scholarly journals MRGPRX2 Activation by Rocuronium: Insights from Studies with Human Skin Mast Cells and Missense Variants

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 156 ◽  
Author(s):  
Chalatip Chompunud Na Ayudhya ◽  
Aetas Amponnawarat ◽  
Saptarshi Roy ◽  
Carole A. Oskeritzian ◽  
Hydar Ali
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Hypersensitivity Reaction ◽  
Neuromuscular Blocking ◽  
Missense Mutations ◽  
Blocking Drug ◽  
Wild Type ◽  
Human Ortholog ◽  
G Protein Coupled ◽  
Type Receptor

Perioperative hypersensitivity (POH) to the neuromuscular blocking drug (NMBD) rocuronium was previously thought to be IgE and mast cell (MC)-mediated. However, the recent seminal observation that rocuronium induces degranulation in murine peritoneal MCs (PMCs) via Mas-related G protein-coupled receptor B2 (MrgprB2) led to the idea that POH to this drug involves the activation of MRGPRX2 (human ortholog of MrgprB2). Furthermore, based on the demonstration that a patient with POH to rocuronium displayed three missense mutations (M196I, L226P and L237P) in MRGPRX2’s transmembrane domains, it was proposed that this hypersensitivity reaction resulted from aberrant activation of this receptor. We found that rocuronium at 20 µg/mL caused degranulation in mouse PMCs via MrgprB2 but required at least 500 µg/mL to induce degranulation in human MCs via MRGPRX2. Furthermore, RBL-2H3 cells transiently expressing M196I, L226P and L237P variants did not display enhanced degranulation in response to rocuronium when compared to the wild-type receptor. These findings provide the first demonstration that rocuronium induces degranulation in human MCs via MRGPRX2. Furthermore, the important differences between MrgprB2 and MRGPRX2 and the inability of rocuronium to induce enhanced response in cells expressing MRGPRX2 variants suggest that the mechanism of its POH is more complex than previously thought.

scholarly journals Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-arrestin Recruitment and Receptor Internalization

2021 ◽  
Vol 22 (10) ◽  
pp. 5318
Author(s):  
Chalatip Chompunud Na Ayudhya ◽  
Aetas Amponnawarat ◽  
Hydar Ali
Keyword(s):  
Substance P ◽  
G Protein ◽  
G Proteins ◽  
Receptor Internalization ◽  
The Novel ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Human Ortholog ◽  
G Protein Coupled ◽  
Type Receptor

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2’s NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.

scholarly journals Pathogenic role of mast cells in experimental eosinophilic esophagitis

2013 ◽  
Vol 304 (12) ◽  
pp. G1087-G1094 ◽  
Author(s):  
Rituraj Niranjan ◽  
Parm Mavi ◽  
Madhavi Rayapudi ◽  
Scott Dynda ◽  
Anil Mishra
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Muscle Cell ◽  
Significant Role ◽  
Eosinophilic Esophagitis ◽  
Brdu Incorporation ◽  
Dependent Manner ◽  
Wild Type ◽  
Cell Hyperplasia ◽  
Cell Accumulation

Eosinophilic esophagitis (EoE) is a chronic allergic disease characterized by esophageal intraepithelial eosinophils, extracellular eosinophil granule deposition, induced mast cell accumulation, and epithelial cell hyperplasia. However, the processes involved in the development of a number of these characteristics are largely unknown. Herein, we tested the hypothesis whether induced mast cell accumulation in the esophagus has a role in promoting EoE pathogenesis. Accordingly, we induced experimental EoE in wild-type mice, mast cell-deficient WWv mice, and mast cell-reconstituted WWv mice. We report that esophageal mast cell numbers increase in parallel with eosinophils in a dose- and time-dependent manner following the induction of allergen-induced EoE. The induced mast cells are localized in the esophageal lamina propria and muscular mucosa but have no influence on promoting esophageal eosinophilia. The 5′-bromodeoxyuridine (BrdU) incorporation analysis indicated that mast cells have a significant role in muscle cell hyperplasia and hypertrophy. In addition, the wild-type and mast cell-reconstituted WWv mice showed a comparable number of BrdU+ cells in the esophageal muscular mucosa following allergen-induced EoE. In conclusion, we provide for the first time direct evidence that mast cell promotes muscle cell hyperplasia and hypertrophy and may have a significant role in promoting esophageal functional abnormalities in EoE.

