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Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.

McNeil BD, Pundir P, Meeker S, Han L, Undem BJ, Kulka M, Dong X - Nature (2014)

Bottom Line: Although they are classically activated by immunoglobulin (Ig)E antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions.The pathogenic roles of these substances have prompted a decades-long search for their receptor(s).Finally, we determine that Mrgprb2 and MRGPRX2 are targets of many small-molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; we show that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice; and we identify a common chemical motif in several of these molecules that may help predict side effects of other compounds.

View Article: PubMed Central - PubMed

Affiliation: The Solomon H. Snyder Department of Neuroscience, Department of Neurosurgery, Center for Sensory Biology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Mast cells are primary effectors in allergic reactions, and may have important roles in disease by secreting histamine and various inflammatory and immunomodulatory substances. Although they are classically activated by immunoglobulin (Ig)E antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions. The pathogenic roles of these substances have prompted a decades-long search for their receptor(s). Here we report that basic secretagogues activate mouse mast cells in vitro and in vivo through a single receptor, Mrgprb2, the orthologue of the human G-protein-coupled receptor MRGPRX2. Secretagogue-induced histamine release, inflammation and airway contraction are abolished in Mrgprb2- mutant mice. Furthermore, we show that most classes of US Food and Drug Administration (FDA)-approved peptidergic drugs associated with allergic-type injection-site reactions also activate Mrgprb2 and MRGPRX2, and that injection-site inflammation is absent in mutant mice. Finally, we determine that Mrgprb2 and MRGPRX2 are targets of many small-molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; we show that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice; and we identify a common chemical motif in several of these molecules that may help predict side effects of other compounds. These discoveries introduce a mouse model to study mast cell activation by basic secretagogues and identify MRGPRX2 as a potential therapeutic target to reduce a subset of drug-induced adverse effects.

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MrgprB2 is the orthologue of human MrgprX2a. Diagram of mouse and human Mrgpr genomic loci. Mouse MrgprA3 and MrgprC11 are orthologues of human MrgprX1, determined by expression and ligand specificity15. The MrgprX2 orthologue MrgprB2 is described in this study.b. Results from a stringent RT-PCR screen identifying MrgprB2 transcript (arrow) in mouse peritoneal mast cells. The negative control (Neg.) omitted reverse transcriptase.c. Example traces of intracellular calcium concentrations [Ca2+]i, measured by ratiometric Fura-2 imaging, from MrgprB2-HEK or MrgprX2-HEK cells exposed to 20 μM PAMP(9-20) (duration indicated by black line). Each trace is a response from a unique cell.d. Representative confocal images from BAC transgenic mouse tissues in which tdTomato expression is controlled by eGFP-Cre expression from the MrgprB2 locus (see methods). Avidin staining was used to identify mast cells. Percentages of avidin-positive mast cells that also were tdTomato-positive: glabrous skin, 97.5%; hairy skin, 90.1%; trachea, 97.2%; heart, 87.1%. Percentages of tdTomato-positive cells that also were avidin-positive: glabrous skin, 99.2%; hairy skin, 100%; trachea, 98.3%; heart, 99%. n=3 mice and >300 cells counted/tissue, except n=2 and >100 cells/heart. Scale bar 20 μm.
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Figure 1: MrgprB2 is the orthologue of human MrgprX2a. Diagram of mouse and human Mrgpr genomic loci. Mouse MrgprA3 and MrgprC11 are orthologues of human MrgprX1, determined by expression and ligand specificity15. The MrgprX2 orthologue MrgprB2 is described in this study.b. Results from a stringent RT-PCR screen identifying MrgprB2 transcript (arrow) in mouse peritoneal mast cells. The negative control (Neg.) omitted reverse transcriptase.c. Example traces of intracellular calcium concentrations [Ca2+]i, measured by ratiometric Fura-2 imaging, from MrgprB2-HEK or MrgprX2-HEK cells exposed to 20 μM PAMP(9-20) (duration indicated by black line). Each trace is a response from a unique cell.d. Representative confocal images from BAC transgenic mouse tissues in which tdTomato expression is controlled by eGFP-Cre expression from the MrgprB2 locus (see methods). Avidin staining was used to identify mast cells. Percentages of avidin-positive mast cells that also were tdTomato-positive: glabrous skin, 97.5%; hairy skin, 90.1%; trachea, 97.2%; heart, 87.1%. Percentages of tdTomato-positive cells that also were avidin-positive: glabrous skin, 99.2%; hairy skin, 100%; trachea, 98.3%; heart, 99%. n=3 mice and >300 cells counted/tissue, except n=2 and >100 cells/heart. Scale bar 20 μm.

