Mast Cell Receptor Research Articles

Histamine and TH2 cytokines regulate the biosynthesis of cysteinyl-leukotrienes and expression of their receptors in human mast cells

IntroductionIn skin lesions of atopic dermatitis (AD), a chronic inflammatory skin disease, mast cells beyond other immune cells are present in increasing numbers. Upon activation, mast cells release a plethora of mediators, in particular histamine and leukotrienes, as well as chemokines and cytokines, which modulate the immune response of cells in their microenvironment and may influence mast cells in an autocrine loop. This study investigated the effects of histamine and TH2 cytokines on the biosynthesis of cysteinyl leukotrienes (CysLTs) as well as CysLT receptor expression on human mast cells from healthy volunteers and patients with AD.MethodsHuman mast cells were generated from CD34+ progenitor cells from peripheral blood. The cultured mast cells were stimulated with IL-4, IL-13, histamine and different histamine receptor selective ligands. Expression of enzymes in the biosynthesis of leukotrienes and expression of CysLT receptors were quantified by real-time PCR. The release of CysLTs was measured by ELISA. ResultsMast cells from AD patients showed higher expression of 5-Lipoxygenase (5-LO) and 5-Lipoxygenase activating protein (FLAP) compared to mast cells from healthy volunteers at baseline and in presence of histamine and TH2 cytokines. Expression of leukotriene C4 synthase (LTC4S), the biosynthesis of CysLTs, and mRNA expression of both CysLT receptors were induced by histamine and TH2 cytokines in mast cells from healthy volunteers and AD patients.ConclusionWe provide evidence that in an acute allergic situation histamine and TH2 cytokines may activate the biosynthesis of pro-allergic cysteinyl leukotrienes and up-regulation of CysLT receptor expression in human mast cells. This suggests a novel mechanism for sustaining mast cell activation through a possible autocrine signalling loop under these conditions.

Scratching promotes allergic inflammation and host defense via neurogenic mast cell activation.

Itch is a dominant symptom in dermatitis, and scratching promotes cutaneous inflammation, thereby worsening disease. However, the mechanisms through which scratching exacerbates inflammation and whether scratching provides benefit to the host are largely unknown. We found that scratching was required for skin inflammation in mouse models dependent on FcεRI-mediated mast cell activation. Scratching-induced inflammation required pain-sensing nociceptors, the neuropeptide substance P, and the mast cell receptor MrgprB2. Scratching also increased cutaneous inflammation and augmented host defense to superficial Staphylococcus aureus infection. Thus, through the activation of nociceptor-driven neuroinflammation, scratching both exacerbated allergic skin disease and provided protection from S. aureus, reconciling the seemingly paradoxical role of scratching as a pathological process and evolutionary adaptation.

Pharmacological blockade of the mast cell MRGPRX2 receptor supports investigation of its relevance in skin disorders.

Because MRGPRX2 is now recognized as the mast cell receptor for basic secretagogues, there is currently a tremendous interest in whether MRGRPX2 could play an important role in various pruritic dermatoses such as chronic spontaneous urticaria. Therefore, we sought to identify new potent and selective antagonists to pharmacologically characterize the biological role of MRGPRX2. Various relevant in vitro, ex vivo, and in vivo model systems were used to investigate the role of MRGPRX2. This included the study of freshly isolated human skin mast cells and human basophils as well as an ex vivo human skin microdialysis preparation. The additivity of MRGPRX2 and FcεR1-mediated degranulation was also investigated. Human MRGPRX2 knock-in mice were generated to interrogate pharmacokinetic/pharmacodynamic relationships because both antagonists studied were shown to be human specific. Two novel and structurally distinct MRGPRX2 antagonists were identified with one, Compound B, being orally active and demonstrating high potency in blocking Substance P-mediated degranulation using freshly isolated human skin mast cells with half maximal inhibitory concentration (IC50) at 0.42 nM. Compound B also potently blocked Substance P-stimulated histamine release from resident mast cells in a human skin explant setup as well as blocking itch in an established behavioral scratching model using MRGPRX2 knock-in mice. Unlike human mast cells, Substance P failed to elicit a functional response in human basophils. These data fully support the investigation of MRGPRX2 receptor antagonists in mast cell-driven allergic skin disorders such as chronic spontaneous urticaria.

