Regulation of NF-kappaB and ICAM-1 expression in human airway epithelial cells.

The aim of this study was to elucidate the redox regulation of cytokine-induced NF-kappaB activation and NF-kappaB mediated gene induction in A549 cells and primary cultures of human airway epithelial cells. In A549 cells, Western blot analysis showed transient depletion of IkappaBalpha after 15 min IL-1beta treatment followed by its reappearance after 60 min, indicating efficient NF-kappaB-driven gene induction. A similar pattern was observed in primary epithelial cells however, the kinetics were slower and depletion was less. In primary airway epithelial cells IkappaBalpha levels were 59.8+/-8.5% of control following 30 min treatment with IL-1beta and in A549 cells 29.1+/-8.5% of control following 15 min IL-1beta treatment. Cytokine-induced IkappaBalpha depletion was associated with NF-kappaB nuclear accumulation and subsequent resynthesis of IkappaBalpha and upregulation of ICAM-1 in both cell types. The antioxidant, NAC (20 mM) had no effect on the kinetics of cytokine-induced IkappaBalpha depletion or NF-kappaB p65 nuclear translocation in either cell type and failed to influence kappaB dependent IkappaBalpha resynthesis. H2O2 treatment alone or in combination with cytokines had no significant effects on IkappaBalpha depletion, NF-kappaB p65 nuclear translocation or ICAM-1 expression in either cell type but did cause significant activation of p38 MAPK. These results suggest that cytokine-induced NF-kappaB activation in cultured human airway epithelial cells does not involve an NAC-sensitive oxidant stress and that H2O2-induced oxidant stress does not result in effective NF-kappaB activation and NF-kappaB mediated gene induction.     

Peroxisome proliferator-activated receptor-gamma agonists inhibit respiratory syncytial virus-induced expression of intercellular adhesion molecule-1 in human lung epithelial cells

Respiratory syncytial virus (RSV) is the major causative agent of severe lower respiratory tract disease and death in infants worldwide. The epithelial cells of the airways are the target cells for RSV infection and the site of the majority of the inflammation associated with the disease. However, despite five decades of intensive RSV research there exist neither an effective active vaccine nor a promising antiviral and anti-inflammatory therapy. Recently, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we hypothesized whether the detrimental increase of intercellular adhesion molecule-1 (ICAM-1) on RSV-infected lung epithelial cells (A549 and primary normal human bronchial epithelial cells (NHBE)) might be modulated by natural and synthetic PPAR-gamma agonists (15d-PGJ2, ciglitazone, troglitazone, Fmoc-Leu). Our data show that all PPAR-gamma agonists under study significantly down-regulated the RSV-induced expression of ICAM-1 on A549- and NHBE cells in a dose-dependent manner resulting in a reduced beta2 integrin-mediated adhesion of monocytic effector cells (U937) to RSV-infected A549 cell monolayers. In contrast, the PPAR-alpha agonist bezafibrate had no impact on the RSV-induced ICAM-1 expression. The reduced ICAM-1 expression was associated with a diminished ICAM-1 mRNA level and binding activity of nuclear factor-kappaB (p65/p50) in A549 cells. These findings suggest that PPARgamma agonists have beneficial effects in the suppression of the inflammatory response during RSV infection and therefore might have clinical efficacy in the course of severe RSV-infection.     

Activation of peroxisome proliferator-activated receptor-gamma in dendritic cells inhibits the development of eosinophilic airway inflammation in a mouse model of asthma

