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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 mouse mast cell basic secretagogue receptora. Left, representative Fluo-4 fluorescence heat map images of mouse peritoneal mast cells showing changes in [Ca2+]i induced by bath application of anti-IgE (5 μg/ml) or 48/80 (10 μg/ml). Middle, representative imaging traces. Each color line represents an individual cell. Black lines in “anti-IgE” panels are average traces for each genotype. Note: [Ca2+]i traces are similar between WT and MUT groups. Right, quantification of responding cells (n=3/genotype; >150 cells counted/condition). Anti-IgE responses were not significantly different. Scale bar 10 μm.b. Histamine release into the supernatant from trachea and abdominal skin from WT and MrgprB2MUT mice after exposure to 48/80 (30 μg/ml) for 30 minutes at 37°C. n = 5/trachea, n= 8/skin.c. Top, representative traces showing contractions of trachea isolated from WT and MrgprB2MUT mice (previously sensitized to ovalbumin (ova), in response to 48/80 (30 μg/ml) or ova (10 μg/ml; i.e. IgE-dependent). Bottom, average data; maximum total contraction determined as response to 10 μM carbamycholine added at the end of the experiment. n=5 for 48/80 WT, 3 for 48/80 MrgprB2MUT.d. Left, representative images of Evans Blue extravasation 15 minutes after intraplantar injection of 48/80 (right, arrow, 10 μg/ml, 5 μl in saline) or saline (left). Right, quantification of Evans Blue leakage into the paw and paw thickness increase after 15 minutes. *, p<0.02 (n=5/ WT, n=6/MrgprB2MUT). Differences after saline injection were not significant.e. Quantification of WT and MrgprB2MUT mast cell responsiveness to MrgprX2 ligands and basic secretagogues, assayed using Fluo-4 imaging. Concentrations of substances (in μM): PAMP(9-20), 20; cortistatin-14 (cort.), 20; Substance P (sub P), 200; kallidin, 200; mastoparan (masto., a component of wasp venom), 20; vespid mastoparan, 20. n=3/genotype; >150 cells counted/secretagogue.Data are presented as mean ± standard error of mean (SEM). Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at p<0.05. *, p < 0.05. **, p<0.01 unless noted.
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Figure 2: MrgprB2 is the mouse mast cell basic secretagogue receptora. Left, representative Fluo-4 fluorescence heat map images of mouse peritoneal mast cells showing changes in [Ca2+]i induced by bath application of anti-IgE (5 μg/ml) or 48/80 (10 μg/ml). Middle, representative imaging traces. Each color line represents an individual cell. Black lines in “anti-IgE” panels are average traces for each genotype. Note: [Ca2+]i traces are similar between WT and MUT groups. Right, quantification of responding cells (n=3/genotype; >150 cells counted/condition). Anti-IgE responses were not significantly different. Scale bar 10 μm.b. Histamine release into the supernatant from trachea and abdominal skin from WT and MrgprB2MUT mice after exposure to 48/80 (30 μg/ml) for 30 minutes at 37°C. n = 5/trachea, n= 8/skin.c. Top, representative traces showing contractions of trachea isolated from WT and MrgprB2MUT mice (previously sensitized to ovalbumin (ova), in response to 48/80 (30 μg/ml) or ova (10 μg/ml; i.e. IgE-dependent). Bottom, average data; maximum total contraction determined as response to 10 μM carbamycholine added at the end of the experiment. n=5 for 48/80 WT, 3 for 48/80 MrgprB2MUT.d. Left, representative images of Evans Blue extravasation 15 minutes after intraplantar injection of 48/80 (right, arrow, 10 μg/ml, 5 μl in saline) or saline (left). Right, quantification of Evans Blue leakage into the paw and paw thickness increase after 15 minutes. *, p<0.02 (n=5/ WT, n=6/MrgprB2MUT). Differences after saline injection were not significant.e. Quantification of WT and MrgprB2MUT mast cell responsiveness to MrgprX2 ligands and basic secretagogues, assayed using Fluo-4 imaging. Concentrations of substances (in μM): PAMP(9-20), 20; cortistatin-14 (cort.), 20; Substance P (sub P), 200; kallidin, 200; mastoparan (masto., a component of wasp venom), 20; vespid mastoparan, 20. n=3/genotype; >150 cells counted/secretagogue.Data are presented as mean ± standard error of mean (SEM). Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at p<0.05. *, p < 0.05. **, p<0.01 unless noted.

