Irene H. Heijink

Down-Regulation of E-Cadherin in Human Bronchial Epithelial Cells Leads to Epidermal Growth Factor Receptor-Dependent Th2 Cell-Promoting Activity

Airway epithelial cells are well-known producers of thymus- and activation-regulated chemokine (TARC), a Th2 cell-attracting chemokine that may play an important role in the development of allergic airway inflammation. However, the mechanism responsible for up-regulation of TARC in allergy is still unknown. In the asthmatic airways, loss of expression of the cell-cell contact molecule E-cadherin and reduced epithelial barrier function has been observed, which may be the result of an inadequate repair response. Because E-cadherin also suppressed multiple signaling pathways, we studied whether disruption of E-cadherin-mediated cell contact may contribute to increased proallergic activity of epithelial cells, e.g., production of the chemokine TARC. We down-regulated E-cadherin in bronchial epithelial cells by small interference RNA and studied effects on electrical resistance, signaling pathways, and TARC expression (by electric cell-substrate impedance sensing, immunodetection, immunofluorescent staining, and real-time PCR). Small interference RNA silencing of E-cadherin resulted in loss of E-cadherin-mediated junctions, enhanced phosphorylation of epidermal growth factor receptor (EGFR), and the downstream targets MEK/ERK-1/2 and p38 MAPK, finally resulting in up-regulation of TARC as well as thymic stromal lymphopoietin expression. The use of specific inhibitors revealed that the effect on TARC is mediated by EGFR-dependent activation of the MAPK pathways. In contrast to TARC, expression of the Th1/Treg cell-attracting chemokine RANTES was unaffected by E-cadherin down-regulation. In summary, we show that loss of E-cadherin-mediated epithelial cell-cell contact by damaging stimuli, e.g., allergens, may result in reduced suppression of EGFR-dependent signaling pathways and subsequent induction of Th2 cell-attracting molecule TARC. Thus, disruption of intercellular epithelial contacts may specifically promote Th2 cell recruitment in allergic asthma.

E-cadherin: gatekeeper of airway mucosa and allergic sensitization

The airway epithelium plays a role in immune regulation during environmental challenge, which is intertwined with its barrier function and capacity to limit submucosal access of environmental factors. In asthma, mucosal barrier function is often compromised, with disrupted expression of the adhesion molecule E-cadherin. Recent progress suggests that E-cadherin contributes to the structural and immunological function of airway epithelium, through the regulation of epithelial junctions, proliferation, differentiation, and production of growth factors and proinflammatory mediators that can modulate the immune response. Here, we discuss this novel role for E-cadherin in mediating the crucial immunological decision between maintenance of tolerance versus induction of innate and adaptive immunity.

Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery.

Cigarette smoking, the major cause of chronic obstructive pulmonary disease (COPD), induces aberrant airway epithelial structure and function. The underlying mechanisms are unresolved so far. We studied effects of cigarette smoke extract (CSE) on epithelial barrier function and wound regeneration in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from COPD patients, nonsmokers and healthy smokers. We demonstrate that CSE rapidly and transiently impairs 16HBE barrier function, largely due to disruption of cell–cell contacts. CSE induced a similar, but stronger and more sustained, defect in PBECs. Application of the specific epidermal growth factor receptor (EGFR) inhibitor AG1478 showed that EGFR activation contributes to the CSE-induced defects in both 16HBE cells and PBECs. Furthermore, our data indicate that the endogenous protease calpain mediates these defects through tight junction protein degradation. CSE also delayed the reconstitution of 16HBE intercellular contacts during wound healing and attenuated PBEC barrier function upon wound regeneration. These findings were comparable between PBECs from smokers, healthy smokers and COPD patients. In conclusion, we demonstrate for the first time that CSE reduces epithelial integrity, probably by EGFR and calpain-dependent disruption of intercellular contacts. This may increase susceptibility to environmental insults, e.g. inhaled pathogens. Thus, EGFR may be a promising target for therapeutic strategies to improve mucosal barrier function in cigarette smoking-related disease.

