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Background: Rhinovirus (RV)-induced chronic obstructive pulmonary disease (COPD) exacerbations exhibit TH2-like inflammation. We hypothesized that RV-infected bronchial epithelial cells (BEC) overproduce TH2-switching hub cytokine, thymic stromal lymphopoietin (TSLP) in COPD. Methods: Primary BEC from healthy (HBEC) and from COPD donors (COPD-BEC) were grown in 12-well plates, infected with RV16 (0.5–5 MOI) or stimulated with agonists for either toll-like receptor (TLR) 3 (dsRNA, 0.1–10 µg/ml) or RIG-I-like helicases (dsRNA-LyoVec, 0.1–10 µg/ml). Cytokine mRNA and protein were determined (RTqPCR; ELISA). Results: dsRNA dose-dependently evoked cytokine gene overproduction of TSLP, CXCL8 and TNF-α in COPD-BEC compared to HBEC. This was confirmed using RV16 infection. IFN-β induction did not differ between COPD-BEC and HBEC. Endosomal TLR3 inhibition by chloroquine dose-dependently inhibited dsRNA-induced TSLP generation and reduced generation of CXCL8, TNF-α, and IFN-β. Stimulation of cytosolic viral sensors (RIG-I-like helicases) with dsRNA-LyoVec increased production of CXCL8, TNF-α, and IFN-β, but not TSLP. Conclusions: Endosomal TLR3-stimulation, by dsRNA or RV16, induces overproduction of TSLP in COPD-BEC. dsRNA- and RV-induced overproduction of TNF-α and CXCL8 involves endosomal TLR3 and cytosolic RIG-I-like helicases and so does the generation of IFN-β in COPD-BEC. RV16 and dsRNA-induced epithelial TSLP may contribute to pathogenic effects at exacerbations and development of COPD.

Asthma is characterized pathologically by structural changes in the airway, termed airway remodeling. These changes are associated with worse long-term clinical outcomes and have been attributed to eosinophilic inflammation. In vitro studies indicate, however, that the compressive mechanical forces that arise during bronchoconstriction may induce remodeling independently of inflammation. We evaluated the influence of repeated experimentally induced bronchoconstriction on airway structural changes in patients with asthma.We randomly assigned 48 subjects with asthma to one of four inhalation challenge protocols involving a series of three challenges with one type of inhaled agent presented at 48-hour intervals. The two active challenges were with either a dust-mite allergen (which causes bronchoconstriction and eosinophilic inflammation) or methacholine (which causes bronchoconstriction without eosinophilic inflammation); the two control challenges (neither of which causes bronchoconstriction) were either saline alone or albuterol followed by methacholine (to control for nonbronchoconstrictor effects of methacholine). Bronchial-biopsy specimens were obtained before and 4 days after completion of the challenges.Allergen and methacholine immediately induced similar levels of bronchoconstriction. Eosinophilic inflammation of the airways increased only in the allergen group, whereas both the allergen and the methacholine groups had significant airway remodeling not seen in the two control groups. Subepithelial collagen-band thickness increased by a median of 2.17 μm in the allergen group (interquartile range [IQR], 0.70 to 3.67) and 1.94 μm in the methacholine group (IQR, 0.37 to 3.24) (P0.001 for the comparison of the two challenge groups with the two control groups); periodic acid-Schiff staining of epithelium (mucus glands) also increased, by a median of 2.17 percentage points in the allergen group (IQR, 1.03 to 4.77) and 2.13 percentage points in the methacholine group (IQR, 1.14 to 7.96) (P=0.003 for the comparison with controls). There were no significant differences between the allergen and methacholine groups.Bronchoconstriction without additional inflammation induces airway remodeling in patients with asthma. These findings have potential implications for management.

Toxic injury can induce squamous metaplasia of respiratory epithelium, which normally is pseudostratified. Terminally differentiated squamous epithelial cells have a flattened, elongated appearance. During differentiation, they have an intermediate phenotype that is difficult to identify and distinguish from tangentially cut columnar cells in tissue sections from endobronchial biopsies, whose small size makes orientation difficult. The aim of our study was to develop a panel of antibodies that could be employed to distinguish normal epithelium from metaplastic epithelium and would be suitable for use on endobronchial biopsies. Nasal polyp tissue and tonsil tissue, which have pseudostratified and squamous epithelia, respectively, were collected from surgical cases and embedded in glycol methacrylate resin. Cut sections were stained immunohistochemically with a panel of antibodies to cytokeratins (CK), whose expression varies with epithelial type and stage of differentiation, and involucrin, a marker of terminal squamous differentiation. Squamous epithelium stained positively for CK5/6, CK13 and involucrin. In the pseudostratified epithelium, basal cells exhibited weak staining for CK13 and strong staining for CK5/6, and columnar cells exhibited strong immunoreactivity for CK7, CK8 and CK18. Application of this panel to endobronchial biopsies from smokers enabled areas of squamous metaplasia to be distinguished from tangentially sectioned epithelium.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells and can initiate allergic inflammation. Since exposure to rhinovirus or double-stranded (ds) RNA (a surrogate of viral infection) induces TSLP expression in bronchial epithelial cells (BECs), this cytokine may link innate antiviral responses and the type 2 adaptive immune response.As BECs from donors with asthma have a deficient interferon (IFN) response to rhinovirus infection, a study was undertaken to test the hypothesis that their antiviral response shows a bias towards TSLP production.Primary BECs were grown from subjects with asthma and healthy volunteers. After exposure to dsRNA, interleukin (IL)-8, IFNbeta and TSLP mRNA and protein expression were measured by RT-qPCR and ELISA, respectively.dsRNA dose-dependently increased IL-8 expression in BECs with no significant difference between the groups. However, BECs from subjects with asthma expressed less IFNbeta and more TSLP mRNA and protein in response to dsRNA than BECs from those without asthma (median (IQR) 57 (38-82) pg/ml vs 106 (57-214) pg/ml for IFNbeta (p0.05) and 114 (86-143) pg/ml vs 65 (32-119) pg/ml for TSLP (p0.05) in response to 10 microg/ml dsRNA for 24 h). Induction of TSLP mRNA by dsRNA was blocked by Toll-like receptor 3 or protein kinase inhibitors or by preventing de novo protein synthesis, but not by neutralisation of type I IFN receptors.BECs from subjects with asthma are biased towards higher TSLP and lower IFNbeta production in response to dsRNA, suggesting that viral infection in asthma may lead to an altered mediator profile that biases towards a Th2 immune response.