Thrombopoietin Regulates Proliferation and Maturation of Murine Mast Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1707-1707
Author(s):  
Giovanni Migliaccio ◽  
Barbara Ghinassi ◽  
Lucia Centurione ◽  
Maria Zingariello ◽  
Lucia Bianchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Mast Cells ◽  
Wild Type ◽  
Connective Tissues ◽  
Immature Cells

Abstract Megakaryocytopoiesis is regulated by extrinsic (interaction of the growth factor thrombopoietin, TPO with its receptor Mpl) and intrinsic (interaction between the trascription factors GATA-1 and Fog-1) factors. The observation that mice impaired for GATA-1 expression (i.e. harbouring the GATA-1low mutation) are defective not only in megakaryocyte maturation but also in mast cell differentiation (Migliaccio et al. J Exp Med197:281, 2003), led us to investigate whether TPO might control mast cell differentiation as well. We first observed that mice genetically unable to responde to TPO (Mplnull mice) express in the connective tissues 5 times more mast cells than their normal littermates. Then, we analysed the effects on mast cell differentiation of in vivo treatment with TPO. Normal mice, and their GATA-1low littermates, were injected i.p. with TPO (100 μg/kg/day per 5 days, kindly provided by Kirin Brewery, Japan) and the number of immature (Toluidinepos) and mature (AlcianBlue/Saphraninepos) mast cells present in the connective tissues of the animals, as well as the frequency of GATA-1pos and TUNELpos mast cells, was evaluated 14 days after treatment. In wild-type animals, TPO reduced the presence of GATA-1 in mast cells (by immuno-histochemistry) and increased the number of immature cells (from 320±28 to 852±60) and of those undergoing apoptosis (from 16±1 to 600±43). In contrast, in GATA-1low animals, TPO-treatment induced the expression of GATA-1 in mast cells while decreased the number of immature cells (from 1100±72 to 427±29) as well as that of apoptotic cells (from 600±45 to 60±2). The role of TPO on mast cell differentiation were further confirmed by the analysis of the effects exerted by the growth factor on in vitro differentiation of bone marrow derived mast cells (BMMC). In these experiments, wild type bone marrow and spleen cells were cultured for 21 days with SCF and IL-3 with or without TPO and BMMC differentiation measured on the basis of the number of cells expressing the phenotype c-kithigh/CD34high and FcεRIpos. In cultures stimulated with SCF and IL-3, all the cells expressed the phenotype c-kithigh/CD34high and FcεRIpos. In contrast, in cultures supplemented also with SCF, IL-3 and TPO, only 25% of the cells were c-kithigh/CD34high and none of them was FcεRIpos. These results establish a role for TPO in the control of mast cell differentiation (possibly by modulating the GATA-1 content of the cells) and unveil further similarities between the mechanism(s) controlling megakaryocyte and mast cell differentiation.

Effects of AMN107, a Novel Aminopyrimidine Tyrosine Kinase Inhibitor, on Human Mast Cells Bearing Wild-Type or Mutated Codon 816 c-kit.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3528-3528 ◽  
Author(s):  
Srdan Verstovsek ◽  
Cem Akin ◽  
Giles J. Francis ◽  
Manshouri Taghi ◽  
Ly Huynh ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Kit Mutation

Abstract Background. Majority of adult patients with systemic mastocytosis (SM) have activating mutation in codon 816 of c-kit (CD117), a receptor on the surface of mast cells. This abnormality is responsible for the pathogenesis of the disease. Methods. We investigated the effects of a newly designed tyrosine kinase inhibitor, AMN107, by comparing its in vitro inhibitory potency on c-kit mutated mast cell lines and patient samples with that of imatinib mesylate, another tyrosine kinase inhibitor, effective in some patients with SM. Two cell lines, subclones of HMC-1 cells, were used: HMC-1560 carrying juxtamembrane domain mutation in codon 560 of c-kit, and HMC-1560, 816 carrying both codon 560 mutation and tyrosine kinase domain mutation in codon 816 of c-kit. Results. In HMC-1560 mast cell line carrying wild-type codon 816, AMN107 was as potent as imatinib in inhibiting cellular proliferation, with IC50 values of 108 and 74 nM respectively, while in HMC-1560, 816 cell line carrying 816 mutation, neither medication had an effect. AMN107 was also as effective as imatinib in inhibiting phosphorylation of c-kit tyrosine kinase in HMC-1560 cells. The inhibition of cellular proliferation was associated with induction of apoptosis in HMC-1560 cells. AMN107 in concentrations up to 1 uM had no effect on bone marrow mast cells carrying D816V c-kit mutation obtained from patients with mastocytosis. Conclusions. Our results suggest similar potency of AMN107 and imatinib in mast cells that carry wild-type codon 816, but no activity against codon 816 mutation carrying cells.

