Although the mechanisms of acute rejection of organ transplants are more apparent, the mechanisms of chronic rejection (CR) are still poorly understood and more complex. Persistent alloantigen shedding from the graft may maintain a host immune response through the indirect pathway of allorecognition, leading to B-cell activation and alloantibody-mediated injury. CR may also represent a general repair and remodeling process initiated by nonspecific innate immune responses associated with tissue ischemia and reperfusion at the time of the transplant. These insults may lead to gradual obliteration of arterial and arterioloar lumina, resulting in allograft arteriosclerosis, tissue ischemia, and ultimately interstitial fibrosis (1). CR is not simply delayed acute rejection although the injury sustained during acute rejection may be a cofactor in CR. In the current clinical era with improved organ preservation and immunosuppression, most organs transplanted into humans are not lost to acute rejection. However, the argument can be made that immunologic long-term graft survival has not improved during the past two decades, and most organs rejected in human organ transplantation are lost to CR (2–4) with retransplantation as the only definitive therapy.
Cause of Chronic Rejection
The mechanisms for CR are believed to be multifactorial, and many risk factors have been identified for this late phase rejection, including immunologic and nonimmunologic. Numerous factors are believed to promote chronic allograft vasculopathy, a pathologic manifestation of CR. However, an exact mechanism or cause is not fully understood yet.
CR of the graft is not only solely due to immune causes but also represents repair processes that are the final common response to many types of tissue injury. Smooth muscle cells (SMC) are believed to be a key component in intimal hyperplasia because SMC migrate from the media to the intima and proliferate (2–4). Proliferative vascular lesions (intimal hyperplasia and neointimal proliferation of SMC) are common features of CR that is actually histologically similar to arteriosclerosis. Both types of vascular lesions are currently believed to involve inflammatory processes because local proinflammatory mediators are increased in these regions (5, 6). Leukocytes recruited in response to inflammation also contribute to development of cardiac allograft vasculopathy by activating SMC. Proinflammatory cytokine and growth factors released by local resident cells and by infiltrating leukocytes have also been implicated in modulating SMC function and proliferation.
Ischemia-reperfusion injury is inevitable in solid organ transplantation and prolongation of ischemia-reperfusion injury has been associated with an increased incidence of acute and CR (7). The observation of vasculopathy after ischemic injury in isografts showed that nonallospecific factors (or nonantigen-specific factors implicating innate immunity) influence CR as well (8). It has been shown that transient damage is sufficient to induce chronic graft vasculopathy without alloimmunity (9, 10). Activated macrophages also can damage graft tissue by releasing mediators such as reactive oxygen intermediates (11, 12). We reported a positive correlation of macrophage infiltrates in kidney grafts with the severity of CR, and infiltration of macrophages in the arterial intimal region was also reported in a nonhuman primate model (13, 14). In the transplantation setting, because antigen is not eliminated, macrophages would remain persistently activated and release cytokines and growth factors that act on mesenchymal cells to promote stromal cell growth and fibrosis.
More recently, a contribution of self-reactivity (autoimmunity) to CR has been proposed. Conditions such as tissue damage induce expression of cryptic self-antigen through matrix metalloprotease-mediated remodeling. In lung transplantation, even when the remodeling process ceases, the matrix remains dysregulated, with ongoing exposure of antigenic collagen V in perivascular and peribronchiolar tissues (15). Interestingly, in this study, patients showed a Th17 response toward collagen V. Mediators released from injured tissues led to elevated pathogenic cytokines such as interferon-γ (IFN-γ) or interleukin-17 by alloreactive T cells (16). A direct role of Th17 cells on allograft rejection and CR has been confirmed in the absence of Th1 responses by using T-bet−/− mice (17). However, the role of interleukin-17/Th17 cells in CR is still unclear in human patients.
