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

Abstract

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 (C(16)- and C(24))-ceramides, which are lipid second messengers of apoptosis and stress signaling. Treatment of neutrophils with fumonisin B(2), a selective inhibitor of the de novo pathway of ceramide synthesis, prevented accumulation of C(16)- and C(24)-ceramides. Moreover, fumonisin B(2) 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 neutrophils, indicating that in these cells, ceramides are not generated from membrane sphingomyelin. Further experiments showed that increasing endogenous C(16)- and C(24)-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 C(16)- and/or C(24)-ceramides were added to neutrophil cultures. Finally, GM-CSF, a cytokine that delays neutrophil apoptosis, abrogated C(16)- and C(24)-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 C(16)- and C(24)-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.

Sphingolipid profile alters in retinal dystrophic P23H-1 rats and systemic FTY720 can delay retinal degeneration

“…CerS1 and CerS4 are specific to synthesis of Cers with chain length C18-20 and CerS2 for C20-26. Numerous cell culture studies suggest that shortchain Cers, especially, C16, are proapoptotic, and long chains such as C22:0, C24:0, and C24:1 are antiapoptotic or pro-proliferative (59)(60)(61)(62). Increases in long-chain species in P23H-1 retina could therefore be a cellular response to counter the apoptosis of photoreceptor cells that is induced by genetic mutation, ER stress, and activation of the unfolded protein response.…”

Palmitate increases sphingosine-1-phosphate in C2C12 myotubes via upregulation of sphingosine kinase message and activity

“…Since various sphingolipids, including diverse ceramide species ( 24 ), S1P ( 25 ), C1P ( 26 ), and complex sphingolipids, including sphingomyelin and glycosphingolipids ( 27,28 ), each play distinct cellular roles, we aimed to determine the impact of PAL oversupply on tissue sphingolipid profi les. These data revealed pleiotropic effects of PAL on cell sphingolipids not limited to ceramides.…”

“…by guest, on May 11, 2018 www.jlr.org Downloaded from never been determined in these systems. Since recent studies indicate different functional roles for different ceramide species ( 24,36,37 ), determining ceramides produced under these conditions may shed light on sphingolipid-dependent pathology associated with excess PAL. Indeed, LC/MS data revealed specifi city in dihycroceramide profi les.…”

Triggering role of acid sphingomyelinase in endothelial lysosome-membrane fusion and dysfunction in coronary arteries

“…Failure of the de novo synthesis of ceramide to activate or promote lysosome fusion to the cell membrane may be related to its enzymatic reaction time and location. In general, it is accepted that the de novo synthesis of ceramide takes more time in response to any stimuli compared with sphingomyelin hydrolysis (7,32,38). In addition, this ceramide de novo synthesis mainly occurs in the endoplasmic reticulum.…”

“…The production of ceramide was measured as described in our laboratory's previous studies (39) and by others (8,32). Briefly, CAECs were grown on 10-cm dishes.…”