Glenn Cruse

Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions

BACKGROUND Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Functional KCa3.1 K+ channels are required for human lung mast cell migration.

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 α 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.

Human Airway Smooth Muscle Promotes Human Lung Mast Cell Survival, Proliferation, and Constitutive Activation: Cooperative Roles for CADM1, Stem Cell Factor, and IL-6

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.

Mast Cells Promote Airway Smooth Muscle Cell Differentiation via Autocrine Up-Regulation of TGF-β1

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 β-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-β1 secretion, α-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-β1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-β1 in response to activation by mast cell β-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of α-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.

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.

Airway wall geometry in asthma and nonasthmatic eosinophilic bronchitis

Background:  Variable airflow obstruction and airway hyperresponsiveness (AHR) are features of asthma, which are absent in nonasthmatic eosinophilic bronchitis (EB). Airway remodelling is characteristic of both conditions suggesting that remodelling and airway dysfunction are disassociated, but whether the airway geometry differs between asthma and nonasthmatic EB is uncertain.

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6

BackgroundMast 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.MethodsPurified 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.ResultsIgE 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.ConclusionThese 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.

Vibratory Urticaria Associated with a Missense Variant in ADGRE2.

Patients with autosomal dominant vibratory urticaria have localized hives and systemic manifestations in response to dermal vibration, with coincident degranulation of mast cells and increased histamine levels in serum. We identified a previously unknown missense substitution in ADGRE2 (also known as EMR2), which was predicted to result in the replacement of cysteine with tyrosine at amino acid position 492 (p.C492Y), as the only nonsynonymous variant cosegregating with vibratory urticaria in two large kindreds. The ADGRE2 receptor undergoes autocatalytic cleavage, producing an extracellular subunit that noncovalently binds a transmembrane subunit. We showed that the variant probably destabilizes an autoinhibitory subunit interaction, sensitizing mast cells to IgE-independent vibration-induced degranulation. (Funded by the National Institutes of Health.).

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.