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MRGPRX2 Activation Causes Increased Skin Reactivity in Patients with Chronic Spontaneous Urticaria

  • Author Footnotes
    5 These authors contributed equally to this work.
    Maria Shtessel
    Correspondence
    Corresponding author
    Footnotes
    5 These authors contributed equally to this work.
    Affiliations
    Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Division of Allergy, Immunology, and Rheumatology, Department of Pediatrics, Columbia University Vagelos College of Physicians and Surgeons, New York, New York, USA
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  • Author Footnotes
    5 These authors contributed equally to this work.
    Nathachit Limjunyawong
    Footnotes
    5 These authors contributed equally to this work.
    Affiliations
    The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Eric T. Oliver
    Affiliations
    Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Kristin Chichester
    Affiliations
    Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Li Gao
    Affiliations
    Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Xinzhong Dong
    Affiliations
    The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Sarbjit S. Saini
    Affiliations
    Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Author Footnotes
    5 These authors contributed equally to this work.
Open ArchivePublished:August 06, 2020DOI:https://doi.org/10.1016/j.jid.2020.06.030

      Abbreviations:

      CSU (chronic spontaneous urticaria), MC (mast cell)
      To the Editor
      Previous studies showed that injection of basic secretagogues such as substance P, compound 48/80, and vasoactive intestinal peptide induced exaggerated responses in the skin of patients with chronic spontaneous urticaria (CSU) versus those of healthy controls through an unknown G protein‒coupled receptor (
      • Borici-Mazi R.
      • Kouridakis S.
      • Kontou-Fili K.
      Cutaneous responses to substance P and calcitonin gene-related peptide in chronic urticaria: the effect of cetirizine and dimethindene.
      ;
      • Bédard P.M.
      • Brunet C.
      • Pelletier G.
      • Hébert J.
      Increased compound 48/80 induced local histamine release from nonlesional skin of patients with chronic urticaria.
      ;
      • Smith C.H.
      • Atkinson B.
      • Morris R.W.
      • Hayes N.
      • Foreman J.C.
      • Lee T.H.
      Cutaneous responses to vasoactive intestinal polypeptide in chronic idiopathic urticaria.
      ). The human receptor for these secretagogues has been identified as MRGPRX2, and in vitro work has shown that MRGPRX2 can activate mast cells (MCs) through a diverse range of drug ligands such as the neuromuscular blocking drug, atracurium, and a bradykinin B2 receptor antagonist, icatibant (
      • McNeil B.D.
      • Pundir P.
      • Meeker S.
      • Han L.
      • Undem B.J.
      • Kulka M.
      • et al.
      Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.
      ). Further in vitro studies have provided evidence of MRGPRX2 drug ligand activation in cell lines and animal models, but no human experiments have been performed (
      • Che D.
      • Rui L.
      • Cao J.
      • Wang J.
      • Zhang Y.
      • Ding Y.
      • et al.
      Cisatracurium induces mast cell activation and pseudo-allergic reactions via MRGPRX2.
      ;
      • Navinés-Ferrer A.
      • Serrano-Candelas E.
      • Lafuente A.
      • Muñoz-Cano R.
      • Martín M.
      • Gastaminza G.
      MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia.
      ;
      • Roy S.
      • Ganguly A.
      • Haque M.
      • Ali H.
      Angiogenic host defense peptide AG-30/5C and bradykinin B2 receptor antagonist icatibant are G protein biased agonists for MRGPRX2 in mast cells.
      ). With the recent discovery that MRGPRX2 protein expression is increased in the MC taken from skin biopsies of patients with severe CSU (
      • Fujisawa D.
      • Kashiwakura J.
      • Kita H.
      • Kikukawa Y.
      • Fujitani Y.
      • Sasaki-Sakamoto T.
      • et al.
      Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria.
      ), we examined whether subjects with milder CSU would show a heightened in vivo functional skin response when tested with MRGPRX2 drug ligands compared with healthy controls. This study is a demonstration of an increased functional skin response supporting MRGPRX2-increased protein expression in patients with CSU. We further demonstrate that drug-induced MC activation requires MRGPRX2 using knockout LAD2 cells and is not occurring through IgE pathways.
