Tumour necrosis factor-alpha and interferon-gamma synergistically activatethe RANTES promoter through nuclear factor kappa B and interferon regulatory factor 1 (IRF-1) transcription factors

Inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synergistically activate expression of the RA NTES(regulated upon activation, normal T-cell expressed and secreted) gene, which plays a crucial role in the chemoattraction of leukocytes during the inflammatory response. To understand at the molecular level the mechanism by which the two cytokines activate RANTES gene expression, we determined t he requirement of cis-acting elements in the RANTES promoter and trans-acti ng factors. The murine RANTES promoter contained one putative interferon re gulatory factor, IRF, and three putative nuclear factor kappa B (NF-kappa B ) binding sites. Specific destruction of the IRF binding site and one of th e three NF-kappa B binding sites abolished the inducibility of promoter act ivity by IFN-gamma and TNF-alpha, respectively. In contrast, mutation of th e other two putative NF-kappa B binding sites did not affect RANTES promote r activity significantly. In addition, the RANTES promoter was stimulated b y co-transfection of plasmids that expressed either p65, an NF-kappa B fami ly protein, or the IRF-1 transcription factor. RANTES promoters with mutati ons in the NF-kappa B or IRF binding sites were not stimulated by p65 or IR F-1 expression, respectively. In electrophoretic mobility-shift and immunol ogic assays, we showed that IRF-1 was induced after cells were treated with IFN-gamma and that NF-kappa B was activated by TNF-alpha treatment. These results demonstrate that both NF-kappa B and IRF-1 transcription factors me diate the induction of RANTES expression via their cognate cis-acting eleme nts when cells are stimulated by TNF-alpha and IFN-gamma.