ACTIVATION OF THE CASPASE CASCADE IS IMPORTANT IN LIVER ALLOGRAFT REJECTION

Abstract

499 Recent studies suggest that apoptosis is an important mechanism of cell death in acute liver allograft rejection. Moreover, molecular and immunohistochemical experiments demonstrate roles for both the Fas/FasL and granule-exocytosis pathways in the induction of apoptosis during graft rejection. The purpose of this study was to determine if apoptosis during graft rejection is dependent on activation of the caspase cascade. Caspases are expressed as inactive precursors which are proteolytically activated during apoptosis. Groups of Lewis recipients received orthotopic liver grafts from either syngeneic (Lewis) or allogeneic (DA) donors. We have determined that on day 7 post-transplant, allograft rejection is severe and that there is a significant increase of apoptosis in allografts, as determined by both TUNEL staining and in vivo imaging with 99mTc-annexin-V. RNA was isolated from liver grafts on day 8 and analyzed by quantitative ribonuclease protection assay (RPA) for caspases 1 (ICE), 2 and 8 (FLICE). As shown in the Table, the caspases were all upregulated in the allogeneic combination. Since the anti-apoptotic gene bcl-2 can prevent processing and activation of caspases, we determined expression of bcl-2 and the pro-apoptotic gene bax in graft tissue. Similar levels of bcl-2 were observed in all groups, however there was a marked increase in bax expression in the allogeneic groups.TableTo determine if CD8+ CTL are responsible for providing the death signal leading to caspase activation and apoptosis, CD8+ cells in recipient rats were depleted by administration of the OX-8 monoclonal antibody. Although there was a trend, there was no significant difference in caspase expression between the allogeneic and CD8 depleted allogeneic groups (Table). Importantly, severe graft rejection was observed on day 8 in H&E stained tissue sections in both groups of animals. Although CD8 depletion did not prevent graft rejection or apoptosis, the initiating death signals may differ. By competitive RT-PCR, we demonstrated a 100-fold increase in granzyme B transcripts in allogeneic graft liver relative to syngeneic grafts. However, there was a marked decrease (10-fold) of granzyme B expression in CD8 depleted graft liver when compared to the allogeneic groups. These data suggest that activation of the caspase cascade, perhaps through multiple pathways, is important in apoptosis and rejection after liver transplantation. Modulation of the expression or proteolytic processing of caspases may prevent apoptosis and promote graft survival.