Urticaria: An update on pathophysiology, diagnosis, and management

Abstract Urticaria is a relatively common clinical skin disorder, and the condition is an inflammatory allergic immune response in the skin mucosa which increases the permeability of the subcutaneous capillaries. Localized, treatment-resistant skin edema is a characteristic symptom of urticaria. The activation of mast cells located in the dermal layer of the skin and consequent release of histamines is thought to be the primary cause of the pathology of urticaria. The broad definition of urticaria also includes contact urticaria and measles vasculitis, and recent studies have shown that almost half of patients with persistent spontaneous urticaria have mast cell receptor autoimmunity. These patients need immediate attention, because they may also have other severe medical or surgical conditions. The low number of studies on chronic urticaria means that thorough serological investigation of patients is not typically advised clinically. There is presently no scientific evidence connecting malignancy or persistent infections to urticaria.

MRGPRX2 antagonist GE1111 attenuated DNFB-induced atopic dermatitis in mice by reducing inflammatory cytokines and restoring skin integrity.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterised by itching, erythema, and epidermal barrier dysfunction. The pathogenesis of AD is complex and multifactorial; however,mast cell (MC) activation has been reported to be one of the crucial mechanisms in the pathogenesis of AD. The MC receptor Mas related G protein-coupled receptor-X2 (MRGPRX2) has been identified as a prominent alternative receptor to the IgE receptor in causing MC activation and the subsequent release of inflammatory mediators. The current study aimed to evaluate the therapeutic effect of a novel small molecule MRGPRX2 antagonist GE1111 in AD using in vitro and in vivo approaches. We developed an in vitro cell culture disease model by using LAD-2 MC, HaCaT keratinocytes and RAW 264.7 macrophage cell lines. We challenged keratinocytes and macrophage cells with CST-14 treated MC supernatant in the presence and absence of GE1111 and measured the expression of tight junction protein claudin 1, inflammatory cytokines and macrophage phagocytosis activity through immunohistochemistry, western blotting, RT-qPCR and fluorescence imaging techniques. In addition to this, we developed a DFNB-induced AD model in mice and evaluated the protective effect and underlying mechanism of GE1111. Our in vitro findings demonstrated a potential therapeutic effect of GE1111, which inhibits the expression of TSLP, IL-13, MCP-1, TNF-a, and IL-1ß in MC and keratinocytes. In addition to this, GE1111 was able to preserve the expression of claudin 1 in keratinocytes and the phagocytotic activity of macrophage cells. The in vivo results demonstrated that GE1111 treatment significantly reduced phenotypic changes associated with AD (skin thickening, scaling, erythema and epidermal thickness). Furthermore, immunohistochemical analysis demonstrated that GE1111 treatment preserved the expression of the tight junction protein Involucrin and reduced the expression of the inflammatory mediator periostin in the mouse model of AD. These findings were supported by gene and protein expression analysis, where GE1111 treatment reduced the expression of TSLP, IL-13, and IL-1ß, as well as downstream signalling pathways of MRGPRX2 in AD skin lesions. In conclusion, our findings provide compelling in vitro and in vivo evidence supporting the contribution of MRGPRX2-MC interaction with keratinocytes and macrophages in the pathogenesis of AD.

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 MβCD, 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.

MRGPRB2/X2 and the analogous effects of its agonist and antagonist in DSS-induced colitis in mice

The mast cell receptor Mrgprb2, a mouse orthologue of human Mrgprx2, is known as an inflammatory receptor and its elevated expression is associated with various diseases such as ulcerative colitis. We aimed to elucidate the role of Mrgprb2/x2 and the effect of its ligands on a chemically induced murine colitis model. We showed that in Mrgprb2-/- mice, there is a differential regulation of cytokine releases in the blood plasma and severe colonic damages after DSS treatment. Unexpectedly, we demonstrated that known Mrgprb2/x2 agonists (peptide P17, P17 analogues and CST-14) and antagonist (GE1111) similarly increased the survival rate of WT mice subjected to 4% DSS-induced colitis, ameliorated the colonic damages of 2.5% DSS-induced colitis, restored major protein mRNA expression involved in colon integrity, reduced CD68+ and F4/80+ immune cell infiltration and restored cytokine levels. Collectively, our findings highlight the eminent role of Mrpgrb2/x2 in conferring a beneficial effect in the colitis model, and this significance is demonstrated by the heightened severity of colitis with altered cytokine releases and inflammatory immune cell infiltration observed in the Mrgprb2 knockout mice. Elevated expression of Mrgprb2 in WT colitis murine models may represent the organism’s adaptive protective mechanism since Mrgprb2 knockout results in severe colitis. On the other hand, both agonist and antagonist of Mrgprb2 analogously mitigated the severity of colitis in DSS-induced colitis model by altering Mrgprb2 expression, immune cell infiltration and inflammatory cytokine releases.