Murine Mast Cells Express Mpl, the Thrombopoietin Receptor, and Thrombopoietin Is a Potent Regulator of Mast Cell Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1335-1335
Author(s):  
Fabrizio Martelli ◽  
Giovanni Amabile ◽  
Barbara Ghinassi ◽  
Rodolfo Lorenzini ◽  
Alessandro M. Vannucchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Progenitor Cells ◽  
Peritoneal Cavity ◽  
Surface Protein ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Progenitor Cell Proliferation

Abstract Mast cells are hematopoietic cells localized in extramedullary sites where they engage themselves in the process of allergic response and in the immune reaction against parasites. Mast cells derive from multilineage c-KitlowCD34lowSca-1pos progenitor cells present in the marrow. These cells give rise to Linnegc-KitposSca-1neg T1/ST2pos mast cell restricted progenitor cells (MCP) whose futher maturation in the marrow remains limited under steady state conditions. MCP migrate through the blood in extramedullary sites were they mature into tissue-retricted c-KitposFceRIpos mast cells characterized by a specific mast cell protease (MMCP) profiling (dermal, mucosal and serosal mast cells in skin, gut and peritoneal cavity, respectively). The molecular mechanism that, in normal mice, restricts the mastocytopoietic potential of progenitor cells to the extramedullary sites, as well as the factors that guide the tissue-restricted differentiation of these cells, are unknown. Thrombopoietin (TPO)-Mpl interactions play an important role in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation in the marrow. Here we report that mast cells, and their precursors, express Mpl (both as mRNA and cell surface protein) (see Table). Furthermore, targeted deletion of this gene (Mplnull mutation) decrease the number of MCP (by 1-log) and increases that of mast cells in dermis (by 3-fold), peritoneal cavity (by 3-fold), bone marrow (2-log) and spleen (2-log). Furthermore, because of their higher (by 2-log) MMCP-7 expression, serosal Mplnull mast cells resemble more wild-type dermal rather than serosal mast cells. On the other hand, either treatment of mice with TPO or addition of TPO to bone marrow-derived mast cell cultures induces mast cell apoptosis (by Tunel and Annexin staining) and severely hampers mast cell differentiation (by expression profiling). These data are consistent with a regulatory mechanism for murine mastocytopoiesis according to which TPO favours the transition from multilineage progenitors to CMP but blocks differentiation of MCP to mature mast cells. We propose TPO as the growth factor that restrict mast cell differentiation to extramedullaty sites and that control the switch between serosal vs dermal mast cell differentiation. Mpl expression mRNA 2-ΔCt Protein (AFU) Cy7-A Protein (AFU) Cy7-AMM2 AFU= arbitrary fluorescence intensity. p< 0.01 with respect to Cy7-A (irrilevant antibody) Wild type Marrow B cells (B220pos) b.d. 120±4 205±4 Wild type Marrow Megakaryocytes (CD61pos/CD41pos) 5.0±0.1 × 10-2 178±3 978±74* Wild type Marrow MCP (cKitpos/T1ST2pos) 1.3±0.01 × 10-2 139±16 1658±73* Wild-type Marrow Mast Cells (cKitpos/Fcε RIpos) 1.9±0.1 × 10-2 110±1 868±71* Serosal Mast Cells (cKitpos/FcεRIpos) 7.2±2.1 × 10-4 393±1 1374±25* Mplnull Marrow Megakaryocytes (CD61pos/CD41pos) b.d. 365±28 469±50 Mplnull Marrow Mast Cells (cKitpos/FcεRIpos) b.d 107±1 109±3

scholarly journals Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

1999 ◽  
Vol 274 (18) ◽  
pp. 12548-12554 ◽  
Author(s):  
Christian Le Gouill ◽  
Jean-Luc Parent ◽  
Carolyn-Ann Caron ◽  
Rémi Gaudreau ◽  
Léonid Volkov ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Selective Modulation ◽  
G Protein Coupled ◽  
Type Receptor

scholarly journals Neuro-immune interactions in chemical-induced airway hyperreactivity

2016 ◽  
Vol 48 (2) ◽  
pp. 380-392 ◽  
Author(s):  
Fien C. Devos ◽  
Brett Boonen ◽  
Yeranddy A. Alpizar ◽  
Tania Maes ◽  
Valérie Hox ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Transient Receptor Potential ◽  
Chinese Hamster ◽  
Transient Receptor Potential Channels ◽  
Airway Hyperreactivity ◽  
Sensory Nerves ◽  
Wild Type ◽  
Immunological Parameters

Asthma may be induced by chemical sensitisers,viamechanisms that are still poorly understood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airway inflammation. Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensory irritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, major chemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves, in chemical-induced AHR.In vitrointracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressing Chinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using anin vivomodel, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, we demonstrated that AHR does not develop, despite successful sensitisation, inTrpa1andTrpv1knockout mice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficientKitWsh/Wshmice, and in wild-type mice pretreated with the mast cell stabiliser ketotifen, without changes in immunological parameters.These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in the development of TDI-elicited AHR.

scholarly journals Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe

1999 ◽  
Vol 163 (3) ◽  
pp. 447-456 ◽  
Author(s):  
B Byrne ◽  
A McGregor ◽  
PL Taylor ◽  
R Sellar ◽  
FE Rodger ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Maximal Response ◽  
Wild Type ◽  
Releasing Hormone ◽  
Agonist And Antagonist ◽  
Gonadotrophin Releasing Hormone ◽  
Antagonist Action ◽  
G Protein Coupled ◽  
Type Receptor ◽  
D Value

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.

