A. J. M. van Oosterhout

MeDALL (Mechanisms of the Development of ALLergy): an integrated approach from phenotypes to systems medicine

To cite this article: Bousquet J, Anto J, Auffray C, Akdis M, Cambon‐Thomsen A, Keil T, Haahtela T, Lambrecht BN, Postma DS, Sunyer J, Valenta R, Akdis CA, Annesi‐Maesano I, Arno A, Bachert C, Ballester F, Basagana X, Baumgartner U, Bindslev‐Jensen C, Brunekreef B, Carlsen KH, Chatzi L, Crameri R, Eveno E, Forastiere F, Garcia‐Aymerich J, Guerra S, Hammad H, Heinrich J, Hirsch D, Jacquemin B, Kauffmann F, Kerkhof M, Kogevinas M, Koppelman GH, Kowalski ML, Lau S, Lodrup‐Carlsen KC, Lopez‐Botet M, Lotvall J, Lupinek C, Maier D, Makela MJ, Martinez FD, Mestres J, Momas I, Nawijn MC, Neubauer A, Oddie S, Palkonen S, Pin I, Pison C, Rancé F, Reitamo S, Rial‐Sebbag E, Salapatas M, Siroux V, Smagghe D, Torrent M, Toskala E, van Cauwenberge P, van Oosterhout AJM, Varraso R, von Hertzen L, Wickman M, Wijmenga C, Worm M, Wright J, Zuberbier T. MeDALL (Mechanisms of the Development of ALLergy): an integrated approach from phenotypes to systems medicine. Allergy 2011; 66: 596–604.

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.

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.

Regulatory T-lymphocytes in asthma

T-helper cell type (Th)2 lymphocytes play an important role in the initiation, progression and persistence of allergic diseases, including asthma. However, little is known about immunoregulatory mechanisms that determine susceptibility to, severity of, or persistence of asthma. The concept of a disturbed Th1/Th2 balance, although having furthered the present understanding of immunoregulation in asthma, has recently been named a “procrustean paradigm”, because of its failure to adequately explain many (pre)clinical observations. In recent years, the general knowledge regarding the regulation of infectious, autoimmune diseases, asthma and allergen immunotherapy by T-regulatory (Treg) cells, has rapidly increased. Many different Treg subsets have been described, including CD8+ Treg cells, natural killer (NK) cells and several different CD4+ Treg cell subsets. In this review, the authors will focus on two major and well-described CD4+ Treg cell subsets. These consist of naturally occurring CD25+ Treg cells and adaptive Treg cells that are postulated to prevent immune responses against self-antigens and adaptive immune responses, respectively. The adaptive T-regulatory cells are further subdivided into T-regulatory cells type 1 and T-helper cell type 3 that mediate suppression exclusively via the cytokines interleukin-10 and transforming growth factor-β, respectively.

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.

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.

Modulation of airway hyperresponsiveness and eosinophilia by selective histamine and 5‐HT receptor antagonists in a mouse model of allergic asthma

