Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and indefinite survival

We have tested the effects of FK506 (FK), a new immunosuppressive agent, on a rat limb allograft model. Histoincompatible BN limb allografts were rejected in untreated F344 hosts within 11 +/- 1 days (mean +/- SD) after operation. A single injection of 2 mg/kg, 10 mg/kg, or 50 mg/kg of FK on the day of limb transplantation (day 0) significantly prolonged graft survival in a dose-dependent manner--i.e., mean limb survival times (MST) based on gross signs of skin rejection were 16 +/- 3 days, 51 +/- 6 days, or 104 +/- 17 days, respectively (P less than 0.01). Delayed treatment with a single injection of 10 mg/kg of FK at when early signs of rejection were visible (day 7 or day 10) reversed the ongoing rejection. The MSTs in these groups were comparable to that of those treated with the same dosage of FK on day 0. The FK-induced unresponsiveness toward limb allografts was donor-specific because limb-allografted. FK-protected rats could not accept the skin grafts from a third-party donor. In the next set of experiments, rats were given a single administration of 10 mg/kg of FK on the day of limb allograft, followed by intermittent injections of 3 mg/kg of FK once a week. This regimen produced complete graft survival for more than 200 days, though Pneumocystis carinii pneumonia occurred in most of the recipients. These results represent the unique effects of FK in preventing or reversing the graft rejection and in inducing indefinite survival in this animal model of composite tissue allografts.     

Evidence that the immunosuppressive effects of FK506 and cyclosporine are identical.

The fungal metabolite FK506 was discovered because it shared an important property, the ability to inhibit production of IL-2, with another well-known immunosuppressive fungal metabolite, CsA. FK506 has, since its isolation, also been shown to share other immunosuppressive effects with CsA. This study was performed to further investigate the in vitro immunological properties of FK506, in comparison with and in combination with CsA, to evaluate the plausibility that their mechanisms of action were identical or similar, in spite of their different molecular structures. The ability to inhibit several responses of human peripheral blood lymphocytes to mitogenic and alloantigenic stimulation was explored. Synergistic effects of the two drugs were extensively studied, since this would provide additional information regarding their mechanisms of action. Also, similar immunosuppressive properties would enable the use of the two drugs in combination. We found that FK506 and CsA had very similar mechanisms of action and additive effects were recorded.     

Evidence that long-term survival of concordant xenografts is achieved by inhibition of antispecies antibody production.

Antibody and complement have been shown to be of primary importance in the rejection of hamster heart xenografts by rats. Very high anti-hamster antibody titers were detected at the time of rejection of hamster hearts transplanted into untreated or T cell deficient rats. This study demonstrates a method of inhibiting this antibody production by pulse therapy with cyclophosphamide (CyP) and continuous cyclosporine treatment, resulting in a median survival of the hamster heart of greater than 100 days. Controls and CsA-treated rats reject the transplanted hamster heart in a median of 3 days. CyP as a sole therapy resulted in a median survival of 14 days. Prolonged CyP therapy when combined with CsA was associated with increased death among rat recipients due to infection. Antispecies antibody production was suppressed during CyP and CsA therapy and did not recur after cessation of CyP therapy. Cessation of CsA therapy at 60 and 100 days posttransplantation resulted in subsequent rejection of the xenografts (median survival after cessation of therapy of 11 and 19.5 days, respectively) and was associated with production of rat anti-hamster antibodies.     

Evidence that LS-2616 (linomide) causes acute rejection of rat allografts protected by cyclosporine but not of long-term surviving allografts

The immunomodulator LS-2616 (Linomide) induces rejection of cyclosporine-protected rat cardiac allografts. The aim of this study was to characterize this rejection in the presence of CsA and to test LS-2616 in other models of permanent graft acceptance in the rat. PVG rat hearts were transplanted heterotopically to Wistar/Kyoto (Wi/Ky) rat recipients on day 0. The recipients were treated orally on days 0-9 with CsA (10-40 mg/kg) and/or with LS-2616 (2.5-160 mg/kg) starting at different times (day -7 -+5) until the day of complete rejection. The addition of LS-2616 (day -1--stop) to CsA (10 mg/kg) resulted in a dose-dependent antagonism of the immunosuppressive effect of CsA with daily doses of 2.5-160 mg/kg. Furthermore, the results were similar, irrespective of whether LS-2616 treatment (160 mg/kg) was started on day -7, -1, +1, +3, or +5. LS-2616 (160 mg/kg) pretreatment of the recipient for 7 days before transplantation was considerably less effective. CsA (20 mg/kg) for 14 days after a PVG to DA transplantation resulted in permanent graft survival. This was not abrogated by LS-2616. Neither was rejection induced in long-term surviving grafts of RT1.C incompatible Lewis recipients. Our data suggest that LS-2616 activates already stimulated and sensitized T cells that are otherwise controlled by CsA.     

