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Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) ( P  < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss ( P  = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P  = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo , SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P  = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

The mechanisms driving corticosteroid insensitivity in asthma are still unclear although evidence points toward a potential role of lung mast cells. Indeed, a number of in vitro studies using various cell types showed that different mediators produced by activated mast cells, including cytokines, have the capacity to interfere with the therapeutic action of corticosteroids. In patients with severe allergic refractory asthma, the anti-IgE monoclonal antibody (mAb), Omalizumab, has been shown to be associated with a marked reduction in inhaled and systemic use of corticosteroids, further suggesting a key role of mast cells in the poor response of patients to these drugs. The present chapter will discuss the possible underlying mechanisms by which mast cells could contribute to reducing corticosteroid sensitivity seen in patients with severe asthma.

The purpose of this study was to investigate whether growth factors produced by activated human lung mast cells (HLMCs) impair β 2 -adrenoceptor (β 2 -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 β 2 -AR response was reproduced by treating ASM cells with TGF-β1 or fibroblast growth factor (FGF)-2, which induced β 2 -AR phosphorylation at tyrosine residues Tyr 141 and Tyr 350 , 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 β 2 -AR phosphorylation at both Tyr 141 and Tyr 350 and a reduced relaxant response to albuterol. This study shows for the first time that abnormal β 2 -AR phosphorylation/function in ASM cells that is induced rapidly by HLMC-derived growth factors, is present constitutively in cells from severe asthmatics., (© 2018 British Society for Immunology.)

Fibrosis is a medical condition characterized by an excessive deposition of extracellular matrix compounds such as collagen in tissues. Fibrotic lesions are present in many diseases and can affect all organs. The excessive extracellular matrix accumulation in these conditions can often have serious consequences and in many cases be life-threatening. A typical event seen in many fibrotic conditions is a profound accumulation of mast cells (MCs), suggesting that these cells can contribute to the pathology. Indeed, there is now substantialv evidence pointing to an important role of MCs in fibrotic disease. However, investigations from various clinical settings and different animal models have arrived at partly contradictory conclusions as to how MCs affect fibrosis, with many studies suggesting a detrimental role of MCs whereas others suggest that MCs can be protective. Here, we review the current knowledge of how MCs can affect fibrosis., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We describe two methods to study CRAC channel function in human lung mast cells. Both methods involve suppression of endogenous channel function. In the first we use Orai-targeting shRNAs to knock down Orai channel mRNA transcripts. In the second we overexpress dominant-negative mutants of the three members of the Orai channel family. To overcome the poor transfection efficiency of mast cells, we employ an adenoviral delivery system for cell transduction. Knockdown of CRAC channel transcripts is assessed initially using quantitative RT-PCR. We describe an assay for β-hexosaminidase release as a measure of mast cell degranulation to assess the effect of overexpression of dominant-negative mutants.

β2-adrenoceptor agonists, often used in combination with corticosteroids, have been extensively used for the treatment of asthma. However, concerns have been raised regarding their adverse effects and safety including poor asthma control, life-threatening exacerbations, exacerbations that often require hospitalization, and asthma-related deaths. The question as to whether these adverse effects relate to the loss of their bronchoprotective action remains an interesting possibility. In the chapter, we will review the experimental evidence that describes the different potential factors and associated mechanisms that can blunt the therapeutic action of β2-adrenoceptor agonists in asthma. We show here evidence that various key inflammatory cytokines, growth factors, some respiratory viruses, certain allergens, unknown factors present in serum from atopic asthmatics have the capacity to impair β2-adrenoceptor function in airway smooth muscle, the main target of these drugs. More importantly, we present our latest research describing the role played by mast cells in impairing β2-adrenoceptor function. Although no definitive conclusion could be made regarding the implication of one single mechanism, receptor uncoupling, or receptor desensitization due to phosphorylation represents the main inhibitory pathways associated with a loss of β2-adrenoceptor function in airway smooth muscle. Targeting the pathways leading to β2-adrenoceptor dysfunction will likely provide novel therapies to improve the efficacy of β2-agonists in asthma., (© 2017 Elsevier Inc. All rights reserved.)

Mast cell infiltration of tumour islets represents a survival advantage in non-small cell lung cancer (NSCLC). The phenotype and activation status of these mast cells is unknown. We investigated the mast cell phenotype in terms of protease content (tryptase-only [MC T ], tryptase + chymase [MC TC ]) and tumour necrosis factor-alpha (TNFα) expression, and extent of degranulation, in NSCLC tumour stroma and islets. Surgically resected tumours from 24 patients with extended survival (ES; mean survival 86.5 months) were compared with 25 patients with poor survival (PS; mean survival 8.0 months) by immunohistochemistry. Both MC T and MC TC in tumour islets were higher in ES (20.0 and 5.6 cells/mm 2 respectively) compared to PS patients (0.0 cells/mm 2 ) (p < 0.0001). Both phenotypes expressed TNFα in the islets and stroma. In ES 44% of MC T and 37% of MC TC expressed TNFα in the tumour islets. MC T in the ES stroma were more degranulated than in those with PS (median degranulation index = 2.24 versus 1.73 respectively) (p = 0.0022), and ES islet mast cells (2.24 compared to 1.71, p < 0.0001). Since both MC T and MC TC infiltrating tumour islets in ES NSCLC patients express TNFα, the cytotoxic activity of this cytokine may confer improved survival in these patients. Manipulating mast cell microlocalisation and functional responses in NSCLC may offer a novel approach to the treatment of this disease.

