Caveolin‐1 (Cav‐1), the principal structural protein of caveolae, is implicated in normal endothelial barrier function, buts its role in lung inflammation is not well understood. Using caveolin‐1 knockout (Cav‐1 −/−) mice, we addressed the role of Cav‐1 in sepsis‐induced lung injury. We assessed lung inflammation in Cav‐1 −/− mice following i.p. injection of LPS. Neutrophil (PMN) binding was measured by an adhesion assay performed on primary cultured mouse endothelial cells. PMN sequestration was assessed by myeloperoxidase activity in the whole lung. Lung microvascular permeability was determined using iodine‐125 radio‐labeled albumin. When compared to wild type (WT) mice, Cav‐1 −/− mice exhibited significantly impaired PMN binding and sequestration after LPS challenge. LPS‐induced increases in lung microvascular permeability and edema formation were also markedly reduced in Cav‐1 −/− mice relative to WT. To address the basis of the reduced lung inflammation, we examined the specific role of nitric oxide (NO). As Cav‐1 is known to sequester eNOS rendering it inactive, we postulated that in Cav‐1 −/− mice there should be increased eNOS activity and eNOS‐derived NO levels in response to LPS challenge. We observed a marked increase in eNOS activity and NO production in Cav‐1 −/− lung relative to WT. Corresponding to the known role of NO in modulating NF‐κB activity, we also noted a time‐dependent suppression of NF‐κB activity in Cav‐1 −/− mice. Thus, Cav‐1 expression and its ability to regulate eNOS‐derived NO production is a crucial determinant of the lung inflammatory response to sepsis. This abstract is funded by: NIH(T32HL007829, HL60678, HL77806)

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host-defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a non-selective and redox-sensitive cation channel, inhibits ROS production in phagocytic cells and prevents endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) showed an increased inflammatory signature and decreased survival compared to controls. TRPM2 functions by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. Since ROS also activates TRPM2, our findings establish a negative feedback mechanism inactivating ROS production through inhibition of the membrane potential-sensitive NADPH oxidase.

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.