Surface charge, but not size, of liposomes is involved in the suppression of rat basophilic leukemia (RBL-2H3) cell degranulation mediated by Akt phosphorylation.

Cationic liposomes composed of cholesteryl-3ß-carboxyamidoethylene-N-hydroxyethylamine (OH-chol) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) inhibit mast cell degranulation mediated via crosslinking of high-affinity IgE receptors (FcεRI). Although the inhibitory efficiency of mast cell degranulation is altered by modifying the ratio of OH-chol and DOPE in cationic liposomes, the manner in which physicochemical properties, such as surface charge and size, influence suppression is not clear. We observed that positive surface charge, but not the size, of liposomes plays a role in suppressing rat basophilic leukemia (RBL-2H3) cell activation. Pretreatment with middle-ratio OH-chol liposomes (zeta potential, 62.2 ± 0.5 mV; diameter, 325.4 ± 7.3 nm) exhibited a larger suppression of RBL-2H3 cell degranulation evoked by FcεRI crosslinking compared with that by low-ratio OH-chol liposomes (zeta potential, 48.6 ± 1.9 mV; diameter, 344.4 ± 25.0 nm), although both liposomes were similarly attached to RBL-2H3 cells. Preparation of middle-ratio OH-chol liposomes, classified roughly by size using an extrusion method, revealed that the liposomal size did not affect the inhibitory efficiency of RBL-2H3 cell activation. Mechanistically, we found that middle-ratio OH-chol liposomes increased the inhibition of antigen-induced Akt phosphorylation compared to low-ratio OH-chol liposomes. We measured the phosphorylation of linker for activation of T cells (LAT) and paxillin, which are important proteins in FcεRI- and focal adhesions (FAs)-mediated signaling, respectively. Middle ratio OH-chol liposomes significantly suppressed antigen-induced paxillin phosphorylation, but did not affect LAT phosphorylation, suggesting that middle-ratio OH-chol liposomes attached to RBL-2H3 cells suppress the degranulation by impairing FA-mediated Akt phosphorylation evoked by FcεRI crosslinking.

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, FcεRIß and a homodimer of FcRγ (originally known as FcεRIγ), with FcεRIα recognizing the Fc region of IgE (Fcε) and FcεRIß-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 role in the receptor's assembly, interacting with FcεRIß and both FcRγ subunits. FcεRIß 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 FcεRIß 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.

Ligand-independent function of ß2-adrenergic receptor affects IgE-mediated Ca2+ influx in mast cells.

BACKGROUND: Mast 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 ß2-adrenergic receptor (Adrb2) on mast cells negatively regulates FcεRI signaling, as demonstrated by the inhibition of IgE/antigen (Ag)-induced activation by Adrb2 agonists. OBJECTIVE: Although ß2-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. METHODS: Adrb2-/- 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. RESULTS: Adrb2-/- 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. CONCLUSION: These results suggest that Adrb2 has a novel ligand-independent function, increasing Ca2+ entry in mast cells when stimulated with IgE/Ag.

Anti-Allergic and Anti-Inflammatory Signaling Mechanisms of Natural Compounds/Extracts in In Vitro System of RBL-2H3 Cell: A Systematic Review.

Various extracts are tested for anti-allergic or anti-inflammatory properties on in vitro models. RBL-2H3 cells are widely used in allergic or immunological studies. FCεRI and its downstream signaling cascades, such as MAPK, NF-κB, and JAK/STAT signaling pathways, are important allergic or inflammatory signaling mechanisms in mast and basophil cells. This systematic review aims to study common signaling pathways of the anti-allergic or anti-inflammatory compounds on RBL-2H3 cells. We selected the relevant research articles published after 2015 from the PubMed, Scopus, Science Direct and Web of Science databases. The risk of bias of the studies was assessed based on the modified CONSORT checklist for in vitro studies. The cell lines, treatments, assay, primary findings, and signaling pathways on RBL-2H3 cells were extracted to synthesize the results. Thirty-eight articles were included, and FCεRI and its downstream pathways, such as Lyn, Sky, PLCγ, and MAPK, were commonly studied. Moreover, the JAK/STAT pathway was a potential signaling mechanism in RBL-2H3 cells. However, the findings based on RBL-2H3 cells needed to be tested along with human mast cells to confirm its relevance to human health. In conclusion, a single plant extract may act as an anti-inflammatory reagent in RBL-2H3 cells via multiple signaling pathways besides the MAPK signaling pathway.

