Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis

We recently demonstrated that the engagement of HLA class I alpha 1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha 1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.