High-affinity Immunoglobulin E Receptor Research Articles

The impact of atmospheric ultrafine particulate matter on IgE-mediated type 1 hypersensitivity reaction

The effect of atmospheric ultrafine particulate matter (UPM) on respiratory allergic diseases has been investigated for decades; however, the precise molecular mechanisms underlying these effects remain poorly understood. In this study, we used a simulated UPM (sUPM) generated via the spark discharge method to refine black carbon, a core particle that closely mimics real-world UPM, including the size (i.e., size of agglomerates: 165 nm) and organic carbon/elemental carbon ratio (i.e., 2.62). When 25 μg/mouse of dispersed sUPM was instilled into the lungs of mice, it promoted the infiltration and degranulation response of pulmonary mast cells, and exposure to sUPM in an immunoglobulin E (IgE)-mediated passive anaphylaxis model intensified the degranulation response of peripheral mast cells. These effects of sUPM were demonstrated to amplify the downstream signaling mechanism of the high-affinity IgE receptor (FcεRI) mediated by IgE when tested using rat basophil leukemia (RBL)−2H3 and mouse bone marrow-derived mast cells (BMMCs) collected from the bone marrow of BALB/c mice. These results indicate that airborne UPM can exacerbate type 1 hypersensitivity reactions by enhancing the IgE-mediated signaling pathways within mast cells. Furthermore, this study provided mechanistic evidence on exacerbated allergic pulmonary diseases induced by UPM inhalation.

Ligand-independent function of β2-adrenergic receptor affects IgE-mediated Ca2+ influx in mast cells

BackgroundMast cells are key effector cells that elicit immunoglobulin E (IgE)-mediated allergic inflammations. Allergen cross-linking of IgE bound to the high-affinity IgE receptor, FcεRI, on mast cells triggers signaling cascades that activate signal proteins and evoke extracellular Ca2+ influx, which are crucial for cytokine production. The β2-adrenergic receptor (Adrb2) on mast cells negatively regulates FcεRI signaling, as demonstrated by the inhibition of IgE/antigen (Ag)-induced activation by Adrb2 agonists. ObjectiveAlthough β2-adrenergic-related reagents are known to influence mast cell functions, the specific intrinsic role of Adrb2 in these cells is not fully understood, potentially because of off-target effects. In this study, the additional roles of Adrb2 in mast cells were investigated, specifically the involvement of Adrb2 in FcεRI signaling, using Adrb2−/− mice. MethodsAdrb2−/− mice were used to investigate the roles of Adrb2 in mast cells by examining bone marrow-derived mast cells (BMMCs) for surface expression of mast cell markers, granule numbers, and gene expression of mast cell proteases. Cytokine production, Ca2+ influx, and nuclear factor of activated T cells (NFAT) nuclear translocation were measured in Adrb2−/− and Adrb2+/+ BMMCs upon IgE/Ag stimulation. ResultsAdrb2−/− did not affect the generation of BMMCs, their surface expression of mast cell markers, granule numbers, or gene expression of mast cell proteases, indicating that the absence of Adrb2 had no adverse effect on mast cell development. However, Adrb2−/− BMMCs exhibited reduced tumor necrosis factor α (TNFα) production and diminished Ca2⁺ influx upon IgE/Ag stimulation, which correlated with decreased NFAT translocation. Restoration of Adrb2 in Adrb2−/− BMMCs rescued cytokine production. Notably, FcεRI-mediated phosphorylation of the phospholipase PLCγ1 and mitogen-activated protein kinases (MAPKs) remained unchanged in the absence of Adrb2. ConclusionThese results suggest that Adrb2 has a novel ligand-independent function, increasing Ca2+ entry in mast cells when stimulated with IgE/Ag.

Structural insights into the high-affinity IgE receptor FcεRI complex.

