Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

Abstract

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2•−) due to the NADPH-dependent univalent reduction of oxygen to O2•− by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O2•− in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can modulate macrophage activation by inhibiting O2•− formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO2 decreased O2•− production in a dose- (IC50=4.1±1.8μM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40phox and p47phox), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47phox was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.