C2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils

Abstract Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C 2 -ceramide modulates some of the effects of N -formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O 2 − ) production. The preincubation of PMN for 15 min with C 2 -ceramide increased by up to almost 3-fold the amounts of O 2 − generated in response to 0.1 and 1 μM fMLP. Priming was detected at C 2 -ceramide concentrations of 2 μM to 4 μM per million PMN. Though less potent than C 2 -ceramide, C 6 -ceramide ( N -hexanoylsphingosine) could prime for O 2 − generated in response to 0.1 μM fMLP, with maximal effects obtained at 10–20 μM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O 2 − generation. As expected, TNF-α (1000 U/ml), also primed for fMLP-induced O 2 − production; however, the combination of TNF-α and C 2 -ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C 2 -ceramide and TNF-α. C 2 -ceramide at 2 μM did not enhance the production of O 2 − induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B 4 (LTB 4 ), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C 2 -ceramide (2 μM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A 2 or D (PLC, PLA 2 , PLD) were involved in the priming effect by C 2 -ceramide. However, C 2 -ceramide inhibited in a dose-related manner the production of O 2 − induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C 2 -ceramide. The priming O 2 − production by C 2 -ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-α have different mechanisms than C 2 -ceramide.