Regulation of mast cells by overlapping but distinct protein interactions of Siglec-6 and Siglec-8.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in exvivo and invivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.

Charge variants of proposed biosimilar to Omalizumab: Isolation, purification and analysis by HPLC methods

Omalizumab (Xolair) is a humanized monoclonal antibody derived by recombinant DNA technology. It binds specifically to immunoglobulin E (IgE) which plays a major role in allergic reaction by releasing histamine and other inflammatory factors from mast cells. Omalizumab binds circulatory IgE with high affinity and prevents from its binding to mast cell receptor. Charge variants are one of the critical quality attributes (CQAs) in biological drug development and sources of heterogeneity which needs to be considered in biosimilarity assessment. In this study, biosimilar product of Xolair was expressed in mammalian cell culture process in laboratory to isolate charge variants (acidic, main peak and basic). Different charge variants were isolated from intermediate purified biosimilar product of Xolair. Isolated charge variants were purified with preparative cation exchange chromatography technique and characterized with different analytical tools includes size exclusion chromatography (SEC-HPLC) and cation exchange chromatography (CEX-HPLC). Purity of acidic, main peak and basic variants was 99.58%, 99.98% and 98.64% respectively as per SEC-HPLC and according to CEX-HPLC purity was 94.25%, 95.58% and 91.33% respectively. The study data indicates that isolated charge variants were purified with desired purity and can be further used for process characterization, in vitro potency and in vivo kinetics studies.

MRGPRX2, drug pseudoallergies, inflammatory diseases, mechanisms and distinguishing MRGPRX2- and IgE/FcεRI-mediated events.

MRGPRX2, a novel Gaq -coupled human mast cell receptor, mediates non-immune adverse reactions without the involvement of antibody priming. Constitutively expressed by human skin mast cells, MRGPRX2 modulates cell degranulation producing pseudoallergies manifesting as itch, inflammation and pain. The term pseudoallergy is defined in relation to adverse drug reactions in general and immune/non-immune-mediated reactions in particular. A list of drugs with MRGPRX2 activity is presented, including a detailed examination of three important and widely used approved therapies: neuromuscular blockers, quinolones and opioids. For the clinician, the significance of MRGPRX2 is considered as an aid in distinguishing and ultimately identifying specific immune and non-immune inflammatory reactions. Anaphylactoid/anaphylactic reactions, neurogenic inflammation and inflammatory diseases with a clear or strongly suspected association with MRGPRX2 activation are examined. Inflammatory diseases include chronic urticaria, rosacea, atopic dermatitis, allergic contact dermatitis, mastocytosis, allergic asthma, ulcerative colitis and rheumatoid arthritis. MRGPRX2- and allergic IgE/FcεRI-mediated reactions may be clinically similar. Importantly, the usual testing procedures do not distinguish the two mechanisms. Currently, identification of MRGPRX2 activation and diagnosis of pseudoallergic reactions is generally viewed as a process of exclusion once other non-immune and immune processes, particularly IgE/FcεRI-mediated degranulation of mast cells, are ruled out. This does not take into account that MRGPRX2 signals via β-arrestin, which can be utilized to detect MRGPRX2 activation by employing MRGPRX2 transfected cells to assess MRGPRX2 activation via two pathways, the G-protein-independent β-arrestin pathway and the G-protein-dependent Ca2+ pathway. Testing procedures, interpretations for distinguishing mechanisms, patient diagnosis, agonist identification and drug safety evaluations are addressed.

IL-10 Modulates the Expression and Activation of Pattern Recognition Receptors in Mast Cells.

