Interleukin-1beta and inducible form of nitric oxide synthase expression in early syngeneic islet transplantation.

Islets are particularly vulnerable in the initial days after transplantation when cell death results in the loss of more than half of the transplanted islet tissue. To determine whether a non-specific inflammation at the grafted site mediated by the local expression of inflammatory cytokines could play a role on the initial damage to transplanted islets, we studied the expressions of interleukin-1beta (IL-1beta) and inducible form of nitric oxide synthase (iNOS) after syngeneic islet transplantation. Insulin-treated streptozotocin-diabetic Lewis rats were syngeneically transplanted with 500 islets. Grafts were harvested 1, 3, or 7 days after transplantation, and the expressions of IL-1beta and iNOS genes were determined by RT-PCR. IL-1beta and iNOS mRNAs were detected in islets immediately after isolation, and were upregulated after transplantation. IL-1beta mRNA was ninefold increased on day 1, was still sevenfold increased on day 3 after transplantation, and declined towards pretransplantation levels on day 7. iNOS mRNA showed a similar pattern of expression to that of IL-1beta: on days 1 and 3 after transplantation it was 14-and 4-fold higher respectively than in freshly isolated islets. In addition, IL-1beta and iNOS were identified in islet grafts and found to be produced mainly by CD68-positive macrophages. A low number of IL-1beta- and iNOS-positive but CD68-negative cells were also identified suggesting that other cell types, in addition to macrophages, were involved in the expression of IL-1beta and NO production in islet grafts. The finding of increased IL-1beta and iNOS gene expressions in the initial days after islet transplantation and the presence of IL-beta and iNOS proteins in the graft confirmed the presence of an early non-specific inflammatory response after islet transplantation. Overall, the data suggest that IL-1beta plays a role in the extensive beta-cell death found in the initial days after islet transplantation.

Role of blood glucose in cytokine gene expression in early syngeneic islet transplantation.

In islet transplantation, local production of cytokines at the grafted site may contribute to the initial nonspecific inflammation response. We have determined whether the metabolic condition of the recipient modulates the cytokine expression in islet grafts in the initial days after transplantation. Normoglycemic and hyperglycemic streptozotocin-diabetic Lewis rats were transplanted with 500 syngeneic islets, an insufficient beta cell mass to restore normoglycemia in hyperglycemic recipients. The expression of IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-10, and IL-4 genes was determined by real-time PCR in freshly isolated islets, in 24-h cultured islets and in islet grafts on days 1, 3, and 7 after transplantation. IL-1beta mRNA was strongly and similarly increased in normoglycemic and hyperglycemic groups on days 1, 3, and 7 after transplantation compared with freshly isolated and cultured islets. TNF-alpha mRNA was also strongly increased on day 1, and it remained increased on days 3 and 7. IL-6 and IL-10 were not detected in freshly isolated islets, but their expression was clearly enhanced in 24-h cultured islets and islet grafts. IL-6 was further increased in hyperglycemic grafts. IL-10 expression was increased in both normoglycemic and hyperglycemic grafts on day 1 after transplantation, and remained increased in hyperglycemic grafts compared to 24-h cultured islets. IFN-gamma mRNA was barely detected in a few grafts, and IL-4 mRNA was never detected. Thus, the inflammatory response in islet grafts was maximal on day 1 after transplantation, it was sustained, although at lower levels, on days 3 and 7, and it was partly enhanced by hyperglycemia.

Beta-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia.

We studied the effects of hyperglycemia on beta-cell death and mass in syngeneically transplanted islets. Six groups of STZ-induced diabetic C57BL/6 mice were transplanted with 100 syngeneic islets, an insufficient beta-cell mass to restore normoglycemia. Groups 1, 2, and 3 remained hyperglycemic throughout the study. Groups 4, 5, and 6 were treated with insulin from day 7 before transplantation to day 10 after transplantation. After insulin discontinuation, group 6 mice achieved definitive normoglycemia. Grafts were harvested at 3 (groups 1 and 4), 10 (groups 2 and 5), and 30 (groups 3 and 6) days after transplantation. On day 3, the initially transplanted beta-cell mass (0.13 +/- 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulin-treated groups (group 1: 0.048 +/- 0.002 mg; group 4: 0.046 +/- 0.007 mg; P < 0.001). Extensive islet necrosis (group 1: 30.7%; group 4: 26.8%) and increased beta-cell apoptosis (group 1: 0.30 +/- 0.05%; group 4: 0.42 +/- 0.07%) were found. On day 10, apoptosis remained increased in both hyperglycemic and insulin-treated mice (group 2: 0.44 +/- 0.09%; group 5: 0.48 +/- 0.08%) compared with normal pancreas (0.04 +/- 0.03%; P < 0.001). In contrast, on day 30, beta-cell apoptosis was increased in grafts exposed to sustained hyperglycemia (group 3: 0.37 +/- 0.03%) but not in normoglycemic mice (group 6: 0.12 +/- 0.02%); beta-cell mass was selectively reduced in islets exposed to hyperglycemia (group 3: 0.046 +/- 0.02 mg; group 6: 0.102 +/- 0.009 mg; P < 0.01). In summary, even in optimal conditions, approximately 60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to beta-cell death. Increased apoptosis and reduced beta-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted beta-cells.

Short-term culture with the caspase inhibitor z-VAD.fmk reduces beta cell apoptosis in transplanted islets and improves the metabolic outcome of the graft.

In the initial days after transplantation islets are particularly vulnerable and show increased apoptosis and necrosis. We have studied the effects of caspase inhibition on this early beta cell death in syngeneically transplanted islets. Streptozotocin-diabetic C57BL/6 mice were transplanted with 150 syngeneic islets, an insufficient mass to restore normoglycemia, preincubated with or without the pan-caspase inhibitor z-VAD. fmk 2 h before transplantation. Beta cell apoptosis was increased in control islets on day 3 after transplantation (0.28 +/- 0.02%) compared with freshly isolated islets (0.08 +/- 0.02%, p < 0.001), and was partially reduced in transplanted islets preincubated with z-VAD.fmk 200 microM (0.14 +/- 0.02%, p = 0.003) or with z-VAD.fmk 500 microM (0.17 +/- 0.01%, p = 0.012), but not with a lower z-VAD.fmk (100 microM) concentration. Diabetic mice transplanted with islets preincubated with z-VAD.fmk 500 microM showed an improved metabolic evolution compared with control and z-VAD.fmk 200 microM groups. The z-VAD.fmk 500 microM group showed an overall lower blood glucose after transplantation (p = 0.02), and at the end of the study blood glucose values were reduced compared with transplantation day (15.7 +/- 3.6 vs. 32.5 +/- 0.5 mmol/L, p = 0.001). In contrast, blood glucose was not significantly changed in control and z-VAD.fmk 200 microM groups. Four weeks after transplantation beta cell mass was higher in z-VAD.fmk 500 microM group (0.15 +/- 0.02 mg) than in the control group (0.10 +/- 0.02 mg) (p = 0.043). In summary, the treatment of freshly isolated islets with the caspase inhibitor z-VAD.fmk reduced the subsequent apoptosis of the islets once they were transplanted and improved the outcome of the graft.