Distinct maturation, glucose metabolism, and inflammatory function of human monocytes-derived IDECs mediated by anti-IgE and Pam3CSK4 alone or in combination.

Background: Cell energy metabolism controls the activation and function of dendritic cells (DCs). Inflammatory dendritic epidermal cells (IDECs) in skin lesions of atopic dermatitis (AD) express high-affinity IgE receptor (FcϵRI) and toll-like receptor 2 (TLR2), which mediate the generation and maintenance of inflammation. However, cellular energy metabolism and effector function of IDECs mediated by FcϵRI and TLR2 have not been fully elucidated. Methods: IDECs in vitro were treated with TLR2 agonist Pam3CSK4 and anti-IgE alone or in combination for 24 h. Further, we analyzed the expression of cell surface activation markers, production of inflammatory factors, and cellular energy metabolism profiles of IDECs by using flow cytometry, multiplex assay, RNA sequencing, targeted energy metabolism, and seahorse assays. Results: Compared to the unstimulated or anti-IgE groups, Pam3CSK4 alone or combined with anti-IgE groups significantly increased the expression of CD80, CD83, and CD86 on IDECs, but did not affect the expression of the above markers in the anti-IgE group. The release of inflammatory cytokines increased in the Pam3CSK4 alone or combined with anti-IgE groups, while there was a weak increasing trend in the anti-IgE group. The glycolysis/gluconeogenesis pathway of carbon metabolism was affected in all treatment groups. Furthermore, compared to the control group, we found a decrease in pyruvic acid, upregulation of PFKM, downregulation of FBP1, and increase in extracellular lactate, glycolysis rate, and glycolysis capacity after all treatments, while there was no difference between each treatment group. However, there was no difference in glycolytic reserve and mitochondrial basic and maximum respiration among all groups. Conclusion: Our results indicate that glycolysis of IDECs may be activated through FcϵRI and TLR2 to upregulate inflammatory factors, suggesting that danger signals from bacteria or allergens might evoke an inflammatory response from AD through the glycolysis pathway.

Allosteric inhibition of IgE-FcεRI interactions by simultaneous targeting of IgE F(ab')2 epitopes.

Immunoglobulin E (IgE) plays pivotal roles in allergic diseases through interaction with a high-affinity receptor (FcεRI). We established that Fab fragments of anti-IgE antibodies (HMK-12 Fab) rapidly dissociate preformed IgE-FcεRI complexes in a temperature-dependent manner and inhibit IgE-mediated anaphylactic reactions, even after allergen challenge. X-ray crystallographic studies revealed that HMK-12 Fab interacts with each of two equivalent epitopes on the Cε2 homodimer domain involved in IgE F(ab')2. Consequently, HMK-12 Fab-mediated targeting of Cε2 reduced the binding affinity of Fc domains and resulted in rapid removal of IgE from the receptor complex. This unexpected finding of allosteric inhibition of IgE-FcεRI interactions by simultaneous targeting of two epitope sites on the Cε2 homodimer domain of IgE F(ab')2 may have implications for the development of novel therapies for allergic disease.

Unexpected Expression and Function of FcεRI in Immortalized Breast Cancer Cells: A Cautionary Null Study.

The high-affinity IgE receptor, FcεRI, is typically associated with type 2 effectors such as mast cells (MC). The relatively unique expression profile of FcεRI and accumulating evidence from pre-clinical and clinical settings, such as MC interactions with tumors, have led us to study MCs as a potential therapeutic target in breast cancer. Our work identified MCs interacting with tumor cells at primary sites using the 4T1 (BALB/c) adenocarcinoma model in vivo. However, this analysis was complicated by a surprising finding that the tumor cells intrinsically and strongly expressed FcεRI. We further studied the expression and function of FcεRI in breast cancer cells in vitro. The 4T1 cells expressed FcεRI to a level similar to mouse bone marrow-derived MC (BMMC). Additionally, two established breast cancer cultures derived from human T-47D cells, one estrogen-dependent (E3) and the other estrogen-withdrawn (EWD8), also expressed FcεRI with EWD8 cells showing the greatest abundance. Functional analyses indicated that IgE-mediated antigen stimulation did not elicit classic Ca2+ flux in breast cancer cells as seen in the respective species' MCs; however, FcεRI crosslinking could stimulate IL-6 production from the T-47D derivatives. Preliminary analysis of primary breast cancer biopsy datasets using R2: Genomics Analysis and Visualization Platform was discordant with our in vivo model and in vitro observations. Indeed, FcεRI mRNA abundance declined in metastatic breast cancers compared to non-cancerous breast tissue. Altogether, we report a previously unidentified and immunologically substantive difference between breast cancer models and human primary tumors. Investigators pursuing FcεRI-relevant therapeutics in this context should be aware of this translational barrier.

