What precedes the initial tyrosine phosphorylation of the high affinity IgE receptor in antigen-activated mast cell?

An interaction of multivalent antigen with its IgE bound to the high-affinity IgE receptor (FcεRI) on the surface of mast cells or basophils initiates a series of signaling events leading to degranulation and release of inflammatory mediators. Earlier studies showed that the first biochemically defined step in this signaling cascade is tyrosine phosphorylation of the FcεRI β subunit by Src family kinase Lyn. However, the processes affecting this step remained elusive. In this review we critically evaluate three current models (transphosphorylation, lipid raft, and our preferential protein tyrosine kinase-protein tyrosine phosphatase interplay model) substantiating three different mechanisms of FcεRI phosphorylation.     

The FcRβ- and γ-ITAMs play crucial but distinct roles in the full activation of mast cells induced by IgEκ and Protein L

Previous studies suggested that Protein L (PpL), the bacterial Ig-binding protein, activates mast cells. PpL presumably performs the activation by interacting with membrane-bound IgEκ, but the underlying mechanisms behind the process remain unclear. In the current study, we found that cell-surface FcεRI expression is a critical factor participant in PpL-mediated full activation of murine mast cells, which includes cytokine production, the degranulation response, and leukotriene C(4) (LTC(4)) release, and that engagement of the FcεRI with IgEκ and PpL is enough to induce tyrosine phosphorylation of ITAM in the FcRβ- and γ-signaling subunits. Introduction of mutations in two canonical tyrosine residues (Y47F/Y58F) of the FcRγ-ITAM completely abolished the above-mentioned mast cell functions, with the exception of LTC(4) release. Importantly, the FcRβ-ITAM acts as a signal transducer that is responsible for LTC(4) release independently of the FcRγ-ITAM. Taken together, our results suggest crucial and distinct functions for the FcRβ- and γ-ITAMs in the FcεRI-dependent full activation of mast cells induced by IgEκ and PpL.     

3-Benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one suppresses FcεRI-mediated mast cell degranulation via the inhibition of mTORC2-Akt signaling

Mast cells express high-affinity IgE receptor (FcεRI) on their surface, cross-linking of which leads to the immediate release of proinflammatory mediators such as histamine but also late-phase cytokine secretion, which are central to the pathogenesis of allergic diseases. Despite the growing evidences that mammalian target of rapamycin (mTOR) plays important roles in the immune system, it is still unclear how mTOR signaling regulates mast cell function. In this study, we investigated the effects of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) as an mTOR agonist on FcεRI-mediated allergic responses of mast cells. Our data showed that administration of 3BDO decreased β-hexosaminidase, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) release in murine bone marrow-derived mast cells (BMMCs) after FcεRI cross-linking, which was associated with an increase in mTOR complex 1 (mTORC1) signaling but a decrease in activation of Erk1/2, Jnk, and mTORC2-Akt. In addition, we found that a specific Akt agonist, SC79, is able to fully restore the decrease of β-hexosaminidase release in 3BDO-treated BMMCs but has no effect on IL-6 release in these cells, suggesting that 3BDO negatively regulates FcεRI-mediated degranulation and cytokine release through differential mechanisms in mast cells. The present data demonstrate that proper activation of mTORC1 is crucial for mast cell effector function, suggesting the applicability of the mTORC1 activator as a useful therapeutic agent in mast cell-related diseases.     

p66Shc is a negative regulator of FcεRI-dependent signaling in mast cells

Aggregation of FcεRI on mast cells activates signaling pathways, resulting in degranulation and cytokine release. Release of mast cell-derived inflammatory mediators is tightly regulated by the interplay of positive and negative signals largely orchestrated by adapter proteins. Among these, the Shc family adapter p52Shc, which couples immunoreceptors to Ras activation, positively regulates FcεRI-dependent signaling. Conversely, p66Shc was shown to uncouple the TCR for the Ras-MAPK pathway and prime T cells to undergo apoptotic death. Loss of p66Shc in mice results in breaking of immunologic tolerance and development of lupus-like autoimmune disease, which includes alopecia among its pathological manifestations. The presence of numerous activated mast cells in alopecic skin areas suggests a role for this adapter in mast cells. In this study, we addressed the involvement of p66Shc in FcεRI-dependent mast cell activation. We showed that p66Shc is expressed in mast cells and that mast cells from p66Shc(-/-) mice exhibit enhanced responses following Ag stimulation of FcεRI. Furthermore, using RBL-2H3 cell transfectants, we showed that aggregation of FcεRI resulted in the recruitment of a p66Shc-SHIP1 complex to linker for activation of T cells. Collectively, our data identified p66Shc as a negative regulator of mast cell activation.     

