Wedi Bettina, Gehring Manuela, Kapp Alexander
Department of Dermatology and Allergy, Comprehensive Allergy Center, Hannover Medical School, Hannover, Germany.
Allergy. 2020 Sep;75(9):2229-2242. doi: 10.1111/all.14213. Epub 2020 Feb 15.
Mas-related G protein-coupled receptor X2 (MRGPRX2) is regarded as a mast cell-specific receptor mediating non-IgE-dependent activation. We aimed to investigate whether human basophils and eosinophils express functional MRGPRX2.
Flow cytometry, immunocytochemistry, immunofluorescence, Western blot, and RT-PCR were performed in highly purified peripheral blood basophils and eosinophils of atopic and nonatopic donors. To assess functional activity, fluorescent avidin-based degranulation assay, calcium mobilization, cytokine production in supernatants, assessment of viability/apoptosis, and tricolor granulocyte activation test were used.
MRGPRX2 was significantly expressed by basophils and eosinophils but not neutrophils. Functional capacity was shown by anti-MRGPRX2 mAb-induced calcium influx and concentration-dependent induction of degranulation. Sequential stimulation in the calcium mobilization assay gave no evidence for desensitization or receptor internalization. Anti-MRGPRX2 mAb significantly promoted survival. Inhibition of apoptosis could be due to released IL-3, IL-5, and GM-CSF found in supernatants. Short-term incubation with IL-3 dose-dependently upregulated MRGPRX2 expression in both, stimulation for 24 hours with anti-IgE, C5a, fMLP, and IL-3 in basophils and by IL-3, IL-5, and IL-33 in eosinophils. Among known mast cell MRGPRX2 agonists ciprofloxacin but not PMX-53 was functional on basophils and eosinophils. In basophils of allergic subjects, tricolor granulocyte activation test using grass pollen demonstrated MRGPRX2 upregulation associated with degranulation and CD63 expression.
Unraveling the regulation and signaling mechanisms of MRGPRX2 on basophils and eosinophils might enable the development of new therapeutic strategies to prevent or inhibit allergic and nonallergic hypersensitivity. Moreover, addressing MRGPRX2 might have potential for diagnostic purposes in (drug) hypersensitivity.
Mas相关G蛋白偶联受体X2（MRGPRX2）被认为是介导非IgE依赖性激活的肥大细胞特异性受体。我们旨在研究人类嗜碱性粒细胞和嗜酸性粒细胞是否表达功能性MRGPRX2。
对特应性和非特应性供体的高度纯化外周血嗜碱性粒细胞和嗜酸性粒细胞进行流式细胞术、免疫细胞化学、免疫荧光、蛋白质印迹和逆转录聚合酶链反应。为评估功能活性，使用了基于荧光抗生物素蛋白的脱颗粒试验、钙动员、上清液中细胞因子产生、活力/凋亡评估以及三色粒细胞激活试验。
嗜碱性粒细胞和嗜酸性粒细胞显著表达MRGPRX2，而中性粒细胞不表达。抗MRGPRX2单克隆抗体诱导的钙内流和浓度依赖性脱颗粒诱导显示了其功能能力。钙动员试验中的顺序刺激未显示脱敏或受体内化的证据。抗MRGPRX2单克隆抗体显著促进存活。凋亡抑制可能归因于上清液中发现的白细胞介素-3、白细胞介素-5和粒细胞-巨噬细胞集落刺激因子的释放。用白细胞介素-3短期孵育剂量依赖性地上调两者中的MRGPRX2表达，嗜碱性粒细胞中用抗IgE、C5a、N-甲酰甲硫氨酸-亮氨酸-苯丙氨酸和白细胞介素-3刺激24小时，嗜酸性粒细胞中用白细胞介素-3、白细胞介素-5和白细胞介素-33刺激。在已知的肥大细胞MRGPRX2激动剂中，环丙沙星而非PMX-53对嗜碱性粒细胞和嗜酸性粒细胞有功能作用。在过敏性受试者的嗜碱性粒细胞中，使用草花粉的三色粒细胞激活试验显示MRGPRX2上调与脱颗粒和CD63表达相关。
阐明MRGPRX2在嗜碱性粒细胞和嗜酸性粒细胞上的调节和信号传导机制可能有助于开发预防或抑制过敏性和非过敏性超敏反应的新治疗策略。此外，针对MRGPRX2可能在（药物）超敏反应的诊断中有潜在应用。
