Aryl hydrocarbon receptor- and calcium-dependent induction of the chemokine CCL1 by the environmental contaminant benzo[a]pyrene

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.     

Polycyclic aromatic hydrocarbons inhibit differentiation of human monocytes into macrophages

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are ubiquitous environmental carcinogenic contaminants exerting deleterious effects toward cells acting in the immune defense such as monocytic cells. To investigate the cellular basis involved, we have examined the consequences of PAH exposure on macrophagic differentiation of human blood monocytes. Treatment by BP markedly inhibited the formation of adherent macrophagic cells deriving from monocytes upon the action of either GM-CSF or M-CSF. Moreover, it reduced expression of macrophagic phenotypic markers such as CD71 and CD64 in GM-CSF-treated monocytic cells, without altering cell viability or inducing an apoptotic process. Exposure to BP also strongly altered functional properties characterizing macrophagic cells such as endocytosis, phagocytosis, LPS-triggered production of TNF-alpha and stimulation of allogeneic lymphocyte proliferation. Moreover, formation of adherent macrophagic cells was decreased in response to PAHs distinct from BP such as dimethylbenz(a)anthracene and 3-methylcholanthrene, which interact, like BP, with the arylhydrocarbon receptor (AhR) known to mediate many PAH effects. In contrast, benzo(e)pyrene, a PAH not activating AhR, had no effect. In addition, AhR was demonstrated to be present and functional in cultured monocytic cells, and the use of its antagonist alpha-naphtoflavone counteracted inhibitory effects of BP toward macrophagic differentiation. Overall, these data demonstrate that exposure to PAHs inhibits functional in vitro differentiation of blood monocytes into macrophages, likely through an AhR-dependent mechanism. Such an effect may contribute to the immunotoxicity of these environmental carcinogens owing to the crucial role played by macrophages in the immune defense.     

Aryl hydrocarbon receptor-dependent induction of the NADPH oxidase subunit NCF1/p47 expression leading to priming of human macrophage oxidative burst

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are toxic environmental contaminants known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that acute treatment by BaP markedly increased expression of the NADPH oxidase subunit gene neutrophil cytosolic factor 1 (NCF1)/p47^phox in primary human macrophages; NCF1 was similarly up-regulated in alveolar macrophages from BaP-instilled rats. NCF1 induction in BaP-treated human macrophages was prevented by targeting AhR, through its chemical inhibition or small interference RNA-mediated down-modulation of its expression. BaP moreover induced activity of the NCF1 promoter sequence, containing a consensus AhR-related xenobiotic-responsive element (XRE), and electrophoretic mobility shift assays and chromatin immunoprecipitation experiments indicated that BaP-triggered binding of AhR to this XRE. Finally, we showed that BaP exposure resulted in p47^phox protein translocation to the plasma membrane and in potentiation of phorbol myristate acetate (PMA)-induced superoxide anion production in macrophages. This BaP priming effect toward NADPH oxidase activity was inhibited by the NADPH oxidase specific inhibitor apocynin and the chemical AhR inhibitor α-naphtoflavone. These results indicated that BaP induced NCF1/p47^phox expression and subsequently enhanced superoxide anion production in PMA-treated human macrophages, in an AhR-dependent manner; such an NCF1/NADPH oxidase regulation by polycyclic aromatic hydrocarbons may participate in deleterious effects toward human health triggered by these environmental contaminants, including atherosclerosis and smoking-related diseases.     

Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.     

Adiponectin differentially regulates cytokines in porcine macrophages

Adiponectin, an adipocyte-derived hormone, attenuates the production of TNFalpha by activated human macrophages. In the present study, we used porcine blood-derived macrophages to test the hypothesis that the anti-inflammatory action of adiponectin includes suppression of IL6 and an induction of IL10. Adiponectin suppressed both TNFalpha and IL6 production in macrophages activated with lipopolysaccharide (P<0.01). In contrast, adiponectin increased IL10 expression (P<0.05) and augmented (P<0.05) the induction of this cytokine by lipopolysaccharide (LPS). Mechanistically, the attenuation of proinflammatory cytokine production by adiponectin was associated with an attenuation of the translocation of NFkappaB to the nucleus. Either adiponectin or inhibition of ERK1/2 with U0126 diminished the induction of IL6 by LPS (P<0.05), but the combination of adiponectin and the inhibitor did not further reduce IL6 production. In contrast, the inhibitory actions of adiponectin and a p38 MAPK inhibitor (SB203580) were additive (P<0.05). These data indicate that the anti-inflammatory actions of adiponectin include suppression of IL6 and induction of IL10. In addition, we provide evidence that some of the anti-inflammatory actions of adiponectin are mediated in part by suppression of NFkappaB signaling and ERK1/2 activity.     