Lack of neurokinin-1 receptor expression affects tissue mast cell numbers but not their spatial relationship with nerves

2005 ◽  
Vol 288 (2) ◽  
pp. R491-R500 ◽  
Author(s):  
Michael R. D'Andrea ◽  
Marcia R. Saban ◽  
Norma P. Gerard ◽  
Barry K. Wershil ◽  
Ricardo Saban
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Spatial Association ◽  
Spatial Relationship ◽  
Genetically Engineered ◽  
Receptor Expression ◽  
Wild Type ◽  
Genetically Engineered Mice ◽  
Neurokinin 1 ◽  
Anatomic Relationship

A spatial association between mast cells and nerves has been described in both the gastrointestinal and genitourinary tracts. However, the factors that influence the anatomic relationship between mast cells and nerves have not been completely defined. It has been suggested that the high-affinity receptor for substance P [neurokinin-1 (NK1)] might modulate this interaction. We therefore assessed mast cell-nerve relationships in tissues isolated from wild-type and NK1 receptor knockout (NK1−/−) mice. We now report that, in the complete absence of NK1 receptor expression, there is a significant increase in the number of mast cells without a change in the anatomic relationship between mast cell and nerves in stomach and bladder tissues at the light microscopic level. We next determined whether transplanted mast cells would maintain their spatial distribution, number, and contact with nerve elements. For this purpose, mast cell-deficient Kit W /Kit W−v mice were reconstituted with wild-type or NK1−/− bone marrow. No differences in mast cell-nerve contact were observed. These results suggest that NK1 receptor expression is important in the regulation of the number of mast cells but is not important in the interaction between mast cells and nerves. Furthermore, the interaction between mast cells and nerves is not mediated through NK1 receptor expression on the mast cell. Further studies are needed to determine the molecular pathway involved in mast cell migration and interaction with nerve elements, but the model of reconstitution of Kit W /Kit W−v mice with mast cells derived from different genetically engineered mice is a useful approach to further explore these mechanisms.

scholarly journals Mast Cell Degranulation Decreases Lipopolysaccharide-Induced Aortic Gene Expression and Systemic Levels of Interleukin-6 In Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-7
Author(s):  
Jason M. Springer ◽  
Vineesh V. Raveendran ◽  
Mingcai Zhang ◽  
Ryan Funk ◽  
Donald D. Smith ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Interleukin 6 ◽  
Systemic Vasculitis ◽  
Mast Cell Degranulation ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Serum Samples ◽  
Cytokine Expression Profile

Mast cells play an important role in immunomodulation and in the maintenance of vascular integrity. Interleukin-6 (IL-6) is one of the key biomarkers and therapeutic target in systemic vasculitis. The objective of the current study is to describe the role of mast cells in arterial IL-6 homeostasis. Eight- to ten-week-old male C57BL/6 (wild-type) mice were injected with either (a) saline, (b) compound 48/80 (a systemic mast cell degranulating agent), (c) lipopolysaccharide (LPS), or (d) a combination of C48/80 and LPS. Twenty-four hours after the injections, mice were sacrificed and serum samples and aortic tissues were analyzed for determining inflammatory response and cytokine expression profile. The results revealed that induction of mast cell degranulation significantly lowers serum IL-6 levels and aortic expression of IL-6 in LPS-treated mice. Significantly higher aortic expression of toll-like receptor-2 (TLR-2) and TNF-α was seen in the LPS and LPS+C48/80 groups of mice compared to controls. Aortic expression of TLR-4 was significantly decreased in LPS+C48/80 compared to C48/80 alone. LPS+C48/80-treated mice presented with a 3-fold higher aortic expression of suppressor of cytokine signaling (SOCS-1) compared to saline-injected groups. The inhibition of LPS-induced increase in serum IL-6 levels by mast cell degranulation was not seen in H1R knockout mice which suggests that mast cell-derived histamine acting through H1R may participate in the regulatory process. To examine whether the mast cell-mediated downregulation of LPS-induced IL-6 production is transient or cumulative in nature, wild-type mice were injected serially over a period of 10 days (5 injections) and serum cytokine levels were quantified. We found no significant differences in serum IL-6 levels between any of the groups. While mice injected with C48/80 or LPS had higher IL-10 compared to vehicle-injected mice, there was no difference between C48/80- and LPS+C48/80-injected mice. In conclusion, in an in vivo setting, mast cells appear to partially and transiently regulate systemic IL-6 homeostasis. This effect may be regulated through increased systemic IL-10 and/or aortic overexpression of SOCS-1.

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