Elaine Wakely

Prevention of murine cardiac allograft rejection with gallium nitrate : Comparison with anti-CD4 monoclonal antibody

Gallium nitrate (GN) was evaluated for its ability to interfere with a cute rejection of DBA/2-->C57BL/6 heterotopic cardiac allografts, in comparison with the depleting anti-CD4 mAb, GK1.5. The administration of GN for 30 days (s.c. 30 mg/kg elemental gallium on days 0 and 3, 10 mg/kg every third day) resulted in >60-day graft survival in 78% (25 of 32) of the graft recipients, whereas 2 perioperative injections of anti-CD4 monoclonal antibody (mAb) resulted in >60-day graft survival in 58% (24 of 41) of the graft recipients. Serum gallium levels peaked at about 2000 ng/ml after 2-3 weeks of treatment and decreased to about 300 ng/ml by day 60, a level that was maintained for at least 30 more days. During the early posttransplant period, 25% of GN-treated grafts, but not anti-CD4 mAb-treated grafts, exhibited an unusual, transient reduction in graft impulse strength, suggesting a transient rejection response. Macroscopically, the long-surviving (>60 days) grafts from either treatment group exhibited none of the features of rejecting allografts. Histologically, they exhibited minor edema and rare epicardial inflammation but no tissue necrosis. However, there were vascular changes in allografts from GN-treated mice, including altered endothelial morphology, associated with moderate intimal hyperplasia and mild perivascular leukocytic infiltration. Allografts from anti-CD4 mAb-treated mice exhibited prominent neointimal hyperplasia associated with endothelial morphologic changes and prominent vascular and perivascular leukocytic infiltration. In general, both GN and anti-CD4 mAb promoted long-term allograft survival, but these allografts displayed the histopathologic signs of ongoing inflammation and chronic allograft rejection.

Prolonged murine cardiac allograft acceptance: characteristics of persistent active alloimmunity after treatment with gallium nitrate versus anti-CD4 monoclonal antibody.

We have treated DBA/2-->C57BL/6 murine cardiac allograft recipients with anti-CD4 monoclonal antibody or with gallium nitrate to promote long-term (>60 days) allograft survival. Within this period, all grafts developed histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and neointimal hyperplasia, which are characteristic of chronic allograft rejection. To evaluate residual alloimmunity associated with the pharmacologic avoidance of acute graft rejection and the development of chronic tissue remodeling, we subjected these graft recipients to a battery of histologic and immunologic tests. Similar test results were obtained for graft recipients treated with either of the two immunosuppressive agents. All long-surviving allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcriptase-polymerase chain reaction analyses demonstrated intragraft expression of mRNAs for interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor, interferon-gamma, and transforming growth factor-beta. All recipients had limiting dilution analysis-detectable, graft-reactive cytolytic T lymphocytes and helper T lymphocytes in their spleens and grafts, and all produced high titers of graft-reactive alloantibodies. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 monoclonal antibody or gallium nitrate was used for antirejection therapy, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes that are qualitatively similar to those observed during acute allograft rejection, and (3) no specific immune responses developed selectively in long-term graft recipients to account for the avoidance of acute graft rejection or the development of chronic tissue remodeling in the graft.

Alloreactive delayed-type hypersensitivity in graft recipients: complexity of responses and divergence from acute rejection.

BACKGROUND Immunocompetent allograft recipients typically exhibit evidence of sensitization to graft antigens through alloantibody production and allograft rejection, as well as delayed-type hypersensitivity (DTH) reactivity to donor antigens. Most previous studies have relied on whole donor splenocytes, which primarily elicit allorestricted allogeneic responses, to test specific DTH responses and overlook the independent element of self-restricted responses in host-allograft interactions. METHODS We tested expression of self-MHC-restricted versus allo-MHC-restricted allogeneic DTH responses in both nonimmunosuppressed and tolerized C57BL/6 mice. Mice were sensitized for allogeneic DTH either by rejection of skin or cardiac allografts, or by subcutaneous injection of intact allogeneic splenocytes. Patterns of alloreactive DTH were compared in allosensitized, tolerant, and naive hosts. RESULTS All three methods of allosensitization resulted in equivalent self-restricted and allorestricted allogeneic DTH responses in nonimmunosuppressed mice. Gallium nitrate blocked acute rejection of cardiac allografts, and also blocked allosensitization of both self-restricted and allorestricted DTH responses, but did not influence the expression of DTH responses in presensitized mice. Gallium nitrate treatment could not block acute rejection of skin allografts, but interfered with sensitization for self-restricted, but not allorestricted, DTH responses in these recipients. This divergence of self- versus allo-MHC-restricted allosensitization for DTH was observed in two additional situations: the rates of allosensitization for self-restricted versus allorestricted DTH, and the acquisition of allorestricted, but not self-restricted, alloreactive DTH responses in cardiac allograft tolerant mice subsequently challenged with a skin allograft. CONCLUSIONS These studies demonstrate that acute rejection correlates generally with allogeneic DTH, whereas tolerance is associated with a lack of alloreactive DTH. However, self-restricted and allorestricted allosensitization can operate independently in allograft recipients. Thus, the relationships between alloreactive DTH and graft-induced allosensitization, acute rejection, or tolerance are more complicated than previously appreciated.

