Published online before print February 28, 2007

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(Journal of Leukocyte Biology. 2007;81:1477-1486.)
© 2007 by Society for Leukocyte Biology

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

Grégory Seumois^*, Marianne Fillet, Laurent Gillet, Céline Faccinetto, Christophe Desmet^*, Cédric François^*, Benjamin Dewals, Cécile Oury, Alain Vanderplasschen, Pierre Lekeux^* and Fabrice Bureau^*,||,1

^* Departments of Physiology,
Analytical Pharmaceutical Chemistry,
Immunology and Vaccinology,
Human Genetics, and
^|| Biochemistry and Molecular Biology, University of Liège, Liège, Belgium

¹ Correspondence: Laboratory of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, University of Liège, Boulevard de Colonster, Bâtiment B42, Sart-Tilman, B-4000, Liège, Belgium. E-mail: fabrice.bureau{at}ulg.ac.be

Neutrophils 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 (C₁₆- and C₂₄)-ceramides, which are lipid second messengers of apoptosis and stress signaling. Treatment of neutrophils with fumonisin B₂, a selective inhibitor of the de novo pathway of ceramide synthesis, prevented accumulation of C₁₆- and C₂₄-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 C₁₆- and C₂₄-ceramide production nor decreased the apoptosis rate in neutrophils, indicating that in these cells, ceramides are not generated from membrane sphingomyelin. Further experiments showed that increasing endogenous C₁₆- and C₂₄-ceramide levels by using DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol and (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, two inhibitors of ceramide metabolism, enhances caspase-3, -8, and -9 activity and increases neutrophil apoptosis. Similarly, apoptosis was induced rapidly when synthetic C₁₆- and/or C₂₄-ceramides were added to neutrophil cultures. Finally, GM-CSF, a cytokine that delays neutrophil apoptosis, abrogated C₁₆- and C₂₄-ceramide accumulation totally in cultured neutrophils, 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 neutrophil apoptosis via caspase activation and that GM-CSF exerts its antiapoptotic effects on neutrophils, at least partly through inhibition of ceramide accumulation.

Key Words: caspases • GM-CSF • granulocytes • survival

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