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Aryl Hydrocarbon Receptor- and Calcium-dependent Induction of the Chemokine CCL1 by the Environmental Contaminant Benzo[a]pyrene

2006; Elsevier BV; Volume: 281; Issue: 29 Linguagem: Inglês

10.1074/jbc.m601192200

ISSN

1083-351X

Autores

Monique N’Diaye, Eric Le Ferrec, Dominique Lagadic‐Gossmann, Sébastien Corre, David Gilot, Valérie Lecureur, Patricia Monteiro, Claudine Rauch, Marie‐Dominique Galibert, Olivier Fardel,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1β and tumor necrosis factor-α. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner. Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1β and tumor necrosis factor-α. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner. Polycyclic aromatic hydrocarbons (PAHs) 3The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; AhR, aryl hydrocarbon receptor; CYP, cytochrome P450; BP, benzo[a]pyrene; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; BeP, benzo[e]pyrene; 2-APB, 2-amino-ethoxydiphenylborate; XRE, xenobiotic-responsive elements; ChIP, chromatin immunoprecipitation assays; 3′M4′NF, 3′-methoxy-4′-nitroflavone; Fura-2-AM, Fura-2-acetoxymethylester; CCL1, CC-chemokine ligand 1; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester; RT, reverse transcription; RT-qPCR, reverse transcription-real-time quantitative-PCR; ELISA, enzyme-linked immunosorbent assay; siRNA, small interference RNA; SiAhR, AhR siRNA; SiAhRmut, AhR-mutated siRNA. such as benzo-[a]pyrene (BP) constitute a major class of chemical contaminants to which humans are widely exposed. They are usually generated through the burning of fossil fuels or wood, and are notably found in diesel exhaust particles, cigarette smoke, charcoal-broiled foods, and industrial waste by-products (1.Hattemer-Frey H.A. Travis C.C. Toxicol. Ind. Health. 1991; 7: 141-157Crossref PubMed Scopus (207) Google Scholar, 2.Knize M.G. Salmon C.P. Pais P. Felton J.S. Adv. Exp. Med. Biol. 1999; 459: 179-193Crossref PubMed Scopus (169) Google Scholar, 3.Hecht S.S. J. Natl. Cancer Inst. 1999; 91: 1194-1210Crossref PubMed Scopus (1621) Google Scholar). These pollutants exert various major toxic effects toward human health, especially development of cancers and inflammatory diseases in various tissues (4.Sorensen M. Autrup H. Moller P. Hertel O. Jensen S.S. Vinzents P. Knudsen L.E. Loft S. Mutat. Res. 2003; 544: 255-271Crossref PubMed Scopus (198) Google Scholar, 5.Wogan G.N. Hecht S.S. Felton J.S. Conney A.H. Loeb L.A. Semin. Cancer Biol. 2004; 14: 473-486Crossref PubMed Scopus (488) Google Scholar). Immunotoxicity, including contact hypersensitivity, also constitutes a key hallmark of PAH exposure (6.Burchiel S.W. Luster M.I. Clin. Immunol. 2001; 98: 2-10Crossref PubMed Scopus (169) Google Scholar, 7.Klemme J.C. Mukhtar H. Elmets C.A. Cancer Res. 1987; 47: 6074-6078PubMed Google Scholar, 8.Anderson C. Hehr A. Robbins R. Hasan R. Athar M. Mukhtar H. Elmets C.A. J. Immunol. 1995; 155: 3530-3537PubMed Google Scholar, 9.Wu M.T. Pan C.H. Huang Y.L. Tsai P.J. Chen C.J. Wu T.N. Environ Mol. Mutagen. 2003; 42: 98-105Crossref PubMed Scopus (55) Google Scholar). This PAH pleiotropic toxicity has been linked, at least in part, to activation of the aryl hydrocarbon receptor (AhR), a ligand-activated basic helix-loop-helix transcription factor. Indeed, PAHs bind to cytoplasmic AhR, thereby triggering its translocation into cell nucleus, its association with the AhR nuclear translocator, and its interaction with xenobiotic responsive elements (XREs) (core sequence: GCGTG) found in the 5′-flanking regions of PAH-regulated genes (10.Hankinson O. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1438) Google Scholar). This usually leads to altered expression of target genes, which are presumed to contribute to PAH toxicity. Thus, CYP1A1, a drug-metabolizing enzyme required for the bioactivation of PAHs into reactive mutagenic electrophilic metabolites, is massively up-regulated in PAH-exposed cells in an AhR-dependent manner. Besides AhR activation, PAHs trigger elevation of intracellular calcium concentration in various cellular types, which has been hypothesized to contribute to their immunotoxic effects (11.Karras J.G. Conrad D.H. Holsapple M.P. Toxicol. Lett. 1995; 75: 225-233Crossref PubMed Scopus (11) Google Scholar, 12.Krieger J.A. Born J.L. Burchiel S.W. Toxicol. Appl. Pharmacol. 1994; 127: 268-274Crossref PubMed Scopus (51) Google Scholar). Moreover, CYP1A1 induction by AhR agonists has been shown to be abolished by inhibition of calcium movements, thus linking AhR target gene regulation with calcium changes (13.Le Ferrec E. Lagadic-Gossmann D. Rauch C. Bardiau C. Maheo K. Massiere F. Le Vee M. Guillouzo A. Morel F. J. Biol. Chem. 2002; 277: 24780-24787Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Among genes regulated by PAHs, pro-inflammatory cytokines constitute an emerging class. Indeed, increased expressions of IL-1β, IL-8, or tumor necrosis factor-α occur in cells exposed to PAHs or to other potent AhR agonists such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which may contribute to the adverse inflammatory or immunotoxic effects due to PAH exposure (14.Lecureur V. Ferrec E.L. N′Diaye M. Vee M.L. Gardyn C. Gilot D. Fardel O. FEBS Lett. 2005; 579: 1904-1910Crossref PubMed Scopus (99) Google Scholar, 15.Lyte M. Bick P.H. Int. J. Immunopharmacol. 1986; 8: 377-381Crossref PubMed Scopus (45) Google Scholar, 16.Lyte M. Life Sci. 1986; 38: 1163-1170Crossref PubMed Scopus (5) Google Scholar, 17.Vandebriel R.J. Meredith C. Scott M.P. Roholl P.J. Van Loveren H. Toxicol. Appl. Pharmacol. 1998; 148: 126-136Crossref PubMed Scopus (30) Google Scholar). To identify new cytokines regulated by PAHs, we have recently analyzed the transcriptome of BP-treated human macrophages using macroarrays. Data from these experiments suggest that CCL1 (CC-chemokine ligand 1, also named I-309), a chemokine triggering Th2 immune response and strongly implicated in cardiovascular diseases, asthma, and allergic inflammation (18.Haque N.S. Zhang X. French D.L. Li J. Poon M. Fallon J.T. Gabel B.R. Taubman M.B. Koschinsky M. Harpel P.C. Circulation. 2000; 102: 786-792Crossref PubMed Scopus (85) Google Scholar, 19.Haque N.S. Fallon J.T. Taubman M.B. Harpel P.C. Blood. 2001; 97: 39-45Crossref PubMed Scopus (79) Google Scholar, 20.Haque N.S. Fallon J.T. Pan J.J. Taubman M.B. Harpel P.C. Blood. 2004; 103: 1296-1304Crossref PubMed Scopus (72) Google Scholar), may be up-regulated by AhR agonists. In the present study, we demonstrate for the first time that BP markedly induces CCL1 production in human macrophages by an AhR-dependent mechanism. Moreover, BP was shown to trigger an early and transient increase of intracellular calcium concentration, which seems essential to CCL1 up-regulation by this environmental contaminant. Reagents—PAHs, α-naphtoflavone (αNF), resveratrol, and 2-aminoethoxydiphenylborate (2-APB) were purchased from Sigma-Aldrich. TCDD was obtained from Cambridge Isotope Laboratories (Cambridge, MA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was obtained from Calbiochem. Pluronic acid and Fura-2 acetoxymethylester (Fura-2-AM) were provided from Molecular Probes (Eugene, OR), and Ficoll was obtained from Amersham Biosciences. Granulocyte macrophage-colony stimulating factor (specific activity, 1.2 × 108 units/mg) was purchased from Schering Plough (Lyon, France). 