      We conducted the study at the Johns Hopkins Asthma and Allergy Center (Baltimore, MD) after approval by the Institutional Review Board. After written informed consent, healthy subjects and subjects with mild CSU between the ages of 18 and 65 years underwent serial intradermal skin titration testing with two MRGPRX2 drug ligands, icatibant (0.01–100 μg/ml, 7 × 10−3–76 μM, physiologic range 974 ± 280 ng/ml [Shire Pharmaceuticals, Lakewood, NJ]) and atracurium (0.001–10 μg/ml, 8 × 10−4–8 μM, physiologic range ∼100–3,000 ng/ml [Hospira, Lake Forest, IL]) and saline control (Hospira) as well as histamine (0.01–100 μg/ml, 9 × 10−2–900 μM [HollisterStier, Spokane, WA]). The drugs chosen were based on the in vitro evidence of the presence of MRGPRX2 activation (icatibant and atracurium) (
      • McNeil B.D.
      • Pundir P.
      • Meeker S.
      • Han L.
      • Undem B.J.
      • Kulka M.
      • et al.
      Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.
      ) and either Food and Drug Administration approval for subcutaneous injection (icatibant) or a history of safe use in skin testing (atracurium) (
      • Leysen J.
      • Uyttebroek A.
      • Sabato V.
      • Bridts C.H.
      • De Clerck L.S.
      • Ebo D.G.
      Predictive value of allergy tests for neuromuscular blocking agents: tackling an unmet need.
      ). A single, unblinded investigator did all the skin testing with the exception of two subjects. By design, all subjects with CSU recruited had a history of pruritus and urticaria for >6 consecutive weeks but were able to withdraw all antihistamines at 7 days before the study for skin testing; therefore, all subjects had a mild phenotype with an average urticaria activity score of 0.5 and typically required a single daily antihistamine (Supplementary Table S1). Subjects who had taken steroids in the last 4 weeks, tricyclics in the last 2 weeks, or immunomodulators were excluded. The healthy controls lacked any atopic or urticaria history (Supplementary Table S2).
      For each drug concentration, a fixed volume of 0.03 ml was injected on the volar aspect of the forearm. Individual wheals were recorded at 15 minutes, and the total area under the curve of wheal diameters was calculated. The wheal diameter for each concentration of the MRGPRX2 ligand was determined for each subject and corrected by the subjects’ saline wheal size to account for possible dermatographism. Comparisons between the subjects with CSU and healthy controls were performed using the student’s t-test using Stata (StataCorp, College Station, TX). For LAD2 studies, similar concentrations of icatibant and atracurium were incubated with wild-type or MRGXPR2-knockout cells for 30 minutes at 37 °C, and supernatants were tested for β-hexosaminidase release (Supplementary Materials and Methods). In selected subjects, basophil activation test was performed in whole blood. Cells were stimulated for 30 minutes with anti-IgE, icatibant, atracurium (at concentrations used for skin testing), or N-formyl-L-methionyl-L-leucyl-phenylalanine (10−6 M) at 37 °C. Cells were then stained with CD63 phycoerythrin antibody (Beckman Coulter, Brea, CA), lyse-fixed, and run on FacsCalibur flow cytometer (BD, Franklin Lakes, NJ) for analysis (Supplementary Materials and Methods).
      Histamine skin test responses were similar in both groups (Figure 1a). Skin responses to the two MRGPRX2 drug ligands, atracurium and icatibant, revealed differences between the subject groups. Overall, subjects with CSU showed significantly greater wheal responses to atracurium at all but the lowest tested doses, with a mean area under the curve of 21.50 ± 3.54 for subjects with CSU versus area under the curve of 9.65 ± 2.32 for control subjects (P = 0.01) (Figure 1b and Supplementary Table S3). Subjects with CSU also showed a significantly greater wheal response to icatibant at concentrations of 1 μg/ml, 10 μg/ml, and 100 μg/ml (P < 0.05) and overall larger area under the curve of 23.16 ± 4.06 than that of the healthy controls (9.79 ± 1.76, P = 0.01) (Figure 1c and Supplementary Table S3). In vitro testing with similar drug concentrations with wild-type and MRGPRX2-knockout LAD2 cells revealed a similar pattern as skin reactivity with a more robust β-hexosaminidase