Although the contribution of alloantigen-independent processes is important for CR, it is generally accepted that the cellular immune system participates in the pathogenesis of CR. CR is associated with the presence of various immune cells. Among these cells, T cells play a crucial role in early acute graft rejection. However, the role of T cell subsets in the development of chronic allograft vasculopathy is not fully understood. CD4 T cells (TH cell) have been implicated in inducing development of CR by directly responding to the graft, generating CD8 effector T cells (Tc cell), thus promoting antibody production from B cells and activating antigen-independent leukocytes such as macrophages (18). Clinical studies show a predominance of Th-1 type immune responses in graft arteriosclerosis (19). Increased Th1 cells both in blood and biopsy were associated with CR (19–21). CD8 T cells may also produce signals that mediate delayed type hypersensitivity response (especially IFN-γ). Previous studies have shown conflicting results on the role of CD8 T cells in CR. Abrogation of CD8 T cells in an major histocompatibility complex (MHC) class I mismatched swine model demonstrated the importance of CD8 T cells, whereas it did not alter the severity of CR in a rat MHC class I and II mismatched model (22, 23). Previous studies have shown that allograft vasculopathy is dependent on the presence of IFN-γ (24, 25). IFN-γ elicited chemokines, such as IP-10 and MIG, may also contribute to inflammation by recruiting IFN-γ producing T cells. We showed that reduced infiltration of CD8 T cells in a CR model using CXCR3−/− mice alleviated CR (26). In accordance with this, induction of IFN-γ inducible chemokine transcripts (such as MIG, IP-10, I-TAC, and RANTES) in endomyocardial biopsies showed an association with the development of graft arteriosclerosis (27–29). Burns et al. (30) showed the infiltration of CXCR3 and CCR5 expressing T cells into human arteries transplanted to severe combined immunodeficiency (CB17; H-2d) mice reconstituted with human alloreactive T cells. These findings suggest a possible role for therapeutic agents targeting chemokines (further discussed below).
Regulatory T cells (TR cell) may help prevent CR by reducing acute rejection episodes. However, a direct role of TR cells in preventing CR is still unclear. Proportional increases of circulating TR cells (CD4+CD25hi and FoxP3+) from patients with long-term allograft survival was reported (31, 32), whereas patient with CR showed a decreased level of TR cells and FoxP3 transcripts (33). An increased ratio of low avidity CD8 TR cells over high avidity T effectors is another example of various types of TR cells controlling CR development (34). TR cell-related gene profiling (FoxP3, CCR7, and CCR4) has been described in clinically tolerant patients as well (35). Taken together, there is an association of TR cells with absence of CR. However, establishing causation versus association will require further investigation.
B Cell and Antibody
CR is often associated with alloantibody formation in renal transplantation (36, 37). A recent review by Terasaki and Cai (38) postulated a strong causal link of human leukocyte antigen (HLA) antibody to the development of CR. A retrospective study showed that patients who develop CR showed higher antibody titers in the first year after transplantation (39). Possible mechanisms of antibody induced CR development include complement fixation by donor endothelium bound to anti-HLA antibody. Although the endothelium repairs itself, repair can be accompanied by endothelial proliferation and thickening. A causal association of antibodies with the incidence of CR has not been shown clinically. However, in our nonhuman primate experience with CD3 immunotoxin, we found that antibody titers were associated with CR. Antibodies have also been associated with injury and early graft failure in lung allografts (40) and CR in cardiac allografts (41). Interestingly, liver allografts in humans may experience antibody-mediated rejection (42), but there are no substantial data linking alloantibody to CR of liver. Interestingly, liver allografts are also known to have a relatively low incidence of CR compared with other vascularized organ grafts, perhaps due to the high regenerative capacity of liver, its unique microvascular structure, and other poorly defined immunological advantages such as its enormous antigen load and its capacity to shed soluble HLA antigen.
