Eosinophilic inflammation: mechanisms regulating IL-5 transcription in human T lymphocytes

Abstract

Background: Interleukin (IL)-5 is a key regulator of eosinophilia in allergic inflammation and parasite infections but the mechanisms regulating IL-5 expression in activated human T lymphocytes are poorly understood. From studies on mouse cells, the activation protein (AP)-1 and GATA-3 sites in the proximal promoter region appear to be important in IL-5 regulation but the significance of an adjacent Ets/nuclear factor of activated T cell (NFAT) site has been less clear. Methods: Interleukin-5 transcriptional activity was measured by transfection of reporter genes into the human HSB-2 cells and normal T lymphocytes. Expression vectors encoding transcription factors were used for transactivation studies and IL-5 expression measured using reporter genes and mRNA levels. Transcription factor binding was shown with chromatin immunoprecipitation (ChIP). Results: HSB-2 cells showed high inducible expression of IL-5 mRNA. Mutation of reporter gene plasmids showed the Ets/NFAT site was of equal importance to the AP-1 and GATA-3 sites in regulating IL-5 transcription. Transactivation by Ets1 increased luciferase expression 15-fold, in the absence of stimulation, and AP-1 (c-Fos/c-Jun) and GATA-3 gave transactivations of 85-fold, and 100-fold, respectively. Synergistic interactions were demonstrated between Ets1, GATA-3 and AP-1. Dominant-negative AP-1 inhibited IL-5 transcription. Transactivation by GATA-3 and synergy between GATA-3, Ets1 and AP-1 were verified measuring IL-5 mRNA levels. Chromatin immunoprecipitation showed increased binding of Ets1 and GATA-3 to the IL-5 promoter after stimulation. The importance of the Ets1 site and of synergistic interactions between the three transcription factors were verified with primary human T cells. Conclusion: Ets1, GATA-3 and AP-1 synergize to regulate IL-5 transcription in human T cells.