Biphasic regulation of leukocyte superoxide generation by nitric oxide and peroxynitrite

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.