Mapping of the CD23 Binding Site on Immunoglobulin E (IgE) and Allosteric Control of the IgE-Fc{epsilon}RI Interaction

IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcεRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcεRI, whereas IgE-CD23 interactions control IgE expression levels. We have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. We show that the CD23 and FcεRI interaction sites are at opposite ends of the Cε3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FcεRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE-FcεRI and IgE-CD23 interactions.     

The Effects of Low-dose Ionizing Radiation in the Activated Rat Basophilic Leukemia (RBL-2H3) Mast Cells

Mast cells play important roles in many biological responses, such as those during allergic diseases and inflammatory disorders. Although laser and UV irradiation have immunosuppressive effects on inflammatory diseases by suppressing mast cells, little is known about the effects of γ-ionizing radiation on mast cells. In this study, we investigated the effects of γ-ionizing radiation on RBL-2H3 cells, a convenient model system for studying regulated secretion by mast cells. Low-dose radiation (<0.1 gray (Gy)) did not induce cell death, but high-dose radiation (>0.5 Gy) induced apoptosis. Low-dose ionizing radiation significantly suppressed the release of mediators (histamine, β-hexosaminidase, IL-4, and tumor necrosis factor-α) from immunoglobulin E (IgE)-sensitized RBL-2H3 cells. To determine the mechanism of mediator release inhibition by ionizing radiation, we examined the activation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, PKCs, and MAPK, and intracellular free calcium concentrations ([Ca(2+)](i)). The phosphorylation of signaling molecules following stimulation of high-affinity IgE receptor I (FcεRI) was specifically inhibited by low-dose ionizing radiation (0.01 Gy). These results were due to the suppression of FcεRI expression by the low-dose ionizing radiation. Therefore, low-dose ionizing radiation (0.01 Gy) may function as a novel inhibitor of mast cell activation.     

Role of reactive oxygen species in mast cell degranulation

Mast cells are a heterogeneous multifunctional cellular population that promotes connective tissue homeostasis by slow release of biologically active substances, affecting primarily the permeability of vessels and vascular tone, maintenance of electrolyte and water balance, and composition of the extracellular matrix. Along with this, they can rapidly release inflammatory mediators and chemotactic factors that ensure the mobilization of effector innate immune cells to fight against a variety of pathogens. Furthermore, they play a key role in initiation of allergic reactions. Aggregation of high affinity receptors to IgE (FcεRI) results in rapid degranulation and release of inflammatory mediators. It is known that reactive oxygen species (ROS) participate in intracellular signaling and, in particular, stimulate production of several proinflammatory cytokines that regulate the innate immune response. In this review, we focus on known molecular mechanisms of FcεRI-dependent activation of mast cells and discuss the role of ROS in the regulation of this pathway.     

Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (FcεRI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with FcεRI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders.     

Immunoglobulin E receptor signaling and asthma

Elevated IgE levels and increased IgE sensitization to allergens are central features of allergic asthma. IgE binds to the high-affinity Fcε receptor I (FcεRI) on mast cells, basophils, and dendritic cells and mediates the activation of these cells upon antigen-induced cross-linking of IgE-bound FcεRI. FcεRI activation proceeds through a network of signaling molecules and adaptor proteins and is negatively regulated by a number of cell surface and intracellular proteins. Therapeutic neutralization of serum IgE in moderate-to-severe allergic asthmatics reduces the frequency of asthma exacerbations through a reduction in cell surface FcεRI expression that results in decreased FcεRI activation, leading to improved asthma control. Our increasing understanding of IgE receptor signaling may lead to the development of novel therapeutics for the treatment of asthma.     