Diagnosis of CR
CR is histologically characterized by sclerosing vasculitis and extensive interstitial fibrosis (3) Monitoring of graft function after transplantation is routinely performed using organ-specific parameters, which are rising sCr, proteinuria, and hypertension in kidney transplantation, bronchiolar obliteration (bronchiolitis obliterans) with falling pO2 in lung transplantation, vanishing bile ducts in liver transplantation and coronary vasculopathy in heart transplantation. CR is confirmed by perivascular inflammation, fibrosis, and allograft vasculopathy histologically. However, this is diagnosed only after irreversible damage has been established in the graft. In both renal and hepatic transplantation, differentiating CR from recurrence of autoimmune disease may be difficult if not impossible. Previous studies have correlated circulating anti-HLA antibodies with the presence of CR in renal allograft biopsies (43, 44). Two recent reports have described an association of C4d deposition with the histopathologic changes of CR in kidney (45, 46). Concomitantly, a high frequency of circulating anti-donor antibody was also identified (46). Subclinical C4d deposition has been reported in preliminary studies (45) and this may precede chronic glomerulopathy. Recently, an association of nondonor-specific antibody/anti-HLA Ab with CR was reported (47). According to several reports, HLA antibodies are ideal markers for impending graft failure (43, 48). These reports would indicate that alloantibody appears in advance of pathological changes in the graft, and that C4d staining in the graft or HLA antibody in the circulation predicts CR early enough to intervene before disease onset. Although histologic diagnosis of CR confirms this entity, if any therapies are to achieve successful intervention in this process, it will be imperative to have earlier indicators and predictors of CR. Thus, the implementation of clinically useful biomarkers of CR will need to accompany our better mechanistic understanding and better therapies.
Various animal models are used as preliminary tools for better understanding the pathogenesis, diagnosis, prevention, and therapy of CR. Early studies used large animals, such as the rabbit, pig, and dog. However, because of the lack of congenic strains and lack of appropriate reagents, larger animals have limitations as experimental models.
Murine Models of CR
Because of the well-defined genetic systems, a number of investigators have used the rodent as an experimental system for CR. The rat aortic transplant model has been used to study rejection-associated vascular lesions. In nonimmunosuppressed recipients, such grafts undergo a short episode of acute inflammation which evolves over weeks into a chronic type of inflammation of lesser intensity (49). Heterotopically transplanted heart grafts also show CR in certain inbred rat strain combinations. Cardiac allografts of Lewis rat into untreated F344 recipients are subject to CR and after at least 6 weeks show the development of diffuse graft arteriosclerosis, interstitial fibrosis, myocytes loss, and hypertrophy of the remaining myocytes (50).
Because of the benefit of transgenic strains and more readily available reagents such as antibodies, mice may be a preferred model to study CR. The MHC II mismatched model (Bm12-B6) with cardiac allografts has been used commonly (24). Transplanting heterotopic cardiac grafts across minor histocompatibility barriers using inbred strains of mice (26, 51) or a fully mismatched combination with treatments such as costimulation blockade, and gallium nitrate (52, 53) allows a reproducible CR model (Fig. 1).
Large Animal Models of CR
Some rodent models may be misleading as models of CR. For instance, some agents successfully induce tolerance without CR in these models, whereas human patients with similar treatment regimens develop CR (54). Therefore, the nonhuman primate model has been proposed as more relevant to human patients given the greater genetic and immunologic similarities between the species. We used anti-rhesus CD3-immunotoxin to deplete T cells in peripheral blood and lymph nodes of nonhuman primates. This approach significantly prolonged kidney allograft survival in recipient rhesus monkeys without the need for maintenance immunosuppression (55–57). However, these monkeys developed CR with clinical and histopathologic findings similar to human renal allograft CR. This model would therefore appear relevant to human patients and represent a reproducible large animal model of CR.
Novel Theraputic Targets of CR
CR is difficult to treat because it is diagnosed after irreversible damage of the graft occurs, and its prognosis is usually poor. More importantly, we currently lack effective definitive preventive or therapeutic strategies for CR. Medical management of patients with CR focuses on limiting progression of injury and managing side effects of CR. For renal transplant patients, this may include discontinuing calcineurin-inhibitors, treating hypertension, and treating any component of associated acute rejection or antibody-mediated injury.