Immunoglobulin E (IgE) plays a pivotal role in allergic responses1,2. The high-affinity IgE receptor, FcεRI, found on mast cells and basophils, is central to the effector functions of IgE. FcεRI is a tetrameric complex, comprising FcεRIα, FcεRIβ and a homodimer of FcRγ (originally known asFcεRIγ), with FcεRIα recognizing the Fc region of IgE (Fcε) and FcεRIβ-FcRγ facilitating signal transduction3. Additionally, FcRγ is a crucial component of other immunoglobulin receptors, including those for IgG (FcγRI and FcγRIIIA) and IgA (FcαRI)4-8. However, the molecular basis of FcεRI assembly and the structure of FcRγ have remained elusive. Here we elucidate the cryogenic electron microscopy structure of the Fcε-FcεRI complex. FcεRIα has an essential rolein the receptor's assembly, interacting with FcεRIβ and both FcRγ subunits. FcεRIβ is structured as a compact four-helix bundle, similar to the B cell antigen CD20. The FcRγ dimer exhibits an asymmetric architecture, and coils with the transmembrane region of FcεRIα to form a three-helix bundle. A cholesterol-like molecule enhances the interaction between FcεRIβ and the FcεRIα-FcRγ complex. Our mutagenesis analyses further indicate similarities between the interaction of FcRγ with FcεRIα and FcγRIIIA, but differences in that with FcαRI. These findings deepen our understanding of the signalling mechanisms of FcεRI and offer insights into the functionality of other immune receptors dependent on FcRγ.

A Comparison of Natural and Therapeutic Anti-IgE Antibodies.

Immunoglobulin E (IgE) plays a critical role for the immune system, fighting against parasites, toxins, and cancer. However, when it reacts to allergens without proper regulation, it can cause allergic reactions, including anaphylaxis, through a process initiated by effector cells such as basophils and mast cells. These cells display IgE on their surface, bound to the high-affinity IgE receptor FcεRI. A cross-linking antigen then triggers degranulation and the release of inflammatory mediators from the cells. Therapeutic monoclonal anti-IgE antibodies such as omalizumab, disrupt this process and are used to manage IgE-related conditions such as severe allergic asthma and chronic spontaneous urticaria. Interestingly, naturally occurring anti-IgE autoantibodies circulate at surprisingly high levels in healthy humans and mice and may thus be instrumental in regulating IgE activity. Although many open questions remain, recent studies have shed new light on their role as IgE regulators and their mechanism of action. Here, we summarize the latest insights on natural anti-IgE autoantibodies, and we compare their functional features to therapeutic monoclonal anti-IgE autoantibodies.

Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Leaves and pseudostems extract of Curcuma longa attenuates immunoglobulin E/bovine serum albumin-stimulated bone marrow-derived cultured mast cell activation and passive cutaneous anaphylaxis in BALB/c mice

Ethnopharmacological relevanceCurcuma longa, known as turmeric, is an herbaceous perennial plant belonging to the genus Curcuma. It is dispersed throughout tropical and subtropical regions worldwide. Since ancient times, turmeric has been used as an ethnomedicinal plant in the Ayurvedic system, particularly in Asian countries. Rhizomes of turmeric possess several pharmacological properties that give high value as a medicinal remedy for treating a range of conditions, including inflammation, pain, allergies, and digestive issues. Moreover, turmeric leaves and pseudostems also contain a variety of health-enhancing secondary metabolites, such as curcumin, flavonoids, and other phenolic compounds, which exhibit anti-inflammatory, antitumor, antibacterial, and antioxidant properties. Aim of the studyAllergic diseases are a group of immune-mediated disorders mainly caused by an immunoglobulin E (IgE)-dependent immunological response to an innocuous allergen. Therefore, this study aimed to investigate the effect of leaves and pseudostems extract of turmeric (TLSWE-8510) on IgE/bovine serum albumin (BSA)-stimulated allergic responses in mouse bone marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in BALB/c mice. Materials and methodsThe effect of TLSWE-8510 on mast cell degranulation has been evaluated by investigating the release of β-hexosaminidase and histamine in IgE/BSA-stimulated BMCMCs. Additionally, anti-allergic properties of TLSWE-8510 on IgE/BSA-stimulated BMCMCs were investigated using suppression of nuclear factor-kappa B (NF-κB), and spleen tyrosine kinase (Syk)-linker for T-cell activation (LAT)-extracellular-signal-regulated kinase (ERK)-GRB2 associated binding protein 2 (Gab2) signaling pathway and downregulation of allergy-related cytokines and chemokines expression. Furthermore, in vivo, studies were conducted using IgE-mediated PCA in BALB/c mice. ResultsTLSWE-8510 treatment significantly inhibited the degranulation of IgE/BSA-stimulated BMCMCs by inhibiting the release of β-hexosaminidase and histamine dose-dependently. Additionally, TLSWE-8510 reduced the expression of high-affinity IgE receptors (Fc epsilon receptor I-FcεRI) on the surface of BMCMCs and the binding of IgE to FcεRI. Besides, the expression of cytokines and chemokines is triggered by IgE/BSA stimulation via activating the allergy-related signaling pathways. TLSWE-8510 dose-dependently downregulated the mRNA expression and the production of allergy-related cytokines (interleukin (IL)-1β, IL-3, IL-4, IL-5, IL-6, IL-13, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ), and chemokines (thymus and activation-regulated chemokine (TARC), and regulated upon activation, normal T cell expressed and secreted (RANTES)) by regulating the phosphorylation of downstream signaling molecules, NF-κB, and Syk, LAT, ERK and Gab2 in IgE/BSA-stimulated BMCMCs. Moreover, PCA reaction in IgE/BSA-stimulated BALB/c mice ears was effectively decreased by TLSWE-8510 treatment in a dose-dependent manner. ConclusionsThese results collectively demonstrated that TLSWE-8510 suppressed mast cell degranulation by inhibiting the release of chemical mediators related to allergies. TLSWE-8510 downregulated the allergy-related cytokines and chemokines expression and phosphorylation of downstream signaling molecules in IgE/BSA-stimulated BMCMCs. Furthermore, in vivo studies with IgE-mediated PCA reaction in the BALB/c mice ears were attenuated by TLSWE-8510 treatment. These findings revealed that TLSWE-8510 has the potential as a therapeutic agent for the treatment of allergic diseases.

Norisoboldine exerts antiallergic effects on IgE/ovalbumin-induced allergic asthma and attenuates FcεRI-mediated mast cell activation.

Allergic asthma is an inflammatory lung disorder, and mast cells play crucial roles in the development of this allergic disease. Norisoboldine (NOR), the major isoquinoline alkaloid present in Radix Linderae, has received considerable attention because it has anti-inflammatory effects. Herein, the aim of this study was to explore the antiallergic effects of NOR on allergic asthma in mice and mast cell activation. In a murine model of ovalbumin (OVA)-induced allergic asthma, oral administration at 5mg/kg body weight (BW) of NOR produced strong reductions in serum OVA-specific immunoglobulin E (IgE) levels, airway hyperresponsiveness, and bronchoalveolar lavage fluid (BALF) eosinophilia, while an increase in CD4+Foxp3+ T cells of the spleen was detected. Histological studies demonstrated that NOR treatment significantly ameliorated the progression of airway inflammation including the recruitment of inflammatory cells and mucus production by decreasing levels of histamine, prostaglandin D2 (PGD2), interleukin (IL)-4, IL-5, IL-6, and IL-13 in BALF. Furthermore, our results revealed that NOR (3∼30μM) dose-dependently reduced expression of the high-affinity receptor for IgE (FcεRI) and the production of PGD2 and inflammatory cytokines (IL-4, IL-6, IL-13, and TNF-α), and also decreased degranulation of bone marrow-derived mast cells (BMMCs) activated by IgE/OVA. In addition, a similar suppressive effect on BMMC activation was observed by inhibition of the FcεRI-mediated c-Jun N-terminal kinase (JNK) signaling pathway using SP600125, a selective JNK inhibitor. Collectively, these results suggest that NOR may have therapeutic potential for allergic asthma at least in part through regulating the degranulation and the release of mediators by mast cells.

Agarose/crystalline nanocellulose (CNC) composites promote bone marrow-derived mast cell integrity, degranulation and receptor expression but inhibit production of de novo synthesized mediators.

Introduction: Mast cells are highly granulated tissue-resident leukocytes that require a three-dimensional matrix to differentiate and mediate immune responses. However, almost all cultured mast cells rely on two-dimensional suspension or adherent cell culture systems, which do not adequately reflect the complex structure that these cells require for optimal function. Methods: Crystalline nanocellulose (CNC), consisting of rod-like crystals 4-15nm in diameter and 0.2-1µm in length, were dispersed in an agarose matrix (12.5% w/v), and bone marrow derived mouse mast cells (BMMC) were cultured on the agarose/CNC composite. BMMC were activated with the calcium ionophore A23187 or immunoglobulin E (IgE) and antigen (Ag) to crosslink high affinity IgE receptors (FcεRI). Results: BMMC cultured on a CNC/agarose matrix remained viable and metabolically active as measured by reduction of sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), and the cells maintained their membrane integrity as analyzed by measuring the release of lactate dehydrogenase (LDH) and propidium iodide exclusion by flow cytometry. Culture on CNC/agarose matrix had no effect on BMMC degranulation in response to IgE/Ag or A23187. However, culture of BMMC on a CNC/agarose matrix inhibited A23187-and IgE/Ag-activated production of tumor necrosis factor (TNF) and other mediators such as IL-1β, IL-4, IL-6, IL-13, MCP-1/CCL2, MMP-9 and RANTES by as much as 95%. RNAseq analysis indicated that BMMC expressed a unique and balanced transcriptome when cultured on CNC/agarose. Discussion: These data demonstrate that culture of BMMCs on a CNC/agarose matrix promotes cell integrity, maintains expression of surface biomarkers such as FcεRI and KIT and preserves the ability of BMMC to release pre-stored mediators in response to IgE/Ag and A23187. However, culture of BMMC on CNC/agarose matrix inhibits BMMC production of de novo synthesized mediators, suggesting that CNC may be altering specific phenotypic characteristics of these cells that are associated with late phase inflammatory responses.

Ligelizumab: A novel molecule in the management of chronic spontaneous urticaria

The next-generation high affinity monoclonal immunoglobulin E (IgE) inhibitor Ligelizumab is being tested in clinical studies to treat chronic spontaneous urticaria (CSU). In 2020, the US Food and Drug Administration (FDA) designated Ligelizumab (QGE031) as a breakthrough therapy for patients with chronic idiopathic urticaria; however, FDA clearance has not yet been granted, despite the phase 3 trial’s completion. Recent clinical investigations have shown that it has a considerable affinity for the high-affinity IgE receptor (FcRI) on the surface of immune cells, which is quite beneficial in the management of the distressing symptoms of CSU. Studies have been done comparing Ligelizumab to Omalizumab, the current standard of care for treating CSU that is refractory. While omalizumab is more effective than Ligelizumab at inhibiting IgE: CD23 interactions, Ligelizumab exhibits stronger inhibition of IgE binding to FcRI, basophil activation, IgE synthesis by B cells, and passive systemic anaphylaxis. To learn about this molecule’s extended characteristics, long-term efficacy and safety, and potential uses in other dermatological disorders, more clinical data is needed.

Regulatory T cells and immunoglobulin E: A new therapeutic link for autoimmunity?

Autoimmune diseases have a prevalence of approximately 7 to 9% and are classified as either organ-specific diseases, including type I diabetes, multiple sclerosis, inflammatory bowel disease and myasthenia gravis, or systemic diseases, including systemic lupus erythematosus, rheumatoid arthritis and Sjögren's syndrome. While many advancements have been made in understanding of the mechanisms of autoimmune disease, including the nature of self-tolerance and its breakdown, there remain unmet needs in terms of effective and highly targeted treatments. T regulatory cells (Tregs) are key mediators of peripheral tolerance and are implicated in many autoimmune diseases, either as a result of reduced numbers or altered function. Tregs may be broadly divided into those generated in the thymus (tTregs) and those generated in the periphery (pTregs). Tregs target many different immune cell subsets and tissues to suppress excessive inflammation and to support tissue repair and homeostasis: there is a fine balance between Treg cell stability and the plasticity that is required to adjust Tregs' regulatory purposes to particular immune responses. The central role of immunoglobulin E (IgE) in allergic disease is well recognized, and it is becoming increasingly apparent that this immunoglobulin also has a wider role encompassing other diseases including autoimmune disease. Anti-IgE treatment restores the capacity of plasmacytoid dendritic cells (pDCs) impaired by IgE- high-affinity IgE receptor (FcεR1) cross-linking to induce Tregs in vitro in atopic patients. The finding that anti-IgE therapy restores Treg cell homeostasis, and that this mechanism is associated with clinical improvement in asthma and chronic spontaneous urticaria suggests that anti-IgE therapy may also have a potential role in the treatment of autoimmune diseases in which Tregs are involved.

Omalizumab for the management of refractory chronic spontaneous urticaria

Chronic spontaneous urticaria (CSU) is a heterogeneous, systemic, primarily mast cell-driven disease with sudden appearance of wheals with or without angioedema with pruritus for more than 6 weeks. It affects 0.3%–0.6% of the general population. CSU is a complex immune-mediated disease due to induction of basophil and mast cell degranulation with more than one mechanism of auto-allergy Type I (mediated by specific immunoglobulin E (IgE) targeting auto-allergens as thyroperoxidase, etc.) and auto-immunity Type II (mediated by IgG auto-antibodies binding to alpha-subunit of high affinity IgE receptor [FcERI]) with increased expression of Type II cytokines and activation of coagulation cascade. Pruritus is due to sensory nerve activation by histaminergic pathway (Histamine receptors- H1R and H4R) and histamine-independent pathway (Substance P, Capsacin, and Reactive oxygen species, etc.). The international guidelines on CSU recommend to treat CSU until it is gone. The use of second-generation anti-histamine in effective dose as the first-line treatment and up-dosing of second generation (SgAH) up to four-fold is recommended in treatment-resistant patients. The humanized anti-IgE monoclonal antibody Omalizumab has been shown to have both excellent safety profiles as the only third-line treatment option in anti-histamine-resistant urticaria (approved by United States Food and Drug Administration in August, 2014). However, there are several questions to be answered with reference to the optimal dose, duration, and the rates of long-term remission with omalizumab. Other biologics as Ligelizumab are less well studied but have an important role in the specific type of omalizumab-resistant CSU. In this review, we will summarize the role of omalizumab in anti-histamine resistant CSU.

FcεRI: A Master Regulator of Mast Cell Functions.

Mast cells (MCs) perform multiple functions thought to underlie different manifestations of allergies. Various aspects of antigens (Ags) and their interactions with immunoglobulin E (IgE) cause diverse responses in MCs. FcεRI, a high-affinity IgE receptor, deciphers the Ag–IgE interaction and drives allergic responses. FcεRI clustering is essential for signal transduction and, therefore, determines the quality of MC responses. Ag properties precisely regulate FcεRI dynamics, which consequently initiates differential outcomes by switching the intracellular-signaling pathway, suggesting that Ag properties can control MC responses, both qualitatively and quantitatively. Thus, the therapeutic benefits of FcεRI-targeting strategies have long been examined. Disrupting IgE–FcεRI interactions is a potential therapeutic strategy because the binding affinity between IgE and FcεRI is extremely high. Specifically, FcεRI desensitization, due to internalization, is also a potential therapeutic target that is involved in the mechanisms of allergen-specific immunotherapy. Several recent findings have suggested that silent internalization is strongly associated with FcεRI dynamics. A comprehensive understanding of the role of FcεRI may lead to the development of novel therapies for allergies. Here, we review the qualitatively diverse responses of MCs that impact the attenuation/development of allergies with a focus on the role of FcεRI toward Ag exposure.

Anti-Allergic Effect of 3,4-Dihydroxybenzaldehyde Isolated from Polysiphonia morrowii in IgE/BSA-Stimulated Mast Cells and a Passive Cutaneous Anaphylaxis Mouse Model.

In this study, we investigated the anti-allergic effects of 3,4-dihydroxybenzaldehyde (DHB) isolated from the marine red alga, Polysiphonia morrowii, in mouse bone-marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-sensitized mice. DHB inhibited IgE/bovine serum albumin (BSA)-induced BMCMCs degranulation by reducing the release of β-hexosaminidase without inducing cytotoxicity. Further, DHB dose-dependently decreased the IgE binding and high-affinity IgE receptor (FcεRI) expression and FcεRI-IgE binding on the surface of BMCMCs. Moreover, DHB suppressed the secretion and/or the expression of the allergic cytokines, interleukin (IL)-4, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF)-α, and the chemokine, thymus activation-regulated chemokine (TARC), by regulating the phosphorylation of IκBα and the translocation of cytoplasmic NF-κB into the nucleus. Furthermore, DHB attenuated the passive cutaneous anaphylactic (PCA) reaction reducing the exuded Evans blue amount in the mouse ear stimulated by IgE/BSA. These results suggest that DHB is a potential therapeutic candidate for the prevention and treatment of type I allergic disorders.

Desensitization for the prevention of drug hypersensitivity reactions.

Drug desensitization is the temporary induction of tolerance to a sensitized drug by administering slow increments of the drug, starting from a very small amount to a full therapeutic dose. It can be used as a therapeutic strategy for patients with drug hypersensitivity when no comparable alternatives are available. Desensitization has been recommended for immunoglobulin E (IgE)-mediated immediate hypersensitivity; however, its indications have recently been expanded to include non-IgE-mediated, non-immunological, or delayed T cell-mediated reactions. Currently, the mechanism of desensitization is not fully understood. However, the attenuation of various intracellular signals in target cells is an area of active research, such as high-affinity IgE receptor (FcɛRI) internalization, anti-drug IgG4 blocking antibody, altered signaling pathways in mast cells and basophils, and reduced Ca2+ influx. Agents commonly requiring desensitization include antineoplastic agents, antibiotics, antituberculous agents, and aspirin/nonsteroidal antiinflammatory drugs. Various desensitization protocols (rapid or slow, multi-bag or one-bag, with different target doses) have been proposed for each drug. An appropriate protocol should be selected with the appropriate concentration, dosage, dosing interval, and route of administration. In addition, the protocol should be adjusted with consideration of the severity of the initial reaction, the characteristics of the drug itself, as well as the frequency, pattern, and degree of breakthrough reactions.

Calcineurin Aα Contributes to IgE-Dependent Mast-Cell Mediator Secretion in Allergic Inflammation

Mast cells (MCs) are key mediators of allergic inflammation through the activation of cross-linked immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcϵRI) on the cell surface, leading to the release of biologically potent mediators, either from preformed granules or newly synthesized. Pharmacological inhibitors have been developed to target a key signaling protein phosphatase in this pathway, calcineurin, yet there is a lack of genetic and definitive evidence for the various isoforms of calcineurin subunits in FcϵRI-mediated responses. In this study, we hypothesized that deficiency in the calcineurin Aα isoform will result in a decreased allergic immune response by the MCs. In a model of passive cutaneous anaphylaxis, there was a reduction in vascular permeability in MC-deficient mouse tissues reconstituted with calcineurin subunit A (CnAα) gene-knockout (CnAα^−/−) MCs, and in vitro experiments identified a significant reduction in release of preformed mediators from granules. Furthermore, released levels of de novo synthesized cytokines were reduced upon FcϵRI activation of CnAα^−/− MCs in vitro. Characterizing the mechanisms associated with this deficit response, we found a significant impairment of nuclear factor of kappa light polypeptide gene enhancer in B cell phosphorylation and impaired nuclear factor kappa-light-chain-enhancer of activated B-cell inhibitor alpha (NF-κB) activation. Thus, we concluded that CnAα contributes to the release of preformed mediators and newly synthesized mediators from FcϵRI-mediated activation of MCs, and this regulation includes NF-κB signaling.

Pruritus, Allergy and Autoimmunity: Paving the Way for an Integrated Understanding of Psychodermatological Diseases?

Pruritus is a key symptom in allergology and dermatology, contributing to the global and huge impact on quality of life related to skin disorders, both those which are not related to a primary dermatosis (illness) and those which are linked with primary skin lesions (disease). This is particularly evident within psychophysiological dermatoses, a group of psychodermatological diseases where there is a primary dermatosis, where psychological stress plays a role, and where pruritus may represent a major and shared symptom. The etiopathogenesis of pruritus in those disorders sheds light on the link among psychopathological features, psychological stress and the subtle interface between allergic and autoimmune mechanisms, where mast cells play a pivotal role. Allergy has long been recognised as an altered reactivity to exogenous antigens (allergens), defined as an immediate hypersensitivity mediated by immunoglobulin E (IgE). In turn, the immunological understanding of atopy is related to an immediate hypersensitivity reaction to environmental antigens involving T-helper 2 (Th2) responses and the IgE production. Mast cells are major cells in the early phase of allergy, releasing the mediators involved in the symptoms associated with the allergic disease, including pruritus, when the allergen cross-links with IgE, whose mechanisms can be observed in acute urticaria and atopy. Some allergic reactions may persist and allergy may eventually lead to autoimmunity, with the development of a T-helper 1 (Th1) and then IgE-independent inflammation. For instance, in chronic spontaneous urticaria, the mast cell activation may include autoimmune mechanisms, where autoantibodies against the extracellular α subunit of the high-affinity IgE receptor (FcεRIα) and to IgE are observed, with the involvement of Th1 lymphocytes and the production of interferon-γ (INF-γ). The role of autoimmunity is also suggested in the etiopathogenesis of other psychophysiological dermatoses, namely psoriasis, atopic dermatitis and alopecia areata. In the latter, for example, mast cells were reported to be linked with the loss of immune privilege and they are the key cells involved in the experience of pruritus, whose intensity was reported to precede and be correlated with the onset of the hair loss. Furthermore, considering that the role of hair and skin is wide, from psychosocial aspects (communication and social interaction) to vital functions (such as, temperature control), it is straightforward that they are central in our interactions and synchronization with others and the world; thereby, we may admit that the psychophysiological dermatoses could represent a loss of such synchronization. Furthermore, they are often linked with psychopathology which strongly connects with the concept of desynchronization, namely, sleep disorders and depressive symptoms, the clinical expression of a dysfunction in the interplay among mast cells, pineal gland and melatonin, thus the circadian rhythm, as well as their connection with the hypothalamic corticotrophin-releasing hormone (CRH), well-known for its key role in stress response. Moreover, increasing evidence has supported the existence of cutaneous equivalents for these mechanisms, connecting with those central pathways. Thereby, taking all these concepts into consideration, this review intends to look into the updated evidence on the shared biological mechanisms between allergy and autoimmunity, underlining pruritus as a core element, then revisiting the key role of mast cells and discussing the connection with melatonin and immune-inflammatory pathways in the physiopathology of psychophysiological dermatoses, thus paving the way for the understanding of their psychosomatic correlates and a comprehensive psychodermatological approach.

Ephedra Herb, Mao, Inhibits Antigen-Induced Mast Cell Degranulation by Induction of the Affinity Receptor for IgE Internalization.

Ephedra herb (Mao) exerts potent anti-allergic effects. This study aimed to examine the underlying mechanisms of Mao on allergic inflammation using in vitro cultured mast cells (MCs) and an in vivo model of MC-dependent anaphylaxis. Bone marrow-derived MCs (BMMCs) were presensitized with anti-2,4-dinitrophenol (DNP) immunoglobulin E (IgE) and challenged with antigens (Ag; DNP-human serum albumin). Degranulation responses and cell surface high-affinity receptor for IgE (FcεRI) expression were assessed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were administered Mao and the pathophysiological responses were evaluated. Mao inhibited Ag-induced BMMC degranulation, but not polyclonal activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, indicating that Mao inhibits IgE-dependent activation of BMMCs. Mao-treated BMMCs exhibited significant reductions in expression of surface IgE and its receptor FcεRI. Analysis of subcellular localization revealed that Mao induces FcεRI internalization in BMMCs without degranulation. In the PSA mouse model, Mao administration prevented antigen-induced hypothermia. Mao administration significantly reduced cell surface expression of IgE-bound FcεRI on peritoneal MCs. Mao induced FcεRI internalization in MCs, thereby inhibiting Ag-induced IgE-dependent degranulation. The inhibitory effects of Mao on MC degranulation may offer a novel therapeutic approach for allergic diseases.

PI3Kγ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Genome-wide Analyses of Chromatin State in Human Mast Cells Reveal Molecular Drivers and Mediators of Allergic and Inflammatory Diseases

Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca2+-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function.

Imaging Membrane Curvature inside a FcεRI-Centric Synapse in RBL-2H3 Cells Using TIRF Microscopy with Polarized Excitation.

Total internal reflection fluorescence microscopy with polarized excitation (P-TIRF) can be used to image nanoscale curvature phenomena in live cells. We used P-TIRF to visualize rat basophilic leukemia cells (RBL-2H3 cells) primed with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) coming into contact with a supported lipid bilayer containing mobile, monovalent DNP, modeling an immunological synapse. The spatial relationship of the IgE-bound high affinity IgE receptor (FcεRI) to the ratio image of P-polarized excitation and S-polarized excitation was analyzed. These studies help correlate the dynamics of cell surface molecules with the mechanical properties of the plasma membrane during synapse formation.