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Summary: The purpose of this study was to investigate whether growth factors produced by activated human lung mast cells (HLMCs) impair β2‐adrenoceptor (β2‐AR) function in human airway smooth muscle (ASM) cells. Protein array analysis confirmed the presence of various growth factors, including transforming growth factor (TGF)‐β1, in the supernatants of high‐affinity IgE receptor (FcεRI)‐activated HLMCs which, when applied to ASM cells, impaired albuterol‐induced cyclic adenosine monophosphate (cAMP) production, an effect that was prevented following neutralization of TGF‐β1. This blunted β2‐AR response was reproduced by treating ASM cells with TGF‐β1 or fibroblast growth factor (FGF)‐2, which induced β2‐AR phosphorylation at tyrosine residues Tyr141 and Tyr350, and significantly reduced the maximal bronchorelaxant responses to isoproterenol in human precision cut lung slices (PCLS). Finally, ASM cells isolated from severe asthmatics displayed constitutive elevated β2‐AR phosphorylation at both Tyr141 and Tyr350 and a reduced relaxant response to albuterol. This study shows for the first time that abnormal β2‐AR phosphorylation/function in ASM cells that is induced rapidly by HLMC‐derived growth factors, is present constitutively in cells from severe asthmatics. Despite their clinical benefits, the use of β2 agonists in asthma has been associated with loss of β2‐adrenoceptor function, deleterious effects and/or worsening of the symptoms and asthma deaths. The present study provided the first evidence that abnormal β2‐adrenoceptor phosphorylation/function in airway smooth muscle can be experimentally induced by growth factors released from activated mast cells, a feature that was constitutively present in airway smooth muscle cells from severe asthmatics. [ABSTRACT FROM AUTHOR]

Background The mechanisms driving glucocorticoid ( GC) insensitivity in patients with severe asthma are still unknown. Recent evidence suggests the existence of GC-insensitive pathways in airway smooth muscle ( ASM) caused by a defect in GC receptor ( GRα) function. We examined whether other mechanisms could potentially explain the reduced sensitivity of ASM cells to GC in severe asthmatics. Methods Airway smooth muscle cells from healthy and severe asthmatic subjects were treated with TNF-α and responses to corticosteroids in both cohorts were compared by ELISA, immunoblot, immunohistochemistry and real-time PCR. Immunohistochemistry and flow cytometry assays were used to assess the expression of the protein phosphatase PP5 in endobronchial biopsies and ASM cells. Results The production of CCL11 and CCL5 by TNF-α was insensitive to both fluticasone and dexamethasone in ASM cells from severe asthmatic compared to that in healthy subjects. Fluticasone-induced GRα nuclear translocation, phosphorylation at serine 211 and expression of GC-induced leucine zipper ( GILZ) were significantly reduced in ASM cells from severe asthmatics compared to responses in healthy subjects. Levels of PP5 were increased in ASM cells from severe asthmatics and PP5 knockdown using si RNA restored fluticasone repressive action on chemokine production and its ability to induce GRα nuclear translocation and GRE-dependent GILZ expression. In vivo PP5 expression was also increased in the ASM bundles in endobronchial biopsies in severe asthmatics. Conclusions PP5-dependent impairment of GRα function represents a novel mechanism driving GC insensitivity in ASM in severe asthma. [ABSTRACT FROM AUTHOR]

Background Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM–mast cell interactions is unknown. Objective We sought to investigate ASM proliferation and survival in asthma and the effects of co-culture with mast cells. Methods Primary ASM cultures were derived from 11 subjects with asthma and 12 non-asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co-culture with the human mast cell line-1, unstimulated human lung mast cells (HLMC) or IgE/anti-IgE-activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release. Results Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co-culture with mast cells did not affect the rate of proliferation or survival of ASM cells. Conclusion Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma. Cite this as: D. Kaur, F. Hollins, R. Saunders, L. Woodman, A. Sutcliffe, G. Cruse, P. Bradding and C. Brightling, Clinical & Experimental Allergy, 2010 (40) 279– 288. [ABSTRACT FROM AUTHOR]

