DeLeo F R, Renee J, McCormick S, Nakamura M, Apicella M, Weiss J P, Nauseef W M
Department of Medicine and the Inflammation Program, Veterans Administration Medical Center and University of Iowa, Iowa City, Iowa 52246, USA.
J Clin Invest. 1998 Jan 15;101(2):455-63. doi: 10.1172/JCI949.
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.
细菌脂多糖（LPS）是多形核中性粒细胞（PMN）的多能激动剂。尽管它不直接激活依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的氧化酶，但LPS使PMN对其他刺激更敏感，这一现象称为“预激活”。由于LPS依赖性预激活的机制尚未完全了解，我们研究了其对NADPH氧化酶组装和激活的影响。LPS预处理使对N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）的反应中超氧化物（O2-）生成增加近10倍。在破碎细胞的O2-生成系统中，LPS处理细胞富含质膜的组分中活性增加，而富含特异性颗粒的组分中活性相应降低。氧化还原光谱和流式细胞术表明LPS增加了黄素细胞色素b558与质膜的结合。LPS处理的PMN质膜囊泡的免疫印迹显示p47-吞噬细胞氧化酶（p47-phox）发生易位，但p67-吞噬细胞氧化酶（p67-phox）或Rac2未发生易位。然而，先用LPS然后用fMLP处理的PMN在质膜相关的p47-phox、p67-phox和Rac2中显示增加了三到六倍（与单独使用任何一种试剂相比），并且易位与完整PMN增强的O2-生成平行。LPS处理导致p47-phox的磷酸化有限，并且LPS和/或fMLP处理细胞的富含质膜的组分中p47-phox的酸性形式比佛波酯（PMA）处理细胞的少。综上所述，这些研究表明NADPH氧化酶组分的重新分布可能是LPS引发呼吸爆发的基础。