Flow Cytometric Identification of Human IgE+ B Lineage Subsets.

The use of flow cytometry for immunophenotyping is contingent on the ability to accurately assign biological relevance to the detected signal. This process has historically been challenging when defining IgE expressing B cells or IgE expressing antibody-secreting cells due to widespread expression of receptors for IgE on various leukocyte subsets, including human B cells. Here we describe our implementation of intracellular staining for human IgE following a blocking step to negate the challenge of surface-bound IgE. We also describe our experience with a human B cell culture system that can be used to robustly validate this approach before application to primary human samples. Orthogonal confirmatory techniques remain essential; these are not described in detail, but several possible strategies are suggested.

Antibody Fc-receptor FcεR1γ stabilizes cell surface receptors in group 3 innate lymphoid cells and promotes anti-infection immunity.

Group 3 innate lymphoid cells (ILC3) are crucial for maintaining mucosal homeostasis and regulating inflammatory diseases, but the molecular mechanisms governing their phenotype and function are not fully understood. Here, we show that ILC3s highly express Fcer1g gene, which encodes the antibody Fc-receptor common gamma chain, FcεR1γ. Genetic perturbation of FcεR1γ leads to the absence of critical cell membrane receptors NKp46 and CD16 in ILC3s. Alanine scanning mutagenesis identifies two residues in FcεR1γ that stabilize its binding partners. FcεR1γ expression in ILC3s is essential for effective protective immunity against bacterial and fungal infections. Mechanistically, FcεR1γ influences the transcriptional state and proinflammatory cytokine production of ILC3s, relying on the CD16-FcεR1γ signaling pathway. In summary, our findings highlight the significance of FcεR1γ as an adapter protein that stabilizes cell membrane partners in ILC3s and promotes anti-infection immunity.

Kamebakaurin Suppresses Antigen-Induced Mast Cell Activation by Inhibition of FcεRI Signaling Pathway.

INTRODUCTION: Kamebakaurin is an active constituent of both Rabdosia japonica and Rabdosia excisa, which are utilized in Chinese traditional medicine for improving symptoms in patients with allergies. We investigated the molecular mechanisms of the anti-allergic effects of kamebakaurin using BMMCs. METHODS: The degranulation ratio, histamine release, and the interleukin (IL)-4, leukotriene B4 (LTB4), and cysteinyl leukotriene productions on antigen-triggered BMMC were investigated. Additionally, the effects of kamebakaurin on signal transduction proteins were examined by Western blot and binding to the Syk and Lyn kinase domain was calculated. The effects of kamebakaurin on antigen-induced hyperpermeability were investigated using mouse model. RESULTS: At 10 µm, kamebakaurin partially inhibited degranulation, histamine release, and IL-4 production. At 30 µm, kamebakaurin partially reduced LTB4 and cysteinyl leukotriene productions and suppressed degranulation, histamine release, and IL-4 production. Phosphorylation of both Syk Y519/520 and its downstream protein, Gab2, was reduced by kamebakaurin, and complete inhibition was observed with 30 µm kamebakaurin. In contrast, phosphorylation of Erk was only partially inhibited, even in the presence of 30 µm kamebakaurin. Syk Y519/520 is known to be auto-phosphorylated via intramolecular ATP present in its own ATP-binding site, and this auto-phosphorylation triggers degranulation, histamine release, and IL-4 production. Docking simulation study indicated kamebakaurin blocked ATP binding to the ATP-binding site in Syk. Therefore, inhibition of Syk auto-phosphorylation by kamebakaurin binding to the Syk ATP-binding site appeared to cause a reduction of histamine release and IL-4 production. Kamebakaurin inhibited antigen-induced vascular hyperpermeability in a dose-dependent fashion but did not reduce histamine-induced vascular hyperpermeability. CONCLUSION: Kamebakaurin ameliorates allergic symptoms via inhibition of Syk phosphorylation; thus, kamebakaurin could be a lead compound for the new anti-allergic drug.

