INTRODUCTION

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INFLAMMATORY RESPONSES elicited by a variety of stimuli in the vascular endothelium have been implicated in the development of atherosclerosis. For example, the recruitment of inflammatory cells such as monocytes and macrophages and their migration throughout the endothelium are thought to be critical early pathological events in atherogenesis. These processes are directly promoted by chemokines, which were shown in recent studies to be closely related to the progression of atherosclerotic processes (14, 28). Chemokines can be divided into two subfamilies, CXC and CC chemokines, based on structural and genetic considerations (2). Human monocyte chemoattractant protein-1 (MCP-1), a 76-amino acid protein with an NH₂-terminal pyroglutamic acid, is a member of the CC chemokine family and plays a crucial role in monocyte chemotaxis and transmigration. A compelling body of evidence indicates the potential role of MCP-1 in the pathogenesis of atherosclerosis. Both MCP-1 protein and mRNA expression have been detected in early atherosclerotic lesions by immunostaining, Northern blot analysis, and in situ hybridization (24, 32, 36, 43). Furthermore, MCP-1 deficiency significantly reduced atherosclerosis in low-density lipoprotein (LDL) receptor-deficient mice fed a high-cholesterol diet (13). In a similar study, the selective absence of CCR2, the receptor for MCP-1, markedly decreased atherosclerotic lesion formation in apolipoprotein E (apoE)-deficient mice (5). On the other hand, Aiello et al. (1) reported that overexpression of MCP-1 accelerated atherosclerosis in apoE-knockout mice. These studies strongly support the idea that the MCP-1-mediated inflammatory environment in the vascular endothelium is critical for the initiation and development of atherosclerosis.

MCP-1 is expressed and released by a variety of cell types, including vascular endothelial cells, smooth muscle cells, monocytes/macrophages, and fibroblasts, in response to various stimuli such as inflammatory cytokines, lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), and interferon- (IFN-) (6, 29, 34, 37, 42, 44). Evidence suggests that the expression of human MCP-1 might be regulated by redox mechanisms. For example, it was demonstrated that red wine with high antioxidant capacity can inhibit MCP-1 expression and reduce neointimal thickening after balloon injury of the aorta in cholesterol-fed rabbits (10).

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 (TH2) lymphocytes, eosinophils, and mast cells (26, 27). IL-4 promotes the differentiation of premature lymphocytes to the TH2 subset and induces immunoglobulin class switching in B lymphocytes. In addition, IL-4 is present at high levels in tissues of patients with chronic inflammatory disease, including atherosclerotic lesions (23, 25, 30). Evidence indicates that IL-4 may play a role in atherogenesis through induction of inflammatory responses, such as upregulation of vascular cell adhesion molecule-1 (VCAM-1) (11, 19, 38) and MCP-1 (29). IL-4 may also be considered as a prooxidative cytokine that can increase the oxidative potential of target cells (7, 19, 20).

Although recent evidence indicates that IL-4 may stimulate the synthesis and secretion of MCP-1 in human endothelial cells, the molecular regulatory mechanism of MCP-1 expression by this cytokine is not yet fully understood. We investigated the molecular signaling pathway of IL-4-mediated upregulation of MCP-1 gene transcription and expression in human vascular endothelial cells. In addition, the present study also focused on the possible involvement of an antioxidant-sensitive mechanism in this process. We demonstrate that IL-4 can trigger transcription factor signal transducers and activators of transcription 1 (STAT1)-mediated molecular signaling pathway in human vascular endothelial cells, leading to overexpression of human MCP-1 production.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Endothelial cell cultures. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described previously (40). HUVEC were cultured in enriched medium 199 (M199) supplemented with 20% fetal calf serum, 1% each of penicillin-streptomycin, glutamine, and antibiotic-antimycotic, heparin (300 µg/ml, GIBCO-BRL; Grand Island, NY), HEPES (6 mg/ml, Sigma Chemical; St. Louis, MO), and endothelial cell growth supplement (40 µg/ml, Collaborative Research; Bedford, MA) in 5% CO₂ at 37°C. Cells were determined to be endothelial by their cobblestone morphology and uptake of fluorescent-labeled acetylated LDL (1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate, Molecular Probes; Eugene, OR). HUVEC from passage 2 were used in all experiments.

