Resveratrol inhibits expression and binding activity of the monocyte chemotactic protein-1 receptor, CCR2, on THP-1 monocytes

Abstract

Monocyte chemotactic protein-1 and its receptor, CCR2, play a key role in atherosclerosis. We determined the effect of the polyphenol, resveratrol, on CCR2 and the mechanisms involved. Resveratrol treatment inhibited 125I-MCP-1 binding to THP-1 cells; 31, 56, 84% decrease for 10, 50 and 100 microM resveratrol, in the absence of any effect on receptor affinity. The inhibitory effect of resveratrol on 125I-MCP-1 binding to THP-1 cells and on CCR2 protein expression determined by FACS analysis was attenuated by treatment with L-NAME (NOS inhibitor), PD98059 (MAPK inhibitor) and LY294002 (PI3K inhibitor), whereas neither X/XO (reactive oxygen species generator) nor ICI182780 (estrogen receptor antagonist) had any effect. Concomitant with a decrease in CCR2 protein expression, resveratrol inhibited THP-1 CCR2 mRNA levels, in the absence of any effect on its stability; 26 and 45% inhibition at 10 and 50 microM resveratrol, respectively. This effect was not altered by co-treatment with L-NAME, PD98059 or ICI182780, but was potentiated by LY294002 and X/XO.

Conclusions: Resveratrol inhibits monocyte CCR2 binding activity in an NO-, MAPK- and PI3K-dependent manner, whereas it inhibits CCR2 mRNA in an NO- and MAPK-independent, PI3K-dependent manner. These inhibitory effects of resveratrol on chemokine receptor binding and expression may contribute, in part, to its cardiovascular protective activity in vivo.

Figure 1

Effect of resveratrol on ¹²⁵I-MCP-1 binding to THP-1 monocytes. Binding of ¹²⁵I-MCP-1 was determined by equilibrium binding assay as described in Methods. THP-1 cells were treated (6h) with or without Resveratrol (1–100 µM) or ethanol (100 mM) before binding was measured. For these 1 point binding assays, ¹²⁵I-MCP-1 was added at a concentration of 0.1 nM (~Kd) in the absence or presence of 100 nM MCP-1 to determine non-specific binding. Data are means ± SEM; n=3. *P<0.05 vs. control.

Figure 2

(a) Displacement of ¹²⁵I-MCP-1 binding by unlabeled MCP-1 in control and resveratrol (50 µM, 6h) treated THP-1 cells. (b) Data expressed as % Bound. Representative experiment shown, mean of duplicate determinations.

Figure 3

THP-1 monocytes were preincubated for 30 min with the indicated pharmacological antagonists before treatment with or without resveratrol for 6h. Western blot analysis was performed on cell lysates and (a) MAPK activity determined by measuring specific phosphorylated pp44ERK-1 and pp42ERK-2 expression, (b) activation of eNOS determined by measuring phosphorylated eNOS, and (c) PI3K activity was assessed by measuring phosphorylated AKT (phospho AKT). Res = resveratrol (50 µM, unless otherwise indicated), PD=10 µM PD98059, L-NAME (100 µM). Representative Western blots are shown together with the cumulative densitometric data of 3 separate experiments. Data are means ± SEM. *P<0.05 vs control.

Figure 4

THP-1 monocytes were preincubated for 30 min with the indicated pharmacological antagonists before treatment with or without resveratrol for 6h. Binding of ¹²⁵I-MCP-1 was then determined as described in Methods. PD=10 µM PD98059; L–N=100 µM L-NAME; LY=10 µM LY294002; X/XO=500 mM//10 miliunits/ml xanthine/xanthine oxidase; ICI=10 µM ICI182780. Data are means ± SEM, n=4–5. ^*P<0.05 vs control; #P<0.05 vs resveratrol alone.

Figure 5

(a) FACS analysis of control (DMSO) and resveratrol (50 µM) treated THP-1 cells using anti-CCR2 antibody (solid line) or isotype control antibody (dashed line). (b) THP-1 monocytes were preincubated for 30 min with the indicated pharmacological antagonists before treatment with or without resveratrol (50 µM) for 6h. The effect of resveratrol ± the inhibitors on surface CCR2 receptor expression was then determined by FACS analysis. Data are mean ± SEM; n=3–4. *p<0.05 vs control, #p<0.05 vs resveratrol alone.

Figure 6

Effect of Resveratrol on CCR2 protein expression; dose-response and time course. THP-1 cells were treated with or without resveratrol (0.1 – 100 µM) for 24h (top), or with 50 µM resveratrol for different times (2–24h) (bottom). CCR2 protein expression was determined in cell lysates by Western blot analysis. Data are mean ± SEM, n=3–4. *P<0.05 vs control.

Figure 7

Resveratrol inhibition of CCR2 mRNA. (a) Representative northern blot showing the effect of resveratrol (R) (1–100µM) on CCR2 mRNA levels in THP-1 cells. The effect of ethanol (EtOH, 50 mM) is shown for comparison purposes. (b) Effect of resveratrol on the stability of CCR2 mRNA expression. THP-1 cells were treated with or without resveratrol (50 µM) and incubated for various periods of time in the presence of actinomycin D (10 µg/ml). Total THP-1 RNA was isolated and northern blots were performed. Top; representative northern blot; bottom, normalized graph (semi-log plot) with values representing means of 3 separate experiments.

Figure 8

THP-1 monocytes were preincubated for 30 min with the indicated pharmacological antagonists before treatment with or without resveratrol for 6h. Total RNA was isolated and CCR2 mRNA levels analyzed by Northern blot. (a) The effect of the inhibitors alone, (b) the effect of resveratrol ± the inhibitors. Graphs represents cumulative densitometric data from 3 separate experiments. Mean ± SEM, *p<0.05 vs control, #p<0.05 vs resveratrol alone.