Peroxisome proliferator-activated receptors (PPARs) are activated by an array of polyunsaturated fatty acid derivatives, oxidized fatty acids, and phospholipids and are proposed to be important modulators of immune and inflammatory responses. Recently, we showed that activation of PPAR-gamma alters the maturation process of dendritic cells (DCs), the most potent antigen-presenting cells. In the present report, we investigated the possibility that, by targeting DCs, PPAR-gamma activation may be involved in the regulation of the pulmonary immune response to allergens. Using a model of sensitization, based on the intratracheal transfer of ovalbumin (OVA)-pulsed DCs, we show that rosiglitazone, a selective PPAR-gamma agonist, reduces the proliferation of Ag-specific T cells in the draining mediastinal lymph nodes but, surprisingly enough, dramatically increases the production of the immunoregulatory cytokine interleukin (IL)-10 by T cells, as compared to control mice sensitized with OVA-pulsed DCs. After aerosol challenge, the recruitment of eosinophils in the bronchoalveolar lavage fluids was strongly reduced compared to control mice. Finally, T cells from the mediastinal lymph nodes produced higher amounts of IL-10 and interferon-gamma. Inhibition of IL-10 activity with anti-IL-10R antibodies partly restored the inflammation. The specificity of the phenomenon was confirmed by treating OVA-pulsed DCs with ciglitazone, another PPAR-gamma agonist, and by using GW9662, a PPAR-gamma antagonist. Our data suggest that PPAR-gamma activation prevents induction of Th2-dependent eosinophilic airway inflammation and might contribute to immune homeostasis in the lung.     

Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues

Objective: Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues.

Methods: Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10?ng/ml) treatment for 4?h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay.

Results: Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB.

Conclusions: The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine

It was reported recently that histamine induced Toll-like receptor (TLR)2 and TLR4 expression in endothelial cells and enhanced their sensitivity to Gram-positive and Gram-negative bacteria; and that TLRs were expressed in airway epithelial cells and that several inflammatory mediators modulated their expression. However, little is known of potential influence of histamine on TLRs in pulmonary epithelial cells. In the present study, effects of histamine on expression of TLRs in both human A549 and NCI-H292 cell lines were examined by using real-time quantitative RT-PCR analysis, flow cytometry and immunofluorescent staining. The results revealed that both cell types constitutively expressed mRNAs for TLR1-TLR10. Histamine up-regulated the expression of TLR3 mRNA by 12.3- and 11.6-fold, respectively in both cell types. The time course showed that histamine induced TLR3 mRNA expression was initiated at 30 min, nearly reached peak levels after 2 h and was sustained at least until 12 h. Histamine also induced TLR3 protein expression in A549 and NCI-H292 cells. Histamine and poly (I:C), a specific TLR3 ligand stimulated interleukin (IL)-8 secretion from both cell types. Moreover, histamine enhanced poly (I:C)-induced IL-8 secretion and phosphorylation of NF-kappaB in the two cell types, and histamine H1 receptor antagonists inhibited the action of histamine. In conclusion, histamine selectively up-regulated expression of TLR3, and stimulated IL-8 secretion from the cells. Histamine also enhanced poly (I:C) induced IL-8 secretion and phosphorylation of NF-kappaB. These observations suggest that histamine might play an important role in enhancing the innate immune responses of airway to viral infection.     

Peroxisome proliferator-activated receptor gamma is expressed in airways and inhibits features of airway remodeling in a mouse asthma model

BACKGROUND: Allergic asthma is associated with persistent functional and structural changes in the airways and involves many different cell types. Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, is predominantly expressed in adipose tissue and plays a major role in regulating adipocyte differentiation and glucose metabolism. Recently, PPAR-gamma has been shown to play an important role in the control of inflammatory responses, including within the lung, acting on both immune and nonimmune cells. OBJECTIVE: Our aim was to assess the anti-inflammatory potential of a PPAR-gamma agonist locally delivered by means of nebulization. METHODS: We used a mouse model of asthma induced by sensitization and airway challenge with ovalbumin. Ciglitazone, a PPAR-gamma agonist, was administered by means of nebulization alone at the time of antigen challenge or by means of gavage and nebulization. Treatments with both ciglitazone and GW9662, a specific antagonist, were also performed to verify that ciglitazone's effects were mediated through PPAR-gamma activation. RESULTS: Our results show that PPAR-gamma is mainly expressed in airway epithelium on antigen sensitization. Treatment with ciglitazone reduced PPAR-gamma levels in the lung, whereas combined treatment with GW9662 abrogated this inhibition. Importantly, nebulization with ciglitazone decreased airway hyperresponsiveness, basement membrane thickness, mucus production, collagen deposition, and TGF-beta synthesis. A significant correlation was also found between airway hyperresponsiveness, basement membrane thickness, and TGF-beta levels. CONCLUSION: These results demonstrate that inhaled agonistic ligands of PPAR-gamma might have new therapeutic potential for airway asthmatic inflammation.     