House Dust Mite-Promoted Epithelial-to-Mesenchymal Transition in Human Bronchial Epithelium

The molecular basis of airway remodeling and loss of epithelial integrity in asthma is still undefined. We aimed to establish if exposure of human bronchial epithelium (16HBE cells) to asthma-related stimuli can induce epithelial-to-mesenchymal transition (EMT), a key process in tissue repair and remodeling associated with loss of intercellular contacts. We studied the effects of fibrogenic cytokine TGF-beta and protease-containing aeroallergen house dust mite (HDM) on mesenchymal and epithelial markers, cytoskeleton organization, and activation of beta-catenin-driven reporter TopFLASH. TGF-beta alone up-regulated vimentin and fibronectin, modestly down-regulated E-cadherin, but did not affect cytokeratin. HDM alone did not affect these markers, but promoted stress fibers. Importantly, when added to TGF-beta-primed epithelium, HDM induced E-cadherin internalization, enhanced beta-catenin-dependent transcription, and down-regulated cytokeratin. Regarding the underlying mechanisms, the stimuli together induced sustained myosin light chain phosphorylation, which was crucial for E-cadherin internalization and beta-catenin-dependent transcription. Previously, we showed that HDM signals through the epidermal growth factor receptor (EGFR). Accordingly, inhibition of EGFR prevented TGF-beta/HDM-induced mesenchymalization. TGF-beta facilitated uncoupling of EGFR from E-cadherin, its negative regulator, and prolonged EGFR signaling. Thus, we show that HDM promotes EMT in TGF-beta-primed epithelium. Analysis of primary epithelium appears consistent with this phenotypic change. We propose that TGF-beta secretion and dysregulated EGFR signaling may increase epithelial vulnerability to allergens and trigger the induction of EMT, a hitherto unrecognized contributor to airway remodeling in asthma.

The composition of house dust mite is critical for mucosal barrier dysfunction and allergic sensitisation

Background House dust mite (HDM) allergens have been reported to increase airway epithelial permeability, thereby facilitating access of allergens and allergic sensitisation. Objectives The authors aimed to understand which biochemical properties of HDM are critical for epithelial immune and barrier responses as well as T helper 2-driven experimental asthma in vivo. Methods Three commercially available HDM extracts were analysed for endotoxin levels, protease and chitinase activities and effects on transepithelial resistance, junctional proteins and pro-inflammatory cytokine release in the bronchial epithelial cell line 16HBE and normal human bronchial cells. Furthermore, the effects on epithelial remodelling and airway inflammation were investigated in a mouse model. Results The different HDM extracts varied extensively in their biochemical properties and induced divergent responses in vitro and in vivo. Importantly, the Greer extract, with the lowest serine protease activity, induced the most pronounced effects on epithelial barrier function and CCL20 release in vitro. In vivo, this extract induced the most profound epithelial E-cadherin delocalisation and increase in CCL20, CCL17 and interleukin 5 levels, accompanied by the most pronounced induction of HDM-specific IgE, goblet cell hyperplasia, eosinophilic inflammation and airway hyper-reactivity. Conclusions This study shows the ability of HDM extracts to alter epithelial immune and barrier responses is related to allergic sensitisation but independent of serine/cysteine protease activity.

Interleukin-17A induces glucocorticoid insensitivity in human bronchial epithelial cells

A subset of asthma patients suffer from glucocorticoid (GC) insensitivity. T-helper cell type 17 cells have an emerging role in GC insensitivity, although the mechanisms are still poorly understood. We investigated whether interleukin (IL)-17A induces GC insensitivity in airway epithelium by studying its effects on responsiveness of tumour necrosis factor (TNF)-&agr;-induced IL-8 production to budesonide in human bronchial epithelial 16HBE cells. We unravelled the underlying mechanism by the use of specific pathway inhibitors, reporter and overexpression constructs and a histone deacetylase (HDAC) activity assay. We demonstrated that IL-17A-induced IL-8 production is normally sensitive to GCs, while IL-17A pre-treatment significantly reduced the sensitivity of TNF-&agr;-induced IL-8 production to budesonide. IL-17A activated the p38, extracellular signal-related kinase (ERK) and phosphoinositide-3-kinase (PI3K) pathways, and the latter appeared to be involved in IL-17A-induced GC insensitivity. Furthermore, IL-17A reduced HDAC activity, and overexpression of HDAC2 reversed IL-17A-induced GC insensitivity. In contrast, IL-17A did not affect budesonide-induced transcriptional activity of the GC receptor, suggesting that IL-17A does not impair the actions of the ligated GC receptor. In conclusion, we have shown for the first time that IL-17A induces GC insensitivity in airway epithelium, which is probably mediated by PI3K activation and subsequent reduction of HDAC2 activity. Thus, blockade of IL-17A or downstream signalling molecule PI3K may offer new strategies for therapeutic intervention in GC-insensitive asthma.