Asthma is an inflammatory disorder of the airways dominated by a Th2-type pattern. Because of this, most research has focused on investigating the role of allergic pathways with the hope of discovering novel therapeutic targets. Unfortunately, this strategy (which has been extended to animal models) has failed to identify any therapeutic modalities other than anti-IgE and leukotriene modifiers directed to targets known about for many years. It seems that the problem lies in placing allergy at the center of disease pathogenesis, when in practice other environmental factors may be equally if not more important in the induction and then progression of asthma. An alternative view is that asthma is primarily a defect of epithelial barrier function that, as in atopic dermatitis, allows greater access of environmental allergens, microorganisms, and toxicants to the airway tissue. Evidence is provided to show that both the physical and functional barrier of the airway epithelium is defective in asthma with disrupted tight junctions, reduced antioxidant activity, and impaired innate immunity. This explains the remarkable susceptibility of asthmatic airways to respiratory viruses and the impact of air pollutants on asthma exacerbations. It also provides a mechanism for programming of dendritic cells to drive a Th2 response in the origins of asthma. Viewing asthma primarily as an epithelial disease with adoption of a chronic wound scenario also provides a route to airway wall remodeling and the varying asthma phenotypes over the life course.

Aims: Mutations in the FERM domain containing 7 (FRMD7) genes are known to cause a significant number of cases of congenital idiopathic nystagmus (CIN). Only limited expression data exist suggesting low levels of expression in all tissues. In this study, we assess the expression profile of the murine homologue of FRMD7(Frmd7) in tissue from three murine organs during development. Methods: cDNA was extracted from heart, lung, and brain tissues of MF-1 mice at 12 developmental time points, embryonic days 11–19, postnatal days 1 and 8, and from adult mice. Relative expression of Frmd7mRNA was calculated using quantitative real-time PCR techniques with two normalising genes (Gapdhand Actb). Results: Expression of Frmd7was low in all tissues consistent with earlier reports. In heart and lung tissues, expression remained very low with an increase only in adult samples. In brain tissue, expression levels were higher at all time points with a significant increase at embryonic day 18, with no gender-specific influence on Frmd7expression. Conclusions: Frmd7is expressed at low levels in all tissues studied suggesting a role in many tissue types. However, higher overall expression and a sharp increase at ED18 in the murine brain suggest a different role in this tissue. Earlier studies have shown that genes expressed in the murine brain during development exhibit temporal functional clustering. The temporal pattern of Frmd7 expression found in this study mirrors that of genes involved in synapse formation/function, and genes related to axon growth/guidance. This suggests a role for Frmd7 in these processes and should direct further expression studies.

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.

Background IL-13 is a key cytokine associated with the asthmatic phenotype. IL-13 signals via its cognate receptor, a complex of IL-13 receptor (IL-13R) α 1 chain with IL-4 receptor α; however, a second protein, IL-13Rα2, also binds IL-13. Recently a polymorphic variant of IL-13 (R110Q) has been shown to be associated with atopy. Objective To investigate the binding properties of this IL-13 variant to its cognate receptors. Methods We used surface plasmon resonance to measure the binding kinetics of R110Q to its receptors. Primary human fibroblasts were grown from endobronchial biopsies obtained from volunteers. Receptor levels were measured by fluorescence-activated cell sorting. Results There was no significant difference in the binding of R110Q with soluble human IL-13Rα1 compared with IL-13 (32 ± 5 nmol/L and 36 ± 7 nmol/L, respectively; P = .625). However, a small but significant difference was observed in the binding of R110Q to soluble human IL-13Rα2 compared with IL-13 (840 ± 87 pmol/L and 1.1 ± .05 nmol/L, respectively; P = .04). We observed that primary human lung fibroblasts expressed different levels of IL-13Rα2. Eotaxin release from fibroblasts expressing low IL-13Rα2 levels was significantly higher in response to R110Q compared with IL-13. This was not evident in cells that had high baseline IL-13Rα2 levels. Conclusion These results suggest that relatively small changes in functional properties of a ligand combined with variation in receptor levels in vivo can result in significant differences in responsiveness. Clinical implications Expression of R110Q and low IL-13Rα2 levels can result in important biological differences that may have clinical relevance in an atopic environment.

Recent advances in the field of innate immunity have driven an important reappraisal of the role of these processes in airway disease. Various strands of evidence indicate that resident cells, such as macrophages and epithelial cells, have central importance in the initiation of inflammation. Macrophage activation has the potential to regulate not just typical aspects of innate immunity but also, via a variety of intricate cell-cell networks, adaptive responses and responses characterized by Th2-type cytokine production. In turn, such adaptive immune processes modify the phenotype and function of the innate immune system. Cooperative responses between monocytic cells and tissue cells are likely to be crucial to the generation of effective inflammatory responses, and a realization of the importance of these networks is providing a new way of identifying antiinflammatory therapies. Importantly, the repeated cycles of allergic and nonallergic inflammation that comprise chronic human airway disease are not necessarily well described by current terminology, and we propose and describe a concept of contiguous immunity, in which continual bidirectional cross-talk between innate and adaptive immunity describes disease processes more accurately.

Background Cysteinyl leukotrienes (cys-LTs) are potent asthma-related mediators that function through their G protein–coupled receptors, cys-LT receptor type 1 (CysLT1R) and cys-LT receptor type 2 (CysLT2R). Objective Because many G protein–coupled receptors transactivate the epidermal growth factor receptor (EGFR) through metalloprotease-mediated ligand shedding, we investigated the effects of cys-LTs on signal transduction and proliferation of bronchial fibroblasts. Methods Human bronchial fibroblasts were grown from biopsy specimens of healthy subjects. Mitogenesis was assessed on the basis of tritiated methylthymidine incorporation. Results Leukotriene (LT) D 4 alone did not increase mitogenesis but dose-dependently increased thymidine incorporation and cell proliferation in the presence of epidermal growth factor (EGF). The enhancement was not prevented by CysLT1R antagonists (MK-571 and montelukast) or by a dual antagonist (BAY u9773), which is consistent with the lack of detectable mRNA for CysLT1R and CysLT2R in bronchial fibroblasts. LTD 4 did not cause EGFR transphosphorylation nor was the synergism blocked by the metalloprotease inhibitor GM6001. The EGFR-selective kinase inhibitor AG1478 suppressed the synergy between LTD 4 and EGF but had no effect on synergistic interactions of LTD 4 with other receptor tyrosine kinase growth factors. The effect of LTD 4 involved a pertussis toxin–sensitive and protein kinase C–mediated intracellular pathway, leading to sustained growth factor–dependent phosphorylation of extracellular signal–regulated kinase 1/2 and protein kinase B (PKB/Akt). Conclusion Cys-LTs do not transactivate EGFR but have a broader capability to synergize with receptor tyrosine kinase pathways. Clinical implications This study implies a critical role of cys-LTs in airway fibrosis in asthma and other chronic airway diseases, which might not be blocked by therapy with current LT receptor antagonists.