Production of matrix metalloproteinase-9 in CaCO-2 cells in response to inflammatory stimuli.

Matrix metalloproteinase-9 (MMP-9) may play an important role in the development of inflammatory bowel disease (IBD). However, the cellular source of MMP-9 in the inflamed mucosa of IBD remains unclear. Here we report that MMP-9 mRNA is expressed in CaCO-2 cells, an intestinal epithelial cell line, and that its expression is upregulated by inflammatory stimuli. Stimulation of CaCO-2 cells with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) led to a dose-dependent increase in expression and secretion of MMP-9. In contrast, bacterial lipopolysaccharide (LPS) failed to induce expression or secretion of MMP-9, suggesting that an inflammatory reaction leading to cytokine release is a necessary step for the induction of MMP-9 release in intestinal epithelial cells. Additional studies show that induction of MMP-9 mRNA peaked at 16 h of IL-1beta stimulation, whereas expression of monocyte chemoattractant protein-1 (MCP-1) and IL-8 both peaked at 3 h of stimulation. Treatment of CaCO-2 cells with rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, significantly reduced secretion of MMP-9, indicating that agents that activate PPAR-gamma may have therapeutic use in patients with IBD.     

Serotoninergic receptors on human airway epithelial cells

There is accumulating evidence that points to a role of serotonin (5-hydroxytryptamine [5-HT]) in the pathophysiology of asthma. Therefore, we analyzed the expression of serotoninergic receptors (5-HTR), its linkage to intracellular calcium homeostasis, and its influence on the production and secretion of IL-6, prostaglandin E(2), the CCL-Chemokine CCL5/Rantes, and the CXC-chemokines CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC in primary alveolar epithelial cells type II and the human lung cell lines A549 and BEAS-2B. Employing a PCR approach we were able to demonstrate mRNA expression of several 5-HTR, such as the heptahelical receptors 5-HTR1A, 5-HTR1B, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR4, 5-HTR6, and 5-HTR7, as well as the ligand-gated ion channel 5-HTR3 in alveolar epithelial cells type II (AEC-II), A549, and BEAS-2B cells. To verify functional expression of 5-HTR subtypes, Ca(2+)-transients were analyzed. This enabled us to show that 5-HT induced an increase in intracellular calcium. Further experiments with isotype-selective receptor agonists allowed us to demonstrate that 5-HT induced calcium transients via activation of 5-HTR1, 5-HTR2, and 5-HTR3 in A549 and BEAS-2B cells. Moreover, we revealed that stimulation of 5-HTR1 and 5-HTR2 induced Ca(2+) mobilization from intracellular stores, whereas activation of 5-HTR3 induced Ca(2+) influx from the extracellular space. Functional studies indicated that activation of 5-HTR1B, 5-HTR1E/F, 5-HTR2, 5-HTR3, 5-HTR4, and 5-HTR7 regulated the release of the cytokine IL-6 and the CXC-chemokine CXCL8/IL-8. Our study shows that 5-HT stimulates different signaling pathways and regulates cytokine release in airway epithelial cells. In summary, our data implicate a pathophysiologic role of 5-HT in the asthmatic inflammatory responses in human airway epithelial cells.     

Regulated expression of the IL-31 receptor in bronchial and alveolar epithelial cells, pulmonary fibroblasts, and pulmonary macrophages.

Interleukin-31 (IL-31), an IL-6 cytokine family member, is proposed to play a role in animal models of airway hyperreactivity. It is produced by activated T cells and signals via a heterodimeric receptor complex composed of IL-31Ralpha and OSMRbeta. Only low levels of IL-31Ralpha expression have been demonstrated in pulmonary epithelial cell lines, however, and little is known about the ability to regulate its expression and signaling. Therefore, primary cultures of human bronchial and alveolar epithelial cells, pulmonary fibroblasts, pulmonary macrophages, and established lines of immortalized bronchial epithelial cells (HBE) and alveolar carcinoma cells (A549) were analyzed by RT-PCR, immunoblotting, and thymidine incorporation. Distinct, cell type-specific regulation of IL-31Ralpha expression was detected. Transforming growth factor-beta (TGF-beta) enhanced IL-31Ralpha mRNA expression in primary cultures and established lines of epithelial cells, but not in macrophages. In contrast, interferon-gamma (IFN-gamma) induced IL-31Ralpha mRNA expression in macrophages. IL-31Ralpha protein expression was below detection threshold in primary epithelial cell cultures but was detectable in A549 cells and increased with TGF-beta treatment. In HBE and A549 cells, TGF-beta pretreatment increased IL-31-mediated Stat3 and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. In A549 cells, TGF-beta magnified IL-31-dependent suppression of proliferation. The data suggest that increased IL-31Ralpha expression correlates with an enhanced response to IL-31.     

Interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus.

The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [IL-1 alpha/beta, TNF-alpha/beta, IL-3, IL-6, interferon-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta 2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-1 alpha/beta and TNF-alpha/beta induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-3, G-CSF, IFN-gamma and TGF-beta 2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.     

Airway epithelial IL-15 transforms monocytes into dendritic cells

IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.     

Extracellular regulation of interleukin (IL)-1beta through lung epithelial cells and defective IL-1 type II receptor expression

Interleukin (IL)-1beta is produced primarily by activated mononuclear phagocytic cells in the lung airway and functions as a potent proinflammatory cytokine. Release of IL-1beta in the airway microenvironment induces the production of proinflammatory factors from parenchymal airway cells, including IL-8. To study the regulation of lung epithelial cell responsiveness to IL-1beta, the human type II-like airway epithelial cell line A549 and primary normal human bronchial epithelial (NHBE) cells were assayed for IL-1-specific response modifiers. Specifically, the IL-1 type I receptor (IL-1RI), IL-1 type II receptor (IL-1RII), IL-1 receptor accessory protein (IL-1RAcP), and IL-1 receptor antagonist (IL-1Ra) were analyzed. Constitutive expression of IL-1RI, IL-1RAcP, and IL-1Ra was detected in both immortalized and primary human airway epithelial cells. Interestingly, a complete absence of IL-1RII expression was demonstrated under all study conditions in both A549 and NHBE cells. Both cell types were responsive to IL-1beta at concentrations as low as 50 to 500 pg/ml when measured by IL-8 release into cell supernatants. IL-1beta-induced chemokine production and release were inhibited by a 10- to 1,000-fold molar excess of recombinant IL-1RII or IL-1Ra, whereas IL-1RI was a less effective inhibitor. On the basis of our results, we propose that human lung epithelial cells lack the ability to downregulate IL-1beta activity extracellularly because of an inability to express IL-1RII. Release of extracellular IL-1 inhibitors, including soluble IL-1Ra and soluble IL-1RII, by other inflammatory cells present in the airway may be critical for regulation of IL-1beta activity in the airway microenvironment.     

(R)-albuterol elicits antiinflammatory effects in human airway epithelial cells via iNOS

Catecholamines can suppress production of inflammatory mediators in different cell types, including airway epithelium, but downstream signaling mechanisms involved in regulation of these antiinflammatory effects are largely unknown. We theorized that acute beta2-adrenergic stimulation of airway epithelial cells with albuterol could suppress the production and release of inflammatory mediators, specifically granulocyte macrophage-colony stimulating factor (GM-CSF) via a pathway involving inducible nitric oxide synthase (iNOS). Normal human bronchial epithelial (NHBE) cells in primary culture were exposed to a cytokine mixture (10 ng/ml each IFN-gamma and IL-1beta) to induce iNOS expression. (R)- and (S)-enantiomers of albuterol, as well as racemic mixtures, were added with these cytokines, and effects on GM-CSF expression and production were assessed. Specific inhibitors and activators of protein kinases (PKs), beta2-adrenergic receptor antagonists, and small interfering RNAs against iNOS were used to delineate signaling pathways involved. iNOS message was significantly upregulated in a concentration-dependent manner by the active (R)-enantiomer of albuterol. (R)-albuterol also attenuated cytokine-induced increases in GM-CSF steady-state mRNA expression and protein release. The (S)-enantomer of albuterol had no effect on these parameters. PKC, specifically, the delta isoform, was required for iNOS message increase, but PKA and PKG were not involved in the pathway. Overall, this study identifies a novel pathway by which beta2-adrenergic agonists may exhibit antiinflammatory effects in airway epithelium and surrounding milieu.