Expression Of FcεRIα Research Articles

Higher House Dust Mite-Specific IgE Levels among Chronic Spontaneous Urticaria Patients May Implicate Higher Basophil Reactivity

Introduction: Allergen-specific IgE (sIgE) sensitization exists in a considerable fraction of chronic spontaneous urticaria (CSU) patients. Basophils have been implicated in the pathogenesis of CSU. This paper aimed to explore the relationship between allergic sensitization and basophil reactivity in CSU and the possible underlying mechanism. Methods: Basophil-enriched leukocytes were isolated from the peripheral blood of 76 CSU patients and 9 healthy controls. Basophil CD63 and FcεRIα (the alpha subunit of the high-affinity IgE receptor) expression in the blood samples with various house dust mite (HDM)-sIgE levels were determined by flow cytometry. Basophil reactivity and SHIP-1 (a molecule related to the IgE/FcεRI signaling pathway) expression were analyzed after stimulation with an HDM allergen or other stimuli. Results: HDM-sIgEstrong positive (≥3.5 kU/L) CSU patients had a significantly higher mean percentage of basophil CD63 and higher baseline levels of FcεRIα expressed by basophils than HDM-sIgEnormal (<0.35 kU/L) CSU patients and healthy controls; the same went for total serum IgE. After stimulation with Dermatophagoides pteronyssinus peptidase 1 (Derp1) alone or together with Derp1-sIgE, the stimulation index of CD63 and levels of FcεRIα expressed by basophils in HDM-sIgEstrong positive CSU patients were significantly higher than those in HDM-sIgEnormal CSU patients and healthy controls. Significantly more SHIP-1 mRNA expression in HDM-sIgEstrong positive CSU patients was induced after the combined stimulation in comparison to other subjects. Conclusion: CSU patients with higher HDM-sIgE levels (≥3.5 kU/L) may have higher CD63 and FcεRIα expression on peripheral blood basophils. Peripheral blood basophils in these CSU patients are more responsive to HDM allergen stimulation. Higher HDM-sIgE levels among CSU patients may implicate higher basophil reactivity.

A Co-Expressed Natural Antisense RNA FCER1A-AS Controls IgE-Dependent Immunity by Promoting Expression of FcεRIα.

As the α-subunit of the high-affinity receptor for the Fc portion of immunoglobulin E (FcεRIα), FcεRIα plays a central role in IgE-mediated allergic disorders and in the immunity and immunopathology of some parasitic infections. FcεRIα is specifically expressed on basophils and mast cells, but the mechanism that controls FcεRIα expression in these cells is poorly understood. In this study, we found that the natural antisense transcript (NAT) of FcεRIα (FCER1A-AS) is co-expressed with the sense transcript (FCER1A-S) in both interleukin (IL)-3-induced FcεRIα-expressing cells and in the high FcεRIα-expressing cell line MC/9. When FCER1A-AS is selectively knocked down by the CRISPR/RfxCas13d (CasRx) approach in MC/9 cells, the expression of both FCER1A-S mRNA and proteins is markedly decreased. Furthermore, FCER1A-AS deficiency was also found to be associated with a lack of FCER1A-S expression in vivo. Correspondingly, homozygous mice deficient in FCER1A-AS demonstrated a similar phenotype to FCER1A knockout mice in Schistosoma japonicum infection and in IgE-FcεRIα-mediated cutaneous anaphylaxis. Thus, we uncovered a novel pathway for the control of FcεRIα expression by its co-expressed natural antisense transcript. IMPORTANCE FcεRIα is responsible for high-affinity binding with the Fc portion of IgE, which is critical for IgE-dependent disease responses such as allergy responses and anti-parasite immunity. FcεRIα is expressed on a few cell types, including mast cells and basophils. Although the expression of FcεRIα is known to be promoted by the IL-3-GATA-2 pathway during its differentiation, the mechanism by which FcεRIα expression is maintained remains unknown. In this study, we discovered that a natural antisense transcript, FCER1A-AS, is co-expressed with the sense transcript. The presence of FCER1A-AS is essential for sense transcript expression in mast cells and basophils, but not for the differentiation of these cells through cis-regulation. Like FcεRIα knockout mice, mice lacking FCER1A-AS also exhibit reduced survival after Schistosoma japonicum infection and a lack of IgE-mediated cutaneous anaphylaxis. Thus, a novel pathway for regulating IgE-mediated allergic diseases through noncoding RNAs has been revealed.