Mentions: Responsiveness to basic secretagogues is conserved among mammals4, and also is found in birds5, indicating an ancient, fundamental role for its mechanism. Many basic secretagogues are endogenous peptides, often linked to inflammation; however, they activate connective tissue mast cells only at high concentrations and independent of their canonical receptors, so another mechanism of stimulation must exist6. Several candidates which bind polycationic compounds have been proposed as basic secretagogue receptors6-9. Among these, MrgprX2 has been screened with the most compounds8,10-14, and siRNA knockdown studies support at least a partial role for MrgprX2 in activation by four non-canonical basic secretagogues11,13. However, no direct in vivo study or knockout model has been employed for any candidate. The investigation of MrgprX2 in mice is complicated because the gene cluster containing the four human MrgprX members is dramatically expanded in mice, consisting of 22 potential coding genes, many with comparable sequence identity to MrgprX2 (Fig. 1a). Therefore, a mouse MrgprX2 orthologue must be determined by expression pattern and pharmacology. A stringent RT-PCR screen in mouse primary mast cells uncovered a band for a single family member, MrgprB2 (Fig. 1b), while MrgprX1 orthologues were not expressed at relevant levels (Extended Data Fig. 1a,b). Functionally, HEK293 cells heterologously expressing MrgprB2 (MrgprB2-HEK) responded to the MrgprX2 agonist PAMP (9-20)14 (Fig. 1c) and Compound 48/80 (48/80), a classical mast cell activator and canonical basic secretagogue (Extended Data Fig. 2). MrgprB2-HEK cells also responded to other MrgprX2 ligands, including the basic secretagogue Substance P, but had no response to the MrgprX1 ligand chloroquine (CQ)15; no closely related family members in mice responded to any compound (Extended Data Fig. 1c, 2a,c). To determine the expression of MrgprB2, we generated MrgprB2 BAC transgenic mice in which the expression of eGFP-Cre recombinase was under the control of the MrgprB2 promoter. Strikingly, Cre expression patterns indicate that MrgprB2 expression is highly specific to connective tissue mast cells (Fig. 1d; Extended Data Fig. 3 and 4). Together the pharmacological and expression data strongly suggest that MrgprB2 is the mouse orthologue of MrgprX2.


Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.

McNeil BD, Pundir P, Meeker S, Han L, Undem BJ, Kulka M, Dong X - Nature (2014)