Background Chemokine receptors play an important role in cell migration and wound repair. In asthma, CCR3 and 7 are expressed by airway smooth muscle (ASM) and CCR7 has been implicated in the development of ASM hyperplasia. The expression profile of other chemokine receptors by ASM and their function needs to be further explored. Objective We sought to investigate ASM chemokine receptor expression and function in asthma. Methods ASM cells were derived from 17 subjects with asthma and 36 non-asthmatic controls. ASM chemokine receptor expression was assessed by flow cytometry and immunofluorescence. The function of chemokine receptors expressed by more than 10% of ASM cells was investigated by intracellular calcium measurements, chemotaxis, wound healing, proliferation and survival assays. Results In addition to CCR3 and 7, CXCR1, 3 and 4 were highly expressed by ASM. These CXC chemokine receptors were functional with an increase in intracellular calcium following ligand activation and promotion of wound healing [CXCL10 (100 ng/mL) 34 ± 2 cells/high-powered field (hpf) vs. control 29 ± 1; P=0.03; n=8]. Spontaneous wound healing was inhibited by CXCR3 neutralizing antibody (mean difference 7 ± 3 cells/hpf; P=0.03; n=3). CXC chemokine receptor activation did not modulate ASM chemotaxis, proliferation or survival. No differences in chemokine receptor expression or function were observed between ASM cells derived from asthmatic or non-asthmatic donors. Conclusions Our findings suggest that the chemokine receptors CXCR1, 3 and 4 modulate some aspects of ASM function but their importance in asthma is uncertain. [ABSTRACT FROM AUTHOR]

Background: Airway smooth muscle hyperplasia is a feature of asthma, and increases with disease severity. CCR3-mediated recruitment of airway smooth muscle progenitors towards the airway smooth muscle bundle has been proposed as one possible mechanism involved in airway smooth muscle hyperplasia. Mast cells are microlocalized to the airway smooth muscle bundle and whether mast cells influence CCR3-mediated migration is uncertain. Methods: We examined the expression of CCR3 by primary cultures of airway smooth muscle cells from asthmatics and nonasthmatics. CCR3 function was examined using intracellular calcium measurements, chemotaxis, wound healing, cell proliferation and survival assays. We investigated the recovery and function of both recombinant and airway smooth muscle-derived CCL11 (eotaxin) after co-culture with β-tryptase and human lung mast cells. Results: Airway smooth muscle expressed CCR3. Airway smooth muscle CCR3 activation by CCL11 mediated intracellular calcium elevation, concentration-dependent migration and wound healing, but had no effect on proliferation or survival. Co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11, and β-tryptase inhibited CCL11-mediated airway smooth muscle migration. Conclusions: CCL11 mediates airway smooth muscle migration. However co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11 and inhibited CCL11-mediated airway smooth muscle migration. Therefore these findings cast doubt on the importance of the CCL11/CCR3 axis in the development of airway smooth muscle hyperplasia in asthma. [ABSTRACT FROM AUTHOR]

Background: Mast cell recruitment and activation are critical for the initiation and progression of inflammation and fibrosis. Mast cells infiltrate specific structures in many diseased tissues such as the airway smooth muscle (ASM) in asthma. This microlocalisation of mast cells is likely to be key to disease pathogenesis. Human lung mast cells (HLMC) express the Ca2+ activated K+ channel KCa3.1 which modulates mediator release, and is proposed to facilitate the retraction of the cell body during migration of several cell types. A study was undertaken to test the hypothesis that blockade of KCa3.1 would attenuate HLMC proliferation and migration. Methods: HLMC were isolated and purified from lung material resected for bronchial carcinoma. HLMC proliferation was assessed by cell counts at various time points following drug exposure. HLMC chemotaxis was assayed using standard Transwell chambers (8 µm pore size). Ion currents were measured using the single cell patch clamp technique. Results: KCa3.1 blockade with triarylmethane-34 (TRAM-34) did not inhibit HLMC proliferation and clotrimazole had cytotoxic effects. In contrast, HLMC migration towards the chemokine CXCL10, the chemoattractant stem cell factor, and the supernatants from tumour necrosis factor c stimulated asthmatic ASM was markedly inhibited with both the non-selective KCa3.1 blocker charybdotoxin and the highly specific KCa3.1 blocker TRAM-34 in a dose dependent manner. Although KCa3.1 blockade inhibits HLMC migration, KCa3.1 is not opened by the chemotactic stimulus, suggesting that it must be involved downstream of the initial receptor-ligand interactions. Conclusions: Since modulation of KCa3.1 can inhibit HLMC chemotaxis to diverse chemoattractants, the use of KCa3.1 blockers such as TRAM-34 could provide new therapeutic strategies for mast cell mediated diseases such as asthma. [ABSTRACT FROM AUTHOR]

