Attenuation of p47phOX and p67Phox Membrane Translocation as the Mechanism

Surfactant has been shown to inhibit the production of reac-tive oxygen intermediates by various cells including alveolar macrophages and peripheral blood neutrophils. Superoxide (O°) production by the respiratory burst oxidase in isolated plasma membranes prepared from PMA-treated human neutrophils was significantly attenuated by prior treatment with native porcine surfactant. The effect was concentration dependent with half-maximal inhibition seen at t 0.050 mg surfactant phospholipid/ml. Kinetic analyses of the mem-brane-bound enzyme prepared from neutrophils stimulated by PMA in the presence or absence of surfactant demon-strated that surfactant treatment led to a decrease in the maximal velocity of 02 production when NADPH was used as substrate, but there was no effect on enzyme substrate affinity. Immunoblotting studies demonstrated that surfac-tant treatment induced a decrease in the association of two oxidase components, p47Phox and p67Plox, with the isolated plasma membrane. In contrast, surfactant treatment of the cells did not alter the phosphorylation of p47P"ox. A mixture of phospholipids (phosphatidylcholine and phosphati-dylglycerol in a 7:3 ratio) showed similar inhibition of the PMA-induced °2 generation. Taken together, these data suggest the mechanism of surfactant-induced inhibition of °2 production by human neutrophils involves attenuation of translocation of cytosolic components of the respiratory burst oxidase to the plasma membrane. The phospholipid components of surfactant appear to play a significant role in this mechanism. (J. Clin. Invest. 1995. 96:2654-2660.) Key words: lung surfactant * neutrophils * NAD(P)H oxido-reductase * superoxide * assembly mechanism