The pseudoallergen receptor MRGPRX2 on peripheral blood basophils and eosinophils: Expression and function

Abstract

Background 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. Methods 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. Results 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. Conclusion 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.

Reply to Sabato V et al “Surface expression of MRGPRX2 expression on resting basophils: An area of controversy”

“…Moreover, using sensitive methods that would have indicated prior cellular activation, such as calcium mobilization assay, we did not find evidence for priming of our cells. In addition, as described in our publication, 3 using different purification methods we found similar results.…”

“…To give a clearer answer to the controversial question of whether only activated basophils show higher expression of MRGPRX2 upon treatment with different stimuli, we re‐analyzed our experiments in which expression of MRGPRX2 and CD63 was assessed in parallel and presents them in another way. For detailed methods, please refer to our original manuscript 3 …”

Surface expression of MRGPRX2 on resting basophils: An area of controversy

“…In their recent publication, Wedi et al state that they are the first to describe basophils to constitutively express the Mas‐related G protein‐coupled receptor MRGPRX2 and that this expression can by upregulated by various stimuli. It seems that the authors have overlooked some of our data about the expression of MRGPRX2 by basophils, and a hypothesis we already postulated about the potential of basophil activation experiments in immediate hypersensitivity reactions originating from MRGPRX2 occupancy by quinolones .…”

“…Furthermore, significant constitutive surface expression of MRGPRX2 by resting basophils, as shown by Wedi et al, is difficult to align with the observation that ciprofloxacin, amongst other quinolones, does not trigger significant basophil degranulation in almost all control individuals tolerant for these antibiotics . We think that the explanation of the divergent observations by Wedi et al could relate to their medium. Panel C of figure 5 of the study by Wedi shows that the applied medium elicits upregulation of the lysosomal degranulation marker CD63 in over 20% of the cells.…”

“…We think that the explanation of the divergent observations by Wedi et al could relate to their medium. Panel C of figure 5 of the study by Wedi shows that the applied medium elicits upregulation of the lysosomal degranulation marker CD63 in over 20% of the cells. By consequence, it is likely the cells in their experiments not to be at rest but to be in a preactivated status, we, like the authors, showed upregulated MRGPRX2 expression.…”