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 indu ces membrane cross linking and apoptosis) induced a 3-fold increase in the mass of endogenous ceramide measured by P-32 labeling by diacylgl ycerol kinase and a 4-fold increase in ceramide as measured by metabol ic labeling with [H-3]palmitate in a B-lymphocyte cell line, WEHI 231. This correlated with the induction of apoptosis. Shorter exposure tim es to anti-IgM (up to 8 h) failed to elicit apoptosis and did not elic it increased ceramide formation, After 8 h, apoptosis occurs concomita ntly with ceramide formation over the next 10 h. Further, we showed th at exogenous ceramide mimicked anti-IgM-induced apoptosis and that apo ptosis was potentiated in serum-free media. Treatment of cells with an inhibitor of ceramide catabolism, N-oleoylethanolamine, increased bot h ceramide formation and apoptosis and accelerated apoptosis induced b y anti-IgM. To examine further how ceramide metabolism is involved in apoptosis, we derived cell lines from a small population of cells resi stant to N-oleoylethanolamine. These cell lines were selected based on an altered ceramide metabolic pathway, were resistant to apoptosis in duced by anti-IgM, and showed no significant increase in ceramide when challenged with anti-IgM. The basis of this resistance was shown to b e the failure to activate neutral sphingomyelinase activity following 24-h treatment with anti-IgM, in contrast to the 2-fold increase in ne utral sphingomyelinase activity observed in wild type cells. We have s hown previously that transfection of WEHI cells with bcl-x(L) conferre d 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 stud y, 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) t ransfectants produced levels of ceramide similar to wild type cells, s uggesting that ceramide formation is upstream of bcl-x(L) and that it is a major determinant of B-cell death.