Ceramide does not mediate the effect of tumour necrosis factor α on superoxide generation in human neutrophils

The effect of tumour necrosis factor α (TNFα) on superoxide generation in human neutrophils was investigated using the Nitro Blue Tetrazolium reduction assay. TNFα stimulated superoxide generation in a time- and concentration-dependent fashion. The maximally effective concentration of TNFα for superoxide generation was 10 nM and maximal response was obtained after 15-20 min. The monoclonal antibody (mAb), utr-1, which was raised against the 75 kDa receptor and behaves as an antagonist, had no effect on superoxide generation, but partially inhibited the response to TNFα. mAb htr-9, which was raised against the 55 kDa receptor and behaves as an agonist, mimicked the effect of TNFα, but with a lower maximal response. As it has been reported that ceramide might act as a second messenger to mediate many of the effects of TNFα, the effects of exogenous sphingomyelinase and the cell-permeable ceramide analogue, C₂-ceramide, on production of superoxide anions, induction of priming in response to formylmethionyl-leucyl-phenylalanine, and cell-shape change were examined. Neither sphingomyelinase nor C₂-ceramide mimicked the effect of TNFα. Ceramide is converted into ceramide 1-phosphate by ceramide kinase and we have measured levels of this metabolite to clarify the effect of TNFα on sphingomyelinase activity in neutrophils. Although exogenous sphingomyelinase increased the amount of ceramide 1-phosphate in a time-dependent manner, and C₂-ceramide was rapidly converted into C₂-ceramide phosphate, TNFα had no effect on the level of ceramide 1-phosphate. These results suggest that TNFα stimulates superoxide generation through both the 55 kDa and 75 kDa receptors, but that ceramide does not act as an intracellular mediator for TNFα in human neutrophils.