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Microbial killing by human neutrophil cytokineplasts: similar suppressive effects of reversible and irreversible inhibitors of nitric oxide synthase

Employing anucleate, granule‐poor, motile fragments from human blood neutrophils (cytokineplasts; CKP), we previously provided evidence for a new staphylococcal killing pathway for human neutrophils involving reactive nitrogen intermediates: the NO synthase inhibitor Nω‐monomethyl‐L‐argmine (NMMA), an analogue of L‐arginine (L‐Arg), substantially decreased the killing capacity of CKP for Staphylococcus aureus (Staph), an effect reversible by excess L‐Arg but not D‐Arg. We have extended these findings to two irreversible NO synthase inhibitors: the first, N‐iminoethyl‐L‐ornithme (L‐N10), is an L‐Arg analogue; the other, diphenyleneiodonium (DPI), is not. After 60 min of incubation with bacteria, despite having taken up somewhat fewer staphylococci than did controls, cytoplasts treated with NO synthase inhibitors had many more live, CKP‐associated bacteria: for NMMA, 6.9 times more (40.0% of the inoculum vs. 5.8%; n = 8, P = 0.003); for L‐N10, 3.6 times more (25.5 vs. 7%; n = 4, P = 0.004); for DPI, 5.8 times more (37.4 vs. 6.4%; n = 7, P = 0.002). Results were similar after only 20 min of incubation. In two experiments in which the Gram‐negative bacterium, Serratia marcescens, was employed instead of Staph, the results were again similar. In contrast, killing of either bacterium by intact neutrophils (PMN) was not inhibited by NMMA, by L‐N10, or by DPI, a failure most likely attributable to their granule content. The irreversible inhibitors of NO synthase will be especially useful in analyzing particular effects on CKP employed in multicellular systems. J. Leukoc. Biol. 60: 753–757; 1996.