Interleukin-5 enhances eosinophil adhesion to bronchial epithelial cells

BACKGROUND: Eosinophil-bronchial epithelial cell interactions are thought to be central to the pathogenesis of asthma, both in terms of the epithelium as a source of pro-inflammatory mediators and as a target for eosinophil-mediated damage. We have therefore investigated adhesion interactions between these two cell types. OBJECTIVES: To determine the role of eosinophil and epithelial activation on eosinophil adhesion to bronchial epithelium and to characterize the adhesion receptors mediating eosinophil adhesion. METHODS: Eosinophils were purified from human peripheral blood by immunomagnetic selection and adhesion to confluent cultures of the airway epithelial cell lines A549 and BEAS-2B was studied. RESULTS: Stimulation of A549 cells with TNFalpha, IFNgamma or a combination of 50 ng/mL of TNFalpha, IFNgamma and IL-1 (cytomix) did not effect eosinophil binding despite an increase in ICAM-1 expression. Similarly stimulation of eosinophils with PAF or IL-5 had no effect on eosinophil binding to medium- or cytokine-treated A549 cells. In contrast stimulation of BEAS-2B cells with cytomix caused a significant increase in eosinophil adhesion. This was associated with an increase in expression of ICAM-1 and induced expression of VCAM-1. Treatment of eosinophils with Mn2+ and IL-5 but not eotaxin, RANTES or PAF also significantly enhanced eosinophil adhesion to medium-treated BEAS-2B cells. Using blocking mAbs we were able to demonstrate that the increased adhesion resulting from stimulation of eosinophils or BEAS-2B cells was in both cases mediated by a combination of CD18 and alpha4 integrins. CONCLUSIONS: This study demonstrates a selective role for IL-5 in mediating integrin-dependent eosinophil adhesion to airway epithelium and once again emphasizes the importance of this cytokine in controlling eosinophil activation in diseases such as asthma.     

Effect of IL-17 on ICAM-1 expression of human bronchial epithelial cells, NCI-H 292]

Bronchial asthma is characterized as a chronic inflammation of the airway infiltrated by eosinophils, lymphocytes, and neutrophils. ICAM-1 expression on airway epithelium facilitates adhesion between these inflammatory cells and bronchial epithelial cells, and induces the activation of inflammatory cells. ICAM-1 expression was affected by various cytokines, such as IL-17. IL-17 is a novel cytokine released by CD4+ activated memory T cells. In this study, we examined the effect of IL-17 on ICAM-1 expression by RT-PCR and flow cytometry. Human bronchial epithelial cells, NCI-H 292 cells, were stimulated with IL-17 (100 ng/ml) and/or IFN-gamma (100 U/ml). ICAM-1 was expressed constitutively. IL-17 alone did not enhance ICAM-1 expression on NCI-H 292 cells. However, IL-17 synergistically enhanced ICAM-1 expression induced by IFN-gamma. These results suggest that IL-17 has an effect on ICAM-1 expression of bronchial epithelial cells in airway inflammation.     

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.     

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.     

Elevated expression of interleukin-9 mRNA in the bronchial mucosa of atopic asthmatics and allergen-induced cutaneous late-phase reaction: relationships to eosinophils, mast cells and T lymphocytes

BACKGROUND: Interleukin-9 is a T cell-derived Th2-type cytokine that has been linked to airway hyper-responsiveness, mucus hypersecretion and mast cell infiltration in animal models. We recently demonstrated the potential for IL-9 to act in human eosinophil development and survival. OBJECTIVES: The aims of this study were: (i) to compare IL-9 mRNA expression in bronchial biopsies between atopic asthmatics and normal controls, (ii) to investigate kinetic expression of IL-9 mRNA in skin biopsies after allergen challenge; and (iii) to relate IL-9 expression to infiltration of eosinophils, mast cell and T lymphocytes in local tissue. METHODS: Bronchial biopsies were obtained from atopic asthmatics (n = 12) and normal non-asthmatics (n = 12) at baseline. Skin biopsies were obtained from atopic subjects (n = 11) at 1, 3, 6, 24, 48 and 72 h after allergen challenge. Diluent challenge sites at 24 h were used as controls. IL-9 mRNA was identified using the technique of in situ hybridization. The numbers of eosinophils, mast cells and T cells were evaluated by immunohistochemistry. RESULTS: The numbers of IL-9 mRNA(+) cells present in the bronchial mucosa were significantly greater in atopic asthmatics than those in normal controls (P = 0.003). The numbers of eosinophils, but not mast cells, were also significantly higher in asthmatics (P < 0.005). The numbers of IL-9 mRNA(+) cells present in the airway of asthmatics significantly correlated with the numbers of eosinophils (r = 0.623, P = 0.03), but not mast cells or T cells. Compared with diluent challenge, the numbers of IL-9 mRNA(+) cells were significantly elevated at all allergen-challenged sites in the skin, with maximal signals at 48 h (P < 0.005). At 72 h, the numbers of IL-9 mRNA(+) cells significantly correlated with the numbers of eosinophils (r = 0.707, P = 0.015). CONCLUSION: Elevated expression of IL-9 in allergic inflammation may contribute to local eosinophil infiltration and survival in asthma and other allergic atopic diseases.     

Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells

BACKGROUND: We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils. OBJECTIVES: To compare and contrast the phagocytic capabilities of monocyte-derived macrophage and primary airway epithelial cells for apoptotic granulocytes. RESULTS: Here we compared phagocytosis of human apoptotic eosinophils and neutrophils by small and large airway epithelial cells (SAEC and LAEC) and monocyte-derived macrophages. Confocal microscopy of F-actin staining and scanning and transmission electron microscopy revealed phagocytic cup formation around apoptotic eosinophils by airway epithelial cells (AEC) membranes with evidence of their digestion. Resting and cytokine-stimulated AEC did not recognize and ingest apoptotic neutrophils. The latter were phagocytosed by macrophages that exhibited greater ingestion of and higher capacity for, apoptotic eosinophils over apoptotic neutrophils. Cytochalasin D completely abolished uptake of apoptotic eosinophils by SAEC, LAEC or macrophage monolayers. Ligation of epithelial cell CD44 receptors for 24 h increased phagocytosis of apoptotic eosinophils by SAEC and LAEC with a potency comparable with that of IL-1. Phagocytosis was a specific receptor-mediated process involving integrin- (alphavbeta3, alphavbeta5, CD36), phosphatidylserine receptor- and lectin-dependent mechanisms. No significant differences were observed in avarice for apoptotic eosinophils by SAEC or LAEC either resting, CD44 monoclonal antibodies- or cytokine- stimulated, or in their usage and expression of recognition receptors. CONCLUSION: These findings further suggest and define an important role for the bronchial epithelium in the selective removal of apoptotic eosinophils from the airways in asthma.     

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.     

The expression of stem cell factor and c-kit receptor in human asthmatic airways

BACKGROUND: Asthmatic airways are characterized by infiltration with a variety of inflammatory cells such as mast cells and eosinophils. Stem cell factor (SCF) is an important activating and chemotactic factor for both mast cells and eosinophils. In addition, it is a critical growth and differentiation factor for mast cells. OBJECTIVES: To investigate the contribution of SCF to the pathogenesis of asthma, we examined the expression of SCF and its receptor c-kit in bronchial biopsies and bronchoalveolar lavage (BAL) specimens obtained from asthmatic subjects (n=13) and non-asthmatic control subjects (n=10). METHODS: SCF and c-kit were detected by in situ hybridization (ISH) and immunocytochemistry (ICC). In order to phenotype the cells expressing SCF and c-kit in asthmatic tissue and BAL cells, combined ISH and ICC were also performed. RESULTS: There was a significant difference (P<0.001) in the SCF mRNA expression in asthmatic airway epithelium (70.38+/-12.33% positive cells) compared with controls (12.7+/-17.21% positive cells). There was also a significant difference in subepithelial SCF-mRNA expression, being higher in asthmatics (P<0.001). A significant difference was also found in c-kit receptor mRNA expression in asthmatic biopsies both in epithelium (P<0.001) and subepithelium (P<0.05) compared with controls. ICC results were consistent with the ISH for both SCF and c-kit receptor from asthmatics and controls. The SCF and c-kit receptor mRNA and immunoreactivity in cells recovered from bronchial washing were also significantly higher in asthmatics compared with controls (P<0.05). While SCF expression was localized predominantly in the epithelial layer in bronchial biopsy tissues, alveolar macrophages were found to be the major source of SCF in bronchial washing from asthmatic subjects. CONCLUSION: The results of this study demonstrate the increased expression of SCF and its receptor, c-kit within human asthmatic airways, which suggests an important role of this cytokine in the pathophysiology of asthma.     

Human alveolar epithelial cells engulf apoptotic eosinophils by means of integrin- and phosphatidylserine receptor-dependent mechanisms: A process upregulated by dexamethasone

BACKGROUND: We previously demonstrated receptor-mediated apoptotic-eosinophil engulfment by human small airway epithelial cells, which may represent a potentially important mechanism in the resolution of allergic and asthmatic inflammation. OBJECTIVE: A549 cells were selected as being representative of alveolar epithelial cells, and their ability to ingest human apoptotic eosinophils was examined in terms of the effects of dexamethasone treatment and the receptor-mediated recognition mechanisms important in this process. METHODS: A549 epithelial-cell expression of alpha(v)beta3, alpha(v)beta5, CD36, and the phosphatidylserine receptor was established by using immunostaining and flow cytometry, and inhibition assays were examined by using the role of these receptors in apoptotic-eosinophil recognition by resting and dexamethasone-treated A549 epithelial cells. Electron microscopy confirmed engulfment of apoptotic eosinophils, and receptor clustering around apoptotic eosinophils was examined by using immunofluorescent labeling. RESULTS: A549 epithelial cells recognized and engulfed apoptotic eosinophils but not freshly isolated cells. Dexamethasone enhanced the number of A549 cells ingesting apoptotic eosinophils and dose dependently increased their capacity for ingestion. The tetrapeptide RGDS significantly inhibited apoptotic-eosinophil uptake by A549 cells, indicating a role for integrins in the recognition process. A549 cells expressed alpha(v)beta3, alpha(v)beta5, beta5, CD36, and the phosphatidylserine receptor, and expression of these receptors was not significantly increased after dexamethasone treatment. Engulfment of apoptotic eosinophils by A549 cells involved alpha(v)beta3-, CD36-, alpha(v)beta5-, and phosphatidylserine receptor-mediated recognition mechanisms and was associated with the formation of integrin focal adhesion complexes around apoptotic eosinophils. CONCLUSIONS: These data further suggest a nonpassive role for airway epithelium in the resolution of eosinophilic inflammation in asthma.     

