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)-1α 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 μg/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-κB (NF-κB) binding site. Gel-mobility shift assays revealed NF-κB binding to the MIP-2 promoter/enhancer sequence was induced by H₂O₂. LPS treatment for 4 hr also significantly activated NF-κB 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.