Mrgprx2 Activation by Rocuronium: Insights From Studies with Human Skin Mast Cells and Missense Variants

Perioperative hypersensitivity (POH) to the neuromuscular blocking drug (NMBD) rocuronium was previously thought to be IgE and mast cell (MC)-mediated. However, the recent seminalobservation 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 causeddegranulation 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 comparedto the wild-type receptor. These findings provide the first demonstration that rocuronium induces degranulation in human MCs via MRGPRX2. Furthermore, the important differences between Mrg-prB2 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

Inhibition of Orai Channel Function Regulates Mas-Related G Protein-Coupled Receptor-Mediated Responses in Mast Cells

Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders. Copyright © 2022 Chaki, Alkanfari, Roy, Amponnawarat, Hui, Oskeritzian and Ali

Mast Cell Survival and Mediator Secretion in Response to Hypoxia

Tissue hypoxia is a consequence of decreased oxygen levels in different inflammatory conditions, many associated with mast cell activation. However, the effect of hypoxia on mast cell functions is not well established. Here, we have investigated the effect of hypoxia per se on human mast cell survival, mediator secretion, and reactivity. Human cord blood derived mast cells were subjected to three different culturing conditions: culture and stimulation in normoxia (21% O2); culture and stimulation in hypoxia (1% O2); or 24 hour culture in hypoxia followed by stimulation in normoxia. Hypoxia, per se, did not induce mast cell degranulation, but we observed an increased secretion of IL-6, where autocrine produced IL-6 promoted mast cell survival. Hypoxia did not have any effect on A23187 induced degranulation or secretion of cytokines. In contrast, cytokine secretion after LPS or CD30 treatment was attenuated, but not inhibited, in hypoxia compared to normoxia. Our data suggests that mast cell survival, degranulation and cytokine release are sustained under hypoxia. This may be of importance for host defence where mast cells in a hypoxic tissue can react to intruders, but also in chronic inflammations where mast cell reactivity is not inhibited by the inflammatory associated hypoxia

Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases

AbstractSphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the “sphingolipid rheostat”, producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases

Role of ABCC1 in export of sphingosine-1-phosphate from mast cells

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that S1P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S1P release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses

Distinct roles of sphingosine kinases 1 and 2 in human mast-cell functions

Sphingosine-1-phosphate (S1P) is now emerging as a potent lipid mediator produced by mast cells that contributes to inflammatory and allergic responses. In contrast to its weak effect on degranulation of murine mast cells, S1P potently induced degranulation of the human LAD2 mast-cell line and cord blood–derived human mast cells (hMCs). S1P also stimulated production and secretion of cytokines, TNF-α and IL-6, and markedly enhanced secretion of a chemokine, CCL2/MCP-1, important modulators of inflammation. S1P is produced in mast cells by the 2 sphingosine kinases, SphK1 and SphK2. SphK1 but not SphK2 plays a critical role in IgE/Ag-induced degranulation, migration toward antigen, and CCL2 secretion from hMCs, as determined by specifically down-regulating their expression. However, both isoenzymes were required for efficient TNF-α secretion. Taken together, our data suggest that differential formation of S1P by SphK1 and SphK2 has distinct and important actions in hMCs

A specific sphingosine kinase 1 inhibitor attenuates airway hyperresponsiveness and inflammation in a mast cell-dependent mouse model of allergic asthma

Background: Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cells and IgE-dependent murine model of allergic asthma has not yet been examined. Objective: We used an isoenzyme-specific SphK1 inhibitor,SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. Methods: Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. Results: SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor-kB (NF-kB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13,IFN-g, and TNF-a and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-kB in the lungs.Fil: Price, Megan M.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Oskeritzian, Carole A.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Falanga, Yves T.. Virginia Commonwealth University. Department of Microbiology and Immunology; Estados UnidosFil: Harikumar, Kuzhuvelil B.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Allegood, Jeremy C.. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Alvarez, Sergio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Conrad, Daniel. Virginia Commonwealth University. Department of Biology; Estados UnidosFil: Ryan, John J.. Virginia Commonwealth University. Department of Microbiology and Immunology; Estados UnidosFil: Milstien, Sheldon. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Spiegel, Sarah. Virginia Commonwealth University. School of Medicine. Department of Biochemistry and Molecular Biology; Estados Unido