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.     

Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a β2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) are widely distributed environmental contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). These chemicals trigger an early and transient increase of intracellular calcium concentration ([Ca(2+)](i)), required for AhR-related effects of PAHs. The mechanisms involved in this calcium mobilization were investigated in the present study. We demonstrated that B(a)P-mediated [Ca(2+)](i) induction was prevented in endothelial HMEC-1 cells by counteracting β2-adrenoreceptor (β2ADR) activity using pharmacological antagonists, anti-β2ADR antibodies, or siRNA-mediated knockdown of β2ADR expression; by contrast, it was strongly potentiated by β2ADR overexpression in human kidney HEK293 cells. B(a)P was shown, moreover, to directly bind to β2ADR, as assessed by in vitro binding assays and molecular modeling. Pharmacological inhibition and/or siRNA-mediated silencing of various signaling actors acting downstream of β2ADR in a sequential manner, such as G protein, adenylyl cyclase, Epac-1 protein, and inositol 1,4,5-trisphosphate (IP(3))/IP(3) receptor, were next demonstrated to prevent B(a)P-induced calcium signal. Inhibition or knockdown of these signaling elements, as well as the use of chemical β-blockers, were finally shown to counteract B(a)P-mediated induction of cytochrome P-450 1B1, a prototypical AhR target gene. Taken together, our results show that B(a)P binds directly to β2ADR and consequently utilizes β2ADR machinery to mobilize [Ca(2+)](i), through activation of a G protein/adenylyl cyclase/cAMP/Epac-1/IP(3) pathway. This β2ADR-dependent signaling pathway activated by PAHs may likely be crucial for PAH-mediated up-regulation of AhR target genes, thus suggesting a contribution of β2ADR to the health-threatening effects of these environmental pollutants.     

Priming of the neutrophil respiratory burst involves p38 mitogen-activated protein kinase-dependent exocytosis of flavocytochrome b558-containing granules

The respiratory burst of human neutrophils is primed by a number of pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS); however, the mechanism of priming remains unknown. LPS has been shown previously to increase membrane expression of flavocytochrome b(558), a component of the NADPH oxidase. This study shows that TNFalpha also increases membrane expression of flavocytochrome b(558). Mitogen-activated protein kinase (MAPK) modules have been implicated in the action of priming agents. Pharmacologic inhibitors of MAPKs, SB203580 and PD098059, revealed that priming of the respiratory burst and up-regulation of flavocytochrome b(558) are dependent on p38 MAPK but not on extracellular-signal regulated kinase (ERK). TNFalpha and LPS primed respiratory burst activity and increased membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules that contain flavocytochrome b(558), with similar time courses and concentration dependences. These processes also required p38 MAPK but were independent of ERK. TNFalpha failed to prime respiratory burst activity or to increase membrane CD35 expression in enucleated neutrophil cytoplasts. These data suggest that one mechanism by which TNFalpha and LPS prime neutrophil respiratory burst activity is by increasing membrane expression of flavocytochrome b(558) through exocytosis of intracellular granules in a process regulated by p38 MAPK.     

Cytochrome P450-dependent toxicity of environmental polycyclic aromatic hydrocarbons towards human macrophages

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are potent immunosuppressive environmental contaminants acting on lymphocytes and monocytes. To establish whether differentiated macrophages, which play a crucial role in innate and acquired immunity, can also constitute major cellular targets, we have characterized PAH effects towards primary human macrophages. BP-treatment was found to dramatically alter their functional capacities and to trigger a caspase- and mitochondrion-related apoptosis, associated with down-regulation of the survival factors c-FLIP(L) and Bcl-X(L) and up-regulation of the pro-apoptotic factor p53. Such deleterious effects were associated with BP metabolite production, whose inhibition by the cytochrome P-450 1A1 inhibitor alpha-naphthoflavone fully abolished BP toxicity. In contrast to BP, the related halogenated arylhydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin, known to be poorly metabolized if any, only minimally affected macrophages. Overall, these data provide evidence for a cytochrome P-450-dependent toxicity of PAHs towards human differentiated macrophages, which may contribute to their immunosuppressive effects.     