3′-Methoxy-4′-nitroflavone (3′M4′NF) was a kind gift from Dr. T. Gasiewicz (Dept. of Environmental Medicine, University of Rochester Medical Center, Rochester, NY). Polyclonal rabbit antibody anti-AhR was obtained from Biomol Research Laboratories (Plymouth, PA), and nonspecific IgG peroxidase-conjugated donkey anti-rabbit was from Amersham Biosciences. All other reagents were commercial products of the highest purity available. Chemicals were commonly used as stock solution in Me2SO. The final concentration of solvent did not exceed 0.2% (v/v); control cultures received the same dose of solvent as for treated counterparts. Cell Culture—Primary human macrophages were obtained from blood monocytes as previously described (21.van Grevenynghe J. Rion S. Le Ferrec E. Le Vee M. Amiot L. Fauchet R. Fardel O. J. Immunol. 2003; 170: 2374-2381Crossref PubMed Scopus (138) Google Scholar). Briefly, mononuclear cells, isolated from blood buffy coats (kindly provided by the Etablissement Français du Sang, Rennes, France) through Ficoll gradient centrifugation, were initially seeded at the density of 2 × 106 cells per cm2 into plastic culture plates for 2 h. Non-adherent cells were then removed, and adherent monocytes were further cultured at 37 °C under 5% CO2 for 6 days in RPMI 1640 medium supplemented with 10% decomplemented fetal bovine serum (Invitrogen), 2 mm glutamine, 100 units/ml penicillin, 10 μg/ml streptomycin, and 400 units/ml granulocyte macrophage-colony stimulating factor. Such a protocol allows the obtainment of pure macrophage cultures with <1% of contaminating cells. Macrophages were then routinely cultured in the medium described above. Human endothelial EAhy-926 cells (22.Edgell C.J. McDonald C.C. Graham J.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737Crossref PubMed Scopus (1360) Google Scholar) (generously provided by Dr. C. J. Edgell, University of North Carolina, Chapel Hill) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 10 μg/ml streptomycin, and 2 mm glutamine. Animal Treatment—Male adult C57bl/6 mice (10 weeks old, weighing 25 g) were treated by intranasal instillation of 500 μg of BP dissolved in tricaprylin (50 μl/mouse) under etomidate anesthesia. Control mice received tricaprylin only. Twenty-four hours after BP treatment, mice were quickly anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg) and underwent removal of lungs, which were stored at –80 °C until analysis. RNA Isolation and Analysis—Total RNAs were extracted from cells or lungs using the TRIzol method (Invitrogen). They were then subjected to semiquantitative reverse transcription (RT)-PCR or to reverse transcription-real-time quantitative-PCR (RT-qPCR) analyses as previously described (23.Laupeze B. Amiot L. Sparfel L. Le Ferrec E. Fauchet R. Fardel O. J. Immunol. 2002; 168: 2652-2658Crossref PubMed Scopus (108) Google Scholar, 24.Jigorel E. Le Vee M. Boursier-Neyret C. Bertrand M. Fardel O. Drug Metab. Dispos. 2005; 33: 1418-1422Crossref PubMed Scopus (96) Google Scholar). For RT-PCR assays, cDNA aliquots were amplified using Master mix (Promega) and 0.1 mm of forward and reverse gene primers listed in Table 1. Each PCR cycle consisted of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. β-Actin primers were used as an internal control. PCR products were finally visualized using ethidium bromide-staining agarose gels. RT-qPCR assays were performed using the fluorescent dye SYBR Green methodology and an ABI Prism 7000 detector (Applied Biosystems, Foster City, CA). Gene-specific primers (Table 1) were designed with the Primer3 software (available at frodo.wi.mit.edu/cgi-bin/mrimer3/primer3_www.cgi). The specificity of each gene amplification was checked up at the end of qPCR reactions through the analysis of dissociation curves of the PCR products. Amplification curves were read with ABI Prism 7000 SDS software