Twelve-hour and twenty four-hour preservation of small bowel allografts by simple hypothermia. Survival utilizing cyclosporine

Canine small bowel was harvested and stored by simple hypothermic technique. After 12- and 24-hr storage, respectively, the small bowel graft was allotransplanted into recipients. All animals receiving 12-hr stored grafts (n = 9) survived beyond 5 days. In the 24-hr storage group, 67% of the animals (n = 9) survived beyond 5 days. Successful storage for such extended periods by simple hypothermia has not been achieved previously. Donor pretreatment with antibiotics as well as extensive intraluminal irrigation of the harvested small bowel are considered to be important technical features in this successful preservation.     

Evidence that liposome incorporation of cyclosporine reduces its toxicity and potentiates its ability to prolong survival of cardiac allografts in mice

Using liposomes, multilamellar lipid vesicles (MLV), we have found that liposome-incorporated cyclosporine is not only less toxic but also more effective in prolonging survival of cardiac allografts in mice. Following intravenous injection of liposome-incorporated drug, cyclosporine levels in blood were shown to decrease rapidly, while concentrations in spleen were higher (when compared with concentration following administration of commercial preparation). In this context, possible mechanisms of the beneficial effect of liposome-incorporated cyclosporine are discussed.     

Ex vivo and systemic transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of FK506 therapy prolongs islet graft survival

Adenovirus-mediated CTLA4Ig gene transfer has been reported to enhance graft survival in several rodent transplantation models. In this study, we investigated the efficacy of ex vivo and systemic transfer of the CTLA4Ig gene by adenoviral vectors in pancreatic islet allo-transplantation. Islet grafts from BN rats were transplanted to chemically induced diabetic LEW rats. First, ex vivo CTLA4Ig gene transfer into isolated islets was performed prior to transplantation. Survival of transduced grafts under the kidney capsule was slightly prolonged (8.6+/-1.3 days) compared with survival of untransduced grafts (6.7+/-1.2 days); when combined with a short course of FK506, graft survival was further extended (32.6+/-10.7 days vs. 13.7+/-1.0 days with FK506 alone). Secondly, systemic gene transfer was accomplished by intravenous administration immediately after the transplantation procedure. In these animals, islet grafts under the kidney capsule survived longer (15.2+/-3.3 days) than in controls (6.7+/-1.2 days), and when FK506 was administered perioperatively, all the islet grafts survived for more than 100 days. In systemically transduced recipients, the survival of islet grafts transplanted into the liver was not significantly different from that of the grafts placed under the kidney capsule. In order to examine organ-specific immunogenicity, heterotopic BN cardiac grafts were transplanted to LEW rats intra-abdominally, with the virus transferred systemically as in the islet model. In contrast to the islet grafts, all the cardiac grafts were accepted for longer than 100 days, even without FK506 therapy. Finally, the LEW recipients with long-surviving islet or cardiac grafts were re-transplanted with islet grafts from the same donor strain (BN) on day 100. The second islet grafts survived longer than 100 days in half of the cardiac recipients, but consistently failed in the islet recipients. We conclude that in this transplant model, CTLA4Ig gene transfer and FK506 treatment synergistically improved islet graft survival, systemic transfer of the gene was more effective than ex vivo transfer to the islets, and donor-specific tolerance could not be achieved for islet transplantation but was achieved for cardiac transplantation.     

Evidence that sensitivity to cyclosporine is influenced by the HLA-DR phenotype of kidney graft recipients.

Clinical as well as experimental studies have shown a great interindividual variability in the immunosuppressive efficacy of CsA. Evaluating previous in vitro findings of a correlation between sensitivity of alloresponsiveness to CsA and the HLA-DR phenotype CsA levels were compared in kidney transplant recipients with and without rejections during the early posttransplant period and tested for a possible relationship to the HLA-DR phenotype of the recipient. In patients treated with CsA and prednisolone only, rejection frequency was significantly higher in HLA-DRw6 positive than in DRw6 negative graft recipients (77% vs. 53%, P = 0.045). In the DRw6 positive group incidence of rejection was independent of CsA blood levels, whereas in DRw6 negative patients frequency of rejection episodes decreased as a function of increasing CsA levels. Therefore the relative risk in developing graft rejection continuously increased in HLA-DRw6 positive patients. In HLA-DR2 positive graft recipients, however, a decrease in the relative risk could be observed with increasing CsA levels. Within patients with bioptically verified rejection episodes HLA-DR2 positive recipients had significantly lower CsA levels than DR2 negative patients (P = 0.01). In other HLA-DR phenotypes no association with CsA blood levels could be assessed. Also no statistically significant difference could be found in nonrejecting patients. These clinical findings demonstrate an association of sensitivity to immunosuppressive treatment and the HLA-DR phenotype of the graft recipient. Our results would indicate a very low CsA sensitivity of HLA-DRw6 positive graft recipients and thus might offer an explanation for previous findings about an increase in the incidence of rejection reported on those patients.     