Extracellular-regulated kinase activation regulates replication of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> intracellularly in primary human monocytes

Mycobacterium avium-intracellulare (MAI) is a ubiquitous environmental pathogen that causes disseminated infection in immunocompromised patients, such as those with human immunodeficiency virus, interleukin-12 deficiency, or interferon-γ receptor mutation. Colony morphotypes are associated with MAI pathogenicity. Our previous studies have reported that smooth-transparent (SmT) morphotypes are more virulent and induce less cytokine (interleukin-1β and tumor necrosis factor-α) production by human monocytes than the smooth-domed (SmD) morphotypes. Mitogen-activated protein (MAP) kinases such as extracellular-regulated kinase (ERK) are activated by the phagocytosis of particle antigens in macrophages, and this ERK activation subsequently influences cytokine expression and the control of intracellular pathogen growth. The influence of MAP kinase activation on MAI replication in human monocytes was examined. Peripheral blood monocytes isolated from healthy subjects by Ficoll-Hypaque sedimentation were infected with virulent SmT or avirulent SmD MAI without or with MAP kinase inhibitors. MAP kinase activities were determined by in vitro kinase assay, intracellular MAI growth by CFU assay, and cytokines by enzyme-linked immunosorbent assay. MAI infection induced ERK and p38 activation. Inhibition of ERK by PD98059, but not p38, significantly increased intracellular MAI growth. Tumor necrosis factor-α release and interleukin-1β production in response to MAI were reduced by MAP kinase inhibition. p38 inhibition tended to reduce cytokine production more substantially. These data suggest that ERK activation limits intra-monocytic MAI replication and enhances monocytic cytokine release, whereas p38 activation influences only cytokine release. The effect of MAP kinases on MAI growth might thus be mediated by the modulation of cytokine production.     

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.     

Protein kinase C zeta plays an essential role for Mycobacterium tuberculosis-induced extracellular signal-regulated kinase 1/2 activation in monocytes/macrophages via Toll-like receptor 2

This study characterized the upstream signalling molecules involved in extracellular signal-regulated kinase (ERK) 1/2 activation and determined their effects on differential tumour necrosis factor (TNF)-alpha expression by monocytes/macrophages infected with virulent or avirulent mycobacteria. The avirulent Mycobacterium tuberculosis (MTB) strain H37Ra (MTBRa) induced higher levels of activation of ERK 1/2 and the upstream MAPK kinase (MEK)1 and, subsequently, higher levels of TNF-alpha expression in human primary monocytes and monocyte-derived macrophages, as compared with MTB strain H37Rv (MTBRv). The MTB-induced activation of ERK 1/2 was not dependent on Ras or Raf. However, inhibition of the activity of atypical protein kinase C (PKC) zeta decreased the in vitro phosphorylation of MEK, ERK 1/2 activation and subsequent TNF-alpha induction caused by MTBRv or MTBRa. Toll-like receptor (TLR) 2 was found to play a major role in MTB-induced TNF-alpha expression and PKCzeta phosphorylation. Co-immunoprecipitation experiments showed that PKCzeta interacts physically with TLR2 after MTB stimulation. Moreover, PKCzeta phosphorylation was increased more in macrophages following MTBRa, versus MTBRv, infection. This is the first demonstration that PKCzeta interacts with TLR2 to play an essential role in MTB-induced ERK 1/2 activation and subsequent TNF-alpha expression in monocytes/macrophages.     

Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2-RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1beta (interleukin-1beta) and TNFalpha (tumour necrosis factor-alpha). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-beta-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38alpha MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38alpha MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1beta and TNFalpha in these cells. Taken together, our results indicate that the MDP-NOD2/RIP2 and LPS (lipopolysaccharide)-TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.     

Regulatory Mechanisms of Vitamin D<Subscript>3</Subscript> on Production of Nitric Oxide and Pro-inflammatory Cytokines in Microglial BV-2 Cells

Inhibition of pro-inflammatory functions of microglia has been considered a promising strategy to prevent pathogenic events in the central nervous system under neurodegenerative conditions. Here we examined potential inhibitory effects of nuclear receptor ligands on lipopolysaccharide (LPS)-induced inflammatory responses in microglial BV-2 cells. We demonstrate that a vitamin D receptor agonist 1,25-dihydroxyvitamin D₃ (VD3) and a retinoid X receptor agonist HX630 affect LPS-induced expression of pro-inflammatory factors. Specifically, both VD3 and HX630 inhibited expression of mRNAs encoding inducible nitric oxide synthase (iNOS) and IL-6, whereas expression of IL-1β mRNA was inhibited only by VD3. The inhibitory effect of VD3 and HX630 on expression of iNOS and IL-6 mRNAs was additive. Effect of VD3 and HX630 was also observed for inhibition of iNOS protein expression and nitric oxide production. Moreover, VD3 and HX630 inhibited LPS-induced activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor κB (NF-κB). PD98059, an inhibitor of ERK kinase, attenuated LPS-induced nuclear translocation of NF-κB and induction of mRNAs for iNOS, IL-1β and IL-6. These results indicate that VD3 can inhibit production of several pro-inflammatory molecules from microglia, and that suppression of ERK activation is at least in part involved in the anti-inflammatory effect of VD3.