S. Mark Duffy

Functional Transient Receptor Potential Melastatin 7 Channels Are Critical for Human Mast Cell Survival

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.

Resting and Activation-Dependent Ion Channels in Human Mast Cells

The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca2+, K+, and Cl− conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca2+-activated K+ current exhibiting weak inward rectification, which polarized the cells to around −40 mV and a smaller outwardly rectifying Ca2+-independent Cl− conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca2+-activated K+ currents and Ca2+-independent Cl− currents developing in ∼50% of cells. In both cell types, the K+ current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca2+-activated K+ channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C4 release, indicating that the Ca2+-activated K+ current may enhance, but is not essential for, the release of these mediators.

Adenosine closes the K+ channel KCa3.1 in human lung mast cells and inhibits their migration via the adenosine A2A receptor

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 β2adrenoceptor through a Gαs‐coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Gαs‐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 Gαs‐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.

β2-Adrenoceptor regulation of the K+ channel iKCa1 in human mast cells

Human mast cells express the intermediate conductance Ca2+‐activated K+ channel iKCa1, which opens following IgE‐dependent activation. This results in cell membrane hyperpolarization and potentiation of both Ca2+ influx and degranulation. Mast cell activation is attenuated following exposure to β2‐adrenoceptor agonists such as salbutamol, an effect postulated to operate via intracellular cyclic AMP. In this study, we show that salbutamol closes iKCa1 in mast cells derived from human lung and peripheral blood. Salbutamol (1–10 µM) inhibited iKCa1 currents following activation with both anti‐IgE and the iKCa1 opener 1‐EBIO, and was reversed by removing salbutamol or by the addition of the selective β2‐adrenoceptor antagonist and inverse agonist ICI 118551. Interestingly, ICI 118551 consistently opened iKCa1 in quiescent cells, suggesting that constitutive β2‐receptor signaling suppresses channel activity. Manipulation of intracellular cAMP, Gαi, and Gαs demonstrates that the β2‐adrenergic effects are consistent with a membrane‐delimited mechanism involving Gαs. This is the first demonstration that gating of the iKCa1 channel is regulated by a G protein‐coupled receptor and provides a clearly defined mechanism for the mast cell “stabilizing” effect of β2‐agonists. Furthermore, the degree of constitutive β2‐receptor “tone” may control the threshold for human mast cell activation through the regulation of iKCa1.

CRACM/Orai ion channel expression and function in human lung mast cells

Background Influx of extracellular Ca2+ 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 Ca2+ influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca2+ release–activated Ca2+ 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 Ca2+-selective current obtained under both conditions was blocked by 10 μM La3+ and Gd3+, known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers—GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca2+ influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C4, 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 Ca2+ 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.

Engagement of the EP2 prostanoid receptor closes the K+ channel KCa3.1 in human lung mast cells and attenuates their migration†

Human lung mast cells (HLMC) express the Ca2+‐activated K+ channel KCa3.1, which plays a crucial role in their migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the β2‐adrenoceptor and the adenosine A2A receptor through a Gs‐coupled mechanism independent of cyclic AMP. Prostaglandin E2 promotes degranulation and migration of mouse bone marrow‐derived mast cells through the Gi‐coupled EP3 prostanoid receptor, and induces LTC4 and cytokine secretion from human cord blood‐derived mast cells. However, PGE2 binding to the Gs‐coupled EP2 receptor on HLMC inhibits their degranulation. We show that EP2 receptor engagement closes KCa3.1 in HLMC. The EP2 receptor‐specific agonist butaprost was more potent than PGE2 in this respect, and the effects of both agonists were reversed by the EP2 receptor antagonist AH6809. Butaprost markedly inhibited HLMC migration induced by chemokine‐rich airway smooth muscle‐conditioned media. Interestingly, PGE2 alone was chemotactic for HLMC at high concentrations (1 µM), but was a more potent chemoattractant for HLMC following EP2 receptor blockade. Therefore, the Gs‐coupled EP2 receptor closes KCa3.1 in HLMC and attenuates both chemokine‐ and PGE2‐dependent HLMC migration. EP2 receptor agonists with KCa3.1 modulating function may be useful for the treatment of mast cell‐mediated disease.

Functional KCa3.1 K+ channels are required for human fibrocyte migration.

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 KCa3.1 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 KCa3.1 ion-channel blockers. Results Human fibrocytes cultured from the peripheral blood of both healthy control subjects and asthmatic patients expressed robust KCa3.1 ion currents together with KCa3.1 mRNA and protein. Two specific and distinct KCa3.1 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 KCa3.1 plays a key role in human fibrocyte migration. Currently available KCa3.1-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.

The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells.

Background The influx of extracellular Ca2+ 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 Ca2+ influx occurs. However the individual role of each of the three members of the Orai channel family in Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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.

Functional KCa3.1 Channels Regulate Steroid Insensitivity in Bronchial Smooth Muscle Cells

Identifying the factors responsible for relative glucocorticosteroid (GC) resistance present in patients with severe asthma and finding tools to reverse it are of paramount importance. In asthma we see in vivo evidence of GC-resistant pathways in airway smooth muscle (ASM) bundles that can be modeled in vitro by exposing cultured ASM cells to TNF-α/IFN-γ. This action drives GC insensitivity via protein phosphatase 5–dependent impairment of GC receptor phosphorylation. In this study, we investigated whether KCa3.1 ion channels modulate the activity of GC-resistant pathways using our ASM model of GC insensitivity. Immunohistochemical staining of endobronchial biopsies revealed that KCa3.1 channels are localized to the plasma membrane and nucleus of ASM in both healthy controls and asthmatic patients, irrespective of disease severity. Western blot assays and immunofluorescence staining confirmed the nuclear localization of KCa3.1 channels in ASM cells. The functional importance of KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNF-α/IFN-γ was assessed using complementary inhibitory strategies, including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific small hairpin RNA delivered by adenoviruses. KCa3.1 channel blockade led to a significant reduction of fluticasone-resistant CX3CL1, CCL5, and CCL11 gene and protein expression. KCa3.1 channel blockade also restored fluticasone-induced GC receptor-α phosphorylation at Ser211 and transactivation properties via the suppression of cytokine-induced protein phosphatase 5 expression. The effect of KCa3.1 blockade was evident in ASM cells from both healthy controls and asthmatic subjects. In summary, KCa3.1 channels contribute to the regulation of GC-resistant inflammatory pathways in ASM cells: blocking KCa3.1 channels may enhance corticosteroid activity in severe asthma.

Counterregulation of β2-adrenoceptor function in human mast cells by stem cell factor

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.