Neuronal Fc\u03b5RI\u03b1 directly mediates ocular itch via IgE-immune complex in a mouse model of allergic conjunctivitis

BackgroundClassical understanding of allergic conjunctivitis (ACJ) suggests that ocular itch results from a mast cell-dependent inflammatory process. However, treatments that target inflammatory mediators or immune cells are often unsatisfying in relieving the stubborn itch symptom. This suggests that additional mechanisms are responsible for ocular itch in ACJ. In this study, we aim to determine the role of neuronal FcεRIa in allergic ocular itch.MethodsCalcium imaging was applied to observe the effect of IgE-immune complex in trigeminal neurons. Genomic FcεRIa knockout mice and adeno-associated virus (AAV) mediated sensory neuron FcεRIa knockdown mice were used in conjunction with behavioral tests to determine ocular itch. In addition, immunohistochemistry, Western blot and quantitative RT-PCR were used for in vitro experiments.ResultsWe found that FcεRIα was expressed in a subpopulation of conjunctiva sensory neurons. IgE-IC directly activated trigeminal neurons and evoked acute ocular itch without detectible conjunctival inflammation. These effects were attenuated in both a global FcεRIa-knockout mice and after sensory neuronal-specific FcεRIa-knockdown in the mouse trigeminal ganglion. In an ovalbumin (OVA) induced murine ACJ model, FcεRIα was found upregulated in conjunctiva-innervating CGRP+ sensory neurons. Sensory neuronal-specific knockdown of FcεRIa significantly alleviated ocular itch in the ACJ mice without affecting the immune cell infiltration and mast cell activation in conjunctiva. Although FcεRIα mRNA expression was not increased by IgE in dissociated trigeminal ganglion neurons, FcεRIα protein level was enhanced by IgE in a cycloheximide-resistance manner, with concordant enhancement of neuronal responses to IgE-IC. In addition, incremental sensitization gradually enhanced the expression of FcεRIα in small-sized trigeminal neurons and aggravated OVA induced ocular itch.ConclusionsOur study demonstrates that FcεRIα in pruriceptive neurons directly mediates IgE-IC evoked itch and plays an important role in ocular itch in a mouse model of ACJ. These findings reveal another axis of neuroimmune interaction in allergic itch condition independent to the classical IgE-mast cell pathway, and might suggest novel therapeutic strategies for the treatment of pruritus in ACJ and other immune-related disorders.

Expression and existence forms of mast cell activating molecules and their antibodies in systemic lupus erythematosus.

IntroductionMast cells are regarded as a kind of classical anaphylaxis cells. However autoimmune diseases and allergic reactions have many similarities or overlaps. A large number of papers have proved that mast cells play a significant role in the pathogenesis of systemic lupus erythematosus (SLE). It is speculated that IgE, anti‐IgE antibodies, FcεRI, and anti‐FcεRI antibodies activate mast cells through autoimmune pathways and participate in the disease process of SLE. Naturally occurring protein molecules not only exist in monomer form, but also in polymer of protein molecules. Therefore, whether IgE, FcεRIα, anti‐IgE antibodies, and anti‐FcεRI antibodies also exist in polymeric forms in the natural state is worthy of further investigation.MethodsThe serum samples and clinical data of 131 patients with SLE were collected from Qilu Hospital (Qingdao). Sixty healthy individuals were collected as the control group. Serum FcεRIα, anti‐IgE, and anti‐FcεRI were detected by enzyme‐linked immunosorbent assay. Serum IgE was detected by rate scatter nephelometry. A Chinese hamster ovarian cancer cell line CHO3D10 transfected with human FcεRIα was cultured and the cell protein extract was prepared. The existence forms of FcεRIα in the cell protein extract were detected by the native‐page method.ResultsThe serum FcεRIα in SLE patients was significantly higher than that in control group (3.52 [2.18, 4.71] µg/ml and 1.87 [1.52, 2.33] µg/ml, respectively; p < .05). Anti‐IgE was significantly lower than that in the control group (0.85 [0.55, 1.21] µg/ml and 1.23 [0.95, 1.58] µg/ml, respectively; p < .05). The CHO3D10 cell line expressed the FcεRIα, which had one kind of monomer (mFcεRIα) and two kinds of polymers (pFcεRIα) in the degeneration conditions.ConclusionIn patients with SLE, the expression of FcεRIα was increased and the level of anti‐IgE was decreased. FcεRIα had one kind of monomer and two kinds of polymers. Mast cell‐associated FcεRIα involved in the inflammatory lesion of SLE.

