Abstract
Chronic damage and repair of the bronchial epithelium are features of asthma. We have previously reported that ex vivo stimulation of normal bronchial epithelial cells with epidermal growth factor (EGF), a key factor of epithelial repair, enhances the mechanisms of neutrophil accumulation, thereby promoting neutrophil defences during acute injury but potentially enhancing inflammation in chronic airway diseases. We have now sought to (i) determine whether this EGF-dependent pro-neutrophil activity is increased in asthma, where EGF and its epithelial receptor are over-expressed, and (ii) elucidate some of the mechanisms underlying this asthmatic epithelial-neutrophil interaction. Primary bronchial epithelial cells (PBEC) from healthy subjects, mild asthmatics and moderate-to-severe asthmatics (Mod/Sev) were stimulated with EGF, a model that mimics a repairing epithelium. Conditioned culture media (EGF-CM) were assessed for neutrophil chemotactic and anti-apoptotic activities and inflammatory mediator production. EGF induced the epithelium to produce soluble mediators with neutrophil chemotactic (p<0.001) and pro-survival (p = 0.021) activities which were related to the clinical severity of asthma (trend p = 0.010 and p = 0.009, respectively). This was associated with enhanced IL-6, IL-8, GM-CSF and TNF-α release, and cytokine-neutralising experiments using EGF-CM from Mod/Sev asthmatics demonstrated a role for GM-CSF in neutrophil survival (p<0.001). Pre-treatment of neutrophils with specific inhibitors of the myeloid-restricted class I phosphatidylinositol-3-OH kinase (PI(3)K) isoforms showed that the EGF-CM from Mod/Sev asthmatics depended on the γ (p<0.021) but not δ isoforms, while neutrophil survival required multiple class I PI(3)Ks. The EGF-induced chemotactic, but not pro-survival activity, involved RhoA signaling in neutrophils (p = 0.012). EGF whose activity is upregulated in asthma induces ex vivo the epithelium from asthmatic patients to produce pro-neutrophil activities; these are related to asthma severity and, in moderate-to-severe asthmatics, involves class IB PI(3)Kγ signaling, providing a potential therapeutic target for neutrophilic forms of asthma.
Highlights
Neutrophilic airway inflammation is a common feature of severe chronic asthma [1,2,3,4] shown to be insensitive to glucocorticoids (GCs) [5,6], but the mechanisms which regulate the accumulation of neutrophils within the inflamed airways are still poorly understood
In an earlier study using the bronchial 16HBE cell line and primary bronchial epithelial cells (PBECs) from healthy individuals, we showed that ex vivo the bronchial epithelium produces an array of neutrophil chemotactic factors, IL-8, granulocytemacrophage colony-stimulating-factor (GM-CSF), tumour necrosis factor (TNF)-a and LTB4 [14]
In order to assess whether any observed effects of epidermal growth factor (EGF)-activated PBECs on neutrophil chemotaxis or anti-apoptotic survival are due to an increase in the numbers or activity of existing epithelial cells following EGF stimulation, an assessment of proliferation of PBECs exposed to EGF was first performed using a colorimetric assay based on methylene blue uptake and release
Summary
Neutrophilic airway inflammation is a common feature of severe chronic asthma [1,2,3,4] shown to be insensitive to glucocorticoids (GCs) [5,6], but the mechanisms which regulate the accumulation of neutrophils within the inflamed airways are still poorly understood. Over-expression of EGF receptor (EGFR) and its ligands (i.e. EGF, amphiregulin, heparin-binding EGF-like growth factor) has been observed in the airways of adult [19,20,21,22] as well as paediatric asthmatics [23,24], with levels of EGF and amphiregulin being significantly elevated following acute exacerbations in the latter patient population [25,26]. This suggests that the pathobiology of asthma involves and may result, in part, from EGFR-mediated dysregulation of injury-repair responses in the airway mucosa. A direct link between the observed effects of EGF on endogenous mediator production by the epithelium and neutrophil accumulation has not been established, nor is there evidence that this is a feature of clinical asthma
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