Induction of apoptosis through B-cell receptor cross-linking occurs via denovo generated C16-ceramide and involves mitochondria

B-cells, triggered via their surface E-cell receptor (BcR), start an apopto tic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been prop osed to be involved in AICD, the contribution of either and the exact molec ular events through which AICD commences are still unknown. Here we show th at in Ramos B-cells, BcR-triggered cell death is associated with an early r ise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate, There was no evidence for activation of sphingomyelinases or hy drolysis of sphingomyelin. Importantly, FB1 inhibited several specific apop totic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragme ntation. Electron microscopy revealed morphological evidence of mitochondri al damage, suggesting the involvement of mitochondria in BcR-triggered apop tosis, and this was inhibited by FB1, Moreover; a loss of mitochondrial mem brane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val- Ala-DL-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentat ion, These results suggest a crucial role for de novo generated C16 ceramid e in the execution of AICD, and, they further suggest an ordered and more s pecific sequence of biochemical events in which de novo generated C16 ceram ide is involved in mitochondrial damage resulting in a downstream activatio n of caspases and apoptosis.