Glucocorticoids enhance airway epithelial barrier integrity

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Abstract

Asthma is a chronic inflammatory disorder of the airways, but its pathogenesis is incompletely understood. While asthma is a complex disease caused by multiple factors, epithelial barrier damage is a cardinal feature. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs in the treatment of asthma. However, the effects of GCs on the airway epithelial barrier have not been evaluated. Epithelial barrier functions were evaluated in cultured human airway epithelial cell monolayers, Calu-3 and 16HBE. Then, the cells were treated with dexamethasone (Dex), fulticasone propionate (FP), or budesonide (BD) for 5 days. Permeability measured by transepithelial electrical resistance was increased by treatment with Dex, FP, and BD in a dose-dependent manner. Permeability to fluorescein isothiocyanate-labeled dextran was markedly reduced by these treatments. Immunocytostaining revealed that Dex treatment potentiated tight junction formation in these polarized epithelial cells. Knockdown of epidermal growth factor receptor (EGFR) by small interference RNA blunted the effects of Dex on barrier integrity. Although EGFR expression was not affected by Dex treatment, EGFR phosphorylation was enhanced in Dex-treated cells. This is suggesting that EGFR are important for this phenomenon. These findings suggest that GC inhalation therapy can improve epithelial barrier integrity and might contribute to the therapeutic effects of GCs for treating asthma.

Highlights

Glucocorticoids potentiate permeability barrier function in airway epithelial cells. ► Budesonide (BUD) and fluticasone propionate (FP) potentiate permeability barrier function in airway epithelial cells. ► Glucocorticoids potentiate tight junction formation in airway epithelial cells. ► Glucocorticoids enhance EGFR signaling in airway epithelial cells.

Introduction

Asthma is characterized by allergic inflammation and remodeling of the airways [1]. Asthma is a highly complex disease and its pathogenesis remains largely unknown [2]. To date, the first-line therapy for the treatment of asthma is an inhaled corticosteroid (ICS) used for long-term control [3], [4].

The therapeutic role of glucocorticoids (GCs) in allergic diseases, including asthma, is mainly attributed to their powerful anti-inflammatory properties [5]. It is well documented that the central mechanism of the anti-inflammatory properties of GCs is their ability to diminish the infiltration of inflammatory cells into affected tissues [6], [7]. Emerging evidence indicates that airway epithelial cells play a central role in asthma pathogenesis by secreting numerous cytokines and chemokines [8], [9]. Early in vitro studies demonstrated that primary airway epithelial cells expressed functional GC receptors to which radiolabeled ligands bound with high affinity [10]. In airway epithelial cells, treatment with GCs activated a Gal4 response element-mediated reporter that inhibited activating protein-1 and nuclear translocation of transcription factor κB activation, which led to the hypothesis that epithelial cells were a target for the anti-inflammatory effects of ICSs [11].

Although the therapeutic effects of GCs in asthma are largely mediated by the inhibition of inflammatory genes [6], other mechanisms of action could be involved in their therapeutic effects in asthma. Damage to the airways epithelium is a prominent feature in chronic asthma. Apical junctional complexes in the airway epithelium consist mostly of apical tight junctions (TJs) and underlying adherence junctions (AJs) [12]. TJs are responsible for the control of paracellular transport between epithelial cells [13], [14]. A recent study showed that the bronchial epithelial barrier in asthma was compromised, and TJ formation in bronchial epithelial cell cultures obtained from asthmatic patients were significantly lower than from non-asthmatic subjects [15].

The inflamed and damaged airways of asthmatics have been implicated in their increased susceptibility to allergens and viral infections. Therefore, improving this vulnerability of the airways by restoring epithelial integrity could be a potential therapeutic approach for asthma [16]. A previous study that evaluated bronchial biopsies before and after GC treatments of different lengths and courses of administration clearly showed that GCs promoted the restoration of epithelial integrity in asthma [17]. However, the precise roles of GCs on the permeability barrier function of airway epithelial cells are mostly unknown. In the present study, we investigated the effects of GCs on the permeability barrier function and development of TJs in cultured human airway epithelial cells.

Section snippets

Cells and reagents

Transformed human bronchial epithelial cells (16HBE140 ; abbreviated as 16HBE cells) [18] were kindly provided by Associate Professor Dieter C. Gruenert (Gene Therapy Center, Cardiovascular Research Institute, Department of Laboratory Medicine, University of California). Calu-3 cells were obtained from the American Type Culture Collection (Rockville, MD). Dexamethasone (Dex), fluticasone propionate (FP) [19], budesonide (BD) [20], and fluorescein isothiocyanate-labeled dextran (FITC-dextran; 4

Dex potentiates permeability barrier formation

First, to clarify the role of GCs in permeability barrier integrity, we examined the effects of Dex on the development of TER in Calu-3 cells. Calu-3 cells were cultured with or without 10 6 M Dex applied to the apical and basolateral surfaces of cell monolayers for 5 days. TER of the cells cultured without Dex reached a plateau on day 4 (Fig. 1A). In contrast, TER of the cells cultured with Dex continued to increase up to day 5 (Fig. 1A). Adding varying concentrations of Dex (10 8, 10 7, 10 6 M)

Discussion

Airway epithelial cells are the first site of contact with inhaled allergens, particulates, and viruses. The damaged airway epithelial barrier in asthmatics has been implicated as a cause for their increased susceptibility to allergens and viral infections. Therefore, improving this vulnerability of the airways by restoring epithelial integrity could be a potential therapeutic approach in asthma [9], [16]. In the present study, we found that GCs could improve epithelial barrier integrity and

Acknowledgments

This work was supported in part by the Grants-in-Aid for Scientific Research (C) program of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government, the Nihon University Joint Research Grant, and the Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

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