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The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca 2+ ) signaling. Ca 2+ is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca 2+ signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca 2+ signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca 2+ stores was decreased in patients with COPD and that the Ca 2+ influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca 2+ release, significant in smoker subjects, and to a decrease in Ca 2+ influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca 2+ signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca 2+ influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca 2+ signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.

Background: Structural changes to the airways are features of severe asthma. The bronchial epithelium facilitates this remodeling process. Learning about the changes that develop in the airway epithelium could improve our understanding of asthma pathogenesis and lead to new therapeutic approaches., Objective: We sought to determine the feasibility and relevance of air-liquid interface cultures of bronchial epithelium derived from endobronchial biopsy specimens of patients with different severities of asthma for studying the airway epithelium., Methods: Human bronchial epithelial cells derived from endobronchial biopsy specimens of patients with mild and severe asthma were maintained in culture for 21 days in an air-liquid interface to reproduce a fully differentiated airway epithelium. Initially, features of remodeling that included epithelial and subepithelial layers, as well as mucus production, were assessed in paraffin-embedded endobronchial biopsy specimens to evaluate morphologic characteristics of asthmatic patients' epithelia. Ex vivo differentiated epithelia were then analyzed for morphology and function based on ultrastructural analysis, IL-8 release, lipoxin A(4) generation, mucin production, and lipoxygenase gene expression., Results: Morphologic and inflammatory imbalances initially observed in endobronchial biopsy specimens obtained from patients with severe or mild asthma persisted in the air-liquid interface reconstituted epithelium throughout the differentiation process to 21 days. Epithelium from patients with severe asthma produced greater levels of mucin, released more IL-8, and produced lower levels of lipoxin A(4) than that from patients with mild asthma. Expression of 15-lipoxygenase 2 was increased in epithelium from patients with severe asthma, whereas expression levels of MUC5AC, MUC5B, 5-lipoxygenase, and 15-lipoxygeanse 1 were similar to those of patients with mild asthma., Conclusion: Ex vivo cultures of fully differentiated bronchial epithelium from endobronchial biopsy specimens maintain inherent phenotypic differences specifically related to the severity of asthma., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: The adipokine leptin is a potential new mediator for bronchial epithelial homeostasis. Asthma is a chronic inflammatory disease characterized by airway remodeling that might affect disease chronicity and severity. TGF-beta is a tissue growth factor the dysregulation of which is associated with airway remodeling., Objective: We sought to determine whether a bronchial epithelial dysfunction of the leptin/leptin receptor pathway contributes to asthma pathogenesis and severity., Methods: We investigated in vitro the presence of leptin/leptin receptor on human bronchial epithelial cells. Then we studied the effect of TGF-beta and fluticasone propionate on leptin receptor expression. Finally, the role of leptin on TGF-beta release and cell proliferation was analyzed. Ex vivo we investigated the presence of leptin/leptin receptor in the epithelium of bronchial biopsy specimens from subjects with asthma of various severities and from healthy volunteers, and some features of airway remodeling, such as reticular basement membrane (RBM) thickness and TGF-beta expression in the epithelium, were assessed., Results: In vitro bronchial epithelial cells express leptin/leptin receptor. TGF-beta decreased and fluticasone propionate increased leptin receptor expression, and leptin decreased the spontaneous release of TGF-beta and increased cell proliferation. Ex vivo the bronchial epithelium of subjects with mild, uncontrolled, untreated asthma showed a decrease expression of leptin and its receptor and an increased RBM thickness and TGF-beta expression when compared with values seen in healthy volunteers. Furthermore, severe asthma was associated with a reduced expression of leptin and its receptor and an increased RBM thickness with unaltered TGF-beta expression., Conclusions: Decreased expression of leptin/leptin receptor characterizes severe asthma and is associated with airway remodeling features.

