Pretreatment Of BEAS-2B Cells Research Articles

Oxidative stress-mediated epidermal growth factor receptor activation regulates PM2.5-induced over-secretion of pro-inflammatory mediators from human bronchial epithelial cells

BackgroundExposure to PM2.5 has been associated with increased morbidity and mortality of lung diseases although the underlying mechanisms have not been fully uncovered. Airway inflammation is a critical event in the pathogenesis of lung diseases. This study aimed to examine the role of oxidative stress and epidermal growth factor receptor (EGFR) in PM2.5-induced pro-inflammatory response in a human bronchial epithelial cell line, BEAS-2B. MethodsBEAS-2B cells were exposed to 0, 20, 50, 100 and 150 μg/ml of PM2.5. Secretion of pro-inflammatory mediators including interleukin-6 (IL-6), IL-8 and IL-1β was determined using enzyme linked immunosorbent assay. Levels of intracellular reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the EGFR was examined with immunoblotting. ResultsPM2.5 exposure increased the secretion of IL-6, IL-8, and IL-1β in a concentration-dependent fashion. Moreover, exposure to PM2.5 elevated intracellular levels of ROS, and phosphorylation of the EGFR (Y1068). Pretreatment of BEAS-2B cells with either an antioxidant or a specific EGFR inhibitor significantly reduced PM2.5-induced IL-6, IL-8 and IL-1β secretion, implying that both oxidative stress and EGFR activation were involved in PM2.5-induced pro-inflammatory response. Furthermore, pre-treatment of BEAS-2B cells with an antioxidant significantly blunted PM2.5-induced EGFR activation, suggesting that oxidative stress was required for PM2.5-induced EGFR activation. ConclusionPM2.5 exposure induces pro-inflammatory response in human bronchial epithelial cells through oxidative stress-mediated EGFR activation.

Antioxidant and anti-genotoxic properties of cerium oxide nanoparticles in a pulmonary-like cell system.

Cerium oxide nanoparticles (CeO2-NP) present two different oxidation states what can suppose an auto-regenerative redox cycle. Potential applications of CeO2-NP to quench reactive oxygen species (ROS) in biological systems are currently being investigated. In this context, CeO2-NP may represent a novel agent to protect cells and tissues against oxidative damage by its regenerative free radical-scavenging properties. In this study, we have used a human epithelial lung cell line, BEAS-2B, as a model to study the possible antioxidant and anti-genotoxic effect of CeO2-NP in a pulmonary-like system. We have assessed the protective effect of CeO2-NP pre-treatment in front of a well-defined oxidative stress-inducing agent (KBrO3). Different endpoints like toxicity, intracellular ROS induction, genotoxicity and DNA oxidative damage (comet assay), and gene expression alterations have been evaluated. The obtained results confirmed the antioxidant properties of CeO2-NP. Thus, its pre-treatment significantly reduced the intracellular production of ROS induced by KBrO3. Similarly, a reduction in the levels of DNA oxidative damage, as measured with the comet assay complemented with formamidopyrimidine DNA glycosylase enzyme, was also observed. Pre-treatment of BEAS-2B cells with CeO2-NP (at 2.5µg/mL) slightly increased the viability of cells treated with KBrO3 as well as down-regulated the expression of the Ho1 and Sod2 genes involved in the oxidative Nrf2 pathway. Our finding would support the potential usefulness of CeO2-NP as a pharmacological agent to be used against diseases caused by oxidative stress.

SRC-mediated EGF receptor activation regulates ozone-induced interleukin 8 expression in human bronchial epithelial cells.