Mast cells (MCs) are involved in several immune-related responses, including those in bacterial infections, autoimmune diseases, inflammatory bowel diseases, and cancer, among others. MCs identify microorganisms by pattern recognition receptors (PRRs), activating a secretory response. Interleukin (IL)-10 has been described as an important modulator of MC responses; however, its role in PRR-mediated activation of MC is not fully understood. We analyzed the activation of TLR2, TLR4, TLR7 and Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mucosal-like MCs (MLMCs) and peritoneum-derived cultured MCs (PCMCs) from IL-10-/- and wild-type (WT) mice. IL-10-/- mice showed a reduced expression of TLR4 and NOD2 at week 6 and TLR7 at week 20 in MLMC. In MLMC and PCMC, TLR2 activation induced a reduced secretion of IL-6 and TNFα in IL-10-/- MCs. TLR4- and TLR7-mediated secretion of IL-6 and TNFα was not detected in PCMCs. Finally, no cytokine release was induced by NOD2 ligand, and responses to TLR2 and TLR4 were lower in MCs at 20 weeks. These findings indicate that PRR activation in MCs depends on the phenotype, ligand, age, and IL-10.

Degranulation of Mast Cells as a Target for Drug Development.

Mast cells act as key effector cells of inflammatory responses through degranulation. Mast cell degranulation is induced by the activation of cell surface receptors, such as FcεRI, MRGPRX2/B2, and P2RX7. Each receptor, except FcεRI, varies in its expression pattern depending on the tissue, which contributes to their differing involvement in inflammatory responses depending on the site of occurrence. Focusing on the mechanism of allergic inflammatory responses by mast cells, this review will describe newly identified mast cell receptors in terms of their involvement in degranulation induction and patterns of tissue-specific expression. In addition, new drugs targeting mast cell degranulation for the treatment of allergy-related diseases will be introduced.

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.

Developments and perspectives in allergology.

Allergology is a key part of dermatological care. This paper reviews current pathophysiological, diagnostic and therapeutic developments in immediate-type allergies. Type-2 inflammation is involved in several allergological diseases such as allergic rhinitis and asthma. Allergen immunotherapy as an important therapeutic procedure is regulated in Germany by an official legal directive (Therapieallergene-Verordnung). Therapeutically, several biologics are already available that target interleukin (IL)-4, -5, -13, -33, or TSLP (thymic stromal lymphopoietin). Collateral efficacy may result in simultaneous treatment of allergological comorbidities. In mast cell mediated diseases (urticaria, anaphylaxis), there is increasing understanding of mast cell activation pathways. Several mast cell receptors such as MRGPRX2 (mas-related G protein coupled receptor X2) and Siglec-8 (sialinic acid binding Ig like lectin-8) as well as intracellular signaling pathways have recently been identified. Clinical trials are underway with drugs acting on mast cell receptors and intracellular signaling, i.e., Bruton's tyrosine kinase inhibitors. Further perspectives on biomarkers, novel therapeutics and unmet needs for future research activities are presented.

Hydroquinone-induced skin irritant reaction could be achieved by activating mast cells via mas-related G protein-coupled receptor X2.

Hydroquinone (HQ) is one of the most effective drugs to treat hyperpigmentary disorders, but often causes skin irritation in clinic. Mast cell plays an important role in contact dermatitis and triggering pseudo-allergic reactions via MRGPRX2. Whether HQ-induced skin irritant reaction through activating mast cells via MRGPRX2 remains unknown. To investigate the role of mast cells in HQ-induced skin irritant reaction and verify whether MRGPRX2 participated in the HQ effect on mast cells which contributed to the pathogenesis of skin irritant reaction, a mouse model of HQ-induced skin irritation was established to observe the local and systemic inflammation associated with mast cell receptor MrgprB2. Human mast cell LAD2 was used to verify the effect of HQ on mast cells via MRGPRX2 by knocking down with siRNA. As a result, mast cells were involved in the development of HQ-induced irritant reaction, and local inflammation is closely related to mast cell receptor MrgprB2. HQ could activate mast cells via MRGPRX2, causing changes in calcium concentration, degranulation and release of inflammatory cytokines which lead to skin irritant reaction. In conclusion, HQ-induced skin irritant reaction could be skin pseudo-allergic reactions achieved by activating mast cells via MRGPRX2.

Rutin ameliorates inflammatory pain by inhibiting P2X7 receptor in mast cells.

