Prolonged (>24 h) exposure to anti-IgM (an antigen surrogate that induces membrane cross-linking and apoptosis) induced a 3-fold increase in the mass of endogenous ceramide measured by 32 P labeling by diacylglycerol kinase and a 4-fold increase in ceramide as measured by metabolic labeling with [ 3 H]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 40 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, Noleoylethanolamine, 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-IgMinduced 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.Apoptosis (1) can be induced readily in lymphocytes by means of irradiation, corticosteroids, or cross-linking of their antigen receptors. The apoptotic response of the murine Blymphoma WEHI 231 cell line to the cross-linking of surface IgM receptors provides a model to study the induction of physiological cell death (apoptosis) in lymphocytes (2-4). In WEHI 231, the response is specifically triggered via surface IgM since cross-linking other surface proteins such as IgD, Fc receptor, or major histocompatibility complex class II have no effect (5). Apoptosis is reversed by both phorbol esters and by bacterial lipopolysaccharide (6). Apoptosis can also be reversed by the removal of the antigen prior to 12 h of exposure, emphasizing its slow acting nature compared with necrosis and some types of apoptosis. We showed recently that the time course and ...

Cylosporin A, and Cyclosporin A (CsA), FK-506 (FK), and rapamycin (Rap) are naturally occurring antifungal agents known to be powerful immunosuppressants (1). These immunosuppressants bind to immunophilins, a family of proteins that have peptidylprolyl cis-trans-isomerase activity (1-3). The effects of these drugs are not due to their inhibition of isomerase activity but to the interaction of immunosuppressant-immunophilin complexes with specific effector molecules (4-12). CsA binds the immunophilin cyclophilin and inhibits the calcium/calmodulindependent protein phosphatase calcineurin (5, 6). Both FK and Rap interact with the immunophilin FK binding protein (FKBP) (4, 7). The complex of FK and FKBP also binds calcineurin (4-6), but the Rap-FKBP complex inhibits the activity of the p70s6 k (4, 8-10), p34cdc2 (11,12), and p33cdk2 (12).Although CsA, FK, and Rap have been extensively used to study T-cell activation, little is known about the effects of these immunosuppressants on B cells (1). In T cells, CsA and FK inhibit antigen-dependent signaling events required for interleukin 2 gene transcription (1, 4, 7), and Rap blocks biochemical events necessary for interleukin 2-dependent progression from G1 to S phase in the cell cycle (4, 7). In addition, CsA and FK, but not Rap, were able to prevent physiological cell death (PCD) in thymocytes and T-cell hybridomas induced by either anti-T-cell receptor antibodies or the combination of phorbol 12-myristate 13-acetate and ionomycin (4,(13)(14)(15)(16) Viability Assay. Cells (2 x 105) were incubated in 24-well plates and treated with CsA (1 pug/ml), FK (10 Mg/ml), Rap (10 pg/ml), or an equal volume of EtOH as the vehicle (t = 0). After the indicated amount of time, the cells were collected and resuspended in PBS containing 1% (wt/vol) bovine serum albumin and 0.01% sodium azide. Propidium iodide was added immediately prior to analysis on a FACScan (Becton Dickinson) using LYSIS II software. Cells excluding propidium iodide were scored as viable.Cell Cycle Analysis. Cells (2 x 105) were incubated in 24-well plates and treated with CsA (1 pg/ml), FK (10 pg/ml), Rap (10 pg/ml), or an equal volume of EtOH as the vehicle. After 24 hr, the cells were collected and resuspended in solution containing propidium iodide (50 ug/ml), 0.3% sodium citrate, and 0.01% Triton X-100; then incubated at Abbreviations: PCD, physiological cell death; CsA, cyclosporin A; FK, FK-506; FKBP, FK binding protein; Rap, rapamycin.tTo whom reprint requests should be addressed. 7350The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Biological activities of monoclonal A/J antibodies to the T15 idiotype in BALB/c mice were compared to heterogeneous antibodies raised by conventional immunization procedures. Two monoclonal antibodies, AB1-2 and GB4-10, which are of the gamma 1, chi class, appeared to have identical specificities by binding criteria and reacted similarly to conventional antibodies in their abilities to induce neonatal suppression, inhibit plaque-forming cell induction by phosphorylcholine (PC) antigens and to inhibit specifically, anti-PC plaque-forming cells. However, in functional analyses of anti-PC responses in various strains of mice, discrepancies were noted in the T15 responses as defined by monoclonal antibodies and conventional antisera. This heterogeneity was also observed in adult mice suppressed with the GB4-10 monoclonal antibody. These animals eventually produced an anti-PC responses of AB1-2 idiotype but lacking the GB4-10 marker. These results show that the T15 IgM anti-PC response in BALB/c and other strains of mice is heterogeneous and probably consists of a family of clones. Particular clones can be precisely eliminated by the use of appropriate monoclonal antibodies, and the anti-PC response that eventually recovers is still T15+ but lacking the suppressed clones.

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