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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 mediates mast cell responsiveness and side effects of small molecule therapeutic drugsa. Structures of 48/80 and a cyclized variant. The THIQ motif is highlighted in blue.b. Structures of representative members of all NMBD classes (see Supplementary Information). THIQ motifs highlighted in blue. Note that only succinylcholine lacks a bulky hydrophobic group.c. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after application of various NMBDs, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): atracurium, 50; mivacurium, 20; tubocurarine, 30; rocuronium, 500. n=3 mice /genotype; >150 cells counted/substance.d. Structure of ciprofloxacin, with the motif common to all fluoroquinolones highlighted in blue. Note nitrogens close to the quinolone motifs.e. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after fluoroquinolone application, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): ciprofloxacin, 200; levofloxacin, 500; moxifloxacin, 160; ofloxacin, 400. n=3 mice/genotype; >150 cells counted/substance.f. Changes in body temperature after intravenous injection of ciprofloxacin (1.5 mg in 125 μl saline) at time 0. n=4 mice/genotype.Data are presented as mean ± SEM. Two-tailed unpaired Student's t test: *, p < 0.05. **, p<0.01.
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Figure 4: MrgprB2 mediates mast cell responsiveness and side effects of small molecule therapeutic drugsa. Structures of 48/80 and a cyclized variant. The THIQ motif is highlighted in blue.b. Structures of representative members of all NMBD classes (see Supplementary Information). THIQ motifs highlighted in blue. Note that only succinylcholine lacks a bulky hydrophobic group.c. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after application of various NMBDs, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): atracurium, 50; mivacurium, 20; tubocurarine, 30; rocuronium, 500. n=3 mice /genotype; >150 cells counted/substance.d. Structure of ciprofloxacin, with the motif common to all fluoroquinolones highlighted in blue. Note nitrogens close to the quinolone motifs.e. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after fluoroquinolone application, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): ciprofloxacin, 200; levofloxacin, 500; moxifloxacin, 160; ofloxacin, 400. n=3 mice/genotype; >150 cells counted/substance.f. Changes in body temperature after intravenous injection of ciprofloxacin (1.5 mg in 125 μl saline) at time 0. n=4 mice/genotype.Data are presented as mean ± SEM. Two-tailed unpaired Student's t test: *, p < 0.05. **, p<0.01.

Mentions: We explored next the possibility that MrgprB2 mediates pseudo-allergic reactions induced by small molecules. We focused on intravenously applied drugs because they often are administered rapidly and in high doses, and thus are more likely to achieve high blood concentrations and rapid tissue distribution than drugs administered through other routes. Symptoms of pseudo-allergic reactions after intravenous administration, which at the most severe are called anaphylactoid, include skin flushing or rash, changes in blood pressure or heart rate, and bronchospasms20. We based our initial search on the structure of 48/80. While the structure-function relationship of 48/80 as an MrgprX2 agonist is unknown, a cyclized variant containing a tetrahydroisoquinoline (THIQ) motif (Fig. 4a) is reported to be seven times more potent than 48/80 as a mast cell degranulator21. A search of FDA-approved drugs containing a THIQ recovered members of the nicotinic receptor antagonist non-steroidal neuromuscular blocking drugs (NMBDs), including tubocurarine and atracurium (Fig. 4b). NMBDs are used routinely in surgery to reduce unwanted muscle movement and allow intratracheal intubation for mechanical ventilation. Intriguingly, NMBDs alone are responsible for nearly 60% of allergic reactions in a surgical setting22, and all except succinylcholine induce histamine release in humans23. We found that members of all NMBD families (Supplemental Information) except succinylcholine activated mast cells in an MrgprB2-dependent manner at concentrations as low as 0.5% of the clinical injection concentration (Fig. 4c; Extended Data Fig. 9d). Interestingly, rocuronium does not contain a THIQ but has a bulky hydrophobic group with a charged nitrogen within several angstroms (Fig. 4b), reminiscent of 48/80. Therefore, we searched using modifications of the THIQ motif and the 48/80 structure, including changes in cyclization and position of the positive or polar nitrogen, limiting our assay to intravenous drugs at high injection concentrations. We identified the fluoroquinolone family of antibiotics as having a similar motif (Fig. 4d). Like NMBDs, these are associated with allergic-type reactions24,25 and can activate mast cells26,27. We found that the four members approved for intravenous use activated MrgprB2-HEK and MrgprX2-HEK cells (Extended Data Fig. 2), and mast cells in an MrgprB2- dependent manner (Fig. 4e; Extended Data Fig. 9e). Correspondingly, atracurium and ciprofloxacin induced histamine release in WT peritoneal mast cells and substantially less in MrgprB2MUT mast cells (Fig. 3c). We selected ciprofloxacin for in vivo tests of anaphylaxis, which in mice is measured most often by a drop in body temperature, likely due to changes in blood pressure and peripheral vasodilation28. Rodents nearly are immune to histamine toxicity at a systemic level, contrary to other experimental organisms4, but can be rendered sensitive to mast cell activators and secreted products by pretreatment with beta-adrenergic blockers29,30. Under these conditions, a high dose of ciprofloxacin induced a rapid drop in body temperature that was very slow to recover, while MrgprB2MUT mice showed a much smaller drop that recovered quickly (Fig. 4f). These results establish that mast cell activation through MrgprB2 is an off-target effect of fluoroquinolones and other drugs.


