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β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy.

Matrix metalloproteinases (MMPs) are the main proteolytic enzymes involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). A radiolabeled MMP inhibitor, [F-18]FB-ML5, was prepared, and its in vivo kinetics were tested in a mouse model of pulmonary inflammation. BALB/c mice were exposed for 4 days to cigarette smoke (CS) or air. On the fifth day, a dynamic microPET scan was made with [F-18]FB-ML5. Standardized uptake values (PET-SUVmean) were 0.19 +/- 0.06 in the lungs of CS-exposed mice (n = 6) compared to 0.11 +/- 0.03 (n = 5) in air-exposed controls (p

Rationale We have previously shown increased expression of the Frizzled-8 receptor of the Wingless/integrase-1 (WNT) signalling pathway in COPD. Here, we investigated if the Frizzled-8 receptor has a functional role in airway inflammation associated with chronic bronchitis.Methods Acute cigarette-smoke-induced airway inflammation was studied in wild-type and Frizzled-8-deficient mice. Genetic association studies and lung expression quantitative trait loci (eQTL) analyses for Frizzled-8 were performed to evaluate polymorphisms in FZD8 and their relationship to tissue expression in chronic bronchitis. Primary human lung fibroblasts and primary human airway epithelial cells were used for in vitro studies.Results Cigarette-smoke-exposure induced airway inflammation in wild-type mice, which was prevented in Frizzled-8-deficient mice, suggesting a crucial role for Frizzled-8 in airway inflammation. Furthermore, we found a significant genetic association (p=0.009) between single nucleotide polymorphism (SNP) rs663700 in the FZD8 region and chronic mucus hypersecretion, a characteristic of chronic bronchitis, in a large cohort of smoking individuals. We found SNP rs663700 to be a cis-eQTL regulating Frizzled-8 expression in lung tissue. Functional data link mesenchymal Frizzled-8 expression to inflammation as its expression in COPD-derived lung fibroblasts was regulated by pro-inflammatory cytokines in a genotype-dependent manner. Moreover, Frizzled-8 regulates inflammatory cytokine secretion from human lung fibroblasts, which in turn promoted MUC5AC expression by differentiated human airway epithelium.Conclusions These findings indicate an important pro-inflammatory role for Frizzled-8 and suggest that its expression is related to chronic bronchitis. Furthermore, our findings indicate an unexpected role for fibroblasts in regulating airway inflammation in COPD.

T helper (Th) 2 lymphocytes play a crucial role in the initiation, progression and persistence of allergic diseases, including asthma. Drugs that interfere with the activation of T-cells or more selectively Th2-specific signaling molecules and drugs that prevent the selective migration into lung tissue are promising novel strategies for the treatment of allergic asthma. Although the mainstay asthma therapy of inhaled glucocorticoids is rather effective, targeting Th2 cells may be an important alternative in childhood. Regulatory T-cells (Treg cells) have a physiological role in protection of unwanted immune responses to auto-antigens and allergens. Literature data indicate that an imbalance between Th2 and Treg cells may underlie development and disease expression of allergic asthma. Drugs or immunotherapies that stimulate these counter-Treg cells may limit aberrant Th2 responses leading to suppression of symptoms. Furthermore, these types of treatments may offer the perspective of disease modification and long-term relief of symptoms. (c) 2006 Elsevier Inc. All rights reserved.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease and is currently the fourth leading cause of death worldwide. Chronic inflammation and repair processes in the small airways are characteristic of COPD. Despite extensive efforts from researchers and industry, there is still no cure for COPD, hence an urgent need for new therapeutic alternatives. MicroRNAs are such an option; they are small noncoding RNAs involved in gene regulation. Their importance has been shown with respect to maintaining the balance between health and disease. Although previous reviews have discussed the expression of microRNAs related to lung disease, a detailed discussion regarding the function of differential miRNA expression in the pathogenesis of COPD is lacking.In this review we link the expression of microRNAs to different features of COPD and explain their importance in the pathogenesis of this disease. We further discuss their potential to contribute to the development of future therapeutic strategies.

