Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-x(L)

Prolonged (>24 h) exposure to anti-IgM tan antigen surrogate that induces membrane cross linking and apoptosis) induced a 3-fold increase in the mass of endogenous ceramide measured by P-32 labeling by diacylglycerol kinase and a 4-fold increase in ceramide as measured by metabolic labeling with [H-3]palmitate in a B-lymphocyte cell line, WEHI 231. This correlated with the induction of apoptosis. Shorter exposure times to anti-IgM (up to 8 h) failed to elicit apoptosis and did not elicit increased ceramide formation, After 8 h, apoptosis occurs concomitantly with ceramide formation over the next 10 h. Further, we showed that exogenous ceramide mimicked anti-IgM-induced apoptosis and that apoptosis was potentiated in serum-free media. Treatment of cells with an inhibitor of ceramide catabolism, N-oleoylethanolamine, increased both ceramide formation and apoptosis and accelerated apoptosis induced by anti-IgM. To examine further how ceramide metabolism is involved in apoptosis, we derived cell lines from a small population of cells resistant to N-oleoylethanolamine. These cell lines were selected based on an altered ceramide metabolic pathway, were resistant to apoptosis induced by anti-IgM, and showed no significant increase in ceramide when challenged with anti-IgM. The basis of this resistance was shown to be the failure to activate neutral sphingomyelinase activity following 24-h treatment with anti-IgM, in contrast to the 2-fold increase in neutral sphingomyelinase activity observed in wild type cells. We have shown previously that transfection of WEHI cells with bcl-x(L) conferred resistance to anti-IgM-induced apoptosis, whereas transfection with bcl-2 did not (Gottschalk, A., Boise, L., Thompson, C., and Quintans, J. (1994) Proc. Natl. Acad. Sci, U. S. A. 91, 7350-7354). In this study, these bcl-x(L) transfectants also displayed increased resistance to exogenous N-acetylsphingosine (C-2-ceramide) or N-hexanoylsphingosine (C-6-ceramide). However, when challenged with anti-IgM the bcl-x(L) transfectants produced levels of ceramide similar to wild type cells, suggesting that ceramide formation is upstream of bcl-x(L) and that it is a major determinant of B-cell death.