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 mediates mast cell responsiveness and side effects of small molecule therapeutic drugsa. Structures of 48/80 and a cyclized variant. The THIQ motif is highlighted in blue.b. Structures of representative members of all NMBD classes (see Supplementary Information). THIQ motifs highlighted in blue. Note that only succinylcholine lacks a bulky hydrophobic group.c. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after application of various NMBDs, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): atracurium, 50; mivacurium, 20; tubocurarine, 30; rocuronium, 500. n=3 mice /genotype; >150 cells counted/substance.d. Structure of ciprofloxacin, with the motif common to all fluoroquinolones highlighted in blue. Note nitrogens close to the quinolone motifs.e. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after fluoroquinolone application, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): ciprofloxacin, 200; levofloxacin, 500; moxifloxacin, 160; ofloxacin, 400. n=3 mice/genotype; >150 cells counted/substance.f. Changes in body temperature after intravenous injection of ciprofloxacin (1.5 mg in 125 μl saline) at time 0. n=4 mice/genotype.Data are presented as mean ± SEM. Two-tailed unpaired Student's t test: *, p < 0.05. **, p<0.01.
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Figure 4: MrgprB2 mediates mast cell responsiveness and side effects of small molecule therapeutic drugsa. Structures of 48/80 and a cyclized variant. The THIQ motif is highlighted in blue.b. Structures of representative members of all NMBD classes (see Supplementary Information). THIQ motifs highlighted in blue. Note that only succinylcholine lacks a bulky hydrophobic group.c. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after application of various NMBDs, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): atracurium, 50; mivacurium, 20; tubocurarine, 30; rocuronium, 500. n=3 mice /genotype; >150 cells counted/substance.d. Structure of ciprofloxacin, with the motif common to all fluoroquinolones highlighted in blue. Note nitrogens close to the quinolone motifs.e. Percentage of responding cells from WT and MrgprB2MUT peritoneal mast cells after fluoroquinolone application, assayed using Fluo-4 imaging. Concentrations of drugs (in μg/ml): ciprofloxacin, 200; levofloxacin, 500; moxifloxacin, 160; ofloxacin, 400. n=3 mice/genotype; >150 cells counted/substance.f. Changes in body temperature after intravenous injection of ciprofloxacin (1.5 mg in 125 μl saline) at time 0. n=4 mice/genotype.Data are presented as mean ± SEM. Two-tailed unpaired Student's t test: *, p < 0.05. **, p<0.01.
Mentions: We explored next the possibility that MrgprB2 mediates pseudo-allergic reactions induced by small molecules. We focused on intravenously applied drugs because they often are administered rapidly and in high doses, and thus are more likely to achieve high blood concentrations and rapid tissue distribution than drugs administered through other routes. Symptoms of pseudo-allergic reactions after intravenous administration, which at the most severe are called anaphylactoid, include skin flushing or rash, changes in blood pressure or heart rate, and bronchospasms20. We based our initial search on the structure of 48/80. While the structure-function relationship of 48/80 as an MrgprX2 agonist is unknown, a cyclized variant containing a tetrahydroisoquinoline (THIQ) motif (Fig. 4a) is reported to be seven times more potent than 48/80 as a mast cell degranulator21. A search of FDA-approved drugs containing a THIQ recovered members of the nicotinic receptor antagonist non-steroidal neuromuscular blocking drugs (NMBDs), including tubocurarine and atracurium (Fig. 4b). NMBDs are used routinely in surgery to reduce unwanted muscle movement and allow intratracheal intubation for mechanical ventilation. Intriguingly, NMBDs alone are responsible for nearly 60% of allergic reactions in a surgical setting22, and all except succinylcholine induce histamine release in humans23. We found that members of all NMBD families (Supplemental Information) except succinylcholine activated mast cells in an MrgprB2-dependent manner at concentrations as low as 0.5% of the clinical injection concentration (Fig. 4c; Extended Data Fig. 9d). Interestingly, rocuronium does not contain a THIQ but has a bulky hydrophobic group with a charged nitrogen within several angstroms (Fig. 4b), reminiscent of 48/80. Therefore, we searched using modifications of the THIQ motif and the 48/80 structure, including changes in cyclization and position of the positive or polar nitrogen, limiting our assay to intravenous drugs at high injection concentrations. We identified the fluoroquinolone family of antibiotics as having a similar motif (Fig. 4d). Like NMBDs, these are associated with allergic-type reactions24,25 and can activate mast cells26,27. We found that the four members approved for intravenous use activated MrgprB2-HEK and MrgprX2-HEK cells (Extended Data Fig. 2), and mast cells in an MrgprB2- dependent manner (Fig. 4e; Extended Data Fig. 9e). Correspondingly, atracurium and ciprofloxacin induced histamine release in WT peritoneal mast cells and substantially less in MrgprB2MUT mast cells (Fig. 3c). We selected ciprofloxacin for in vivo tests of anaphylaxis, which in mice is measured most often by a drop in body temperature, likely due to changes in blood pressure and peripheral vasodilation28. Rodents nearly are immune to histamine toxicity at a systemic level, contrary to other experimental organisms4, but can be rendered sensitive to mast cell activators and secreted products by pretreatment with beta-adrenergic blockers29,30. Under these conditions, a high dose of ciprofloxacin induced a rapid drop in body temperature that was very slow to recover, while MrgprB2MUT mice showed a much smaller drop that recovered quickly (Fig. 4f). These results establish that mast cell activation through MrgprB2 is an off-target effect of fluoroquinolones and other drugs.

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