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.     

Evidence that formation of vimentin mitogen-activated protein kinase (MAPK) complex mediates mast cell activation following FcεRI/CC chemokine receptor 1 cross-talk

Accumulating evidence points to cross-talk between FcεRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcεRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using β,β'-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcεRI and CCR1 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.     

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.     

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.     

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.     

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.