Caspase 8 activity in membrane blebs after anti-Fas ligation

Previous studies of thymocyte apoptosis using a series of cell-permeable fl uorogenic peptide substrates showed that Fas crosslinking triggered a caspa se cascade in which cleavage of the IETDase (caspase 8-selective) substrate was the earliest caspase activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when apoptosis was i nduced by anti-Fas in CTL and the caspase cascade examined by this approach , IETDase activation followed increases in LEHDase, YVHDase, and VEIDase ac tivities (selective for caspases 9, 1, and 6, respectively). When examined by confocal microscopy, anti-Fas-treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface, followed by activation of other caspase activities in the cell in terior. Since these vesicles were not included in the flow cytometry analys is, the early IETDase activity had been underestimated. In contrast to anti -Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in ea rly IETDase activity in the cytoplasm, with no plasma membrane vesiculation . Thus, anti-Fas-induced initiation of caspase activity at the plasma membr ane may In some cells result in local proteolysis of submembrane proteins, leading to generation of membrane vesicles that are highly enriched in acti ve caspase 8.