RT-PCR. Total RNA was extracted with the use of TRI reagent (Sigma) and reverse transcribed at 42°C for 60 min in 20 µl of 5 mM MgCl₂, 10 mM Tris · HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1 mM dNTP, 1 U/µl of recombinant RNasin ribonuclease inhibitor, 15 U/µg of avian myeloblastosis virus reverse transcriptase, and 0.5 µg of oligo(dT)₁₅ primer (40). For amplification of MCP-1 and of -actin (a housekeeping gene), the following primer combinations were used: 5'-CAGCCAGATGCAATCAATGC-3' and 5'-GTGGTCCATGGAATCCTGAA-3' (MCP-1; expected 198-bp fragment; R&D Systems; Minneapolis, MN) and 5'-AGCACAATGAAGATCAAGAT-3' and 5'-TGTAACGCAACTAAGTCATA-3' (-actin; expected 188-bp fragment) (3). The PCR mixture consisted of a Taq PCR Master Mix Kit (Qiagen; Valencia, CA), 2 µl of the reverse transcriptase reaction, and 20 pmol of primer pairs in a total volume of 50 µl. Thermocycling was performed according to the following profile: 94°C for 4 min before the first cycle, 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s, repeated 20 times followed by a final extension at 72°C for 10 min. Amplification was linear within the range of 15-25 cycles. PCR amplification of MCP-1 and -actin mRNA was performed in separate tubes. PCR products were separated by 2% agarose gel electrophoresis, stained with SYBR Green I (Molecular Probes; Eugene, OR), and visualized using phosphoimaging technology (FLA-2000, Fuji; Stamford, CT).

Measurement of MCP-1 production. MCP-1 concentrations in cell culture supernatants were determined by using a Quantikine Human MCP-1 Immunoassay kit (R&D Systems) according to the manufacturer's recommendations. This assay employs the quantitative sandwich enzyme immunoassay technique using a murine monoclonal antibody against human MCP-1 and a polyclonal secondary antibody conjugated with horseradish peroxidase. The minimum detectable concentration of MCP-1 was <5.0 pg/ml.

Electrophoretic mobility shift assay. Nuclear extracts from HUVEC were prepared according to the method of Beg et al. (4). Binding reactions were performed in a 20-µl volume containing 4 µg of nuclear protein extracts, 10 mM Tris · HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, 2 µg of poly[dI-dC] (nonspecific competitor), and 40,000 counts/min of ³²P-labeled specific oligonucleotide probe. Double-stranded oligonucleotides containing the GAS sequence from human MCP-1 promoter (5'-CCTTAAGCTTTTCCTGGAAATCCACAGGATGC-3') (44) were radiolabeled with [-³²P]ATP (Amersham Pharmacia Biotech; Piscataway, NJ) using T4 polynucleotide kinase. Resultant protein-DNA complexes were resolved on native 5% polyacrylamide gels using 0.25× TBE buffer (consisting of 50 mM Tris · HCl, 45 mM boric acid, and 0.5 mM EDTA; pH 8.4). Competition studies were performed by the addition of a molar excess of unlabeled oligonucleotide to the binding reaction. Rabbit polyclonal anti-STAT1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and employed in supershift experiments. The intensity of the bands corresponding to specific STAT1-DNA binding was determined using UN-SCAN-IT gel image analysis software (Silk Scientific; Orem, UT). The values of relative pixel intensity were given below each image.

