell apoptosis and prolonged rat cardiac allograft essential for in vivo allogeneic cell rejection (32). survival by the phosphorylation of T-cell-specific residues lead- The molecules that choreograph apoptosis and lymphocyte ing to the cleavage downstream of caspase-3 and caspase-9 (22). cytolysis are generally considered to be rejection-promoting The caspase cascades therefore might represent novel targets for effector molecules. However, in other circumstances, they can immunosuppressive intervention, if they can be pinpointed be protective. For example, it has been suggested that perforin with adequate specificity and without untoward side effects. from donor cells can down regulate the alloimmune response Along with the GE pathway, CD8þ CTLs can also utilize the by inducing apoptosis of recipient immune cells (33). In this Fas-dependent pathway to induce cytolysis and apoptosis paradigm, the donor perforin may overwhelm the recipient (23). In the Fas-dependent pathway, FasL is either packaged T-cell-expressed cathepsin B that normally serves to protect the into cytotoxic granules with perforins and granzymes or is recipient effector cells from the actions of perforin. Similarly, trafficked directly to the activated effector cell surface for it has been shown that expression of FasL confers immune binding to the target cell. Binding of FasL on the effector cell privilege to the allograft by inducing Fas-dependent apoptosis to Fas on the target cell membrane triggers apoptosis ultim- in the recipient immune effector cells. For instance, murine ately through the same caspase effector mechanisms as in the testis tissue transplanted under the kidney capsule of an allo- GE pathway. Expression of Fas and FasL can be detected in geneic recipient survives indefinitely due to the expression of rejecting allografts in humans, but their presence is not always FasL on Sertoli cells (34). In a more recent series of human specific for rejection (24, 25). CD4þ effector cells may also pre-implantation renal allograft biopsies, FasL expression was eliminate cells expressing MHC class II antigen through a inversely correlated to subsequent AR (35). In another study, Fas-dependent or Fas-independent mechanism (26). over-expression of FasL on a thyroid allograft prevented rejec- Although early studies with perforin- or Fas-deficient mice tion and resulted in suppressed donor-specific CTL activity demonstrated the importance of both the GE- and Fas-depend- (36). Finally, murine recipients of allogeneic bone marrow ent pathways to cell-mediated cytotoxicity (27, 28), the fol- that had undergone vector-mediated transduction with FasL lowing studies suggest that the GE pathway plays the had enhanced short-term engraftment relative to controls dominant role in apoptosis induction in allograft rejection. (37). Despite these promising studies, a beneficial role for Krupnick et al. (29) showed that in vitro CTL-mediated killing donor FasL in human transplantation has yet to be exploited. of donor vascular endothelial cells deficient in Fas and FasL proceeded with only mild impairment, indicating that the GE Natural killer cells pathway is the dominant contributor to cytotoxicity in this system. Similarly, co-incubation of graft-infiltrating T cells The natural killer (NK) cell is a large granular lymphocyte that from human renal allografts undergoing rejection with a GE acts as part of the innate immune system to kill virally and pathway inhibitor (concanamycin A) substantially reduced in parasitically infected cells (38). NK cells also provide surveil- vitro lysis and apoptosis of proximal tubular epithelial cells, lance in preventing the growth of some tumors. The NK cell whereas incubation with a Fas inhibitor did not (30). does not rearrange TCR or immunoglobulin (Ig) genes to Immunological Reviews 196/2003 53
  4. 4. Rocha et al Á Effector mechanisms in transplant rejection facilitate binding to a specific antigen. Rather, the NK cell be quite low, even in florid rejection. Thus, the CTL pathway expresses numerous activating (i.e. NKp46, NKp44, and appears to be one of several mechanisms that contribute to NKp30) and inhibitory receptors (i.e. Ly49 class), the ligation transplant injury. Its relative contribution varies depending on of which regulate NK-cell activation (39, 40). The ligands for immunosuppression, the type of graft, and the nature of MHC these receptors are not all well described, but in the prevailing disparity between donor and recipient. Interestingly, compon- theory, self-MHC class I molecules bind to Ly49 receptors to ents of the CTL system might actually be protective in some provide an overriding inhibitory signal that prevents NK-cell circumstances. activation (40). Thus, the absence of self-MHC class I mol- ecules on a cell due to down regulation by infection or due to Delayed-type hypersensitivity allogeneic phenotype can result in its lysis by an NK cell. NK cells lyse targets solely through the GE pathway with perforin- Another major effector limb of the T-cell response to an organ containing granules that are preformed during development graft is the DTH response. DTH is primarily mediated by rather than upon activation, as in the case of T lymphocytes. alloantigen-specific CD4þ T-helper 1 (Th1) cells. After trig- However, similar to T lymphocytes, GB in the NK cell is gering by alloantigen, these Th1 cells secrete cytokines, such as required for the induction of rapid apoptosis in the target