Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly.
Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.
DeLeo
F R
FR
Department of Medicine and the Inflammation Program, Veterans Administration Medical Center and University of Iowa, Iowa City, Iowa 52246, USA.
Renee
J
J
McCormick
S
S
Nakamura
M
M
Apicella
M
M
Weiss
J P
JP
Nauseef
W M
WM
D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000737:chemistry / Q000187:drug effects*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D016897:Respiratory Burst / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism
D003573:Cytochrome b Group; D008070:Lipopolysaccharides; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; C036888:cytochrome b558; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1