Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.

BACKGROUND: Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. OBJECTIVES: To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. METHODS: Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; RT ) and permeability to fluorescent dextran. RESULTS: Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. CONCLUSION: This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma.

Impaired airway epithelial cell responses from children with asthma to rhinoviral infection.

BACKGROUND: The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood. OBJECTIVE: To explore the relationship between apoptotic, innate immune and inflammatory responses to HRV infection in airway epithelial cells (AECs) obtained from children with asthma and non-asthmatic controls. In addition, to test the hypothesis that aberrant repair of epithelium from asthmatics is further dysregulated by HRV infection. METHODS: Airway epithelial brushings were obtained from 39 asthmatic and 36 non-asthmatic children. Primary cultures were established and exposed to HRV1b and HRV14. Virus receptor number, virus replication and progeny release were determined. Epithelial cell apoptosis, IFN-ß production, inflammatory cytokine release and epithelial wound repair and proliferation were also measured. RESULTS: Virus proliferation and release was greater in airway epithelial cells from asthmatics but this was not related to the number of virus receptors. In epithelial cells from asthmatic children, virus infection dampened apoptosis, reduced IFN-ß production and increased inflammatory cytokine production. HRV1b infection also inhibited wound repair capacity of epithelial cells isolated from non-asthmatic children and exaggerated the defective repair response seen in epithelial cells from asthmatics. Addition of IFN-ß restored apoptosis, suppressed virus replication and improved repair of airway epithelial cells from asthmatics but did not reduce inflammatory cytokine production. CONCLUSIONS: Collectively, HRV infection delays repair and inhibits apoptotic processes in epithelial cells from non-asthmatic and asthmatic children. The delayed repair is further exaggerated in cells from asthmatic children and is only partially reversed by exogenous IFN-ß.

Suppression of adrenomedullin contributes to vascular leakage and altered epithelial repair during asthma.

BACKGROUND: The anti-inflammatory peptide, adrenomedullin (AM), and its cognate receptor are expressed in lung tissue, but its pathophysiological significance in airway inflammation is unknown. OBJECTIVES: This study investigated whether allergen-induced airway inflammation involves an impaired local AM response. METHODS: Airway AM expression was measured in acute and chronically sensitized mice following allergen inhalation and in airway epithelial cells of asthmatic and nonasthmatic patients. The effects of AM on experimental allergen-induced airway inflammation and of AM on lung epithelial repair in vitro were investigated. RESULTS: Adrenomedullin mRNA levels were significantly (P < 0.05) reduced in acute ovalbumin (OVA)-sensitized mice after OVA challenge, by over 60% at 24 h and for up to 6 days. Similarly, reduced AM expression was observed in two models of chronic allergen-induced inflammation, OVA- and house dust mite-sensitized mice. The reduced AM expression was restricted to airway epithelial and endothelial cells, while AM expression in alveolar macrophages was unaltered. Intranasal AM completely attenuated the OVA-induced airway hyperresponsiveness and mucosal plasma leakage but had no effect on inflammatory cells or cytokines. The effects of inhaled AM were reversed by pre-inhalation of the putative AM receptor antagonist, AM ((22-52)) . AM mRNA levels were significantly (P < 0.05) lower in human asthmatic airway epithelial samples than in nonasthmatic controls. In vitro, AM dose-dependently (10(-11) -10(-7) M) accelerated experimental wound healing in human and mouse lung epithelial cell monolayers and stimulated epithelial cell migration. CONCLUSION: Adrenomedullin suppression in T(H) 2-related inflammation is of pathophysiological significance and represents loss of a factor that maintains tissue integrity during inflammation.

Comparison of techniques for obtaining lower airway epithelial cells from children.

Airway epithelial cells (AECs) are important in asthma as they are the first cells to encounter pathogens/allergens. In children, AECs can be obtained using a "blind" nonbronchoscopic technique through an endotracheal tube. However, due to the increasing use of laryngeal masks the number of children in whom this technique is applicable has become limited. Recently, the present authors began to use a portable "bronchoscope-directed" technique to sample AECs. The current study compares both techniques in both asthmatic and nonasthmatic children. A total of 81 children undergoing elective surgery, were grouped according to atopic status and respiratory symptoms. Cellular yield of blind and bronchoscope-directed brushings were compared and immunocytochemistry performed. AECs were cultured and cytokine analysis of culture supernatant undertaken. Both techniques were equally well-tolerated, with the only adverse effect being a cough in 10% of the subjects. The mean+/-SD cell yield was higher in bronchoscope-directed than blind brushings (5.1+/-2.4 versus 3.1+/-1.4x10(6) cells). Immunocytochemistry confirmed an epithelial cell lineage. Culture supernatant cytokine concentrations were similar regardless of sampling technique with patterns preserved between asthmatic and healthy nonatopic phenotypes. Compared with blind brushing portable bronchoscope-directed brushing is well-tolerated, yields significantly more cells and is a potentially quick and useful technique for obtaining airway epithelial cells for research into childhood respiratory disease, specifically asthma.

Dysregulated repair in asthmatic paediatric airway epithelial cells: the role of plasminogen activator inhibitor-1.

BACKGROUND: Asthma is associated with structural changes to airways such as extracellular matrix deposition and epithelial damage. Evidence suggests that asthmatic airway epithelial repair is abnormal and that elevated plasminogen activator inhibitor-1 levels observed in asthma may be involved in the epithelial repair process and in excessive matrix accumulation. OBJECTIVE: To assess the ability of asthmatic airway epithelial cells (AECs) to repair mechanically induced wounds and to investigate the role that plasminogen activator inhibitor-1 plays in the repair process. METHODS: AECs were isolated from atopic asthmatic and healthy non-atopic children by bronchial brushing, subcultured and wound repair experiments were performed. Plasminogen activator inhibitor-1 gene expression was assessed using real-time PCR while protein activity was measured in cell lysates as well as plasma. The role of plasminogen activator inhibitor-1 in epithelial proliferation and wound repair was investigated using siRNA. RESULTS: Cells from asthmatic children have a significantly longer repair time in comparison with cells from otherwise healthy donors. Plasminogen activator inhibitor-1 mRNA expression was up-regulated 68-fold in freshly isolated asthmatic cells compared with normal cells, and protein levels were also significantly elevated in the asthmatic cell lysates, but plasma levels were similar in both groups. Plasminogen activator inhibitor-1 cells expression increased in both cohorts during culture. Gene silencing substantially reduced the rate of proliferation in asthmatic and healthy cells. Mechanical wounding of epithelial monolayers induced plasminogen activator inhibitor-1 expression in asthmatic and non-asthmatic cohorts, while gene silencing delayed wound repair of healthy cell, with minimal effect on those from asthmatics. CONCLUSION: Asthmatic AECs are inherently dysfunctional in their ability to repair wounds; plasminogen activator inhibitor-1 mRNA and protein activity are constitutively up-regulated in asthmatic epithelium and play functional roles in both proliferation and repair of healthy cells. In asthmatic cells, elevated plasminogen activator inhibitors-1 levels fail to stimulate epithelial repair.