Distinct effects of antigen and compound 48/80 in the guinea pig trachea
Authors: Jielu Liu, Johan Kolmert, Jesper Säfholm, Anna‐Karin Johnsson, Javier Zurita, Craig E. Wheelock, Sven‐Erik Dahlén, Gunnar Nilsson, Mikael Adner
Journal: Allergy (2021)

Mast cells (MCs) can be activated immunologically by allergen and non-immunologically via the Mas-related G protein-coupled receptor member X2 (MRGPRX2).1 With the emerging interest of MRGPRX2, we compared the airway smooth muscle (ASM) response and release of mediators in airways exposed to the human MRGPRX2 agonist compound 48/80 (C48/80) and house dust mite (HDM). A guinea pig model was selected for greatest similarity with human airways.2 We measured ASM contraction using organ baths. Both C48/80 and HDM induced marked constriction in trachea from sensitized guinea pigs (Figure 1). The effects of HDM were most likely due to specific IgG which, in addition to IgE, has been shown to induce smooth muscle contractions in guinea pig trachea3 (Figure S1). Antagonizing histamine H1 receptors inhibited the concentration-response curves and the initial 15 minutes of responses induced by submaximal bolus dose challenges, indicating that the early response of both treatments was mediated by histamine. In contrast, the whole phase responses could only be dampened by inhibition of both histamine and 5-lipoxygenase (5-LOX) pathways, suggesting that both C48/80 and HDM contractions were mediated through histamine and 5-LOX products (Figure 1). For measuring mediator release induced by C48/80 and HDM, a bolus dose of the strongest concentration of each stimulus was given. Histamine and lipid mediators in the bath fluids were measured by ELISA and a recently developed mass-spectrometry platform with high specificity and sensitivity for detecting lipid mediators.4 Both C48/80 and HDM released histamine to similar levels (Figure 2). In addition, 29 lipid mediators were changed after 60 minutes stimulation; 15 were elevated by both C48/80 and HDM challenge, and an additional 13 only by C48/80, while LTE4 was only elevated by HDM (Figure S2 and Table S1). The prostanoids were the most abundant bioactive lipid mediators detected in the organ baths after challenges. Prostaglandin (PG) D2, a major lipid mediator from MCs, together with PGD1 and PGD3, were elevated in bath fluids challenged by C48/80 and HDM to similar levels. On the other hand, thromboxane (TX) B2, was to a greater extent released by C48/80 than HDM, and PGE metabolites, PGF2α, 6-keto-PGF1α, and TXB3 were solely increased by C48/80 (Figure 2 and Figure S3). Both C48/80 and HDM increased LTB4, whereas only HDM triggered the release of cysteinyl leukotrienes (CysLTs) reflected by the elevation of LTE4 (LTC4 0%, LTD4 1.7%, and LTE4 98.3% of measured products by an additional analysis). In addition to prostanoids and leukotrienes, low levels of other lipid mediators were primarily increased by C48/80 (Figure 2 and Figure S3). The strong histamine release induced by C48/80 and HDM indicated that the main effect of the stimuli is most likely due to direct activation of MCs. The different responses can be due to distinct intracellular signaling pathways for G protein-coupled receptors and Fc-receptors.5, 6 However, since the receptor responsible for C48/80 responses in the guinea pig trachea is not completely defined, we cannot rule out a release from other cells present in the airways. But it is likely that the response is mediated through MRGPRX2 orthologues existing in guinea pigs, as other known MRGPRX2 agonists also induced ASM contractions that were blocked by histamine H1 receptor antagonist (Figure S4). In conclusion, C48/80 and HDM demonstrated distinct patterns of MC activation and smooth muscle contraction in guinea pig trachea. Both stimuli induced release of histamine and PGD2, while only HDM challenge caused biosynthesis of CysLTs, C48/80 produced a greater release of several prostanoids. Thus, these findings support the need to characterize the differential release of MC synthesized lipid mediators and their effects in asthma. Dr. Kolmert reports personal fees from Gesynta Pharma AB, outside the submitted work. All the other authors declare that there is no conflict of interest. JL, SED, GN, and MA conceived and designed the studies. JL, JK, AKJ, and JZ carried out the studies. JL and JK acquired and analyzed the data. JL, JK, JS, CEW, SED, GN, and MA interpreted the data. JL, JK, JS, and MA wrote the manuscript draft. All authors reviewed, critically revised, and approved the final manuscript. 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.