Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47^phox and p67 ^phox and can be rescued by exogenous arachidonic acid

Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutropliils. In this study, rac2-/- neutropliils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47^phox and p67^phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47^phox and p67^phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCζ, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cyloplasmic phospholipase A₂ expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47^phox and p67^phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.