Neuronal substance P-driven MRGPRX2-dependent mast cell degranulation products differentially promote vascular permeability

Abstract

Mas-related G protein-coupled receptor b2 (Mrgprb2) binding to its cationic endogenous and exogenous ligands induces mast cell degranulation and promotes inflammation in mice. However, the physiological roles of its human homologue MRGPRX2 remain unclear. Here we aimed to elucidate the mechanisms by which MRGPRX2 regulates vascular permeability, and generated MRGPRX2 knock-in (MRGPRX2-KI) and Mrgprb2 knockout (Mrgprb2-KO) mice. Substance P (SP) and ciprofloxacin strongly degranulated MRGPRX2-KI peritoneal mast cells (PMCs) better than WT PMCs, whereas Dermatophagoides pteronyssinus (Der p) extract and phenol-soluble modulin α3 (PSMα3) did not degranulate PMCs. SP-stimulated MRGPRX2-KI PMCs released large amounts of histamine and mast cell protease 4 (MCPT4) chymase. Der p extract, PSMα3, and MCPT4, but not histamine, induced SP release from dorsal root ganglion (DRG) cells. However, this effect of Der p extract/PSMα3 was suppressed by a transient receptor potential vanilloid 1 (TRPV1) antagonist. SP-, ciprofloxacin-, Der p extract-, PSMα3-, and MCPT4-induced vascular permeability was highest in MRGPRX2-KI mice, which depended on SP. In addition, SP-, ciprofloxacin- and PSMα3-induced MRGPRX2-dependent vascular hyperpermeability was suppressed by antihistamine and chymase inhibitor. TRPV1 antagonist also inhibited PSMα3-induced MRGPRX2-dependent vascular hyperpermeability. Both Mrgprb2-KO and MRGPRX2-KI did not influence the histamine-induced murine vascular hyperpermeability. Overall, our results suggest that neuronal SP induces MRGPRX2-dependent mast cell degranulation, releasing histamine and chymase, which promote vascular hyperpermeability directly or indirectly via DRG cell activation. Importantly, the worsening cycle (MRGPRX2 → mast cell degranulation → chymase → DRG activation → SP → MRGPRX2) seems to play an important role in human MRGPRX2-depdendent inflammation.

Keywords: MRGPRX2; chymase; degranulation; histamine; mast cell; sensory neuron; substance P; vascular permeability.

Figure 1

Generation of MRGPRX2-KI mice. (A, C, E) Surface expression levels of FcεRIα and c-Kit (upper panel) in the peritoneal (A), skin (C), and small intestinal (E) cells. Expression levels of tdTomato (middle panel) and surface expression levels of MRGPRX2 (lower panel) in FcεRIα⁺c-Kit⁺ peritoneal mast cells (A), skin mast cells (C), and small intestinal mast cells (E) in the WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice. Data are representative of three independent experiments. Control staining is shown in the shaded histograms. (B, D) Total cell numbers and percentages of FcεRIα⁺c-Kit⁺ mast cells in the peritoneal cavity (B) and numbers of toluidine blue-positive mast cells in the ear or back skin (D) of WT, Mrgprb2-KO, and MRGPRX2-KI mice. n = 3-6; ± SD.

Figure 2

Binding of compound 48/80 or ciprofloxacin to MRGPRX2 strongly promotes PMC degranulation and murine vascular permeability more than the binding to Mrgprb2. (A) Surface expression levels of FcεRIα and c-Kit (upper panel) and MRGPRX2 (middle panel) and expression levels of tdTomato (lower panel) in the PMCs from WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice. Control staining is shown in the shaded histograms. (B, D, F) Percentages of surface CD63-positive cells in WT, Mrgprb2-KO, and MRGPRX2-KI PMCs and BMMCs (B) and PMCs (D, F) after treatment with the indicated concentrations of compound 48/80 (B), 10 μg/mL ciprofloxacin (D), or 10 μg/mL icatibant (F). Data are representative of three independent experiments and indicate the mean ± SD. *P < 0.05 and **P < 0.01. (C, E, G) Quantification of the Evans blue dye that extravasated into the ear skin in WT, Mrgprb2-KO, and MRGPRX2-KI mice after treatment with indicated amounts of compound 48/80 (C), ciprofloxacin (E), and 1.75 μg icatibant (G) or phosphate-buffered saline (PBS). n = 5-9; ± SD. *P < 0.05 and **P < 0.01.

