CCN1 Secretion Induced by Cigarette Smoking Extracts Augments IL-8 Release from Bronchial Epithelial Cells

Inflammation involves in many cigarette smoke (CS) related diseases including the chronic obstructive pulmonary disease (COPD). Lung epithelial cell released IL-8 plays a crucial role in CS induced lung inflammation. CS and cigarette smoke extracts (CSE) both induce IL-8 secretion and subsequently, IL-8 recruits inflammatory cells into the lung parenchyma. However, the molecular and cellular mechanisms by which CSE triggers IL-8 release remain not completely understood. In this study, we identified a novel extracellular matrix (ECM) molecule, CCN1, which mediated CSE induced IL-8 secretion by lung epithelial cells. We first found that CS and CSE up-regulated CCN1 expression and secretion in lung epithelial cells in vivo and in vitro. CSE up-regulated CCN1 via induction of reactive oxygen spices (ROS) and endoplasmic reticulum (ER) stress. p38 MAPK and JNK activation were also found to mediate the signal pathways in CSE induced CCN1. CCN1 was secreted into ECM via Golgi and membrane channel receptor aquaporin4. After CSE exposure, elevated ECM CCN1 functioned via an autocrine or paracrine manner. Importantly, CCN1 activated Wnt pathway receptor LRP6, subsequently stimulated Wnt pathway component Dvl2 and triggered beta-catenin translocation from cell membrane to cytosol and nucleus. Treatment of Wnt pathway inhibitor suppressed CCN1 induced IL-8 secretion from lung epithelial cells. Taken together, CSE increased CCN1 expression and secretion in lung epithelial cells via induction of ROS and ER stress. Increased ECM CCN1 resulted in augmented IL-8 release through the activation of Wnt pathway.     

Long non-coding RNA MEG3 regulates CSE-induced apoptosis and inflammation via regulating miR-218 in 16HBE cells

Chronic obstructive pulmonary disease (COPD) is a prevalent disease worldwide, mainly caused by cigarette smoking. Maternally expressed gene 3 (MEG3) functions as the lncRNA and is upregulated in COPD patients and human bronchial epithelial cells after fine particulate matter (PM2.5) treatment. However, the molecular mechanism of MEG3 in COPD remains unknown. The expression of MEG3 and miR-218 in COPD tissues and cigarette smoke extract (CSE)-treated 16HBE cells was detected by RT-qPCR. The effects of MEG3 and miR-218 on proliferation and apoptosis in (CSE)-treated 16HBE cells were analyzed by CCK-8 and flow cytometry assay, respectively. The protein levels of inflammatory cytokines (IL-1β IL-6 and TNF-α) were detected in 16HBE cells by ELISA. MEG3 and miR-218 binding interaction was predicted by LncBase Predicted v.2 and further confirmed by dual luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. MEG3 was upregulated in COPD tissues and inversely related to FEV1%. MEG3 was upregulated in (CSE)-treated 16HBE cells, and knockdown of MEG3 mitigated CSE-repressed proliferation and CSE-triggered apoptosis or inflammation. MiR-218 was demonstrated as a target miRNA of MEG3. MiR-218 was downregulated in COPD tissues and (CSE)-treated or MEG3 overexpressed 16HBE cells. MiR-218 overexpression attenuated CSE-blocked proliferation and CSE-induced apoptosis or inflammation. Deficiency of MEG3 counteracted CSE-blocked proliferation CSE-induced apoptotic rate and inflammatory cytokine (IL-1β IL-6 and TNF-α) levels, while introduction of anti-miR-218 reversed these effects. MEG3 regulated CSE-inhibited proliferation and CSE-induced apoptosis or inflammation by targeting miR-218, providing a possible therapeutic target for treatment of CSE-induced COPD.     

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

Comparison of nasal and bronchial epithelial cells obtained from patients with COPD

For in vitro studies of airway pathophysiology, primary epithelial cells have many advantages over immortalised cell lines. Nasal epithelial cells are easier to obtain than bronchial epithelial cells and can be used as an alternative for in vitro studies. Our objective was to compare nasal and bronchial epithelial cells from subjects with COPD to establish if these cells respond similarly to pro-inflammatory stimuli. Cell cultures from paired nasal and bronchial brushings (21 subjects) were incubated with cigarette smoke extract (CSE) prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 (TLR-4) message and expression by RT-PCR and FACS respectively. IL-8 release correlated significantly between the two cell types. IL-6 secretion was significantly less from bronchial compared to nasal epithelial cells and secreted concentrations did not correlate. A 4 h CSE incubation was immunosuppressive for both nasal and bronchial cells, however prolonged incubation for 24 h was pro-inflammatory solely for the nasal cells. CSE reduced TLR-4 expression in bronchial cells only after 24 h, and was without effect on mRNA expression. In subjects with COPD, nasal epithelial cells cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies.     