The incidence of long-term human renal allograft loss has not improved in recent decades (58), suggesting that current immunosuppressive agents do not effectively prevent CR. Among the approved immunosuppressive agents, sirolimus has been proposed as potentially useful for preventing CR. Prevention of CR has been surmised from the fact that sirolimus decreases proliferation of SMC. In one liver transplant study (59), 50% of patients with biopsy-confirmed CR (8/16 patients) showed resolution of their CR under sirolimus treatment, assessed by using levels of total bilirubin and transaminases as endpoints. Sirolimus may also decrease B-cell antibody production and lipopolysaccharide-induced proliferation of B cell (60). However, clinical data supporting this contention is difficult to find. It has also been suggested in renal transplant patients that converting from a calcineurin inhibitors (CNI) to sirolimus, withdrawing a CNI from a sirolimus-based regimen or using a CNI-free strategy may improve long-term outcomes by reducing CNI-related nephrotoxicity (61, 62). On the other hand, the best reported series of clinical trials with respect to long-term outcomes in virtually all solid organ transplants rely on long-term CNI use.
Several T-cell depletional therapies (Table 1) have been used as a context for tapering CNI dose. Despite dose reductions and shortened treatment schedules for CNIs, no regimen tested to date yields durable remissions of allograft rejection, acute or chronic. T-cell depletional therapies in animal models have been associated with homeostatic proliferation of disease-associated memory/effector T cells (63). From our nonhuman primate studies, we found that profound T-cell depletion had little effect on antibody production in treated monkeys compared with nondepleted monkeys. Antidonor alloantibody could be detected in the majority of animals tested. We noted that in immunotoxin-treated monkeys, 5 of 5 rejecting between 40 and 90 days had donor-specific antibody. In animals with graft survival greater than 90 days, 7 of 10 had donor-specific antibody at 3 months posttransplant. This rise in antibody at 3 months is also associated with subsequent development of CR assessed histologically and by rising creatinine (data not shown). Although the sample size is too small to make satisfactory statistical conclusions, data does support the hypothesis that prevention of alloantibody in T-cell depleting regimens is essential for preventing late CR. Currently, several strategies are being evaluated to safely suppress the B-cell alloimmune response.
There is growing awareness that B cells and alloantibodies are important mediators of CR. Theoretically, B cells should be a cell population somewhat familiar with tolerance mechanisms. Up to 30% of B cells are autoreactive in the human body, yet they normally do not cause autoimmune disease. It is also notable that the B cell is the most abundant antigen-presenting cell.
Tolerance strategies based on T-cell depletion commonly face problems with the humoral response in human patients (64). Curiously, the use of alemtuzumab which targets and depletes not only T cells but also B cells does not appear to solve this problem and neither does addition of rituximab for B cell depletion. Rituximab is a humanized chimeric monoclonal antibody that binds to CD20 (65). Although the significance of CD20 infiltration into the graft is still controversial (66, 67), anti-CD20 therapy using Rituximab caused rapid and durable depletion of B cells from acutely rejected renal allografts (68) and may improve the outcome of antibody-mediated rejection in renal transplant patients (69). Even though previous studies suggests that anti-CD20 therapy led to decreased serum alloantibody levels in sensitized recipients (70), it is less likely that rituximab plays a critical role in the prevention of ongoing antibody production because CD20 is expressed on B cells from the early pre-B to mature B cell stage of differentiation, but not on terminally differentiated plasma cells (71). A recent study showed intragraft B cell survival in the absence of circulating B cells after rituximab therapy (72). More interestingly, in this study, BAFF (also termed BlyS) was found to be up-regulated in chronically rejected renal allografts. The affect of Rituximab (or depleting CD20 B cells) in preventing CR has not been evaluated yet. However, according to a gene profile study, tolerant (or long-term stable graft function) patients showed increased B cell related gene expression compared with the patients who developed CR (Personal communication, Dr. Ken Newell). We found that the appearance of a naïve phenotype of B cell in the periphery was associated with stable graft function (64). In this regard, depleting the entire B cells population might abolish regulatory or tolerogenic function from B cells, resulting in more detrimental outcomes by increasing alloimmunity and infection.