Mentions: Next, we determined whether MrgprB2 is the basic secretagogue receptor in mouse mast cells. The MrgprB2 genomic locus contains too much repetitive sequence to permit gene targeting through homologous recombination (Extended Data Fig. 5a). Therefore, we used a zinc finger nuclease-based strategy to generate a mouse line with a 4 base pair deletion in the MrgprB2 coding region (MrgprB2MUT mice), resulting in a frameshift mutation and early termination shortly after the first transmembrane domain (Extended Data Fig. 5b-d). The mutation was stable and inheritable (Extended Data Fig. 5c), so we regard MrgprB2MUT as a functional . Mast cell numbers were comparable in tissues of wild-type (WT) and MrgprB2MUT mice, indicating that MrgprB2 is not essential for mast cell survival or targeting to tissue (Extended Data Fig. 6a). Responsiveness of peritoneal mast cells to anti-IgE antibodies (Fig. 2a) and endothelin (Extended Data Fig. 7) also was comparable, demonstrating that MrgprB2 mutation does not globally impair IgE or GPCR-mediated mast cell signaling. However, 48/80-induced mast cell activation (Fig. 2a) and tissue histamine release essentially was abolished in mutant mast cells (Fig. 2b; Extended Data Fig. 6b). Further, we found that 48/80-evoked tracheal contraction (Fig. 2c) and hindpaw inflammation (extravasation and swelling; Fig. 2d) were almost completely absent in an MrgprB2MUT background, while antigen (Fig. 2c) and anti-IgE evoked responses (Extended Data Fig. 8) were comparable to WT mice. Finally, we found that four additional basic secretagogues, as well as MrgprX2 agonists PAMP (9-20) and cortistatin10, strongly activated WT but not MrgprB2MUT mast cells (Fig. 2e; Extended Data Fig. 9a). HEK293 cells expressing MrgprB2 or MrgprX2 (MrgprX2-HEK) also responded to these secretagogues (Extended Data Fig. 2). Taken together, we conclude that MrgprB2 is the mouse mast cell basic secretagogue receptor. It is likely that the list of small, basic peptides that activate MrgprB2 is greater than the number in this study; indeed, dozens of such peptides have been shown to activate mast cells3,6,16,17. Notably, human MrgprX2 is much more sensitive to Substance P than mouse MrgprB2 (Extended Data Fig. 2c), suggesting a potential species-specific role for Substance P in mast cell signaling.


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 mouse mast cell basic secretagogue receptora. Left, representative Fluo-4 fluorescence heat map images of mouse peritoneal mast cells showing changes in [Ca2+]i induced by bath application of anti-IgE (5 μg/ml) or 48/80 (10 μg/ml). Middle, representative imaging traces. Each color line represents an individual cell. Black lines in “anti-IgE” panels are average traces for each genotype. Note: [Ca2+]i traces are similar between WT and MUT groups. Right, quantification of responding cells (n=3/genotype; >150 cells counted/condition). Anti-IgE responses were not significantly different. Scale bar 10 μm.b. Histamine release into the supernatant from trachea and abdominal skin from WT and MrgprB2MUT mice after exposure to 48/80 (30 μg/ml) for 30 minutes at 37°C. n = 5/trachea, n= 8/skin.c. Top, representative traces showing contractions of trachea isolated from WT and MrgprB2MUT mice (previously sensitized to ovalbumin (ova), in response to 48/80 (30 μg/ml) or ova (10 μg/ml; i.e. IgE-dependent). Bottom, average data; maximum total contraction determined as response to 10 μM carbamycholine added at the end of the experiment. n=5 for 48/80 WT, 3 for 48/80 MrgprB2MUT.d. Left, representative images of Evans Blue extravasation 15 minutes after intraplantar injection of 48/80 (right, arrow, 10 μg/ml, 5 μl in saline) or saline (left). Right, quantification of Evans Blue leakage into the paw and paw thickness increase after 15 minutes. *, p<0.02 (n=5/ WT, n=6/MrgprB2MUT). Differences after saline injection were not significant.e. Quantification of WT and MrgprB2MUT mast cell responsiveness to MrgprX2 ligands and basic secretagogues, assayed using Fluo-4 imaging. Concentrations of substances (in μM): PAMP(9-20), 20; cortistatin-14 (cort.), 20; Substance P (sub P), 200; kallidin, 200; mastoparan (masto., a component of wasp venom), 20; vespid mastoparan, 20. n=3/genotype; >150 cells counted/secretagogue.Data are presented as mean ± standard error of mean (SEM). Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at p<0.05. *, p < 0.05. **, p<0.01 unless noted.