IL-33 mediates inflammatory responses in human lung tissue cells

IL-33 is a member of the IL-1 family and mediates its biological effects via the ST2 receptor, which is selectively expressed on Th2 cells and mast cells. Although polymorphic variation in ST2 is strongly associated with asthma, it is currently unclear whether IL-33 acts directly on lung tissue cells at sites of airway remodeling. Therefore, we aimed to identify the IL-33-responsive cells among primary human lung tissue cells. ST2 mRNA was expressed in both endothelial and epithelial cells but not in fibroblasts or smooth muscle cells. Correspondingly, IL-33 promoted IL-8 production by both endothelial and epithelial cells but not by fibroblasts or smooth muscle cells. Transfection of ST2 small interference RNA into both endothelial and epithelial cells significantly reduced the IL-33-dependent upregulation of IL-8, suggesting that IL-33-mediated responses in these cells occur via the ST2 receptor. Importantly, Th2 cytokines, such as IL-4, further enhanced ST2 expression and function in both endothelial and epithelial cells. The IL-33-mediated production of IL-8 by epithelial cells was almost completely suppressed by corticosteroid treatment. In contrast, the effect of corticosteroid treatment on the IL-33-mediated responses of endothelial cells was only partial. IL-33 induced activation of both ERK and p38 MAPK in endothelial cells but only ERK in epithelial cells. p38 MAPK was required for the IL-33-mediated responses of endothelial cells, whereas ERK was required for IL-33-mediated IL-8 production by epithelial cells. Taken together, these findings suggest that IL-33-mediated inflammatory responses of lung tissue cells may be involved in the chronic allergic inflammation of the asthmatic airway.     

Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle

Background

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.

Methods

HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni''s t test

Results

CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.

Conclusion

Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.     

SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro‐inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF‐κB nuclear accumulation, SIRT1 deacetylase activity, IL‐8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF‐κB binding to the IL‐8 gene promoter thus increasing IL‐8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF‐κB. FoxO3 siRNA treatment increased IL‐8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF‐κB activity and inducing pro‐inflammatory responses.     

Cigarette smoke alters IL-33 expression and release in airway epithelial cells

Airway epithelium is a regulator of innate immune responses to a variety of insults including cigarette smoke. Cigarette smoke alters the expression and the activation of Toll Like Receptor 4 (TLR4), an innate immunity receptor. IL-33, an alarmin, increases innate immunity Th2 responses. The aims of this study were to explore whether mini-bronchoalveolar lavage (mini-BAL) or sera from smokers have altered concentrations of IL-33 and whether cigarette smoke extracts (CSE) alter both intracellular expression (mRNA and protein) and release of IL-33 in bronchial epithelial cells. The role of TLR4 in the expression of IL-33 was also explored.Mini-BALs, but not sera, from smokers show reduced concentrations of IL-33. The expression of IL-33 was increased also in bronchial epithelium from smokers. 20% CSE reduced IL-33 release but increased the mRNA for IL-33 by real time PCR and the intracellular expression of IL-33 in bronchial epithelial cells as confirmed by flow cytometry, immunocytochemistry and western blot analysis. The effect of CSE on IL-33 expression was also observed in primary bronchial epithelial cells. IL-33 expression was mainly concentrated within the cytoplasm of the cells. LPS, an agonist of TLR4, reduced IL-33 expression, and an inhibitor of TLR4 increased the intracellular expression of IL-33. In conclusion, the release of IL-33 is tightly controlled and, in smokers, an altered activation of TLR4 may lead to an increased intracellular expression of IL-33 with a limited IL-33 release.     

Anti-inflammatory role of the cAMP effectors Epac and PKA: implications in chronic obstructive pulmonary disease

Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (ASM) cells, may contribute to the development of chronic obstructive pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little is known about its exact mechanism of action. We report here that next to the β(2)-agonist fenoterol, direct and specific activation of either exchange protein directly activated by cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBα-degradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) phosphorylation, which was selectively reduced by PKA activation. CSE decreased Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 expression was also reduced in lung tissue from COPD patients. In conclusion, Epac and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF-κB and ERK, respectively, pointing at these cAMP effectors as potential targets for anti-inflammatory therapy in COPD. However, cigarette smoke exposure may reduce anti-inflammatory effects of cAMP elevating agents via down-regulation of Epac1.     

