trans-Resveratrol, an extract of red wine, inhibits human eosinophil activation and degranulation

Abstract

Background and purpose: trans-Resveratrol, a non-flavonoid polyphenol found abundantly in red wine possesses antiproliferative and anti-inflammatory activity in various inflammatory disease conditions. However, the effect of trans-resveratrol on eosinophil activation in relation to allergy has not been investigated.

Experimental approach: Human eosinophils were isolated and purified from whole blood and incubated for 16 h with trans-resveratrol. Eosinophil chemotaxis, activation and degranulation, and apoptosis were investigated. The effect of trans-resveratrol on the inhibition of p38 and ERK1/2 activation was examined.

Key results: Treatment of human eosinophils with trans-resveratrol at concentrations <100 microM for 16 h did not induce eosinophil apoptosis. Similar results were seen after 24 h and 48 h incubations. trans-Resveratrol (<100 microM) significantly inhibited eosinophil peroxidase release after activation with IL-5 (IC(50)=2.9+/-0.9 microM) or C5a (IC(50)=3.9+/-0.5 microM) after 5 min priming with cytochalasin B (CB). Similarly, the production of leukotriene C4 after stimulation with calcium ionophore, and eosinophil chemotaxis in response to eotaxin, as well as CD11b upregulation and CD62 L shedding was also significantly reduced by trans-resveratrol, at concentrations above 5 microM. All the activators induced p38 and ERK1/2 phosphorylation maximal at 2 min of activation. trans-Resveratrol potently inhibited p38 and ERK1/2 activation after calcium ionophore and CB and C5a activation.

Conclusions and implications: trans-Resveratrol is effective at inhibiting human eosinophil activation and degranulation at concentrations <100 microM, while not inducing apoptosis. This potent anti-inflammatory activity of trans-resveratrol and possibly its metabolites on eosinophils may be worth investigating for the treatment of eosinophil-related allergic diseases.

Figures

Figure 1

Effect of 16 h trans-resveratrol treatment on cell viability and apoptosis. Eosinophils were treated with or without trans-resveratrol (0–500 μM) and with or without human recombinant interleukin (IL)-5 (0–10 ng mL−1), after which the cells were immediately stained with annexin-V-FITC and propidium iodide (PI) and analysed by flow cytometry. (a) Cell viability was expressed as the percentage of viable (annexin-V-FITC negative and PI negative) in the test sample. (b) Early apoptosis was expressed as the percentage of early apoptotic cells (annexin-V-FITC positive and PI negative) in the test sample. Data are means±s.e.mean percent of n=5–7 per treatment group relative to the vehicle control. *P<0.05 and **P<0.01 versus untreated dimethylsulphoxide control.

Figure 2

Effect of longer (24 and 48 h) trans-resveratrol treatment on eosinophil apoptosis. Eosinophils were treated with or without trans-resveratrol (0–100 μM) and treated as indicated in Figure 1 for (a) 24 or 48 h (b). Data are means±s.e.mean percent of n=4 per treatment group relative to the vehicle control. Values were not statistically significant versus dimethylsulphoxide control.

Figure 3

Effect of trans-resveratrol treatment on caspase-3 activity Eosinophils were treated with trans-resveratrol (0–500 μM) for 16 h and cell lysates were obtained for caspase-3 activity assay. The results were expressed in relative fluorescence unit (RFU) and normalized against the corresponding total protein concentration (mg) in each sample. All measurements were performed in duplicate. Data are means±s.e.mean RFU mg−1 of n=5 per treatment group. **P<0.001 versus untreated dimethylsulphoxide control.

Figure 4

trans-Resveratrol inhibits degranulation and mediator production from activated eosinophils. Eosinophils were treated with trans-resveratrol (0–100 μM) for 16 h and activated with (a) cytochalasin B (CB, 5 μg mL−1) for 5 min followed by C5a (10−8 M) or interleukin (IL)-5 (20 ng mL−1) and eosinophil peroxidase (EPO) release was measured and normalized against total cellular EPO. (b) 10−5 M calcium ionophore A23187 for 3 h and the supernatants were collected for LTC4 ELISA. Data are expressed in the amount of LTC4 released (pg mL−1). Data are means±s.e.mean percent of n=5 per treatment group in triplicate. *P<0.05, **P<0.01 versus the untreated activated control after ANOVA analysis (P<0.01).

Figure 5

trans-Resveratrol reduced eosinophil chemotaxis towards eotaxin. Eosinophils were treated with trans-resveratrol (0–100 μM) or dexamethasone (1 μM) for 16 h before the chemotaxis assay was performed with the ChemoTx chemotaxis system. Eotaxin (10 ng mL−1) was used as the chemoattractant. All measurements were performed in triplicate. The data were expressed as the migration index, which was normalized against the number of randomly migrated cells in the vehicle control sample. Data are means±s.e.mean of the migration index of n=5 per treatment group. *P<0.05 and **P<0.01 versus untreated vehicle control after ANOVA analysis (P<0.01).

Figure 6

trans-Resveratrol inhibits CD11b upregulation and CD62L shedding in activated eosinophils. Eosinophils were treated with trans-resveratrol (0–100 μM) or dexamethasone (1 μM) for 16 h and eosinophils were either immunostained immediately or activated with 10−5 M calcium ionophore A23187 for 1 h before (a) CD11b-PE or (b) CD62L-PE immunostaining and analysed by flow cytometry. The data are represented in mean fluorescence intensity (MFI) after isotype control was subtracted. Data are means±s.e.mean of n=7 per treatment group. *P<0.05, **P<0.01 versus the untreated activated control after ANOVA analysis (P<0.01).

Figure 7

p38 and ERK1/2 MAP kinase phosphorylation in activated eosinophils. Eosinophils were cultured in vitro for 16 h and stimulated with (a) CB+10−8 M C5a (as described in Figure 3), (b) 10−5 M calcium ionophore A23187or (c) 10 ng mL−1 eotaxin for 0, 0.5, 2, 5 or 10 min. After the indicated length of activating time, the cells were harvested and lysed for western blot analysis, as described in the Materials and methods. The same membrane was used to detect phospho- and total p38, phospho- and total ERK1/2 and β-actin. Representative images of five independent sets of experiments are shown. The bar graphs below each blot summarize (n=5) the fold change of phospho p38 (d) or ERK1/2 (e) versus no activation control after normalization to total p38 or ERK1/2, respectively. *P<0.05, **P<0.01 versus the untreated activated sample after ANOVA analysis (P<0.01).

Figure 8

Effect of trans-resveratrol on p38 and ERK1/2 MAP kinase phosphorylation. Eosinophils were treated for 16 h with the indicated concentrations of trans-resveratrol before stimulation with CB+C5a (a,d), A23187 (b,e) or eotaxin (c,f) for 2 min (as described in Figure 7). The same membrane was used to detect phospho-p38/total p38, phospho-ERK1/2/total ERK1/2 and β-actin. Representative images of four independent sets of experiments are shown. The bar graphs below each blot summarize (n=4) the fold change of phospho p38 or ERK1/2 versus no activation control after normalization to total p38 or ERK1/2, respectively. #P<0.05, ##P<0.01 versus the negative control after ANOVA analysis (P<0.01); *P<0.05, **P<0.01 versus the untreated activated sample after ANOVA analysis (P<0.01).