MrgprB2 is the orthologue of human MrgprX2a. Diagram of mouse and human Mrgpr genomic loci. Mouse MrgprA3 and MrgprC11 are orthologues of human MrgprX1, determined by expression and ligand specificity15. The MrgprX2 orthologue MrgprB2 is described in this study.b. Results from a stringent RT-PCR screen identifying MrgprB2 transcript (arrow) in mouse peritoneal mast cells. The negative control (Neg.) omitted reverse transcriptase.c. Example traces of intracellular calcium concentrations [Ca2+]i, measured by ratiometric Fura-2 imaging, from MrgprB2-HEK or MrgprX2-HEK cells exposed to 20 μM PAMP(9-20) (duration indicated by black line). Each trace is a response from a unique cell.d. Representative confocal images from BAC transgenic mouse tissues in which tdTomato expression is controlled by eGFP-Cre expression from the MrgprB2 locus (see methods). Avidin staining was used to identify mast cells. Percentages of avidin-positive mast cells that also were tdTomato-positive: glabrous skin, 97.5%; hairy skin, 90.1%; trachea, 97.2%; heart, 87.1%. Percentages of tdTomato-positive cells that also were avidin-positive: glabrous skin, 99.2%; hairy skin, 100%; trachea, 98.3%; heart, 99%. n=3 mice and >300 cells counted/tissue, except n=2 and >100 cells/heart. Scale bar 20 μm.
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Figure 1: MrgprB2 is the orthologue of human MrgprX2a. Diagram of mouse and human Mrgpr genomic loci. Mouse MrgprA3 and MrgprC11 are orthologues of human MrgprX1, determined by expression and ligand specificity15. The MrgprX2 orthologue MrgprB2 is described in this study.b. Results from a stringent RT-PCR screen identifying MrgprB2 transcript (arrow) in mouse peritoneal mast cells. The negative control (Neg.) omitted reverse transcriptase.c. Example traces of intracellular calcium concentrations [Ca2+]i, measured by ratiometric Fura-2 imaging, from MrgprB2-HEK or MrgprX2-HEK cells exposed to 20 μM PAMP(9-20) (duration indicated by black line). Each trace is a response from a unique cell.d. Representative confocal images from BAC transgenic mouse tissues in which tdTomato expression is controlled by eGFP-Cre expression from the MrgprB2 locus (see methods). Avidin staining was used to identify mast cells. Percentages of avidin-positive mast cells that also were tdTomato-positive: glabrous skin, 97.5%; hairy skin, 90.1%; trachea, 97.2%; heart, 87.1%. Percentages of tdTomato-positive cells that also were avidin-positive: glabrous skin, 99.2%; hairy skin, 100%; trachea, 98.3%; heart, 99%. n=3 mice and >300 cells counted/tissue, except n=2 and >100 cells/heart. Scale bar 20 μm.
Mentions: Responsiveness to basic secretagogues is conserved among mammals4, and also is found in birds5, indicating an ancient, fundamental role for its mechanism. Many basic secretagogues are endogenous peptides, often linked to inflammation; however, they activate connective tissue mast cells only at high concentrations and independent of their canonical receptors, so another mechanism of stimulation must exist6. Several candidates which bind polycationic compounds have been proposed as basic secretagogue receptors6-9. Among these, MrgprX2 has been screened with the most compounds8,10-14, and siRNA knockdown studies support at least a partial role for MrgprX2 in activation by four non-canonical basic secretagogues11,13. However, no direct in vivo study or knockout model has been employed for any candidate. The investigation of MrgprX2 in mice is complicated because the gene cluster containing the four human MrgprX members is dramatically expanded in mice, consisting of 22 potential coding genes, many with comparable sequence identity to MrgprX2 (Fig. 1a). Therefore, a mouse MrgprX2 orthologue must be determined by expression pattern and pharmacology. A stringent RT-PCR screen in mouse primary mast cells uncovered a band for a single family member, MrgprB2 (Fig. 1b), while MrgprX1 orthologues were not expressed at relevant levels (Extended Data Fig. 1a,b). Functionally, HEK293 cells heterologously expressing MrgprB2 (MrgprB2-HEK) responded to the MrgprX2 agonist PAMP (9-20)14 (Fig. 1c) and Compound 48/80 (48/80), a classical mast cell activator and canonical basic secretagogue (Extended Data Fig. 2). MrgprB2-HEK cells also responded to other MrgprX2 ligands, including the basic secretagogue Substance P, but had no response to the MrgprX1 ligand chloroquine (CQ)15; no closely related family members in mice responded to any compound (Extended Data Fig. 1c, 2a,c). To determine the expression of MrgprB2, we generated MrgprB2 BAC transgenic mice in which the expression of eGFP-Cre recombinase was under the control of the MrgprB2 promoter. Strikingly, Cre expression patterns indicate that MrgprB2 expression is highly specific to connective tissue mast cells (Fig. 1d; Extended Data Fig. 3 and 4). Together the pharmacological and expression data strongly suggest that MrgprB2 is the mouse orthologue of MrgprX2.

Bottom Line: Although they are classically activated by immunoglobulin (Ig)E antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions.The pathogenic roles of these substances have prompted a decades-long search for their receptor(s).Finally, we determine that Mrgprb2 and MRGPRX2 are targets of many small-molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; we show that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice; and we identify a common chemical motif in several of these molecules that may help predict side effects of other compounds.

View Article: PubMed Central - PubMed

Affiliation: The Solomon H. Snyder Department of Neuroscience, Department of Neurosurgery, Center for Sensory Biology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Mast cells are primary effectors in allergic reactions, and may have important roles in disease by secreting histamine and various inflammatory and immunomodulatory substances. Although they are classically activated by immunoglobulin (Ig)E antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions. The pathogenic roles of these substances have prompted a decades-long search for their receptor(s). Here we report that basic secretagogues activate mouse mast cells in vitro and in vivo through a single receptor, Mrgprb2, the orthologue of the human G-protein-coupled receptor MRGPRX2. Secretagogue-induced histamine release, inflammation and airway contraction are abolished in Mrgprb2- mutant mice. Furthermore, we show that most classes of US Food and Drug Administration (FDA)-approved peptidergic drugs associated with allergic-type injection-site reactions also activate Mrgprb2 and MRGPRX2, and that injection-site inflammation is absent in mutant mice. Finally, we determine that Mrgprb2 and MRGPRX2 are targets of many small-molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; we show that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice; and we identify a common chemical motif in several of these molecules that may help predict side effects of other compounds. These discoveries introduce a mouse model to study mast cell activation by basic secretagogues and identify MRGPRX2 as a potential therapeutic target to reduce a subset of drug-induced adverse effects.

Show MeSH
Related in: MedlinePlus