Transfection and dual luciferase assays. Transient transfections of HUVEC were performed using pFx-7 (Invitrogen; Carlsbad, CA) as described earlier (18) with modifications (19). Cells were transfected with 10 µg of the firefly luciferase (Luc) reporter plasmids containing the human MCP-1 promoter (pMCP) sequences with wild-type or mutated GAS (mGAS) site (pMCP[213]Luc and pMCP[213, mGAS]Luc, respectively) (generous gifts from Dr. Yulong Han, Cleveland Clinic Foundation). Generation of the pMCP[213]Luc and pMCP[213, mGAS]Luc plasmid constructs was described and characterized earlier (44). HUVEC were cotransfected with 0.5 µg of the Renilla luciferase control vector (pRL-SV40; Promega, WI) to normalize for transfection efficiency. After the transfections, cultures were maintained in normal growth medium for 24 h and then exposed to IL-4 for additional 16 h in M199 enriched with 10% fetal bovine serum. All reactions of firefly and Renilla luciferase were performed using the Dual Luciferase Reporter Assay System (Promega). Briefly, the cells were washed with phosphate-buffered saline and lysed with Passive Lysis Buffer. Cell lysates were mixed with Luciferase Assay Reagent II, and the firefly luminescence was measured using a luminometer with dual automatic injector (Turner Designs, CA). The samples were then mixed with the Stop & Glo reagent, and the Renilla luciferase activity was measured as an internal control.

Statistical analysis. Routine statistical analysis of data was completed using SYSTAT 7.0 (SPSS, Chicago, IL). One-way ANOVA was used to compare mean responses among the treatments. The treatment means were compared by using Bonferroni least-significant difference procedure. Statistical probability of P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IL-4-induced MCP-1 gene expression can be inhibited by antioxidants. Figure 1 shows the effects of IL-4 on MCP-1 mRNA expression in HUVEC using a semiquantitative RT-PCR technique. As indicated, low levels of MCP-1 mRNA were observed in control cell cultures. In addition, treatment of HUVEC with 10 ng/ml of IL-4 significantly, and in a time-dependent way, increased accumulation of MCP-1 mRNA (Fig. 1A). Upregulation of the MCP-1 mRNA expression was already detected 3 h after IL-4 treatment and reached the maximal level at 12 h. Figure 1B indicates that IL-4-induced stimulation of the MCP-1 gene is dose dependent. In these experiments, HUVEC were treated with different doses of IL-4 for 4 h. Maximal stimulation of the MCP-1 mRNA expression was detected in HUVEC exposed to 1.0 ng/ml of IL-4. An increase in the IL-4 dose to 10 ng/ml did not further affect the MCP-1 mRNA levels.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   A: time-dependent upregulation of monocyte chemoattractant protein-1 (MCP-1) mRNA expression by interleukin-4 (IL-4) in human umbilical vein endothelial cells (HUVEC). HUVEC were exposed to 10 ng/ml of IL-4 for 1, 3, 6, 12, and 24 h. Level of MCP-1 mRNA was determined by RT-PCR as described in MATERIALS AND METHODS. PCR products were analyzed by 2% agarose gel electrophoresis and visualized by using phosphoimaging. Predicted sizes of RT-PCR products for MCP-1 and -actin (represented by arrows) are 198 bp and 188 bp, respectively. M, molecular weight markers (100-bp DNA ladder). B: dose-dependent upregulation of MCP-1 mRNA expression by IL-4 in human endothelial cells. HUVEC were exposed to IL-4 at concentrations of 0.1, 1.0, and 10 ng/ml for 4 h. Level of MCP-1 mRNA was determined by RT-PCR as described in A. C: antioxidants inhibit the induction of MCP-1 mRNA in IL-4-treated human endothelial cells. HUVEC were pretreated with indicated amounts of antioxidants, pyrrolidine dithiocarbamate (PDTC, 10, 50, and 100 µM) or N-acetylcycteine (NAC, 10, 50, and 100 mM), for 30 min before a 4-h treatment with 10 ng/ml of IL-4 and assayed for MCP-1 mRNA by RT-PCR as described in A.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   PDTC attenuates transcriptional activation of the human MCP-1 promoter in IL-4-treated human endothelial cells. HUVEC were transfected with the firefly luciferase reporter plasmids containing the human MCP-1 promoter sequences (pMCP[213]Luc construct), pretreated with PDTC at concentrations of 10 or 100 µM for 1 h, and treated with 10 ng/ml of IL-4 for 16 h. MCP-1 promoter activity was analyzed by dual luciferase assay as described in MATERIALS AND METHODS. Data are means ± SD of 4 determinations. * Statistically significant compared with the control group (P < 0.05). #Values in the groups treated with IL-4 plus PDTC are significantly different from the IL-4-treated group (P < 0.05).

