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  • Azithromycin suppresses interleukin-12p40 expression in lipopolysaccharide and interferon-gamma stimulated macrophages.

Azithromycin suppresses interleukin-12p40 expression in lipopolysaccharide and interferon-gamma stimulated macrophages.

International journal of biological sciences (2009-11-07)
Keiko Yamauchi, Yoko Shibata, Tomomi Kimura, Shuichi Abe, Sumito Inoue, Daisuke Osaka, Michiko Sato, Akira Igarashi, Isao Kubota
ABSTRACT

Azithromycin (AZM), a 15-member macrolide antibiotic, possesses anti-inflammatory activity. Macrophages are important in innate and acquired immunity, and produce pro-inflammatory cytokines such as interleukin (IL)-12, which are composed of subunit p40 and p35. The key function of IL-12 is the induction and maintenance of T-helper-1 responses, which is associated with the pathogenesis of chronic inflammatory diseases. We investigated the effect of azithromycin on IL-12p40 production in macrophages after lipopolysaccharide (LPS)/interferon (IFN)-gamma stimulation. RAW264.7 macrophage cell line was pre-treated with vehicle or AZM, followed by the stimulation with LPS/IFN-gamma. We measured IL-12 production by RT-PCR and ELISA. IL-12 transcriptional regulation was assessed by electrophoretic mobility shift assay and reporter assay. Phosphorylation of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP) was assessed by immunoprecipitation using phosphotyrosine antibody, and immunoblotting using specific antibodies against JunB and ICSBP. AZM reduced the induction of IL-12p40 by LPS/IFN-gamma in a dose dependent manner. AZM inhibited the binding of AP-1, nuclear factor of activated T cells (NFAT), and ICSBP, to the DNA binding site in the IL-12p40 promoter. AZM also reduced LPS/IFN-gamma-induced IL-12p40 promoter activity. Phosphorylation of JunB and ICSBP was inhibited by azithromycin-treatment in stimulated cells. In conclusion, AZM reduced IL-12p40 transcriptional activity by inhibiting the binding of AP-1, NFAT, and ICSBP to the promoter site. This may represent an important mechanism for regulating the anti-inflammatory effects of AZM in macrophages.

MATERIALS
Product Number
Brand
Product Description

Supelco
Azithromycin, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Lipopolysaccharides from Pseudomonas aeruginosa 10, purified by phenol extraction
Sigma-Aldrich
Azithromycin dihydrate, ≥98% (HPLC)
USP
Azithromycin, United States Pharmacopeia (USP) Reference Standard
Azithromycin, European Pharmacopoeia (EP) Reference Standard
Azithromycin for peak identification, European Pharmacopoeia (EP) Reference Standard
Azithromycin for system suitability, European Pharmacopoeia (EP) Reference Standard