FIG 5.

PgLPS₁₆₉₀ has no effect on MRGPRX2 expression but inhibits degranulation through its association with LL-37. (A) RBL-MRGPRX2 (left) or LAD2 (right) cells were incubated at 37°C for 5 min with buffer (control), PgLPS₁₆₉₀ (50 μg/ml), or PgLPS_1435/1449 (50 μg/ml). The reaction was stopped, and the cell surface MRGPRX2 level was determined by flow cytometry as described in Materials and Methods. MFI, mean fluorescence intensity. Untransfected RBL cells and LAD2 cells incubated with an isotype-matched antibody were used as controls. ns, not significant. (B and C) RBL-MRGPRX2 cells were preincubated with buffer, PgLPS₁₆₉₀, or PgLPS_1435/1449 at 37°C for 5 min and then exposed to LL-37 (3 μM, 30 min) (B) or hBD3 (300 nM, 30 min) (C), and degranulation (β-hexosaminidase release) was determined. Results are the means and SEM for three independent experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's posttest (**, P < 0.001). (D) Untransfected RBL cells (first panel) and RBL-MRGPRX2 cells (second panel) were exposed to FAM-LL-37 (1 μM) and analyzed by confocal microscopy. RBL-MRGPRX2 cells were preincubated with PgLPS₁₆₉₀ (50 μg/ml) (third panel) or PgLPS_1435/1449 (50 μg/ml) (last panel), exposed to FAM-LL-37 (1 μM, 30 min), and analyzed by confocal microscopy. Bars = 20 μm.