interferon-g (IFN-g) and TNF (45). These cytokines have cell (41). multiple pro-inflammatory actions, including activation of Although early research implicated NK cells in the rejection monocytes and macrophages that are a prominent component of bone marrow allografts (42), more recent studies have also of the cellular infiltrate in allograft rejection. This activation suggested a role for NK cells in solid organ transplant rejec- causes a further amplification of cytokine and chemokine tion. For instance, Ogura and associates (43) reported that production, along with generation of proteolytic enzymes, transplantation of rat livers into CD8þ T-cell-depleted recipi- nitric oxide, and other soluble factors that perpetuate and ents resulted in rejection and intra-graft expression of GB and shape the local inflammatory response. These factors also FasL similar to that of unmanipulated allograft recipient con- directly impact the physiological functioning of the graft trols. Although these authors demonstrate, in a separate study, through effects on vascular tone, permeability, and integrity. infiltration of the liver allografts by recipient NK cells, their Finally, soluble mediators of the DTH response act in an findings do not preclude the contribution to rejection of antigen-independent fashion to promote chemotaxis and another effector arm of the recipient’s immune system, such further activation of immune cells. as CD4þ effector cells or an antibody-mediated response. Slightly more provocative is a murine cardiac allograft model DTH and rejection in which the removal of the CD28-costimulatory signal did not afford long-term graft acceptance unless accompanied by Adoptive transfer experiments have suggested that an allospe- depletion of recipient NK-receptor-bearing cells (44). In this cific DTH response alone is sufficient to mediate skin graft study therefore NK cells were sufficient to mediate solid allo- rejection. Dalloul and associates (46) showed that adoptive graft rejection in the absence of T-cell costimulation. Deple- transfer of CD4þ lymphocytes from CD8–/– mice could induce tion of NK cells alone did not prevent allograft rejection. Taken rejection of MHC class I- or II-disparate skin grafts in SCID together, these studies suggest that NK cells might play a role mice. In this circumstance, skin grafts were rejected in the in the alloimmune response, but their importance in a host absence of a detectable CTL response. Likewise, Valujskikh et al. with normal T-cell function has not been clearly demon- (47) showed that transfer of a Th1 alloreactive cell line that strated. recognizes donor MHC peptides via the indirect pathway was Generation of allospecific CTLs represents an immune effector sufficient to cause rejection of skin grafts in SCID mice. In this pathway that can deliver precise, antigen-specific cell kill- circumstance, direct, donor-specific CTL responses were not ing. The capacity of this system in rejection is reflected by the possible. Histologically, these grafts had a predominant identification of apoptotic cells in biopsies of rejecting allo- macrophage infiltrate consistent with a DTH-type effector grafts and the ability of adoptive transfer of CD8þ CTLs to mechanism, and donor antigens elicited a typical DTH cause transplant rejection. However, the requirements for this response when injected subcutaneously (47). pathway are not absolute, as depletion of CD8þ cells might The intensity of the donor-specific DTH response can be have little effect on the course of rejection. Moreover, the assessed in transplanted animals in vivo by injecting donor relative number of apoptotic cells in rejecting allografts may splenocytes or splenocyte lysates into either the ear or footpad 54 Immunological Reviews 196/2003
  5. 5. Rocha et al Á Effector mechanisms in transplant rejection of the recipient (48). The reaction is characterized by a typical suppressed by these mechanisms and that attenuation of DTH response with exudates, edema, and an intense cellular the intra-graft DTH response might contribute to long-term infiltrate. The intensity of response is proportional to the graft survival. degree of edema formation assessed as thickness of the pinnae Whereas Th1 cells are associated with the production and or footpad. In rodent transplant models, as well in humans release of pro-inflammatory cytokines, TNF and IFN-g, with with organ transplants, graft loss is associated with a vigorous subsequent activation of macrophages, Th2-type cells produce systemic DTH response. By contrast, long-term graft accept- cytokines such as IL-4, IL-5, IL-10, and IL-13. Although the ance is characterized by a blunted or absent DTH response Th2 cytokine profile generally inhibits cell-mediated immun- (49–51). ity and the DTH response, this cytokine profile can induce Bickerstaff and associates (52) have studied the regulation of antibody-mediated rejection (discussed elsewhere in this this systemic DTH response in mice that have accepted heart article), and recent evidence suggests that IL-4 and IL-5 allografts after treatment with gallium nitrate. These studies promote eosinophil-mediated rejection. Eosinophils are indicate that the DTH response, assessed by injecting alloanti- recruited to the graft by IL-4, IL-5, and IL-13 released by gen subcutaneously, is suppressed by the actions of tissue Th2 cells. Upon recruitment and activation, eosinophils growth factor-b (TGF-b) and interleukin (IL)-10. Injecting elaborate