Expression of FcεRIα and tryptase in human lung tissue during drug-induced anaphylactic death

The diagnosis of drug-induced anaphylactic death is an important component of forensic medicine. The purpose of this study was to explore the expression of FcεRIα and tryptase in human lung tissue during drug-induced anaphylactic death and its value for forensic medicine. The expression of FcεRIα and tryptase in lung tissues of the drug-induced anaphylactic death group (n = 30) and control group (who died due to sudden cardiac death, falling from a height, or traffic accidents, n = 30) was detected by immunohistochemistry and immunofluorescence staining. Thedifference in FcεRIα and tryptase expression in the lung tissue between the drug-induced anaphylactic death group and the control group wasstatisticallysignificant (p < 0.05). The levels of FcεRIα and tryptase expression greatly increased in the lung tissue of the drug-induced anaphylactic death group, which may provide morphological evidence and a reference for the diagnosis of drug-induced anaphylactic death in forensic medicine.

Response to Comment on "Tumor-initiating cells establish an IL-33-TGF-β niche signaling loop to promote cancer progression".

Kamphuis et al argue that macrophages accumulated in the proximity of tumor-initiating cells do not express the high-affinity immunoglobulin E receptor FcεRIα. Although we cannot exclude the possibility of nonspecific binding of anti-FcεRIα antibody (clone MAR-1), we provide evidence that macrophages in squamous cell carcinomas express FcεRIα and that IL-33 induces FcεRIα expression in bone marrow cell-derived macrophages.

Expression and mechanism of mast cell activating molecules and their antibodies in allergic rhinitis and nasal polyps

Objective:To analyze the relationship and mechanism of mast cell with allergic rhinitis and nasal polyps. Method:From January 2018 to June 2018, all the inpatients in Otolaryngology Department were examined by nasal endoscopy and CT before operation. They were divided into three groups: 30 patients with allergic rhinitis（AR group） and 30 patients with nasal polyps（NP group） as the experimental group; 30 patients with nasal septum correction after partial inferior turbinectomy alone as the control group（N group）. Clinical data, peripheral blood and nasal mucosa were collected. The percentage of eosinophils in peripheral blood was detected by automatic hematology analyzer. The levels of anti-IgE antibody, FcεRIα and anti-FcεRIα antibody in serum were detected by ELISA. The levels of serum IgE were detected by velocity scattering turbidimetry. HE was used to observe the histopathological changes in the three groups. Immunohistochemical method was used to analyze the expression of tryptase and FcεRIα in the experimental group and the control group. Result:The percentage of eosinophils, IgE, anti-IgE antibody, FcεRIα and anti-FcεRIα antibodies in AR group were significantly higher than those in control group（P<0.05）; the percentage of eosinophils, FcεRIα and anti-FcεRIα antibodies in NP group were significantly higher than those in healthy control group（P<0.05）. There was no significant difference in IgE and anti-IgE antibodies among three groups（P>0.05）. The eosinophil percentage, IgE and anti-IgE antibody levels in AR group were significantly higher than that in NP group（P<0.05）; there were no differences in FcεRIα and anti-FcεRIα antibody levels between AR group and NP group（P>0.05）; There was no significant difference in eosinophil infiltration between the two groups; The brown tryptase-positive mast cells and FcεRIα-positive mast cells were found in the submucosa and interstitium of nasal mucosa. Conclusion:The high expression of FcεRIα and its antibody in the serum of patients with allergic rhinitis and nasal polyps indicated that this antibody system was involved in the occurrence and development of allergic rhinitis and nasal polyps. Local allergy with mast cells as the core might play an important role in the occurrence and development of nasal polyps. The possible role of mast cells in the formation of nasal polyps and the specific mechanism deserve further study.

The Impact of Integrin β2 on Granulocyte/Macrophage Progenitor Proliferation.

Previously, we reported that although the HSPC frequency in bone marrow cells (BMC) was comparable between β2−/− and β2+/+ mice, transplantation of β2−/− BMC into lethally irradiated CD45.1 recipient resulted in more myeloid cell production than β2+/+ BMC. The objective of this study is to address if integrin β2 deficiency skews granulocyte/macrophage progenitor (GMP) proliferation. FACS analysis demonstrated that GMP frequency and cell number were higher and megakaryocyte/erythrocyte progenitor frequency and cell number were lower in β2−/− mice than β2+/+ mice. However, the common myeloid progenitors (CMP) frequency and cell number were similar between the two groups. The increased GMP number was due to GMP proliferation as evidenced by the percentage of BrdU‐incorporating GMP. Whole genome transcriptome analysis identified increased FcεRIα expression in β2−/− CMP compared to β2+/+ CMP. FcεRIα expression on β2−/− GMP was detected increased in β2−/− mice by qRT‐PCR and FACS. Although transplantation of FcεRIαhi GMP or FcεRIαlo GMP into lethally irradiated CD45.1 recipient resulted in comparable myeloid cell production, transplantation of β2 deficient FcεRIαhi GMP generated more myeloid cells than β2+/+ FcεRIαhi GMP. GATA2 expression was increased in β2−/− GMP. Using a luciferase reporter assay, we demonstrated that mutation of the GATA2 binding site in the FcεRIα promoter region diminished FcεRIα transcription. In vitro, the addition of IgE, the ligand of FcεRIα, promoted GMP expansion, which was abrogated by inhibition of JNK phosphorylation. Integrin β2 deficiency promoted GMP proliferation and myeloid cell production, which was mediated via FcεRIα/IgE‐induced JNK phosphorylation in GMP. Stem Cells 2019;37:430–440

Effects of a selective casein kinase 1δ and ε inhibitor on FcεRI expression and IgE-mediated immediate-type cutaneous reactions in dogs.

The molecular clock network in mast cells has been shown to be a factor responsible for circadian regulation of allergic inflammation. PF670462 is a selective inhibitor of casein kinase 1δ and ε (CK1δ/ε) that control the posttranslational modification of clock proteins. The aims of this study were to evaluate the effects of PF670462 on gene and protein expression of FcεRI, the high-affinity IgE receptor, in canine mast cells and on IgE-mediated immediate-type cutaneous reactions in dogs. PF670462 decreased mRNA expression of FcεRIα and β, but not γ, and protein expression of FcεRI in a canine mast cell line. Furthermore, PF670462 suppressed IgE-mediated immediate-type cutaneous erythema in dogs. These findings indicate that CK1δ/ε function as regulators for FcεRI expression and IgE-mediated cutaneous reactions in dogs.

Enhanced plasmacytoid dendritic cell antiviral responses after omalizumab

Atopy and viral respiratory tract infections synergistically promote asthma exacerbations. IgE cross-linking inhibits critical virus-induced IFN-α responses of plasmacytoid dendritic cells (pDCs), which can be deficient in patients with allergic asthma. We sought to determine whether reducing IgE levels invivo with omalizumab treatment increases pDC antiviral IFN-α responses in inner-city children with asthma. PBMCs and pDCs isolated from children with exacerbation-prone asthma before and during omalizumab treatment were stimulated exvivo with rhinovirus and influenza in the presence or absence of IgE cross-linking. IFN-α levels were measured in supernatants, and mRNA expression of IFN-α pathway genes was determined by using quantitative RT-PCR (qRT-PCR) in cell pellets. FcεRIα protein levels and mRNA expression were measured in unstimulated cells by using flow cytometry and qRT-PCR, respectively. Changes in these outcomes and associations with clinical outcomes were analyzed, and statistical modeling was used to identify risk factors for asthma exacerbations. Omalizumab treatment increased rhinovirus- and influenza-induced PBMC and rhinovirus-induced pDC IFN-α responses in the presence of IgE cross-linking and reduced pDC surface FcεRIα expression. Omalizumab-induced reductions in pDC FcεRIα levels were significantly associated with a lower asthma exacerbation rate during the outcome period and correlated with increases in PBMC IFN-α responses. PBMC FcεRIα mRNA expression measured on study entry significantly improved an existing model of exacerbation prediction. These findings indicate that omalizumab treatment augments pDC IFN-α responses and attenuates pDC FcεRIα protein expression and provide evidence that these effects are related. These results support a potential mechanism underlying clinical observations that allergic sensitization is associated with increased susceptibility to virus-induced asthma exacerbations.

Experimental study on substance P in the regulation of degranulation of cultured murine mast cells

Objective: To investigate the effect of interleukin-4 (IL-4) stimulation on the expression of FcεRⅠα and NK-1R on mature mast cells(MC) cultured and differentiated from mouse bone marrow stem cells, and then to study if these MC also respond to substance P (SP) both in FcεRⅠα and NK-1R dependent manners. Methods: Bone marrow cells were aseptically flushed from BALB/c mouse femurs into complete RPMI 1640, followed by culture with stem cell factor (SCF 100 μg/L), IL-3 (15 μg/L) and IL-4 (0, 10, 15, 20 and 25 μg/L, respectively). The culture medium was changed once a week. The morphological changes of culture cells were observed under inverted microscope. After 4 weeks culture, the cells were collected and appraised by toluidine blue staining and flow cytometry. The expressions of surface CD117, FcεRⅠα and NK-1R on these cells were detected by flow cytometry and Western blot. Bone marrow MC were activated with SP (0, 0.01, 0.1, 1.0 and 10 mg/L, respectively) for 30 min. The histamine released into the supernatant and stored in the protoplasm was quantified by enzyme linked immunosorbent assay (ELISA). The percentage of histamine release was calculated as a percent of total histamine content. Results: When different concentrations of IL-4 (0, 10, 15, 20, 25 μg/L)were added into RPMI 1640, the positive rates of CD117 on MC surface were expressed as (94.8±1.3)%, (95.7±2.5)%, (94.1±1.3)%, (96.6±1.0)%, and (96.6±1.1)%, respectively, and there was no significant difference among these groups (F=8.51, P>0.05). The positive rates of FcεRⅠα were expressed as (81.5±2.6)%, (84.2±1.8)%, (91.8±2.0)%, (91.6±1.6)%, and (93.0±2.6)%, respectively, and there was statistically increasing among these groups (F=15.76, P<0.05). Then MC were activated by SP (0, 0.01, 0.1, 1.0, 10 mg/L), histamine from 20 μg/L IL-4 group were released (20.08±1.50)%, (32.76±2.99)%, (42.90±3.36)%、(50.21±1.29)%, (56.10±3.60)%, as similar as from 0 μg/L IL-4 were (19.37±2.02), (19.50±1.50), (21.77±1.91), (32.00±2.50), (33.56±1.25), there was significantly different when compared with each other (all P<0.05). Bone marrow MC were shown to have the highest expression of FcεRⅠα and NK-1R in culture of 20 μg/L IL-4 by the detection of Western blot, meanwhile these MC could be activated to degranulate by a lower concentration of SP (0.01 mg/L), with the release rate of histamine from MC showing a positive correlation with SP concentrations. On the other hand, MC with high expression of FcεRⅠα and little expression of NK-1R cultured with 0 μg/L IL-4, could also be activated by a much higher concentration of SP (1.0 mg/L). Conclusions: Bone marrow mast cells were shown to be successfully differentiated and to express NK-1R and FcεRⅠα upon co-culture with SCF and IL-3 or SCF, IL-3 and IL-4.When IL-4 was added into RPMI 1640, bone marrow MC could highly produce FcεRⅠα and NK-1R, thus building a better model of MC degranulation regulated by SP. And SP-controlled MC degranulation may be mediated through both FcεRⅠα (immunologically) and NK-1R (non-IgE mediated or non-immunologically) pathway.

Innate immune responses to rhinovirus are reduced by the high-affinity IgE receptor in allergic asthmatic children

Children with allergic asthma have more frequent and severe human rhinovirus (HRV)-induced wheezing and asthma exacerbations through unclear mechanisms. We sought to determine whether increased high-affinity IgE receptor (FcεRI) expression and cross-linking impairs innate immune responses to HRV, particularly in allergic asthmatic children. PBMCs were obtained from 44 children, and surface expression of FcεRI on plasmacytoid dendritic cells (pDCs), myeloid dendritic cells, monocytes, and basophils was assessed by using flow cytometry. Cells were also incubated with rabbit anti-human IgE to cross-link FcεRI, followed by stimulation with HRV-16, and IFN-α and IFN-λ1 production was measured by Luminex. The relationships among FcεRI expression and cross-linking, HRV-induced IFN-α and IFN-λ1 production, and childhood allergy and asthma were subsequently analyzed. FcεRIα expression on pDCs was inversely associated with HRV-induced IFN-α and IFN-λ1 production. Cross-linking FcεRI before HRV stimulation further reduced PBMC IFN-α (47% relative reduction; 95% CI, 32% to 62%; P< .0001) and IFN-λ1 (81% relative reduction; 95% CI, 69% to 93%; P< .0001) secretion. Allergic asthmatic children had higher surface expression of FcεRIα on pDCs and myeloid dendritic cells when compared with that seen in nonallergic nonasthmatic children. Furthermore, after FcεRI cross-linking, allergic asthmatic children had significantly lower HRV-induced IFN responses than allergic nonasthmatic children (IFN-α, P= .004; IFN-λ1, P= .02) and nonallergic nonasthmatic children (IFN-α, P= .002; IFN-λ1, P= .01). Allergic asthmatic children have impaired innate immune responses to HRV that correlate with increased FcεRI expression on pDCs and are reduced by FcεRI cross-linking. These effects likely increase susceptibility to HRV-induced wheezing and asthma exacerbations.

Inhibitory effect of heat‐killed Lactobacillus strain on immunoglobulin E‐mediated degranulation and late‐phase immune reactions of mouse bone marrow‐derived mast cells

This study investigated the in vitro effect of Lactobacillus strains, a major group of probiotic lactic acid bacteria, on immunoglobulin E (IgE)- and antigen-induced mast cell degranulation and subsequent gene expression. Bone marrow-derived mast cells (BMMCs) from DBA/2 mice were cultured with heat-killed Lactobacillus strains for 24 h. Some strains significantly inhibited IgE- and antigen-induced β-hexosaminidase release from BMMCs. Furthermore, Lactobacillus reuteri NBRC 15892, which exhibited the strongest inhibitory activity, significantly reduced the elevated interleukin (IL)-4, IL-13, tumor necrosis factor-α, and cyclooxygenase-2 expression levels that was induced by 1-2 h of stimulation with IgE and antigens. The suppressive effect of NBRC 15892 strain on BMMC degranulation was significantly reduced in the presence of a toll-like receptor (TLR)2-neutralizing antibody. In addition, downregulation of cell surface FcεRIα expression was observed after 6 h of NBRC 15892 treatment. These results suggest that some Lactobacillus strains inhibited IgE-mediated mast cell degranulation and subsequent late-phase reactions involving mast cells via a TLR2-dependent mechanism with FcεRIα downregulation.