Piperine inhibits type II phosphatidylinositol 4-kinases: a key component in phosphoinositides turnover

Piperine has been shown to have anti-inflammatory properties. The molecular mechanisms by which it mediates anti-inflammatory activities remain elusive. Type II phosphatidylinositol 4-kinase(s) are key components in FcεRI receptor-mediated signaling leading to inflammatory mediators release in RBL-2H3 cells. The effects of piperine on IgE-mediated signaling and mast cell degranulation were investigated. Pretreatment of RBL-2H3 cells with piperine inhibited IgE-induced activation of type II PtdIns 4-kinase(s). In vitro lipid kinase assays showed piperine-inhibited type II PtdIns 4-kinase activity in a dose-dependent fashion with no effect on PtdIns 3-kinase activity. Concomitantly, pretreatment of RBL-2H3 cells with piperine also inhibited IgE-induced β-hexosaminidase release in RBL-2H3 cells. These results suggest that type II PtdIns 4-kinases are part of piperine-mediated anti-inflammatory signaling mechanisms.     

Cyclical mechanical stretch enhances degranulation and IL‐4 secretion in RBL‐2H3 mast cells

Mast cells are widely distributed in the body and affect their surrounding environment through degranulation and secretion of cytokines. Conversely, mast cells are influenced by environmental stimuli such as cyclical mechanical stretch (CMS), such as that induced by heartbeat and respiration. Peripherally distributed mast cells are surrounded by extracellular matrix, where they bind IgE on their surface by expressing the high‐affinity Fc receptor for IgE (FcεRI), and they release mediators after cross‐linking of surface‐bound IgE by allergen. To analyse how CMS affects mast cell responses, we examined the effect of applying CMS on the behaviour of IgE‐bound mast cells (RBL‐2H3 cell line) adhering to fibronectin as a substitute for extracellular matrix. We found that CMS enhanced FcεRI‐mediated secretion in the presence of antigen (2,4‐dinitrophenol–bovine serum albumin). CMS increased expression of IL‐4 mRNA and secretion of IL‐4 protein. Western blot analysis showed that CMS changes the signal transduction in mitogen‐activated protein kinases and AKT, which in turn alters the regulation of IL‐4 and increases the secretion of IL‐4. These results suggest that CMS modulates the effect of mast cells on inflammation and resultant tissue remodelling. Understanding how CMS affects mast cell responses is crucial for developing therapies to treat mast cell‐related diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Human intestinal mast cells are a potent source of multiple chemokines

Mast cells are key effector cells of immediate type allergic reactions. Upon activation they release a broad array of pre-stored and de novo synthesized mediators including immunoregulatory cytokines and chemokines. Here, we analyzed the chemokine profile expressed by mature human mast cells. Human mast cells were isolated from intestinal tissue and cultured with stem cell factor (SCF) in the presence or absence of IL-4 for 10d. Cells were stimulated by cross-linking of the high affinity IgE receptor (FcεRI) and/or by SCF. Chemokine and chemokine receptor mRNA expression was determined by real-time RT-PCR and chemokine release was measured by multiplex bead immunoassay. Out of 43 chemokines and 19 chemokine receptors human intestinal mast cells express 27 chemokines and nine chemokine receptors. Twelve chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL18, CCL20, CXCL2, CXCL3, CXCL8, and XCL1) were more than four-fold up-regulated in response to FcεRI cross-linking. Combination of pre-culture with IL-4 and/or stimulation with SCF in addition to FcεRI cross-linking further increased the antigen-dependent expression of mRNA for most chemokines. In contrast, the expression of CCL20, CXCL2, and CXCL3 was strongly inhibited by IL-4 treatment. In conclusion, human intestinal mast cells express a broad spectrum of different chemokines underlining their important role as immunoregulatory cells. Furthermore, combined treatment with IL-4 and SCF increases the antigen-mediated expression and release of multiple chemokines, but IL-4 priming inhibits the expression of CCL20, CXCL2, and CXCL3.     