Background: Mast cell microlocalisation within the airway smooth muscle (ASM) bundle is an important determinant of the asthmatic phenotype. We hypothesised that mast cells migrate towards ASM in response to ASM derived chemokines.Methods: Primary ASM cultures from subjects with and without asthma were stimulated with interleukin (IL)-1beta, IL-4, and IL-13 alone and in combination. Mast cell chemotaxis towards these ASM supernatants was investigated, and the chemotaxins mediating migration by using specific blocking antibodies for stem cell factor (SCF) and the chemokine receptors CCR3, CXCR1, 3 and 4 as well as the Gi inhibitor pertussis toxin and the tyrosine kinase inhibitor genistein were defined. The concentrations of CCL11, CXCL8, CXCL10, TGF-beta, and SCF in the supernatants were measured and the effect of non-asthmatic ASM supernatants on the mast cell chemotactic activity of asthmatic ASM was examined.Results: Human lung mast cells and HMC-1 cells migrated towards Th2 stimulated ASM from asthmatics but not non-asthmatics. Mast cell migration was mediated through the combined activation of CCR3 and CXCR1. CCL11 and CXCL8 expression by ASM increased markedly after stimulation, but was similar in those with and without asthma. ASM supernatants from non-asthmatics inhibited mast cell migration towards the asthmatic ASM supernatant.Conclusion: Th2 stimulated ASM from asthmatics is chemotactic for mast cells. Non-asthmatic ASM releases a mediator or mediators that inhibit mast cell migration towards stimulated asthmatic ASM. Specifically targeting mast cell migration into the ASM bundle may provide a novel treatment for asthma. [ABSTRACT FROM AUTHOR]

Asthma is a condition characterized by variable airflow obstruction, airway hyperresponsiveness (AHR) and airway inflammation which is usually, but not invariably, eosinophilic. Current thoughts on the pathogenesis of asthma are focused on the idea that it is caused by an inappropriate response of the specific immune system to harmless antigens, particularly allergens such as cat dander and house dust mite, that result in Th2-mediated chronic inflammation. However, the relationship between inflammation and asthma is complex with no good correlation between the severity of inflammation, at least as measured by the number of eosinophils, and the severity of asthma. In addition there are a number of conditions such as eosinophilic bronchitis and allergic rhinitis in which there is a Th2-mediated inflammatory response, but no asthma as measured by variable airflow obstruction or AHR. When we compared the immunopathology of eosinophilic bronchitis and asthma the only difference we observed was that in asthma the airway smooth muscle (ASM) was infiltrated by mast cells, suggesting that the airways obstruction and AHR was due to an ASM mast cell myositis. This observation emphasizes that the features that characterize asthma, as opposed to bronchitis, are due to abnormalities in smooth muscle responsiveness which could be intrinsic or acquired and that inflammation is only relevant in that it leads to those abnormalities. It also emphasizes the importance of microlocalization as an organizing principle in physiological responses to airway inflammation. The factors controlling the compartmentalization of leucocyte subsets within the bronchial mucosa are therefore of paramount importance and are discussed. [ABSTRACT FROM AUTHOR]

