We addressed the role of O⨪2 generated by the NADPH oxidase complex in the mechanism of polymorphonuclear leukocyte (PMN) accumulation and transalveolar migration and lung microvascular injury. Studies were made in mice lacking the p47phox and gp91phox subunits of NADPH oxidase (p47phox−/− and gp91phox−/−) in which PMN are incapable of the respiratory burst. The mice were challenged i.p. with live Escherichia coli to induce sepsis. We observed time-dependent increases in PMN sequestration and migration from 1 to 6 h after challenge with 2 × 108 E. coli. The responses in knockout mice were greater post-E. coli challenge compared with control mice; i.e., transalveolar PMN migration post-E. coli challenge increased by ∼50% in the null mice above values in wild type. The increased PMN infiltration was associated with decreased lung bacterial clearance. The generation of the chemoattractant macrophage-inflammatory protein-2 in lung tissue was greater in NADPH oxidase-defective mice after E. coli challenge than control mice; moreover, macrophage-inflammatory protein-2 Ab pretreatment prevented the PMN infiltration. We also observed that E. coli failed to increase lung microvascular permeability in p47phox−/− and gp91phox−/− mice despite the greater lung PMN sequestration. Thus, O⨪2 production is required for the induction of sepsis-induced lung microvascular injury. We conclude that NADPH oxidase-derived O⨪2 generation has an important bactericidal role, such that an impairment in bacterial clearance in NADPH oxidase-defective mice results in increased chemokine generation and lung tissue PMN infiltration.

NO is a crucial mediator of the inflammatory response, but its in vivo role as a determinant of lung inflammation remains unclear. We addressed the in vivo role of NO in regulating the activation of NF-κB and expression of inflammatory proteins using an in vivo mouse model of sepsis induced by i.p. injection of Escherichia coli. We observed time-dependent degradation of IκB and activation of NF-κB accompanied by increases in inducible NOS, macrophage inflammatory protein-2 (MIP-2), and ICAM-1 expression after E. coli challenge, which paralleled the ability of lung tissue to produce high-output NO. To determine the role of NO in this process, mice were pretreated with the NO synthase (NOS) inhibitor NG-methyl-l-arginine. Despite having relatively modest effects on NF-κB activation and ICAM-1 or inducible NOS expression, the NOS inhibitor almost completely inhibited expression of MIP-2 in response to E. coli challenge. These responses were associated with the inhibition of migration of neutrophils in lung tissue and increased permeability induced by E. coli. In mice pretreated with NG-methyl-l-arginine, coadministration of E. coli with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate substantially restored MIP-2 expression but decreased ICAM-1 expression. The results suggest that NO generated after administration of E. coli serves as an important proinflammatory signal to up-regulate MIP-2 expression in vivo. Thus, NO production in high quantities may be important in the mechanism of amplification of the lung inflammatory response associated with sepsis.

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