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.

Intrasplenic liver parenchymal cells in conjunction with low-dose rapamycin and cyclosporine induce a unique and specific prolongation of rat cardiac and small bowel allograft survival.

These experiments investigated the immunosuppressive properties of liver tissue. Brown Norway (BN; RT1n) rat heart allografts survived in untreated control Wistar Furth (WFu; RTl(u)) rat recipients for 6.2 +/- 1.5 days, while allografts in animals that received rapamycin (RAPA) 0.0075 mg/kg/day and cyclosporine (CsA) 0.375 mg/kg/day delivered for 14 days by continuous intravenous infusion (civi) using osmotic pumps in conjunction with intrasplenic (i.s.) saline survived to 18.4 +/- 1.3 days. i.s. addition of 3 M-KCl extracted BN hepatic antigen or unpurified BN hepatocytes (liver parenchymal cells-5 x 10(7)/kg), which exhibited a 4.8% class II antigen expression, and which alone failed to prolong allograft survival (MST = 6.0 +/- 1.4 days), increased heart allograft survival to 25.3 +/- 2.3 and 27.2 +/- 1.9 days, respectively (p < 0.01). Hepatocyte purification using Dynabeads and Percoll reduced class II expression to 0.9% and increased allograft survival to 32.8 +/- 1.6 days (p < 0.01). In contrast, the effect of 5 x 10(8)/kg BN erythrocytes, exhibiting only 0.1% class II expression, was much less (23.8 +/- 1.9 days). Administration i.s. of BN splenocytes or nonparenchymal liver cells, demonstrated by flow cytometry to exhibit a 47.3 or 55.1% expression of class II antigen, respectively, failed to induce any significant increase in allograft survival (18.4 +/- 4.6 and 19.4 +/- 0.5 days, respectively). Survival of BN rat small bowel allografts was increased in Lewis (LEW; RTl1) rat recipients treated with RAPA, CsA, and unfractionated BN hepatocytes from 10.2 +/- 1.9 to 21.2 +/- 1.5 days. Pretreatment with i.s. BN hepatocytes, 14 days prior to harvesting, reduced WFu lymphocyte responses to allogeneic stimulation with BN or ACI spleen cells by 75 and 70%, respectively. Addition of 1 x 10(5) unpurified donor-specific BN or third-party Buffalo (BUF; RTl(b)) hepatocytes, but not supernatant, to the responder wells of MLCs resulted in a 61 and 40% suppression, respectively, of the WFu lymphocyte response induced by BN allogeneic stimulation. These findings suggest that while class I MHC expression has a significant role to play in exerting the immunosuppressive effects of hepatocytes, other influences more specific to liver may also prevail.

15 AU81, a prostacyclin analog, potentiates immunosuppression and mitigates renal injury due to cyclosporine.

15 AU81, a synthetic prostacyclin analog, inhibits human mixed lymphocyte culture responses in a dose-dependent manner at concentrations within a range [0.1 to 10.0 micrograms/ml] similar to that of cyclosporine, methylprednisolone, antilymphocyte serum, and prostaglandin E1 and E2 analogs. Using the median-effect analysis to dissect immunosuppressive interactions, 15 AU81 and cyclosporine were shown to produce synergistic effects on human peripheral blood lymphocyte activation upon phytohemagglutinin or anti-CD3 monoclonal antibody stimulation. In vivo, 15 AU81 potentiated the effect of cyclosporine to prolong the survival of rabbit renal allografts using concentrations of each agent that were individually ineffective. Furthermore, addition of 15 AU81 mitigated functional damage of rabbit kidneys due to high-dose cyclosporine therapy. These observations suggest that the addition of 15 AU81 may broaden the therapeutic window of cyclosporine by potentiating immunosuppression and mitigating nephrotoxic effects.

Variable rejection patterns of cultured keratinocyte allografts in the rabbit.

There are many conflicting reports on the survival of cultured keratinocyte allografts (CKAs). Studies were performed in the rabbit model to elucidate further the fate of CKAs. Of 24 CKAs, 7 were identified as technical failures, 13 displayed classical macroscopic evidence of rejection with a prolonged mean survival time of 2.8 days, compared to non-cultured allograft controls (p < 0.01), and 4 failed to display typical macroscopic evidence of rejection. Furthermore, the time to eventual wound healing of the classically rejected CKAs were delayed by 6.0 days (p < 0.0001), compared to identical non-grafted control wounds.

T suppressor cells induced by transfusions and cyclosporine. Studies in the murine cardiac allograft model.

Pregraft transfusion combined with immunosuppression at the time of grafting improves the survival of clinical and experimental allografts. The mechanisms responsible for this effect were investigated in the murine model of cardiac transplantation, combining transfusions 7 to 30 days prior to transplantation with cyclosporine 100 mg/kg, 7 to 20 days pregraft or on days 0, 4, and 6 after grafting. Pregraft DST, third-party blood, and CsA all improved graft survival in the BALB/c-to-CBA donor-recipient combination. In animals treated with DST at 14 days pregrafting, 4/9 grafts survived for greater than 100 days. In those given C57BL/6 blood, or CsA on days 0, 4, 6 postgraft, 1/9 grafts survived for greater than 100 days. When 10(7) spleen cells from DST-treated CBA mice with long-surviving BALB/c heart grafts were transferred to naive CBA mice that then received a BALB/c heart 24 hr later, the transferred cells prolonged graft survival, with all grafts functioning at greater than 40 days, and 4/7 at greater than 100 days. Selective removal of T cells from the spleen cell population prior to transfer showed that L3T4+ T cells, but not Ly-2+ T cells, were required to maintain BALB/c allografts. Combining a short course of CsA with DST was more effective than either treatment alone. The most effective combined treatment was DST at day -14 with 100 mg/kg CsA given on days 0, 4, and 6 postgrafting (8/10 grafts survived greater than 100 days). This treatment also induced splenic suppressor T cells of the L3T4+ Ly-2- phenotype. These results clearly show that L3T4+ splenic T suppressor cells are induced by donor-specific blood transfusion with or without CsA treatment, and that these cells play a role in maintaining long-term tolerance to allografts in the mouse heart transplant model.

The immunosuppressive properties of new oral prostaglandin E1 analogs.

Prostaglandins play an important role in cell-mediated immune responses. Their clinical use has been limited by poor oral bioavailability, short half-lives, and significant toxicity profiles. We studied the immunosuppressive properties of new, synthetic, prostaglandin E1 (PGE1) methyl ester analogs (misoprostol, enisoprost) with oral bioavailability using an allogeneic in vitro immunoassay. Our results show that the PGE1 analogs suppress alloproliferative responses and supplement the immunosuppressive activity of cyclosporine and methylprednisolone. Moreover, we demonstrate that addition of recombinant interleukin-2 to the PGE1 analogs restores alloimmune responsiveness and the expression of surface class II antigen and IL-2 receptors on responder lymphocytes. These studies, together with preliminary in vivo data in rodents and man, suggest that the new synthetic oral PGE1 analogs may provide therapeutic efficacy in clinical transplantation and a variety of immunologically mediated diseases.