Mast cells (MCs) are present in connective tissue and at mucosal surfaces in all classes of vertebrates. In health, they contribute to tissue homeostasis, host defense, and tissue repair via multiple receptors regulating the release of a vast stockpile of proinflammatory mediators, proteases, and cytokines. However, these potentially protective cells are a double-edged sword. When there is a repeated or long-term stimulus, MC activation leads to tissue damage and dysfunction. Accordingly, MCs are implicated in the pathophysiologic aspects of numerous diseases covering all organs. Understanding the biology of MCs, their heterogeneity, mechanisms of activation, and signaling cascades may lead to the development of novel therapies for many diseases for which current treatments are lacking or are of poor efficacy. This review will focus on updates and developments in MC biology and their clinical implications, with a particular focus on their role in respiratory diseases., (Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Mast cells and their activation contribute to lung health via innate and adaptive immune responses to respiratory pathogens. They are also involved in the normal response to tissue injury. However, mast cells are involved in disease processes characterized by inflammation and remodeling of tissue structure. In these diseases mast cells are often inappropriately and chronically activated. There is evidence for activation of mast cells contributing to the pathophysiology of asthma, pulmonary fibrosis, and pulmonary hypertension. They may also play a role in chronic obstructive pulmonary disease, acute respiratory distress syndrome, and lung cancer. The diverse mechanisms through which mast cells sense and interact with the external and internal microenvironment account for their role in these diseases. Newly discovered mechanisms of redistribution and interaction between mast cells, airway structural cells, and other inflammatory cells may offer novel therapeutic targets in these disease processes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mast cells are major effector cells of inflammation and there is strong evidence that mast cells play a significant role in asthma pathophysiology. There is also a growing body of evidence that mast cells contribute to other inflammatory and fibrotic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. This review discusses the role that mast cells play in airway diseases and highlights how mast cell microlocalisation within specific lung compartments and their cellular interactions are likely to be critical for their effector function in disease., (Published by Elsevier B.V.)

Mast cells (MCs) play a central role in tissue homoeostasis, sensing the local environment through numerous innate cell surface receptors. This enables them to respond rapidly to perceived tissue insults with a view to initiating a co-ordinated programme of inflammation and repair. However, when the tissue insult is chronic, the ongoing release of multiple pro-inflammatory mediators, proteases, cytokines and chemokines leads to tissue damage and remodelling. In asthma, there is strong evidence of ongoing MC activation, and their mediators and cell-cell signals are capable of regulating many facets of asthma pathophysiology. This article reviews the evidence behind this., (© 2015 John Wiley & Sons Ltd.)

Human lung mast cells (HLMCs) play a central role in asthma pathogenesis through their relocation to the airway smooth muscle (ASM) bundles. β2 adrenoceptor (β2-AR)-agonists are used to relieve bronchoconstriction in asthma, but may reduce asthma control, particularly when used as monotherapy. We hypothesized that HLMC and human ASM cell (HASMC) responsiveness to β2-AR agonists would be attenuated when HLMCs are in contact with HASMCs. Cells were cultured in the presence of the short-acting β2-agonist albuterol, and the long-acting β2-agonists formoterol and olodaterol. Constitutive and FcεRI-dependent HLMC histamine release, HASMC contraction, and β2-AR phosphorylation at Tyr(350) were assessed. Constitutive HLMC histamine release was increased in HLMC-HASMC coculture and this was enhanced by β2-AR agonists. Inhibition of FcεRI-dependent HLMC mediator release by β2-agonists was greatly reduced in HLMC-HASMC coculture. These effects were reversed by neutralization of stem cell factor (SCF) or cell adhesion molecule 1 (CADM1). β2-AR agonists did not prevent HASMC contraction when HLMCs were present, but this was reversed by fluticasone. β2-AR phosphorylation at Tyr(350) occurred within 5 min in both HLMCs and HASMCs when the cells were cocultured, and was inhibited by neutralizing SCF or CADM1. HLMC interactions with HASMCs via CADM1 and Kit inhibit the potentially beneficial effects of β2-AR agonists on these cells via phosphorylation of the β2-AR. These results may explain the potentially adverse effects of β2-ARs agonists when used for asthma therapy. Targeting SCF and CADM1 may enhance β2-AR efficacy, particularly in corticosteroid-resistant patients., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Orai/CRACM1 ion channels provide the major Ca(2+) influx pathway for FcεRI-dependent human lung mast cell (HLMC) mediator release. The Ca(2+)-activated K(+) channel KCa3.1 modulates Ca(2+) influx and the secretory response through hyperpolarisation of the plasma membrane. We hypothesised that there is a close functional and spatiotemporal interaction between these Ca(2+)- and K(+)-selective channels., Results: Activation of FcεRI-dependent HLMC KCa3.1 currents was dependent on the presence of extracellular Ca(2+), and attenuated in the presence of the selective Orai blocker GSK-7975A. Currents elicited by the KCa3.1 opener 1-EBIO were also attenuated by GSK-7975A. The Orai1 E106Q dominant-negative mutant ablated 1-EBIO and FcεRI-dependent KCa3.1 currents in HLMCs. Orai1 but not Orai2 was shown to co-immunoprecipitate with KCa3.1 when overexpressed in HEK293 cells, and Orai1 and KCa3.1 were seen to co-localise in the HEK293 plasma membrane using confocal microscopy., Conclusion: KCa3.1 activation in HLMCs is highly dependent on Ca(2+) influx through Orai1 channels, mediated via a close spatiotemporal interaction between the two channels.