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Figure 2: MrgprB2 is the mouse mast cell basic secretagogue receptora. Left, representative Fluo-4 fluorescence heat map images of mouse peritoneal mast cells showing changes in [Ca2+]i induced by bath application of anti-IgE (5 μg/ml) or 48/80 (10 μg/ml). Middle, representative imaging traces. Each color line represents an individual cell. Black lines in “anti-IgE” panels are average traces for each genotype. Note: [Ca2+]i traces are similar between WT and MUT groups. Right, quantification of responding cells (n=3/genotype; >150 cells counted/condition). Anti-IgE responses were not significantly different. Scale bar 10 μm.b. Histamine release into the supernatant from trachea and abdominal skin from WT and MrgprB2MUT mice after exposure to 48/80 (30 μg/ml) for 30 minutes at 37°C. n = 5/trachea, n= 8/skin.c. Top, representative traces showing contractions of trachea isolated from WT and MrgprB2MUT mice (previously sensitized to ovalbumin (ova), in response to 48/80 (30 μg/ml) or ova (10 μg/ml; i.e. IgE-dependent). Bottom, average data; maximum total contraction determined as response to 10 μM carbamycholine added at the end of the experiment. n=5 for 48/80 WT, 3 for 48/80 MrgprB2MUT.d. Left, representative images of Evans Blue extravasation 15 minutes after intraplantar injection of 48/80 (right, arrow, 10 μg/ml, 5 μl in saline) or saline (left). Right, quantification of Evans Blue leakage into the paw and paw thickness increase after 15 minutes. *, p<0.02 (n=5/ WT, n=6/MrgprB2MUT). Differences after saline injection were not significant.e. Quantification of WT and MrgprB2MUT mast cell responsiveness to MrgprX2 ligands and basic secretagogues, assayed using Fluo-4 imaging. Concentrations of substances (in μM): PAMP(9-20), 20; cortistatin-14 (cort.), 20; Substance P (sub P), 200; kallidin, 200; mastoparan (masto., a component of wasp venom), 20; vespid mastoparan, 20. n=3/genotype; >150 cells counted/secretagogue.Data are presented as mean ± standard error of mean (SEM). Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at p<0.05. *, p < 0.05. **, p<0.01 unless noted.
Mentions: Next, we determined whether MrgprB2 is the basic secretagogue receptor in mouse mast cells. The MrgprB2 genomic locus contains too much repetitive sequence to permit gene targeting through homologous recombination (Extended Data Fig. 5a). Therefore, we used a zinc finger nuclease-based strategy to generate a mouse line with a 4 base pair deletion in the MrgprB2 coding region (MrgprB2MUT mice), resulting in a frameshift mutation and early termination shortly after the first transmembrane domain (Extended Data Fig. 5b-d). The mutation was stable and inheritable (Extended Data Fig. 5c), so we regard MrgprB2MUT as a functional . Mast cell numbers were comparable in tissues of wild-type (WT) and MrgprB2MUT mice, indicating that MrgprB2 is not essential for mast cell survival or targeting to tissue (Extended Data Fig. 6a). Responsiveness of peritoneal mast cells to anti-IgE antibodies (Fig. 2a) and endothelin (Extended Data Fig. 7) also was comparable, demonstrating that MrgprB2 mutation does not globally impair IgE or GPCR-mediated mast cell signaling. However, 48/80-induced mast cell activation (Fig. 2a) and tissue histamine release essentially was abolished in mutant mast cells (Fig. 2b; Extended Data Fig. 6b). Further, we found that 48/80-evoked tracheal contraction (Fig. 2c) and hindpaw inflammation (extravasation and swelling; Fig. 2d) were almost completely absent in an MrgprB2MUT background, while antigen (Fig. 2c) and anti-IgE evoked responses (Extended Data Fig. 8) were comparable to WT mice. Finally, we found that four additional basic secretagogues, as well as MrgprX2 agonists PAMP (9-20) and cortistatin10, strongly activated WT but not MrgprB2MUT mast cells (Fig. 2e; Extended Data Fig. 9a). HEK293 cells expressing MrgprB2 or MrgprX2 (MrgprX2-HEK) also responded to these secretagogues (Extended Data Fig. 2). Taken together, we conclude that MrgprB2 is the mouse mast cell basic secretagogue receptor. It is likely that the list of small, basic peptides that activate MrgprB2 is greater than the number in this study; indeed, dozens of such peptides have been shown to activate mast cells3,6,16,17. Notably, human MrgprX2 is much more sensitive to Substance P than mouse MrgprB2 (Extended Data Fig. 2c), suggesting a potential species-specific role for Substance P in mast cell signaling.

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