PrP is cleaved from the surface of mast cells by ADAM10 and proteases released during degranulation.

While several functions of the endogenous prion protein have been studied, the homeostatic function of prion protein is still debated. Notably, prion protein is highly expressed on mast cells, granular immune cells that regulate inflammation. When activated, mast cells shed prion protein, although the mechanism and consequences of this are not yet understood. First, we tested several mast cell lines and found that, while prion protein was almost always present, the total amount differed greatly. Activation of mast cells induced a cleavage of the N-terminal region of prion protein, and this was reduced by protease inhibitors. Exogenous mast cell proteases caused a similar loss of the prion protein N-terminus. Additionally, mast cells shed prion protein in an ADAM10-dependent fashion, even in the absence of activation. Our results suggest that prion protein is cleaved from resting mast cells by ADAM10 and from activated mast cells by mast cell proteases. Prion protein also appears to affect mast cell function, as Prnp-/- bone marrow-derived mast cells showed lower levels of degranulation and cytokine release, as well as lower levels of both FcεRI and CD117. Finally, we sought to provide clinical relevance by measuring the levels of prion protein in bodily fluids of asthmatic patients, a disease that involves the activation of mast cells. We found an N-terminal fragment of prion protein could be detected in human sputum and serum, and the amount of this prion protein fragment was decreased in the serum of patients with asthma.

SR-BI regulates the synergistic mast cell response by modulating the plasma membrane-associated cholesterol pool.

The high-affinity IgE receptor FcεRI is the mast cell (MC) receptor responsible for the involvement of MCs in IgE-associated allergic disorders. Activation of the FcεRI is achieved via crosslinking by multivalent antigen (Ag) recognized by IgE resulting in degranulation and proinflammatory cytokine production. In comparison to the T- and B-cell receptor complexes, for which several co-receptors orchestrating the initial signaling events have been described, information is scarce about FcεRI-associated proteins. Additionally, it is unclear how FcεRI signaling synergizes with input from other receptors and how regulators affect this synergistic response. We found that the HDL receptor SR-BI (gene name: Scarb1/SCARB1) is expressed in MCs, functionally associates with FcεRI, and regulates the plasma membrane cholesterol content in cholesterol-rich plasma membrane nanodomains. This impacted the activation of MCs upon co-stimulation of the FcεRI with receptors known to synergize with FcεRI signaling. Amongst them, we investigated the co-activation of the FcεRI with the receptor tyrosine kinase KIT, the IL-33 receptor, and GPCRs activated by adenosine or PGE2. Scarb1-deficient bone marrow-derived MCs showed reduced cytokine secretion upon co-stimulation conditions suggesting a role for plasma membrane-associated cholesterol regulating respective MC activation. Mimicking Scarb1 deficiency by cholesterol depletion employing MßCD, we identified PKB and PLCγ1 as cholesterol-sensitive proteins downstream of FcεRI activation in bone marrow-derived MCs. When MCs were co-stimulated with stem cell factor (SCF) and Ag, PLCγ1 activation was boosted, which could be mitigated by cholesterol depletion and SR-BI inhibition. Similarly, SR-BI inhibition attenuated the synergistic response to PGE2 and anti-IgE in the human ROSAKIT WT MC line, suggesting that SR-BI is a crucial regulator of synergistic MC activation.

Novel IgE crosslinking-induced luciferase expression method using human-rat chimeric IgE receptor-carrying mast cells.

BACKGROUND: The measurement of antigen-specific serum IgE is common in clinical assessments of type I allergies. However, the interaction between antigens and IgE won't invariably trigger mast cell activation. We previously developed the IgE crosslinking-induced luciferase expression (EXiLE) method using the RS-ATL8 mast cell line; however, the method may not be sensitive enough in some cases. METHODS: In this study, we introduced an NF-AT-regulated luciferase reporter gene into the RBL-2H3 rat mast cell line and expressed a chimeric high-affinity IgE receptor (FcεRI) α chain gene, comprising an extracellular domain from humans and transmembrane/intracellular domains from rats. RESULTS: We generated multiple clones expressing the chimeric receptor. Based on their responsiveness and proliferation, we selected the HuRa-40 clone. This cell line exhibited significantly elevated human α chain expression compared to RS-ATL8 cells, demonstrating a 10-fold enhancement of antigen-specific reactivity. Reproducibility across different batches and operators was excellent. Moreover, we observed a detectable response inhibition by an anti-allergy drugs (omalizumab and cyclosporin A). CONCLUSIONS: HuRa-40 cells-which carry the human-rat chimeric IgE receptor-comprise a valuable reporter cell line for the EXiLE method. Their versatility extends to various applications and facilitates high-throughput screening of anti-allergy drugs.