using the comparative cycle threshold method. Relative quantification of the steady-state target mRNA levels was performed after normalization of the total amount of cDNA tested to an 18 S RNA endogenous reference.TABLE 1Primer sequencesGenesPrimersForwardReverseRT-PCR CCL15′-GTTGCTTCTCATTTGCGGAG-3′5′-GGTGTAGGGCTGGTAGTTTGC-3′ CCL25′-CTGAAGCTCGCACTCTCGCC-3′5′-GGTGTCTGGGGAAAGCTAGG-3′ CCL55′-CCCCATATTCCTCGGACAC-3′5′-GTTGTTGTTGTTGTGACGG-3′ CCL225′-GAAGCTGGCTGTGGTAGCG-3′5′-CCTGGATGACACTGAGCTGG-3′ CYP1A15′-TCACAGACAGCCTGATTGAGC-3′5′-GATGGGTTGACCCATAGCTT-3′ IL-1β5′-GATGGCGGCATCCAGCTAGC-3′5′-CTTTCAACACGCAGGACAGG-3′ β-actin5′-GCCAGACAAGAGAG-3′5′-GGCCATCTCTTGCTGC-3′RT-qPCR CCL15′-AGGCCTCTTTGCCTCTCTTC-3′5′-ATGCAGATCATCACCACAGC-3′ TCA35′-GGATGTTGACAGCAAGAGCA-3′5′-TGGAGGACTGAGGGAAACTG-3′ 18 S5′-CGCCGCTAGAGGTGAAATTC-3′5′-TTGGCAAATGCTTTCGCT-3′CHIP assay Primers spanning the XRE-28035′-TTGCCTGTGCTGGTCTGACT-3′5′-GTGGGATTGCTGGGTCAAAT-3′ Primers spanning the XRE-3125′-TGCTATTTGCATTTTGTGTGAATATG-3′5′-TCGTGGTGGTGATGGATTGA-3′ Open table in a new tab Determination of Cytokine Levels—Levels of CCL1 and IL-1β in the supernatants of macrophage cultures were quantified using Duoset ELISA development system kits obtained from R&D systems (Abington, UK). Briefly, 96-well plates, initially coated with 1 μg/ml mouse anti-human CCL1 or IL-1β antibody overnight at room temperature, were incubated for 2 h with macrophage culture supernatants or recombinant human CCL1 or IL-1β standard. After washes, plates were processed according to the manufacturer's instructions. Reporter Gene Constructs and Luciferase Assays—The 2975-bp region of the CCL1 gene promoter (GenBank™ under accession number AC002482), spanning –3038 to –63, was obtained through PCR amplification and cloned into the firefly luciferase reporter vector pGL3 basic (Promega); the resulting plasmid construct was designed as pCCL1-FL(–2975). The pCCL1-FL.Mut(–2975) plasmid corresponds to the pCCL1-FL(–2975), with three mutations located at positions –312, –311, and –310 on the XRE consensus site located at –312 (consensus XRE-312: GCGTG; mutated XRE-312: GCCAA). The pCYP1A1-FL(–1639) construct containing a 1639-bp region (from –1566 to +73) of the human CYP1A1 gene upstream of the firefly luciferase reporter gene had been previously described (25.Morel Y. Barouki R. J. Biol. Chem. 1998; 273: 26969-26976Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and was a kind gift from Dr. R. Barouki (INSERM U490, Paris, France). EAhy-926 cells cultured in 24-well plates were transiently co-transfected with one of the firefly luciferase promoter-containing plasmids described above and the Renilla luciferase-encoding pRL-TK vector (Promega), used as an internal control, according to the Lipofectamine™ 2000 method (Invitrogen). Briefly, 400 μl of transfection medium (Opti-MEM) containing 200 ng of luciferase reporter plasmid was added per well of confluent EAhy-926 cells along with 20 ng of the pRL-TK plasmid and 0.4 μl of Lipofectamine™ 2000. After a 24-h period, cells were exposed to PAHs for another 24-h period. Dual luciferase assays (firefly and Renilla) were then performed with a Promega kit according to the manufacturer's instructions. pGL3 basic (a promoterless pGL3-luciferase construct) and pGL3 SV40 (a pGL3-luciferase construct with the SV40 promoter) vectors were used in parallel as negative and positive firefly luciferase controls, respectively. Design and Transfection of AhR-targeted Specific siRNA—Small interfering RNA (siRNA) oligonucleotide sequences, designed as recommended (26.Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar) with two 2′-deoxythymidine overhands on each strand, were: AhR siRNA (SiAhR), 5′-AAGUCGGUCUCUAUGCCGCTT-3′, and control AhR-mutated siRNA (SiAhRmut), 5′-AACUCGGUCUCUAUGCCGCTT-3′. The absence of matching of these sequences with that of any other human gene was checked by using the NCBI standard nucleotide-nucleotide BLAST program. Transfection of siRNAs was performed using the HiPerFect Transfection kit (Qiagen). Macrophages, cultured in 24-well plates, were incubated with the transfection mix containing 5 nm siRNA and 4.5 μl of HiPerFect reagent in 500 μl of culture medium, in agreement with the manufacturer's instructions. After a 40-h incubation, the transfection medium was removed, and cells were thereafter exposed to BP for an additional 8-h period. Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays were performed essentially as previously described (27.Weinmann A.S. Farnham P.J. Methods. 2002; 26: 37-47Crossref PubMed Scopus (302) Google Scholar, 28.Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (714) Google Scholar). Briefly, macrophages either untreated or exposed to BP or TCDD for 30 min, were crossed-linked with 1% formaldehyde in phosphate-buffered saline at room temperature for 10 min. After washing and suspension in cell lysis buffer, chromatin was digested using the BamH1 restriction enzyme at 37 °C overnight. Protein-DNA complexes were then immunoprecipitated using rabbit polyclonal antibody against AhR or nonspecific IgG anti-rabbit and protein G-Sepharose as described by Corre et al. (29.Corre S. Primot A. Sviderskaya E. Bennett D.C. Vaulont S. Goding C.R. Galibert M.D. J. Biol. Chem. 2004; 279: 51226-51233Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The recovered DNA was then subjected to PCR using primers listed in Table 1 and covering XRE sequences found in CCL1 and CYP1A1 promoter (positive control) (30.Hestermann E.V. Brown M. Mol. Cell. Biol. 2003; 23: 7920-7925Crossref PubMed Scopus (120) Google Scholar) or an XRE-free sequence of the tyrosinase gene (negative control) (29.Corre S. Primot A. Sviderskaya E. Bennett D.C. Vaulont S. Goding C.R. Galibert M.D. J. Biol. Chem. 2004; 279: 51226-51233Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Electrophoretic Mobility Shift Assays—Nuclear extracts were isolated from cultured macrophages using the BD™ Transfactor extraction kit (BD Biosciences). Binding of nuclear extracts to [γ-32P]ATP (Amersham Biosciences) end-labeled double-stranded oligonucleotides was then analyzed with the Promega gel shift assay system (Promega). Two double-stranded oligonucleotides, corresponding to (–326 to –294) region of the CCL1 promoter were used: the wild-type CCL1–312-WT (5′-ATCTTGAAAAAGTGCGTGGGAACTGTCCTGGT-3′) (underlined characters represent the core XRE) and the mutated CCL1–312-Mut (5′-ATCTTGAAAAAGTGCCAAGGAACTGTCCTGGT-3′). DNA-protein complexes were separated by electrophoresis on a polyacrylamide gel and detected by autoradiography. Intracellular Calcium Measurements—Variations in intracellular concentrations of calcium were analyzed by microspectrofluorometry using the Ca2+-sensitive probe Fura-2-AM as previously reported (13.Le Ferrec E. Lagadic-Gossmann D. Rauch C. Bardiau C. Maheo K. Massiere F. Le Vee M. Guillouzo A. Morel F. J. Biol. Chem. 2002; 277: 24780-24787Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Briefly, macrophages were incubated at 37 °C in cell suspension buffer (134.8 mm NaCl; 4.7 mm KCl; 1.2 mm K2HPO4; 1 mm MgCl2; 1 mm CaCl2; 10 mm glucose; 10 mm HEPES; pH 7.4) supplemented with 1.5 μm Fura-2-AM and 0.006% pluronic acid. Following probe loading, cells, placed in a continuously perfused recording chamber mounted on the stage of an epifluorescence microscope (Nikon Diaphot), were irradiated alternately with 340 and 380 nm light, and fluorescence from the trapped dye was measured at 510 nm. The ratio of fluorescence intensities recorded after excitation at 