Rutin is a natural anti-inflammatory ingredient widely found in medicinal plants. Studies have shown that rutin inhibits mast cell degranulation and the release of inflammatory mediators. Mast cell P2X7 receptor mediates mast cell degranulation and serves as a therapeutic target for inflammatory pain. Herein, the aim of this study was to investigate whether the anti-inflammatory mechanism of rutin is related to the mast cell P2X7 receptor. Our results showed that rutin could inhibit [Ca2+]i elevation induced by 5mM ATP or 30μM BZATP in a concentration-dependent manner in mouse peritoneal mast cells. Rutin also suppressed the inward current mediated by P2X7 receptor. In vivo, rutin could significantly inhibit the mechanical hypersensitivity induced by 100mM ATP that is associated with P2X7 receptor in mast cells. Moreover, molecular docking revealed the high affinity between rutin and the P2X7 receptor crystal structure. Collectively, this study demonstrated that rutin attenuated inflammatory pain by inhibiting the activity of P2X7 receptor in mast cells.

Opioid toxicity: histamine, hypersensitivity, and MRGPRX2.

Insights into the pathophysiology of many non-immune-mediated drug reactions referred to as toxicities, sensitivities, intolerances, or pseudoallergies have resulted from research identifying the mastocyte-related G-protein-coupled receptor (GPCR) member X2 (MRGPRX2), a human mast cell receptor mediating adverse reactions without the involvement of antibody priming. Opioid-induced degranulation of mast cells, particularly morphine, provoking release of histamine and other preformed mediators and causing hemodynamic and cutaneous changes seen as flushing, headache and wheal and flare reactions in the skin, is an example of results of MRGPRX2 activation. Opioids including morphine, codeine, dextromethorphan and metazocine as well as endogenous prodynorphin opioid peptides activate MRGPRX2 at concentrations causing mast cell degranulation. Unlike the canonical opioid receptors, MRGPRX2 shows stereochemical recognition preference for dextro rather than levo opioid enantiomers. Opioid analgesic drugs (OADs) display a range of histamine-releasing potencies from the strong releaser morphine to doubtful releasers like hydromorphone and the non-releaser fentanyl. Whether there is a correlation between histamine release by individual OADs, MRGPRX2 activation, and presence or absence of adverse cutaneous effects is not known. To investigate the question, ongoing research with recently pursued methodologies and strategies employing basophil and mast cell tests resulting from MRGPRX2 insights should help to elucidate whether or not an opioid's histamine-releasing potency, and its property of provoking an adverse reaction, are each a reflection of its activation of MRGPRX2.

The Number of MRGPRX2-Expressing Cells Is Increased in Skin Lesions of Patients With Indolent Systemic Mastocytosis, But Is Not Linked to Symptom Severity.

BackgroundRecently, the expression of the mast cell (MC) receptor Mas-related G protein–coupled receptor X2 (MRGPRX2) has been detected in lesional skin of adult patients with cutaneous mastocytosis. As of yet, little is known about the clinical relevance of MRGPRX2 and its agonists in patients with mastocytosis, including indolent systemic mastocytosis (ISM).MethodsMRGPRX2 and MRGPRX2 agonists, cortistatin (CST), and major basic protein (MBP) were analyzed in lesional and non-lesional skin of patients with ISM and skin of healthy controls by immunohistochemistry. Co-localization of MRGPRX2 and MRGPRX2-mRNA with the MC marker tryptase was assessed by immunofluorescence microscopy and in situ hybridization, respectively. We assessed clinical, demographic, and laboratory data, including mastocytosis activity score (MAS), serum tryptase, and KIT D816V allele burden.ResultsThe number of MRGPRX2-expressing (MRGPRX2+) cells, MRGPRX2-mRNA+ MCs, and CST-expressing (CST+) and MBP-expressing (MBP+) cells was significantly higher in lesional skin as compared to non-lesional skin and/or skin of healthy controls (all p < 0.05). Increased numbers of MRGPRX2+ cells, MRGPRX2-mRNA+ MCs, and CST+ and MBP+ cells were not associated with clinical and laboratory features of ISM, including disease burden, symptom severity, evidence of anaphylaxis, and tryptase levels.ConclusionsSkin lesions of patients with ISM showed high numbers of MRGPRX2+ cells, although they were not linked to symptom severity. Clinical relevance of the MRGPRX2-mediated pathway of MC activation in ISM remains unclear and should be investigated in further studies.

