SummaryIntraceilular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed ceil surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monodonal antibody was produced by immunizing mice with human CD95-transfected L ceils. Crosslinkiug of CD95 with DX2 resulted in the activation of a sphingomydinase (SMase) in promydocytic U937 ceils, as well as in other human tumor ceil lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomydin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labded SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hy&olysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to ceil-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be rdevant in defining the biochemical nature of intraceilular messengers leading to apoptotic cell death.

The early signals generated following cross‐linking of Fas/APO‐1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO‐1 cross‐linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine‐specific phospholipase C (PC‐PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC‐PLC inhibitor D609 blocked Fas/APO‐1‐induced aSMase activation, but not Fas/APO‐1‐induced neutral sphingomyelinase (nSMase) activation. Fas/APO‐1 cross‐linking resulted also in ERK‐2 activation and in phospholipase A2 (PLA2) induction, independently of the PC‐PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain‐defective Fas/APO‐1 splice isoform which blocks Fas/APO‐1 apoptotic signalling in a dominant negative fashion. Fas/APO‐1 cross‐linking in these clones fails to activate PC‐PLC and aSMase, while nSMase, ERK‐2 and PLA2 activates are induced. These results strongly suggest that a PC‐PLC/aSMase pathway contributes directly to the propagation of Fas/APO‐1‐generated apoptotic signal in lymphoid cells.

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The expression and function of Fas (CD95 ր APO-1), a cell surface receptor directly responsible for triggering cell death by apoptosis, was investigated on human T lymphocytes resident within the intestinal lamina propria, a major site of antigen challenge and persistent lymphocyte activa-

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