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Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USADivision of Allergy, Immunology, and Rheumatology, Department of Pediatrics, Columbia University Vagelos College of Physicians and Surgeons, New York, New York, USA
The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAHoward Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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 (
). 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 (
), 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) (
). 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 (
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 (
), 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.
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.
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.
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.
Supplementary Table S1Characteristics of Chronic Urticaria Subjects