Regulation of IL-1-induced selective IL-6 release from human mast cells and inhibition by quercetin

Abstract

Mast cells are involved in allergic reactions, but also in innate immunity and inflammation. Crosslinkage of mast cell Fc immunoglobulin E receptors (FcvarepsilonRI) by multivalent antigen triggers secretion of granule-stored mediators, as well as de novo synthesis of cytokines, including interleukin (IL)-6. We showed recently that the proinflammatory cytokine IL-1 stimulates human leukemic mast cells (HMC-1) and human umbilical cord blood-derived cultured mast cells (hCBMCs) to release newly synthesized IL-6 without tryptase in the absence of degranulation. Here, we investigated several signal-transduction pathways activated by IL-1 leading to IL-6 production by HMC-1 and hCBMCs. We also investigated the effect of the flavonol quercetin that was recently shown to strongly inhibit IL-6 secretion in response to allergic stimulation from hCBMCs.IL-1 stimulated p38, but did not activate extracellular signal-regulated kinase (ERK) or c-jun N-terminal kinase (JNK); it also did not activate protein kinase C (PKC) isozymes alpha, beta, mu and zeta, except for PKC-theta, which was phosphorylated. The p38 inhibitor SB203580 and the PKC inhibitors Calphostin C and Gö6976 completely inhibited IL-1-induced IL-6 production. Quercetin 1-100 microM inhibited IL-1-induced IL-6 secretion, p38 and PKC-theta phosphorylation in a dose-dependent manner. These results indicate that IL-1-stimulated IL-6 production from human mast cells is regulated by biochemical pathways distinct from IgE-induced degranulation and that quercetin can block both IL-6 secretion and two key signal transduction steps involved.

Figure 1

IL-1 selectively stimulates phosphorylation of p38 MAP kinase in hCBMCs. The phosphorylation status of MAP kinases was determined in unstimulated and IL-1α (50 ng ml⁻¹)- or anti-IgE (10 μg ml⁻¹)-treated hCBMCs by using ELISA. Amounts of phosphorylated and total MAPKs were determined; data were normalized and expressed as fold-induction. Cells were stimulated for 3 min for p38 (a; n=5), 5 min for ERK (b; n=8) and 15 min for JNK (c; n=4) assay, conditions for optimal anti-IgE activation of MAPKs. Data is mean±s.e.m. of the number of experiments shown in the parentheses above, ^*P<0.05 for each experimental condition compared to control; spont=spontaneous; a-IgE=anti-IgE.

Figure 2

Kinetics of p38 phosphorylation stimulated by IL-1. The phosphorylation of p38 in IL-1-treated (a) hCBMCs (50 ng ml⁻¹) or (b) HMC-1 cells (10 ng ml⁻¹) determined by ELISA. p-p38, phosphorylated p38. Representative experiment from four experiments in duplicate is shown; data are normalized against total p38 expression.

Figure 3

Inhibition of IL-1-stimulated IL-6 production by the p38 inhibitor SB203580. Cells were pretreated with SB203580 for 30 min prior to stimulation. (a) hCBMCs were stimulated for 6 h with 50 ng ml⁻¹ of IL-1 (n=5) and (b) HMC-1 cells were stimulated for 18 h with 10 ng ml⁻¹ of IL-1 (n=5). Data=mean±s.e.m., P<0.05 for all concentrations, except for 1 μM SB253080, compared to IL-1 alone. Please note IL-6 units differ as they are listed as pg per 5 × 10⁵ for hCBMCs, but as pg per 10⁶ for HMC-1 cells due to the abundance of the latter. MC=mast cells.

Figure 4

Inhibition of IL-1-stimulated IL-6 production by PKC inhibitors. Cells were pretreated with different concentrations of Calphostin C (a, b) for 30 min prior to stimulation with IL-1 (50 ng ml⁻¹) for 6 h for hCBMCs or 18 h for HMC-1 cells (10 ng ml⁻¹). Results are expressed as mean±s.e.m., n=4, P<0.05 for all concentrations. MC=mast cells.

Figure 5

Effect of IL-1 on activation of PKC isozymes in HMC-1 cells. (a, b) HMC-1 cells were stimulated with IL-1 (10 ng ml⁻¹), PMA (25 ng ml⁻¹) or PMA and calcium ionophore A23187 (0.5 μg ml⁻¹); the cytosolic (C) and membrane (M) fractions were separated and immunoblotted by antibodies to PKC-α, PKC-β or PKC-ζ. (c) HMC-1 cells were stimulated with IL-1 (10 ng ml⁻¹) or PMA (25 ng ml⁻¹) for 60 min. Lysates were immunoblotted with antibodies to p-PKC-α, p-PKC-μ, p-PKC-ζ, p-PKC-θ or actin; P=phospho; P+I=PMA and ionophores. The arrows indicate the respective phospho proteins. There was only one loading control for both cytosolic and membrane fractions for all blots. Each gel is representative of three similar ones.

Figure 6

Effect of quercetin on IL-1-induced (a) IL-6 release (n=4), (b) PKC-θ phosphorylation. Incubation of HMC-1 cells with IL-1 (10 ng ml⁻¹) stimulated phosphorylation of PKC-θ (Thr538). HMC-1 cells were preincubated with quercetin (1 or 10 μM) for 15 min prior to incubation with IL-1 (10 ng ml⁻¹) for 30 min. Equal loading was verified by immunoblotting the same membrane with antibody to actin (representative gel of three similar ones). Q=quercetin.

Figure 7

Effect of quercetin on IL-1-induced p38 phosphorylation. HMC-1 cells were preincubated with quercetin (0.1, 1 or 10 μM) for 15 min prior to stimulation with IL-1 (10 ng ml⁻¹) in the presence or absence of quercetin (0.1, 1 or 10 μM) for another 3 min. Cell lysates were then prepared and the phosphorylation status of p38 MAPK was determined by ELISA. Amounts of phosphorylated and total p38 was determined; data were normalized and expressed as fold-induction. Q=quercetin. Data represented as mean±s.e.m. (n=3). ^*P=0.05 versus control, ^**P<0.05 versus IL-1.