title: Azithromycin suppresses interleukin-12p40 expression in lipopolysaccharide and interferon-gamma stimulated macrophages.
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.
authors.affiliation: Department of Cardiology, Pulmonology, and Nephrology, Yamagata University School of Medicine, Yamagata, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Yamauchi
authors.forename: Yoko
authors.initials: Y
authors.lastname: Shibata
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kimura
authors.forename: Shuichi
authors.initials: S
authors.lastname: Abe
authors.forename: Sumito
authors.initials: S
authors.lastname: Inoue
authors.forename: Daisuke
authors.initials: D
authors.lastname: Osaka
authors.forename: Michiko
authors.initials: M
authors.lastname: Sato
authors.forename: Akira
authors.initials: A
authors.lastname: Igarashi
authors.forename: Isao
authors.initials: I
authors.lastname: Kubota
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017963:Azithromycin / Q000008:administration & dosage / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D050835:Interferon Regulatory Factors / Q000378:metabolism; D007371:Interferon-gamma / Q000276:immunology*; D053762:Interleukin-12 Subunit p40 / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology*; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D050778:NFATC Transcription Factors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D000099185:Interferon Regulatory Factor-8
chemical_list: D000900:Anti-Bacterial Agents; D017963:Azithromycin; D050835:Interferon Regulatory Factors; D007371:Interferon-gamma; D053762:Interleukin-12 Subunit p40; D008070:Lipopolysaccharides; D050778:NFATC Transcription Factors; D019000:Phosphotyrosine; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D000099185:Interferon Regulatory Factor-8