PDTC attenuates IL-4-stimulated induction of MCP-1 protein expression. The quantitative sandwich enzyme immunoassay technique was employed to determine whether IL-4-mediated induction of the MCP-1 gene is paralleled by a concomitant production of MCP-1 protein. Concentration of MCP-1 protein was determined in culture supernatants from HUVEC treated with different doses of IL-4 for 16 h (Fig. 3). Consistent with the data on MCP-1 gene expression, treatment with IL-4 resulted in a dose-dependent upregulation of MCP-1 protein levels (Fig. 3A). In addition, PDTC markedly and in a dose-dependent manner attenuated this effect (Fig. 3B). In fact, MCP-1 protein levels in cultures treated with IL-4 in the presence of 100 µM PDTC were in the range of control values.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   A: IL-4 increases production of MCP-1 protein in human endothelial cells. HUVEC were treated with indicated amounts of IL-4 for 16 h. Aliquots of culture media were collected, and the concentration of MCP-1 was measured by ELISA. Data are means ± SD of 4 determinations. * Statistically significant compared with the control group (P < 0.05). B: PDTC inhibits production of MCP-1 protein in IL-4-stimulated human endothelial cells. HUVEC were pretreated with PDTC at concentrations of 10 or 100 µM for 1 h before a 16-h treatment with 10 ng/ml of IL-4. Aliquots of culture media were collected, and the concentration of MCP-1 was measured by ELISA. Data are means ± SD of 4 determinations. * Statistically significant compared with the control group (P < 0.05). #Values in the groups treated with IL-4 plus PDTC are significantly different from the IL-4-treated group (P < 0.05).

PDTC blocks IL-4-activated DNA binding activity of transcription factor STAT1. Protective effects of antioxidants on IL-4-induced upregulation of the MCP-1 gene and protein production suggest a redox-related regulatory mechanism(s) of MCP-1 expression. Putative binding sites for several transcription factors, such as nuclear factor (NF)-B, activation protein (AP)-1, and STAT1, exist in the 5'-flanking region of the human MCP-1 gene. However, IL-4 does not activate NF-B or AP-1 in HUVEC (19, 20). Therefore, to elucidate the possible molecular signaling pathway of IL-4-mediated upregulation of MCP-1 expression, we examined the effects of IL-4 treatment on the DNA binding activity of transcription factor STAT1. This transcription factor specifically interacts with the IFN--activated site (GAS) in the human MCP-1 gene promoter.

View larger version (72K): [in this window] [in a new window]   Fig. 4.   A: IL-4 enhances binding of signal transducers and activators of transcription 1 (STAT1) to GAS element of the MCP-1 promoter in human endothelial cells. HUVEC were either untreated (lane 2) or treated with IL-4 (0.1, 1.0, and 10 ng/ml, lanes 3-5) for 30 min. Nuclear extracts were prepared and analyzed by electrophoretic mobility shift asssay (EMSA). Competition study and supershift analysis were performed by the addition of excess unlabeled oligonucleotide (lane 6) and anti-STAT1 antibody (lane 7), using nuclear extracts from HUVEC stimulated by 10 ng/ml of IL-4 for 30 min. B: PDTC attenuates DNA binding activity of transcription factor STAT1 in IL-4-treated human endothelial cells. HUVEC were pretreated with 10 or 100 µM PDTC (lanes 4 and 5) for 30 min and exposed to 10 ng/ml of IL-4 (lanes 3-5) for 30 min. Nuclear extracts were prepared and analyzed by EMSA.

