Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane.     

Basis of the 1:1 stoichiometry of the high affinity receptor FcεRI-IgE complex

A soluble fragment of the high-affinity IgE receptor FcεRI α-chain (sFcεRIα) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the Cε2, Cε3 and Cε4 domains of the ε-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG₁, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the Cε2 and Cε3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging Cε2 domains. To test this hypothesis we have expressed a recombinant ε-chain fragment containing Cε3 and Cε4. This product, Fcε3–4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface FcεRI. Titration experiments, together with molecular mass measurements of the Fcε3–4/sFcεRIα complex, reveal that Fcε3–4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by Cε2 accounts for the unexpected stoichiometry. Received: 31 July 1996 / Accepted: 1 December 1996     

Lipid Raft-Dependent FcεRI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcεRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcεRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcεRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.     

CFFM4: a new member of the CD20/FcεRIβ family

Proteins with transmembrane domains are classified in different families based on their structure, amino acid homology, and function. In this study, we report the identification, sequence, and expression profile of a new member of the CD20/FcepsilonRIbeta family, CD20/FcepsilonRIbeta family member 4 (CFFM4). The CFFM4 gene contains seven exons and six introns and is transcribed into an mRNA encoding a 240-amino acid protein with four hydrophobic regions. The CFFM4 protein shares a high degree of homology with the other members of the family, especially in the hydrophobic regions where several amino acids are conserved. However, the CFFM4 protein can be distinguished from the other members of the family based on the length of the second extracellular loop and the absence of an immunoreceptor tyrosine-based activation motif signal. Another distinct characteristic is that CFFM4 mRNA expression is not limited to the hematopoietic lineage. CFFM4 was detected by Northern dot blot in a variety of normal and cancerous tissues. CFFM4 expression was also compared in developmentally early hematopoietic human bone marrow CD34+ stem cells versus peripheral blood-derived CD14+ mature monocytes, in the undifferentiated versus differentiated myelomonocytic U937 cell line, and in acute myelogenous leukemia FAB1 versus FAB5. In each of these systems, cellular myelomonocytic differentiation correlated with an increase in CFFM4 mRNA expression. Such results indicate that CFFM4 is associated with mature cellular function in the monocytic lineage and like CD20 and FcepsilonRIbeta, it may be a component of a receptor complex involved in signal transduction.     

Tec family kinases: regulation of FcεRI-mediated mast-cell activation

Mast cells express the high-affinity receptor for IgE (FcεRI) and are key players in type I hypersensitivity reactions. They are critically involved in the development of allergic rhinitis, allergic asthma and systemic anaphylaxis, however, they also regulate normal physiological processes that link innate and adaptive immune responses. Thus, their activation has to be tightly controlled. One group of signaling molecules that are activated upon FcεRI stimulation is formed by Tec family kinases, and three members of this kinase family (Btk, Itk and Tec) are expressed in mast cells. Many studies have revealed important functions of Tec kinases in signaling pathways downstream of the antigen receptors in lymphocytes. This review summarizes the current knowledge about the function of Tec family kinases in FcεRI-mediated signaling pathways in mast cell.     

Analysis of Proteins Binding to the ITAM Motif of the β-Subunit of the High-Affinity Receptor for IgE (FcεRI)

Aggregation of the multichain (α β γ₂) high-affinity IgE receptor (Fcε RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the Fcε RI-β and -γ subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the γ ITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcε RI crosslinking. Phosphorylation of the β ITAM is insufficient to mediate cell activation. The rat β ITAM contains three tyrosines (Tyr²¹⁸, Tyr²²⁴, and Tyr²²⁸) with an intermediate noncanonical tyrosine. Synthetic peptides based on the ITAM of the Fcε RI-β subunit were used to investigate the role of each phosphotyrosine in the binding of signaling proteins to this motif. Among the proteins that bind to phosphorylated β ITAM are Syk, Grb2, Shc, SHIP, and SHP-1, and binding does not depend on previous cell activation. Nonphosphorylated peptides do not bind these proteins. Syk binding to β -peptides is dependent on the number and position of phosphotyrosines in the ITAM. Phosphorylation of Tyr²¹⁸ seems to be most important for Syk binding. Recruitment of Syk and other signaling proteins to the β -subunit might be important for its amplifier role.     