Since both histamine and 5‐hydroxytryptamine (5‐HT) can be released by murine mast cells, we investigated the possible role of these autacoids on airway hyperresponsiveness (AHR), eosinophil infiltration and serum‐IgE levels in a murine model of allergic asthma. Ovalbumin‐sensitized mice were exposed to either ovalbumin (2 mg ml−1) or saline aerosols on 8 consecutive days. Starting one day before the challenge, animals were injected i.p. twice a day with a 5‐HT‐type 1 (5‐HT1) or type 2 (5‐HT2) receptor antagonist (methiotepine, 1.25 or 2.0 mg kg−1 and ketanserin, 12 mg kg−1, respectively) or a histamine‐type 1 (H1) or type 2 (H2) receptor antagonist (mepyramine, 12 or 20 mg kg−1 and cimetidine, 10 or 25 mg kg−1, respectively). Furthermore, animals were injected with a combination of cimetidine and ketanserin or with an α‐adrenoceptor antagonist (phentolamine, 5 mg kg−1). In vehicle‐treated ovalbumin‐challenged animals airway responsiveness to intravenous injections of methacholine in vivo was significantly (9 fold increase, P<0.01) increased when compared to vehicle‐treated saline‐challenged animals. Furthermore, ovalbumin challenge of vehicle‐treated animals induced a significant increase in both eosinophil numbers in bronchoalveolar lavage (BAL) fluid (0±0, vehicle/saline and 15.0±5.9×104 cells vehicle/ovalbumin, P<0.05) and ovalbumin‐specific IgE levels in serum (157±69 and 617±171 units ml−1, respectively, P<0.05) compared to saline‐challenged mice. Virtually no eosinophils could be detected in saline‐challenged animals after all different treatments. Treatment with ketanserin or cimetidine resulted in a partial but significant decrease of the ovalbumin‐induced AHR compared to ovalbumin‐challenged controls (P<0.05) and reduced eosinophil infiltration after ovalbumin challenge by 60% and 58%, respectively. The combination of cimetidine and ketanserin almost completely abolished AHR whereas eosinophilia was decreased by 49%. No effects of these antagonists were observed on IL‐16 levels in BAL fluid or on serum antigen‐specific IgE levels. Treatment with either the H1‐receptor, the 5‐HT1‐receptor or the α‐adrenoceptor antagonist, did not decrease the observed ovalbumin‐induced airway responsiveness or eosinophilia in vehicle‐treated animals. Higher doses of either methiotepine (2.0 mg kg−1) or mepyramine (20 mg kg−1) did decrease ovalbumin‐induced eosinophil infiltration (by 67%, P<0.05 and 73%, respectively), whereas no effects of these antagonists were observed on ovalbumin‐specific IgE levels in serum. From these data it can be concluded that both histamine and 5‐HT play a role in antigen‐induced AHR and eosinophilia in the mouse.

Effect of dexamethasone and endogenous corticosterone on airway hyperresponsiveness and eosinophilia in the mouse

1 Mice were sensitized by 7 intraperitoneal injections of ovalbumin without adjuvant (10 μg in 0.5 ml of sterile saline) on alternate days and after 3 weeks exposed to either ovalbumin (2 mg ml−1 in sterile saline) or saline aerosol for 5 min on 8 consecutive days. One day before the first challenge, animals were injected intraperitoneally on a daily basis with vehicle (0.25 ml sterile saline), dexamethasone (0.5 mg kg−1) or metyrapone (30 mg kg−1). 2 In vehicle‐treated ovalbumin‐sensitized animals ovalbumin challenge induced a significant increase of airway responsiveness to metacholine both in vitro (27%, P < 0.05) and in vivo (40%, P < 0.05) compared to saline‐challenged mice. Virtually no eosinophils could be detected after saline challenge, whereas the numbers of eosinophils were significantly increased (P < 0.01) at both 3 and 24 h after the last ovalbumin challenge (5.48 ± 3.8 × 103 and 9.13 ± 1.7 × 103 cells, respectively). Furthermore, a significant increase in ovalbumin‐specific immunoglobulin E level (583 ± 103 units ml−1, P < 0.05) was observed after ovalbumin challenge compared to saline challenge (201 ± 38 units ml−1). 3 Plasma corticosterone level was significantly reduced (−92%, P < 0.001) after treatment with metyrapone. Treatment with metyrapone significantly increased eosinophil infiltration (17.4 ± 9.93 × 103 and 18.7 ± 2.57 × 103 cells, P < 0.05 at 3 h and 24 h, respectively) and potentiated airway hyperresponsiveness to methacholine compared to vehicle‐treated ovalbumin‐challenged animals. Dexamethasone inhibited both in vitro and in vivo hyperresponsiveness as well as antigen‐induced infiltration of eosinophils (0, P < 0.05 and 0.7 ± 0.33 × 103 cells, P < 0.05 at 3 h and 24 h, respectively). Metyrapone as well as dexamethasone did not affect the increase in ovalbumin‐specific immunoglobulin E levels after ovalbumin challenge (565 ± 70 units/ml−1; P < 0.05; 552 ± 48 units ml−1, P < 0.05 respectively). 4 From these data it can be concluded that exogenously applied corticosteroids can inhibit eosinophil infiltration as well as airway hyperresponsiveness. Vise versa, endogenously produced corticosteroids play a down‐regulating role on the induction of both eosinophil infiltration and airway hyperresponsiveness.

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.