STAT1 is the critical transcription factor in IL-4-induced MCP-1 gene. To further prove the critical role of transcription factor STAT1 in the regulation of the MCP-1 gene expression induced by IL-4, HUVEC were transfected either with the luciferase construct of normal human MCP-1 promoter (pMCP[213]Luc) or with the construct of MCP-1 promoter with mutated GAS sequence (pMCP[213, mGAS]Luc). In addition, HUVEC were cotransfected with pRL-SV40 to normalize transfection rates. In agreement with Fig. 2, exposure to IL-4 significantly induced luciferase activity (2.9-fold) only in cells transfected with the construct of the normal MCP-1 promoter. On the other hand, mutation in GAS sequence completely inhibited IL-4-mediated stimulation of luciferase activity in transfected HUVEC (Fig. 5).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Functional analysis of the STAT1 binding site of the human MCP-1 promoter in IL-4-treated human endothelial cells. HUVEC were transfected with the pMCP[213]Luc or pMCP[213, mGAS]Luc constructs and either untreated or treated with 10 ng/ml of IL-4 for 16 h. Promoter activities were analyzed by dual luciferase assay as described in MATERIALS AND METHODS. Mutation of the STAT1 binding site in the MCP-1 promoter construct completely inhibited IL-4-induced luciferase activity. Data are means ± SD of 4 determinations. * Values in the group treated with IL-4 are statistically significant compared with the untreated control (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we demonstrated that stimulation of cultured human vascular endothelial cells with IL-4 leads to an enhanced MCP-1 gene transcription and expression via an antioxidant-sensitive mechanism. More interestingly, this effect was associated with the activation of the transcription factor STAT1, which interacts specifically with the IFN--activated site (GAS) in the human MCP-1 gene promoter.

Evidence indicates that MCP-1 expression can be induced in response to a variety of proinflammatory stimuli (29, 33, 34, 37, 41, 44); however, detailed mechanisms of this process remain unknown. To study transcriptional regulatory mechanisms of IL-4-induced MCP-1 mRNA and protein expression in human endothelial cells, we first examined whether IL-4 can induce the transcription of MCP-1 gene in HUVEC by using RT-PCR and reporter gene assay. As shown in Fig. 1, A and B, IL-4 increased the MCP-1 mRNA accumulation in a time- and dose-dependent manner. The marked increase in MCP-1 mRNA levels was already detected as early as 3 h and reached a peak at 12 and 24 h after IL-4 stimulation. This kinetic expression was quite different from a previous report on IL-4-induced MCP-1 gene expression in endothelial cells (29). To further prove the transcriptional induction of MCP-1 by IL-4 treatment, transient transfection and reporter gene assay were performed by using a promoter/reporter plasmid construct containing upstream elements of the human MCP-1 gene fused to a reporter, luciferase. A significant increase in MCP-1 promoter transcriptional activity by IL-4 was observed (Fig. 2). These data strongly indicate that IL-4-induced MCP-1 expression in human endothelial cells is regulated at the transcriptional level.

To elucidate the molecular signaling pathway of IL-4-induced MCP-1 gene expression, we studied IL-4-mediated activation of nuclear transcription factors for which binding sites were previously identified in the 5'-flanking region of the human MCP-1 gene. The MCP-1 promoter has been shown to contain specific binding sequences for the redox-responsive transcription factors NF-B and AP-1 (33). Indeed, NF-B and AP-1 have been known to be activated in response to alterations of cellular redox status in a wide range of cells, leading to the upregulation of a number of proinflammatory genes, including MCP-1 (12, 33, 41, 42). However, we reported that treatment of HUVEC with IL-4 does not result in activation of NF-B or AP-1 (19, 20), and induction of the inflammatory genes in response to IL-4 is independent of these transcription factors (19). Thus the transcriptional regulation of MCP-1 expression by IL-4 in human vascular endothelial cells appears to be unique among a variety of biological systems.