Transforming growth factor-beta and interleukin-10 subvert alloreactive delayed type hypersensitivity in cardiac allograft acceptor mice.

We have previously reported that temporary treatment of cardiac allograft recipients with gallium nitrate (GN) results in indefinite graft survival, and the inability to mount donor-reactive delayed type hypersensitivity (DTH) responses. We report that antibodies to either transforming growth factor-beta (TGFbeta) or interleukin-10 (IL10) can uncover DTH responses to donor alloantigens in cardiac allograft acceptor mice. The DTH responses uncovered with TGFbeta-reactive antibodies can be blocked by exogenous IL10, and those uncovered with IL10-reactive antibodies can be blocked by exogenous TGFbeta. These data demonstrate that allograft acceptor mice are fully allosensitized, and poised to make donor-reactive cell-mediated immune responses. However, such responses are subverted by a donor alloantigen-dependent mechanism that involves TGFbeta and IL10, which in turn interfere with local cell-mediated immune responses.

Cardiac allograft tolerance: failure to develop in interleukin-4-deficient mice correlates with unusual allosensitization patterns.

BACKGROUND The development of long-term allograft survival and understanding the mechanism(s) by which it is induced are major goals of experimental transplantation. Studies by several different investigators have provided conflicting evidence for the role of interleukin (IL)-4 in the process of allograft rejection or long-term allograft survival. These studies examine the role of IL-4 in experimental cardiac allograft rejection and in inducing long-term allograft survival. A possible mechanism for long-term allograft maintenance via alternative allosensitization is discussed. METHODS Adult IL-4-intact or IL-4-deficient (knockout, KO) C57BL/6 (B6) mice were transplanted with heterotopic DBA/2 cardiac allografts and immunosuppressed either with gallium nitrate (GN), or the anti-CD4 monoclonal antibody, GK1.5. Cellular allosensitization was assessed by testing the allograft recipients for donor-reactive delayed-type hypersensitivity (DTH) responses. The presence of antigen-driven suppressive mechanisms was assessed using a linked unresponsiveness (bystander suppression) DTH assay. RESULTS In general, the results were the same with either GN or GK1.5. We observed that (1) IL-4 is not required for acute allograft rejection or allogeneic DTH responses in nonsuppressed mice, (2) IL-4 is required for long-term allograft survival in immunosuppressed mice, (3) immunosuppression creates a requirement for IL-4 in major histocompatibility complex self-restricted, but not allorestricted, DTH responses, and (4) the development of alloantigen-dependent linked DTH unresponsiveness (bystander suppression) in allograft recipients requires IL-4. CONCLUSION In summary, these studies demonstrate a common requirement for IL-4 during the development of long-term allograft survival and the concurrent development of alloantigen-dependent DTH down-regulation in cardiac allograft recipients after immunosuppression.

Acute Versus Chronic Graft Rejection: Related Manifestations of Allosensitization in Graft Recipients