Background Fibrocytes are bone marrow–derived CD34 + collagen I–positive cells present in peripheral blood that develop α-smooth muscle actin expression and contractile activity in tissue culture. They are implicated in the pathogenesis of tissue remodeling and fibrosis in both patients with asthma and those with idiopathic pulmonary fibrosis. Targeting fibrocyte migration might therefore offer a new approach for the treatment of these diseases. Ion channels play key roles in cell function, but the ion-channel repertoire of human fibrocytes is unknown. Objective We sought to examine whether human fibrocytes express the K Ca 3.1 K + channel and to determine its role in cell differentiation, survival, and migration. Methods Fibrocytes were cultured from the peripheral blood of healthy subjects and patients with asthma. Whole-cell patch-clamp electrophysiology was used for the measurement of ion currents, whereas mRNA and protein were examined to confirm channel expression. Fibrocyte migration and proliferation assays were performed in the presence of K Ca 3.1 ion-channel blockers. Results Human fibrocytes cultured from the peripheral blood of both healthy control subjects and asthmatic patients expressed robust K Ca 3.1 ion currents together with K Ca 3.1 mRNA and protein. Two specific and distinct K Ca 3.1 blockers (TRAM-34 and ICA-17043) markedly inhibited fibrocyte migration in transwell migration assays. Channel blockers had no effect on fibrocyte growth, apoptosis, or differentiation in cell culture. Conclusions The K + channel K Ca 3.1 plays a key role in human fibrocyte migration. Currently available K Ca 3.1-channel blockers might therefore attenuate tissue fibrosis and remodeling in patients with diseases such as idiopathic pulmonary fibrosis and asthma through the inhibition of fibrocyte recruitment. [ABSTRACT FROM AUTHOR]

Introduction and Objectives Cell adhesion molecule 1 (CADM1) contributes to cell-cell adhesion, viability and adhesion-induced degranulation in mast cells. We found that it is expressed as three alternatively spliced isoforms (major SP4, SP1 and minor SP6) in human lung mast cells (HLMCs). Here we investigated the role of CADM1 isoforms in the adhesion of mast cells to primary airway smooth muscle cells (ASM) and lung fibroblasts (LF). Methods Modulation of CADM1 expression was investigated in transient transfection or adenoviral delivery in the mast cell line HMC-1 and HLMCs. Cells with overexpressed CADM1 isoforms or downregulated CADM1 were examined in cell adhesion assays. Results CADM1 RNA interference in HMC-1 resulted in 60% reduction of surface CADM1 and complete loss of CADM1 determined by FACS and Western blotting, respectively, 6 days after transduction. This decrease in CADM1 expression reduced adhesion of transduced HMC-1 cells to ASM and LF by 42% and 50%, respectively. Downregulation of CADM1 in HLMCs reduced adhesion to ASM by 39%. HMC-1 cells transfected with SP1 (exon 8/9/11) and SP6 (exon 8/9/10/11) showed lower adhesion to LF compared to cells transfected with SP4 (exon 8/11). No differences in adhesion to ASM were found. When SP4 and SP1 isoforms were overexpressed up to 206% and 148% on the cell surface 6 days post transduction by viral delivery, this CADM1 overexpression did not change adhesion to ASM. However, increased levels of SP4 isoform increased mast cell adhesion to LF by 37%. Thus, CADM1 isoforms affected adhesion to LF differently. The CADM1 counter-receptors, examined by Western blotting, are nectin-3 on ASM and CADM1 +nectin-3 on LF . Conclusions CADM1 contributes significantly to mast cell adhesion to ASM and LF . Mast cell adhesion to ASM is likely to be limited by the number of counter-receptors on ASM. In contrast, mast cell adhesion to LF is determined by the levels of SP4 isoform on mast cells. Increased levels of other CADM1 isoforms do not increase adhesion to LF . It is likely that increased levels of longer SP1 and SP6 isoforms in proportion to the CADM1 pool decrease mast cell adhesion to LF . [ABSTRACT FROM AUTHOR]