Diclofenac binding to human polymorphonuclear neutrophils: Effect on respiratory burst and N-formylated peptide binding

Abstract

The respiratory burst of human polymorphonuclear neutrophils (PMN) induced by particle or soluble stimuli was measured in the presence of the nonsteroidal anti-inflammatory drug, diclofenac sodium (Voltaren). Diclofenac (25–100 μg/ml) inhibited the oxygen consumption of PMN stimulated by 5 × 10 −7M of N-formyl-methionyl-leucyl-phenylalanine (FMLP). The inhibition was linearly correlated to diclofenac concentration. By contrast, diclofenac did not affect the rate of heat-killed Klebsiella pneumoniae ingestion of PMN, or the PMN O 2-uptake induced by (0.67 μg/ml) serum-opsonized zymosan or (1 μg/ml) phorbol myristate acetate (PMA). The PMN production of Superoxide anion induced by various FMLP concentrations (10 −7, 10 −6 and 10 −5 M) was also decreased by diclofenac. However, this inhibition declined when the formylated peptide concentration was raised suggesting that diclofenac could alter FMLP binding to the PMN membrane. Binding experiments of tritiated FMLP to intact PMN performed at 22° and 4° showed high- and low-affinity FMLP sites with dissociation constant ( K d) values of approximately 2 × 10 −8 M and 10 −5 M respectively. Diclofenac did not significantly alter the low-affinity component but induced modifications of the high-affinity component which were different at 22° and 4°. At 22° only the dissociation constant value was enhanced by diclofenac (competitive inhibition) whereas at 4° both binding parameters (i.e. dissociation constant and number of available binding sites) were modified (mixed inhibition). Diclofenac was also shown to bind to PMN with a low affinity. This binding was not diminished at 4° by various concentrations of FMLP which even increased the number of diclofenac binding sites on PMN at 22°. These data suggest that diclofenac binding to PMN may decrease FMLP-induced PMN respiratory burst by interfering with the peptide recognition by specific FMLP receptors.