Figure 3

SP-stimulated MRGPRX2-KI PMCs released larger amounts of amines and proteases than the WT counterparts. (A–E) Percentages of surface CD63⁺ PMCs (A) and levels of histamine (B), serotonin (C), mMCPT6 (D), and mMCPT4 (E) in the culture supernatants of anti-TNP IgE-sensitized WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) PMCs after treatment with the indicated concentrations of TNP-BSA and SP. Data are representative of three independent experiments. *P < 0.05 and **P < 0.01.

Figure 4

Intradermal injection of SP induces higher vascular permeability in MRGPRX2-KI mice compared to that in WT mice. (A, F) Quantification of the Evans blue dye that extravasated into the ear skin in WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice after treatment with indicated amounts of SP (A) and anti-DNP IgE plus DNP-HSA (F). (B-E) WT, Mrgprb2-KO, and MRGPRX2-KI mice 6 and 24 h (B) or 6 h (D, E) after the intradermal injection of 25 pmol SP. (B) Ear thickness. (C) Representative images of toluidine blue (pH 4.1)-stained mast cells in the ear skin sections (Scale bar; 20 μm). (D) Total number of mast cells (left panel) and percentage of degranulated mast cells (right panel) in the ear skin. (E) Percentage of neutrophils. (A, B, D-F) n = 4-8; ± SD. *P < 0.05 and **P < 0.01.

Figure 5

Stimulation with the Der P extract and PSMα3 increases vascular permeability via SP-driven MRGPRX2 signal in the skin mast cells. (A, D) Quantification of the Evans blue dye that extravasated into the ear skin in the WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice intradermally injected with the indicated amounts of the Der p extract (A) and PSMα3 (D). (B, E) Percentages of degranulated skin mast cells in the WT, Mrgprb2-KO and MRGPRX2-KI 6 h after the intradermal injection of 10 μg Der p extract (B) and 2 μg PSMα3 (E). (C, F) Percentages of surface CD63⁺ PMCs from the WT, Mrgprb2-KO, and MRGPRX2-KI mice after stimulation with the indicated concentrations of Der p extract (C) and PSMα3 (F). (G, H) Quantification of the Evans blue dye that extravasated into the ear skin in the WT, Mrgprb2-KO, and MRGPRX2-KI mice intradermally injected with 10 μg Der p extract (G) and 2 μg PSMα3 (H) along with the anti-SP or control serum. (A, B, D, E, G, H) n = 3-10; ± SD. *P < 0.05 and **P < 0.01. (C, F) Data are representative of three independent experiments.

Figure 6

SP-, ciprofloxacin- or PSMα3-stimulated MRGPRX2-dependent vascular hyperpermeability is suppressed by antihistamine or chymase inhibitor. (A-K) Quantification of the Evans blue dye that extravasated into the ear skin. (A, B, F, G) WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice were intradermally injected with 25 pmol SP (A, B) and 2 μg PSMα3 (F, G). (C-E, H-J) MRGPRX2-KI mice were intradermally injected with 40 μg ciprofloxacin (C-E). WT mice were intradermally injected with anti-DNP IgE, followed by intravenous injection of DNP-HSA (H-J). Effects of 600 μg cetirizine or vehicle (A, C, F, H), 50 μg TY-51469 or vehicle (B, D, G, I), and 15 μL of anti-SP or control serum (E, J) on vascular permeability. (K) WT, Mrgprb2-KO, and MRGPRX2-KI mice were intradermally injected with 100 μg histamine. n = 4-10; ± SD. *P < 0.05 and **P < 0.01. Data are representative of two independent experiments.

Figure 7

Der p extract, PSMα3, and chymase, but not histamine, stimulated the release of SP from DRG cells. (A-D) Levels of SP in the culture supernatants of DRG cells stimulated with the indicated concentrations of Der p extract, PSMα3, capsaicin, and vehicle (A), 30 μg/mL Der p extract, 30 μg/mL PSMα3, 100 μM capsaicin, and PBS in the presence of AMG517 or vehicle (B), 5 μg/mL of mMCPT1, mMCPT4, and mMCPT6, 1 μM histamine, and vehicle (C), and 5 μg/mL of mMCPT1, mMCPT4, MCPT6, and PBS in the presence of TY-51469 or vehicle (D). *P < 0.05 and **P < 0.01. Data are representative of three independent experiments. (E-G) Quantification of the Evans blue dye that extravasated into the ear skin in the WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice intradermally injected with 200 ng mMCPT4 or vehicle (E), MRGPRX2-KI mice intradermally injected with 200 ng mMCPT4 along with anti-SP or control serum (F), or WT, Mrgprb2-KO, and MRGPRX2-KI mice intradermally injected with 2 μg PSMα3 before oral administration of AMG517 or vehicle (G). n = 4-6; ± SD. *P < 0.05 and **P < 0.01. Data are representative of two independent experiments.