STAT factors are latent cytoplasmic proteins that are activated by phosphorylation of a specific tyrosine residue and transduce a signal from a cytokine receptor. Phosphorylated STATs dimerize and rapidly translocate into the nucleus, where they bind to specific DNA elements, activating transcription of target genes. To date, seven mammalian STAT family proteins have been identified, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6, and each protein has been shown to be activated by distinct cytokines (16, 31). It has been hypothesized that activation of the STAT transcription factors can be regulated by cellular redox status (22). Recently, Takeda et al. (35) indicated the essential roles of each STAT family protein in cytokine-mediated biological responses through studies of gene-targeted knockout mice, suggesting that STAT transcription factors act as critical intermediates in cytokine-dependent gene induction. Indeed, biological effects of IL-4 might be mediated by the activation of transcription factors of the STAT family. For example, it was demonstrated that IL-4 can specifically increase the STAT6-DNA binding activity (20, 31), which appears to be a critical mechanism of IL-4-induced upregulation of 15-lipoxygenase-I expression (15). However, the possible relation between IL-4 and other STAT family proteins is not well defined. Specifically, the role of STAT1 activation in IL-4-induced alteration of endothelial cell metabolism remains unclear.

Structural analysis of the 5'-flanking region of human MCP-1 gene reveals the existence of a potential binding site for the STAT1 transcription factor (33, 44). Therefore, the present study was focused on the role of STAT1 in IL-4-stimulated MCP-1 gene expression in HUVEC. As indicated in Fig. 4A, dose-dependent increases in STAT1-DNA binding activity were detected in nuclear extracts prepared from HUVEC stimulated by IL-4 treatment. These results are in agreement with the report by Chang et al. (8), who demonstrated that IL-4 can activate STAT1 in colon cancer cell lines, leading to growth inhibition. The role of STAT1 in MCP-1 gene expression was further confirmed by transient transfection experiments with the reporter plasmid constructs of the MCP-1 promoter (Fig. 5). Indeed, these results provide the first evidence that STAT1 signaling pathways may be critically involved in the transcriptional regulatory mechanisms of IL-4-induced MCP-1 expression. However, from the kinetic data of STAT1 activation (which occurred within 30 min of IL-4 treatment) and MCP-1 mRNA expression (which reached the maximum increase after 12 h of IL-4 exposure), we cannot exclude possible involvement of other redox-sensitive pathways in IL-4-induced MCP-1 expression.

It is generally accepted that oxidative stress plays a crucial role in induction of endothelial cell inflammatory genes. For example, we have previously described that IL-4 treatment of HUVEC enhanced the intracellular oxidizing potential as indicated by an increase in 2',7'-dichlorofluorescein fluorescence, leading to the upregulation of VCAM-1 expression (19). Therefore, in the present study, we investigated effects of PDTC and NAC on IL-4-stimulated MCP-1 expression in HUVEC. As indicated in Fig. 1C, pretreatment of HUVEC with PDTC and NAC significantly attenuated IL-4-induced MCP-1 mRNA expression in a dose-dependent manner. This effect correlated with PDTC-mediated effects on MCP-1 promoter transcriptional activity and protein expression (Fig. 2 and 3B), indicating that MCP-1 transcription and expression by IL-4 is regulated by antioxidant-sensitive mechanisms. In addition, PDTC efficiently blocked IL-4-activated DNA binding activity of STAT1 (Fig. 4B). To our knowledge, this is the first report to demonstrate that IL-4-mediated STAT1 activation is regulated through an antioxidant-sensitive pathway.

In conclusion, the present study provides strong evidence that antioxidants can inhibit IL-4-induced MCP-1 gene transcription and expression in human vascular endothelial cells by blocking activation of STAT1. These data may contribute to a clinical strategy for the prevention of atherosclerotic lesion development specifically targeted against MCP-1 expression.