340 nm (F340) and at 380 nm (F380) was used to estimate intracellular concentrations ([Ca2+]i). The monochromator and the photometers, which allow emission and detection of fluorescence from ∼3 to 5 cells in the field of view, were part of a DeltaRAM system from Photon Technology International (PTI, Birmingham, UK), which also provided software systems to acquire and process data. Statistical Analysis—Results are presented as mean ± S.D. Significant differences were routinely evaluated with the non-parametric Wilcoxon test except for the variations of intracellular calcium concentration for which the Mann-Whitney U test was used. The level of significance was p < 0.05. Effect of BP on CCL1 Expression and Production in Human Macrophages and on TCA3 Expression in Mouse Lung—CCL1-chemokine expression was investigated in primary human macrophages exposed to various BP concentrations (from 0.1 to 20 μm) for 24 h (Fig. 1A). As for CYP1A1, used here as positive control of treatment, the BP effect on CCL1 mRNA levels was dose-dependent: CCL1 mRNA up-regulation was detected after 0.1 μm BP, reaching a maximum after 10 μm BP. Such data, obtained with semiquantitative RT-PCR assays, were fully confirmed using RT-qPCR analyses, which revealed a 3.9 ± 2.2-, 12.6 ± 6.6-, and 27.9 ± 9.9-fold induction of CCL1 mRNA levels in response to 0.1, 1, and 10 μm BP treatment, respectively (n = 4). CCL1 secretion in supernatants of macrophagic cultures was concomitantly enhanced by BP in a dose-dependent manner as assessed by ELISA measurements, reaching a value of 12.8 ± 3.4 ng/ml in the supernatant of cultures exposed to 10 μm BP (Fig. 1A). Kinetic analysis of CCL1 gene induction by BP showed a maximum rate of mRNA level at 12–24 h, as for CYP1A1 up-regulation and a maximum of CCL1 secretion from 24-h exposure (Fig. 1B). Using shorter BP treatment time, CCL1 mRNA induction was detected as soon as 90-min exposure to BP (data not shown). Alveolar macrophages are the main cellular source of CCL1 in the lung (31.Bishop B. Lloyd C.M. J. Immunol. 2003; 170: 4810-4817Crossref PubMed Scopus (53) Google Scholar). To determine whether in vivo BP treatment could also be effective on CCL1 levels, the expression of TCA3, the mouse CCL1 orthologue gene (32.Goya I. Gutierrez J. Varona R. Kremer L. Zaballos A. Marquez G. J. Immunol. 1998; 160: 1975-1981PubMed Google Scholar), was determined using RT-qPCR assays. For this purpose, mice were treated by intranasal instillation of BP. As shown in Fig. 1C, BP exposure increased TCA3 mRNA levels in lung tissue (mean induction of 3.9-fold). Induction of lung CYP1A1 used as a validity control of mice BP treatment was concomitantly observed (data not shown) (33.Ma Q. Curr. Drug Metab. 2001; 2: 149-164Crossref PubMed Scopus (192) Google Scholar). Lack of Contribution of IL-1β to BP-mediated CCL1 Up-regulation—IL-1β has been previously identified as a PAH target (34.Tamaki A. Hayashi H. Nakajima H. Takii T. Katagiri D. Miyazawa K. Hirose K. Onozaki K. Biol. Pharm. Bull. 2004; 27: 407-410Crossref PubMed Scopus (55) Google Scholar), and it was consequently found to be up-regulated at both mRNA and protein level in BP-exposed macrophagic cultures (Fig. 2A). Because CCL1 secretion has been shown to be triggered by IL-1β (35.Selvan R.S. Zhou L.J. Krangel M.S. Eur. J. Immunol. 