release with icatibant (Figure 2). To further establish that these drug ligands were activating skin MCs through non-IgE pathways, we performed the basophil activation testing with the same concentrations used for skin testing in three subjects with evidence of skin test reactivity to both atracurium and icatibant (Supplementary Figure S1). We failed to observe significant basophil activation at any of the drug concentrations tested but did observe expected basophil activation with anti-IgE stimulation, supporting that an IgE-independent pathway is involved in skin MC activation for both atracurium and icatibant. Whereas a recent report demonstrated MRGPRX2 presence and activation by ciprofloxacin but not PMX-53 on basophils (
      • Wedi B.
      • Gehring M.
      • Kapp A.
      The pseudoallergen receptor MRGPRX2 on peripheral blood basophils and eosinophils: expression and function.
      ), others have not found basophil activation with ligands such as 48/80, and this remains an area of active research (
      • Ishizaka T.
      • Dvorak A.M.
      • Conrad D.H.
      • Niebyl J.R.
      • Marquette J.P.
      • Ishizaka K.
      Morphologic and immunologic characterization of human basophils developed in cultures of cord blood mononuclear cells.
      ;
      • Lawrence I.D.
      • Warner J.A.
      • Cohan V.L.
      • Hubbard W.C.
      • Kagey-Sobotka A.
      • Lichtenstein L.M.
      Purification and characterization of human skin mast cells. Evidence for human mast cell heterogeneity.
      ;
      • Van Gasse A.L.
      • Elst J.
      • Bridts C.H.
      • Mertens C.
      • Faber M.
      • Hagendorens M.M.
      • et al.
      Rocuronium hypersensitivity: does off-target occupation of the MRGPRX2 receptor play a role?.
      ).
      Figure thumbnail gr1
      Figure 1Skin test responses to MRGPRX2 ligands in healthy subjects and subjects with CSU. Skin test responses to (a) histamine, (b) atracurium, and (c) icatibant. The mean saline wheal in subjects with CSU was 1.5 mm ± 2.59 versus 5.53 mm ± 2.74 in healthy controls, P < 0.05. The results represent the arithmetic mean ± SEM of the wheal size at each concentration. AUC was calculated for each dose-response curve (). Asterisks represent significant dose comparisons (P < 0.05). AUC, area under the curve; CSU, chronic spontaneous urticaria.
      Figure thumbnail gr2
      Figure 2MRGPRX2 ligand‒induced degranulation of human WT and MRGPRX2-deficient LAD2 MCs. (a) Atracurium and (b) icatibant degranulation was measured by β-hexosaminidase release. Data represent the mean ± SEM of the percentage release at each concentration. ∗P < 0.05; ∗∗∗P < 0.001. The experiments were independently repeated three times. KO, knockout; MC, mast cell; WT, wild type.
      Our results support differentiation in skin reactivity to both drug ligands at almost every dose in subjects with mild CSU compared with healthy controls. The LAD2 knockout results confirm that these two ligands stimulate mediator release through the MRGPRX2 receptor, with icatibant appearing to be a stronger ligand on the basis of both the in vivo and in vitro studies. Whereas the results of increased MRGPRX2 ligand skin reactivity in subjects with CSU need to be replicated, they carry implications as a potential disease target in CSU as well as various other MC-mediated disorders.
      Limitations of this study include small sample size and the variability of intradermal skin testing. Although MRGPRX2 antagonists have been reported (
      • Azimi E.
      • Reddy V.B.
      • Shade K.C.
      • Anthony R.M.
      • Talbot S.
      • Pereira P.J.S.
      • et al.
      Dual action of neurokinin-1 antagonists on Mas-related GPCRs.
      ;
      • Ogasawara H.
      • Furuno M.
      • Edamura K.
      • Noguchi M.
      Novel MRGPRX2 antagonists inhibit IgE-independent activation of human umbilical cord blood-derived mast cells.
      ), there are no Food and Drug Administration‒approved MRGPRX2 antagonists currently available to confirm that the skin test response is fully mediated through MRGPRX2. However, we were able to exclude IgE pathway utilization in the subjects and demonstrate the dependence of MC activation by these drugs to be selective for MRGPRX2. We present a heightened skin sensitivity to MRGPRX2 ligands in subjects with CSU, which conveys a functional consequence to the past report of heightened protein expression of MRGPRX2 in CSU skin MCs.

