Protease priming of neutrophil superoxide production effects on membrane lipid order and lateral mobility

Phagocyte superoxide (O₂^-) response is primed by a variety of physiologic compounds including the neutrophil secretory proteases cathepsin G and elastase. To study whether protease priming of neutrophil O₂^- response is related to changes in membrane physical state, we examined enzyme effects on the order and lateral mobility of lipid probes in intact neutrophil membranes. Exposure to cathepsin G (5 μg/ml) or elastase (10 μg/ml) caused a significant decrease in fluorescence anisotropy of the probe trimethylammonium diphenylhexatriene in neutrophil plasma membranes (0.279 to 0.256 for cathepsin G, 0.274 to 0.256 for elastase, p < 0.02 for both), indicating a decrease in phospholipid chain order in the surface membrane bilayer. Cathepsin G and elastase also caused significant increases in membrane lipid lateral mobility as measured by excimer formation of the fluorescent probe 1-pyrenedecanoic acid (for cathepsin G, a 107% increase, and for elastase, a 44% increase in excimer/monomer fluorescence ratio, p < 0.001). Enzyme effects on membrane structure were dependent on intact proteolytic activity, and were cell specific; the proteases had no effect on lipid order or lateral mobility in liposomes. In corollary studies, the possible association between the physical state of the polymorphonuclear leukocyte membrane and O₂^- generation was analyzed with the membrane modifying compounds, linoleic acid, ethanol, and cholesterol. Cell exposure to linoleic acid (1 μM) caused a significant decrease in lipid order and an increase in lipid lateral mobility along with increased O₂^- production to N-formyl-Met-Leu-Phe (fMLP) (191%) and phorbol myristate acetate (PMA) (39%), p < 0.02 for each. 3 mM ethanol also augmented O₂^- response to fMLP (31%) and PMA (48%) and caused a significant decrease in lipid order, but did not affect lipid lateral mobility. Treatment with cholesteryl hemisuccinate (100 μg/ml) resulted in increased lipid order and decreased lipid lateral mobility, as well as decreased neutrophil superoxide response to fMLP (-61%, p < 0.001) and PMA (-50%, p < 0.02). We then examined whether modulation of membrane physical state may explain the mechanism of action of a known priming agent by studying the effects of low concentrations of a diacylglycerol. Cells treated with 10 μM. l-oleoyl-2-acetyl-sn-glycerol had a greater than 8-fold increase in superoxide response to fMLP (p < 0.001) while demonstrating a significant decrease in lipid order (0.289 to 0.281, p < 0.01) and a 50% increase in lipid lateral mobility (p < 0.001). Although proteases and the membrane modifying compounds may have other cellular actions which affect superoxide production, these results suggest that changes in membrane structure can modify oxidative burst activity and may, in part, underlie protease priming of neutrophil superoxide response.