substances such as leukotrienes (LTs) (discussed neutralizing antibodies to TGF-b and IL-10 along with the below), superoxides, major basic protein, eosinophil cationic donor antigen can restore the DTH response. Similar blunting protein, and eosinophil peroxidase. In several models, Th2- of DTH responses is observed in mouse recipients of dominant alloresponsive T cells can mediate allograft rejection. spontaneously accepted kidney allografts. However, in this Histologic examination of many of these models reveals an circumstance, suppression of DTH is mediated primarily by intense infiltrate of eosinophils (54). Using IFN-g and IL-2 TGF-b (49). Enhanced activity of TGF-b in recipients of long- double knockout (KO) mice, Zand et al. (55) demonstrated surviving allografts appears to be due to activation of TGF-b that these animals rapidly rejected cardiac allografts, and the by the protease plasmin rather than to enhanced production of intra-graft cytokine profile was characteristic of a Th2 TGF-b. In this regard, DTH responses can be restored in response. Le Moine et al. (56, 57) evaluated the role of cardiac allograft acceptors by co-injecting antigen with eosinophils in a model of chronic skin allograft rejection. In antibodies against tissue-specific plasminogen activator (53). this model, MHC class II-disparate skin grafts were applied VanBuskirk et al. (51) used a ‘trans vivo’ model to measure after generalized T-cell depletion. The skin grafts survived for DTH responses in human transplant recipients. Peripheral more than 60 days before they were eventually rejected, and blood mononuclear cells (PBMCs) were harvested from three there was skewing toward a Th2 cytokine profile in the reject- human transplant recipients (two kidney, one liver), who had ing grafts with marked increases in IL-4 and IL-5 but not IFN-g. well-functioning allografts despite discontinuing their imm- Histologically, these grafts had predominant eosinophil unosuppressive therapy. PBMCs from the patients were then infiltrates, and they developed an impressive obliterative arter- co-injected with donor antigen into the footpad of SCID mice. iolopathy. When neutralizing antibodies to IL-4 were admin- Similar to the rodent models, all three patients with long- istered, both graft vasculopathy and eosinophil infiltration surviving allografts had suppressed DTH responses to donor were abolished. Using neutralizing antibodies to IL-5 or IL-5 antigens. By contrast, the response to tetanus toxoid was gene KO mice, eosinophil graft infiltration was inhibited, but intact. However, when tetanus toxoid was injected with vasculopathy was unaffected. These studies suggest a critical alloantigen, the DTH response was blunted, suggesting active role for eosinophils in settings where rejection is mediated by suppression of bystander antigen responses. As in the mouse a predominant Th2-type response. It is worth noting that experiments, antibodies against either TGF-b or IL-10 rescued significant eosinophil infiltrates are sometimes seen in severe DTH responsiveness. Thus, the immunoregulatory cytokines allograft rejection in humans (58, 59). TGF-b and IL-10 play an important role in the impaired DTH As discussed above, the DTH response is perpetuated and response that is associated with allograft acceptance (51). shaped by soluble inflammatory mediators. These mediators Furthermore, these studies suggest that inhibition of the act in several capacities to promote and amplify the inflamma- DTH response by ‘acceptors’ is an active process that is tory response to an allograft. They recruit immune cells to the mediated, at least in part, by the anti-inflammatory cytokines, graft and can promote the activation and differentiation of TGF-b and IL-10. It is attractive to speculate that DTH antigen-specific T cells. Finally, they directly affect the physio- responses in the long-surviving allografts might also be logical functions of the allograft through effects on vascular Immunological Reviews 196/2003 55
  6. 6. Rocha et al Á Effector mechanisms in transplant rejection tone and integrity. Among the wide range of inflammatory reduced in splenocytes or isolated T cells from mice lacking mediators that contribute to the pathogenesis of allograft thromboxane prostanoid (TP) receptors. In addition, survival rejection, lipid mediators generated by the metabolism of of cardiac allografts was prolonged in TP–/– recipients treated arachidonic acid (AA) pathway play a prominent role (60). with sub-therapeutic doses of cyclosporine compared to Eicosanoids are generated by the enzymatic metabolism of cyclosporine-treated wildtype controls. Similarly, survival of AA. In the first step of these metabolic pathways, AA is elabor- kidney allografts transplanted into TP–/– animals is likewise ated from membrane-bound phospholipids through the significantly prolonged compared to wildtype controls (our actions of phospholipases. AA can then be further metabolized unpublished observation). Thus, the COX metabolite, TXA2, to a variety of biologically active products including prosta- acting via the TP receptor promotes allograft rejection. noids, LTs, P450 metabolites (HETEs and EETs), and the iso- Compared to the pro-inflammatory actions of TXA2, PGE2 prostanes. Most eicosanoid products are rapidly metabolized tends to inhibit or suppress immune responses. In transplant- and, therefore, must work in an autocrine or paracrine fash- ation models, administration of PGE