Fig. 3: RASF can induce mast cell activation through MRGPRX2.

To imitate RA synovial microenvironment in vitro, LAD2 cells were stimulated with synovial fluid from RA patients (RASF). a LAD2 cells were loaded with Indo-1 and applied with RASF. The ratio of Ca²⁺ bound Indo-1 violet to Ca²⁺ free Indo-1 blue was plotted against time and was monitored for nearly five minutes by flow cytometry. The black arrow indicated the time when RASF was added. b Intracellular calcium flux (fold change of Indo-1 ratio compared with baseline) of LAD2 cells in response to RASF or PBS was shown (n = 3 for each group, pooled from three independent experiments, P = 0.0002). c Statistical analysis of degranulation rate, indicated by the proportion of CD63⁺ cells in LAD2 after being stimulated with different concentrations of RASF for 20 min (n = 3 for each group, pooled from three independent experiments, P = 0.0139). d Phosphorylated levels of signaling pathways in LAD2 cells treated with 10% RASF for different time intervals. Data are representative of three independent experiments. e Western blots of MRGPRX2 and Tubulin in total protein extracts of synovial tissues from OA and RA patients. Data are representative of two independent experiments. f Quantification of relative MRGPRX2 expression by densitometry (n = 10 biologically independent samples for each group, P = 0.0068). g Statistical analysis of LL37 concentration in synovial tissues between OA and RA patients (n = 10 biologically independent samples for each group, P = 0.0023). Intracellular calcium flux (h), degranulation rate (i), and representative blots (j) for LAD2 treated with siRNA against MRGPRX2 or control siRNA (n = 3 for each group, pooled from three independent experiments, P < 0.0001 for h, P = 0.0362 for i) after RASF stimulation. Data are representative of three independent experiments (j). Data are presented as the mean ± SEM and analyzed using two-way ANOVA (b, h, i), Kruskal-Wallis test (c), two-tailed Mann Whitney test (f), two-tailed unpaired t test (g). *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are provided as a Source Data file.