Background: Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells., Methods: We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins., Results: Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling., Conclusion: Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.

MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1-enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases., (© 2015 Cruse et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

Background: The influx of extracellular Ca(2+) into mast cells is critical for the FcεR1-dependent release of preformed granule-derived mediators and newly synthesised autacoids and cytokines. The Orai(CRACM) ion channel family provide the major pathway through which this Ca(2+) influx occurs. However the individual role of each of the three members of the Orai channel family in Ca(2+) influx and mediator release has not been defined in human mast cells., Objective: To assess whether there might be value in targeting individual Orai family members for the inhibition of FcεRI-dependent human lung mast cells (HLMC) mediator release., Methods: We used an adenoviral delivery system to transduce HLMCs with shRNAs targeted against Orai1 and Orai2 or with cDNAs directing the expression of dominant-negative mutations of the three known Orai channels., Results: shRNA-mediated knockdown of Orai1 resulted in a significant reduction of approximately 50% in Ca(2+) influx and in the release of β-hexosaminidase (a marker of degranulation) and newly synthesized LTC4 in activated HLMCs. In contrast shRNA knockdown of Orai2 resulted in only marginal reductions of Ca(2+) influx, degranulation and LTC4 release. Transduced dominant-negative mutants of Orai1, -2 and -3 markedly reduced Orai currents and completely inhibited HLMC degranulation suggesting that Orai channels form heteromultimers in HLMCs, and that Orai channels comprise the dominant Ca(2+) influx pathway following FceRI-dependent HLMC activation. Inhibition of Orai currents did not alter HLMC survival. In addition we observed a significant down-regulation of the level of CRACM3 mRNA transcripts together with a small increase in the level of CRACM1 and CRACM2 transcripts following a period of sustained HLMC activation., Conclusion and Clinical Relevance: Orai1 plays an important role in Ca(2+) influx and mediator release from HLMCs. Strategies which target Orai1 will effectively inhibit FcεRI-dependent HLMC activation, but spare off-target inhibition of Orai2 in other cells and body systems.

Human linkage analyses have implicated the MS4A2-containing gene locus (encoding FcεRIβ) as a candidate for allergy susceptibility. We have identified a truncation of FcεRIβ (t-FcεRIβ) in humans that contains a putative calmodulin-binding domain and thus, we sought to identify the role of this variant in mast cell function. We determined that t-FcεRIβ is critical for microtubule formation and degranulation and that it may perform this function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals. Mutagenesis studies suggest that calmodulin binding to t-FcεRIβ in the presence of Ca2+ could be critical for t-FcεRIβ function. In addition, gene targeting of t-FcεRIβ attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals. Therefore, t-FcεRIβ mediates Ca2+ -dependent microtubule formation, which promotes degranulation and cytokine release. Because t-FcεRIβ has this critical function, it represents a therapeutic target for the downregulation of allergic inflammation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mast cells play a central role in the pathophysiology of asthma and related allergic conditions. Mast cell activation leads to the degranulation of preformed mediators such as histamine and the secretion of newly synthesised proinflammatory mediators such as leukotrienes and cytokines. Excess release of these mediators contributes to allergic disease states. An influx of extracellular Ca2+ is essential for mast cell mediator release. From the Ca2+ channels that mediate this influx, to the K+ , Cl- and transient receptor potential channels that set the cell membrane potential and regulate Ca2+ influx, ion channels play a critical role in mast cell biology. In this review we provide an overview of our current knowledge of ion channel expression and function in mast cells with an emphasis on how channels interact to regulate Ca2+ signalling., (© 2012 Blackwell Publishing Ltd.)

Background: Mast cells (MCs) play a central role in the development of many diseases including asthma and pulmonary fibrosis. Interactions of human lung mast cells (HLMCs) with human airway smooth muscle cells (HASMCs) are partially dependent on adhesion mediated by cell adhesion molecule-1 (CADM1), but the adhesion mechanism through which HLMCs interact with human lung fibroblasts (HLFs) is not known. CADM1 is expressed as several isoforms (SP4, SP1, SP6) in HLMCs, with SP4 dominant. These isoforms differentially regulate HLMC homotypic adhesion and survival., Objective: In this study we have investigated the role of CADM1 isoforms in the adhesion of HLMCs and HMC-1 cells to primary HASMCs and HLFs., Methods: CADM1 overexpression or downregulation was achieved using adenoviral delivery of CADM1 short hairpin RNAs or isoform-specific cDNAs respectively., Results: Downregulation of CADM1 attenuated both HLMC and HMC-1 adhesion to both primary HASMCs and HLFs. Overexpression of either SP1 or SP4 isoforms did not alter MC adhesion to HASMCs, whereas overexpression of SP4, but not SP1, significantly increased both HMC-1 cell and HLMC adhesion to HLFs. The expression level of CADM1 SP4 strongly predicted the extent of MC adhesion; linear regression indicated that CADM1 accounts for up to 67% and 32% of adhesion to HLFs for HMC-1 cells and HLMCs, respectively. HLFs supported HLMC proliferation and survival through a CADM1-dependent mechanism. With respect to CADM1 counter-receptor expression, HLFs expressed both CADM1 and nectin-3, whereas HASMCs expressed only nectin-3., Conclusion and Clinical Relevance: Collectively these data indicate that the CADM1 SP4 isoform is a key receptor mediating human MC adhesion to HASMCs and HLFs. The differential expression of CADM1 counter-receptors on HLFs compared to HASMCs may allow the specific targeting of either HLMC-HLF or HLMC-HASMC interactions in the lung parenchyma and airways.