Background:Human exposure to ozone (O3) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation.ObjectiveWe examined the role of EGFR activation in O3-induced expression of the chemokine interleukin 8 (IL-8) in human bronchial epithelial cells (HBEC).MethodsWe detected phosphorylated EGFR using immunoblotting. EGFR dimerization was examined through cross-linking reaction and immunoblotting, and levels of IL-8 protein were measured using ELISA.ResultsExposure to O3 (0.25–1.0 ppm) induced rapid and marked increase in EGFR phosphorylation at the autophosphorylation site Y1068 and the transphosphorylation site Y845, implicating the involvement of Src kinase. Further investigation showed that O3 stimulation induced phosphorylation of Src at Y416, indicative of Src activation. Pharmacological inhibition of Src kinase activity abrogated O3-induced EGFR phosphorylation at tyrosines 1068 and 845. Moreover, pretreatment of BEAS-2B cells with inhibitor of either EGFR or Src kinase activities significantly blocked O3-induced IL-8 expression.ConclusionConclusion: O3 exposure increased IL-8 expression through Src-mediated EGFR transactivation in HBEC.Citation>Wu W, Wages PA, Devlin RB, Diaz-Sanchez D, Peden DB, Samet JM. 2015. Src-mediated EGF receptor activation regulates ozone-induced interleukin 8 expression in human bronchial epithelial cells. Environ Health Perspect 123:231–236; http://dx.doi.org/10.1289/ehp.1307379

Vam3, a resveratrol dimer, inhibits cigarette smoke-induced cell apoptosis in lungs by improving mitochondrial function.

To investigate the effects of Vam3 (a resveratrol dimer extracted from Vitis amurensis Rupr) on cigarette smoke (CS)-induced cell apoptosis in lungs in vitro and in vivo and the underlying mechanisms of action. Human bronchial epithelial cell line BEAS-2B was exposed to cigarette smoke condensate (CSC, 300 mg/L), and cell apoptosis was determined using flow cytometry and Hoechst staining. Mitochondrial membrane potential was examined with TMRE staining. ROS and ceramide levels were detected with DCFH-DA fluorescence and HPLC-MS/MS, respectively. Cytochrome c release was detected using immunofluorescence. Caspase-9 and neutral sphingomyelinase 2 expression was measured with Western blotting. The breast carcinoma cell line MCF7 stably expressing GFP-tagged Bax was used to elucidate the role of mitochondria in CS-induced apoptosis. For in vivo study, male mice were exposed to CS for 5 min twice a day for 4 weeks. The mice were orally administered Vam3 (50 mg·kg(-1)·d(-1)) or resveratrol (30 mg·kg(-1)·d(-1)) each day 1 h before the first CS exposure. Pretreatment of BEAS-2B cells with Vam3 (5 μmol/L) or resveratrol (5 μmol/L) significantly suppressed CSC-induced apoptosis, and prevented CSC-induced Bax level increase in the mitochondria, mitochondrial membrane potential loss, cytochrome c release and caspase-9 activation. Furthermore, pretreatment of BEAS-2B cells with Vam3 or resveratrol significantly suppressed CSC-stimulated intracellular ceramide production, and CSC-induced upregulation of neutral sphingomyelinase 2, the enzyme responsible for ceramide production in bronchial epithelial cells. Similar results were obtained in C6-pyridinium ceramide-induced apoptosis of GFP-Bax-stable MCF7 cells in vitro, and in the lungs of CS-exposed mice that were treated with oral administration of Vam3 or resveratrol. Vam3 protects bronchial epithelial cells from CS-induced apoptosis in vitro and in vivo by preventing mitochondrial dysfunction.

Transcriptional and posttranscriptional regulation and endocytosis were involved in zinc oxide nanoparticle-induced interleukin-8 overexpression in human bronchial epithelial cells

