G protein coupled receptor specificity for C3a and compound 48/80-induced degranulation in human mast cells: roles of Mas-related genes MrgX1 and MrgX2

Abstract

Although human mast cells express G protein coupled receptors for the anaphylatoxin C3a, previous studies indicated that C3a causes mast cell degranulation, at least in part, via a C3a receptor-independent mechanism similar to that proposed for polycationic molecules such as compound 48/80. The purpose of the present study was to delineate the receptor specificity of C3a-induced degranulation in human mast cells. We found that C3a, a C3a receptor "superagonist" (E7) and compound 48/80 induced Ca(2+) mobilization and degranulation in a differentiated human mast cell line, LAD2. However, C3a and E7 caused Ca(2+) mobilization in an immature mast cell line, HMC-1 but compound 48/80 did not. We have previously shown that LAD2 cells express MrgX1 and MrgX2 but HMC-1 cells do not. To delineate the receptor specificity for C3a and compound 48/80 further, we generated stable transfectants expressing MrgX1 and MrgX2 in a rodent mast cell line, RBL-2H3 cells. We found that compound 48/80 caused degranulation in RBL-2H3 cells expressing MrgX1 and MrgX2 but C3a did not. By contrast, E7 activated RBL-2H3 cells expressing MrgX2 but not MrgX1. These findings demonstrate that in contrast to previous reports, C3a and compound 48/80 do not use a shared mechanism for mast cell degranulation. It shows that while compound 48/80 utilizes MrgX1 and MrgX2 for mast cell degranulation C3a does not. It further reveals the novel finding that the previously characterized synthetic peptide, C3a receptor "superagonist" E7 activates human mast cells via two mechanisms; one involving the C3a receptor and the other MrgX2.

Figure 1. Effects of C3a receptor agonists and compound 48/80 on degranulation in human mast cells

(A) LAD2 mast cells were stimulated with different concentrations of C3a, C3 desArg, C3aP, C3a receptor superagonist E7 and scrambled E7 (E7S) for 30 min and percent degranulation (β-hexosaminidase release) was determined. (B) LAD2 cells were stimulated with different concentrations of compound 48/80 and degranulation was determined. (C) CD34⁺-derived human mast cells were exposed to buffer (control), C3a (100 nM), E7 or E7S (1 µM) for 30 min and percent degranulation was determined. Data are mean ± S.E.M of 3 – 4 experiments. Statistical significance was tested using two way (A) or one way (B and C) ANOVA with Bonferroni’s post test. * indicates P<0.05

Figure 2. Ca²⁺ mobilization by C3a, E7, E7S and compound 48/80 in LAD2 mast cells and HMC-1 cells

(A– D) LAD2 mast cells (0.2 × 10⁶/ml) and (E– H) HMC-1 cells (1 × 10⁶/ml) were incubated with Indo-1AM and, as indicated by arrows, were stimulated with C3a (10 nM; A and E), E7 (1 µM, B and F), E7S (1 µM; C and G), compound 48/80 (1 µg/ml, D and H) and intracellular Ca²⁺ mobilization was determined. For panels G and H first arrow (from the left) indicates E7S or compound 48/80 but the second arrow indicates C3a (10 nM). Data shown are representative of 3 similar experiments.

Figure 3. Effects of Pertussis toxin (PTx) on degranulation induced by C3a, E7, E7S, compound 48/80 and antigen in LAD2 mast cells

Mast cells were cultured with human IgE (1 µg/ml) in the presence or absence of PTx for 16 h. Cells were then washed and stimulated with C3a (100 nM), E7 (1 µM), E7S (1 µM), compound 48/80 (100 ng/ml) or anti-human IgE (1 µg/ml) for 30 min. Degranulation is expressed as percent of total β-hexosaminidase present in granules. Data are mean ± S.E.M of 3 experiments. Statistical significance was tested using two way ANOVA with Bonferroni’s post test. * indicates P<0.05 comparing vehicle to PTx-treated cells.

Figure 4. Roles of MrgX1 and MrgX2 on C3a,E7 and compound 48/80-induced mast cell degranulation

(A) Mock transfected RBL-2H3 cells (B) cells stably expressing MrgX1 or (C) MrgX2 were incubated with anti-DNP specific IgE (1 µg/ml, 16 hours) and stimulated with C3a (100 nM) or the indicated peptides (1 µM), compound 48/80 (100 ng/ml) or antigen (DNP-BSA, 30 ng/ml) for 30 min and β-hexosaminidase release was measured. Data shown are representative of 3 experiments. Statistical significance was tested using one way ANOVA with Bonferroni’s post test. * indicates P<0.05.