Bicalutamide, an androgen receptor antagonist, effectively alleviate allergic rhinitis via suppression of PI3K–PKB activity

ObjectivesTo investigate the therapeutic effect of Bicalutamide, an androgen receptor antagonist on the onset and development of allergic rhinitis in an animal model.Methods40 male BALB/c mice were randomly divided into five groups (eight mice per group). Aluminum hydroxide powder was used as an adjuvant, combined with Ovalbumin (OVA) to establish the mouse model of allergic rhinitis via ultrasonic nebulization of OVA to stimulate the nasal cavity. Mice in Bica#1 group were intraperitoneally injected with 0.02 mg Bicalutamide/0.5 ml of normal saline daily for 7 consecutive days; mice in Bica#2 group were administered 0.02 mg Bicalutamide/0.5 ml of normal saline via intraperitoneal injection for 5 consecutive days, and then the same amount of normal saline was injected intraperitoneally for 2 consecutive days. Enzyme-linked immunosorbent assay was adopted to detect the serological levels of IgE, IL-4, and IL-6 production. Eosinophil infiltration was observed under microscope after hematoxylin and eosin staining of nasal mucosa. Quantitative PCR and Western blot were employed for determination of histamine receptors mRNA expression and PI3K/PKB associated protein levels, respectively.ResultsHistological analysis shown that allergic lesion was induced after OVA sensitization. Intraperitoneal injection with 0.02 mg Bicalutamide daily for 7 consecutive days significantly reduced the allergic lesion; however, mice injected with the same amount of normal saline at the same time demonstrated no allergic rhinitis symptoms. In addition, there was a significant reduction in eosinophils number in Bicalutamide treated mice (n = 8) compared to the OVA group (n = 8) (OVA: 19.6 ± 5.3 vs. Bica#1: 7.7 ± 0.8 vs. Bica#2: 9.4 ± 1.2, both p < 0.01). Furthermore, ELISA results revealed that the serological levels of IgE (OVA: 17.3 ± 1.7 µg/ml vs. Bica#1: 9.2 ± 0.6 vs. Bica#2: 10.4 ± 2.3, both p < 0.05), IL-4 (OVA: 164.3 ± 5.1 pg/ml vs. Bica#1: 110.2 ± 3.1 vs. Bica#2: 115.3 ± 4.1, both p < 0.05) and IL-6 (OVA: 167.3 ± 3.7 pg/ml vs. Bica#1: 117.5 ± 6.5 vs. Bica#2: 114.8 ± 2.4, both p < 0.05) were significantly decreased after two different dosage of Bicalutamide treatment. Similarly, histamine receptors in mast cells were significantly reduced after two different dosage of Bicalutamide treatment. More importantly, p-PKB protein was notably reduced after two different dosage of Bicalutamide treatment compared to the OVA group, mTOR protein levels were also down regulated after two different dosage of Bicalutamide treatment.ConclusionsOur data demonstrated that androgen receptor antagonist Bicalutamide can significantly alleviate allergic rhinitis lesion in the animal model. PI3K/PKB activity in mast cells was suppressed after Bicalutamide injection. Our results provide important implication in allergic rhinitis prevention and treatment.

PLC-IP3-ORAI pathway participates in the activation of the MRGPRB2 receptor in mouse peritoneal mast cells

A novel mast cell-specific G-protein-coupled receptor (GPCR), known as Mas-related G protein-coupled receptor-B2 (MRGPRB2), plays important roles in immune response. However, the opening of ion channels mediated by MRGPRB2 activation remains unclear. In this study, we found that [Ca2+]i elevation and voltage-dependent current generated by MRGPRB2 activation were correlated with extracellular calcium concentration. The increases in [Ca2+]i and voltage-dependent current caused by MRGPRB2 activation were blocked by U73122 (PLC blocker) or 2-APB (IP3 blocker) or synta66 (ORAI blocker). The voltage-dependent current induced by MRGPRB2 was inhibited by calcium-activated chlorine channel (CACCS) blockers, DIDS, or NPPB. Our results indicated the involvement of the PLC-IP3-ORAI signaling pathway and CACCS in MRGPRB2-mediated mast cell activation.