Cell adhesion molecule 1 (CADM1) is implicated in the pathogenesis of several diseases and is responsible for adhesion and survival of mast cells (MCs). Differential expression of CADM1 isoforms was found in different species. We previously cloned SP4, SP1, SP6 and a dysfunctional isoform from human lung MCs (HLMCs) and the MC line HMC-1. The aim of this study was to identify all isoforms expressed in human MCs. The functional isoforms SP4, SP1, SP6 and SP3, with alternative splicing between exons 7/11, were detected in human MCs by RT-PCR. Two dysfunctional isoforms with alternative splicing of cryptic exons A and B between exons 1/2, leading to premature termination of translation, were found in ∼40% of MC specimens. Sequencing of genomic DNA showed that splicing of cryptic exon B did not result from specific SNPs within this exon or its putative splice branch point. Highly glycosylated CADM1 (∼105 kDa) was detected by western blotting, but an extracellular domain (∼95 kDa) was found only in the culture medium from HLMCs, but not HMC-1 cells, indicating differential protein expression. Transfection of SP1 and SP6, but not SP4, reduced adhesion of HMC-1 cells to human lung fibroblasts but not airway smooth muscle cells. Hence, dysfunctional and functional CADM1 isoforms are found in human MCs. The longer SP1 and SP6 were most evident in differentiated HLMCs and displayed differential adhesion compared to SP4. These multiple isoforms are likely to contribute to MC function in both health and disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cell adhesion molecule 1 (CADM1), expressed by human lung mast cells (HLMCs), mediates their adhesion to airway smooth muscle (ASM), and contributes to ASM-dependent HLMC proliferation and survival. CADM1 is expressed in alternatively spliced isoforms, but those present in HLMCs and their function are not known. We cloned three functional and one cryptic non-functional isoform with alternative splicing between exons 7/11 and 1/2, respectively, from HLMCs and human MC lines (HMC-1 and LAD2). Differentiated HLMCs and LAD2 cells expressed the functional isoform SP4 containing exons 7/8/11 (~80% of clones), as well as SP1 (exons 7/8/9/11) and a novel SP6 (exons 7/8/9/10/11). In contrast, immature HMC-1 cells expressed only functional SP4. SP4 overexpression in HMC-1 cells and HLMCs augmented homotypic adhesion to a greater extent than SP1 in various conditions. In contrast, CADM1 downregulation abolished homotypic adhesion, indicating that CADM1 is the sole receptor mediating mast cell aggregation. CADM1-mediated adhesion was enhanced by the presence of cell survival factors. SP1 overexpression in HMC-1 cells compromised survival compared to SP4 overexpression or control. CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs. This coincided with decreased basal Kit levels in HLMCs. In summary, human MCs express multiple CADM1 isoforms which exhibit differential regulation of survival and homotypic adhesion. The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.

Background: Influx of extracellular Ca(2+) into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. However, the identity of the ion channels underlying this Ca(2+) influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca(2+) release-activated Ca(2+) current are candidates., Objectives: To investigate the expression and function of CRACM channels in HLMCs., Methods: CRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus., Results: CRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detectable in HLMCs. A CRACM-like current was detected following FcεRI-dependent HLMC activation and also in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca(2+)-selective current obtained under both conditions was blocked by 10 μM La(3+) and Gd(3+), known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers-GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca(2+) influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C(4), and cytokines (IL-5/-8/-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue., Conclusions: The presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca(2+) influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are therefore a potential therapeutic target in the treatment of asthma and related allergic diseases., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Introduction: Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism., Methods: HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures., Results: Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G(0)/G(i) receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolving D1 and D2 and lipoxin A(4) attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants., Conclusions: Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G(0)/G(i) receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.

Background: Mast cells (MCs) and eosinophils (Eos) are the key effector cells of the allergic reaction. Although classically associated with different stages of the response, the cells co-exist in the inflamed tissue in the late and chronic phases in high numbers and are likely to cross-talk. While some mediators of MCs are known to affect Eos biology and vice versa, paracrine and physical interplay between the two cells has not been described yet. We aimed to investigate whether intercellular MC-Eos communication could take place in the allergic response and exert functional bidirectional changes on the cells., Methods: Tissue sections from various allergic disorders were specifically stained for both cells. Human cord blood-derived MCs and peripheral blood Eos, co-cultured under different conditions, were studied by advanced microscopy and flow cytometry., Results: Several co-localized MC-Eos pairs were detected in human nasal polyps and asthmatic bronchi, as well in mouse atopic dermatitis. In vitro, MCs and Eos formed stable conjugates at high rates, with clear membrane contact. In the presence of MCs, Eos were significantly more viable under several co-culture conditions and at both IgE-activated and steroid-inhibited settings. MC regulation of Eos survival required communication through soluble mediators but was even more dependent on physical cell-cell contact., Conclusions: Our findings provide the first evidence for a complex network of paracrine and membrane interactions between MCs and Eos. The prosurvival phenotype induced by this MC-Eos interplay may be critical for sustaining chronic allergic inflammation., (© 2010 John Wiley & Sons A/S.)