Long non‐coding RNA TUG1 promotes airway remodelling by suppressing the miR‐145‐5p/DUSP6 axis in cigarette smoke‐induced COPD

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is primarily caused by cigarette smoke (CS)‐induced chronic inflammation. In this study, we investigated the function and mechanism of action of the long non‐coding RNA (lncRNA) taurine‐up‐regulated gene 1 (TUG1) in CS‐induced COPD. We found that the expression of TUG1 was significantly higher in the sputum cells and lung tissues of patients with COPD as compared to that in non‐smokers, and negatively correlated with the percentage of predicted forced expiratory volume in 1 second. In addition, up‐regulation of TUG1 was observed in CS‐exposed mice, and knockdown of TUG1 attenuated inflammation and airway remodelling in a mouse model. Moreover, TUG1 expression was higher in CS extract (CSE)‐treated human bronchial epithelial cells and lung fibroblasts, whereas inhibition of TUG1 reversed CSE‐induced inflammation and collagen deposition in vitro. Mechanistically, TUG1 promoted the expression of dual‐specificity phosphatase 6 (DUSP6) by sponging miR‐145‐5p. DUSP6 overexpression reversed TUG1 knockdown‐mediated inhibition of inflammation and airway remodelling. These findings suggested an important role of TUG1 in the pathological alterations associated with CS‐mediated airway remodelling in COPD. Thus, TUG1 may be a promising therapeutic target in CS‐induced airway inflammation and fibroblast activation.     

Shp2 plays an important role in acute cigarette smoke-mediated lung inflammation

Cigarette smoke (CS), the major cause of chronic obstructive pulmonary disease, contains a variety of oxidative components that were implicated in the regulation of Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) activity. However, the contribution of Shp2 enzyme to chronic obstructive pulmonary disease pathogenesis remains unclear. We investigated the role of Shp2 enzyme in blockading CS-induced pulmonary inflammation. Shp2 levels were assessed in vivo and in vitro. Mice (C57BL/6) or pulmonary epithelial cells (NCI-H292) were exposed to CS or cigarette smoke extract (CSE) to induce acute injury and inflammation. Lungs of smoking mice showed increased levels of Shp2, compared with those of controls. Treatment of lung epithelial cells with CSE showed elevated levels of Shp2 associated with the increased release of IL-8. Selective inhibition or knockdown of Shp2 resulted in decreased IL-8 release in response to CSE treatment in pulmonary epithelial cells. In comparison with CS-exposed wild-type mice, selective inhibition or conditional knockout of Shp2 in lung epithelia reduced IL-8 release and pulmonary inflammation in CS-exposed mice. In vitro biochemical data correlate CSE-mediated IL-8 release with Shp2-regulated epidermal growth factor receptor/Grb-2-associated binders/MAPK signaling. Our data suggest an important role for Shp2 in the pathological alteration associated with CS-mediated inflammation. Shp2 may be a potential target for therapeutic intervention for inflammation in CS-induced pulmonary diseases.     

Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a clinical complication caused by primary or secondary lung injury, as well as by systemic inflammation. Researches regarding molecular pathophysiology of ALI/ARDS are immerging with an ultimate aim towards developing prognostic molecular biomarkers and molecule-based therapy. However, the molecular mechanisms concerning ALI/ARDS are still not completely understood. The purpose of the present study was to identify a crucial role of CCN1 in inflammatory microenvironment during ALI/ARDS and focus on a potential communication between CCN1 and interleukin-6 (IL-6) in the airway epithelial cells. Our data illustrated that the expression levels of CCN1 and IL-6 in bronchoalveolar lavage fluid (BALF) in a lipopolysaccharide (LPS)-induced ALI mouse model were significantly elevated and the pulmonary expression of CCN1 was restricted to bronchial epithelial cells. Interestingly, both endogenous and exogenous CCN1 stimulated IL-6 production in vitro. Furthermore, LPS-induced IL-6 production in a bronchial epithelial cell line was blocked by CCN siRNA whereas CCN1 induced by LPS was sensitive to PI3K inhibition. Together, our data indicate a linear signal pathway, LPS-CCN1-IL-6, existing in bronchial epithelial cells after LPS exposure. This finding may represent an additional mechanism and a novel target for development of therapy and biomarker on ALI/ARDS.     

Upregulation of IL-17A/F from human lung tissue explants with cigarette smoke exposure: implications for COPD

Background

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with diminished responses to steroids. Increasing evidence supports elevated IL-17 expression in the lung of COPD subjects. However, whether cells of the immune system (systemic) and/or local lung cells are contributing to the elevated IL-17 remains unclear. To address this issue, we utilized a human parenchymal lung tissue explant culture system with cigarette smoke exposure to investigate the expression of IL-17 and the mechanisms involved.

Methods

Parenchymal lung tissue removed from 10 non-COPD and 8 COPD patients was sectioned and cultured with different concentrations of cigarette smoke extract (CSE) for 3 or 6 hours. Tissue viability was evaluated by LDH (lactate dehydrogenase) in culture supernatants. Western blot and real-time PCR were performed to evaluate IL-17A/F expression. To investigate the mechanisms, pharmacological inhibitors for MAPK p38, ERK1/2, NF-κB and PI3K pathways were added into the culture media.