analogs inhibits rejection ion. Among the eicosanoids, roles for prostanoids and LTs in and prolongs survival (60). The diverse biological actions of the alloimmune response have been most clearly defined (60). PGE2 are mediated via four distinct GPCRs, the E prostanoid These systems provide a prototypical example of the role of (EP) receptors (EP1,2,3,4) (70). EP receptor isoforms are soluble mediators in rejection and, therefore, are reviewed in expressed on immune cells including macrophages and some detail below. T cells. However, until recently, the precise EP receptor iso- forms mediating the immunosuppressive actions of PGE2 were not known. To address this question, we examined responses Prostanoids and rejection to PGE2 in splenocytes and purified T cells from mice lacking Prostanoids are generated from AA by the cyclooxygenase each of the individual four EP receptors. These studies indi- (COX) pathway (61). There are two isoforms of COX that cated that the Gs-coupled EP2 receptor mediates the inhibitory have identical biochemical functions, but regulation and pat- effects of PGE2 upon T cells, whereas both the EP2 and EP4 terns of their expression are quite different. COX-1 is consti- receptors regulate macrophage functions (71). As the clinical tutively expressed in most nucleated cells, whereas COX-2 use of PG analogs has been hindered by their lack of potency expression is markedly up regulated in response to injury or and specificity, the identification of the relevant immuno- inflammation. These enzymes are the targets of widely used modulatory receptors may facilitate exploring this pathway as a conventional non-steroidal anti-inflammatory drugs (NSAIDs), therapeutic target. which inhibit both COX isoforms, and coxibs, which are select- ive for COX-2. The actions of the prostanoids, including effects Leukotrienes in rejection on inflammation and immunity, are mediated via G protein- coupled receptors (GPCRs) (62). Although there is evidence LTs are another class of AA metabolites that contribute to the suggesting a role for various prostanoids in transplantation, inflammatory response to an allograft. In this pathway, AA is the actions of prostaglandin E2 (PGE2) and thromboxane (TX) metabolized to LTA4 via the actions of 5-lipoxygenase (5-LO). A2 have been most thoroughly characterized (60). LTA4 can be hydrolyzed to form LTB4, or can be conjugated A role for the prostanoid TXA2 in rejection was first sug- with glutathione to form LTC4. LTC4 can be further metab- gested by Foegh and associates (63), who reported elevated TX olized to LTD4 and LTE4 by extracellular metabolism. LTC4 and metabolites in the urine of patients with rejecting kidney its metabolites are collectively referred to as the cysteinyl- transplants. These findings were subsequently confirmed in leukotrienes (CysLTs) and were previously known as the animal models of rejection (64–66). TX is a potent vasocon- slow reacting substance of anaphylaxis (72). strictor, and as a hemodynamic mediator, it can have a detri- mental effect on allograft function (64, 67). However, TXA2 Leukotriene B4 may contribute to rejection by influencing cellular immune responses (68, 69). LTB4 is primarily synthesized by neutrophils and macrophages Recently, we have demonstrated that TXA2, acting through and is a potent chemotactic and chemokinetic factor for neu- its receptor TP, directly influences cellular immune responses trophils. There are two receptors for LTB4: BLT1 and BLT2. (Thomas et al., manuscript submitted). Proliferative responses These GPCRs are found in highest concentrations on leuko- to alloantigens or anti-CD3 antibody were significantly cytes. BLT1 expression is highest in monocytes, whereas BLT2 56 Immunological Reviews 196/2003
  7. 7. Rocha et al Á Effector mechanisms in transplant rejection is most highly expressed in lymphocytes (73). LTB4 increases number of circulating lymphocytes, accompanied by seques- leukocyte adhesion to endothelial cells and extravasation into tration of lymphocytes in peripheral lymph nodes, mesenteric tissues (74). LTB4 promotes the production of pro-inflamma- lymph nodes, and Peyer’s patches (88). Honig and associates tory cytokines by T cells and monocytes, such as IL-1, IL-2, (85) found that FTY720 enhances CCL19- and CCL21-induced and IFN-g (75–77). In addition, LTB4 upregulates the chemotaxis by activating the multidrug transporters, Abcb1 expression of integrins such as CD11b (78). In a mouse and Abcc1, thereby promoting peripheral lymphocyte seques- heterotopic heart transplant model, Weringer et al. (79) tration. One function of Abcc1 is to transport LTC4 to the demonstrated a clear role for LTB4 in allograft rejection, as extracellular space. In the setting of FTY720 administration, mice treated with an LTB4 antagonist had significantly pro- 5-LO deficiency or inhibition renders T cells unresponsive to longed graft survival. These findings were associated with a CCL19 and CCL21; however, the addition of exogenous CysLT marked reduction in cellular staining for CD11b and a delayed (LTD4) restores responsiveness (85). These studies demon- peak in graft reactive serum IgG levels (79). strate a clear role for CysLTs in lymphocyte migration, and The CysLTs are primarily synthesized by eosinophils, mast they suggest a mechanism whereby altering CysLT release cells, and macrophages. They stimulate smooth muscle con- results in sequestration of lymphocytes in peripheral lymph traction, contributing