Resveratrol attenuates CXCL11 expression induced by proinflammatory cytokines in retinal pigment epithelial cells

Abstract

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1β and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.

Keywords: Age-related macular degeneration; CXCL11; Nuclear factor-kappa B; Resveratrol; Retinal pigment epithelium.

Fig. 1

Effect of resveratrol on the production of chemokine CXCL11 by RPE cells exposed to IFN-γ, IL-1β and TNF-α. (A) ARPE-19 cells were treated for 16 h with different combinations of proinflammatory cytokines (IFN-γ, 10 u/ml; IL-1β, 1 ng/ml and TNF-α, 1 ng/ml) as indicated, and CXCL11 mRNA expression was analyzed by real-time PCR. (B) The cells were treated for 16 h with different concentrations of proinflammatory cytokines (1X = IFN-γ, 10 u/ml + IL-1β, 1 ng/ml + TNF-α, 1 ng/ml) and the CXCL11 mRNA expression was analyzed by real-time PCR. (C) The CXCL11 protein secretion into culture medium from the cells treated with increasing concentrations of proinflammatory cytokines was analyzed by ELISA. (D) The cells were treated with the proinflammatory cytokine mixture (Cyt) consisting of IFN-γ (10 u/ml), IL-1β (1 ng/ml) and TNF-α (1 ng/ml) for 16 h in the presence or absence of 50 μM resveratrol (Res) and the CXCL11 mRNA expression analyzed by real-time PCR. (E) The secretion of CXCL11 protein into culture medium under conditions described above was estimated by ELISA. (F) Cell extracts (20 μg protein) prepared from ARPE-19 cells treated for 16 h with proinflammatory cytokines in the presence or absence of resveratrol were analyzed by Western immunoblotting using anti-phospho-NF-κB p65 antibody. Actin was used as the gel loading control. The histogram shows the relative signal intensities of pNF-κB immunoreactive bands from three separate blots after normalization with actin. (G) NF-κB p65 transcription factor assay was performed using cell extracts (12 μg protein) prepared from ARPE-19 cells treated for 16 h with the proinflammatory cytokines in the presence or absence of resveratrol. * = p < 0.05 when compared to control; ** = p < 0.05 when compared to Cyt; n = 4 for histograms A, B, C, D, E & G while n = 3 for histogram F.

Fig. 2

Effect of resveratrol on the production of chemokines CXCL9, CXCL10, CCL2 and CCL5 by RPE cells exposed to IFN-γ, IL-1β and TNF-α. ARPE-19 cells pre-incubated with or without 50 μM resveratrol (Res) were treated with cytokine mixture (Cyt) consisting of IFN-γ (10 u/ml), IL-1β (1 ng/ml) and TNF-α (1 ng/ml) for 16 h. Chemokine mRNA expression was analyzed by real-time PCR while the chemokine protein secretion into the culture medium was estimated by ELISA. (A) CXCL9 mRNA expression. (B) CXCL9 protein secretion. (C) CXCL10 mRNA expression. (D) CXCL10 protein secretion. (E) CCL2 mRNA expression. (F) CCL2 protein secretion. (G) CCL5 mRNA expression. (H) CCL5 protein secretion. * = p < 0.05 when compared to control, ** = p < 0.05 when compared to Cyt; n = 4.