Quercetin blocks airway epithelial cell chemokine expression

Abstract

Quercetin (3,3',4',5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase-dependent mechanisms. Pretreatment with quercetin and the PI 3-kinase inhibitor LY294002 each reduced TNF-alpha-induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-alpha-induced PI 3-kinase activity, Akt phosphorylation, intracellular H(2)O(2) production, NF-kappaB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-kappaB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-alpha-induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2alpha, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen-sensitized and -challenged mice. These mice show MCP-1-dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways.

Figure 1.

Effect of quercetin on human bronchial epithelial cell chemokine expression. 16HBE14o- (A and B) and primary cells (C and D) were pretreated with either quercetin, LY294002, or carrier, and stimulated with TNF-α. Release of IL-8 and MCP-1 was assessed by ELISA. Quercetin concentrations as low as 0.1 μM blocked TNF-α–induced IL-8 protein abundance (A and C). Quercetin blocked MCP-1 protein abundance in a similar manner (B and D) (n = 3–4, mean ± SEM; *different from TNF-α, P < 0.001, ANOVA).

Figure 2.

Quercetin inhibits PI 3-kinase/Akt signaling and intracellular reactive oxygen intermediates. Lysates from TNF-α–stimulated 16HBE14o- cells were immunoprecipitated with anti-phosphotyrosine antibody and the precipitates incubated with PI and [γ-³²P]-ATP. Lipids were separated by thin layer chromatography. Both quercetin and LY294002 reduced phosphorylation of PI, indicative of PI 3-kinase activity (A). Quercetin also inhibited Akt phosphorylation in a dose-dependent manner (B). These results were typical of three separate experiments. Pretreatment with LY294002 and quercetin (concentrations as low as 0.1 μM) each decreased TNF-α–induced H₂O₂ concentration (C) (n = 3, mean ± SEM; *different from TNF-α, P < 0.001, ANOVA).

Figure 3.

Effect of quercetin on NF-κB signaling. We examined the effect of quercetin on TNF-α–induced IκB phosphorylation and degradation in 16HBE14o- cells by immunoblotting (A). Quercetin only minimally reduced IκBα Ser32/36 phosphorylation, and only at high concentrations. Subsequently, there was no change in the loss of IκBα protein abundance. Under these circumstances, one would not expect quercetin to attenuate translocation of NF-κB to the nucleus or DNA binding (B). Quercetin and LY294002 in concentration of 10 μM significantly reduced NF-κB promoter activity (C) (n = 3, mean ± SEM; *P < 0.001 compared with TNF-α, ANOVA).

Figure 4.

Effect of quercetin on chemokine transcription. 16HBE14o- bronchial epithelial cells transiently transfected with the −162/+44 fragment of the full-length human IL-8 promoter subcloned into a luciferase reporter, and pretreated either with quercetin (A), LY294002 or vehicle were incubated with TNF-α for an additional 24 h. Quercetin (1μM) significantly reduced IL-8 promoter activity (n = 4, mean ± SEM; *different from TNF-α, P < 0.05, ANOVA). Northern analysis of 16HBE14o- cells pretreated with either quercetin, LY294002, or vehicle, and stimulated with TNF-α for 6 h (B). Quercetin and LY294002 each significantly decreased IL-8 and MCP-1 mRNA levels. 28 and 18 S rRNAs are shown as a loading control. Data are representative of two independent experiments.

Figure 5.

Effect of quercetin on IL-8 and MCP-1 translation. 16HBE14o- bronchial epithelial cells stimulated with TNF-α overnight and treated with actinomycin D in presence or absence of quercetin, LY294002, and vehicle. Quercetin in low concentrations significantly decreased both IL-8 and MCP-1 release (A) (n = 5, mean ± SEM; *P < 0.01 compared with TNF-α, ANOVA). Immunoblot analysis of 16HBE14o- bronchial epithelial cells treated either with quercetin or LY294002 or vehicle in presence of TNF-α (B). Quercetin induced phosphorylation of eIF-2α. Data are representative of three independent experiments.

Figure 6.

Quercetin blocks airways hyperresponsiveness and lung MCP-1 protein abundance in cockroach-sensitized mice. (A) Female adult BALB/c mice were sensitized with cockroach antigen. On the morning of Days 20–22 of the protocol, animals were given 0.06– 2 mg quercetin dihydrate in propylene glycol by mouse gavage feeding needle. On Day 21, all mice underwent intratracheal administration of cockroach allergen. On Day 22, mice were killed for measurement of lung MCP-1 and airway reactivity. Quercetin reduced MCP-1 levels in a concentration-dependent manner (A). Because of inter-experiment differences in ovalbumin challenge–induced MCP-1 levels, data are represented as a fraction of the mean MCP-1 level in challenged mice (n = 3–10, mean ± SEM; *different from vehicle alone, P ⩽ 0.011, ANOVA). Quercetin also significantly reduced peak airways resistance in response to methacholine (250 μg/kg), a measure of airway reactivity (n = 4– 11, mean ± SEM; *different from vehicle alone, P = 0.029, ANOVA). (B) Because of inter-experiment differences in methacholine-induced peak airways resistance, data from two cohorts of mice are shown separately.

Figure 7.

Measurement of quercetin metabolites. (A) The separation and quantification of flavonoids was performed by HPLC with coulometric electrochemical detection. A typical chromatogram including samples from treated mice and a standard is shown. Analysis revealed the presence of one large peak corresponding to a quercetin standard and minor peaks that may represent O-methylated or oxidation products of quercetin. (B) Serum quercetin concentrations, group mean data (n = 3–5, mean ± SEM; *different from vehicle alone, P = 0.008, ANOVA).