β-Defensins activate human mast cells via Mas-related gene X2

Abstract

Human β-defensins (hBDs) stimulate degranulation in rat peritoneal mast cells in vitro and cause increased vascular permeability in rats in vivo. In this study, we sought to determine whether hBDs activate murine and human mast cells and to delineate the mechanisms of their regulation. hBD2 and hBD3 did not induce degranulation in murine peritoneal or bone marrow-derived mast cells (BMMC) in vitro and had no effect on vascular permeability in vivo. By contrast, these peptides induced sustained Ca(2+) mobilization and substantial degranulation in human mast cells, with hBD3 being more potent. Pertussis toxin (PTx) had no effect on hBD-induced Ca(2+) mobilization, but La(3+) and 2-aminoethoxydiphenyl borate (a dual inhibitor of inositol 1,4,5-triphosphate receptor and transient receptor potential channels) caused substantial inhibition of this response. Interestingly, degranulation induced by hBDs was substantially inhibited by PTx, La(3+), or 2-aminoethoxydiphenyl borate. Whereas human mast cells endogenously express G protein-coupled receptor, Mas-related gene X2 (MrgX2), rat basophilic leukemia, RBL-2H3 cells, and murine BMMCs do not. Silencing the expression of MrgX2 in human mast cells inhibited hBD-induced degranulation, but had no effect on anaphylatoxin C3a-induced response. Furthermore, ectopic expression of MrgX2 in RBL-2H3 and murine BMMCs rendered these cells responsive to hBDs for degranulation. This study demonstrates that hBDs activate human mast cells via MrgX2, which couples to both PTx-sensitive and insensitive signaling pathways most likely involving Gαq and Gαi to induce degranulation. Furthermore, murine mast cells are resistant to hBDs for degranulation, and this reflects the absence of MrgX2 in these cells.

Fig 1. hBDs do not induce degranulation in murine mast cells in vitro or in vivo

(A) Amino acid sequences of hBD2, hBD3, CST, LL-37 and mCRAMP. Postively charged amino acids are underlined. (B) Murine peritoneal mast cells (PMC) or (C) bone marrow-derived mast cells (BMMC) were incubated with mouse IgE (1 μg/ml, 16 h) and then stimulated with hBD2 (5 μM), hBD3 (3 μM) or antigen (DNP-BSA, 100 ng/ml) and degranulation was determined. (D); For IgE-mediated passive cutaneous anaphylaxis, C57BL/6 mice or mast cell deficient W^sh/W^sh mice were passively sensitized in the ear with PBS (open white bars) or IgE (closed black bars) (20 ng for 16 h) and challenged with an intravenous injection of a 100 μg antigen (DNP-BSA) in 200 μl of PBS containing 1% Evans blue. For defensin, C57BL/6 mice were intravenously injected with 200 μl of 0.5% Evans blue before intradermal injection of hBD3 (closed black bars) (150 ng) into one side of the ear, and vehicle PBS (open white bars) into the other ear. The mice were killed 30 min after injection, and Evans blue dye contents in the ear tissues were measured. The results are expressed as OD_A650/g tissue. Data shown are representative of 3 similar experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. * indicates p<0.01.

Fig. 2. hBDs induce degranulation and Ca²⁺ mobilization in LAD2 and CD34⁺-derived human mast cells

LAD2 mast cells were stimulated with different concentrations of (A) hBD2 or (C) hBD3 and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± SEM of three experiments. (B, D) LAD2 cells were loaded with Indo-1AM and Ca²⁺ mobilization in response hBD2 or hBD3 was determined. (E) CD34⁺ cell-derived primary mast cells were exposed to different concentrations of hBD3 and degranulation was determined. (F), CD34⁺ cell-derived mast cells were loaded with indo-1 and Ca²⁺ mobilization in response to hBD3 was determined. Data shown are representative of 3 similar experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001.

Fig. 3. Effects of Pertussis toxin and La³⁺ on C3a, hBD2 and hBD3-induced Ca²⁺ mobilization and degranulation in human mast cells

(A, E) Indo-1 loaded LAD2 cells were exposed to C3a, followed by hBD2 or hBD3 and intracellular Ca²⁺ mobilization was determined. (B, F), Cells were treated with medium or pertussis toxin (PTx; 100 ng/ml, 16 h) and effects of C3a, hBD2 or hBD3 on Ca²⁺ mobilization was determined. (C, G), Indo-1 loaded LAD2 cells were exposed to La³⁺ (lanthanum chloride, 1 μM) and C3a, hBD2 or hBD3-induced Ca²⁺ mobilization was determined. Traces are representative of 3 independent experiments. LAD2 mast cells were exposed to (D), Pertussis toxin (PTx; 100 ng/ml, 16 h) or (H) La³⁺ (lanthanum chloride, 1 μM, 30 min) and C3a, hBD2 and hBD3-induced degranulation was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. ** indicates p<0.001.

Fig. 4. Effects of 2-APB and GFX on hBD3-induced Ca²⁺ mobilization and degranulation in human mast cells

(A-C) Indo-1 loaded LAD2 cells were left untreated or pretreated with 2-APB (100 μM)) or GFX (10 μM) and hBD3-induced Ca²⁺ mobilization was determined. Traces are representative of 3 independent experiments. (D) LAD2 mast cells were pretreated with buffer control or 2-APB or GFX and degranulation response to hBD3 was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01.

Fig. 5. Knockdown of MrgX2 inhibits hBD2, hBD3 and cortistatin but not C3a-induced mast cell degranulation

LAD2 mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against MrgX2. (A) Western blotting was performed to determine MrgX2 expression in control and MrgX2 knockdown (KD) cells. (B) shRNA control and MrgX2 KD cells were stimulated with hBD2, hBD3, cortistatin (CST) or C3a and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001.

Fig 6. hBDs activate RBL-2H3 and HEK293 cells expressing MrgX2

(A) RBL-2H3 cells stably expressing MrgX2 were stimulated with buffer, hBD2, hBD3 or cortistatin (CST) for 30 min and β-hexosaminidase release was measured. Data shown are representative of 3 similar experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001. RBL-2H3 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca²⁺ mobilization in response to (B) hBD2, (C) hBD3 or (D) CST was determined. HEK293 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca²⁺ mobilization in response to (E) hBD3 or (F) CST was determined. Traces shown are representative of 3 individual experiments.

Fig 7. hBD3 and mCRAMP activate murine BMMCs expressing MrgX2

(A) BMMCs were transiently transfected with HA tagged MrgX2 (solid line) or control plasmid vector (broken line) and MrgX2 receptor expression level was analyzed using flow cytometry. A representative histogram is shown. (B) Control and MrgX2 expressing BMMCs were incubated with DNP specific mouse IgE (1 μg/mL, 16 h). Cells were exposed to buffer (control), CST, hBD3, mCRAMP or DNP-BSA (10 ng/mL) for 30 minutes and β-hexosaminidase release was measured. LAD2 cells were stimulated with mCRAMP and (C) intracellular Ca²⁺ mobilization or (D) degranulation was determined. Traces are representative of 3 independent experiments. Bar graphs represent mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01.