      Data availability statement

      All the data for this study are displayed in the manuscript.

      Conflict of Interest

      ETO has served as a consultant for Eli Lilly and has received research support from the National Institutes of Health; American College of Allergy, Asthma, and Immunology; Regeneron; Novartis; and AstraZeneca. None of the remaining authors have any potential financial conflict of interest related to this manuscript.

      Acknowledgments

      This work was supported by the National Institutes of Health AI116658 (SSS), 1R01AI116658-S1 (ETO), American College of Allergy, Asthma, and Immunology (ETO), and R01NS054791 (XD).
      Wild-type LAD2 cells were obtained from the lab of Dr. Dean Metcalfe at NIAID/NIH.

      Author Contributions

      Conceptualization: MS, SSS; Formal Analysis: MS, NL, SSS; Funding Acquisition: XD, SSS; Investigation: MS, NL, ETO, KC, XD, SSS; Methodology: MS, NL, LG, XD, SSS; Project Administration: XD, SSS; Resources: XD, SSS; Supervision: XD, SSS; Validation: MS, NL, SSS; Visualization: MS, NL, SSS; Writing - Original Draft Preparation: MS, SSS; Writing - Review and Editing: MS, NL, ETO, KC, LG, XD, SSS

      Supplementary Material and Methods

      Generation of MRGPRX2 knockout LAD2 cells

      Two single-guide RNAs with 2′-O-methyl 3′-phosphorothioate-modified nucleotides at three terminal positions at both the 5′ and 3′ ends targeting the beginning of exon 2 of MRGPRX2 gene were purchased from Synthego (Menlo Park, CA). The single-guide RNA sequences are 5′-UAGUUCAUGACGGCCGGAGG-3′ and 5′-UGGAGAAGCCGAAAAACCAG-3′. Single-guide RNAs were complexed with Streptococcus pyogenes Cas9 nuclease (Synthego) to form ribonucleoprotein. Ribonucleoprotein complexes were transfected into the LAD2 cells using Lipofectamine CRISPRMAX transfection reagent (Thermo Fisher, Waltham, MA). After 2 weeks of transfection, cells were stained with MRGPRX2 antibody (BioLegend, San Diego, CA) and subjected to FACS for selection of populations that lack the expression of MRGPRX2. Sorted cells were expanded, and their deficiency in MRGPRX2 expression was confirmed by DNA sequencing, flow cytometry, and nonresponse to C48/80 and substance P.

