PMX-53 as a dual CD88 antagonist and an agonist for Mas-related gene 2 (MrgX2) in human mast cells

Abstract

Human mast cells express the G protein coupled receptor (GPCR) for C5a (CD88). Previous studies indicated that C5a could cause mast cell degranulation, at least in part, via a mechanism similar to that proposed for basic neuropeptides such as substance P, possibly involving Mas-related gene 2 (MrgX2). We therefore sought to more clearly define the receptor specificity for C5a-induced mast cell degranulation. We found that LAD2, a human mast cell line, and CD34(+) cell-derived primary mast cells express functional MrgX1 and MrgX2 but the immature human mast cell line HMC-1 does not. A potent CD88 antagonist, PMX-53 (10 nM) inhibited C5a-induced Ca(2+) mobilization in HMC-1 cells, but at higher concentrations (≥30 nM) it caused degranulation in LAD2 mast cells, CD34(+) cell-derived mast cells, and RBL-2H3 cells stably expressing MrgX2. PMX-53 did not, however, activate RBL-2H3 cells expressing MrgX1. Although C5a induced degranulation in LAD2 and CD34(+) cell-derived mast cells, it did not activate RBL-2H3 cells expressing MrgX1 or MrgX2. Replacement of Trp with Ala and Arg with dArg abolished the ability of PMX-53 to inhibit C5a-induced Ca(2+) mobilization in HMC-1 cells and to cause degranulation in RBL-2H3 cells expressing MrgX2. These findings demonstrate that C5a does not use MrgX1 or MrgX2 for mast cell degranulation. Moreover, it reveals the novel finding that PMX-53 functions as a potent CD88 antagonist and a low-affinity agonist for MrgX2. Furthermore, Trp and Arg residues are required for the ability of PMX53 to act as both a CD88 antagonist and a MrgX2 agonist.

Fig. 1.

C5a, C5aP, and CD88 antagonist PMX-53 induce degranulation in human mast cells. LAD2 mast cells were stimulated with different concentrations of native C5a, C5aP, PMX-53, PMX-53S, or PMX-53C and percentage degranulation (β-hexosaminidase release) was determined. Data are mean ± S.E.M. of n = 3. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. *, p < 0.05.

Fig. 2.

CD88 antagonist PMX-53 induces Ca²⁺ mobilization in LAD2 mast cells. Cells were incubated with Indo-1AM and stimulated with increasing concentrations of PMX-53 (A), PMX-53S (B), and PMX-53C (C) and intracellular Ca²⁺ mobilization was determined. Data shown are representative of 3 similar experiments.

Fig. 3.

PMX-53 inhibits C5a-induced responses in HMC-1 and RBL-2H3 cells expressing CD88. A, HMC-1 cells were incubated with Indo-1AM and stimulated sequentially with 10 nM C5a and 10 nM C3a, and intracellular Ca²⁺ mobilization was determined. Cells were exposed to 10 nM PMX-53 (B), PMX-53C (C), or PMX-53S (D) 100 s before stimulation with C5a and C3a, and Ca²⁺ mobilization was determined. E, RBL-2H3 cells stably expressing CD88 were pretreated with vehicle or 10 nM PMX-53 and exposed to 1 nM C5a, and percentage degranulation (β-hexosaminidase release) was determined. Data shown are representative of three similar experiments. Data are mean ± S.E.M. of n = 3. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. *, p < 0.05.

Fig. 4.

PMX-53 causes degranulation in human mast cells via GPCR. LAD2 mast cells were pretreated with vehicle, GPA-2 (1 μM, 30 min; A), or PTx (100 ng/ml, 16 h; B). Cells were stimulated with 10 nM C5aP and 100 nM PMX-53, and degranulation was determined. In all experiments, LAD2 cells were also stimulated by IgE/anti-IgE as a non-GPCR control. Data are mean ± S.E.M. of n = 3. Statistical significance was determined by one-way ANOVA with Dunnett's post test to compare differences between vehicle and GPA-2 or PTx treatment. *, p < 0.05.

Fig. 5.

LAD2 cells express functional MrgX1 and MrgX2 receptors but HMC-1 cells do not. A, MrgX1 and MrgX2 receptor expression was determined in LAD2 and HMC-1 cells by RT-PCR. Ligands for MrgX1 (BAM-22P; 1 μM) and MrgX2 (CST; 1 μM), induced sustained Ca²⁺ mobilization (B) and degranulation (C) in LAD2 mast cells. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. *, p < 0.05. D, BAM-22P did not induce Ca²⁺ mobilization, and CST induced modest Ca²⁺ mobilization in HMC-1 cells. 10 nM C3a was used as a positive control. Data shown are representative of three similar experiments.

Fig. 6.

Human CD34⁺-derived primary mast cells express MrgX1 and MrgX2 and respond to PMX-53 for degranulation and Ca²⁺ mobilization. Human CD34⁺-derived primary mast cells were stimulated with C5aP, PMX-53, PMX-53S, and PMX-53C (1 μM), and percentage degranulation (β-hexosaminidase release) was determined (A). MrgX1 and MrgX2 receptor expression was determined in CD34⁺-derived primary mast cells by RT-PCR (A, inset). C5a (B), PMX-53 (C), and PMX-53S but not PMX-53C (D) induced Ca²⁺ mobilization in CD34⁺-derived mast cells. Data shown are representative of three similar experiments. Statistical significance was determined by one-way ANOVA with Dunnett's post test. *, p < 0.05.

Fig. 7.

PMX-53 uses MrgX2 to induce mast cell degranulation. RBL-2H3 cells stably expressing MrgX1 (A) or MrgX2 (B) were incubated with anti-DNP specific IgE (1 μg/ml, 16 h) and stimulated with the indicated peptides (1 μM), substance P (SP; 1 μM), or antigen (DNP-BSA; 30 ng/ml) for 30 min, and β-hexosaminidase release was measured. C and D, MrgX1 and MrgX2-expressing cells were also incubated with Indo-1AM and stimulated with selected peptides (1 μM), and intracellular calcium mobilization was determined. Data shown are representative of three similar experiments. Statistical significance was determined by one-way ANOVA with Dunnett's post test. *, p < 0.05.

Fig. 8.

PMX-53 and CST do not activate murine peritoneal and bone marrow-derived mast cells. A, murine bone marrow-derived and peritoneal mast cells were incubated with DNP-specific mouse IgE (1 μg/ml, 16 h). Cells were exposed to buffer (control), 1 μM PMX-53, 1 μM CST, or 30 ng/ml DNP-BSA (Ag) for 30 min, and β-hexosaminidase release was measured. Data are mean ± S.E.M. of n = 3. B, murine bone marrow-derived mast cells were incubated with DNP-specific mouse IgE (1 μg/ml, 16 h). Cells were incubated with Indo-1AM and were exposed to 1 μM PMX-53, 1 μM CST, or 100 ng/ml DNP-BSA, and intracellular Ca²⁺ mobilization was determined. Data shown are representative of three similar experiments. Statistical significance was determined by one-way ANOVA with Dunnett's post test. *, p < 0.05.