Introduction: Respiratory viruses infect the airway epithelial barrier and cause inflammation. This process involves cellular crosstalk between different cell types. By using a dynamic 3D co-culture model of airway epithelial cells (AECs) and endothelial cells we aimed to analyse the cellular crosstalk regulating airway inflammation during viral infections. Methods: Human AECs were cultured on the apical side of permeable filter supports while endothelial cells were cultured on the basolateral side facing the microfluidic flow. The 3D model was challenged apically with double stranded RNA (Poly(I:C)), a mimic of viral infections. Basal supernatants were collected in 2h intervals and mediator secretion determined by ELISA. Results: In contrast to static cultures, the microfluidic systemallowed analysis of airway barrier responses time-dependently at shorter intervals and with greater sensitivity. Poly(I:C) induced release of TNF-α by epithelial cells with a peak 4h after stimulation; this was followed temporally by release of fractalkine (CX3 CL1), a chemokine regulating immune cell migration, from the endothelial cells. Release of fractalkine could be prevented by neutralisation of TNF-α. Conclusion: Using adynamic 3D model offers the potential to investigate mechanisms of airway inflammation and cellular crosstalk under conditions closer to the in vivo situation. During viral infections, endothelial-derived CX3 CL1 release is triggered by epithelial derived TNF-α. This cellular crosstalk might play an important role in the regulation of immune cell infiltration.

The airway epithelium is an important barrier interface for the lungs and plays a key role in various lung inflammatory disorders. MS4A8B, a member of the MS4A (membrane-spanning, 4 domains, subfamily A) family, contains a putative immunoreceptor tyrosine-based inhibitory motif (ITIM) and we have previously shown that MS4A8B is expressed on the cilia of airway epithelial cells. Our study aimed to determine the expression and function of MS4A8B in the human airways and to clarify its role in lung inflammatory disorders. Real-time PCR was performed on healthy and COPD primary bronchial epithelial cells (PBECs) grown at an air-liquid interface (ALI) to examine differences in MS4A8B expression. MS4A8B, and an ITIM mutant form, were transfected into the BEAS-2B and HEK293 cell lines to examine its effects on cellular function and signaling. MS4A8B gene expression increased over time as PBECs underwent complete mucociliary differentiation. Compared to healthy donors, MS4A8B gene expression was reduced in ALI cultured PBECs obtained from COPD patients. Transfected BEAS-2B cells stably expressing MS4A8B showed a reduced proinflammatory cytokine production and enhanced proliferation that was reversed by tyrosine mutation of the proteins ITIM motif. In signaling studies, MS4A8B was shown to be tyrosine phosphorylated in transfected HEK293 cells following sodium pervanadate treatment with this effect being lost upon MS4A8B ITIM mutation. Our combined data suggests that MS4A8B is an important control mechanism in the airway epithelium and that its ITIM motif is critical for this effect. The loss of MS4A8B expression in airway disease such as COPD may thus enhance airway inflammation and worsen disease.

Aims: Investigate the effect of the HADCI, FK228, on IPF fibroblasts differentiation and migration. Background: IPF is the most severe form of interstitial pneumonias with an unknown aetiology and without a successful therapy. Recent studies have demonstrated that HDAC inhibitors are good candidate for the treatment of fibrosis. These enzymes are specifically druggable, providing important therapeutic targets for IPF. Methods: The expression of FAK in IPF fibroblasts during FK228 treatment was assessed by western blot and real time PCR. The migration ability of IPF fibroblasts was assessed by scratch wound assay and transwell migration assay. Results: During FK228 treatment, FAK protein levels are down regulated in IPF fibroblasts resulting in the reduction of cell motility. Conclusions: FK228 is a good candidate for the treatment of IPF exhibiting a potent inhibition of fibroblast migration. By target FAK expression, FK228 could reduce fibroblasts dissemination resulting in a slower progression of lung fibrosis.

Bronchial epithelial cells (BECs) and fibroblasts (FB) form an epithelial mesenchymal trophic unit (EMTU) that drives responses to disruptions in homeostasis such as viral infection. Evidence suggests that viral infection of BECs releases mediators which activate FBs. We hypothesised that epithelial virus exposure activates the EMTU. The EMTU was modelled using polarised BECs (16HBE14o-) and FBs (MRC5) maintained on the apical and basolateral surface of a nanoporous membrane respectively. Cytokine responses to dsRNA (a viral mimetic) were determined by ELISA. A primary EMTU model was established using primary BECs differentiated at an air-liquid interface, co-cultured with FBs and challenged with rhinovirus (RV16). dsRNA activation of the EMTU model induced significant IL-6, IL-8, GM-CSF and IP-10 release which was synergistically enhanced basolaterally compared to BEC or FB monocultures. dsRNA dependent IL-1αrelease was also detected in the EMTU model and BECs.IL-1 receptor (IL-1R) antagonist abrogated dsRNA-dependent basolateral IL-6, IL-8 and GM-CSF responses.However, only FBs were responsive to stimulation with exogenous IL-α. In the primary EMTU model, RV16 induced IL-1α,IL-6, IL-8 and IP-10 release and following IL-1R antagonism there was a significant reduction in RV16-dependent IL-6 and IL-8. In the EMTU cross-talk between BECs and FBs led to synergistic enhancement in viral-induced responses. Viral-induced basolateral inflammatory responses were mediated by BEC-derived IL-1αacting on FBs.IL-1α may therefore have important consequences in promoting inflammation in viral exacerbations of chronic lung diseases such as asthma.

The bronchial epithelium plays a key role in providing a protective barrier against many environmental substances of anthropogenic or natural origin which enter the lungs during breathing. Appropriate responses to these agents are critical for regulation of tissue homeostasis, while inappropriate responses may contribute to disease pathogenesis. Here, we compared epithelial barrier responses to different pollen species, characterized the active pollen components and the signaling pathways leading to epithelial activation. Polarized bronchial cells were exposed to extracts of timothy grass (Phleum pratense), ragweed (Ambrosia artemisifolia), mugwort (Artemisia vulgaris), birch (Betula alba) and pine (Pinus sylvestris) pollens. All pollen species caused a decrease in ionic permeability as monitored trans-epithelial electrical resistance (TER) and induced polarized release of mediators analyzed by ELISA, with grass pollen showing the highest activity. Ultrafiltration showed that the responses were due to components

Therapeutic options to treat virus-induced asthma exacerbations are limited and urgently needed. Therefore, we tested Pim1 kinase as potential therapeutic target in human rhinovirus (HRV) infections. We hypothesised that inhibition of Pim1 kinase reduces HRV replication by augmenting the interferon-induced anti-viral response due to increased activity of the janus kinase–signal transducer and activator of transcription (JAK-STAT) pathway.Air–liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) from healthy individuals and moderate-to-severe asthmatic volunteers were infected with HRV-16 with or without a specific Pim1 inhibitor; viral replication and induction of anti-viral responses were measured using RT-qPCR. Viral titres were measured by 50% tissue culture infective dose and release of interferon-γ-induced protein 10 (IP-10) and RANTES protein assessed by ELISA. Phosphorylation of STAT-1 was determined using western blotting.Viral replication was reduced in ALI cultures of healthy and asthmatic PBECs treated with the Pim1 inhibitor. Using cultures from healthy donors, enhanced STAT-1 phosphorylation upon inhibition of Pim1 kinase activity resulted in increased mRNA expression of interferon-β, interleukin-29, IP-10 and RANTES 12 h after infection and increased protein levels of IP-10 and RANTES 24 h after infection.We have identified Pim1 kinase as novel target to reduce viral replication in ALI cultures of PBECs. This may open new avenues for therapeutic interventions in virus-induced asthma exacerbations.

BackgroundEpidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.ObjectivesSince primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.MethodsPBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF).ResultsExposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.ConclusionOur results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.

Background IL-13 and IL-4 share many functional properties as a result of their use of a common receptor complex comprising IL-13 receptor α 1 (IL-13Rα1) and IL-4 receptor α (IL-4Rα). The nonsignaling receptor IL-13 receptor α 2 (IL-13Rα2) binds IL-13 with high affinity and specificity and is believed to be a decoy receptor for IL-13. Objective We sought to examine the inhibitory effects of soluble and membrane-bound IL-13Rα2 on IL-13– and IL-4–mediated effects. Methods Primary human fibroblasts were grown from endobronchial biopsy specimens obtained from volunteers. Upregulation of IL-13Rα2 mRNA was measured by means of RT-PCR, and the level of surface expression was measured by means of FACS. Results We found that a recombinant soluble form of IL-13Rα2 blocked the effects of IL-13, but not IL-4, in fibroblasts in vitro . However, we found that the transmembrane form of IL-13Rα2 could attenuate both IL-13 and IL-4 responses, even though the response to TNF-α was unaffected. Furthermore, we found that IL-13Rα2 became associated with IL-4Rα in the presence of IL-4. Addition of a blocking antibody to the extracellular domain of IL-13Rα2 restored responses of both IL-13 and IL-4. Conclusion The ability of IL-13Rα2 to regulate IL-4 was previously unrecognized in primary airway cells. These data reveal a novel role for IL-13Rα2 as a negative regulator of both IL-13 and IL-4 signaling in human bronchial fibroblasts. Clinical implications It appears that IL-13Rα2 might be a powerful suppressor of T H 2-mediated responses and thus represents a potential therapeutic target for the treatment of asthma.

Induced sputum is a readily accessible biological fluid whose composition may alter as a consequence of disease. To date, however, the proteins that routinely populate this biofluid are largely unknown, in part due to the technical difficulties in processing such mucin-rich samples. To provide a catalogue of sputum proteins, we have surveyed the proteome of human-induced sputum (sputome). A combination of 2-D gel analysis and GeLC-MS/MS allowed a total of 191 human proteins to be confidently assigned. In addition to the expected components, several hitherto unreported proteins were found to be present, including three members of the annexin family, kallikreins 1 and 11, and peroxiredoxins 1, 2 and 5. Other sets of proteins identified included four proteins previously annotated as hypothetical or conserved hypothetical. Taken together, these data represent the first extensive survey of the proteome of induced sputum and provide a platform for future identification of biomarkers of lung disease.

IL-4 is a key cytokine associated with allergy and asthma. Induction of cell signaling by IL-4 involves interaction with its cognate receptors, a complex of IL-4Rα with either the common γ-chain or the IL-13R chain α1 (IL-13Rα1). We found that IL-4 bound to the extracellular domain of IL-4Rα (soluble human (sh)IL-4Rα) with high affinity and specificity. In contrast with the sequential mechanism of binding and stabilization afforded by IL-4Rα to the binding of IL-13 to IL-13Rα1, neither common γ-chain nor IL-13Rα1 contributed significantly to the stabilization of the IL-4:IL-4Rα complex. Based on the different mechanisms of binding and stabilization of the IL-4R and IL-13R complexes, we compared the effects of shIL-4Rα and an IL-4 double mutein (R121D/Y124D, IL-4R antagonist) on IL-4- and IL-13-mediated responses. Whereas IL-4R antagonist blocked responses to both cytokines, shIL-4Rα only blocked IL-4. However, shIL-4Rα stabilized and augmented IL-13-mediated STAT6 activation and eotaxin production by primary human bronchial fibroblasts at suboptimal doses of IL-13. These data demonstrate that IL-4Rα plays a key role in the binding affinity of both IL-13R and IL-4R complexes. Under certain conditions, shIL-4Rα has the potential to stabilize binding IL-13 to its receptor to augment IL-13-mediated responses. Thus, complete understanding of the binding interactions between IL-4 and IL-13 and their cognate receptors may facilitate development of novel treatments for asthma that selectively target these cytokines without unpredicted or detrimental side effects.

Background: Transforming growth factor beta (TGFβ) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. As asthma is characterised by an increase in airway smooth muscle, we postulated that TGFβ 2 favours differentiation of asthmatic (myo)fibroblasts towards a smooth muscle phenotype. Methods: Primary fibroblasts were grown from bronchial biopsy specimens from normal (n = 6) and asthmatic (n = 7) donors and treated with TGFβ 2 to induce myofibroblast differentiation. The most stable genes for normalisation were identified using RT-qPCR and the geNorm software applied to a panel of 12 “housekeeping” genes. Expression of α-smooth muscle actin (αSMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and γ-actin were measured by RT-qPCR. Protein expression was assessed by immunocytochemistry and western blotting. Results: Phospholipase A2 and ubiquitin C were identified as the most stably expressed and practically useful genes for normalisation of gene expression during myofibroblast differentiation. TGFβ 2 induced mRNA expression for all five smooth muscle related transcripts; αSMA, HCM and CPN 1 protein were also increased but desmin protein was not detectable. Although there was no difference in basal expression, HCM, CPN 1 and desmin were induced to a significantly greater extent in asthmatic fibroblasts than in those from normal controls (p = 0.041 and 0.011, respectively). Conclusions: Although TGFβ 2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGFβ 2 is unable to induce a bona fide smooth muscle cell phenotype, it may “prime” (myo)fibroblasts for further differentiation, especially if the cells are derived from asthmatic airways.

Although asthma is defined in terms of reversibility of airflow obstruction, as the disease becomes more severe and chronic, it adopts different characteristics, including a degree of fixed airflow obstruction and corticosteroid refractoriness. Underlying these phenotypes is evidence of airway wall remodeling, which should be distinguished from the increase in smooth muscle linked to airways hyperresponsiveness. Aberrant epithelial-mesenchymal communication leads to a chronic wound scenario, which is characterized by activation of the epithelial-mesenchymal trophic unit, epithelial damage, the laying down of new matrix, and greater involvement of neutrophils in the inflammatory response. In allergic asthmatic patients who remain symptomatic despite high-dose corticosteroid therapy, blockade of IgE with omalizumab confers appreciable clinical benefit. Chronic severe asthma is also accompanied by a marked increase in TNF-α production that might contribute to corticosteroid refractoriness. Based on this, TNF blockade with the soluble fusion protein entanercept produces improvement in asthma symptoms, lung function, and quality of life paralleled by a marked reduction in airways hyperresponsiveness. Identification of novel susceptibility genes, such as a disintegrin and metalloprotease 33 (ADAM33), will provide further targets against which to direct novel therapies for asthma, especially at the more severe end of the disease spectrum.

