J Elst1, V Sabato1,2, M A Faber1, C H Bridts1, C Mertens1, M Van Houdt1, A L Van Gasse1,3, M M Hagendorens1,3, V Van Tendeloo4, M Maurer5, D Campillo-Davo4, J P Timmermans6, I Pintelon6, D G Ebo1,2. 1. Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium and Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp, B. 2. Department of Immunology, AZ Jan Palfijn Hospital Gent, Ghent, Belgium. 3. Department of Pediatrics, Faculty of Medicine and Health Sciences, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium. 4. Laboratory of Experimental Haematology, Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Science, University of Antwerp, Antwerp, Belgium. 5. Dermatological Allergology, Allergie-Centrum-Charité, Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin, Berlin, Germany. 6. Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
Abstract
BACKGROUND AND OBJECTIVES: Mast cell (MC) degranulation via activation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. METHODS: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. RESULTS: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. CONCLUSION: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing.
BACKGROUND AND OBJECTIVES: Mast cell (MC) degranulation via activation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. METHODS: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. RESULTS: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. CONCLUSION: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing.
Authors: Maud A W Hermans; Astrid C van Stigt; Sanne van de Meerendonk; Benjamin Schrijver; Paul L A van Daele; Petrus M van Hagen; Marloes van Splunter; Willem A Dik Journal: Front Immunol Date: 2021-03-15 Impact factor: 7.561