Nishihara M

Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts

Summary Fas ligand (FasL) is highly expressed in testicular tissues and thought to be responsible for protection from allograft rejection by inducing apoptosis of anti-graft activated T cells. FasL-expressing islets have been shown to induce a granulocyte-mediated inflammatory reaction. We investigated whether a graft can be protected from alloimmune responses by manipulating the Fas/FasL-system. We transplanted allogeneic islets under the kidney capsule of streptozotocin-induced diabetic mice together with testicular tissue. Significant prolongation of survival of C3H islet allograft was observed in C57BL/6 (B6) recipients transplanted with C3H testicular tissue, but not in those transplanted with C3H-gld testicular tissue expressing non-functional FasL. No significant prolongation was observed in B6-lpr recipients expressing non-functional Fas. Immunohistochemical staining of C3H testicular tissue in the composite graft showed a high expression of FasL, but not that of the C3H-gld testicular tissue. In situ terminal deoxynucleotidyl transferase-mediated dUDP-biotin catalysed DNA nick-end labelling (TUNEL) staining of a composite graft of C3H islet and testicular tissue in B6 recipients demonstrated extensive apoptosis of infiltrating mononuclear cells around the graft. The protective effect of C3H testicular tissue was abrogated when anti-FasL monoclonal antibody was administered i. p. postoperatively. Our results suggest that FasL-positive testicular allografts protect composite islet allografts and indicate that manipulation of Fas/FasL mediated apoptosis is a suitable strategy for controlling rejection of islet allografts. [Diabetologia (1998) 41: 315–321]

Pretreatment of crude pancreatic islets with mitomycin C prolongs graft survival time in xenogeneic rat-to-mouse model.

BACKGROUND Rejection of pancreatic islet grafts is still a serious problem. We evaluated the effect of mitomycin C (MMC) on the survival of crude islets grafts after xenogeneic islet transplantation. METHODS WS (RT1k) rat islets pretreated with various concentrations of MMC (0, 1, 3.2, 10, 32, 50, 100, 320, and 1,000 microg/ml) were transplanted into C57BL/6 mice with streptozotocin-induced diabetes. In vivo graft function was assessed by a daily measurement of nonfasting blood glucose concentration in each animal. We also examined the separate effect of MMC on purified islets and contaminants present in the crude islet preparation. RESULTS MMC at doses of 10, 32, 50, and 100 microg/ml resulted in a significant prolongation of the mean graft survival time from a control of 12.4+/-2.5 days to 23+/-7.4, 17.5+/-5.4, 25.5+/-14.7, and 26.7+/-8.9 days, respectively. Deterioration of glucose metabolism was noted when the dose exceeded 32 microg/ml, whereas at 320 microg/ ml, MMC failed to restore normoglycemia. Prolongation of survival time of crude islets was the result of its effect on islets and contaminant components of the crude islet preparation. In vitro study showed that MMC treatment at a higher concentration than 10 microg/ml reduces the stimulatory as well as proliferative capacity of lymph node cells. CONCLUSIONS Pretreatment of pancreatic islets with MMC at 10 microg/ml prolongs xenograft survival without deterioration of in vivo graft function. This novel treatment modality represents a new strategy for the modulation of immunity of islets and contaminants in crude islet preparations.

Direct antigen presentation through binding of donor intercellular adhesion molecule-1 to recipient lymphocyte function-associated antigen-1 molecules in xenograft rejection.

Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P<0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.

Pretreatment of crude pancreatic islets with mitomycin C (MMC) prolongs islet graft survival in a xenogeneic rat-to-mouse model.

In this study, we examined the effect of mitomycin C (MMC) treatment on graft survival and evaluated its efficacy in immunomodulation of islet graft for transplantation. Male WS rats were used as islet donors and streptozotocin-induced diabetic C57BL/6 mice as recipients. The isolated islets were treated with MMC at concentrations of 0, 0.1, 1, 3.2, 10, 32, 100, 320, and 1000 microg/mL for 30 min, and were cultured for 20 h. Then, 300-400 islets were transplanted into the renal subcapsular space of diabetic mice. Significant prolongation of graft survival was obtained when the islets were treated with MMC at a concentration of 10, 32, or 100 microg/mL (MST 23 +/- 7.4, 17.5 +/- 5.4, 29.6 +/- 9.7 days: p < 0.003, p < 0.012, p < 0.001, respectively, vs. 12.3 +/- 2.7 days for culturing alone). Islets treated with MMC at a concentration of 320 microg/mL or more failed to restore normoglycemia in the diabetic recipient mice after transplantation. Viability of islets incubated with doses up to 100 microg/mL, assessed under the confocal microscope after propidium iodide and Hoechst 33342 staining, was maintained well comparable to that of freshly isolated islets, while those treated at 320 microg/mL was significantly decreased. Thus, a therapeutic window for MMC efficacy was found at concentrations from 10 microg/mL to 100 microg/mL. This modality is simple and effective and underlying molecular mechanisms need to be determined in the future.