Activation-induced cell death of splenocytes requires ASMase for efficient killing. (A) Representative images and (B) quantification of ceramide-rich platforms (arrows) formed on the surface of C57BL/6asmase+/+ and C57BL/6asmase−/− C57BL/6 splenic T cells 4 hours after induction of AICD with 10 ng/mL anti-CD3 as described in “Activation-induced cell death.” Cells were fixed with 4% formalin-buffered phosphate and stained with DAPI and FITC-labeled anticeramide mAb as in Figure 4C. Images were acquired as in Figure 4C. AICD induces a 2.0- ± 0.1-fold increase in the overall ceramide signal as determined by mean fluorescence intensity in asmase+/+ T cells (P < .005 compared with unstimulated controls), not evident in asmase−/− T cells, accounting for the difference in overall staining between panels. (C) Apoptotic response of C57BL/6asmase+/+ or C57BL/6asmase−/− splenic T cells after AICD apoptotic fratricide was induced as in panel A. Apoptosis was quantified 16 hours thereafter after nuclear bisbenzimide staining. (D) AICD was initiated in C57BL/6asmase−/− splenic T cells as in panel A, in the presence of 500 nM C16-ceramide or C16-dihydroceramide. Apoptosis was quantified 16 hours thereafter after nuclear bisbenzimide staining. Data (mean ± SEM) represent triplicate samples from 3 independent experiments for panels B through D.