Inhaled zinc oxide nanoparticles (ZnO-NPs) can induce lung inflammation through released inflammatory mediators, such as interleukin 8 (IL-8), from airways. However, the mechanisms underlying ZnO-NP-induced IL-8 gene expression have not been fully characterized. The transcription inhibitor actinomycin D (Act D) and the BEAS-2B cells stably overexpressing wild-type or mutated IL-8 promoter at the NFκB or C/EBPβ binding site were used to determine the involvement of transcriptional mechanisms. The effect of ZnO-NPs on IL-8 mRNA stability was examined using mRNA decay assay. The phagocytosis inhibitor cytochalasin B (CB) was utilized to define the role of endocytosis in ZnO-NP-induced IL-8 expression. In addition, the solubility of ZnO-NPs in culture medium was assessed using atomic absorption spectroscopy. Exposure to ZnO-NPs significantly increased the expression of IL-8 mRNA and protein in a dose-dependent manner. Pretreatment with Act D blocked ZnO-NP-induced IL-8 expression. Both NFκB and C/EBPβ transcription factors were required for ZnO-NP-induced IL-8 transcription. mRNA decay assay showed that ZnO-NP stimulation delayed IL-8 mRNA degradation in BEAS-2B cells. Pretreatment of BEAS-2B cells with CB blocked ZnO-NP-induced IL-8 expression by 30%. Exposure to ZnO-NPs induced IL-8 gene expression through transcriptional activation and mRNA stabilization. Internalization of nanoparticles was partially involved in ZnO-NP-induced IL-8 expression.

Double-stranded RNA increases kinin B1 receptor expression and function in human airway epithelial cells

Increased levels of kinins have been detected within the airways during upper respiratory viral infections (URIs). Rhinovirus, the major URI associated with acute exacerbations of asthma, is an ssRNA virus that primarily infects the airway epithelium and produces dsRNA during replication. We asked whether dsRNA could increase the expression of kinin receptors in airway epithelial cells, thereby potentiating the inflammatory consequences of kinin generation. Human airway epithelial cell line BEAS-2B was stimulated with the dsRNA analog Poly I:C and kinin receptor expression detected by quantitative RT-PCR as well as radioligand binding. Poly I:C induced an increase in B1 and B2 receptor mRNA levels in BEAS-2B and primary human normal bronchial epithelial cells. At the cell surface, only B1 receptor expression was increased by Poly I:C. Furthermore, pretreatment of BEAS-2B cells with Poly I:C enhanced the induction of phospho-ERK following B1 receptor ligand stimulation. To investigate whether these finding had potential in vivo relevance, we assessed B1 receptor expression in nasal tissue obtained from 8 normal human subjects with URIs and 3 control subjects. Five of the URI subjects demonstrated increased B1 receptor mRNA compared to the 3 control subjects. We suggest that increased expression of B1 receptor in the human airway following a URI could increase the risk of an exacerbation of asthma by contributing to increased inflammation in the airway.

Upregulation of interleukin-4 receptor by interferon-gamma: enhanced interleukin-4-induced eotaxin-3 production in airway epithelium.

Airway epithelial cells produce a number of chemokines, including eotaxins. Among the three known eotaxins, T helper (Th) type 2 cytokines have been observed to induce the expression of eotaxin-3 mRNA. This study investigated the effect of interferon (IFN)-gamma, a Th1 cytokine, on Th2 cytokine-induced eotaxin-3 production in a bronchial epithelial cell line, BEAS-2B. BEAS-2B cells produced eotaxin-3 after stimulation with the Th2 cytokines interleukin (IL)-13 and IL-4. When BEAS-2B cells were cultured with varying concentrations of IFN-gamma for 24 h, dose-dependent inhibition of Th2 cytokine-induced eotaxin-3 mRNA expression and protein production was observed. This was associated with downregulation of signal transducer and activator of transcription 6 activation. On the other hand, 2-d pretreatment of BEAS-2B cells with IFN-gamma dose-dependently enhanced Th2 cytokine-induced eotaxin-3 mRNA expression and production. IFN-gamma also increased the mRNA expression and protein production of IL-4 receptor (R) alpha in a time- and dose-dependent manner. In addition, IL-2Rgamma, a component of the type 1 IL-4R, was also upregulated by IFN-gamma. These results indicate that IFN-gamma has opposite effects on Th2 cytokine-induced eotaxin-3 production in BEAS-2B cells, depending on the length of exposure. Because high levels of IFN-gamma are produced during viral infection, airway viral infection may affect allergic airway inflammation in vivo by modulation of eotaxin-3 production.