1997; 27: 687-694Crossref PubMed Scopus (40) Google Scholar), it was therefore tempting to speculate that up-regulation of CCL1 expression in BP-treated macrophages might have been a consequence of primary induction of IL-1β. To test this hypothesis, we evaluated the effect of IL1-RA, an antagonist of IL-1 receptor (36.Arend W.P. Gabay C. Arthritis Res. 2000; 2: 245-248Crossref PubMed Scopus (91) Google Scholar), on BP-mediated induction of CCL1. IL1-RA, used at doses (50 and 100 ng/ml) that fully abolished IL-1β-mediated CCL1 mRNA up-regulation (Fig. 2B), failed to counteract CCL1 induction in BP-exposed macrophages (Fig. 2C), making a direct contribution of IL-1β to CCL1 regulation by BP very unlikely. Effects of AhR Agonists and/or Antagonists and of AhR Knockdown on CCL1 Up-regulation—We analyzed the effects of hydrocarbons such as benzo[e]pyrene (BeP) and TCDD, known as very weak and potent activators of AhR, respectively (37.Sterling K. Raha A. Bresnick E. Toxicol. Appl. Pharmacol. 1994; 128: 18-24Crossref PubMed Scopus (23) Google Scholar, 38.Mandal P.K. J. Comp. Physiol. [B]. 2005; 175: 221-230Crossref PubMed Scopus (469) Google Scholar), on CCL1 expression levels. As shown in Fig. 3A, 10 μm BeP failed to significantly increase CCL1 mRNA level and CCL1 secretion in macrophage cultures; by contrast, 10 nm TCDD markedly enhanced CCL1 expression at both mRNA and protein levels, similarly to BP. We next investigated the effects of 3′M4′NF, a potent antagonist of AhR (39.Nazarenko D.A. Dertinger S.D. Gasiewicz T.A. Toxicol. Sci. 2001; 61: 256-264Crossref PubMed Scopus (18) Google Scholar, 40.Lu Y.F. Santostefano M. Cunningham B.D. Threadgill M.D. Safe S. Arch. Biochem. Biophys. 1995; 316: 470-477Crossref PubMed Scopus (97) Google Scholar), on BP-triggered induction of CCL1. As shown in Fig. 3B, 3′M4′NF, used either at 1 or 5 μm, markedly counteracted the up-regulation of CCL1 mRNA and secretion in BP-exposed macrophagic cell cultures. It concomitantly blocked induction of the AhR-regulated gene CYP1A1 at mRNA levels, fully confirming that 3′M4′NF was active under our experimental conditions. Similar inhibition of BP-mediated up-regulation of CCL1 expression was obtained using resveratrol or αNF as AhR antagonists instead of 3′M4′NF (data not shown). Besides pharmacological AhR inhibition, we analyzed whether AhR knockdown, using specific AhR-targeted siRNAs, may impair CCL1 induction in response to PAHs. Transfection of SiAhR allowed a strong reduction of AhR mRNA and AhR protein levels in primary macrophages (data not shown) and, as expected, CYP1A1 induction by BP was also prevented by transfection of SiAhR (Fig. 3C). Similarly, AhR knocking down markedly counteracted the inducing effects of BP toward CCL1 mRNA levels in macrophagic cultures when compared with transfection of control siRNA (SiAhR-mut) (Fig. 3C). Effects of AhR Agonists and/or Antagonists on CCL1 Promoter Activity—We next studied the effects of BP, BeP, TCDD, and/or 3′M4′NF on CCL1 promoter activity. For these experiments, we performed transient transfection assays in EAhy-926 cells with the pCCL1-FL(–2975) reporter vector. This construct contained 2975 bp of the CCL1 gene 5′-flanking region. As shown in Fig. 4A, potent AhR agonists as BP and TCDD significantly enhanced luciferase activity of pCCL1-FL(–2975), unlike the very weak AhR activator BeP. Similar effects were observed with the pCYP1A1-FL(–1639) plasmid, a CYP1A1 promoter construct used here as positive control. Moreover, the AhR antagonist 3′M4′NF was able to abrogate the increase of CCL1 and CYP1A1 promoter activity due to TCDD (Fig. 4B). Direct AhR Interaction with CCL1 Promoter in BP- or TCDD-treated Macrophages—Analysis of the CCL1 promoter sequence indicated the presence of two AhR-related consensus XRE located at –2803 (XRE-2803) and –312 (XRE-312) (Fig. 5A), in agreement with a previous report (41.Sun Y.V. Boverhof D.R. Burgoon L.D. Fielden M.R. Zacharewski T.R. Nucleic Acids Res. 2004; 32: 4512-4523Crossref PubMed Scopus (183) Google Scholar). To investigate whether AhR interacts in vivo with these consensus XREs, we performed a ChIP assay using either untreated or

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