Phospholipases and the activation and priming of neutrophils by peritoneal dialysis effluent

Objective: To investigate the role of phospholipases during the activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by peritoneal dialysis effluent (PDE). Design: Examine the action of 4-hour dwell PDE upon phospholipase activation in the circulating neutrophils obtained from healthy individuals. Results: We have previously reported that PDE stimulated superoxide release by the NADPH oxidase of human neutrophils and primed the response to the bacterial peptide, fMLP (fMetLeuPhe). To elucidate the biochemical mechanisms underlying these observations, we have examined the roles of phospholipases (PL) C, D, and A₂, whose activation causes the release of a range of intracellular secondary messengers. Following fMLP stimulation, we observed a rapid activation of both PLC and PLD as well as a small but nonsignificant increase in PLA₂ activity. Peritoneal dialysis effluent alone failed to stimulate either PLC or PLD, while pre-incubation with PDE had no affect upon fMLP-induced PLC and PLD activation. However, PDE caused a small but nonsignificant increase in PLA₂ activity (which was comparable to that observed with fMLP) and primed the fMLP-induced response. In common with a role for PLA₂ and the subsequent release of arachidonic acid (AA), we have demonstrated dose-dependent inhibition of PDE-induced superoxide release by the PLA₂ inhibitor mepacrine, as well as activation and priming of the fMLP-induced superoxide generation by AA. Conclusions: These results imply that PDE-induced NADPH-oxidase activation and priming in human neutrophils is mediated via a PLA₂-dependent but PLC- and PLD-independent mechanism.