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

BackgroundCigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.MethodsWe studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.ResultsWe observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.ConclusionThe observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

Characterisation of cell adhesion in airway epithelial cell types using electric cell–substrate impedance sensing

Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell–substrate impedance sensing technique was used to monitor cell adhesion/spreading, barrier function and wound healing. Primary bronchial epithelium was compared with airway epithelial cell lines 16HBE14o-, BEAS-2B, NCI-H292 and A549. BEAS-2B, A549 and primary cells form a confluent monolayer more rapidly than do 16HBE14o- cells. In contrast, 16HBE14o- cells form stronger intercellular contacts, with a 10-fold higher resistance than BEAS-2B, A549 and NCI-H292 cells and a five-fold increase over primary cells. Accordingly, expression of the adhesion molecules zona occludens-1 and E-cadherin was highest in 16HBE14o- cells. These molecules were localised in intercellular junctions in both 16HBE14o- and primary cells. Finally, restoration of barrier function upon injury was impaired in BEAS-2B compared to 16HBE14o- cells. In conclusion, epithelial cell types display remarkable phenotypic differences and should, accordingly, be used to address specific research questions. 16HBE14o- cells appear most suitable for studies on barrier formation, whereas resemblance in attachment of primary and BEAS-2B and A549 cells makes the latter more important for translational research on cell–matrix contact.

Epidermal growth factor receptor signalling contributes to house dust mite-induced epithelial barrier dysfunction

Impaired airway epithelial barrier function has emerged as a key factor in the pathogenesis of allergic asthma. We aimed to discern the involvement of the epidermal growth factor receptor (EGFR) in allergen-induced epithelial barrier impairment, as we previously observed that house dust mite (HDM) signals through EGFR. We investigated the junctional integrity of human bronchial epithelial cells using electric cell-substrate impedance sensing and immunofluorescent staining. HDM induced a rapid, transient fall in epithelial resistance, concomitant with delocalisation of E-cadherin and zona occludens (ZO)-1, and proteolytic cleavage of the latter. EGFR inhibition by AG1478 reduced the HDM-triggered decrease in epithelial resistance and improved restoration of epithelial junctions. Similarly, AG1478 increased epithelial barrier recovery upon electroporation-induced injury, although it delayed the migration phase of the wound healing response. HDM-promoted redistribution of E-cadherin was mediated via EGFR-dependent activation of protease-activated receptor (PAR)-2, while the concomitant ZO-1 degradation was PAR-2/EGFR-independent. Importantly, the fibrogenic cytokine transforming growth factor (TGF)-&bgr; prolonged HDM-induced EGFR phosphorylation and inhibited ligand-induced EGFR internalisation/degradation, which resulted in sustained E-cadherin and ZO-1 redistribution. Thus, allergen-induced, PAR-2/EGFR-mediated signalling decreases epithelial resistance and promotes junction disassembly. The TGF-&bgr;-enhanced EGFR signalling may be an important contributor to barrier dysfunction and increased epithelial vulnerability in response to HDM.

DAMPs activating innate and adaptive immune responses in COPD.

Chronic obstructive pulmonary disease (COPD), a progressive lung disease characterized by sustained neutrophilic airway inflammation, is caused by chronic exposure to noxious stimuli, e.g., cigarette smoke. This chronic exposure can induce immunogenic cell death of structural airway cells, inducing the release of damage-associated molecular patterns (DAMPs). Levels of several DAMPs, including S100 proteins, defensins, and high-mobility group box-1 (HMGB1), are increased in extracellular lung fluids of COPD patients. As DAMPs can attract and activate immune cells upon binding to pattern recognition receptors, we propose that their release may contribute to neutrophilic airway inflammation. In this review, we discuss the novel role of DAMPs in COPD pathogenesis. Relevant DAMPs are categorized based on their subcellular origin, i.e. cytoplasm, endoplasmic reticulum, nucleus, and mitochondria. Furthermore, their potential role in the pathophysiology of COPD will be discussed.