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)
Highlights
From the Departments of Medicine and Surgery, Case Western Reserve University, University Hospitals of Cleveland, and MetroHealth Medical Center, Cleveland, Ohio 44106
Superoxide Productionin Protease-primed Neutrophils-As decrease in anisotropy was maximaalt 5 min and anisotropy previously reported [13], exposureof PMN to cathepsin G or remained lower than the pretreatment value throughoutthe elastase resulted in augmented superoxide production upon 30-min experimental period
G pretreatment produceda 64% and elastase a 32% in super- separate experiments, measurements were madaet 90-s interoxide generation (5-min accumulation) compared to control vals, and an 8% decells treated with buffer alone (Fig. 1,A and B, p < 0.05 for crease in the membrane lipid order occurred 90 s after caeach)
Summary
To measure the effects of cathepsin G and elastase on the order incubated for 10-15 min at 37 "C in a shaking water bath, followed by centrifugation and washing of the cells with calcium- and magnesium-free HBSS. Ethanol and was 0.53 p~ for the superoxide assay and 0.25 p~ for anisotropy and excimer fluorescence measurements (mean of five individual determinations for each) This level of ethanol did not alter superoxide production or TMA-DPH anisotropy compared with cells processed in the absence of ethanol (nanomole of superoxide with fMLP stimulation: +ethanol = 3.81, -ethanol = 3.88; anisotropy: superoxide generation (9-min accumulation, Fig., C and D, p
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