to bronchiolar and arteriolar constric- nodes. In this circumstance, FTY720 appears to impair the tion, and increase vessel permeability, promoting plasma alloimmune response by augmenting the release of CysLTs. extravasation. The actions of the CysLTs are mediated by two receptors, CysLT1 and CysLT2. These GPCRs are expressed in a Antibodies and complement wide variety of tissues and cell types, including the spleen, lungs, eosinophils, and monocytes/macrophages (80). CysLTs Generation of CTL and DTH are the principal effector limbs of may also contribute to allograft rejection as LTC4 levels are the T-cell response to an allograft. The third major element enhanced in rejecting rat kidneys correlating with the devel- contributing to graft injury and rejection is the development opment of cellular infiltrates, and administration of a CysLT of an alloantibody response to the transplant. Although anti- receptor antagonist decreases vascular rejection (81, 82). body production is ultimately a B-cell function, the contribu- Consistent with the apparent benefits of inhibiting indi- tion of T cells cannot be overlooked. As will be discussed vidual LT receptors discussed above, global inhibition of LT below, B cells require help from alloreactive CD4þ T cells to synthesis using 5-LO inhibitors improves function and pro- grow, differentiate, and secrete antibodies. The binding of longs allograft survival in various models of transplantation, alloantibodies to ABO or MHC antigens expressed on endothe- including kidney, heart, and pancreas allografts. In a rat kidney lial cells triggers a complex response involving the comple- transplant model, inhibition of 5-LO improved allograft sur- ment and coagulation pathways that activate and recruit vival, diminished MHC class II expression, and preserved inflammatory cells, ultimately resulting in graft injury. Allo- allograft morphology (81). Despite the beneficial effects antibodies can also mediate antibody-dependent cellular cyto- observed with pharmacological inhibition of 5-LO, transplant toxicity (ADCC). In this case, NK cells or macrophages bind to outcomes in mice with targeted deletion of the 5lo gene were the Fc region of antibody molecules promoting lysis of target quite different. In a mouse model of kidney transplantation, cells. The cross-linking of Fc receptors on NK cells triggers allografts transplanted into 5-LO-deficient recipients had sig- perforin/granzyme-mediated cytotoxicity, whereas in macro- nificantly reduced survival (83). Similarly, 5-LO deficiency phages this cross-linking promotes the release of mediators accelerated the course and severity of autoimmune disease in such as nitric oxide (NO), TNF-a, and reactive oxygen species. MRL-lpr mice, consistent with an unexpected role for 5-LO to The actions of alloantibodies and complement to promote ameliorate immune injury (84). graft injury produce distinct clinical manifestations in hyper- Recent studies by Honig and associates (85) support the acute, acute humoral, and chronic rejection. In addition, there view that 5-LO products can inhibit the immune response. are some circumstances, in which, antibodies and complement These studies suggest that LTC4 contributes to the efficacy of may have beneficial effects. FTY720, a sphingosine-derived immunosuppressant. Although its mechanism of action is not clearly understood, Hyperacute rejection FTY720 prolongs survival in heart and skin grafts without impairing T-cell and B-cell activation (86, 87). Following Hyperacute allograft rejection (HAR) is the classic and most administration of FTY720, there is a marked reduction in the exuberant example of antibody-mediated rejection. In this Immunological Reviews 196/2003 57
  8. 8. Rocha et al Á Effector mechanisms in transplant rejection process, large quantities of preformed antibodies against ABO interstitial hemorrhage, and severe injury to endothelial cells; or MHC bind these antigens on endothelial cells and activate immunostaining reveals Ig and complement deposits along the classic complement pathway (Fig. 2). endothelial surfaces of graft blood vessels. Given the lack of The split products of the early complement components, effective treatment, HAR almost invariably leads to allograft loss. such as C3a and C5a, function as anaphylatoxins attracting With the advent of blood typing and T-cell cross-match inflammatory cells and platelets to the target area. The late testing, this type of rejection has become a rare event in the complement components, namely C5b-9, form the membrane clinical arena. However, HAR remains a major barrier to attack complex (MAC) that, in turn, activates and damages the xenotransplantation. For example, when a pig organ is trans- endothelium (89, 90). Activated endothelial cells produce IL-8 planted into primates, ‘xenoreactive natural antibodies’ bind and monocyte chemotactic protein-1 (MCP-1) to recruit neu- the carbohydrate galactose-a-1,3-galactose (Gala1, 3Gal) trophils and monocytes to the site of injury (91). Other expressed in pig endothelial cells and cause HAR (94). More- inflammatory cytokines, such as IL-1, are also secreted and over, xenografts appear to be particularly susceptible to function to upregulate the expression of tissue factor in complement-mediated injury, because porcine complement endothelial cells. There is release of preformed von Willebrand regulatory proteins fail to dampen the activation of human factor (vWF) and P selectin from cytoplasmic