title: Interleukin-4 induction of the CC chemokine TARC (CCL17) in murine macrophages is mediated by multiple STAT6 sites in the TARC gene promoter.
abstract: BACKGROUND
  Macrophages (Mtheta) play a central role in the innate immune response and in the pathology of chronic inflammatory diseases. Macrophages treated with Th2-type cytokines such as Interleukin-4 (IL-4) and Interleukin-13 (IL-13) exhibit an altered phenotype and such alternatively activated macrophages are important in the pathology of diseases characterised by allergic inflammation including asthma and atopic dermatitis. The CC chemokine Thymus and Activation-Regulated Chemokine (TARC/CCL17) and its murine homologue (mTARC/ABCD-2) bind to the chemokine receptor CCR4, and direct T-cell and macrophage recruitment into areas of allergic inflammation. Delineating the molecular mechanisms responsible for the IL-4 induction of TARC expression will be important for a better understanding of the role of Th2 cytokines in allergic disease.
  
  
  RESULTS
  We demonstrate that mTARC mRNA and protein are potently induced by the Th2 cytokine, Interleukin-4 (IL-4), and inhibited by Interferon-gamma (IFN-gamma) in primary macrophages (Mtheta). IL-4 induction of mTARC occurs in the presence of PI3 kinase pathway and translation inhibitors, but not in the absence of STAT6 transcription factor, suggesting a direct-acting STAT6-mediated pathway of mTARC transcriptional activation. We have functionally characterised eleven putative STAT6 sites identified in the mTARC proximal promoter and determined that five of these contribute to the IL-4 induction of mTARC. By in vitro binding assays and transient transfection of isolated sites into the RAW 264.7 Mtheta cell-line, we demonstrate that these sites have widely different capacities for binding and activation by STAT6. Site-directed mutagenesis of these sites within the context of the mTARC proximal promoter revealed that the two most proximal sites, conserved between the human and mouse genes, are important mediators of the IL-4 response.
  
  
  CONCLUSIONS
  The induction of mTARC by IL-4 results from cooperative interactions between STAT6 sites within the mTARC gene promoter. Significantly, we have shown that transfer of the nine most proximal mTARC STAT6 sites in their endogenous conformation confers potent (up to 130-fold) IL-4 inducibility on heterologous promoters. These promoter elements constitute important and sensitive IL-4-responsive transcriptional units that could be used to drive transgene expression in sites of Th2 inflammation in vivo.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK. kateliddiard@lycos.co.uk <kateliddiard@lycos.co.uk>
authors.forename: Kate
authors.initials: K
authors.lastname: Liddiard
authors.forename: John S
authors.initials: JS
authors.lastname: Welch
authors.forename: Jean
authors.initials: J
authors.lastname: Lozach
authors.forename: Sven
authors.initials: S
authors.lastname: Heinz
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Glass
authors.forename: David R
authors.initials: DR
authors.lastname: Greaves
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D054414:Chemokine CCL17; D019742:Chemokines, CC / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D015847:Interleukin-4 / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050800:STAT6 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C499538:Ccl17 protein, mouse; D054414:Chemokine CCL17; D019742:Chemokines, CC; D009841:Oligonucleotides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D050800:STAT6 Transcription Factor; D015847:Interleukin-4; D008156:Luciferases