Lin-CD117+CD34+FcεRI+ progenitor cells are increased in chronic spontaneous urticaria and predict clinical responsiveness to anti-IgE therapy.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a common, debilitating skin disorder characterized by recurring episodes of raised, itchy and sometimes painful wheals lasting longer than 6 weeks. CSU is mediated by mast cells which are absent from peripheral blood. However, lineage-CD34hiCD117int/hiFcεRI+ cells in blood have previously been shown to represent a mast cell precursor. METHODS: We enumerated FcεRI-, FcεRI+ and FcεRIhi lineage-CD34+CD117+ cells using flow cytometry in blood of patients with CSU (n = 55), including 12 patients receiving omalizumab and 43 not receiving omalizumab (n = 43). Twenty-two control samples were studied. Disease control and patient response to omalizumab was evaluated using the urticaria control test. We performed single-cell RNA sequencing (scRNA-Seq) on lineage-CD34hiCD117hi blood cells from a subset of patients with CSU (n = 8) and healthy controls (n = 4). RESULTS: CSU patients had more lineage-CD34+CD117+FcεRI+ blood cells than controls. Lineage-CD34+CD117+FcεRI+ cells were significantly higher in patients with CSU who had an objective clinical response to omalizumab when compared to patients who had poor disease control 90 days after initiation of omalizumab. scRNA-Seq revealed that lineage-CD34+CD117+FcεRI+ cells contained both lymphoid and myeloid progenitor lineages, with omalizumab responsive patients having proportionally more myeloid progenitors. The myeloid progenitor lineage contained small numbers of true mast cell precursors along with more immature FcεRI- and FcεRI+ myeloid progenitors. CONCLUSION: Increased blood CD34+CD117+FcεRI+ cells may reflect enhanced bone marrow egress in the setting of CSU. High expression of these cells strongly predicts better clinical responses to the anti-IgE therapy, omalizumab.

Mast cell signaling and its role in urticaria.

Chronic urticaria is a mast cell (MC)-driven disease characterized by the development of itching wheals and/or angioedema. In the last decades, outstanding progress has been made in defining the mechanisms involved in MC activation, and novel activating and inhibitory receptors expressed in MC surface were identified and characterized. Besides an IgE-mediated activation through high-affinity IgE receptor cross-linking, other activating receptors, including Mas-related G-protein-coupled receptor-X2, C5a receptor, and protease-activated receptors 1 and 2 are responsible for MC activation. This would partly explain the reason some subgroups of chronic spontaneous urticaria (CSU), the most frequent form of urticaria in the general population, do not respond to IgE target therapies, requiring other therapeutic approaches for improving the management of the disease. In this review, we shed some light on the current knowledge of the immunologic and nonimmunologic mechanisms regulating MC activation in CSU, considering the complex inflammatory scenario underlying CSU pathogenesis, and novel potential MC-targeted therapies, including surface receptors and cytoplasmic signaling proteins.

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

The Role Played by Autophagy in FcεRI-Dependent Activation of Mast Cells.

The significant role of mast cells in the development of allergic and inflammatory diseases is well-established. Among the various mechanisms of mast cell activation, the interaction of antigens/allergens with IgE and the subsequent binding of this complex to the high-affinity IgE receptor FcεRI stand out as the most studied and fundamental pathways. This activation process leads to the rapid exocytosis of granules containing preformed mediators, followed by the production of newly synthesized mediators, including a diverse array of cytokines, chemokines, arachidonic acid metabolites, and more. While conventional approaches to allergy control primarily focus on allergen avoidance and the use of antihistamines (despite their associated side effects), there is increasing interest in exploring novel methods to modulate mast cell activity in modern medicine. Recent evidence suggests a role for autophagy in mast cell activation, offering potential avenues for utilizing low-molecular-weight autophagy regulators in the treatment of allergic diseases. More specifically, mitochondria, which play an important role in the regulation of autophagy as well as mast cell activation, emerge as promising targets for drug development. This review examines the existing literature regarding the involvement of the molecular machinery associated with autophagy in FcεRI-dependent mast cell activation.

