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 hunan blood ne utrophils (cytokine-plasts; CKP), we previously provided evidence for a new staphylococcal killing pathway for human neutrophils involving r eactive nitrogen intermediates: the NO synthase inhibitor N-omega-mono methyl-L-arginine (NMMA), an analogue of L-arginine (L-Arg), substanti ally decreased the killing capacity of CKP for Staphylococcus aureus ( Staph) an effect reversible by excess L-Arg but not D-Arg. We have ext ended these findings to two irreversible NO synthase inhibitors: the f irst, N-iminoethyl-L-ornithine (L-NIO), is an L-Arg analogue; the othe r, diphenyleneiodonium (DPI), is not. After 60 min of incubation with bacteria, despite having taken up somewhat fewer staphylococci than di d controls, cytoplasts treated with NO synthase inhibitors had many mo re live, CKP-associated bacteria: for NMMA, 6.9 times more (40.0% of t he inoculum vs, 5.8%; n = 8, P = 0.003); for L-NIO, 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 incubatio n, In two experiments in which the Gram-negative bacterium, Serratia m arcescens, was employed instead of Staph, the results were again simil ar, In contrast, killing of either bacterium by intact neutrophils (PM N) was not inhibited by NMMA, by L-NIO, or by DPI, a failure most like ly attributable to their granule content, The irreversible inhibitors of NO synthase will be especially useful in analyzing particular effec ts on CKP employed in multicellular systems.