We investigated whether adenosine, a potent contributor to the regulation of pulmonary function, can modulate human lung mast cell (HLMC) fibrinolytic activity. Tissue plasminogen activator (tPA) activity and tPA transcript expression levels from a human mast cell line (HMC-1) and HLMC were monitored following adenosine application. Adenosine potentiated mast cell tPA activity and tPA gene expression in a dose-dependent manner. Adenosine effects were abolished in the presence of adenosine deaminase. HMC-1 cells and HLMC predominantly expressed adenosine A(2A) and A(2B) receptor transcripts (A(2B) ≈ A(2A) > A(3) >> A(1)). Pharmacological and signaling studies suggest that the A(2A) receptor is the major subtype accounting for adenosine-induced mast cell tPA activity. Finally, the supernatant from HMC-1 cells and HLMC treated with adenosine (for 24 h) significantly increased fibrin clot lysis, whereas ZM241385, an A(2A) receptor antagonist, abolished this effect. To our knowledge, this study provides the first data to demonstrate the potentiating effect of adenosine on mast cell tPA activity and fibrin clot lysis.

Mast cells, traditionally regarded as effector cells of the immune system, have more recently been demonstrated to be key figures in initiating, developing and sustaining complex pathophysiological processes underlying asthma and other allergic diseases. Asthma is characterised by airway inflammation alongside a disturbance to airway physiology manifesting as variable airflow obstruction and airway hyper-responsiveness (AHR). Evidence has emerged that mast cells influence airway function by forming close intercellular relationships with different structural components of the airway wall. In asthma, mast cells are seen to localise to the airway epithelium, to mucous glands and to the airway smooth muscle (ASM). It is mast cell-ASM interaction that is most fundamental to the asthma phenotype and many mast cell mediators have been demonstrated to have important effects on ASM function. In asthma, alongside the inflammatory and physiological changes, structural changes occur to the airway wall in the form of denudation of the epithelium, goblet cell and mucous gland hyperplasia, subepithelial fibrosis, abnormal extracellular matrix (ECM) deposition, vascular proliferation and increased ASM mass. There are many ways in which mast cells can contribute to these structural changes through direct cell to cell communication and more indirectly through mediator release. Mast cells exhibit an array of diverse functions and roles and are fundamental to our current understanding of asthma pathogenesis including severe asthma. Novel targeting of mast cells and their mediators therefore should offer significant therapeutic potential in the treatment of asthma.

Mast cells play an important role in the lung in both health and disease. Their primary role is to initiate an appropriate program of inflammation and repair in response to tissue damage initiated by a variety of diverse stimuli. They are important for host immunity against bacterial infection and potentially in the host immune response to non small cell lung cancer. In situations of ongoing tissue damage, the sustained release of numerous pro-inflammatory mediators, proteases and cytokines, contributes to the pathophysiology of lung diseases such as asthma and interstitial lung disease. A key goal is the development of treatments which attenuate adverse mast cell function when administered chronically to humans in vivo. Such therapies may offer a novel approach to the treatment of many life-threatening diseases.

Mast cell microlocalization to the airway smooth muscle (ASM) bundle is a key feature of asthma, but whether these mast cells have an altered phenotype is uncertain. In this paper, we report that in vivo, mast cells within the ASM bundle, in contrast to mast cells in the bronchial submucosa, commonly expressed fibroblast markers and the number of these cells was closely related to the degree of airway hyperresponsiveness. In vitro human lung mast cells and mast cell lines cultured with fibronectin or with primary human ASM cells acquired typical fibroblastic markers and morphology. This differentiation toward a fibroblastoid phenotype was mediated by ASM-derived extracellular matrix proteins, independent of cell adhesion molecule-1, and was attenuated by α5β1 blockade. Fibroblastoid mast cells demonstrated increased chymase expression and activation with exaggerated spontaneous histamine release. Together these data indicate that in asthma, ASM-derived extracellular matrix proteins mediate human mast cell transition to a fibroblastoid phenotype, suggesting that this may be pivotal in the development of airway dysfunction in asthma.

Mast cells contribute to allergy through IgE-dependent activation via the high-affinity IgE receptor FcεRI. The role of the FcεRIβ chain (MS4A2) in mast cell function is not understood fully, although it serves to amplify FcεRI-dependent signaling. We demonstrate the expression of a novel MS4A2 truncation lacking exon 3 in human mast cells termed MS4A2(trunc). MS4A2(trunc) gene expression was regulated negatively by the mast cell growth factor stem cell factor (SCF), and its expression was not detected in the SCF receptor gain-of-function human mast cell line HMC-1. Unlike MS4A2, MS4A2(trunc) did not traffic to the cytoplasmic membrane but instead was associated with the nuclear membrane. Overexpression of MS4A2(trunc) induced human lung mast cell death and profoundly inhibited HMC-1 cell proliferation by inducing G(2)-phase cell cycle arrest and apoptosis. Thus, we have identified a novel splice variant of MS4A2 that might be important in the regulation of human mast cell proliferation and survival. This finding demonstrates that the MS4A2 gene has multiple roles, extending beyond the regulation of acute allergic responses. By understanding the mechanisms regulating its function, it might be possible to induce its expression in mast cells in vivo, which could lead to better treatments for diseases such as mastocytosis and asthma.