Asthma is a complex disorder in which major genetic and environmental factors interact to both initiate the disease and modify its progression. While asthma is recognised as a disorder of the conducting airways characterised by Th2-directed inflammation, it is being increasingly apparent that alteration of the structural cells of the airways (airway remodelling) is also fundamental to disease chronicity and severity. The gene ADAM33, encoding a novel member of a identified as an asthma susceptibility gene as the result of a positional cloning effort in a cohort of families recruited form the UK and USA. Subsequent genetic studies have now provided evidence that ADAM33 may be involved in determining lung function throughout life, associated with early life lung function as well as increased decline therapeutic intervention in asthma and future work will focus on the mechanisms by which it alters lung function and bronchial hyperresponsiveness.

Asthma is increasing in prevalence in the developing world, affecting approximately 10% of the world's population. It is characterised by chronic lung inflammation and airway remodelling associated with wheezing, shortness of breath, acute bronchial hyperresponsiveness to a variety of innocuous stimuli and a more rapid decline in lung function over time. Airway remodelling, involving proliferation and differentiation of mesenchymal cells, particularly myofibroblasts and smooth muscle cells, is generally refractory to corticosteroids and makes a major contribution to disease chronicity. Transforming growth factor-beta is a potent profibrogenic factor whose expression is increased in the asthmatic airways and is a prime candidate for the initiation and persistence of airway remodelling in asthma. This review highlights the role of transforming growth factor-beta in the asthmatic lung, incorporating biosynthesis, signalling pathways and functional outcome. In vivo, however, it is the balance between transforming growth factor-beta and other growth factors, such as epidermal growth factor, which will determine the extent of fibrosis in the airways. A fuller comprehension of the actions of transforming growth factor-beta, and its interaction with other signalling pathways, such as the epidermal growth factor receptor signalling cascade, may enable development of therapies that control airway remodelling where there is an unmet clinical need.

Asthma has a high heritability of up to 75%, involving a few genes with moderate effects rather than many genes with small effects. Two methods most frequently used to search for disease-causing genes are the candidate gene approach and positional cloning (reverse genetics). The candidate gene approach selects genes that seem to be relevant to the pathophysiology of a particular disease. Polymorphism in the genes is then directly tested for the genes’ involvement in the disease by association analyses. A second method is positional cloning, incorporating genome-wide scans. Here the entire genome is screened in a family-based cohort using a panel of DNA markers spaced at regular intervals across the genome to identify specific chromosomal regions that seem to be linked to specific disease phenotypes or partial phenotypes. This is followed by identification of a gene or genes that underlie the region of linkage and detection of single-nucleotide polymorphisms (SNPs) within these (candidate) genes that may predispose to disease. SNPs are used in populations and family-based association studies to strengthen the confidence that a specific gene is causally related to the disease or partial phenotype. Many genome screens for atopy and atopic disorders have been completed, with multiple regions of the genome being linked to varying phenotypes in different population cohorts, reflecting the heterogeneity and complexity of this condition.

The embryonic Wnt/β-catenin (‘canonical’) pathway has been implicated in epithelial regeneration. To investigate the role of Wnt signal transduction in the airways, we characterised the expression of key pathway components in human bronchial epithelial cells (HBEC) and studied the influence of cell density on pathway activity, using sub-confluent cells in log-phase growth as a simple model of repairing epithelium. Primary HBEC and H292 bronchial epithelial cells were found to express TCF-4, TCF-3 and isoforms of LEF-1, transcription factors that are regulated by Wnt signalling. The cells also had the potential to respond to Wnt signalling through expression of several members of the Frizzled receptor family, including FZD-5 and -6. In confluent H292 cells, 20 mM lithium and 25% v/v Wnt-3a conditioned medium induced 4.5-fold (p = 0.008) and 1.4-fold (p = 0.006) increases in TOPflash activity, respectively. Under conditions of reduced cell density, TOPflash activity increased 1.8-fold (p = 0.002) in association with increased nuclear localisation of hypophosphorylated (active) β-catenin and increased cell proliferation. This up-regulation in reporter activity occurred independently of EGF receptor activation and could not be recapitulated by use of low-calcium medium to disrupt cadherin-mediated cell–cell adhesion, but was associated with changes in FZD-6 expression. We conclude that reactivation of this embryonic pathway may play an important role in bronchial epithelial regeneration, and that modulation of Fzd-6 receptors may regulate Wnt signalling at confluence. Recognising that many chronic inflammatory disorders of the airways involve epithelial damage and repair, altered Wnt signalling might contribute to disease pathogenesis or progression.

Background: In adult asthma the bronchial epithelium shows high expression of the epidermal growth factor receptor (EGFR) and the cyclin dependent kinase inhibitor, p21waf, linked to ongoing stress and injury. Methods: To determine if these are early markers of disease, sections of bronchial specimens obtained post mortem or by bronchoscopy from non-asthmatic (n = 7), moderate (n = 7), or severe (n = 9) asthmatic children aged 5–15 years were examined immunohistochemically. All severe and one moderately asthmatic children were receiving inhaled corticosteroids. Results: The lamina reticularis of the asthmatic biopsy sections was found to be thicker (p = 0.01) than normal with increased deposition of collagen III (p = 0.007); submucosal eosinophil numbers did not differ between groups. As in adults, there was an asthma-related increase in epithelial EGFR (p

Polymorphic variation in ADAM33 (A Disintegrin And Metalloprotease) is strongly associated with asthma and bronchial hyperresponsiveness in different populations.To study the role of ADAM33 in asthma, we investigated its expression in normal, asthmatic, and embryonic airways using reverse transcriptase-quantitative polymerase chain reaction and immunochemistry.Several ADAM33 mRNA splice variants were detected in bronchial biopsies and embryonic lung; however, the beta-isoform and variants encoding the metalloprotease domain were rare transcripts. Western blotting of bronchial biopsies confirmed the presence of multiple isoforms of ADAM33, which had molecular weights of 22, 37, 55, and 65 kD. Immunohistochemistry and laser confocal microscopy of adult bronchial biopsies showed that alpha-smooth muscle actin and ADAM33 immunoreactivity were mostly colocalized to smooth muscle and isolated cells in the submucosa. There was no significant difference in ADAM33 mRNA amplicons or protein in subjects with asthma compared with control subjects. In developing lung, ADAM33 was found around bronchial tubes; however, immunoreactivity was more widely distributed than alpha-smooth muscle actin within undifferentiated mesenchyme; on Western blots, an additional 25-kD ADAM33 variant was detected.Several ADAM33 protein isoforms occur in adult bronchial smooth muscle and in human embryonic bronchi and surrounding mesenchyme, strongly suggesting its importance in smooth muscle development and/or function, which could explain its genetic association with bronchial hyperresponsiveness. The occurrence of ADAM33 in embryonic mesenchymal cells suggests that it may be involved in airway wall "modeling" that contributes to the early life origins of asthma.