1. The adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP) system is known to negatively regulate transcriptional activity of T cells, thereby possibly modulating T-cell-mediated responses at the sites of inflammation. Effects of cAMP have been widely studied in freshly isolated T cells and T-cell clones; yet, effects in differentiated Th1 and Th2 cells are largely unknown. 2. To obtain differentiated T helper cells, we activated naive T cells for 1 week in the presence of IL-12 plus alpha-IL-4 to generate Th1-type cells and in the presence of IL-4 plus alpha-IL-12 to generate Th2-type cells. 3. We demonstrate that, in contrast to freshly isolated T cells, the production of Th1 (IFN-gamma) and Th2 (IL-4, IL-5) cytokines in polarized T helper cells is not strictly controlled by the activation of AC/cAMP-linked beta(2)-adrenergic and prostaglandin (PG)E(2) receptors. 4. In Th2 cells, PGE(2) could still activate the G(s) protein-coupled AC/cAMP system and subsequently induce CREB phosphorylation, whereas PGE(2) was unable to activate the cAMP-dependent pathway in Th1 cells. In both Th1 and Th2 cells, the induction of CREB phosphorylation by beta(2)-agonist fenoterol was impaired. 5. The loss of control over cytokine production by cAMP elevating agents in differentiated Th1 and Th2 subsets may have important implications for the regulation of Th1- and Th2-mediated diseases, in particular those associated with the ongoing immune responses.

Rationale: Cigarette smoke is the major risk factor in the development of chronic obstructive pulmonary disease (COPD). Lipidomics is a novel and emerging research field that may provide new insights in the origins of chronic inflammatory diseases, such as COPD.Objectives: To investigate whether expression of the sputum lipidome is affected by COPD or cigarette smoking.Methods: Lipid expression was investigated with liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry in induced sputum comparing smokers with and without COPD, and never-smokers. Changes in lipid expression after 2-month smoking cessation were investigated in smokers with and without COPD.Measurements and Main Results: More than 1,500 lipid compounds were identified in sputum. The class of sphingolipids was significantly higher expressed in smokers with COPD than in smokers without COPD. At single compound level, 168 sphingolipids, 36 phosphatidylethanolamine lipids, and 5 tobacco-related compounds were significantly higher expressed in smokers with COPD compared with smokers without COPD. The 13 lipids with a high fold change between smokers with and without COPD showed high correlations with lower lung function and inflammation in sputum. Twenty (glyco)sphingolipids and six tobacco-related compounds were higher expressed in smokers without COPD compared with never-smokers. Two-month smoking-cessation reduced expression of 26 sphingolipids in smokers with and without COPD.Conclusions: Expression of lipids from the sphingolipid pathway is higher in smokers with COPD compared with smokers without COPD. Considering their potential biologic properties, they may play a role in the pathogenesis of COPD.

To the Editor: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality, with a worldwide prevalence of 9–10% [1]. COPD is associated with chronic, neutrophilic inflammation in the lungs, causing destruction of lung parenchyma (emphysema) and/or remodelling of the airways with mucus hypersecretion (bronchitis) [2]. Chronic exposure to noxious particles and gases, such as cigarette smoke, is the major risk factor for COPD, while susceptibility to the disease has a strong genetic component [2]. While the activity of the innate immune system increases with disease progression during early stages of COPD, the precise nature of the factors that trigger innate immune responses in COPD is currently unknown. Cell damage and death upon exposure to cigarette smoke in COPD may induce the release of damage associated molecular patterns (DAMPs) [3]. Elevated levels of several prototypic DAMPs, including high-mobility group box (HMGB)1, heat shock proteins (HSPs) and S100A8, have been observed in bronchoalveolar lavage (BAL) fluid, serum and epithelial lining fluid of COPD patients [4–6]. DAMPs activate cells of the innate immune system upon binding to pattern recognition receptors, such as toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE). Importantly, the AGER gene, which encodes the RAGE receptor, is a genome-wide association study susceptibility gene for COPD [7]. To date, studies investigating the role of DAMPs in COPD have focused on the analysis of a single DAMP. However, while each individual DAMP triggers …

Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium, thereby potentially inducing allergic sensitization at the expense of inhalation tolerance. We hypothesized that the proteolytic activity of allergens may play an important factor in the allergenicity to house dust mite and is essential to overcome airway tolerance. Here, we aimed to investigate the role of PAR-2 activation in allergic sensitization and HDM-induced allergic airway inflammation. In our study, Par-2 deficient mice were treated with two different HDM extracts containing high and low serine protease activities twice a week for a period of 5 weeks. We determined airway inflammation through quantification of percentages of mononuclear cells, eosinophils and neutrophils in the bronchial alveolar lavage fluid and measured total IgE and HDM-specific IgE and IgG1 levels in serum. Furthermore, Th2 and pro-inflammatory cytokines including IL-5, IL-13, Eotaxin-1, IL-17, KC, Chemokine (C-C motif) ligand 17 (CCL17) and thymic stromal lymphopoietin (TSLP), were measured in lung tissue homogenates. We observed that independent of the serine protease content, HDM was able to induce elevated levels of eosinophils and neutrophils in the airways of both wild-type (WT) and Par-2 deficient mice. Furthermore, we show that induction of pro-inflammatory cytokines by HDM exposure is independent of Par-2 activation. In contrast, serine protease activity of HDM does contribute to enhanced levels of total IgE, but not HDM-specific IgE. We conclude that, while Par-2 activation contributes to the development of IgE responses, it is largely dispensable for the HDM-induced induction of pro-inflammatory cytokines and airway inflammation in an experimental mouse model of HDM-driven allergic airway disease.

BACKGROUND: A core feature of chronic obstructive pulmonary disease (COPD) is the accelerated decline in forced expiratory volume in one second (FEV1). The recent Groningen and Leiden Universities study of Corticosteroids in Obstructive Lung Disease (GLUCOLD) study suggested that particular phenotypes of COPD benefit from fluticasone±salmeterol by reducing the rate of FEV1 decline, yet the underlying mechanisms are unknown.METHODS: Whole-genome gene expression profiling using the Affymetrix Gene ST array (V.1.0) was performed on 221 bronchial biopsies available from 89 COPD patients at baseline and after 6 and 30 months of fluticasone±salmeterol and placebo treatment in GLUCOLD.RESULTS: Linear mixed effects modelling revealed that the expression of 138 genes decreased, whereas the expression of 140 genes significantly upregulated after both 6 and 30 months of treatment with fluticasone±salmeterol versus placebo. A more pronounced treatment-induced change in the expression of 50 and 55 of these 278 genes was associated with a lower rate of decline in FEV1 and Saint George Respiratory Questionnaire, respectively. Genes decreasing with treatment were involved in pathways related to cell cycle, oxidative phosphorylation, epithelial cell signalling, p53 signalling and T cell signalling. Genes increasing with treatment were involved in pathways related to focal adhesion, gap junction and extracellular matrix deposition. Finally, the fluticasone-induced gene expression changes were enriched among genes that change in the airway epithelium in smokers with versus without COPD in an independent data set.CONCLUSIONS: The present study suggests that gene expression in biological pathways of COPD is dynamic with treatment and reflects disease activity. This study opens the gate to targeted and molecular phenotype-driven therapy of 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.