Weibel-Palade complement on xenogeneic cells (95). In fact, much effort has bodies to the surface of endothelial cells; vWF promotes the been concentrated on generating transgenic pigs that either do formation and stabilization of the platelet plug, whereas P not synthesize Gala1,3Gal (96) or that express human com- selectin, a member of the family of adhesion molecules, regu- plement regulatory proteins as potential organ donors for lates interactions between endothelial cells and leukocytes humans (97). Although these recent advances of research on (92). P selectin interacts with its natural ligand, P-selectin xenotransplantation are still far from translating into clinical glycoprotein ligand-1 (PSGL-1), present on neutrophils and practice, they have undoubtedly provided invaluable insights monocytes and mediates the adherence of these cells to the into the pathogenetic roles of antibodies and complement in endothelium for subsequent extravasation into the tissue (93). HAR (98). In essence, the actions of complement and inflammatory medi- ators transform the endothelium from a protective barrier between the blood and extravascular tissues into a pro- Acute rejection coagulant, chemoattractive, and adhesive interface that promotes inflammation. Once endothelial cells are damaged, the under- In contrast to HAR, the role of antibodies in acute allograft lying matrix is exposed, and there is release of tissue factor rejection is controversial. Acute rejection (AR) is typically into the circulation, which binds factor VIIa and activates the viewed as a T-cell-mediated process. Mice lacking T cells extrinsic coagulation cascade. The end result of these processes cannot reject fully mismatched allografts, and reconstitution is widespread intra-vascular thrombosis, hemorrhage, and tis- of these animals with T cells restores the rejection process. sue injury manifested grossly by a mottled and cyanotic graft. Moreover, T cells can be readily seen in histologic sections as Pathologically, HAR is characterized by obstruction of small the principal components infiltrating the allograft during AR. vessels (including glomerular capillaries) by platelet thrombi, Current anti-rejection protocols directly target T cells and are ADCC Complement cascade C3a + Coagulation cascade + MØ C1q IL-8 NK Platelets Fc MCP-1 VIIa Fc P selectin + TF IL-1 MAC TF vWF Fig. 2. Mechanisms of antibody-induced graft damage. ADCC, antibody-dependent cellular cytotoxicity; MCP-1, monocyte chemotactic protein-1; vWF, von Willebrand Activated endothelial cells factor. 58 Immunological Reviews 196/2003
  9. 9. Rocha et al Á Effector mechanisms in transplant rejection able to prevent or treat the vast majority of AR episodes. On Rocha et al. (106) compared the outcome of 16 patients the other hand, B cells and antibodies do not appear to be with AHR treated with PP þ IVIG with that of 43 patients essential for graft rejection, as agammaglobulinemic mice are with acute cellular rejection diagnosed and treated during able to reject the first and second set skin transplants at control the study period. The one-year graft survival by Kaplan rate (99). Finally, antibody and complement deposition are Meier analysis was 81% in the AHR and 84% in the not typical findings in most AR biopsies. ACR group (P ¼ NS) (106). Prospective trials comparing Over the last decade, however, repeated clinical observa- PP þ IVIG with other regimens are needed. Given the low tions have suggested a central role for antibodies in at least a incidence of humoral rejection, a multicenter approach will subset of patients with AR. These patients are typically pre- likely be required to recruit the necessary number of sensitized and present with severe allograft dysfunction early patients for such studies. after transplant that is resistant to anti-T-cell therapy (100). There is a series of studies in animal models supporting an The histologic features of antibody-mediated acute humoral important contribution of antibodies to the pathogenesis of rejection (AHR) are distinct from those of typical acute cellular AR. Brandle et al. (107) used B-cell-deficient mice to show that rejection (ACR). In AHR, neutrophils constitute a large pro- donor-specific antibodies contribute to the pathogenesis of portion of the cellular infiltrate, which appears to preferen- acute allograft rejection. In a model of cardiac allograft rejec- tially target the peritubular capillaries. Sensitive flow tion in the mouse using subtherapeutic doses of cyclosporine cytometry techniques can detect circulating donor-specific to dampen cellular immune responses, graft survival was sig- alloantibodies (DSAs) in the majority of AHR cases. nificantly prolonged in B-cell-deficient mice compared to A recent study showed that 95% of allograft biopsies from controls (107). Using a different donor-recipient strain com- recipients with DSAs at the time of rejection had positive bination, Wasowska et al. (91) documented prolonged staining for the complement split product C4d (101). It has cardiac allograft survival in Ig KO mice, despite an intact been suggested that staining for C4d might have some advan- cellular immune response. When complement-activating anti- tages as a marker of complement activation, as C4d remains bodies to donor antigens were passively transferred to Ig KO covalently bound to the endothelium, serving as a footprint of mice 10 days after transplant, AR was rapidly restored. Trans- complement activation