Mast cells are emerging as contributors to innate immunity. Mouse mast cells have a pivotal role in protection against bacterial infection, and human cord blood-derived mast cells reduce bacterial viability in culture. The objectives of this study were to determine whether human lung mast cells (HLMCs) might be protective against pneumococcal lung infection through direct antimicrobial activity. Tissue-derived HLMCs and the human mast cell lines HMC-1 and LAD2 were cocultured with wild-type and mutant pneumococci, and viability and functional assays were performed. Mast cells were also stimulated with purified pneumolysin. HLMCs killed wild-type serotype-2 (D39) pneumococci in coculture but had no effect on an isogenic pneumolysin-deficient (PLN-A) pneumococcus. D39 wild-type, but not PLN-A pneumococci, induced the release of leukotriene C4 from human mast cells in a dose-dependent manner, which was not accompanied by histamine release. Stimulation of mast cells with sublytic concentrations of purified pneumolysin replicated this effect. Furthermore, pneumolysin induced the release of the cathelicidin LL-37 from HLMCs, purified LL-37 reduced pneumococcal viability, and neutralizing Ab to LL-37 attenuated mast cell-dependent pneumococcal killing. In addition, at high concentrations, all pneumococcal strains tested reduced HLMC viability through a combination of pneumolysin and H2O2-dependent mechanisms. HLMCs exhibit direct antimicrobial activity to pneumococci through their activation by pneumolysin. This antimicrobial activity is mediated, in part, by the release of LL-37 from HLMCs. This suggests that mast cells provide an early warning system and potentially limit pneumococcal dissemination early in the course of invasive pulmonary pneumococcal disease.

Background: In human subjects platelet-activating factor (PAF) concentrations are markedly increased in the plasma after anaphylactic reactions, and these correlate strongly with the severity of the response. The mechanism for the systemic spread of mast cell (MC) activation in anaphylaxis is often assumed to relate to the hematogenous spread of allergen, but this is implausible, and amplification mechanisms need to be considered., Objective: We have investigated the ability of PAF to induce human MC degranulation using skin, lung, and peripheral blood (PB)-derived cultured MCs and the signaling pathways activated in PB-derived MCs in response to PAF., Methods: The expression of PAF receptor was investigated by means of RT-PCR and Western blot analysis. Cell-signaling pathways in PB-derived MCs in response to PAF were investigated by analyzing the effect of various inhibitors and the silencing of phospholipase C (PLC) mRNA on PAF-mediated histamine release., Results: We show for the first time that PAF induces histamine release from human lung MCs and PB-derived MCs but not skin MCs. Activation of PAF receptor-coupled G(alphai) leads to degranulation through PLCgamma1 and PLCbeta2 activation in human MCs. PAF-induced degranulation was rapid, being maximal at 5 seconds, and was partially dependent on extracellular Ca(2+)., Conclusion: Our findings provide a mechanism whereby PAF mediates an amplification loop for MC activation in the generation of anaphylaxis., (Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

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.

Background: Mast cells contribute to the pathophysiology of asthma with the sustained release of both preformed and newly generated mediators in response to allergens and other diverse stimuli. Stem cell factor (SCF) is the key human mast cell growth factor, but also primes mast cells for mediator release. SCF expression is markedly increased in asthmatic airways. Short-acting beta(2)-adrenoceptor drugs such as albuterol inhibit human lung mast cell (HLMC) degranulation in vitro in the absence of SCF, but their effect in the presence of SCF is not known., Objective: The aim of this study was to elucidate the effects of albuterol on HLMC function in the presence of SCF., Methods: Mediator release and K(Ca)3.1 ion channel activity were analyzed in purified HLMC. Intracellular signalling and beta(2)-adrenoceptor phosphorylation and internalization were analyzed in the HMC-1 human mast cell line., Results: beta(2)-Adrenoceptor agonist-dependent inhibition of K(Ca)3.1 ion channels and HLMC mediator release was markedly attenuated in the presence of SCF. Remarkably, albuterol actually potentiated IgE-induced histamine release in a dose-dependent manner when both SCF and IgE were present. These effects were related to the SCF-dependent phosphorylation of Tyr350 on the beta(2)-adrenoceptor with immediate uncoupling of the receptor followed by receptor internalization., Conclusion: The potentially beneficial effects of beta(2)-adrenoceptor agonists in asthmatic airways may be blunted as a result of the high concentrations of SCF present., (Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background and Purpose: P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs)., Experimental Approach: Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools., Key Results: RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 microM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by alphabetamethylene ATP (10 microM, 94% cells, n= 31) and was antagonized in LAD2 cells by NF 449 (1 microM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 microM). A P2X7-like non-desensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n= 24; 54% HLMCs, n= 24) which was antagonized by AZ11645373 (1 microM). P2X7-like responses were also evoked in LAD2 cells by 2'(3')-0-(4-benzoylbenzoyl)ATP (300 microM). A P2X4-like current was evoked by 100 microM ATP (80% LAD2, n= 10; 21% HLMCs, n= 29), the amplitude and duration of which was potentiated by ivermectin (3 microM)., Conclusion and Implications: Our data confirmed the presence of functional P2X1, P2X4 and P2X7 receptors in LAD2 cells and HLMCs.