Betulinic Acid Potentiates Mast Cell Degranulation by Compromising Cell Membrane Integrity and Without Involving Fcεri Receptors.

Mast cells play important role in acquired and natural immunity making these favorable therapeutic targets in various inflammatory diseases. Here we observed that, pentacyclic tri terpenoid betulinic acid (BA) treatment resulted in a significantly high number (9%) of cells positive for Hoechst and negative for annexin-V indicating that BA could interfere with plasma membrane integrity. The degranulation of both activated and non-activated mast cells was enhanced upon treatment with BA. The pre-treatment of BA had remarkable effect on calcium response in activated mast cells which showed increased calcium influx relative compared to untreated cells. The results also showed potentially less migration of BA treated mast cells signifying the possible effect of BA on cell membrane. BA treatment resulted in a significant increase in mRNA levels of IL-13 while as mRNA levels of other target cytokines, IL-6 and TNF-α seem to be not affected. Moreover, there was global Increase in phosphorylation of signaling proteins and no significant change in phosphorylation of FcεRI receptors indicating that the effect of BA was independent of signaling cascade or FcεRI receptor mediated mast cell aggregation. Overall, these results portray BA potentiates mast cell effector functions by compromising the membrane integrity and independent of FcεRI involvement.

Biomarkers of Autologous Whole Blood Injection Efficacy in Patients with Chronic Spontaneous Urticaria with Autoreactivity: A Preliminary Study.

INTRODUCTION: Chronic spontaneous urticaria (CSU) with autoreactivity is often resistant to antihistamines. Autologous whole blood injection (AWBI) has shown potential efficacy in the treatment of this disease, but it is controversial. It is necessary to screen patients who are suitable for this therapy in advance. This study aimed to identify biomarkers that predict the efficacy of AWBI treatment in CSU patients with autoreactivity. METHODS: A total of 30 patients with autologous serum skin test-positive CSU treated with AWBI were included in this study; urticaria activity score (UAS7) was recorded and the treatment response was judged based on it. Levels of total serum IgE, anti-high-affinity IgE receptor (FcεRI) IgG, and basophils CD63 and FcεRI expressions, and D-dimer of all patients were determined and analyzed. RESULTS: Baseline levels of total IgE, D-dimer, basophil FcεRI and CD63 expressions showed good correlations with UAS7 variations. D-dimer, basophil FcεRI and CD63 expressions changed significantly before and after AWBI treatment in AWBI responders, and the basophil FcεRI and CD63 expressions consistently and dynamically decreased in AWBI responders during the treatment. Baseline levels of total IgE, D-dimer, basophil FcεRI and CD63 expressions showed certain predictive values for AWBI response. CONCLUSIONS: Baseline levels of total IgE, D-dimer, basophil FcεRI and CD63 expressions could be biomarkers of predicting AWBI efficacy in patients with CSU with autoreactivity.

Cannabidiol Inhibits IgE-Mediated Mast Cell Degranulation and Anaphylaxis in Mice.

SCOPE: Cannabidiol (CBD), the most abundant non-psychoactive constituent of the plant Cannabis sativa, is known to possess immune modulatory properties. This study investigates the effects of CBD on mast cell degranulation in human and mouse primary mast cells and passive cutaneous anaphylaxis in mice. METHODS AND RESULTS: Mouse bone marrow-derived mast cells and human cord-blood derived mast cells are generated. CBD suppressed antigen-stimulated mast cell degranulation in a concentration-dependent manner. Mechanistically, CBD inhibited both the phosphorylation of FcεRI downstream signaling molecules and calcium mobilization in mast cells, while exerting no effect on FcεRI expression and IgE binding to FcεRI. These suppressive effects are preserved in the mast cells that are depleted of type 1 (CB1) and type 2 (CB2) cannabinoid receptors, as well as in the presence of CB1 agonist, CB2 agonist, CB1 inverse agonist, and CB2 inverse agonist. CBD also inhibited the development of mast cells in a long-term culture. The intraperitoneal administration of CBD suppressed passive cutaneous anaphylaxis in mice as evidenced by a reduction in ear swelling and decrease in the number of degranulated mast cells. CONCLUSION: Based on these results, the administration of CBD is a new therapeutic intervention in mast cell-associated anaphylactic diseases.