title: Protease priming of neutrophil superoxide production. Effects on membrane lipid order and lateral mobility.
abstract: 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 micrograms/ml) or elastase (10 micrograms/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 less than 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 less than 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 microM) 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 less than 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 micrograms/ml) resulted in increased lipid order and decreased lipid lateral mobility, as well as decreased neutrophil superoxide response to fMLP (-61%, p less than 0.001) and PMA (-50%, p less than 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 microM 1-oleoyl-2-acetyl-sn-glycerol had a greater than 8-fold increase in superoxide response to fMLP (p less than 0.001) while demonstrating a significant decrease in lipid order (0.289 to 0.281, p less than 0.01) and a 50% increase in lipid lateral mobility (p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, Case Western Reserve University, University Hospitals of Cleveland, Ohio 44106.
authors.forename: D J
authors.initials: DJ
authors.lastname: Kusner
authors.forename: J N
authors.initials: JN
authors.lastname: Aucott
authors.forename: D
authors.initials: D
authors.lastname: Franceschi
authors.forename: M M
authors.initials: MM
authors.lastname: Sarasua
authors.forename: P J
authors.initials: PJ
authors.lastname: Spagnuolo
authors.forename: C H
authors.initials: CH
authors.lastname: King
mesh_terms: D000328:Adult; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D010450:Endopeptidases / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D010196:Pancreatic Elastase / Q000494:pharmacology; D012697:Serine Endopeptidases; D013481:Superoxides / Q000378:metabolism*
chemical_list: D008563:Membrane Lipids; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D002403:Cathepsins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase