De novo C16- and C24-ceramide generation contributes to spontaneous neutrophil apoptosis

Nentrophils rapidly undergo spontaneous apoptosis following their release from the bone marrow. Although central to leukocyte homeostasis, the mechanisms that regulate neutrophil apoptosis remain poorly understood. We show here that apoptosis of cultured neutrophils is preceded by a substantial increase in the intracellular levels of 16 and 24 carbon atom (C-16- and C-24)-ceramides, which are lipid second messengers of apoptosis and stress signaling. Treatment of neutrophils with fumonism B-2, a selective inhibitor of the de novo pathway of ceramide synthesis, prevented accumulation of C-16- and C-24-ceramides. Moreover, fumonisin B2 significantly reduced caspase-3, -8, and -9 activation and apoptosis in these cells. Conversely, 3-O-methylsphingomyelin and fantofarone, which are specific inhibitors of neutral and acid sphingomyelinases, respectively, neither inhibited C-16- and C-24-ceramide production nor decreased the apoptosis rate in nentrophils, indicating that in these cells, ceramides are not generated from membrane sphingomyclin. Further experiments showed that increasing endogenous C-16- and C-24-ceramide levels by using DL-threo-l-phenyl-2-palmitoylaniino-3-inorpholino-l-propanol and (1S,2R)-D-erythro-2-(N-myristoylamino)-1-ph,enyl-l-propanol, two inhibitors of ceramide metabolism, enhances caspase-3, -8, and -9 activity and increases neutrophil apoptosis. Similarly, apoptosis was induced rapidly when synthetic C-16- and/or C-24-ceramides were added to nentrophil cultures. Finally, GM-CSF, a cytokine that delays nentrophil apoptosis, abrogated C-16- and C-24-ceramide accumulation totally in cultured nentrophils, whereas Fas ligation accelerated apoptosis in these cells without affecting de novo ceramide production. We conclude that de novo generation Of C16- and C24-ceramides contributes to spontaneous nentrophil. apoptosis via caspase activation and that GM-CSF exerts its antiapoptotic effects on neutrophils, at least partly through inhibition of ceramide accumulation.