Expression of a functional high-affinity IgG receptor, Fc gamma RI, on human mast cells: Up-regulation by IFN-gamma

Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor Fc epsilon RI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor Fc gamma RI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for Fc gamma RI. We next found that IFN-gamma up-regulated the expression of Fc gamma RI. This was confirmed by flow cytometry, where Fc gamma RI expression on human mast cells was increased from approximately 2 to 44% by IFN-gamma exposure. Fc epsilon RI, Fc gamma RII, and Fc gamma RIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4-5 x 108 M-1) increased from approximately 2,400 to 12,100-17,300 per cell. Aggregation of Fc gamma RI on human mast cells, and only after IFN-gamma exposure, led to significant degranulation as evidenced by histamine release (24.5 +/- 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-alpha, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases.     

STIM1-directed reorganization of microtubules in activated mast cells

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca(2+) controlled by store-operated Ca(2+) entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca(2+). Changes in cytosolic Ca(2+)concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca(2+) response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca(2+) plays an important role in the formation of microtubule protrusions in BMMCs.     

Pentagalloylglucose down-regulates mast cell surface FcεRI expression in vitro and in vivo

Mast cell activation by immunoglobulin E (IgE)-mediated stimuli is a central event in the pathogenesis of allergic disorders. The present report shows that treatment with pentagalloylglucose (PGG) resulted in a down-regulation of FcεRI surface expression on mucosal-type murine bone marrow-derived mast cells (mBMMCs), which correlated with a reduction in IgE-mediated activation of mBMMCs. Furthermore, PGG prevented development of allergic diarrhea in a food-allergy mouse model and suppressed the up-regulated FcεRI surface expression on mast cells derived from the food-allergy mouse colon. These findings on PGG suggest its therapeutic potential for allergic diseases through suppressing the FcεRI surface expression.     

A comparison of mediators released or generated by IFN-gamma-treated human mast cells following aggregation of Fc gamma RI or Fc epsilon RI

The high affinity receptor for IgG (Fc gamma RI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-gamma and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-gamma-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of Fc gamma RI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via Fc epsilon RI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-gamma-treated cultured human mast cells after Fc gamma RI or Fc epsilon RI aggregation. Aggregation of Fc gamma RI resulted in histamine release and PGD(2) and LTC(4) generation. These responses were qualitatively indistinguishable from responses stimulated via Fc epsilon RI. Aggregation of Fc epsilon RI or Fc gamma RI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of Fc gamma RI compared with Fc epsilon RI. TNF-alpha mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-alpha data using IFN-gamma-treated purified human lung mast cells. Thus, aggregation of Fc gamma RI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-alpha, were released and generated to a greater degree after Fc gamma RI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through Fc gamma RI in an IFN-gamma-rich environment.     

The FcεRIβ homologue,MS4A4A,promotes FcεRI signal transduction and store-operated Ca2+ entry in human mast cells

Members of the membrane spanning 4A (MS4A) gene family are clustered around 11q12-13, a region linked to allergy and asthma susceptibility. Other than the known functions of FcεRIβ (MS4A2) and CD20 (MS4A1) in mast cell and B cell signaling, respectively, functional studies for the remaining MS4A proteins are lacking. We thus explored whether MS4A4A, a mast cell expressed homologue of FcεRIβ, has related functions to FcεRIβ in FcεRI signaling. We establish in this study that MS4A4A promotes phosphorylation of PLCγ1, calcium flux and degranulation in response to IgE-mediated crosslinking of FcεRI. We previously demonstrated that MS4A4A promotes recruitment of KIT into caveolin-1-enriched microdomains and signaling through PLCγ1. Caveolin-1 itself is an important regulator of IgE-dependent store-operated Ca²⁺ entry (SOCE) and promotes expression of the store-operated Ca²⁺ channel pore-forming unit, Orai1. We thus further report that MS4A4A functions through interaction with caveolin-1 and recruitment of FcεRI and KIT into lipid rafts. In addition to proximal FcεRI signaling, we similarly show that MS4A4A regulates Orai1-mediated calcium entry downstream of calcium release from stores. Both MS4A4A and Orai1 had limited effects with compound 48/80 stimulation, demonstrating some degree of selectivity of both proteins to FcεRI receptor signaling over Mas-related G Protein coupled receptor X2 signaling. Overall, our data are consistent with the conclusion that MS4A4A performs a related function to the homologous FcεRIβ to promote PLCγ1 signaling, SOCE, and degranulation through FcεRI in human mast cells and thus represents a new target in the regulation of IgE-mediated mast cell activation.