FcεRI‐ and MRGPRX2‐evoked acute degranulation responses are fully additive in human skin mast cells

Abstract

To the Editor, Mast cell (MC) activation by FcεRI-aggregation is the root of hypersensitivity reactions. However, the recent discovery of MRGPRX2 (Mas-Related G Protein-Coupled Receptor-X2) as a major IgE-independent stimulation pathway has also attracted much interest.1-4 Indeed, MRGPRX2 is activated by a plethora of ligands, which can be constantly present in tissues, but the low-affinity nature of MRGPRX2 (requiring micromolar agonist concentrations) likely impedes its permanent stimulation; it is, therefore, controversial whether clinically significant reactions can be triggered by MRGPRX2 (alone).2, 3 Combinatorial scenarios are an interesting option, but the sequels following concurrent activation of FcεRI and MRGPRX2 remain unresolved. We tested this possibility in skin MCs stimulated by FcεRI-aggregation or the MRGPRX2 agonists compound 48/80 (c48/80), Substance P (SP), or codeine, individually or jointly. MRGPRX2 agonists were also combined. Degranulation was assessed by histamine or β-hexosaminidase release. Activated MCs were quantified by CD107a exteriorization (detailed methods can be found in Appendix S1). Using cultured skin-derived MCs, more responsive to FcεRI-aggregation than to MRGPRX2 ligation,5 we found that histamine release on combinatorial stimulation surpassed the one elicited by each receptor individually (Figure 1A). Still more, joint stimulation of the routes was fully additive since measured release by the two stimuli was at least equal to the calculated secretion when both were applied separately (i.e., sum of “stimulus 1 + stimulus 2”) (Figure 1B). Results were the same for β-hexosaminidase (Figure S1). For CD107a upregulation, barely any increase was noted after 2 min in cells stimulated via the FcεRI-route, while MRGPRX2 ligation led to exteriorization at this point (Figures 1C and S2a). Conversely, FcεRI-aggregation resulted in a well-separated CD107a+ population after 30 min (Figures 1E and S2b). Differential kinetics were as expected since signaling by MRGPRX2 is more rapid.1, 3, 6 Importantly, the proportions of CD107+ cells were additive on combined stimulation (Figure 1D, F). In ex vivo skin MCs, simultaneous activation of MRGPRX2 and FcεRI likewise elevated the response over each stimulus alone (Figure 2A–C). Additivity was ascertained by contrasting the sum of individual stimuli against the measured response on joint stimulation (Figure 2D–F). Activating the routes at different stimulus concentrations revealed additivity also at suboptimal conditions, even trending towards synergism (Figure S4a–d). Collectively, in skin MCs, signals from the two receptor systems unite to produce strong and, indeed, additive responses, a finding of substantial clinical relevance. For example, allergens reportedly contain stimuli activating both receptors (e.g., hymenoptera venoms); moreover, drug classes (opiates, antibiotics), that activate MRGPRX2, can also elicit IgE responses in susceptible individuals. When co-activated, the anaphylactic threshold may be more readily exceeded. We tested whether low concentrations of MRGPRX2 agonists can amplify each other. Indeed, SP and codeine at suboptimal concentrations resulted in increased responsiveness over each individual stimulus (Figure S3a). In contrast, joining SP and codeine at saturating concentrations did not enhance degranulation vis-à-vis each substance (Figure S3b).6, 7 Accordingly, the sum was equivalent to the measured value in the suboptimal setting (Figure S3c), but far above under optimal conditions (Figure S3d). Collectively, we demonstrate that IgE-dependent and IgE-independent pathways unite to boost exocytosis of human skin MCs. The same degree of mediator release and clinical response may thus be achieved by strong activation of FcεRI or MRGPRX2 individually or by weaker activation of the two simultaneously, the latter likely the common scenario in vivo. Multiple combinations of elicitors are conceivable, also encompassing low levels of different MRGPRX2 agonists, which likewise amplify each other. Considering the wide repertoire of ligands, MRGPRX2 may plausibly contribute to anaphylactic reactions despite its low affinity. The study has also implications for urticaria, mastocytosis, and atopic dermatitis. We thank Ms. von Grüner and Ms. Heßler for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (BA-3769/4-1) to MB. ZW was funded by scholarships from CSC and Charité. ZL was funded by a scholarship from CSC. The study also received funding from ECARF (European Center for Allergy Research Foundation) to TZ. The authors declare they have no relevant conflicts of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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