Asthma is an inflammatory disease, and the epithelial mesenchymal unit appears to be of importance in regulating the disease mechanisms. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is commonly mediated by cadherins, a family of Ca(2+)-dependent transmembrane adhesion receptors. The cadherin family is involved in control of the cellular architecture. Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha are involved in asthma and may interfere with epithelial integrity. In the present study, we investigated the role of TNF-alpha and dexamethasone on the expression of E-cadherin, beta-catenin, and gamma-catenin. We used two bronchial epithelial cell models: primary small airway epithelial cell cultures and primary culture obtained from human bronchial tubes. After 48 h of TNF-alpha stimulation with or without dexamethasone expression of E-cadherin, beta-catenin and gamma-catenin were analyzed using Western blot analysis and immunofluorescence. This study showed a decrease in the expression of adhesion molecules in both epithelial cell cultures after stimulation. Dexamethasone and anti-TNF-alpha inhibited this effect. In unstimulated cells, E-cadherin and beta- and gamma-catenin expression was membranous, expressed only on the lateral cell wall with minimal cytoplasmic expression. Immunoreactivity was cytoplasmic in stimulated cells. We demonstrated, using Western blot analysis and immunofluorescence, that proinflammatory cytokines could be responsible for structural damage to the epithelium and that this process was potentially reversed by steroids.

The expression of the glucocorticoid receptor (GR) in untreated or in steroid-dependent asthmatic patients is poorly understood. We therefore studied GR mRNA and protein levels in bronchial biopsies obtained from seven untreated asthmatic patients, seven control volunteers, and seven patients with chronic bronchitis. We also studied in bronchial epithelial cells obtained by brushing from 13 untreated asthmatics, 18 steroid-dependent asthmatics, 11 control volunteers, and 12 patients with chronic bronchitis, GR and heat shock protein 90 kD (hsp90) mRNA as well as the immunoreactivity of GR, intercellular adhesion molecule (ICAM-1), and granulocyte macrophage-colony-stimulating factor (GM-CSF). GR mRNA and protein level was similar in all subject groups in both biopsies and bronchial epithelial cells. Hsp90 mRNA level was also similar in all subject groups. ICAM-1 expression was significantly increased in bronchial epithelial cells from untreated asthmatics, but ICAM-1 was not expressed in those from steroid-dependent asthmatic patients. GM-CSF expression was significantly increased in bronchial epithelial cells from untreated and steroid-dependent asthmatic patients. GR expression within the airways is unaltered by oral long-term steroid treatment in asthma, but the expression of some but not all specific markers for asthma is modified by oral steroid.

Bronchial epithelial cells are the first cells to come into contact with inhaled pneumoallergens. It has been suggested that these cells may play an important role in the allergic response, and indeed bronchial epithelial cells of some atopic asthmatic subjects have been shown to express the low-affinity receptor for IgE on their surface. In this report we demonstrate, using bronchial biopsies, that bronchial epithelial cells of some asthmatic subjects express both the alpha and gamma chains of the high-affinity receptor for IgE (Fcepsilon RI) on their surface and that they are capable of fixing IgE. Second, using reverse transcription-polymerase chain reaction, we show that both control and asthmatic subjects have messenger RNA for Fcepsilon RI. Finally, we demonstrate that this receptor may be functional since stimulation of the cells with the antibody to the alpha chain of Fcepsilon RI results in the liberation of 15-hydroxyeicosatetraenoic acid from epithelial cells of asthmatic, but not control, subjects or subjects suffering from chronic bronchitis. These data suggest that bronchial epithelial cells from at least some asthmatic subjects express a functional high-affinity receptor for IgE and it is therefore possible that these cells may be able to interact directly with inhaled allergens.

The study of cellular events in the bronchi of asthmatic patients shows significant epithelial destruction. The ciliated cells are more often destroyed than others in the respiratory epithelium. Using highly specific monoclonal antibodies, we detected that the epithelial cells were positive for HLA-DR antigen expression, whereas those from healthy subjects were negative. The aberrant expression could be explained by the presence of T lymphocytes in the asthmatic bronchial mucosa. The activated lymphocytes (CD4+) are known to release gamma interferon, which is a lymphokine that induces or enhances the expression of HLA-DR antigens. These cells, which are known to mediate cytotoxicity in an Ag-specific and Ia-restricted way, could take part in the shedding process of epithelial cells in asthma.