Pellino-1 has recently been identified as a regulator of interleukin-1 (IL-1) signaling, but its roles in regulation of responses of human cells to human pathogens are unknown. We investigated the potential roles of Pellino-1 in the airways. We show for the first time that Pellino-1 regulates responses to a human pathogen, rhinovirus minor group serotype 1B (RV-1B). Knockdown of Pellino-1 by small interfering RNA (siRNA) was associated with impaired production of innate immune cytokines such as CXCL8 from human primary bronchial epithelial cells in response to RV-1B, without impairment in production of antiviral interferons (IFN), and without loss of control of viral replication. Pellino-1 actions were likely to be independent of interleukin-1 receptor-associated kinase-1 (IRAK-1) regulation, since Pellino-1 knockdown in primary epithelial cells did not alter responses to IL-1 but did inhibit responses to poly(I·C), a Toll-like receptor 3 (TLR3) activator that does not signal via IRAK-1 to engender a response. These data indicate that Pellino-1 represents a novel target that regulates responses of human airways to human viral pathogens, independently of IRAK signaling. Neutralization of Pellino-1 may therefore provide opportunities to inhibit potentially harmful neutrophilic inflammation of the airways induced by respiratory viruses, without loss of control of the underlying viral infection.

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.

Asthma and chronic obstructive pulmonary disease (COPD) are the two most prominent chronic inflammatory lung diseases, and their prevalence is still increasing. Although both diseases are associated with genetic and environmental factors, their precise etiologies are still unclear. Recent advances in biology and medicine have introduced new technologies to study the genetics and function of resident cells of the lungs, and the data demonstrate a much broader range of impact than previously envisaged. The function of resident cells of the lung may be crucial to fully understand the pathology of asthma and COPD. Knowledge and understanding of resident cells will lead to the development of improved animal models, more successful therapies, novel tools to characterize asthma, and COPD and provide better care to patients. In our present special issue, several outstanding research groups present their original research articles that will stimulate the continuing efforts to understand the molecular pathology underlying asthma and COPD, the development of strategies to treat these conditions, and the evaluation of outcomes. Moreover, several important novel insights on the role of resident cells in asthma and COPD are being reviewed in this special issue. Y. Matsumura reviews the proteinase activity of many common allergens and shows their interaction with lung epithelial cells; they induce disruption of the tight junctions between epithelial cells, activate the protease-activated receptor-2, and lead to the production of thymic stromal lymphopoietin. Hence, allergen source-derived proteases are a potentially critical factor in the development of allergic sensitization and appear to be strongly associated with heightened allergenicity. S. Kolahian and R. Gosens describe a novel role of the parasympathetic neurotransmitter acetylcholine in the regulation of airway remodeling and inflammation in respiratory disease. Recent data have indicateed that nonneuronal cells, including smooth muscle cells, secrete acetylcholine and express receptors for acetylcholine. S. Kolahian and R. Gosens focus on the role of acetylcholine in smooth muscle cell function and review data to show that the activation of acetylcholine receptors in these cells can lead to proliferation, production of growth factors, inflammatory mediators, and deposition of extra-cellular matrix proteins. Moreover, it can lead to increased smooth muscle mass, contractility, mucus gland remodeling and airway inflammation in vivo in a guinea pig model of asthma. Similarly, tiotropium studies show that acetylcholine may contribute to cigarette smoke-induced neutrophilic airway inflammation in a murine model of COPD. In addition to the new role of acetylcholine, N. Miglino et al. present data on another novel target to reduce bronchial smooth muscle remodeling in their original research article. They show that calreticulin, which has been described as a negative regulator of C/EBPα [1], is also able to reduce airway smooth muscle (ASM) cell proliferation and may thus provide beneficial effects on airway remodeling. This pathway was insensitive to corticosteroids, which may explain why airway remodeling in asthma patients is refractory to corticosteroid therapy [2]. In particular subpopulations of asthma patients do not respond well to the current therapy available for asthma. To identify more effective treatments, it is of importance to unlock the underlying mechanisms and understand the genetic background. A. Faiz and K. Burgess review the history of expression microarray technologies, that is, genome-wide association studies (GWASs)/locus fine mapping, gene candidate approaches, and gene expression studies (gene-expression microarrays, including the Gene-Chip), and their contribution to increase our understanding of asthma pathology. For instance, GWASs have been essential in the discovery of many asthma-associated genes, including disintegrin and