CERAMIDE DOES NOT MEDIATE THE EFFECT OF TUMOR-NECROSIS-FACTOR-ALPHA ON SUPEROXIDE GENERATION IN HUMAN NEUTROPHILS

The effect of tumour necrosis factor alpha (TNFalpha) on superoxide ge neration in human neutrophils was investigated using the Nitro Blue Te trazolium reduction assay. TNFalpha stimulated superoxide generation i n a time- and concentration-dependent fashion. The maximally effective concentration of TNFalpha for superoxide generation was 10 nM and max imal response was obtained after 15-20 min. The monoclonal antibody (m Ab), utr-1, which was raised against the 75 kDa receptor and behaves a s an antagonist, had no effect on superoxide generation, but partially inhibited the response to TNFalpha. mAb htr-9, which was raised again st the 55 kDa receptor and behaves as an agonist, mimicked the effect of TNFalpha, but with a lower maximal response. As it has been reporte d that ceramide might act as a second messenger to mediate many of the effects of TNFalpha, the effects of exous C2-sphingomyelinase and the cell-permeable ceramide analogue, C2-ceramide, on production of super oxide anions, induction of priming in response to formylmethionyl-leuc yl-phenylalanine, and cell-shape change were examined. Neither sphingo myelinase nor C2-ceramide mimicked the effect of TNFalpha. Ceramide is converted into ceramide 1-phosphate by ceramide kinase and we have me asured levels of this metabolite to clarify the effect of TNFalpha on sphingomyelinase activity in neutrophils. Although exogenous sphingomy elinase increased the amount of ceramide 1-phosphate in a time-depende nt manner, and C2-ceramide was rapidly converted into C2-ceramide phos phate, TNFalpha had no effect on the level of ceramide 1-phosphate. Th ese results suggest that TNFalpha stimulates superoxide generation thr ough both the 55 kDa and 75 kDa receptors, but that ceramide does not act as an intracellular mediator for TNFalpha in human neutrophils.