Impaired FcεRI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

A key event and potential therapeutic target in allergic and asthmatic diseases is signaling by the IgE receptor FcεRI, which depends on its interactions with Src family kinases (SFK). Here we tested the hypothesis that glycosylphosphatidylinositiol-anchored proteins (GPI-AP) are involved in FcεRI signaling, based on previous observations that GPI-AP colocalize with and mediate activation of SFK. We generated mice with a hematopoietic cell-specific GPI-AP deficiency by targeted disruption of the GPI biosynthesis gene PigA. In these mice, IgE-mediated passive cutaneous anaphylaxis was largely abolished. PigA-deficient mast cells cultured from these mice showed impaired degranulation in response to stimulation with IgE and antigen in vitro, despite normal IgE binding and antigen-induced FcεRI aggregation. On stimulation of these cells with IgE and antigen, coprecipitation of the FcεRI α-chain with the γ-chain and β-chain was markedly reduced. As a result, IgE/antigen-induced FcεRI-Lyn association and γ-chain tyrosine phosphorylation were both impaired in PigA-deficient cells. These data provide genetic evidence for an unanticipated key role of GPI-AP in FcεRI interchain interactions and early FcεRI signaling events, necessary for antigen-induced mast cell degranulation.     

Allergens in the Pathogenesis of Asthma

Evidence suggests that allergy is a significant triggering factor in asthma in children and adults alike. In immunoglobulin (Ig) E-mediated allergic reactions, sensitization occurs when allergen-specific B cells are stimulated and switched to IgE antibody production by interleukin (IL)-4 and IL-13 provided by helper T cells type 2 (Th2). The IgE antibodies act by arming cells bearing either the high-affinity (FcεRI) or low-affinity (FcεRII or CD23) receptor. The subsequent interaction of allergen with IgE-FcεRI complexes on mast cells and basophils causes cross-linking of receptors that triggers the release of a variety of inflammatory mediators, cytokines and chemokines. Therefore, the ability to lower circulating free IgE levels is desirable because most individuals are exposed to multiple allergens to which they are sensitive at any given time.Omalizumab (formerly known as rhuMAb-E25) is a recently developed humanized monoclonal anti-IgE antibody directed at the FcεRI binding domain of human IgE. It inhibits binding of IgE to mast cells without provoking mast cell activation. Preliminary clinical data from randomized controlled trials have shown that the addition of omalizumab to standard asthma therapy reduces asthma exacerbations and decreases inhaled corticosteroid and rescue medication use. The compound is also well tolerated. Omalizumab represents a novel therapeutic approach in the management of asthma.     

NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc epsilon RI aggregation

Aggregation of high-affinity receptors for immunoglobulin E (Fc epsilon RI) on the surface of mast cells results in degranulation, a response that is potentiated by binding of stem cell factor (SCF) to its receptor Kit. We observed that one of the major initial signaling events associated with Fc epsilon RI-mediated activation of human mast cells (HuMCs) is the rapid tyrosine phosphorylation of a protein of 25 to 30 kDa. The phosphorylation of this protein was also observed in response to SCF. This protein was identified as non-T-cell activation linker (NTAL), an adaptor molecule similar to linker for activated T cells (LAT). Unlike the Fc epsilon RI response, SCF induced NTAL phosphorylation in the absence of detectable LAT phosphorylation. When SCF and antigen were added concurrently, there was a marked synergistic effect on NTAL phosphorylation, however, SCF did not enhance the phosphorylation of LAT induced by Fc epsilon RI aggregation. Fc epsilon RI- and SCF-mediated NTAL phosphorylation appear to be differentially regulated by Src kinases and/or Kit kinase, respectively. Diminution of NTAL expression by silencing RNA oligonucleotides in HuMCs resulted in a reduction of both Kit- and Fc epsilon RI-mediated degranulation. NTAL, thus, appears to be an important link between the signaling pathways that are initiated by these receptors, culminating in mast cell degranulation.     

Regulation of mast cell survival and function by tuberous sclerosis complex 1

Mast cells play critical roles in allergic disorders and asthma. The importance of tuberous sclerosis complex 1/2-mammalian target of rapamycin (TSC1/2-mTOR) signaling in mast cells is unknown. Here, we report that TSC1 is a critical regulator for mTOR signaling in mast cells downstream of FcεRI and c-Kit, and differentially controls mast cell degranulation and cytokine production. TSC1-deficiency results in impaired mast cell degranulation, but enhanced cytokine production in vitro and in vivo after FcεRI engagement. Furthermore, TSC1 is critical for mast cell survival through multiple pathways of apoptosis including the down-regulation of p53, miR-34a, reactive oxygen species, and the up-regulation of Bcl-2. Together, these findings reveal that TSC1 is a critical regulator of mast cell activation and survival, suggesting the manipulation of the TSC1/2-mTOR pathway as a therapeutic strategy for mast cell-mediated diseases.     

