CHRONIC LIVER ALLOGRAFT REJECTION IS NOT ASSOCIATED WITH CASPASE ACTIVATION

Abstract

975 Previous studies have demonstrated correlations between acute liver allograft rejection and apoptosis of hepatocytes and bile duct cells. Furthermore, Fas ligand, perforin and the granzymes are upregulated during acute allograft rejection suggesting both the cytotoxic granule-exocytosis and the Fas-mediated pathways may be involved in the induction of apoptotic cell death. In recent studies, we have determined that apoptosis during acute liver allograft rejection involves activation of the caspase cascade. Taken together, these studies suggest a prominent role for apoptosis in the tissue damage that occurs during acute liver allograft rejection. To date, however, there have been few studies which directly examine apoptosis during chronic liver allograft rejection. To determine if caspase-dependent apoptosis occurs during chronic liver allograft rejection, RNA was isolated from liver tissue of patients with a diagnosis of chronic liver allograft rejection (n=5) and from liver tissue classified as normal (n=4), and analyzed by a specific and quantitative ribonuclease protection assay (RPA). Our data suggest that there are similar levels of the caspases 1 (ICE), 2, 5, 6, 7 and 9 in both chronic rejection and normal liver tissue. Interestingly, there is a marked decrease in the expression of both caspase 8 (FLICE) and caspase 3 (CPP-32) during chronic liver allograft rejection. Fas trimerization recruits the death-domain containing protein FADD, which in turn recruits caspase 8 and thus activates the cascade of caspases leading to apoptosis. Decreased expression of caspase 8 in chronic rejection may indicate that the Fas-associated death-inducing signaling complex is not active in this tissue. Caspase 3 can be directly activated by granzyme B and has been shown to induce the proteolysis of death substrates. Both RT-PCR and RPA analyses indicate similar expression of granzyme B in chronic rejection and normal liver. TUNEL staining for apoptotic cells of sections from acute and chronic liver allograft rejection and normal liver demonstrated abundant numbers of apoptotic hepatocytes (3-7 per high power field (HPF)) during acute rejection with only minimal numbers of apoptotic hepatocytes detected during chronic rejection (0-1/HPF) and in normal liver (0-1/HPF). In contrast, TUNEL staining of the endothelium, in both large and small vessels, was specifically detected in the chronic rejection specimens. Therefore, while the caspase cascade is prominent in acute rejection, our data suggest that apoptosis of endothelium, in chronic rejection is independent of caspase expression.