Resveratrol Suppresses Tumor Necrosis Factor- -Induced Fractalkine Expression in Endothelial Cells

Up-regulation of fractalkine is involved in vascular and tissue damage in inflammatory conditions. Resveratrol has been shown to have anti-inflammatory, antioxidant, and antitumor activities. Its regulatory effects on expression of fractalkine in vascular endothelial cells and fractalkine receptor CX3CR1 in monocytes have not been studied. We evaluated the effects of resveratrol on fractalkine expression in human umbilical vein endothelial cells and CX3CR1 expression in THP-1 cells in response to treatment with tumor necrosis factor (TNF). TNFsignificantly induced fractalkine mRNA and protein expression in endothelial cells. Resveratrol strongly suppressed TNF-induced fractalkine expression in endothelial cells through suppression of nuclear factorB and Sp1 activities. Resveratrol decreased the number of TNF-induced fractalkine-positive endothelial cells and CX3CR1-positive cells determined by flow cytometric analysis. Resveratrol suppressed TNF-stimulated monocytes adhesion to human umbilical vein endothelial cells. Immunohistochemical analysis revealed that resveratrol suppressed TNF-induced arterial endothelial fractalkine expression in heart, kidney, and intestine and decreased ED-1-positive cell infiltration in intestinal villi. Resveratrol may provide a new pharmacological approach for suppressing fractalkine/CX3CR1-mediated injury in inflammatory conditions. Adherence of circulating inflammatory cells and migration to the subendothelial space is an initial process in inflammatory conditions (Gimbrone et al., 1997; Cines et al., 1998). Chemokines in endothelial cells and those receptors in inflammatory cells are critical in the initiation, maintenance, and resolution of inflammation (Fujiwara and Kobayashi, 2005). Fractalkine (CX3CL1) is the only CX3C-chemokine in which a soluble chemokine-like domain is fused to a mucin stalk that extends across the cell membrane into the cytoplasm (Bazan et al., 1997; Pan et al., 1997). The expression of membrane-bound fractalkine can be markedly induced on primary endothelial cells by inflammatory cytokines, such as interferon, interleukin-1 and tumor necrosis factor (TNF)(Garcia et al., 2000). Fractalkine can function as both a chemoattractant and an adhesion molecule for its receptor, CX3CR1 (Imai et al., 1997; Haskell et al., 1999). Expression of CX3CR1 and migration toward fractalkine have been demonstrated for monocytes/macrophages, some T cells, and natural killer cells (Imai et al., 1997; Fong et al., 1998; Harrison et al., 1998; Foussat et al., 2000). Thus, fractalkine expressed on inflamed endothelium may be a vascular gateway for CX3CR1-expressing cells by rapidly capturing them from the blood and promoting their migration into tissue. Because up-regulation of fractalkine is involved in vascular and tissue damage in various diseases, such as atherosclerosis (Lesnik et al., 2003), glomerulonephritis (Segerer et al., 2002), cardiac allograft rejection (Robinson et al., 2000), HIV infection (Foussat et al., 2001), and rheumatoid arthritis (Nanki et al., 2004), down-regulation of fractalkine expression can be important in preventing and treating these diseases. Only a few substances—the soluble form of interleukin-6 receptor(Matsumiya et al., 2001) and 15deoxy-prostaglandin J2 (Imaizumi et al., 2002)—and hypoxia (Yamashita et al., 2003) were reported to inhibit This work was supported by grants from the National Research Laboratory Program of Korea Science and Engineering Foundation (S.K.P.) and Korea Research Foundation grant KRF-2004-041-E00031 (W.K.). S.O.M. and W.K. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.022392. ABBREVIATIONS: TNF, tumor necrosis factor; HIV, human immunodeficiency virus; NF, nuclear factor; HUVEC, human umbilical vein endothelial cell; PD98059, 2 -amino-3 -methoxyflavone; MG-132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; H-89, N-[2-(4-bromocinnamylamino)ethyl]-5isoquinoline; RPA, RNase protection assay; EMSA, electrophoretic mobility shift assay. 0026-895X/06/7001-112–119$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 1 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 22392/3121936 Mol Pharmacol 70:112–119, 2006 Printed in U.S.A. 112 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from fractalkine expression in endothelial cells. We have reported that -lipoic acid reduces fractalkine-mediated inflammatory processes in endotoxemia (Ahn et al., 2004; Sung et al., 2005). It is well known that CX3CR1 is expressed in monocytes/ macrophages and is the mechanism of monocytes/macrophages capture, firm adhesion, and activation (Fong et al., 1998). A polymorphism in the CX3CR1 gene was also reported to be associated with low CX3CR1 expression and reduced risk of acute coronary disease in humans (Combadiere et al., 2003). Because fractalkine and CX3CR1 are critical to inflammation, therapeutic interventions that target fractalkine in endothelial cells and CX3CR1 in monocytes/macrophages may open new avenues for controlling inflammatory diseases. Resveratrol (trans-3,4 ,5-trihydroxy-trans-stilbene), a polyphenolic compound present in grapes and red wine, has been reported to have a cardioprotective effect via an antioxidant effect (Frankel et al., 1993; Fauconneau et al., 1997), cancer chemopreventive activity (Jang et al., 1997), suppression of cellular smooth muscle proliferation/migration (Araim et al., 2002), and inhibition of platelet aggregation (PaceAsciak et al., 1996). At concentrations present in human plasma after moderate wine consumption, resveratrol inhibits the expression of adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin by agonist-stimulated endothelial cells (Bertelli et al., 2001; Pendurthi and Rao, 2002). Ferrero et al. (1998) also have reported that resveratrol reduces granulocyte and monocyte adhesion to endothelial cells. The anti-inflammatory activity of resveratrol may be related to interference with the nuclear factor (NF)B signaling pathway, which regulates the expression of various genes involved in inflammation (Tsai et al., 1999; Manna et al., 2000). However, effects of resveratrol on fractalkine expression and its signal pathway in endothelial cells and on CX3CR1 expression in monocytes/macrophages have not been examined. In this study, we examined whether resveratrol decreases the expressions of TNF-induced fractalkine in human umbilical vein endothelial cells (HUVECs) and TNF-induced CX3CR1 in THP-1 cells. Furthermore, we evaluated the role of resveratrol in TNF-induced endothelial fractalkine expression in vivo. Our results show that resveratrol prevents TNF-induced up-regulation of fractalkine in endothelial cells and CX3CR1 in monocytes/macrophages, identifying a novel mechanism of resveratrol for preventing vascular inflammation. Materials and Methods Materials and Cell Culture. Anti-fractalkine antibody (fulllength) and anti-CX3CR1 antibody were purchased from Torrey Pines BioLabs (Houston, TX). Anti-phospho-p65 (Ser536) and antip65 antibodies were from Cell Signaling Technology (Beverly, MA). Anti-p65, anti-p50, and anti-Sp1 for gel supershift assay were from Santa Cruz Biotechnology (Santa Cruz, CA). Mitogen-activated protein kinase kinase 1/2 inhibitor PD98059, NFB inhibitor MG-132, protein kinase C inhibitor calphostin C and H-89 were purchased from Calbiochem (San Diego, CA). Resveratrol, the phosphatidylinositol 3 -kinase inhibitor wortmannin, the NFB inhibitor pyrrolidine dithiocarbamate, TNF, media, sera, and most other biochemical reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. HUVECs were prepared from human umbilical cords by collagenase digestion as described previously (Ahn et al., 2004). THP-1 cells were from American Type Culture Collection