Counterregulation between the FcεRI Pathway and Antiviral Responses in Human Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) play essential roles in directing immune responses. These cells may be particularly important in determining the nature of immune responses to viral infections in patients with allergic asthma as well those with other atopic diseases. The purposes of this study were 1) to compare the functional capacity of pDCs in patients with one type of allergic disorder, allergic asthma, and controls; 2) to determine whether IgE cross-linking affects antiviral responses of influenza-exposed pDCs; and 3) to determine whether evidence of counterregulation of FcepsilonRIalpha and IFN-alpha pathways exists in these cells. pDC function was assessed in a subset of asthma patients and in controls by measuring IFN-alpha production after exposure of purified pDCs to influenza viruses. FcepsilonRIalpha expression on pDCs was determined by flow cytometry in blood samples from patients with allergic asthma and controls. pDCs from patients with asthma secreted significantly less IFN-alpha upon exposure to influenza A (572 versus 2815; p = 0.03), and secretion was inversely correlated with serum IgE levels. Moreover, IgE cross-linking prior to viral challenge resulted in 1) abrogation of the influenza-induced pDC IFN-alpha response; 2) diminished influenza and gardiquimod-induced TLR-7 upregulation in pDCs; and 3) interruption of influenza-induced upregulation of pDC maturation/costimulatory molecules. In addition, exposure to influenza and gardiquimod resulted in upregulation of TLR-7, with concomitant downregulation of FcepsilonRIalpha expression in pDCs. These data suggest that counterregulation of FcepsilonRI and TLR-7 pathways exists in pDCs, and that IgE cross-linking impairs pDC antiviral responses.

The Signal Peptide of the IgE Receptor α-Chain Prevents Surface Expression of an Immunoreceptor Tyrosine-based Activation Motif-free Receptor Pool

The high affinity receptor for IgE, Fc epsilon receptor I (FcepsilonRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcepsilonRI alpha-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcepsilonRI gamma-chains. Here we show that the human FcepsilonRI alpha-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K(b). This single-chain isoform of FcepsilonRI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of FcepsilonRI-alpha. Once the FcepsilonRI alpha-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcepsilonRI gamma-chain, this single-chain FcepsilonRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcepsilonRI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the FcepsilonRI alpha-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.

Circadian clock gene expression in bone marrow derived mast cells and effects on FcϵRI expression (86.12)

Abstract Many functions in the immune system are associated with time-of-day. For instance, blood histamine levels in mastocytosis patients display a diurnal variation. In addition, pulmonary function and inflammation in patients with nocturnal asthma is more severe during the night. However, it is largely unknown if local circadian clocks in immune cells directly control physiological outcomes. We hypothesized that a circadian clock in murine bone marrow derived mast cells (BMMCs) modulates their activation in vitro. Mature BMMCs were synchronized with serum rich media (50% horse serum). Total RNA was harvested at 4 hour intervals for 72 hours following synchronization and expression of circadian genes (mPer1, mPer2, Bmal1, Rev-erbα, and Dbp) and fcer1a were amplified by qPCR. The surface expression of FcϵRIα was analyzed by flow cytometry every 4 hours for 72 hours following synchronization. Serum shock induced rhythmic expression of circadian genes (mPer2, Bmal1, Rev-erbα, and Dbp) with a mean period of 25.9 hours in BMMCs. The expression of fcer1a gene and FcϵRIα protein displayed expression changes following serum shock with mean periods of 26.3 and 28.6 hours respectively as calculated by Single Cosinor Time Series Analysis. These findings demonstrate that BMMCs express circadian clock genes and FcϵRI expression is modulated by synchronization with serum rich media. Serum shock of BMMCs may provide an in vitro model to study the circadian biology of allergic disease.

Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia

Respiratory viral infections are associated with an increased risk of asthma, but how acute Th1 antiviral immune responses lead to chronic inflammatory Th2 disease remains undefined. We define a novel pathway that links transient viral infection to chronic lung disease with dendritic cell (DC) expression of the high-affinity IgE receptor (FcεRIα). In a mouse model of virus-induced chronic lung disease, in which Sendai virus triggered a switch to persistent mucous cell metaplasia and airway hyperreactivity after clearance of replicating virus, we found that FceRIa−/− mice no longer developed mucous cell metaplasia. Viral infection induced IgE-independent, type I IFN receptor–dependent expression of FcεRIα on mouse lung DCs. Cross-linking DC FcεRIα resulted in the production of the T cell chemoattractant CCL28. FceRIa−/− mice had decreased CCL28 and recruitment of IL-13–producing CD4+ T cells to the lung after viral infection. Transfer of wild-type DCs to FceRIa−/− mice restored these events, whereas blockade of CCL28 inhibited mucous cell metaplasia. Therefore, lung DC expression of FcεRIα is part of the antiviral response that recruits CD4+ T cells and drives mucous cell metaplasia, thus linking antiviral responses to allergic/asthmatic Th2 responses.

Inhibition of IgE-mediated phosphorylation of FcεRIγ protein by antiallergic drugs in rat basophilic leukemia (RBL-2H3) cells: A novel action of antiallergic drugs

We examined the effect of antiallergic drugs, azelastine and epinastine, on the expression of FcεRIα, β, and γ chains and phosphorylation of the γ chains in rat basophilic leukemia (RBL-2H3) cells. The cells were cultured for 24 h with IgE treatment in the presence of azelastine or epinastine at the concentration of 10 − 5 M. The FcεRIα mRNA expression was determined by northern blot analysis. The protein level of FcεRI expressed on the plasma membrane was examined following IgE treatment by immunoprecipitation with anti-IgE light chain, followed by western blot analysis with anti-γ chain of FcR. Azelastine and epinastine had no effect on the FcεRIα, β and γ mRNA levels. Although the amount of γ chain assembled into IgE-bound FcεRI was not changed by treatment with azelastine nor epinastine, phosphorylation levels of γ chains of IgE-bound FcεRI were inhibited by azelastine. The inhibitory effect of azelastine on the IgE-mediated expression of FcεRIγ protein is not due to their inhibition of mRNA and protein expression, but due to abrogating phosphorylation of the γ chains, which is important for initiation of FcεRI signaling cascade elicited by IgE interaction.

Omalizumab treatment downregulates dendritic cell FcεRI expression

Background Dendritic cells (DCs) are potent antigen-presenting cells that express FcεRI, the high-affinity IgE receptor. Although the downregulation of basophil FcεRI during anti-IgE therapy with omalizumab is well documented, its effect on FcεRI expression by DCs has not been reported. Objective We hypothesized that IgE regulates surface FcεRI expression by DCs in vivo and that, consequently, anti-IgE therapy decreases FcεRI expression by DCs. Methods In a randomized, double-blind, placebo-controlled clinical trial 24 subjects (16 receiving omalizumab and 8 receiving placebo) with seasonal allergic rhinitis received the study drug on days 0 and 28. Serial blood samples drawn on days 0, 7, 14, 28, and 42 were analyzed for precursor DC1 (pDC1) and pDC2 surface expression of FcεRIα by using flow cytometry. Results Omalizumab caused a significant decrease in surface FcεRI expression at all time points examined in both the pDC1 and pDC2 subsets. No significant change was seen with placebo. The maximum decrease in FcεRI expression in the omalizumab group was 52% and 83%, respectively, for the pDC1 and pDC2 subsets. The decrease in FcεRI expression by both pDC subsets correlated with the decrease in serum-free IgE and was of a similar magnitude to that found in basophils. A 10-fold decrease in IgE corresponded to a 42% and 54% decrease in surface FcεRI expression by the pDC1 and pDC2 subsets, respectively. Conclusion These results demonstrate that anti-IgE therapy causes a rapid decrease in DC surface FcεRI expression and establish that IgE is an important regulator of FcεRI expression by DCs.