NEUTROPHILS EXPOSED TO BACTERIAL LIPOPOLYSACCHARIDE UP-REGULATE NADPHOXIDASE ASSEMBLY

Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more r esponsive to other stimuli, a phenomenon known as ''priming.'' Since t he mechanism of LPS-dependent priming is incompletely understood, we i nvestigated its effects on assembly and activation of the NADPH oxidas e. LPS pretreatment increased superoxide (O-2(-)) generation nearly 10 -fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). I n a broken-cell O-2(-)-generating system, activity was increased in pl asma membrane-rich fractions and concomitantly decreased in specific g ranule-rich fractions from LPS-treated cells. Oxidation-reduction spec troscopy and flow cytometry indicated LPS increased plasma membrane as sociation of flavocytochrome b(558). Immunoblots of plasma membrane ve sicles from LPS-treated PMNs demonstrated translocation of p47-phox bu t not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three-to sixfold increase (compared with either age nt alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O-2(-) generation by intact PMN s. LPS treatment caused limited phosphorylation of p47-phox, and plasm a membrane-enriched fractions from LPS- and/or fMLP-treated cells cont ained fewer acidic species of p47-phox than did those from cells treat ed with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respirato ry burst.