by alloantibodies (102). Initial studies fer of antibodies at earlier time points led to a slower onset of depicted C4d staining as a very sensitive and specific diagnos- rejection, suggesting a significant interaction between anti- tic tool that could reliably distinguish AHR from cellular bodies and the cellular components of the immune response rejection or calcineurin toxicity (102, 103). However, emer- (91). This interaction might occur in several ways. First, ging data have raised questions about the sensitivity and spe- antibodies can coat donor endothelial cells and bind recipient cificity of this assay in the diagnosis of AHR. For example, macrophages and NK cells to promote target cell lysis Bohmig et al. (104) showed that a substantial proportion of via ADCC. Second, it is well documented that B cells require patients with AHR (defined by AR and circulating DSAs T-cell help for antibody production (108). T-cell and detected by flow cytometry) had negative C4d staining, result- B-cell interactions via CD40L–CD40 and CD28–B7 pathways ing in a sensitivity of 31%. Moreover, Nickeleit (105) are essential for B-cell growth, differentiation, and Ig-class demonstrated C4d positivity in 40–50% of biopsies with switching (109). Interruption of these pathways inhibits histologic signs of acute cellular rejection as well as in some both cellular and humoral responses prolonging allograft cases that did not require any anti-rejection therapy. There- survival (110, 111). fore, treatment decisions should probably not be made based The apparent actions of antibody to enhance T-cell solely on C4d-staining results. Rather, clinicians should use responses to an allograft might also involve activation of C4d staining in conjunction with the clinical presentation, complement. In this regard, Pratt and associates (112) have flow cytometry results, and histology before deciding whether provided clear evidence of collaboration between complement antibodies or T cells should be the primary targets of anti- components and cellular immunity during allograft rejection. rejection therapy. The authors transplanted wildtype or C3-gene-disrupted Historically, AHR was associated with poor allograft (C3–/–) kidneys into MHC-mismatched recipients and showed prognosis, but recent studies have suggested that regimens that recipients of C3–/– grafts experienced long-term graft that target antibody removal and resynthesis such as plas- survival; in contrast, wildtype kidneys were rapidly rejected mapheresis with intravenous Ig (PP þ IVIG), immuno- with a mean graft survival of 12.5 days. When wildtype adsorption, or rituximab, might improve clinical outcomes. kidneys were transplanted into C3–/– recipients, there was Immunological Reviews 196/2003 59
  10. 10. Rocha et al Á Effector mechanisms in transplant rejection only a mild prolongation in graft survival (16.2 Æ 1.2, mean In an elegant study using a murine model of cardiac trans- Æ SEM) indicating that locally synthesized C3 had a greater plantation, Hancock et al. (111) demonstrated that adminis- impact on rejection than circulating C3. T cells were isolated tration of anti-CD4 monoclonal antibody induced long-term from recipients of wildtype or C3–/– grafts and were graft survival but did not prevent pathological findings of re-challenged with donor antigen in vitro. In these experiments, chronic rejection. By contrast, when anti-CD40L antibody proliferative responses to donor antigens were lower, if the was administered to disrupt the interactions between B and T recipient had received a C3–/– kidney. Taken together, these cells, allograft survival was indefinite and histologic evidence findings suggest that C3 produced locally by the rejecting renal of CR, including transplant arteriosclerosis, was abolished. allograft augments the alloimmune response by apparently This treatment also led to increased vascular expression of contributing to T-cell priming. ‘protective’ genes such as heme oxygenase-1 (HO-1), Bcl-xL, These data reinforce the notion that the immune response to and A20. When these genes were induced in endothelial cells an allograft comprises cellular and humoral components that by in vitro culture in the presence of Th2 cytokines prior to interact to produce graft injury. Although the central role of alloantibody exposure, there was marked protection against T cells during acute allograft rejection remains unquestioned, endothelial cell activation, as evidenced by decreased E-selectin significant contributions from antibodies and complement expression. In vivo treatment with anti-CD4 antibody along are being slowly uncovered. Moreover, there are important with agents that induce HO-1 (metalloporphyrins) resulted interactions between these components of the immune system in almost complete protection from CR and significantly during the alloimmune response. Therapeutic interventions decreased intra-graft apoptosis (111). These data are in keep- aimed at interrupting each of these effector pathways or their ing with recent findings by Plissonnier et al. (115), which interactions could have a major influence on the fate of organ indicate that apoptosis might be an important mechanism of transplants. antibody-mediated injury during CR. The authors transplanted MHC-mismatched aortic grafts into rats that had been pre- sensitized against donor antigens by skin transplant and Chronic rejection demonstrated that alloantibodies induced apoptosis of graft The pathogenesis