metalloproteinase domain-containing protein 33 (ADAM33). In addition, the 3′ expression arrays have shown that treatment with IL-13 caused deregulation of a number of asthma-related genes in different cell types of the airway, including smooth muscle cells and airway epithelium. The ASM cell has been recognized as a critical effecter cell in the pathophysiology of asthma for almost a century. The interplay between the smooth muscle and the pathophysiology of asthma is reviewed by A. Ozier and colleagues. Specifically they discuss the mechanisms driving airway hyper-responsiveness in asthma, such as mediator release, altered excitation/contraction coupling, and the role of airway smooth muscle in bronchial inflammation and remodeling. T. Chiba et al. prove a compelling review on the role of the arylhydrocarbon receptor (AhR), a nuclear receptor which responds to dioxins and dioxin-like compounds in cigarette smoke and environmental pollutants. In their review they discuss the role of AhR in asthma and COPD, focusing upon how AhR modulates the immunological responses in allergic and inflammatory diseases such as bronchitis, asthma, and chronic obstructive pulmonary disease (COPD) and the crosstalk of AhR signaling with other ligand-activated transcription factors such as peroxisome proliferator-activated receptors (PPARs). In addition to the role of mesenchymal cells in innate immunity, mesenchymal cells also have the ability to direct and in-turn can be influenced by the adaptive immune system. One novel example of this interaction is the role of immunoglobulin receptors found upon the surface of mesenchymal cells. In this issue, N. Roth reviews the role of immunoglobulin receptors upon mesenchymal cell function and importantly addresses some of the basic questions in this field: why do tissue-forming cells express immunoglobulin receptors and do tissue-forming cells process immunoglobulin receptor-bound particles? Several drugs are currently in use targeting the inflammatory component of the asthmatic disorder, including steroids and beta-mimetics [3]. It is well-established that rhinovirus (RV)-induced asthma exacerbations account for high asthma-related health costs and morbidity [4]. D. van Ly and coworkers studied the molecular mechanism underlying this pathology involving RV-induced nuclear factor kappa B (NF-κB)-dependent inflammation. To establish the role of NF-κB inhibitors in RV-induced IL-6 and IL-8 and RV replication, they used pharmacological inhibitors of NF-κB, and steroids and/or β2 agonists were used as comparison. Their data suggest that targeting NF-κB alone is unlikely to be an effective treatment compared to current asthma therapeutics. Q. Ge and coworkers examined the effect of combined corticosteroids and long acting beta2-agonists on in vitro extracellular matrix protein production by TGF-stimulated airway smooth muscle (ASM) cells present in isolated bronchial rings of nonasthmatic individuals. They conclude that current combination asthma therapies are unable to prevent or reverse remodeling events that are regulated by ASM cells. Current asthma therapies have also been in the spotlight of J. C. Allen et al. who investigated corticosteroid insensitivity in vitro and were the first to examine the effect of dexamethasone on the mitogen PDGF-BB-induced cyclin D1 upregulation in ASM cells from both nonasthmatics and asthmatics. They showed that cyclin D1 mRNA and protein up-regulation in cells from asthmatic patients cannot be totally inhibited. These results agree with earlier evidence that corticosteroid-inhibited proliferation occurs only in ASM cells from nonasthmatics [5] and suggest that there are corticosteroid insensitive proliferative pathways in asthmatics that need to be further investigated towards new efficacious antiremodeling strategies. A. Soltani and coworkers have had substantial experience in using Collagen IV antibody as a blood vessel marker in bronchial biopsies [6]. Therefore, they compared the utility of anti-Collagen IV and anti-Factor VIII antibodies as markers for blood vessels in bronchial biopsies from COPD versus normal subjects and investigated the differences in the vessels' profiles that they stained. They showed that anti-Collagen IV antibody tends to stain more vessels in the Rbm and bigger vessels overall in both the Rbm and LP, while anti-Factor VIII antibody stains relatively smaller vessels. A novel finding of this study was the increased leakiness of the vessels in the Rbm in current smokers with COPD which is ripe for speculation and further study and could be factored into the concepts of the pathogenesis of smoking-related airway disease pathophysiology. The complex pathophysiology of COPD and asthma can be clarified by the use of recent advances in CT imaging. G. Dournes et al. present a thorough yet concise review of quantitative measurements of airway wall and lung parenchyma in both COPD and asthma by using CT. Quantification of airway wall and lung parenchyma has demonstrated strong correlations with clinical, functional, and pathological features in humans or animal models. Taken together, this special issue provides a focused overview and state-of-the-art science related to the role and function of resident cells of the lung, and how they may be involved in developing and maintaining asthma and COPD pathologies. Peter Borger Brian Oliver Irene Heijink Georgia Hardavella

