Mechanisms regulating macrophage production of inflammatory mediators in the lung following ozone inhalation: Role of inflammatory mediators in toxicity

Inhalation of ozone (O₃) causes lung injury and inflammation, which is mediated in part by lung macrophages (AM). In the present studies mechanisms regulating AM activity following O₃ inhalation and the role of inflammatory mediators in tissue injury were examined. Treatment of mice with O₃ (0.8 ppm, 3 h) resulted in increased expression of inducible nitric oxide synthase (NOSII) and production of nitric oxide by AM. These effects were not observed in AM from transgenic mice with a targeted disruption of the NOSII gene or in mice overexpressing superoxide dismutase (SOD). Moreover, O₃ toxicity was prevented in these mice demonstrating the importance of reactive nitrogen intermediates in this model. The generation of nitric oxide, is regulated by the redox sensitive transcription factor, NF-κB, as well as by CCAAT/enhancer-binding proteins (C/EBP). Treatment of mice with O₃ resulted in a time-related increase in NF-κB and C/EBP nuclear binding activity in AM. This was not evident in mice with a targeted disruption of the NF-κB p50 subunit. AM from these mice did not generate nitric oxide and NF-κB p50−/− mice were also protected from toxicity, demonstrating that NF-κB is a critical signaling molecule. O₃ inhalation also resulted in increased TNF-β expression in the lung. TNF-β is known to activate phosphatidylinositol 3-kinase (PI3K) and its downstream target, protein kinase B (PKB), which play a role in activation of NF-κB. Both PI3K and PKB protein increased following O₃. Pretreatment of AM with the PI3K inhibitors decreased NF-κB nuclear binding activity and nitric oxide production by AM from O₃ treated mice. This suggests a potential mechanism regulating NOSII activity in the lung after O₃ exposure. Caveolin-1 (Cav-1) is a membrane protein known to negatively regulate PI3K. O₃ inhalation resulted in decreased Cav-1 expression in AM. Treatment of AM with TNF-β resulted in a 2-fold decrease in Cav-1 expression. Thus it appears that O₃-induced TNF-β production may be a critical early event in the pathogenesis of injury induced by O₃. This is supported by our findings that AM from mice with a targeted disruption of TNF-β do not generate nitric oxide and are protected from the toxicity of O₃. The results of the present studies provide mechanistic data on biochemical pathways involved in O₃ toxicity. These data may help elucidate novel targets for therapy aimed at reducing or preventing O₃-induced tissue injury.