曲古菌素A对FcεRI介导的肥大细胞活化的影响

曲古菌素A（TSA）是一种组蛋白去乙酰化酶抑制剂,在变态反应等免疫性疾病和抗肿瘤方面显示了潜在的治疗效应。主要观察了TSA对变态反应的效应细胞—肥大细胞活化的影响。TSA可诱导小鼠骨髓来源的肥大细胞（BMMC）凋亡,并抑制表面FcεRI的表达。TSA处理的BMMC经anti-IgE/IgE刺激之后,脱颗粒及分泌TNF-α、IL-6和IL-13生物活性介质的能力显著受到抑制。由此提示,TSA可能通过诱导BMMC凋亡和/或下调FcεRI表达,进而抑制FcεRI介导的BMMC活化,这为TSA进一步在肥大细胞相关的变态反应性疾病中的应用提供了实验数据。     

miR-142-3p enhances FcεRI-mediated degranulation in mast cells

Mast cells are immune cells derived from hematopoietic progenitors. When they are activated by stimuli, they immediately release granule-associated mediators, leading to allergic inflammation. Several factors controlling mediator release have been identified; however, little is known whether microRNAs (miRNAs) are involved in this process. miRNAs are a small class of non-coding RNAs that negatively regulate gene expression. In this study, we investigated the relationship between miRNAs and degranulation in LAD2 cells, a human mast cell line. We demonstrated that silencing of Dicer, a key enzyme of miRNA biogenesis, attenuates degranulation, indicating that miRNAs are involved in mast cell degranulation. We furthermore discovered that the overexpression of miR-142-3p enhances FcεRI-mediated degranulation and that miR-142-3p rescues the reduction of degranulation by silencing Dicer. Similar effects were observed in bone marrow-derived mast cells obtained miR-142-3p-deficient mice. Our studies suggest that miR-142-3p is a potential therapeutic target in pathological conditions caused by mast cells, such as mastocytosis and allergies.     

Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. Here, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chip flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn.     

Inhibitory effects of phloroglucinol derivatives isolated from Ecklonia stolonifera on FcεRI expression

Two bioactive phloroglucinol derivatives, dioxinodehydroeckol (DHE) and phlorofucofuroeckol A (PFF-A) were isolated from edible marine brown alga, Ecklonia stolonifera, and evaluated for effects on cell surface FcεRI expression in KU812F cells. DHE and PFF-A were found to reduce the cell surface expression, and total cellular protein and mRNA levels for the FcεRI α chain. Moreover, both compounds exerted inhibitory effects against the elevation of intracellular calcium concentration [Ca²⁺]_i and histamine release from anti-FcεRI α chain antibody (CRA-1)-stimulated cells. These inhibitory effects were stronger for PFF-A than for DHE. These results show that two phloroglucinol derivatives, DHE and PFF-A, may exert anti-allergic effects via the inhibition of FcεRI expression, calcium influx, and degranulation in basophils, and contributes to the pharmacological activities of marine brown alga, including E. stolonifera.     

Molecular hydrogen suppresses FcεRI-mediated signal transduction and prevents degranulation of mast cells

Molecular hydrogen ameliorates oxidative stress-associated diseases in animal models. We found that oral intake of hydrogen-rich water abolishes an immediate-type allergic reaction in mice. Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of the FcεRI-associated Lyn and its downstream signal transduction, which subsequently inhibits the NADPH oxidase activity and reduces the generation of hydrogen peroxide. We also found that inhibition of NADPH oxidase attenuates phosphorylation of Lyn in mast cells, indicating the presence of a feed-forward loop that potentiates the allergic responses. Hydrogen accordingly inhibits all tested signaling molecule(s) in the loop. Hydrogen effects have been solely ascribed to exclusive removal of hydroxyl radical. In the immediate-type allergic reaction, hydrogen exerts its beneficial effect not by its radical scavenging activity but by modulating a specific signaling pathway. Effects of hydrogen in other diseases are possibly mediated by modulation of yet unidentified signaling pathways. Our studies also suggest that hydrogen is a gaseous signaling molecule like nitric oxide.     

