Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages.

Abstract

Chemokines are important mediators in the development of inflammation. Our previous work demonstrated that an oxidative stress can up-regulate mRNA expression of a CC chemokine macrophage inflammatory protein (MIP)-1alpha in rat alveolar macrophages. In the present study, we further investigate whether an oxidative stress can regulate the gene expression of a related CXC chemokine MIP-2, involved in both neutrophil chemotaxis and activation. A rat alveolar macrophage cell line (NR8383) was exposed to 10 microg/ml bacterial lipopolysaccharide (LPS) and MIP-2 mRNA levels dramatically increased after 4 hr of stimulation. This increase by LPS was attenuated by co-treatment with the antioxidants N-acetylcysteine and dimethylsulphoxide, suggesting that the induction of MIP-2 mRNA is mediated via the generation of reactive oxygen species. To assess directly the role of oxidative stress on regulation of MIP-2 mRNA expression, macrophages were exposed to H2O2. MIP-2 mRNA levels had significantly increased after 1 hr exposure to 0.5 mm H2O2, were maximally increased after 4 hr and decreased after 6 hr. Co-treatment of macrophages with the transcriptional inhibitor actinomycin D eliminated the H2O2-induction of MIP-2 mRNA, implicating a role for transcriptional activation in increased expression of MIP-2. Genomic cloning of the rat MIP-2 gene 5'-flanking region has identified a consensus nuclear factor-kappaB (NF-kappaB) binding site. Gel-mobility shift assays revealed NF-kappaB binding to the MIP-2 promoter/enhancer sequence was induced by H2O2. LPS treatment for 4 hr also significantly activated NF-kappaB binding, which could also be attenuated by pretreatment with N-acetylcysteine at the doses that reduced MIP-2 mRNA expression. The half-life of MIP-2 mRNA transcripts was also increased by H2O2 treatment. These observations indicate that MIP-2 gene expression is subject to both transcriptional and post-transcriptional control in response to an H2O2 oxidative stress.