Sadamori, Hiroshi

Controversy exists over whether the predominant cell death of hepatocytes is due to apoptosis or necrosis after ischemia/reperfusion injury. In this study we investigated the predominant cell death of hepatocytes after cold ischemia/reperfusion injury using the Annexin V-based assay, and evaluated the anti-apoptotic effect of ascorbic acid 2-glucoside (AA-2G) added to the University of Wisconsin solution (UW solution) in rat liver transplantation. The retrieved liver was preserved in 4 UW solution for 24 h, and then transplanted orthotopically to the syngeneic Wistar recipient. The animals were divided into 2 groups, a control group (n=10), in which liver grafts were preserved in UW solution (4), and an AA-2G group (n=10), in which liver grafts were preserved in UW solution (4) with AA-2G (100 ug/ml). The serum AST level 4 h after reperfusion in the control group was significantly suppressed in the AA-2G group, and the bile production of the liver graft in the AA-2G group was well recovered. The mean survival time in the AA-2G group was significantly improved compared with that in the control group. Annexin-V and Propidium iodide staining 4 h after reperfusion showed a significantly higher percentage of viable hepatocytes in the AA-2G group compared with the control group (93.4 +/- 2.0 vs. 80.3 +- 2.1%, P<0.05). In the control group, the main cell death of hepatocytes was apoptosis (early apoptosis: 10.0 +- 4.7%, late apoptosis: 6.4 +/- 1.7%). The addition of AA-2G to the UW solution significantly inhibited both early and late apoptotic cell death 4 h after reperfusion (early apoptosis: 0.98 +/- 0.88%, late apoptosis: 2.2 +/- 1.1%). The expression of caspase 9 in the immunostaining of the liver graft was suppressed in the AA-2G group compared with in the control group. Our study using the Annexin V-based assay provided evidence that the predominant cell death of hepatocytes was apoptosis after 24 h cold ischemia/reperfusion injury in rat liver transplantation. The addition of AA-2G to the UW solution attenuated 24 h cold ischemia/reperfusion injury by inhibiting the apoptosis of hepatocytes.

Tacrolimus (FK-506) and cyclosporin A (CsA) are calcineurin antagonists used widely as T-cell immunosuppressants; however, their relative efficacy on the production of interleukin-18 (IL-18) remains undefined. We have examined the effects of FK-506 and CsA on the cytokine generation of human peripheral blood mononuclear cells (PBMCs) in mixed lymphocyte reaction (MLR) with lipopolysaccharide (LPS). We studied the levels of interleukin-18 (IL-18), IL-12, IL-10, IL-6, IL-2 and interferon-gamma (IFN-gamma) in the supernatant in allo-MLR by ELISA assay. Supernatant levels of IFN-gamma, IL-2, IL-6, IL-10 and IL-12 were detected 12 h after MLR and markedly increased thereafter. In contrast, production of IL-18 was detected at 12 h, reached a near maximum level at 24 h and decreased at 72 h. These results suggested that IFN-gamma production depended on IL-18, IL-12 and IL-2 in the early phase of MLR and depended mainly on IL-12 and IL-2 in the late phase. Both calcineurin antagonists inhibit the generation of IL-18, which plays a large role in allogeneic cell interactions, in macrophages and they also promote an equivalent down-regulation of T helper 1 (Th1) and Th2 responses in a concentration-dependent manner. About 90% of IFN-gamma production induced by MLR was inhibited by an anti-IL-18 antibody, showing that IL-18 can trigger IFN-gamma production in MLR. These results suggest that dual signaling consisting of antigen-driven nuclear factor of activated T cells (NFAT) activation and LPS-mediated NF-kappaB activation is crucial for IL-18 production in macrophages, and that IL-18 can trigger IFN-gamma production in T-cells by MLR.