Unlike alemtuzumab or rituximab, atacicept (TACI-Ig) blocks B-cell survival factors. The BlyS family of TNF ligands and their receptors governs survival and differentiation within B cell subsets. This family includes two ligands (BAFF and APRIL) and three receptors (BR3 [also termed BAFF-R], TACI, and BCMA). Downstream signal of these ligands crosstalks with inputs from other B cell receptors, modulating their integration in homeostatic/differentiation process. BAFF is the sole known ligand for the BAFF receptor (BAFF-R) even though interaction with two other receptors (TACI and BCMA) is also essential for B cell/plasma cell survival, T-dependent/independent antibody responses, and T cell costimulation (73). We have recently studied the expression of BAFF and APRIL in human renal transplant patients treated with alemtuzumab. Interestingly, we found that BAFF is substantially upregulated in these patients, although APRIL is not (74). It would follow that a natural consequence of substantial T- or B-cell depletion is the upregulation of these B-cell regulatory elements. A safer and more effective way to prevent alloantibody production would therefore be to target BAFF. In addition, new agents such as the proteasome inhibitor bortezomib (Velcade, Millennium Pharmaceuticals, Cambridege, MA) have been shown to be effective treatment of the plasma cell malignancy multiple myeloma (75, 76). Initial experience with this drug showed that bortezomib in renal transplant patients showed decreased levels of serum alloantibody (77, 78). Combination therapy with rituximab and bortezomib might further blunt antibody production because of the combined depletion of mature, antibody-secreting plasma cells, and their precursors.
Selectively blocking chemokines or their receptors may also ameliorate CR possibly by interfering with immune cell transmigration. Although the role of chemokines in manifestations of atherosclerotic vascular disease has been studied for a long time, the effect of targeting chemokine/chemokine receptors on CR is still unclear. Many redundant chemokines were found in atherosclerotic lesions, including CXCR3 and CCR5 ligand (79–85). In accordance with this, several groups showed a beneficial effect of targeting these chemokine receptors on CR (86–90). We also showed that Cxcr3−/− knockout mouse recipients of cardiac allografts showed reduced neointimal hyperplasia with less CD8 T-cell infiltration (26). Combined blockade of Cxcr3/Ccr5 showed a beneficial effect on chronic allograft vasculopathy in the setting of a full MHC full mismatch (91). Interestingly, studies by Schroder et al. (92) showed that CCR5 blockade attenuates vasculopathy when used in combination with a limited course of cyclosporine in heterotopic cynomologous monkey heart transplants. We also found that a CCR5 antagonist prolonged graft survival when used in combination with a sphingosine-1-phosphate (S-1-P) analog (Table 2). The observation is novel in that neither the S-1-P analog nor CCR5 inhibitor alone effectively prevented rejection. Using flow cross match, no anti-donor IgG or IgM was found in the sera of the three recipients in the combination group with greater than 30 days graft survival. Although a definite mechanism is still unclear, CCR5 may block T cell help for alloreactive B cells. Because of the importance of the CCR5 receptor in a variety of immune functions, several pharmaceutical companies have developed small molecule inhibitors of this target. This includes the recently FDA-approved CCR5 small molecule inhibitor Selzentry (Miraviroc, Pfizer).
Although we are unaware of any published reports demonstrating the use of this drug in nonhuman primates in vivo, studies conducted on rhesus lymphocytes in vitro suggest that Selzentry may effectively block CCR5 in an experimental model (93, 94).
CR remains an intransigent detractor from successful long-term outcomes in solid organ transplantation. Although its mechanisms may yet avoid complete understanding, there is solid evidence that both innate immune mechanisms are involved in nonspecific inflammatory, injury after the initial ischemia-reperfusion associated with the transplant, and that B cell, activation leading to alloantibody production is associated with insidious late graft dysfunction. Diagnostics that identify this process before it is histologically established will be crucial to our, prevention of CR. As our mechanistic understanding of the biology of CR grows, so will our, ability to design appropriate therapies to prevent or perhaps reverse this process. Several, candidate therapies aimed at reducing alloantibody after transplantation are being evaluated in, experimental models and in the clinic at this time, and some of these we have discussed earlier. However, these therapies are in their infancy at this time and CR remains an opportune, challenge for the clinician to treat and the researcher to solve.
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