Asthma is a major cause of morbidity and mortality worldwide. It is characterized by airway dysfunction and inflammation. A key determinant of the asthma phenotype is infiltration of airway smooth muscle bundles by activated mast cells. We hypothesized that interactions between these cells promotes airway smooth muscle differentiation into a more contractile phenotype. In vitro coculture of human airway smooth muscle cells with beta-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-beta1 secretion, alpha-smooth muscle actin expression and agonist-provoked contraction. This promotion to a more contractile phenotype was inhibited by both the serine protease inhibitor leupeptin and TGF-beta1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-beta1 in response to activation by mast cell beta-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of alpha-smooth muscle actin expression was strongly related to the number of mast cells within or adjacent to an airway smooth muscle bundle. These findings suggest that mast cell localization in the airway smooth muscle bundle promotes airway smooth muscle cell differentiation into a more contractile phenotype, thus contributing to the disordered airway physiology that characterizes asthma.

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 beta-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 beta-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11, and beta-tryptase inhibited CCL11-mediated airway smooth muscle migration., Conclusions: CCL11 mediates airway smooth muscle migration. However co-culture with beta-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.

Human lung mast cells (HLMC) express the Ca(2+)-activated K(+) channel K(Ca)3.1, which plays a crucial role in their migration to a variety of diverse chemotactic stimuli. K(Ca)3.1 activation is attenuated by the beta(2)-adrenoceptor and the adenosine A(2A) receptor through a G(s)-coupled mechanism independent of cyclic AMP. Prostaglandin E(2) promotes degranulation and migration of mouse bone marrow-derived mast cells through the G(i)-coupled EP(3) prostanoid receptor, and induces LTC(4) and cytokine secretion from human cord blood-derived mast cells. However, PGE(2) binding to the G(s)-coupled EP(2) receptor on HLMC inhibits their degranulation. We show that EP(2) receptor engagement closes K(Ca)3.1 in HLMC. The EP(2) receptor-specific agonist butaprost was more potent than PGE(2) in this respect, and the effects of both agonists were reversed by the EP(2) receptor antagonist AH6809. Butaprost markedly inhibited HLMC migration induced by chemokine-rich airway smooth muscle-conditioned media. Interestingly, PGE(2) alone was chemotactic for HLMC at high concentrations (1 microM), but was a more potent chemoattractant for HLMC following EP(2) receptor blockade. Therefore, the G(s)-coupled EP(2) receptor closes K(Ca)3.1 in HLMC and attenuates both chemokine- and PGE(2)-dependent HLMC migration. EP(2) receptor agonists with K(Ca)3.1 modulating function may be useful for the treatment of mast cell-mediated disease.

The microlocalization of mast cells within specific tissue compartments is thought to be critical for the pathophysiology of many diverse diseases. This is particularly evident in asthma where they localize to the airway smooth muscle (ASM) bundles. Mast cells are recruited to the ASM by numerous chemoattractants and adhere through CADM1, but the functional consequences of this are unknown. In this study, we show that human ASM maintains human lung mast cell (HLMC) survival in vitro and induces rapid HLMC proliferation. This required cell-cell contact and occurred through a cooperative interaction between membrane-bound stem cell factor (SCF) expressed on ASM, soluble IL-6, and CADM1 expressed on HLMC. There was a physical interaction in HLMC between CADM1 and the SCF receptor (CD117), suggesting that CADM1-dependent adhesion facilitates the interaction of membrane-bound SCF with its receptor. HLMC-ASM coculture also enhanced constitutive HLMC degranulation, revealing a novel smooth muscle-driven allergen-independent mechanism of chronic mast cell activation. Targeting these interactions in asthma might offer a new strategy for the treatment of this common disease.

Background: Mast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, via IgE in the absence of antigen. In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival., Methods: Purified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA., Results: IgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells., Conclusion: These data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect.

Mast cells play a significant role in the pathophysiology of many diverse diseases such as asthma and pulmonary fibrosis. Ca2+ influx is essential for mast cell degranulation and release of proinflammatory mediators, while Mg2+ plays an important role in cellular homeostasis. The channels supporting divalent cation influx in human mast cells have not been identified, but candidate channels include the transient receptor potential melastatin (TRPM) family. In this study, we have investigated TRPM7 expression and function in primary human lung mast cells (HLMCs) and in the human mast cell lines LAD2 and HMC-1, using RT-PCR, patch clamp electrophysiology, and RNA interference. Whole cell voltage-clamp recordings revealed a nonselective cation current that activated spontaneously following loss of intracellular Mg2+. The current had a nonlinear current-voltage relationship with the characteristic steep outward rectification associated with TRPM7 channels. Reducing external divalent concentration from 3 to 0.3 mM dramatically increased the size of the outward current, whereas the current was markedly inhibited by elevated intracellular Mg2+ (6 mM). Ion substitution experiments revealed cation selectivity and Ca2+ permeability. RT-PCR confirmed the presence of mRNA for TRPM7 in HLMC, LAD2, and HMC-1 cells. Adenoviral-mediated knockdown of TRPM7 in HLMC with short hairpin RNA and in HMC-1 with short interfering RNA markedly reduced TRPM7 currents and induced cell death, an effect that was not rescued by raising extracellular Mg2+. In summary, HLMC and human mast cell lines express the nonselective cation channel TRPM7 whose presence is essential for cell survival.