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.     

Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr–296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr–296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr–296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr–296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr–296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr–296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.     

Comparison of the anti-allergic activity of Syk inhibitors with optimized Syk siRNAs in FcεRI-activated RBL-2H3 basophilic cells

Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcεRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcεRI-mediated signal transduction, degranulation and TNFα secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcεRI-mediated signal transduction, degranulation and TNFα secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.     

The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

Background

IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium.

Methodology/Principal Findings

FcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains.

Conclusions/Significance

Our data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested.     

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.     

Down-Regulation of FcεRI-Mediated CD63 Basophil Response during Short-Term VIT Determined Venom-Nonspecific Desensitization

Background

We recently showed a desensitization of FcεRI-mediated basophil response after short-term VIT. Our aim was to evaluate the allergen specificity of this desensitization.

Methods

In 11 Hymenoptera-venom double positive subjects, basophil threshold sensitivity (CD-sens) to anti-FcεRI, honeybee, and Vespula venom was assessed at the beginning and just before the first maintenance dose (MD) of single ultra-rush VIT. In some patients we also monitored CD-sens to rApi m 1 and/or rVes v 5 or other co-sensitizations (i.e., grass pollen). In additional 7 patients, basophils were stripped and sensitized with house dust mite (HDM) IgEs at the same time points.

Results

We demonstrated a marked reduction of CD-sens to anti-FcεRI and VIT-specific venom before the first MD in all 18 subjects included. Furthermore, in 10 out of 11 double positive subjects, a significant and comparable decrease before the first MD was also evident for non-VIT venom; this nonspecific decrease was further supported by the opposite recombinant species-specific major allergen. In one subject with additional grass pollen allergy, a decrease of CD-sens to grass allergen was also demonstrated. Similarly, in 7 cases of patients with passively HDM-sensitized basophils, a significant reduction of CD-sens was also evident to de novo sensitized HDM allergen.

Conclusions

Short-term VIT induced basophil desensitization to VIT-specific as well as to VIT-nonspecific venom. As opposed to long-term VIT, which induces venom-specific changes, the effect of short-term VIT seems to be venom-nonspecific.     

A Rapid Method of Detecting Autoantibody against FcεRIα for Chronic Spontaneous Urticaria

Background

Chronic spontaneous urticaria (CU) is a common skin disorder, with an estimated prevalence of 0.5–1.8% in most populations. Around 30–50% of CU patients have an autoimmune etiology, with autoantibodies (autoAbs) against IgE, FcεRIα, and FcεRII/CD23. Although the in vivo autologous serum skin test (ASST) and in vitro histamine release/activation assay are the most frequently used screening methods, these two have many limitations and do not directly measure susceptible autoAbs. This study aimed to establish an in vitro rapid screening test using recombinant autoantigen FcεRIα(rFcεRIα) to improve the diagnosis of autoimmune urticaria.

Methods

Forty patients with CU and 20 healthy individuals were enrolled. After PCR-based cloning and the production of extracellular fragments of the FcεRIαprotein using the E. coli expression system, serum autoAb to rFcεRIαwas evaluated using in-house ELISA and rapid immunodot test.

Results

In ELISA-based detection, 14 out of 20 CU-ASST(+) patients exhibited anti- FcεRIαresponses, whereas five of the 20 CU-ASST(-) and two of the 20 non-CU patients showed autoantibody background in the assay. For the immunodot test, 55% (11/20) of the CU-ASST(+) sera exhibited anti-FcεRIαreactivity. There was no false positive among the CU-ASST(-) and non-CU groups. Using clinical urticaria plus ASST(+) as the gold standard, in-house ELISA had 70% sensitivity, 82.5% specificity, and positive likelihood ratio of 4, while immunodot had 55% sensitivity, 100% specificity, and positive likelihood ratio >55.

Conclusions

This study has developed a rapid immunodot method with high specificity for detecting autoAb to FcεRIαin patients with CU. Preliminary data indicates that this immunodot technique has the potential to be a routine diagnostic assay for autoimmune CU.