Summary Asthma is a disorder of the conducting airways in which Th-2 mediated inflammation interacts with structural changes to cause variable airflow obstruction. Fundamental to disordered function is the concept of bronchial hyperresponsiveness (BHR), in which the airways construct too much and too easily. It has been stated that BHR represents the ‘Holy Grail’ of asthma, yet our understanding of its underlying causes(s) is poorly developed [1]. Measurement of BHR has found clinical utility as a diagnostic test to help differentiate asthma from other lung disorders, but unfortunately its specificity is low. However, it is the severity of BHR and its relative independence from measures of baseline airway calibre that differentiates BHR encountered in asthma from that of other diseases such as chronic obstructive pulmonary disease (COPD), sarcoidosis and heart failure.

Asthma is a heterogeneous disease in which various cytokines orchestrate airway inflammation. Tumor necrosis alpha (TNF-alpha) is a proinflammatory cytokine that has been implicated in the modulation of inflammation in various diseases, including asthma. Although TNF-alpha blocking strategies have been an effective therapeutic modality in diseases such as rheumatoid arthritis, their role in asthma and the effects of the blockade in asthma is poorly understood. This article examines the role of TNF-alpha in asthma and the effects of blocking TNF-alpha as a possible therapeutic option in patients with severe corticosteroid-dependent asthma.

Background: Considerable research has been conducted into the nature of airway inflammation in chronic obstructive pulmonary disease (COPD) but the relationship between proximal airways inflammation and both dynamic collapse of the peripheral airways and HRCT determined emphysema severity remains unknown. A number of research tools have been combined to study smokers with a range of COPD severities classified according to the GOLD criteria. Methods: Sixty five subjects (11 healthy smokers, 44 smokers with stage 0–IV COPD, and 10 healthy non-smokers) were assessed using lung function testing and HRCT scanning to quantify emphysema and peripheral airway dysfunction and sputum induction to measure airway inflammation. Results: Expiratory HRCT measurements and the expiratory/inspiratory mean lung density ratio (both indicators of peripheral airway dysfunction) correlated more closely in smokers with the severity of airflow obstruction ( r = −0.64, p

Asthma is a complex disorder in which major genetic and environmental factors interact to initiate the disease and propagate it as a chronic relapsing disorder. Until recently, genetic factors implicated in the disease pathogenesis have been restricted to variants in known molecules involved in the inflammatory or remodelling pathways. This review discusses evidence for a new susceptibility gene for asthma, ADAM33, which was identified by positional cloning and shown to be selectively expressed in mesenchymal but not immune or inflammatory cells. ADAM33 belongs to a family of membrane–anchored metalloproteinases that also have fusagenic, adhesion and intracellular signalling properties. ADAM33 might play a key role in predisposing to the reduced lung function characteristic of asthma, possibly by influencing airway wall remodelling.

The ADAM (A Disintegrin and Metalloprotease) family of Zn++-dependent metalloproteases are multidomain proteins involved in diverse cellular activities. Polymorphic variation in ADAM33 is strongly associated with asthma and bronchial hyperresponsiveness. Identification of those isoforms of ADAM33 that are expressed in airways is fundamental to dissecting the role of ADAM33 in asthma. Analysis of primary human airways fibroblasts has shown the presence of a number of alternatively spliced forms of ADAM33, including one encoding a putative secreted variant, and many transcripts lacking the metalloproteinase domain. The relative abundance of these transcripts has been quantified using reverse transcription real-time polymerase chain reaction, in both nuclear and cytoplasmic fractions of RNA. These results demonstrate that a number of splice variants of ADAM33 are transported into the cytoplasm. Ninety percent of ADAM33 mRNA is retained in the nucleus and the subtle differences in the composition of nuclear and cytoplasmic RNA suggest important events in the splicing and selection of ADAM33 transcripts. Western blot analysis confirmed that several protein isoforms of ADAM33 are expressed in primary airways fibroblasts. These findings demonstrate that ADAM33 exists in multiple isoforms, suggesting that it is a complex molecule that plays multiple roles within mesenchymal cells.

The CC chemokine eotaxin-1 (CCL11) is chemotactic for eosinophils, basophils, and type 2 helper T cells and may play a role in allergic inflammation. We investigated its contribution as an eosinophil chemoattractant in asthmatic airway secretions (sampled as induced sputum), which possess chemotactic activity for eosinophils and T cells. Sputum samples collected from healthy subjects and subjects with mild, stable-moderate, unstable-moderate, and severe asthma were processed with phosphate-buffered saline and assayed for eotaxin by ELISA and for eosinophil chemotactic activity by fluorescence-based chemotaxis assay. The contribution of eotaxin to chemotactic activity was studied by using a high-affinity neutralizing human anti-eotaxin antibody, CAT-213. Sputum eotaxin concentration was significantly raised in moderate and severe asthma (p < 0.05 versus healthy control subjects) but not in mild asthma. Chemotactic activity was significantly increased in all asthmatic groups relative to healthy subjects (p < 0.05) and was significantly inhibited by CAT-213 (100 nM) in subjects with moderate and severe asthma, with median inhibition of 52% (p < 0.05), 78% (p < 0.0001), and 86% (p < 0.0001), respectively, in samples representing stable-moderate, unstable-moderate, and severe asthma. Eotaxin contributed to the eosinophil chemotactic activity of sputum from subjects with more severe forms of asthma but not mild asthma, suggesting that its contribution is more important in more severe disease. This activity is inhibited significantly by CAT-213.

Background Induced sputum is widely used in asthma research; however, for many mediators, the detection methods have not been validated. Objective We sought to optimize the method of detection of eotaxin, an important chemokine acting through the CCR3 receptor on eosinophils, basophils, and T H 2 cells. Methods Induced sputum from normal and asthmatic subjects was processed with dithioerythritol (DTE) or PBS; recovery of eotaxin was assessed by means of ELISA before and after spiking with recombinant eotaxin. Furthermore, the effects of removing DTE by means of ultrafiltration or the addition of protease inhibitors and high-speed centrifugation on endogenous levels and spiking recovery of eotaxin were assessed. Results Endogenous eotaxin was undetectable in DTE-processed samples, with a mean of only 30% (SD, 13%) spike recovery. DTE had no effect on the immunoassay capture antibody but dramatically reduced the detection of recombinant eotaxin. Removal of DTE from sputum before immunoassay did not improve detection, although it restored the recovery of a subsequent eotaxin spike. In contrast, PBS-processed sputum resulted in an eotaxin spike recovery of 101% (SD, 20%). Addition of protease inhibitors or high-speed centrifugation had no effect on eotaxin detection. By using this optimized protocol, eotaxin levels in PBS-processed sputum samples were found to be significantly increased in asthmatic sputum ( P Conclusion Measurement of eotaxin by means of immunoassay is adversely affected by DTE, possibly through irreversible denaturation of epitopes, which makes eotaxin undetectable by using the immunoassay antibody. Sputum samples should be processed into PBS for assessment of eotaxin, which is present at increased levels in asthmatic sputum.

