Role of caspases 3 (CPP-32) and 8 (FLICE) as apoptotic signal transducers as exemplified by spontaneous tolerance after orthotopic liver transplantation (OLT)

Background: In some mouse and rat strains, the transplantation of allogenei c liver grafts into fully MHC-mismatched recipients induces spontaneous tol erance. One essential factor related to tolerance involves the elimination of intra-graft alloreactive lymphocytes after linkage to Fas-ligand express ed on hepatocytes of tolerized liver organs. This study served to character ize intracellular Fas-related signal pathways via caspases 8 (FLICE) and 3 (CPP-32) in more detail. Methods: Arterialized orthotopic liver transplantation (OLT) was performed between male inbred rat strains as follows (n=10): (i) LEW[RT1.(1)] --> LEW ; (ii) DA[RT1.(av1)] --> LEW; (iii) LEW --> LEW. Liver allografts were harv ested electively on postoperative days (POD) 3, 5, 7, 9, 11, and 21 for imm unohistochemical and mRNA (northern-blot) evaluation of Fas, Fas-L, caspase 8, and caspase 3. Simultaneously, cytospins taken from peripheral blood we re prepared and the frequency of TUNEL+ and proliferating (KiS3R(+)) cells was determined. Results: While in the DA --> LEW strain combination recipient rats died aft er a mean of : 1.2 +/- 1.0 days, DA rats carrying a LEW liver graft survive d long-term, > 100 days. Tolerance was associated with the expression of Fa s-L on hepatocytes of tolerized grafts. Simultaneously, protein expression of Fas and its associated caspase 8 was downregulated in these organs. Conv ersely, programmed cell. death of intra-graft alloreactive lymphocytes indu ced the mRNA transcription of caspase 3. Conclusions: Our results underline the importance of a Fas-induced up-regul ation of caspase 8 during the process of programmed cell death transmitted via binding of alloreactive lymphocytes in conjunction with acute rejection after allogeneic OLT. Conversely, spontaneously tolerized liver grafts bea r the possibility to suppress the Fas-induced activation of downstream loca lized FLICE and subsequently circumvent programmed cell death after binding of Fas-L+ lymphocytes. Noteworthy, a strict correlation between activation of caspase 8 and downstream activation of CPP-32 could not be found.