Urokinase induces its own expression in Beas2B lung epithelial cells.

The urokinase-type plasminogen activator (uPA) interacts with its receptor (uPAR) to promote local proteolysis as well as cellular proliferation and migration. These functions contribute to the pathogenesis of lung inflammation and remodeling as well as the growth and invasiveness of lung neoplasms. In this study, we sought to determine if uPA alters its own expression in lung epithelial cells. Using immunoprecipitation and Western and Northern blotting techniques, we found that uPA treatment enhanced uPA expression in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The induction of uPA expression is mediated through its cell surface receptor uPAR and does not require uPA enzymatic activity. The amino-terminal fragment of uPA, lacking the catalytic domain, is sufficient to induce uPA expression. The serine protease plasmin and the protease inhibitor aprotinin failed to alter uPA-mediated uPA expression, whereas alpha-thrombin potentiated the response. Pretreatment of Beas2B cells with a tyrosine kinase inhibitor, herbimycin, suggests that activation of tyrosine kinase(s) is involved in the uPA-mediated uPA expression. Induction of uPA expression by exposure of lung-derived epithelial cells to uPA is a newly defined pathway by which this protease could influence expression of local fibrinolytic activity and other uPA-dependent cellular responses germane to lung inflammation or neoplasia.

Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells.

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe(3+) concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.

Particulate Matter Initiates Inflammatory Cytokine Release by Activation of Capsaicin and Acid Receptors in a Human Bronchial Epithelial Cell Line

Recent experiments have shown that human bronchial epithelial cells (i.e., BEAS-2B) release pro-inflammatory cytokines (i.e., IL-6 and TNFα) in a receptor-mediated fashion in response to the neuropeptides, substance P (SP), calcitonin gene-related protein (CGRP), and the prototype botanical irritant capsaicin. In the present experiments, we examined the relevance of these receptors to particulate matter (PM)-associated cellular inflammation. BEAS-2B cells, exposed to residual oil fly ash particles (ROFA), responded with an immediate (<30 s) increase in intracellular calcium levels ([Ca2+]i), increases of key inflammatory cytokine transcripts (i.e., IL-6, IL-8, TNFα) within 2 h exposure, and subsequent release of IL-6 and IL-8 cytokine protein after 4 h exposure. Pretreatment of BEAS-2B cells with pharmacological antagonists selective for the SP or CGRP receptors reduced the ROFA-stimulated IL-6 cytokine production by ∼25 and 50%, respectively. However, pretreatment of these cells with capsazepine (CPZ), an antagonist for capsaicin (i.e., vanilloid) receptors, inhibited the immediate increases in [Ca2+]i, diminished transcript (i.e., IL-6, IL-8, TNFα) levels and reduced IL-6 cytokine release to control levels. BEAS-2B cells exposed to ROFA in calcium-free media failed to demonstrate increases of [Ca2+]iand showed reduced levels of cytokine transcript (i.e., IL-6, IL-8, TNFα) and IL-6 release, suggesting that ROFA-stimulated cytokine formation was partially dependent on extracellular calcium sources. A final set of experiments compared the inflammatory properties of the soluble and acidic insoluble components of ROFA. BEAS-2B cells, exposed to ROFA or ROFA that had been filtered through a 0.2-μm pore filter, produced equivocal IL-6. BEAS-2B cells exposed to pH 5.0 media for 15 min released moderate amounts of IL-6, 4 h later. This cytokine release could be blocked by amiloride, a pH receptor antagonist, but not by CPZ. BEAS-2B cells, pretreated with amiloride before ROFA exposure, showed a partial (∼25%) reduction of IL-6. Together, these data indicate that the acidic, soluble components of ROFA initiate cytokine release in BEAS-2B cells through activation of both capsaicin- and pH-sensitive irritant receptors.