I n the earlier years of clinical transplantation, there was a tendency among clinicians and investigators to focus on the immediate problem of acute graft rejection, which occurred soon arter transplantation in a large number or transplant recipients and was a major cause ofgraft loss. In the current clinical era, acute rejection can be pharmacologically controlled in most patients, and clinical concerns have begun to focus on the less immediate problem of chronic graft rejection. This phenomenon occurs in about half of all transplant patients, and is a majot cause of late graft loss. Chronic rejection is generally unresponsive to therapies that are effective against acute rejection, and to date, there is no accepted clinical therapy for chronic graft rejection. As a result, chronic rejection now claims more grafts than acute rejection. Chronic rejection is not simply delayed acute rejection. Indeed, acute and chronic rejection are diKerent in many ways. Acute rejection is histologically characterized by a rapid, intense and destructive leukocytic inliltration of graft tissues, and generally represents a pathologic inflammatory response.‘” In contrast, chronic rejection is characterized by a slower development within the graft or interstitial librosis and neointimal formation within the large1 vessels, and seems to represent a pathologic tissue remodeling response .+a For some time, these disparate characteristics led clinicians and investigators to consider acute and chronic rejection as separate and unrelated entities. However, there is developing cvidence that the two pathologies are closely related manifestations of post-transplant cellular and humoral sensitization to graft alloantigens. provided by several investigators,‘* who indcpendently showed that long-term (5-10 year) graft survival decreases signilicantly in direct relation to the number of acute rejection episodes experienced by the patients. For esample, Matas et al” have calculated that the half-life of kidney allografts in patients with no clinical acute rejection episodes is 733 years, compared with 5 1 years in patients with one rejection episode, and 6 years in patients with more than one acute rejection episode. Conversely, these data indicate that late graft loss due to chronic rejection is relatively rare among patients who do not esperience an early acute rejection episode. This suggests that the two types of graft rejection are somehow related. It also suggests that control of chronic rejection in transplant patients might be achieved by developing immunosuppressive strategies that efTectively avoid, rather than reverse, acute rejection episodes. Indeed, the current clinical tendency to accept acute rejection, because it is treatable, may indirectly promote chronic rejection development. The purpose of this discussion is to review some current concepts regarding the related mechanisms of acute and chronic grart rejection in the light of some key observations in experimental transplant models. This is not meant to be a comprehensive review of the subject, and many interesting studies will not be cited for the sake of clarity and brevity. What should emerge from this discussion is a broadened view of transplant biology, and a working model of the continuum of pathologic events associated with acute and chronic graft rejection.

ELIMINATION OF ACUTE GVHD AND PROLONGATION OF RAT PANCREAS ALLOGRAFT SURVIVAL WITH DST, CYCLOSPORINE, AND SPLEEN TRANSPLANTATION

Allogeneic spleen transplantation has been shown to have a tolerizing effect on pancreas allograft survival in rats. In this study we examined the effect of blood transfusions and cyclosporine administration on both rat pancreas allograft survival and acute graft-versus-host disease in BN recipients of Lewis pancreas-spleen allografts. We found that in this strain combination significant pancreas allograft prolongation occurred when the spleen was included en bloc with the pancreas graft. However, 50% of these recipients developed GVHD and died. A single donor-specific transfusion delivered to BN recipients of Lewis pancreas and pancreas-spleen allografts did not extend graft survival, but precluded the development of GVHD. A short, 6-day, and long, 26-day, course of CsA to recipients of pancreas and pancreas-spleen allografts extended graft and animal survival times, although not indefinitely. All pancreas-spleen recipients of both CsA protocols died following acute GVHD. Combined DST and CsA (short and long-course) administration in pancreas-only allograft recipients was deleterious to graft survival, compared with CsA administration alone. However, in pancreas-spleen recipients, combined DST and CsA had an additive effect. Moreover, in DST and long-course CsA-treated pancreas-spleen recipients, indefinite graft survival occurred with no signs of acute GVHD. The beneficial effect of the spleen allograft on pancreas graft survival was not dependent upon GVHD, since splenic-induced prolongation of pancreas graft survival was observed in the F1-donor to parent-recipient combination.

Evidence that epidermal alloantigen Epa-1 is an immunogen for murine heart as well as skin allograft rejection.

Epa-1 is a non-H-2 mouse alloantigen defined by MHC-restricted, CD8+ cytotoxic T cells. In vitro it is a strong determinant for the lysis of epidermal cells, fibroblasts, and macrophages but not lymphocytes, and in vivo it functions as a target for skin allograft rejection and cutaneous graft-versus-host reactions. Genetically, Epa-1 appears to be the nonpolymorphic manifestation of a loss mutation. The establishment of C3H.Epa-1 (Epa), an Epa-1+ congenic strain on the Epa-1- C3H/HeJ (C3H) inbred strain background, facilitated the investigation of the role of Epa-1 in skin and heart allograft rejection. C3H females and males rejected first-set Epa skin grafts with median survival times (MSTs) of 20 and 30 days, respectively. However, there was a strong factor of immunization, because all second-set skin allografts were rejected by hosts of both sexes within 10 days. In contrast, all Epa hosts of both sexes permanently accepted C3H skin allografts, consistent with Epa-1 arising from a loss mutation. C3H hosts of both sexes rejected primarily vascularized first-set Epa heart allografts in similar tempo to first-set Epa skin allografts, with MSTs of about 30 days. However, in contrast to the accelerated rejection of skin allografts, sensitized C3H hosts rejected Epa heart allografts in chronic fashion, with some transplants showing very prolonged survival. Thus, Epa-1 is a relatively strong determinant of skin allograft rejection but a weaker determinant of heart allograft rejection.