interleukin-8 and 1-nitropyrene

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are widespread environmental pollutants, generated from reactions between PAHs and nitrogen oxides during combustion processes. In the present study we have investigated the mechanisms of CXCL8 (IL-8) responses induced by 1-nitropyrene (1-NP) in human bronchial epithelial BEAS-2B cells, with focus on the possible importance of Ca(2+)-signaling and activation of β2-adrenergic receptors (β2AR). Ca(2+)-chelator treatment obliterated 1-NP-induced CXCL8 (IL-8) responses. 1-NP at 10μM (but not 1μM) induced a rapid and sustained increase in intracellular Ca(2+)-levels ([Ca(2+)]i). The early but not the later, sustained phase of 1-NP-induced [Ca(2+)]i was suppressed by beta-blocker treatment (carazolol). Moreover, inhibition of β2AR by blocking-antibody, beta-blocker treatment (ICI 118551) or siRNA transfection attenuated CXCL8 responses induced by 1-NP. The results confirm that PAHs may induce Ca(2+)-signaling also in BEAS-2B cells, at least partly through activation of β2AR, and suggest that both β2AR- and Ca(2+)-signaling may be involved in 1-NP-induced CXCL8 responses in bronchial epithelial cells.

Topics: Calcium; Calcium Signaling; Cell Line; Gene Expression; Humans; Interleukin-8; Pyrenes; Receptors, Adrenergic, beta-2; RNA, Small Interfering

The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B).. Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C.. AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Topics: Air Pollutants; Aryl Hydrocarbon Receptor Nuclear Translocator; Benzoflavones; Bronchi; Cell Line, Tumor; Chemokine CCL5; Epithelial Cells; Humans; Interleukin-8; NF-kappa B; Phosphorylation; Poly I-C; Pyrenes; Receptors, Aryl Hydrocarbon; Serine; Signal Transduction; Tumor Necrosis Factor-alpha

We have previously observed that 1-nitropyrene (1-NP) and its amine metabolite 1-aminopyrene (1-AP) induce differential chemokine responses in human bronchial epithelial cells (BEAS-2B) characterized by maximum responses for CXCL8 (IL-8) and CCL5 (RANTES), respectively. In the present study, we further explored the effects of 1-NP and 1-AP on chemokine responses. The results suggest that the differential effect of 1-NP and 1-AP on CXCL8 and CCL5 in BEAS-2B cells was mainly related to effects at higher concentrations, which in the case of 1-NP seemed to be linked to ROS-formation and/or metabolic activation by CYP-enzymes. However, at a low concentration (1 μM) where neither 1-NP, 1-AP nor unsubstituted pyrene had any effect on chemokine responses, we found that all three PAHs potentiated CXCL8 and CCL5 responses induced by the TLR3 ligand polyinosinic:polycytidylic acid (Poly I:C) in BEAS-2B cells. As neither benzo[a]pyrene nor β-naphthoflavone induced a similar effect in Poly I:C-primed cells, the response seemed independent of aryl hydrocarbon receptor-mediated mechanisms. The results show that priming cells with an inflammogenic stimuli like Poly I:C sensitizes the cells toward additional pro-inflammatory effects of certain PAHs. The study underscores that testing on healthy cells or animals may not be sufficient to fully evaluate chemokine responses and the pro-inflammatory potential of organic chemicals.

Topics: Blotting, Western; Bronchi; Cell Line; Chemokine CCL5; Chemokines; Cytochrome P-450 Enzyme System; Epithelial Cells; Flow Cytometry; Gene Silencing; Humans; Interleukin-8; Poly I-C; Polycyclic Aromatic Hydrocarbons; Pyrenes; Reactive Oxygen Species; RNA, Small Interfering; Toll-Like Receptor 3

1-nitropyrene (1-NP), a common PAH in diesel exhaust, and its amine metabolite 1-aminopyrene (1-AP) induce distinctly different chemokine-responses in bronchial epithelial cells (BEAS-2B) characterized by increases in CXCL8 and CCL5, respectively. Tumor necrosis factor-α converting enzyme (TACE), which cleaves membrane-bound transforming growth factor (TGF)-α, activating the epidermal growth factor receptor (EGFR), may regulate pro-inflammatory responses induced by a variety of endogenous and exogenous agents. The present results suggest that CXCL8, but not CCL5 responses in 1-NP- or 1-AP-exposed cells required TACE/TGF-α/EGFR-signaling. The findings strengthen the notion that TACE/TGF-α/EGFR-signaling is central in epithelial CXCL8-regulation upon exposure to multiple airborne pollutants.

Topics: ADAM Proteins; ADAM17 Protein; Bronchi; Cells, Cultured; Chemokines; Enzyme Inhibitors; Epithelial Cells; ErbB Receptors; Humans; Imidazoles; Interleukin-8; Pyrenes; Pyridines; Signal Transduction; Transforming Growth Factor alpha