Background Cigarette smoke, the major risk factor for COPD, is known to activate matrix metalloproteinases in airway epithelium. We investigated whether metalloproteinases, particularly A Disintegrin and Metalloproteinase (ADAM)17, contribute to increased pro-inflammatory epithelial responses with respect to the release of IL-8 and TGF-α, cytokines implicated in COPD pathogenesis. Methods We studied the effects of cigarette smoke extract (CSE) and metalloproteinase inhibitors on TGF-α and IL-8 release in primary bronchial epithelial cells (PBECs) from COPD patients, healthy smokers and non-smokers. Results We observed that TGF-α was mainly shed by ADAM17 in PBECs from all groups. Interestingly, IL-8 production occurred independently from ADAM17 and TGF-α shedding, but was significantly inhibited by broad-spectrum metalloproteinase inhibitor TAPI-2. CSE did not induce ADAM17-dependent TGF-α shedding, while it slightly augmented the production of IL-8. This was accompanied by reduced endogenous inhibitor of metalloproteinase (TIMP)-3 levels, suggesting that CSE does not directly but rather indirectly alter activity of ADAM17 through the regulation of its endogenous inhibitor. Furthermore, whereas baseline TGF-α shedding was lower in COPD PBECs, the early release of IL-8 (likely due to its shedding) was higher in PBECs from COPD than healthy smokers. Importantly, this was accompanied by lower TIMP-2 levels in COPD PBECs, while baseline TIMP-3 levels were similar between groups. Conclusions Our data indicate that IL-8 secretion is regulated independently from ADAM17 activity and TGF-α shedding and that particularly its early release is differentially regulated in PBECs from COPD and healthy smokers. Since TIMP-2-sensitive metalloproteinases could potentially contribute to IL-8 release, these may be interesting targets to further investigate novel therapeutic strategies in COPD.

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)-beta 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-beta-enhanced EGFR signalling may be an important contributor to barrier dysfunction and increased epithelial vulnerability in response to HDM.

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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 Top-FLASH. 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 HIDM 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 HIDM 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.

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.

The β2-adrenergic receptor (β2-AR) negatively regulates T cell activity through the activation of the Gs/adenylyl cyclase/cAMP pathway. β2-AR desensitization, which can be induced by its phosphorylation, may have important consequences for the regulation of T cell function in asthma. In the present study we demonstrate that the C-C chemokine thymus and activation-regulated chemokine (TARC) impairs the ability of β2-agonist fenoterol to activate the cAMP downstream effector cAMP-responsive element binding protein (CREB) in freshly isolated human T cells. The TARC-induced activation of Src kinases resulted in membrane translocation of both G protein-coupled receptor kinase (GRK) 2 and β-arrestin. Moreover, TARC was able to induce Src-dependent serine phosphorylation of the β2-AR as well as its association with GRK2 and β-arrestin. Finally, in contrast to CREB, phosphorylation of Src and extracellular signal-regulated kinase was enhanced by fenoterol upon TARC pretreatment. In summary, we show for the first time that TARC exposure impairs β2-AR function in T cells. Our data suggest that this is mediated by Src-dependent activation of GRK2, resulting in receptor phosphorylation, binding to β-arrestin, and a switch from cAMP-dependent signaling to activation of the MAPK pathway. We propose that aberrant T cell control in the presence of endogenous β-agonists promotes T cell-mediated inflammation in asthma.