IgE高亲和力受体在树突状细胞和单核细胞的表达和意义

IgE高亲和力受体(high affinity IgE receptor,FcεRI)是Fc受体的一种,可在肥大细胞、嗜碱性粒细胞、嗜酸性粒细胞、树突状细胞(dendritic cells,DCs)、单核细胞及血小板等细胞表面表达,在变态反应的发生和发展中有重要作用.FcεRI在DCs和单核细胞的表达具有不同于其他免疫...     

Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor Fc{varepsilon}RI

The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FcεRI on mast cells and basophils, and CD23 (FcεRII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen-presenting cells, contribute to allergen uptake and presentation to the immune system. We have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for cross-linking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single FcεRI binding site, which lies at the opposite ends of the Cε3 domains. Structural comparisons with both free IgE-Fc and its FcεRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcεRI binding, but also that the converse is true. The two binding sites are allosterically linked. We demonstrate experimentally the reciprocal inhibition of CD23 and FcεRI binding in solution and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.     

Differences in mast cell-bound IgE in the nasal epithelial layer and lamina propria

Mast cells in the allergic nasal mucosa are reported to have higher concentrations of high affinity type I Fce receptors (FcεRI) than mast cells in the non-allergic mucosa. IgE binds to FcεRI on mast cells. However, it is not clear whether different zones within the nasal mucosa harbor different quantities of IgE-bound mast cells. We stained IgE and mast cells from the nasal mucosa of patients with allergic and non-allergic rhinitis using a double-labeling technique with anti-IgE and antitryptase antibodies and compared the intensities of IgE staining of mast cells. In the allergic nasal mucosa, mast cells displayed more IgE staining in the lamina propria than in the epithelial layer. Mast cells displayed more IgE-staining in the allergic nasal mucosa than in the non-allergic mucosa. We speculate that the quantity of IgE that binds to mast cells depends on the quantity of IgE in nasal mucosal tissue.     

Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release.

Antigen-induced cross-linking of IgE bound to its receptors at the surface of basophils or mast cells initiates a number of biochemical events culminating in the release of histamine-containing granules. In the present study, we investigated the possible involvement of tyrosine phosphorylation in signaling by the high-affinity IgE receptor (Fc epsilon RI). Cross-linking of Fc epsilon RI in rat basophilic leukemia cells (RBL-2H3) led to the phosphorylation of several proteins on tyrosine, the most prominent having a mass of 72 kDa. Tyrosine phosphorylation was rapid, detectable 1 min after stimulation, and correlated with both the time course and antigen dose for histamine release. Reversal of Fc epsilon RI cross-linking prevented continuation of the degranulation process and resulted in rapid loss of tyrosine phosphorylation. The receptor-mediated tyrosine phosphorylation was still induced in the absence of calcium in the medium. Depletion of protein kinase C with phorbol 12-myristate 13-acetate did not dramatically affect the tyrosine phosphorylation signal or the release of histamine. In contrast, the calcium ionophore A23187 induced histamine release in the absence of a perceptible increase in protein tyrosine phosphorylation. Thus, tyrosine phosphorylation is an early signal following Fc epsilon RI aggregation, independent of the exocytotic process itself. Taken together, our findings functionally link protein phosphorylation on tyrosine residues to Fc epsilon RI-mediated signal transduction leading to histamine release.     

Polyclonal IgE Induces Mast Cell Survival and Cytokine Production

BackgroundAg-dependent activation of IgE-bearing mast cells is a critical first step in immediate hypersensitivity and other allergic responses. Recent studies have revealed Ag-independent effects of monoclonal mouse IgE molecules on mast cell survival and activation. However, no studies have been performed on the effects of polyclonal IgE molecules. Here, we tested whether polyclonal mouse and human IgE molecules affect survival and cytokine production in mast cells.MethodsMast cells were cultured in the presence of polyclonal mouse and human IgE molecules, and cell survival and cytokine production were analyzed.ResultsPolyclonal mouse IgE molecules in sera from mice with atopic dermatitis-like allergic skin inflammation, enhanced survival and cytokine production in mast cell cultures. Similar to the effects of monoclonal IgE, the polyclonal IgE effects were mediated by the high-affinity IgE receptor, FcεRI. Human polyclonal IgE molecules present in sera from atopic dermatitis patients were also capable of activating mast cells, and inducing IL- 8 production in human cord blood-derived mast cells.ConclusionsThese results imply that polyclonal IgE in atopic dermatitis and other atopic conditions might modulate mast cell number and function, thus amplifying the allergic response.     

