Suppressive Effects of T-412, a Flavone on Interleukin-4 Production in T Cells

Abstract

Flavonoids are comprised of a large group of low molecular weight polyphenolic compounds having diverse pharmacological effects, 1,2) and among them their possible use as anti-allergic agents have been proposed. [3][4][5] Intakes of naturally occurring flavonoids have been reported to reduce the allergic symptoms and severities in human 6) as well as development of allergic diseases in animals, 7) and it was suggested that the effects might be mediated by the suppression of immune responses caused by Th2 dominance. 5-7)Interleukin (IL)-4 is a representative Th2 cytokine, which is mainly produced by activated Th2 cells. IL-4 not only initiates the development of Th2 cells from Th0 cells but also induces IgE class switching in B cells, resulting in unbalanced Th2 skewing immune system. 8) Furthermore, it plays a central role in the pathogenesis of various allergic inflammations by promoting production of proinflammatory cytokines such as 10) The expression of IL-4 is tightly regulated at the transcriptional level. The inducible transcription factors including nuclear factor of activated T cells (NF-AT), activating protein-1 (AP-1), nuclear factor-kappa B (NF-kB) as well as c-Maf have been identified to control IL-4 and their cooperative interactions on the promoter appears to be critical to induce IL-4 expression in T cells.11,12) Upon T cell activation by PMA and ionomycin (PI), cytoplasmic NF-ATs are dephosphorylated and translocated to the nucleus; the dephosphorylated proteins show increased affinity for P sites (from P0-P4), DNA binding sites of NF-ATs, [13][14][15] which is mediated by increased calcium concentration and protein kinase C (PKC)-RAS signaling pathways.16) NF-AT and AP-1 protein (c-Fos and c-Jun) coordinately bind the P1 site, which is required for full activity of the IL-4 promoter.17-19) NF-kB, generally consisting of p65-p50 dimer, is sequestered into an inactive form by the cytoplasmic inhibitor of NF-kB (IkB) in resting T cells, however active forms of NF-kB translocate to nucleus, where the IL-4 expression is regulated together with other transcription factors.20) Furthermore, the proto-oncogene c-Maf, which is expressed in Th2 cells but not in Th1 cells, binds to a c-Maf response element (MARE) adjacent to a P0 site in synergy with the NF-AT to activate IL-4 production. 21,22)As an effort to find out novel therapeutics from natural sources we screened the down-regulatory effects of several novel flavonoids on IL-4 using PI-activated EL-4 thymoma, a murine T cell line. Among them a flavone, named T-412 (7,8,4Ј-trihydroxyflavone), had significant down-regulatory effects on IL-4 production in a dose-and time-dependent manner, which was accompanied by the suppression of the expression level of IL-4 mRNA. Furthermore, the proximal promoter activities of IL-4 were also suppressed. To understand the mode of action for T-412 at the molecular level when we performed Western blot analysis for selective transcription factors involving in the activation of proximal promoter, the expression of NF-ATc...

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