The type 2 T-helper (Th2) lymphocyte can be regarded as an important target cell for the treatment of allergic asthma as it plays a crucial role in the initiation, progression and persistence of disease. Several strategies to target Th2 cells can be envisioned. Drugs that prevent Th2-cells from migrating into the lung tissue, such as antibodies to the chemokine receptor CCR4 and inhibitors of the adhesion molecule VLA-4, are promising for the treatment of asthma. To inhibit Th2-cell activation, novel asthma drugs that act on Th2-selective transciption factors such as GATA3 are being developed. Although initial strategies aimed to block the action of Th2-derived cytokines, the generation of counter-regulatory Th1 lymphocytes and regulatory T cells is currently being explored.

Background Airway inflammation in asthma is orchestrated by recruitment of T helper (Th)2 lymphocytes to the lung and subsequent production of Th2-like cytokines upon allergen challenge.Objective To examine whether allergen-induced dysfunction of the beta(2)-adrenergic receptor (beta(2)-AR) contributes to the enhanced T(h2) cell activity in asthma.Methods beta(2)-adrenergic regulation of cytokine mRNA expression was studied in alpha-CD3/alpha-CD28-activated peripheral blood lymphocytes from seven asthma patients before and 6 h after allergen challenge, in conjunction with the effects of beta(2)-agonist fenoterol on T cell chemotaxis and signalling pathways.Results A complete loss of beta(2)-AR control over expression of the Th2 cytokines IL-4, IL-5 and IL-13, but not of the Th1 cytokine IFN-gamma, was observed after allergen challenge. Furthermore, we found impaired beta(2)-AR regulation of T cell migration as well as signal transduction pathways, i.e. the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein and the inhibition of the mitogen-activated protein kinase pathway. The loss of beta(2)-AR control was associated with increased beta-adrenergic receptor kinase expression, which might be involved in beta(2)-AR desensitization. In addition, we demonstrate for the first time that T cells exposed to the chemokine thymus and activation-regulated chemokine show hyporesponsiveness to fenoterol.Conclusion Our results suggest that allergen-induced loss of beta(2)-AR control, possibly mediated by chemokine release, plays an important role in enhanced Th2-like activity in asthma.

The cAMP-dependent pathway plays an important role in the regulation of T cell-mediated immune responses by inhibition of T cell proliferation, activation and production of Th1-like cytokines. Depending on costimulatory signals and on the activation status of T cells, cAMP also regulates the production of Th2-like cytokines, yet the mechanism is not completely defined. We investigated the effect of costimulation with IL-2 on cAMP-mediated inhibition of IL-5 secretion and the signaling pathways involved in these effects in freshly isolated, alpha-CD3/alpha-CD28-stimulated human T lymphocytes. We demonstrate that IL-2 counteracts the cAMP-mediated inhibitory effects on IL-5 secretion by the modulation of phosphoinositide 3-kinase (PI3-K)-dependent signaling. Our results indicate that phosphorylation of cAMP-responsive element-binding protein (CREB) and the activity of the small GTPase Rap1 are unlikely involved in the protective effect of IL-2. Instead, the effect of IL-2 may be mediated by the PI3-K-dependent inactivation of the forkhead-related transcription factor FKHR-L1, down-regulation of p27(kip) and abrogation of the cAMP-mediated inhibition of activator protein (AP)-1 binding activity. Together, our results indicate that increased IL-2-dependent PI3-K signaling leads to impaired negative feedback control of the production of Th2-type cytokine IL-5 by the cAMP-dependent pathway.