15AU81, a prostacyclin analog, enhances donor-specific hepatocytes to prolong the survival of rat heart but not small bowel allografts

Prostacyclin analogs have previously been shown to have not only cytoprotective but also independent immunosuppressive effects. The effect of one such analog, 15AU81, to enhance the immunosuppressive effects of liver was investigated. We have previously demonstrated that cyclosporine (CsA) in conjunction with rapamycin (RAPA) potentiates class I+, class II- donor-specific hepatocytes to prolong rat cardiac and small bowel allograft survival. Brown Norway (BN; RT1n) hepatocytes alone (5 x 10(7)/kg, administered intrasplenically) failed to prolong the survival of BN heart allografts in Wistar Furth (WFu; RT1u) recipients, beyond that of untreated controls (MST = 7.2 +/- 0.8 days). Survival of BN hearts was increased to 11.4 +/- 1.7 days in WFu recipients treated with BN hepatocytes and 50 microg/kg/day 15AU81 administered by continuous s.c. infusion for 14 days using osmotic pumps (p < 0.05). The further addition of RAPA 0.0075 mg/kg/day and CsA 0.375 mg/kg/day delivered for 14 days by continuous i.v. infusion (CIVI) using osmotic pumps (a combination that alone prolonged BN heart allografts in WFu hosts to 18.4 +/- 1.3 days and in conjunction with BN hepatocytes prolonged survival to 27.2 +/- 1.9 days) prolonged allograft survival to 35.2 +/- 5.2 days. In contrast, the survival of small bowel allografts was not enhanced by 15AU81 administration. Survival of BN small bowel transplants in LEW recipients treated with hepatocytes alone (MST = 11.6 +/- 1.5 days) or hepatocytes plus 15AU81 (MST = 10.0 +/- 1.0 days) was similar to controls (MST = 10.2 +/- 1.9 days). Treatment with hepatocytes and RAPA/CsA increased survival to 21.2 +/- 1.5 days. The further addition of 15AU81 failed to augment this (MST = 17.0 +/- 1.9 days). In vitro WFu lymphocyte proliferative responses from animals pretreated with BN hepatocytes, 15AU81, or both treatments, for 2 weeks prior to harvesting, exhibited a reduction of at least 50%, compared to untreated controls upon allostimulation with irradiated BN or ACI spleen cells. These findings demonstrate that 15AU81 interacts favorably with hepatocytes either alone or in conjunction with RAPA and CsA to enhance their immunosuppressive effects on rat heart allograft survival. The failure to enhance small bowel allograft survival may be explained by the inability at this low dosage of 15AU81 to influence the intense graft versus host reaction elicited by small bowel transplants.     

Immunosuppression with either cyclosporine a or FK506 supports survival of transplanted fibroblasts and promotes growth of host axons into the transplant after spinal cord injury

Fibroblasts that have been genetically modified to secrete neurotrophins can stimulate axonal regeneration, rescue injured neurons, and improve function when grafted into a spinal cord injury site. These grafts are usually allografts that require immunosuppression to prevent rejection. In this study, we compared the effects of two immunophilin-ligands (cyclosporine A [CsA] and FK506) that are used clinically to prevent transplant rejection on protection of grafted fibroblasts. As there are risks associated with prolonged immunosuppression, we compared the effects of 2 or 8 weeks of administration of these drugs, in combination with our standard methylprednisolone protocol, in animals that survived for 8 weeks, to determine whether a shorter course of immunosuppression would be effective. Outcome measures included fibroblast survival, infiltration of activated macrophages and microglia into the graft, final lesion size, and growth of host axons into the graft. The graft consisted of a Vitrogen matrix into which fibroblasts were suspended; the graft was placed into a C3/C4 lateral funiculus lesion. The fibroblasts were isolated from a transgenic strain of Fischer rats that produce the marker alkaline phosphatase (Fb/AP). This enabled us to track the grafted fibroblasts and to evaluate the extent of their survival. The grafted matrix filled the lesion cavity. The density of fibroblasts within the matrix differed according to treatment. Fibroblast survival was most robust in animals that received 8 weeks of immunophilin-ligand treatment. FK506 supported greater Fb/AP survival than CsA. ED-1 immunostaining for activated microglia and macrophages showed an inverse correlation between AP immunoreactivity and the density of immune cells within the graft. Thus, prolonged administration of either FK506 or CsA was necessary for maximal fibroblast survival and for limiting the macrophage invasion of the graft. None of the FK506 or CsA protocols modified the size of the lesion, indicating that these immunophilin-ligands had little effect on secondary enlargement of the lesion and therefore little neuroprotective effect. Because immunophilin-ligands have been shown to be neurotrophic, we used RT-97 immunostaining for neurofilaments and calcitonin gene related protein (CGRP) staining for dorsal root axons to visualize axons that grew into the graft. Some axons grew into the matrix even in the absence of immunophilin-ligand treatment, suggesting that the Vitrogen matrix itself is permissive, but all of the immunophilin-ligand protocols were much more effective in eliciting axonal growth. Growth of axons into the transplants was equally increased by drug treatment for 2 or 8 weeks. Thus, both treatments improved fibroblast survival, diminished immune cell invasion, and promoted axonal growth, and a 2-week course of treatment with either immunophilin-ligand was as effective as 8 weeks in stimulating axonal growth.