Human lung mast cells (HLMC) express the Ca2+-activated K+ channel KCa3.1, which opens following IgE-dependent activation. This hyperpolarises the cell membrane and potentiates both Ca2+ influx and degranulation. In addition, blockade of KCa3.1 profoundly inhibits HLMC migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the beta2adrenoceptor through a Galphas-coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Galphas-coupled A2A and A2B adenosine receptors, respectively. We show that at concentrations that inhibit HLMC degranulation (10(-5)-10(-3) M), adenosine closes KCa3.1 both dose-dependently and reversibly. KCa3.1 suppression by adenosine was reversed partially by the selective adenosine A2A receptor antagonist ZM241385 but not by the A2B receptor antagonist MRS1754, and the effects of adenosine were mimicked by the selective A2A receptor agonist CGS21680. Adenosine also opened a depolarising current carried by non-selective cations. As predicted from the role of KCa3.1 in HLMC migration, adenosine abolished HLMC chemotaxis to asthmatic airway smooth muscle-conditioned medium. In summary, the Galphas-coupled adenosine A2A receptor closes KCa3.1, providing a clearly defined mechanism by which adenosine inhibits HLMC migration and degranulation. A2A receptor agonists with channel-modulating function may be useful for the treatment of mast cell-mediated disease.

Rationale: Airway smooth muscle (ASM) hyperplasia is a feature of asthma, and increases with disease severity. We hypothesized that this results from migration of ASM or progenitors in response to chemokines derived from ASM or mast cells within the ASM bundle., Objectives: To examine expression of the chemokine receptor, CC chemokine receptor (CCR) 7, in vivo by ASM in patients with asthma and healthy control subjects, and by primary cultures of ASM and fibroblasts; to define expression of its ligands, CC chemokine ligand (CCL) 19 and CCL21, in bronchial biopsies, and primary cultures of ASM and mast cells; and to investigate CCR7's role in ASM migration and repair., Methods: ASM was isolated from bronchoscopy and resection tissue. Receptor and chemokine expression was examined by immunohistochemistry, immunofluorescence, flow cytometry, ELISA, and reverse transcriptase-polymerase chain reaction. CCR7 function was examined by intracellular calcium measurements, chemotaxis, wound healing assays, and measurement of cell proliferation., Measurements and Main Results: ASM, myofibroblasts, and fibroblasts expressed CCR7. CCL19, but not CCL21, was highly expressed in bronchial biopsies by mast cells and vessels in asthma of all severities, ASM in severe disease, and ex vivo ASM and mast cells. ASM CCR7 activation by CCL19-mediated intracellular calcium elevation and concentration-dependent migration, but not proliferation. Importantly, mast cell and ASM-derived CCL19 mediated ASM migration and repair., Conclusions: The CCL19/CCR7 axis may play an important role in the development of ASM hyperplasia in asthma.

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 K(Ca)3.1 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 K(Ca)3.1 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 microm pore size). Ion currents were measured using the single cell patch clamp technique., Results: K(Ca)3.1 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 alpha stimulated asthmatic ASM was markedly inhibited with both the non-selective K(Ca)3.1 blocker charybdotoxin and the highly specific K(Ca)3.1 blocker TRAM-34 in a dose dependent manner. Although K(Ca)3.1 blockade inhibits HLMC migration, K(Ca)3.1 is not opened by the chemotactic stimulus, suggesting that it must be involved downstream of the initial receptor-ligand interactions., Conclusions: Since modulation of K(Ca)3.1 can inhibit HLMC chemotaxis to diverse chemoattractants, the use of K(Ca)3.1 blockers such as TRAM-34 could provide new therapeutic strategies for mast cell mediated diseases such as asthma.

Background: The Th2 cytokine interleukin (IL)-13 is implicated in the development of various allergic diseases including asthma. The IL-13 receptor, IL-13Ralpha1, is expressed on most leukocytes, except T-cells. Evidence to support IL-13Ralpha1 expression on mast cells is limited., Methods: We investigated: (i) IL-13Ralpha1 expression by human lung mast cells (HLMC); (ii) the number of IL-13Ralpha1+ bronchial submucosal mast cells in subjects with asthma and normal controls and (iii) the effect of IL-13 priming on HLMC expression of high-affinity IgE receptor (FcepsilonRI), stem cell factor receptor (CD117), histamine release, proliferation, and survival., Results: Human lung mast cell expressed IL-13Ralpha1 mRNA. IL-13Ralpha1 was highly expressed on the surface HLMC (82+/-9%). Bronchial submucosal mast cell IL-13Ralpha1 expression was higher in asthmatics (86+/-2%) than normal controls (78+/-2%; P=0.015). IL-13 priming for 30 min did not increase HLMC histamine release, in the presence or absence of SCF or in response to IgE/anti-IgE activation. IL-13 priming for 5 days upregulated HLMC FcepsilonRI expression (22% increase in fluorescent intensity; P=0.003), increased histamine release following IgE/anti-IgE activation by 56% (P=0.03) and increased proliferation by 50% (P=0.003) without affecting cell survival or CD117 expression. The IL-13 specific neutralizing antibody CAT-354 inhibited all IL-13 mediated effects., Conclusion: Human lung mast cell express IL-13Ralpha1 and activation by IL-13 for 5 days increased FcepsilonRI expression and proliferation. Histamine release was not affected by short-term priming with IL-13, but was upregulated by priming for 5 days suggesting that this effect was mediated by the increased FcepsilonRI expression. These data support the view that targeting IL-13 may be beneficial in the treatment of asthma.