Constitutive NADPH oxidase and increased mitochondrial respiratory chain activity regulate chemokine gene expression

Alveolar macrophages, which generate high levels of reactive oxygen species, especially O-2(center dot-), are involved in the recruitment of neutrophils to sites of inflammation and injury in the lung, and the generation of chemotactic proteins triggers this cellular recruitment. In this study, we asked whether O-2(center dot-) generation in alveolar macrophages had a role in the expression of chemokines. Specifically, we hypothesized that O-2(center dot-) generation is necessary for chemokine expression in alveolar macrophages after TNF- alpha stimulation. We found that alveolar macrophages have high constitutive NADPH oxidase activity that was not increased by TNF-alpha, but TNF-alpha increased the activity of the mitochondrial respiratory chain. In addition, the mitochondrial respiratory chain increased O-2(center dot-) generation if the NADPH oxidase was inhibited. O-2(center dot-) generation was necessary for macrophage inflammatory protein2 (MIP-2) gene expression, because inhibition of NADPH oxidase or the mitochondrial respiratory chain or overexpression of Cu, Zn-superoxide dismutase significantly inhibited expression of MIP-2. TNF-alpha activated the ERK MAP kinase, and ERK activity was essential for chemokine gene expression. In addition, overexpression of the MEK -> ERK pathway significantly increased IL-8 expression, and a small interfering RNA to the NADPH oxidase inhibited ERK-and TNF-alpha induced chemokine expression. Collectively, these results suggest that in alveolar macrophages, O-2(center dot-) generation mediates chemokine expression after TNF-alpha stimulation in an ERK-dependent manner.