Abstract
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1beta and tumor necrosis factor-alpha. 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.
Highlights
This Polycyclic aromatic hydrocarbons (PAHs) pleiotropic toxicity has been linked, at least in part, to activation of the aryl hydrocarbon receptor (AhR), a ligandactivated basic helix-loop-helix transcription factor
Sions of IL-1, IL-8, or tumor necrosis factor-␣ 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 –17)
Effect of BP on CCL1 Expression and Production in Human Macrophages and on TCA3 Expression in Mouse Lung—CCL1chemokine expression was investigated in primary human macrophages exposed to various BP concentrations for 24 h (Fig. 1A)
Summary
This PAH pleiotropic toxicity has been linked, at least in part, to activation of the aryl hydrocarbon receptor (AhR), a ligandactivated basic helix-loop-helix transcription factor. 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 5Ј-flanking regions of PAH-regulated genes (10). This usually leads to altered expression of target genes, which are presumed to contribute to PAH toxicity. To identify new cytokines regulated by PAHs, we have recently analyzed the transcriptome of BPtreated human macrophages using macroarrays Data from these experiments suggest that CCL1 (CC- chemokine ligand 1, named I-309), a chemokine triggering Th2 immune response and strongly implicated in cardiovascular diseases, asthma, and allergic inflammation (18 –20), may be up-regulated by AhR agonists. 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
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