CYTOKINE PRODUCTION BY CELL CULTURES FROM BRONCHIAL SUBEPITHELIAL MYOFIBROBLASTS

Myofibroblasts have been previously described beneath the bronchial epithelium and were found to increase in number proportional to the accumulation of extracellular matrix in the bronchial lamina reticularis in asthma. The aim of this study was to assess further the contribution of these structural cells to allergic inflammation in the bronchial mucosa through their cytokine expression. Cell cultures were established from the lamina reticularis of human bronchial biopsies from asthmatic and non-asthmatic subjects. Cytokine secretion was measured by ELISA in supernatants of cultures with or without tumour necrosis factor-α (TNF- α). The mRNA levels for granulocyte–macrophage colony-stimulating factor (GM-CSF) in the cultures were examined by ribonuclease protection assays (RPAs). Bronchial myofibroblasts grown from bronchial biopsies were capable of producing GM-CSF, interleukin-6 (IL-6), interleukin-8 (IL-8), and stem cell factor (SCF) constitutively. The GM-CSF production by myofibroblasts was significantly increased in response to TNF-α simulation with a corresponding increase in GM-CSF mRNA expression. The enhancement of GM-CSF production by TNF-α in myofibroblasts was blocked by the inhibition of RNA synthesis. Prednisolone abolished the GM-CSF production. This study provides evidence for the role of bronchial myofibroblasts in the regulation of inflammatory cell recruitment and activation by interaction in the cytokine network in the bronchial mucosa.     

Essential role of transcription factor nuclear factor-kappaB in regulation of interleukin-8 gene expression by nitrite reductase from Pseudomonas aeruginosa in respiratory epithelial cells.

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airways of patients with chronic airway diseases. Recently, we have reported that nitrite reductase from P. aeruginosa induces the production of IL-8 in respiratory cells, including bronchial epithelial cells. To determine the molecular mechanism(s) of nitrite reductase-induced IL-8 expression in respiratory cells, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5'-flanking region of the IL-8 gene and then exposed to nitrite reductase. Nitrite reductase significantly enhanced IL-8 gene promoter-driven reporter activity. This increased IL-8 gene expression was inhibited by mutating the nuclear factor-kappaB (NF-kappaB) binding element. Nitrite reductase enhanced nuclear localization of the NF-kappaB binding complex. Furthermore, nitrite reductase induced the degradation of IkappaBalpha, the major cytoplasmic inhibitor of NF-kappaB, and the expression of IkappaBalpha mRNA. These data support the critical role of the activation of NF-kappaB in nitrite reductase-induced IL-8 gene expression in airway epithelium.     

Involvement of IL-9 in the bronchial phenotype of patients with nasal polyposis

BACKGROUND: Nasal polyposis (NP) is frequently associated with asthma. In this disease, asymptomatic bronchial hyperresponsiveness (BHR) is thought to precede the development of asthma. IL-9 and its receptor have been reported as candidate genes for asthma and to be associated with BHR. OBJECTIVE: The objective of this study was to assess the contribution of 11-9 to the pathogenesis of BHR in NP by comparing the expression of IL-9 and its receptor in bronchial biopsy specimens from three groups of patients with NP: NP without BHR, NP with asymptomatic BHR, and NP with BHR and asthma. METHODS: Bronchial biopsy specimens were examined in terms of cellular infiltration and in terms of expression of IL-9 protein and mRNA as well as of its receptor by using immunohistochemistry and in situ hybridization. RESULTS: Patients with NP with asthma as compared with the two other groups exhibited an increased bronchial infiltration of basophils, eosinophils, and T cells that correlated with the asthma score. The two groups of patients with NP with BHR showed an increased expression in IL-9 protein and mRNA as well as an increase in the expression of IL-9R mRNA at the epithelial level. These modifications were inversely correlated with the airway responsiveness to methacholine, producing a 20% fall in FEV1. There was a close association between IL-9+ cells, IL-5 mRNA expression, and eosinophil infiltration that correlated with each other. CONCLUSIONS: These results suggest an important role for IL-9 in the pathogenesis of BHR and a causal relation between IL-9 and the development of bronchial eosinophilia in asthma.