      β-hexosaminidase release assay

      LAD2 cells were harvested and resuspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 0.4% BSA. Then 20,000 cells were seeded per well and stimulated with icatibant, atracurium, or other known MRGPRX2 agonists (C48/80, substance P, cortistatin-14, or proadrenomedullin N-terminal 20 peptide 8–20) at the indicated concentrations for 30 minutes at 37 °C and 5% carbon dioxide. After a 30-minute incubation, the supernatant was collected, and cells were lysed using 0.1% Triton X-100. The supernatant and cell lysates were incubated with p-nitrophenyl-N-acetyl-β-D glucosamine in 0.04 M citrate buffer (pH 4.5) for 90 minutes at 37 °C. The reactions were developed by adding 0.4 M glycine (pH 10.7). Absorbance was measured at 405 nm and 570 nm as the reference using FlexStation 3 plate reader (Molecular Devices, San Jos, CA). The β-hexosaminidase release into cell supernatant was reported as a percent of total β-hexosaminidase content.

      Basophil activation test

      In select subjects, basophil activation test was performed in whole blood. Cells were stimulated for 30 minutes with anti-IgE (1 × 10−4–1 μg/ml), icatibant, atracurium (at concentrations mentioned above), or N-formyl-L-methionyl-L-leucyl-phenylalanine (10−6 M) at 37 °C. Cells were gated by scatter and CD123+/CCR3+ as well as CD63 antibody, lyse-fixed, and run on FacsCalibur flow cytometer (BD, Franklin Lakes, NJ) for analysis.
      Figure thumbnail fx1
      Supplementary Figure S1BAT with icatibant, atracurium, and anti-IgE in three healthy control subjects with skin test reactivity to icatibant and atracurium. BAT, basophil activation test; Pt., patient.
      Supplementary Table S1Characteristics of Chronic Urticaria Subjects
      Patient IDAge, ySexRaceDuration of Illness, yUAS

      Score
      Sum of itch 0–3 and 0–3 for hives, with 0 = no hive, 1 = 1–6 hives, 2 = 7–12, and 3 = > 12 hives.
      TreatmentAtopic History
      153FCau11CetirizineSAR, AD
      220MAA20CetirizineNone
      325FCau22Fexofenadine + cetirizineSAR, AD, AS
      454MCau30CetirizineSAR, AD, AS
      563FAA61FexofenadineSAR
      635MCau100FexofenadineSAR, AD
      737FCau70LoratadineAS
      851FAA101FexofenadineSAR
      964FCau40LevocetirizineNone
      1023MCau50CetirizineSAR, FA, AS
      Abbreviations: AA, African American; AD, atopic dermatitis; AS, asthma; Cau, Caucasian; F, female; FA, food allergy; ID, identifier number; M, male; SAR, seasonal allergic rhinitis; UAS, urticaria activity score.
      1 Sum of itch 0–3 and 0–3 for hives, with 0 = no hive, 1 = 1–6 hives, 2 = 7–12, and 3 = > 12 hives.
      Supplementary Table S2Characteristics of Nonatopic Control Subjects
      Patient IDAge, ySexRaceOther DiseaseAtopic History
      145FCauHypothyroidismNo
      235MCauHTNNo
      346FCauHTN and/or CKDNo
      428FCauNoneNo
      544FCauMigrainesNo
      637MAANoneNo
      751FCauDepression and/or HTN and/or rosaceaNo
      827FCauNoneNo
      938MAsianNoneNo
      1019FCauNoneNo
      Abbreviations: AA, African American; Cau, Caucasian; CKD, chronic kidney disease; F, female; HTN, hypertension; ID, identifier number; M, male.
      Supplementary Table S3Comparison of the AUC between Healthy Subjects and Subjects with CSU
      ReagentsHealthy (n = 10)CSU (n = 10)P-value
      Histamine40.9 ± 1.5239.67 ± 1.700.59
      Atracurium-saline control9.65 ± 2.3221.50 ± 3.540.01
      Icatibant-saline control9.79 ± 1.7623.1 ± 4.060.01
      Abbreviations: AUC, area under the curve; CSU, chronic spontaneous urticaria.
      Atracurium and icatibant are saline corrected. Mean is reported with SEM.

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