T helper 2 (Th2) cytokines [interleukin (IL)-4 and IL-5] play a central role in the development of allergic immune responses. After allergen provocation, the expression of Th2 cytokines is rapidly up-regulated in atopy and asthma. IL-6 is a multifunctional cytokine that is able to direct Th2 immune responses and is secreted by multiple tissue cell types. This study shows that IL-6 induces up-regulation of IL-4 and IL-5 after short (5 min) preincubation periods in freshly isolated, alpha-CD3/alpha-CD28-stimulated T cells. After longer preincubation periods with IL-6 (12 and 24 hr), the priming effect on IL-4 production gradually disappears, whereas the effect on IL-5 becomes more pronounced. In contrast, a small but significant inhibitory effect is found on the production of the Th1 cytokine interferon-gamma. Additional experiments indicate that the long-term priming effect of IL-6 on IL-5 production is dependent on IL-2 signalling. This is not the case for the short-term IL-6 effect on IL-5 secretion, where the p38 mitogen-activated protein kinase-dependent induction of activator protein-1 DNA-binding activity is involved, independent of signal transducer and activator of transcription 3 phosphorylation. In summary, these data demonstrate that the short-term and long-term priming effects of IL-6 on Th2 cytokine production are regulated by different mechanisms.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with pulmonary and extra-pulmonary manifestations. Although COPD is a complex disease, diagnosis and staging are still based on simple spirometry measurements. Different COPD phenotypes exist based on clinical, physiological, immunological and radiological observations. Cigarette smoking is the most important risk factor for COPD, but only 15-20% of smokers develop the disease, suggesting a genetic predisposition. Unfortunately, little is known about the pathogenesis of COPD, and even less on the very first steps that are associated with an aberrant response to smoke exposure. This study aims to investigate the underlying local and systemic inflammation of different clinical COPD phenotypes, and acute effects of cigarette smoke exposure in individuals susceptible and non-susceptible for the development of COPD. Furthermore, we will investigate mechanisms associated with corticosteroid insensitivity. Our study will provide valuable information regarding the pathogenetic mechanisms underlying the natural course of COPD.METHODS AND ANALYSIS: This cross-sectional study will include young and old individuals susceptible or non-susceptible to develop COPD. At a young age (18-40 years) 60 'party smokers' will be included who are called susceptible or non-susceptible based on COPD prevalence in smoking family members. In addition, 30 healthy smokers (age 40-75 years) and 110 COPD patients will be included. Measurements will include questionnaires, pulmonary function, low-dose CT scanning of the lung, body composition, 6 min walking distance and biomarkers in peripheral blood, sputum, urine, exhaled breath condensate, epithelial lining fluid, bronchial brushes and biopsies. Non-biased approaches such as proteomics will be performed in blood and epithelial lining fluid.ETHICS AND DISSEMINATION: This multicentre study was approved by the medical ethical committees of UMC Groningen and Utrecht, the Netherlands. The study findings will be presented at conferences and will be reported in peer-reviewed journals.TRIAL REGISTRATION: ClinicalTrials.gov, NCT00807469 (study 1) and NCT00850863 (study 2).

Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients’ quality of life. The transcription factor SAM-pointed domain–containing Ets-like factor ( SPDEF) plays a critical role in the regulation of mucus production and, therefore, represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing SPDEF using the novel strategy, epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the SPDEF promoter. All constructs were able to effectively suppress both SPDEF mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes [anterior gradient 2 ( AGR2), mucin 5AC ( MUC5AC)]. For the histone methyltransferase G9A, and not its mutant or other effectors, the obtained silencing was mitotically stable. These results indicate efficient SPDEF silencing and downregulation of mucus-related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition.

Background Cigarette 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. Methods We 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. Results We 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. Conclusion The 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. © 2013 Hoffmann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.