Divergent effects of nitric oxide on airway epithelial cell activation

Nitric oxide (NO*) is a gaseous mediator synthesized by nitric oxide synthases. NO* is involved in the modulation of inflammation, but its role in airway inflammation remains controversial. We investigated the role of NO* in the synthesis of the chemokines interleukin-8 and monocyte chemotactic protein-1, and of intercellular adhesion molecule-1 by human airway epithelial cells. normal human bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used. interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and intercellular adhesion molecule-1 (ICAM-1) expression were measured by ELISA. mRNA was assessed by semiquantitative RTI-PCR. Interleukin-8 secretion was significantly reduced after 24h incubation with the NO* donor, sodium nitroprusside. The effect was dose-dependent. Similar results were obtained with S-nitroso-N-D,L-penicillamine and S-nitroso-L-glutathione. Inhibition of endogenous NO* with the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl-ester caused an increase in IL-8 secretion by lipopolysaccharide- and cytokine-stimulated BEAS-2B cells. Sodium nitroprusside also caused a reduction in monocyte chemotactic protein-1 secretion by both cell types. In contrast, intercellular adhesion molecule-1 expression was upregulated by sodium nitroprusside. RTI-PCR results indicate that the modulation of protein levels was paralleled by modification in mRNA levels. NO* has divergent effects on the synthesis of different inflammatory mediators in human bronchial epithelial cells.     

Biosynthesized matrix provides a key role for survival signaling in bronchial epithelial cells

The extracellular matrix (ECM) influences a variety of cellular functions, including survival, adhesion molecule expression, differentiation, and migration. The ECM composition of the epithelial basement membrane is altered in asthmatics. In this study, we elucidate the major survival signals received by bronchial epithelial cells in vitro by studying the effects of a variety of ECM factors and soluble growth factors on bronchial epithelial cell survival. Our findings indicate that the insulin family of soluble growth factors provides important survival signals but also that adhesion to ECM is a crucial determinant of bronchial epithelial cell survival. In the BEAS-2B bronchial epithelial cell line, collagens I and IV, laminin, fibronectin, and vitronectin provide significant levels of protection from apoptosis. Tenascin-C has no effect, whereas elastin and collagen V increase apoptosis to above control levels. BEAS-2B cells secrete their own biosynthesized matrix (BSM), which also provides rescue from apoptosis. Protection by collagen I, fibronectin, and vitronectin was found to be via an RGD domain. Laminin-, collagen IV-, and BSM-mediated survival is not RGD dependent. Primary bronchial epithelial cells exhibit a similar pattern of apoptosis rescue to the BEAS-2B cell line, although we did not observe any vitronectin-mediated protection in the primary cells. These data indicate that bronchial epithelial cell survival is dependent both on soluble growth factors and on a variety of ECM-derived signals.     

Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.     

Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells

Peroxynitrite, formed by the reaction of nitric oxide (NO. ) with superoxide anions (O(2)(-).), may play a role in the pathophysiology of inflammation. The effects of 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, on the human bronchial epithelial cell line BEAS-2B, were examined. SIN-1 exposure resulted in cell death in a time- and dose-dependent manner. Depletion of intracellular glutathione increased the vulnerability of the cells. Pretreatment with Mn(III)tetrakis(N-methyl-4'-pyridyl)porphyrin (MnTMPyP) or hydroxocobalamin (HC), O(2)(-). and NO. scavengers, respectively, reduced significantly SIN-1-induced cell death (18.66 +/- 3.57 vs. 77.01 +/- 14.07 or 82.20 +/- 9.64, % cell viability SIN-1 vs. MnTMPyP or HC). Moreover, the mitogen-activated protein kinases (MAPK) p44/42 (ERK), p38, and p54/46 (JNK) were also activated in a time- and concentration-dependent manner. PD-98059 and SB-239063, specific inhibitors of ERK and p38 MAPK pathways, failed to protect cells against 1 mM SIN-1. However, PD-98059 partially inhibited (60% cell survival) SIN-1 effects at < or =0.25 mM, and this was increased with the inclusion of SB-239063. Therefore, MAPKs may mediate signal transduction pathways induced by peroxynitrite in lung epithelial cells leading to cell death.     

Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity.

The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2 (cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11.cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2 immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10(-6) M). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.     

Pneumococci induced TLR- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter in lung epithelial cells

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia. The respiratory epithelium constitutes the first line of defense against invading lung pathogens, including pneumococci. We analyzed the involvement of Toll-like receptors (TLR) and Rho-GTPase signaling in the activation of human lung epithelial cells by pneumococci. S. pneumoniae induced release of interleukin-8 (IL-8) by human bronchial epithelial cell line BEAS-2B. Specific inhibition of Rac1 by Nsc23766 or a dominant-negative mutant of Rac1 strongly reduced cytokine release. In addition, pneumococci-related cell activation (IL-8 release, NF-kappaB-activation) depended on MyD88, phosphatidylinositol 3-kinase, and Cdc42 but not on RhoA. Pneumococci enhanced TLR1 and TLR2 mRNA expression in BEAS-2B cells, whereas TLR4 and TLR6 expression was constitutively high. TLR1 and 2 synergistically recognized pneumococci in cotransfection experiments. TLR4, TLR6, LPS-binding protein, and CD14 seem not to be involved in pneumococci-dependent cell activation. At the IL-8 gene promoter, recruitment of phosphorylated NF-kappaB subunit p65 was blocked by inhibition of Rac1, whereas binding of the phosphorylated activator protein-1 subunit c-Jun to the promoter was not diminished. In summary, these results suggest that S. pneumoniae activate human epithelial cells by TLR1/2 and a phosphatidylinositol 3-kinase- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter.     

Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16 production by BEAS-2B cells, a bronchial epithelial cell line

Previous studies have suggested that bronchial epithelial cells may perpetuate airway inflammation. We have reported that the bronchial epithelial cell line BEAS-2B can produce interleukin (IL)-16, a potent chemoattractant for CD4+ T cells. IL-16 is thought to regulate airway inflammation in asthmatics. Recent studies showed that IL-4 induces inflammatory cytokines in bronchial epithelial cells and that IL-9 is a candidate gene for development of asthma. The present study demonstrated that BEAS-2B cells produced specifically IL-16 by synergistic effects of IL-4 + IL-16, or IL-9 + IL-16, and that the synthesized IL-16 induced migration of CD4+ T cells. This study is a first report indicating that IL-16 production may be maintained by an autocrine machinery by epithelial cell-derived IL-16 with IL-4 and IL-9 in asthma.     

Expression and roles of Cl- channel ClC-5 in cell cycles of myeloid cells

This study investigated the effect of exogenous nitric oxide (NO) on endothelial glucocorticoid receptor (GR) function. The NO donor diethylenetriamine NONOate (DETA, 50-500microM) caused concentration dependent nuclear localization of transfected chimeric green fluorescent protein GFP-GR and elevated expression of secreted alkaline phosphatase (SEAP) from a glucocorticoid response element (GRE) promoter construct in bovine aortic endothelial cells. Other weaker NO donors (S-nitroso-N-acetylpenicillamine and spermine NONOate) failed to induce GFP-GR nuclear localization, but all the NO donors activated GRE-SEAP expression, a response unaffected by the antioxidant N-acetyl-L-cysteine. Overall, exogenous NO from high concentration donors can directly activate GR, suggesting a potential feedback mechanism for NO to regulate endothelial inducible nitric oxide synthase (iNOS) expression.     

Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene

A satisfactory model describing the airway surface fluid (ASF) in the airways of persons with cystic fibrosis (CF) remains to be established due to theoretical challenges to both the "Hydration Hypothesis" and the "Salt Hypothesis." Irrespective of these models, inhaled hypertonic saline is often used to facilitate clearance of inspissated secretions. Hypertonicity induces interleukin-8 (IL-8) expression, a potent chemokine for neutrophils. The objectives of this study were: (i) to determine the relative contribution of three potential cis-regulatory elements in the regulation of NaCl-induced IL-8 production in BEAS-2B human bronchial epithelial cells, (ii) to compare NaCl-induced IL-8 expression in IB3-1 bronchial epithelial cells, which have the DeltaF508/W1282X mutation of the CF transmembrane conductance regulator (CFTR) gene, with that in C38 cells, which are IB3-1 cells stably transfected with a truncated but functional CFTR gene, and (iii) to compare equal osmolar concentrations of NaCl and D-sorbitol in the induction of IL-8 in all three cell types. In human bronchial epithelial cells, binding sites for NFkappaB, AP-1, and NF-IL6 in the 5'-flanking region of the IL-8 promoter are necessary for optimal NaCl induction of IL-8. Human bronchial epithelial cells with the DeltaF508/W1282X CFTR mutation produce an exaggerated amount of basal and NaCl-induced IL-8.     

Perilla frutescens Leaf Extract Inhibits Mite Major Allergen Der p 2-induced Gene Expression of Pro-Allergic and Pro-Inflammatory Cytokines in Human Bronchial Epithelial Cell BEAS-2B

Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens.     

Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils

Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.     

Transiently, paralleled upregulation of arginase and nitric oxide synthase and the effect of both enzymes on the pathology of asthma

Changes in the expression of arginase and their association with nitrosative stress were investigated using an asthmatic model previously established in NC/Nga mice with mite extract. Mite crude extract (100 microg/day) from Dermatophagoides farinae was administered intranasally for 5 consecutive days (day 0-4), and a single challenge was performed on day 11. On day 12, upregulation of the mRNA expression of inducible types of nitric oxide synthase (iNOS) and increases in immunohistochemical staining for iNOS and nitrotyrosine were observed. However, the level of nitrite + nitrate was unchanged. An increase in enzymatic activity, upregulation of mRNA expression, and immunostaining for arginase I was detected in the lung tissue and serum. Moreover, increases in both arginase I and II were revealed by immunoblotting. Goblet cell hyperplasia in bronchial epithelial cells and increasing collagen synthesis around the bronchus were also observed. These results suggested that an increase in arginase may lead to decreased availability of arginine for nitric oxide synthase and may contribute to the remodeling of the lung.