Asthma is known to be a Th2 inflammatory syndrome that leads to intermittent airway obstruction. However, the mechanisms involved in development of the clinical features remain enigmatic, although genetic elements clearly are involved. Recently, based on a large genome wide screen involving families in the United Kingdom and the United States with at least two siblings with asthma, a locus was identified that encoded for a family of proteases. This group of proteins is now known as the ADAM superfamily. In this review, we discuss the ADAM superfamily and, in particular, ADAM 33, a member of a family of genes which encode a subgroup of zinc dependent metalloproteinase (metzincin). The potential for therapeutic intervention with ADAM 33 is extremely attractive and further work will not only focus on the specific domains of ADAM 33, but also the mechanisms by which they lead to bronchial hyperreactivity.

BACKGROUND: The extent of epithelial injury in asthma is reflected by expression of the epidermal growth factor receptor (EGFR), which is increased in proportion to disease severity and is corticosteroid refractory. Although the EGFR is involved in epithelial growth and differentiation, it is unknown whether it also contributes to the inflammatory response in asthma. OBJECTIVES: Because severe asthma is characterized by neutrophilic inflammation, we investigated the relationship between EGFR activation and production of IL-8 and macrophage inhibitory protein-1 alpha (MIP-1alpha) using in vitro culture models and examined the association between epithelial expression of IL-8 and EGFR in bronchial biopsies from asthmatic subjects. METHODS: H292 or primary bronchial epithelial cells were exposed to EGF or H2O2 to achieve ligand-dependent and ligand-independent EGFR activation; IL-8 mRNA was measured by real-time PCR and IL-8 and MIP-1alpha protein measured by enzyme-linked immunosorbent assay (ELISA). Epithelial IL-8 and EGFR expression in bronchial biopsies from asthmatic subjects was examined by immunohistochemistry and quantified by image analysis. RESULTS: Using H292 cells, EGF and H2O2 increased IL-8 gene expression and release and this was completely suppressed by the EGFR-selective tyrosine kinase inhibitor, AG1478, but only partially by dexamethasone. MIP-1alpha release was not stimulated by EGF, whereas H2O2 caused a 1.8-fold increase and this was insensitive to AG1478. EGF also significantly stimulated IL-8 release from asthmatic or normal primary epithelial cell cultures established from bronchial brushings. In bronchial biopsies, epithelial IL-8, MIP-1alpha, EGFR and submucosal neutrophils were all significantly increased in severe compared to mild disease and there was a strong correlation between EGFR and IL-8 expression (r = 0.70, P < 0.001). CONCLUSIONS: These results suggest that in severe asthma, epithelial damage has the potential to contribute to neutrophilic inflammation through enhanced production of IL-8 via EGFR- dependent mechanisms.

Asthma is regarded as an inflammatory disorder of the conducting airways characterized by a mast cell, eosinophil and T lymphocyte inflammatory response that is responsive to anti-inflammatory therapy, such as corticosteroids. In more severe and chronic disease, corticosteroids become less effective. As in other chronic inflammatory diseases, the tissue in which the cellular and mediator processes occur plays a major role in maintaining the response and creating a basis for disease persistence. Herein, we describe evidence that the airway epithelium interacting with the underlying mesenchymal cells recapitulates branching morphogenesis, as observed in the developing lung, to create airway wall remodeling. The reciprocal signaling between the susceptible epithelium and responsive mesenchyme (epithelial mesenchymal trophic unit) offers a new paradigm for asthma and creates new opportunities for developing therapeutics based on reversing the 'chronic wound' phenotype of asthmatic airways.

Abnormal apoptotic mechanisms are associated with disease pathogenesis. Because the asthmatic bronchial epithelium is characteristically damaged with loss of columnar epithelial cells, we postulated that this is due to unscheduled apoptosis. Using an antibody directed toward the caspase cleavage product of poly(ADP-ribose) polymerase, immunohistochemistry applied to endobronchial biopsies showed higher levels of staining in the bronchial epithelium of subjects with asthma as compared with normal control subjects (% epithelial staining [median (range) = 10.5 (1.4-24.5) versus 0.4 (0.0-9.7)]; P < 0.001). Because we were unable to determine whether this difference was due to ongoing inflammation in vivo, cultures of normal and asthmatic bronchial epithelial cells were used to study apoptosis in vitro. In complete growth medium, these cells showed no difference in their rate of proliferation or viability. However, cells from subjects with asthma were more susceptible to the apoptotic effects of H2O2 than cells from normal control subjects (% apoptotic cells = 32.2 [8.8-54.9] versus 14.3 [6.4-24.7]; P < 0.05), even though both were similarly affected by treatment with actinomycin D. These data indicate that the susceptibility of asthmatic bronchial epithelium to oxidants is greater than normal. This susceptibility may contribute to the rising trends in asthma associated with air pollution and diets low in antioxidants.

The sharp increase in the prevalence of asthma over the past three decades suggests an important contribution of environmental factors in the generation of this disease, and compels a search for molecular pathways by which such factors could facilitate Th2 immune-inflammatory airway responses; granulocyte–macrophage colony-stimulating factor (GM-CSF) might be one such signal. In this review, we appraise the evidence with respect to the presence of GM-CSF in asthma, the roles played by GM-CSF in these immune responses and environmental triggers that can induce GM-CSF expression. Further, we propose a paradigm that unites these divergent observations, and postulate that GM-CSF produced in response to environmental agents can establish an airway microenvironment that promotes the initiation, influences the evolution and supports the maintenance of an aeroallergen-specific adaptive Th2 immune response.

In sensitized individuals, exposure to allergens such as Dermatophagoides pteronyssinus (Der p) causes Th2 polarization and release of cytokines, including IL-4 and IL-13. Because Der p extracts also have direct effects on epithelial cells, we hypothesized that allergen augments the effects of Th2 cytokines by promoting mediator release from the bronchial epithelium in allergic asthma. To test our hypothesis, primary bronchial epithelial cultures were grown from bronchial brushings of normal and atopic asthmatic subjects. RT-PCR showed that each culture expressed IL-4Rα, common γ-chain, and IL-13Rα1, as well as IL-13Rα2, which negatively regulates IL-13 signaling; FACS analysis confirmed IL-13Rα2 protein expression. Exposure of epithelial cultures to either Der p extracts, TNF-α, IL-4, or IL-13 enhanced GM-CSF and IL-8 release, and this was partially suppressible by corticosteroids. Simultaneous exposure of the epithelial cultures to IL-4 or IL-13 together with Der p resulted in a further increase in cytokine release, which was at least additive. Release of TGF-α was also increased by TNF-α and combinations of IL-4, IL-13, and Der p; however, this stimulation was only significant in the asthma-derived cultures. These data suggest that, in an allergic environment, Th2 cytokines and allergen have the potential to sustain airway inflammation through a cooperative effect on cytokine release by the bronchial epithelium. Our novel finding that IL-4, IL-13, and allergen enhance release of TGF-α, a ligand for the epidermal growth factor receptor that stimulates fibroblast proliferation and goblet cell differentiation, provides a potential link between allergen exposure, Th2 cytokines, and airway remodelling in asthma.

Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

The bronchial epithelium is constantly exposed to a wide range of environmental materials present in inhaled air, including noxious gases and anthropogenic and natural particulates, such as gas and particles from car emissions, tobacco smoke, pollens, animal dander, and pathogens. As a fully differentiated, pseudostratified mucociliary epithelium, the bronchial epithelium protects the internal milieu of the lung from these agents by forming a physical barrier involving adhesive complexes and a chemical barrier involving secretion of mucus, which traps inhaled particles that can be cleared by the mucociliary escalator. It is a testament to the effectiveness of these two barriers that most environmental challenges are largely overcome without the need to develop an inflammatory response. However, as the initial cell of contact with the environment, the bronchial epithelium also plays a pivotal role in immune surveillance and appropriate activation of immune effector cells and antigen presenting cells in the presence of pathogens or other danger signals. Thus, the bronchial epithelium plays a central role in controlling tissue homeostasis and innate immunity. This review will discuss these barrier properties and how dysregulation of these homeostatic mechanisms can contribute to disease pathologies such as asthma.

Rationale: Ex vivo, bronchial epithelial cells from people with asthma are more susceptible to rhinovirus infection caused by deficient induction of the antiviral protein, IFN-β. Exogenous IFN-β restores antiviral activity. Objectives: To compare the efficacy and safety of inhaled IFN-β with placebo administered to people with asthma after onset of cold symptoms to prevent or attenuate asthma symptoms caused by respiratory viruses. Methods: A total of 147 people with asthma on inhaled corticosteroids (British Thoracic Society Steps 2–5), with a history of virus-associated exacerbations, were randomized to 14-day treatment with inhaled IFN-β (n = 72) or placebo (n = 75) within 24 hours of developing cold symptoms and were assessed clinically, with relevant samples collected to assess virus infection and antiviral responses. Measurements and Main Results: A total of 91% of randomized patients developed a defined cold. In this modified intention-to-treat population, asthma symptoms did not get clinically significantly worse (mean change in six-item Asthma Control Questionnaire

Background Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology. Objective We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. Methods Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. Results Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release. Conclusions Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.

Background In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation. Methods Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID50. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways. Results The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone. Conclusion Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.

Background Diesel exhaust is associated with cardiovascular and respiratory mortality and morbidity. Acute exposure leads to increased IL-8 expression and airway neutrophilia, however the mechanism of this response is unknown. Objectives: As cigarette smoke-induced IL-8 expression by epithelial cells involves transactivation of the epidermal growth factor receptor (EGFR), we studied the effects of diesel exhaust particles (DEP) on IL-8 release and the role of the EGFR. Methods Primary bronchial epithelial cells (PBEC) were exposed to DEPs or carbon black. IL-8 and EGFR ligand expression (transforming growth factor alpha (TGFα), heparin-binding EGF-like growth factor, and amphiregulin (AR)) were assessed by quantitative RT-PCR and ELISA. Results DEP, but not carbon black, caused a dose-dependent increase in mitogen-activated protein kinase (MAPK) activation and IL-8 expression, however above 50 μg/ml there was an increase in cytotoxicity. At 50 μg/ml, DEPs stimulated transcription and release of IL-8 and EGFR ligands. IL-8 release was blocked by EGFR neutralizing antibodies, an EGFR-selective tyrosine kinase inhibitor and by the metalloprotease inhibitor, GM6001, which blocks EGFR ligand shedding. Neutralizing antibodies to AR, TGFα and heparin-binding (HB)-EGF reduced DEP-induced IL-8 by >50%. Conclusion Expression of IL-8 in response to DEPs is dependent on EGFR activation and that autocrine production of EGFR ligands makes a substantial contribution to this response. Capsule Summary: This study identifies a mechanism whereby diesel particles stimulates IL-8 release from bronchial epithelial cells. This mechanism may help to explain the recruitment of neutrophils into the airways of people exposed to particulate air pollution.

Asthma exacerbations are among the most frequent causes of hospitalization during childhood, but the underlying mechanisms are poorly understood. We performed a genome-wide association study of a specific asthma phenotype characterized by recurrent, severe exacerbations occurring between 2 and 6 years of age in a total of 1,173 cases and 2,522 controls. Cases were identified from national health registries of hospitalization, and DNA was obtained from the Danish Neonatal Screening Biobank. We identified five loci with genome-wide significant association. Four of these, GSDMB, IL33, RAD50 and IL1RL1, were previously reported as asthma susceptibility loci, but the effect sizes for these loci in our cohort were considerably larger than in the previous genome-wide association studies of asthma. We also obtained strong evidence for a new susceptibility gene, CDHR3 (encoding cadherin-related family member 3), which is highly expressed in airway epithelium. These results demonstrate the strength of applying specific phenotyping in the search for asthma susceptibility genes.

The solution structure of the growth factor chimera mEGF/TGF ?(44-50) has been determined using an extended version of the DYANA procedure for calculating structures from NMR data. The backbone fold and preferred orientation of the domains of the chimera are similar to those found in previous studies of EGF structures, and several H-bonds used as input constraints in those studies were found independently in the chimera. This shows that the modified activity of the chimera does not result from a major structural change. However, the improved precision of the structure presented here allows the origin of some unusual chemical shifts found in all of these compounds to be explained, as well as the results obtained from some site-specific mutants. Further studies of the properties of this chimeric growth factor should help to elucidate the mechanism(s) of hetero- and homodimerization of the c-erbB receptors.

Although patients with severe, steroid-refractory asthma represent a minor proportion of the asthmatic population, they consume a disproportionate amount of healthcare costs and have a greatly impaired quality of life. They respond poorly to conventional anti-inflammatory therapy and frequently exhibit a component of fixed airflow obstruction that has been linked to airway wall remodeling. In addition to its classic barrier function, the bronchial epithelium responds to changes in the external environment by secreting cytoprotective molecules and mediators that signal to cells of the immune system. In asthma, the bronchial epithelium is stressed and damaged, with shedding of the columnar cells into the airway lumen. This damage and ensuing repair responses are proposed to orchestrate airway inflammation and remodeling via activation of myofibroblasts in the underlying lamina reticularis. This allows the two cell types to work as a trophic unit, propagating and amplifying the response at the cell surface into the submucosa. Because wound healing involves inflammation, repair, and remodeling processes, this review considers the evidence that exaggerated inflammation and remodeling of the airways arise as a consequence of abnormal injury and repair responses coordinated by the bronchial epithelium, highlighting, where possible, steroid-insensitive components.