We studied the effects of hyperglycemia on ␤-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 ␤-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 ␤-cell mass (0.13 ؎ 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulintreated 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 ␤-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, ␤-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%); ␤-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, ϳ60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to ␤-cell death. Increased apoptosis and reduced ␤-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted ␤-cells.

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 nonspecific 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-1b (IL-1b) and inducible form of nitric oxide synthase (iNOS) after syngeneic islet transplantation. Insulintreated 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-1b and iNOS genes were determined by RT-PCR. IL-1b and iNOS mRNAs were detected in islets immediately after isolation, and were upregulated after transplantation. IL-1b 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-1b: on days 1 and 3 after transplantation it was 14-and 4-fold higher respectively than in freshly isolated islets. In addition, IL-1b and iNOS were identified in islet grafts and found to be produced mainly by CD68-positive macrophages. A low number of IL-1b-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-1b and NO production in islet grafts. The finding of increased IL-1b and iNOS gene expressions in the initial days after islet transplantation and the presence of IL-b 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-1b plays a role in the extensive b-cell death found in the initial days after islet transplantation.

The naturally occurring inhibitor of interleukin-1 (IL-1) action, interleukin-1 receptor antagonist protein (IRAP), binds to the type 1 IL-1 receptor but does not initiate IL-1 signal transduction. In this study, we have determined the effects of IL-1b and IRAP overexpression on adult b-cell replication and viability. IL-1b reduced dramatically b-cell replication in adult rat islets both at 5.5 mM (control: 0.2970.04%; IL-1b: 0.0270.02%, Po0.05) and 22.2 mM glucose (control: 0.8470.2%; IL-1b: 0.0570.05%, Po0.05). This effect was completely prevented in islets overexpressing IRAP after adenoviral gene transfer at 5.5 mM (Ad-IL-1Ra+IL-1b: 0.8470.1%, Po0.05) and 22.2 mM glucose (Ad-IL-1Ra+IL-1b: 1.2270.2%, Po0.05). Moreover, overexpression of IRAP increased glucose-stimulated b-cell replication in the absence of IL-1b exposure (Ad-IL-1Ra: 1.5970.5%, Po0.05). b-Cell death (TUNEL technique) was increased in IL-1b-exposed islets but not in Ad-IL-1Ra-infected islets (control: 0.8270.2%; control+IL-1b: 1.7770.2; IRAP: 0.6170.2%; IRAP+IL-1b: 0.8670.1%, Po0.05). Comparable results were obtained by flow cytometry. To determine the effect of IRAP overexpression on b-cell replication in vivo, Ad-IL-1Ra-transduced islets were transplanted into streptozotocin diabetic rats. b-Cell replication was significantly increased in IRAP-overexpressing islet grafts (0.9870.3%, Po0.05) compared to normal pancreas (0.3570.02%), but not in control islet grafts (0.5070.1%). This study shows that in addition to the effects of IL-1b on bcell viability, this cytokine exerts a deleterious action on b-cell replication, which can be prevented by IRAP overexpression, and provides support for the potential use of IRAP as a therapeutic tool. Gene Therapy (2005) 12, 120-128.