Biphasic regulation of leukocyte superoxide generation by nitric oxide and peroxynitrite

Abstract

Activation of the NADPH oxidase-derived oxidant burst of polymorphonuclear leukocytes (PMNs) is of critical importance in inflammatory disease. PMN-derived superoxide (O2/ -·) can be scavenged by nitric oxide (NO.) with the formation of peroxynitrite (ONOO-); however, questions remain regarding the effects and mechanisms by which NO. and ONOO- modulate the PMN oxidative burst. Therefore, we directly measured the dose-dependent effects of NO. and ONOO- on O2/-· generation from human PMNs stimulated with phorbol 12-myristate 13-acetate using EPR spin trapping. Pretreatment with low physiological (μM) concentrations of NO. from NO. gas had no effect on PMN O2/-· generation, whereas high levels (≥50 μM) exerted inhibition. With ONOO- pretreatment, however, a biphasic modulation of O2/-· generation was seen with stimulation by μM levels, but inhibition at higher levels. With the NO. donor NOR-1, which provides more sustained release of NO. persisting at the time of O2/ -· generation, a similar biphasic modulation of O2/-· generation was seen, and this was inhibited by ONOO- scavengers. The enhancement of O2/-· generation by low concentrations of ONOO- or NOR-1 was associated with activation of the ERK MAPKs and was blocked by their inhibition. Thus, low physiological levels of NO. present following PMN activation are converted to ONOO-, which enhances O2/-· generation through activation of the ERK MAPK pathway, whereas higher levels of NO. or ONOO- feed back and inhibit O2/-· generation. This biphasic concentration-dependent regulation of the PMN oxidant burst by NO.derived ONOO- may be of critical importance in regulating the process of inflammation.