of chronic rejection (CR) remains incom- vascular cells in vitro and in vivo (115). pletely understood. However, there are data that suggest key Production of alloantibodies in association with episodes of roles for antibodies and complement in the pathogenesis of CR. AR has been long recognized. The pathogenetic role of these Russell and Ley (113) performed cardiac transplants between antibodies, however, remains in question. Recent evidence inbred mice, where the recipients were depleted of CD4þ and suggests that the development of anti-MHC class II alloantibod- CD8þ T cells. This maneuver was sufficient to induce long- ies after transplantation may be a risk factor for CR, indepen- term graft survival but did not prevent the development of dent of AR (116). In a recent report, all kidney transplant obstructive coronary lesions typical of CR. The authors showed failures due to CR were preceded by the development of that transplants between strains that produced antibodies to anti-human leukocyte antigen (HLA) antibodies (117). donor cells (B10.A to B10.BR) developed more intense cor- Mauiyyedi et al. (118) tested the role of C4d antibody staining onary vasculopathy than those in the reverse combination, in in kidney biopsies of patients with chronic allograft nephro- which antibodies were not detected. In the latter strain com- pathy (CAN) and showed that 23/38 CAN biopsies (61%) bination, the severity of the coronary lesions could be had peritubullar capillaries (PTC) staining for C4d, compared increased in a dose-dependent fashion by the administration with 1 of 46 (2%) of controls (P < 0.001). The authors of antibodies against donor antigens. Similarly, continuing concluded that antibodies play a pivotal role in the pathogenesis injections of antidonor antibodies were sufficient to of 61% of cases of CAN and suggested classifying this subset of induce striking coronary lesions in SCID recipients of patients under the label of ‘chronic humoral rejection’. Subse- heart allografts (113). Further evidence that antibodies are quent studies by Theruvath (119) and Regele (120), however, required for the full pathologic expression of CR stems from have shown much lower rates of C4d positivity (13 and 34%, more recent experiments from the same group, in which respectively) among biopsies performed for CAN. Moreover, B-cell-deficient mice failed to develop the typical arterial Nickeleit et al. (105) suggested that C4d is a marker of active lesions of CR (114). Others have shown that transferring AR and not CR. Given the inconsistency of these observations, it antibodies specific to donor antigen into Ig-deficient mice is quite clear that more studies are needed before a role for C4d, can restore these lesions (111). as marker of humoral immunity during CR, can be established. 60 Immunological Reviews 196/2003
  11. 11. Rocha et al Á Effector mechanisms in transplant rejection Potential beneficial effects of antibody and complement in prolonging graft survival independent of the strain combina- transplantation tion. This finding is in stark contrast with the results obtained with a single injection of antibodies after transplantation The data presented so far depict antibodies and complement (121). The mechanisms of antibody-mediated graft enhance- as important effectors of allograft damage during rejection. ment are unclear; proposed mechanisms involve the produc- However, there is evidence suggesting that, in special circum- tion of anti-idiotypic or blocking antibodies. Administration stances, both antibodies and complement might have benefi- of F(ab0 )2 fragments alone does not promote enhancement of cial effects on graft survival. Earlier studies indicated that graft survival, suggesting that this process is Fc dependent (122). transfer of cytotoxic alloantibodies might have in vivo effects This finding is further supported by the fact that non-opsonizing that are both damaging and protective to the allograft. IgM antibodies are unable to induce enhancement (123). Oluwole and coworkers (121) demonstrated that the donor- Sohn and coworkers (124) recently showed that the devel- recipient strain combination and the timing of administration opment of antigen-specific tolerance after intra-ocular are crucial in determining the outcome of antibody transfer. For (immune privileged site) injection is dependent on the com- example, when ACI rats (‘low responders’) are the recipients plement fragment iC3b binding to APCs. Ligation of iC3b to its of Wistar Furth hearts, transfer of antibodies leads to enhance- receptor on APCs resulted in the sequential production of TGF- ment of graft survival, but when the donor-recipient combin- b and IL-10, which is essential for the induction of tolerance ation is reversed and Wistar Furth rats (‘high responders’) in this model (124). The extent to which this elegant observa- become the recipients, antibody transfer results in HAR. More- tion extends to the development of tolerance to alloantigens over, the transfer of 1 mL of alloantibodies for several days still needs to be elucidated. prior to and on the day of transplantation is very effective in References 1. Arakelov A, Lakkis FG. The alloimmune 10. Jones ND, et al. CD40-CD40 ligand- 17. Sutton VR, et al. Initiation of apoptosis by response and effector mechanisms of allograft independent activation of CD8þ T cells can granzyme B requires direct cleavage of bid, rejection. 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