IL-33 stimulates human mast cell release of CCL5 and CCL2 via MAPK and NF-κB, inhibited by methoxyluteolin

Abstract

Mast Cells (MCs) are critical for allergic reactions but also play important roles in inflammation, following stimulation by non-allergic triggers such as cytokines. Upon stimulation, MCs secrete numerous newly synthesized mediators, but the mechanism of the release of chemokines, which are important in the pathogenesis of allergic and inflammatory diseases, remains unknown. IL-33 is an “alarmin”, known to augment allergic stimulation of MCs, but its effect on the release of chemokines is not known. The present work investigated the action of IL-33 on the release of the chemokines CCL5 and CCL2 from human MCs, as well as the inhibitory effect of the flavonoid 3′,4′,5,7-tetramethoxyflavone (methoxyluteolin). Stimulation of cultured human MCs (LAD2) and primary MCs (hCBMCs) by IL-33 (1–100 ng/ml) increased the gene expression and the release of CCL5 ( P < 0.0001) and CCL2 ( P < 0.01). Stimulation with IL-33 (10 ng/ml) activated MAPK components, as shown by phosphorylation of p38α MAPK, JNK, and c-Jun using Western blot analysis. Inhibition of these responses by known inhibitors confirmed that CCL5 and CCL2 are stimulated by the activation of p38α MAPK, JNK, and IκB-α. The gene expression and the release of CCL5 and CCL2 stimulated by IL-33 were significantly inhibited by 2 h pre-treatment with methoxyluteolin (10, 50, 100 μM). The inhibition by methoxyluteolin (50 μM) was not mediated via MAPK inhibition as phosphorylated p38α MAPK and JNK expression were not affected. In conclusion, IL-33 plays an important role in chemokine release from human MCs and that is by activation of more than one signaling pathway. The inhibitory effect of methoxyluteolin may indicate that it can be developed as a novel treatment for inflammatory diseases. Diagrammatic Representation of IL-33 stimulates MCs release of CCL5 and CCL2 and the Proposed Points of Inhibition by Methoxyluteolin. IL-33 binds to the ST2 and IL-1 receptor accessory protein (IL-1RAcP) receptors leading to recruitment of MyD88 and TRAF6, which leads to activation of NF-κB and MAPK and the release of CCL5 and CCL2. Inhibitors of p38 MAPK, JNK, or IκB-α reduces release of CCL5 and CCL2. Pre-treatment with methoxyluteolin also inhibits CCL5 and CCL2 gene expression and release. However, inhibition of the release of CCL5 and CCL2 does not occur via the MAPK pathway but rather via NF-κB as previous work by our laboratory has shown the inhibitory effect of methoxyluteolin on Iκβ-α and NF-κB induction at both the transcriptional and translational levels in LAD2.