Meglumine gadoterate induces immunoglobulin‐independent human mast cell activation via <scp>MRGPRX2</scp>

Gadolinium-based contrast agents (GBCA) are intravenous drugs used to enhance resolution in magnetic resonance imaging. They can induce immediate hypersensitivity reactions, yet their pathogenic mechanisms remain poorly characterized. This hampers the ability to predict which patients are at risk of developing them.1 In fact, affected patients usually show negative skin tests and can react upon the first known GBCA exposure, which implies that IgE-independent mechanisms might be driving this inflammatory response. The Mas-related G protein-coupled receptor member X2 (MRGPRX2) has been recently associated with non-IgE-mediated immediate hypersensitivity reactions.2 Some drugs, such as fluoroquinolones, vancomycin, neuromuscular-blocking agents, icatibant, morphine, leuprolide, and iodinated contrast media, have been reported to activate MRGPRX2, which is highly expressed in mast cells (MCs).3 To assess the ability of GBCA to induce non-IgE-mediated hypersensitivity reactions, we stimulated the human MC line LAD2 with several commercial GBCA, namely, meglumine gadoterate, gadobutrol, gadoxetate disodium, and gadoteridol. Then, we determined cell viability and degranulation by flow cytometry4 (see a detailed material and methods section in this article's Appendix S1). Of the GBCA tested, meglumine gadoterate was able to induce significant MC activation as compared to unstimulated MCs (Figure 1A; Figure S1A). It did it without reducing cell viability (Figure 1B), which can induce nonspecific activation. Moreover, we confirmed the ability of meglumine gadoterate to induce MC activation without affecting viability in LADR cells, a different MC line (Figure S1B). Meglumine gadoterate is an ionic macrocyclic paramagnetic contrast media. It is composed of gadolinium, which together with the chelating agent tetraxetan (also known as DOTA), yields gadoteric acid. The base meglumine and gadoteric acid form the salt meglumine gadoterate (Figure 1C). We ascertained the ability of the different components of meglumine gadoterate to induce MC activation. Meglumine and DOTA induced MC activation significantly (Figure 1D), but only meglumine did it without affecting cell viability, as compared to untreated cells (Figure 1E). Interestingly, meglumine caused MC activation at lower concentrations than meglumine gadoterate, according to the half maximal effective concentration (EC50) of both substances (Figure 1F). The logarithmically transformed EC50 for meglumine gadoterate was 2.04 (R2 = 0.75), and for meglumine was about one order of magnitude lower (1.06; R2 = 0.71). Next, we decided to explore the involvement of MRGPRX2 in meglumine gadoterate-mediated MC activation. We assessed its expression on LAD2 cells by flow cytometry following stimulations with either meglumine gadoterate or vancomycin (a known MRGPRX2 agonist).5 Under basal conditions, LAD2 cells expressed high levels of MRGPRX2 (Figure 2A). Following vancomycin provocation, MRGPRX2 expression levels were reduced, as compared to untreated LAD2 cells (Figure 2B), while the levels of FcεRI, used as a control, remained unchanged (Figure S1C). Interestingly, we observed a similar decrease in MRGPRX2 expression levels upon meglumine gadoterate challenges, suggestive of both the signaling and the internalization of this receptor (Figure 2B).6 On the contrary, a reduction in MRGPRX2 expression following meglumine stimulation was not detected (data not shown), which implies that meglumine induces MC activation by a different mechanism. Finally, to better understand the role of MRGPRX2 in MC activation by meglumine gadoterate, we pretreated MCs with a MRGPRX2 antagonist-1 (HY-145191) prior stimulation with meglumine gadoterate or vancomycin, which prevented MC activation (Figure 2C). In conclusion, our study demonstrates the ability of meglumine gadoterate to induce immunoglobulin-independent MC activation via MRGPRX2. Furthermore, we have delved into the meglumine gadoterate components that may be involved in MC activation and identified meglumine as a potential causative of non-IgE mediated hypersensitivity reactions. Further studies should be performed to define clinically relevant interactions between diverse radiological contrast media and MRGPRX2. RJS reports grants by the FSE/FEDER through the Instituto de Salud Carlos III (CP20/00043; PI22/00236; Spain), The Nutricia Research Foundation (NRF-2021-13; The Netherlands), New Frontiers in Research Fund (NFRFE-2019-00083; Canada), and SEAIC (BECA20A9; Spain). PHR is supported by the INVESTIGO Program of the Community of Madrid (Spain), which is funded by “Plan de Recuperación, Transformación y Resiliencia” and “NextGenerationEU” of the European Union (09-PIN1-00015.6/2022). FV, RJS, and CB conceived and designed the study. PHR, CLS, and RJS performed the experiments and analyzed and discussed the data. PHR and CLS prepared figures. RJS wrote the paper. RJS and CB oversaw the project and raised funding. All the authors read, provided comments, and approved the final version of the manuscript. We thank all the members of the Jiménez-Saiz Lab for technical help and scientific input. All the authors have no significant conflicts of interest to declare in relation to this manuscript. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1 Figure S1 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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