Results

No tissue damage was observed after the cigarette smoke exposure for 3 h or 6 h compared with the control media. At the protein level, the expression of both IL-17A (2.4 ± 0.6 fold) and IL-17 F (3.7 ± 0.7 fold) in the tissue from non-COPD subjects was significantly increased by 5% of CSE at 3 h. For COPD subjects, IL-17A/F expression were significantly increased only at 6 h with 10% of CSE (IL-17A: 4.2 ± 0.8 fold; IL-17 F: 3.3 ± 0.8 fold). The increased expression of IL-17A/F is also regulated at the mRNA level. The inhibitors for NF-κB and PI3K pathways significantly inhibited CSE-induced IL-17A/F expression from lung tissue of non-COPD subjects.

Conclusions

We found the evidence that the expression of both IL-17A and IL-17 F is increased by the cigarette smoke exposure in explants from both non-COPD and COPD subjects, supporting that local lung cells contribute IL-17 production. The elevated IL-17A/F expression is dependent on NF-κB and PI3K pathways. These observations add to the growing evidence which suggests that Th17 cytokines play a significant role in COPD.     

Involvement of GSK-3β in attenuation of the cardioprotective effect of ischemic preconditioning in diabetic rat heart

ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.     

Klotho Reduction in Alveolar Macrophages Contributes to Cigarette Smoke Extract-induced Inflammation in Chronic Obstructive Pulmonary Disease

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Klotho, an anti-aging protein, has an anti-inflammatory function. However, the role of Klotho has never been investigated in COPD. The aim of this study is to investigate the possible role of Klotho by alveolar macrophages in airway inflammation in COPD. Klotho levels were assessed in the lung samples and peripheral blood mononuclear cells of non-smokers, smokers, and patients with COPD. The regulation of Klotho expression by cigarette smoke extract (CSE) was studied in vitro, and small interfering RNA (siRNA) and recombinant Klotho were employed to investigate the role of Klotho on CSE-induced inflammation. Klotho expression was reduced in alveolar macrophages in the lungs and peripheral blood mononuclear cells of COPD patients. CSE decreased Klotho expression and release from MH-S cells. Knockdown of endogenous Klotho augmented the expression of the inflammatory mediators, such as MMP-9, IL-6, and TNF-α, by MH-S cells. Exogenous Klotho inhibited the expression of CSE-induced inflammatory mediators. Furthermore, we showed that Klotho interacts with IκBα of the NF-κB pathway. Dexamethasone treatment increased the expression and release level of Klotho in MH-S cells. Our findings suggest that Klotho plays a role in sustained inflammation of the lungs, which in turn may have therapeutic implications in COPD.     

Roflumilast N-Oxide Prevents Cytokine Secretion Induced by Cigarette Smoke Combined with LPS through JAK/STAT and ERK1/2 Inhibition in Airway Epithelial Cells

Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD). Airway epithelial cells and macrophages are the first defense cells against cigarette smoke and these cells are an important source of pro-inflammatory cytokines. These cytokines play a role in progressive airflow limitation and chronic airways inflammation. Furthermore, the chronic colonization of airways by Gram-negative bacteria, contributes to the persistent airways inflammation and progression of COPD. The current study addressed the effects of cigarette smoke along with lipolysaccharide (LPS) in airway epithelial cells as a representative in vitro model of COPD exacerbations. Furthermore, we evaluated the effects of PDE4 inhibitor, the roflumilast N-oxide (RNO), in this experimental model. A549 cells were stimulated with cigarette smoke extract (CSE) alone (0.4% to 10%) or in combination with a low concentration of LPS (0.1 µg/ml) for 2 h or 24 h for measurement of chemokine protein and mRNAs and 5–120 min for protein phosphorylation. Cells were also pre-incubated with MAP kinases inhibitors and Prostaglandin E₂ alone or combined with RNO, before the addition of CSE+LPS. Production of cytokines was determined by ELISA and protein phosphorylation by western blotting and phospho-kinase array. CSE did not induce production of IL-8/CXCL8 and Gro-α/CXCL1 from A549 cells, but increase production of CCL2/MCP-1. However the combination of LPS 0.1 µg/ml with CSE 2% or 4% induced an important production of these chemokines, that appears to be dependent of ERK1/2 and JAK/STAT pathways but did not require JNK and p38 pathways. Moreover, RNO associated with PGE₂ reduced CSE+LPS-induced cytokine release, which can happen by occur through of ERK1/2 and JAK/STAT pathways. We report here an in vitro model that can reflect what happen in airway epithelial cells in COPD exacerbation. We also showed a new pathway where CSE+LPS can induce cytokine release from A549 cells, which is reduced by RNO.