奥马珠单抗治疗儿童中重度难治性过敏性哮喘的研究进展

难治性过敏性哮喘是指使用高剂量吸入皮质类固醇(含或不含其他控制性药物)治疗,病情仍得不到控制的、较为顽固的过敏性哮喘。作为重组人源化IgE单克隆抗体的奥马珠单抗,是目前唯一被授权应用于6～18岁中、重度难治性过敏性哮喘患儿的生物制剂。其对血清游离IgE具有高度特异性,能有效阻断IgE与肥大细胞、嗜碱粒细胞等炎症细胞膜表...     

Human FcγRIIA induces anaphylactic and allergic reactions

IgE and IgE receptors (FcεRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcεRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans.     

Stem cell factor and interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.     

Activated steady status and distinctive FcεRI-mediated responsiveness in basophils of atopic dermatitis

BackgroundAlthough basophils are considered to play an important role for maintenance of type 2 inflammation in atopic dermatitis (AD), studies on basophils in AD patients are limited. Some studies have reported the activation status, including CD203c and CD63, of peripheral blood basophils in AD patients.MethodsWe examined the features of circulating basophils in AD patients, assessed cell surface marker expressions and total serum IgE, and compared basophil responsiveness to stimulation between AD patients and healthy controls (HCs). In addition, the correlations among AD severity, laboratory factors, and features of basophils were examined. Blood samples from 38 AD patients and 21 HCs were analyzed. Basophil response markers CD203c and CD63, and expression of surface-bound IgE and FcεRI on basophils were measured. CD203c and CD63 expressions induced by stimulation with anti-IgE and anti-FcεRI antibodies were measured. Clinical/laboratory factors including total serum IgE were examined for correlations with these basophil parameters.ResultsBaseline CD203c and CD63 expression on basophils were significantly higher in AD patients compared with HCs. The CD203c/CD63 response ratio to anti-FcεRI stimulation was higher than that to anti-IgE stimulation in AD patients, but not HCs. FcεRI expression on basophils was higher in AD patients than in HCs, although surface-bound IgE on basophils was equivalent. Total serum IgE had negative correlations with surface-bound IgE and CD63 responsiveness to anti-IgE stimulation.ConclusionsBasophils were spontaneously activated under steady-state conditions in AD patients and responsiveness to anti-IgE stimulation was lower than in HCs. Despite high serum IgE and high basophil FcεRI expression, surface-bound IgE on basophils remained relatively low. Basophils might be suppressed or exhausted regarding FcεRI signaling via IgE in severe AD.     

The high affinity IgE receptor (FcεRI) expression and function in airway smooth muscle

The airway smooth muscle (ASM) is no longer considered as merely a contractile apparatus and passive recipient of growth factors, neurotransmitters and inflammatory mediators signal but a critical player in the perpetuation and modulation of airway inflammation and remodeling. In recent years, a molecular link between ASM and IgE has been established through Fc epsilon receptors (FcεRs) in modulating the phenotype and function of these cells. Particularly, the expression of high affinity IgE receptor (FcεRI) has been noted in primary human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthmatic subjects. The activation of FcεRI on ASM cells suggests a critical yet almost completely ignored network which may modulate ASM cell function in allergic asthma. This review is intended to provide a historical perspective of IgE effects on ASM and highlights the recent updates in the expression and function of FcεRI, and to present future perspectives of activation of this pathway in ASM cells.     

Kinetics of the appearance of Fc epsilon RI-bearing cells in interleukin-3-dependent mouse bone marrow cultures: correlation with histamine content and mast cell maturation.

While it is known that mast cells arise from pluripotential hematopoietic cells and express their mature phenotypes in tissues, the sequence of events in maturation is incompletely understood. To study early mast cells, we sorted cells from interleukin-3 (IL-3)-dependent mouse bone marrow cultures on the basis of Fc epsilon RI and examined their morphology, histamine content, and growth characteristics. Flow cytometric analysis and sort showed that the Fc epsilon RI-bearing (Fc epsilon RI+) cells increased from 0% on day 0 to 90% by day 21 and that the total number of Fc epsilon RI+ cells increased from 0 at the start of culture to 3.75 x 10(5) cells by day 21 from an initial population of 1 x 10(5) cells. The dissociation rate of 125I-labeled IgE from early cultured cells resembled the dissociation rate of mouse IgE from mature murine mast cells. Mean fluorescence intensity increased over time, reflecting an increase in IgE receptor density. Fc epsilon RI+ cells were also positive for Fc gamma RII/III. Morphologic studies showed gradual acquisition of metachromatic granules in the Fc epsilon RI+ cells, which was paralleled by an increase in histamine content. Sorted Fc epsilon RI+ cells, when placed in liquid suspension culture, gave rise to pure mast cell populations. Fc epsilon RI+ cells sorted at day 3 and cultured in agarose with IL-3 gave rise to 4,800 small and 150 medium-size mast cell colony-forming units per 10(6) cells, while Fc epsilon RI- cells gave rise to 23 medium-size and 49 large mast cell colony-forming units per 10(6) cells. Fc epsilon RI+ cells grown in granulocyte-macrophage colony-stimulating factor (CSF) or macrophage-CSF did not give rise to colony-forming units. These results show that Fc epsilon RI+ cells have proliferative potential, but that there also is a population of mast cell progenitor cells that have not yet expressed Fc epsilon RI, and such individual progenitor cells have greater potential for proliferation than cells that express Fc epsilon RI.     

Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation.

When aggregated, the high-affinity receptors for IgE on mast cells (Fc epsilon RI) launch a series of phosphorylations, particularly of protein tyrosines. We have analyzed how aggregation initiates this cascade. We examined Fc epsilon RI from unstimulated cells and from cells exposed to a polyvalent hapten conjugate that aggregates the Fc epsilon RI via the receptor-bound anti-hapten IgE. We also examined the latter receptors after they had been disaggregated in vitro with monovalent hapten. By an in vitro kinase assay: (i) Unaggregated and disaggregated receptors are associated with a kinase that phosphorylates an exogenous (peptide) substrate but minimally, or not at all, the subunits of Fc epsilon RI or associated proteins (endogenous substrates). After aggregation, phosphorylation of the exogenous substrate is linear with time, but the modification of the endogenous substrates reaches a plateau, presumably because only those endogenous substrates that are adjacent to the kinase are phosphorylated. (ii) Aggregated receptors and disaggregated receptors have enhanced kinase activity toward exogenous substrate. The state of phosphorylation of the receptor correlates strongly with the yield of enhanced kinase activity. We propose that upon aggregation of Fc epsilon RI, a constitutively associated kinase phosphorylates endogenous substrates by transphosphorylation. As a result, additional kinase activity becomes manifest and this promotes further transphosphorylation. In view of the homology between Fc epsilon RI and other receptors central to the immune response, the latter receptors likely utilize a similar transphosphorylation mechanism.     

Transmembrane signaling by the high-affinity IgE receptor on membrane preparations.

Aggregating the receptor with high affinity for IgE (Fc epsilon RI) stimulates a variety of phenomena in mast cells. Previous efforts to reproduce some of these events in broken-cell preparations such as isolated membranes have had limited success, possibly because the phenomena being monitored were too distal from the initial events. One of the earliest responses is now known to be the phosphorylation of tyrosine residues on several proteins, including the beta and gamma subunits of Fc epsilon RI. We show that in cell sonicates or on partially purified membranes derived from tumor mast cells, aggregating Fc epsilon RI stimulates phosphorylation of receptor tyrosine residues. As in the intact cells, receptor-mediated phosphorylation occurs only on receptors that are themselves aggregated. Because even in the unfractionated sonicates the phosphorylation of other cellular components was not detectably enhanced, and because the evidence is against the receptor itself being a kinase, our results suggest that phosphorylation of Fc epsilon RI is one of the earliest events stimulated by the receptor--an event that can now be investigated on simpler biological preparations than previously available.     

A novel detection method for cross-linking of IgE-receptors by autoantibodies in chronic spontaneous urticaria

BackgroundAutoantibodies (AAbs) against immunoglobulin E (IgE) antibodies (Abs) and their high-affinity receptor alpha subunits (FcεRIα) are key factors in the elicitation of type IIb autoimmune chronic spontaneous urticaria (type IIb aiCSU). In this study, we aimed to develop a new method to detect functional anti-FcεRIα and anti-IgE AAbs, which can crosslink the plural FcεR?α molecules and IgE Abs on the surface of mast cells and basophils, in sera from aiCSU patients using the amplified luminescence proximity homogeneous assay (Alpha).MethodsSera were obtained from 14 aiCSU patients, as diagnosed by recurrent chronic spontaneous urticaria episodes and positive results for the autologous serum skin test and/or histamine release test (HRT). The AAbs to FcεRIα and IgE Abs were determined in sera from aiCSU patients using enzyme-linked immunosorbent assay (ELISA) and Alpha by cross-linking (AlphaCL) of IgE Abs and/or FcεR?α.ResultsSerum anti-FcεRIα and anti-IgE AAb levels were not significantly different between aiCSU patients and healthy subjects in ELISA. Anti-FcεRIα AAbs were detected in 10 of 14 aiCSU patients who displayed positive (5/5) and negative (5/9) results in the HRT for anti-FcεRIα AAbs by AlphaCL, whereas no signals were observed in healthy subjects. Additionally, anti-IgE AAbs were detected in two of four aiCSU patients who displayed positive results in the HRT for anti-IgE AAbs.ConclusionsA new assay method using AlphaCL can detect anti-FcεRIα and anti-IgE AAbs with FcεRIα- and IgE-crosslinking abilities in sera from aiCSU patients. This simple and practical assay method may be available as a diagnostic tool for urticaria patients.