Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells

Abstract

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E₂ (PGE₂) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE₂ and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE₂ were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE₂-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE₂ on MCs may be involved in Ag-independent hypersensitivity in vivo.

Keywords: Ca2+ influx; EP3 receptor; P2X4 receptor; PI3 kinase; bone marrow-derived mast cell; extracellular ATP; mast cell degranulation; prostaglandin E2.

Figure 1

Synergistic effects of ATP and prostaglandin (PG)E₂ on mast cell (MC) degranulation. (A) Bone marrow-derived MCs (BMMCs) were stimulated with sphingosine-1-phosphate (S1P) (1 μM), PGE₂ (1 μM), histamine (100 μM), C5a (10 nM), PGD₂ (1 μM), and UDP-glucose (100 μM) with or without ATP (100 μM) (n = 3, mean ± SEM). ** p < 0.01 indicates a significant difference from the control. (B) BMMCs were stimulated for 1–30 min with ATP (100 μM, ▲) and PGE₂ (1 μM, ■) alone or simultaneously (♦; n = 3, mean ± SEM). (C) BMMCs were stimulated with different concentrations (0.01–1 μM) of PGE₁ (△, ▲) and PGE₂ (□, ■) with (△, □) or without (▲, ■) ATP (100 μM) (n = 3, mean ± SEM). * p < 0.05 and ** p < 0.01 indicate significant differences compared to ATP alone. (D) BMMCs were stimulated with different concentrations of ATP (1–100 μM) with (▲) or without (■) PGE₂ (1 μM) (n = 3, mean ± SEM). * p < 0.05 and ** p < 0.01 indicate significant differences compared to PGE₂ alone.

Figure 2

Involvement of EP3 receptor activation in the synergistic effect of prostaglandin (PG)E₂ and ATP on mast cell (MC) degranulation. (A) Bone marrow-derived MCs were preincubated with a vehicle, ONO-8713 (EP1 antagonist), ONO-AE3-240 (EP3 antagonist), and ONO-AE3-208 (EP4 antagonist) at 1 μM for 5 min and then stimulated with vehicle (-) or ATP (100 μM) with or without PGE₂ (1 μM) for 5 min. Data are shown as the mean ± SEM (n = 3). * p < 0.05 indicates a significant difference from the control. (B) BMMCs were stimulated with PGE₂, ONO-DI-004 (EP1 agonist), ONO-AE1-259 (EP2 agonist), ONO-AE-248 (EP3 agonist), or ONO-AE1-329 (EP4 agonist) at 1 μM with or without ATP (100 μM). Data are shown as the mean ± SEM (n = 3). * p < 0.05 and ** p < 0.01 indicate significant differences from the response without ATP (none). (C) BMMCs were treated with or without pertussis toxin (PTX, 50 ng/mL) overnight and stimulated with ATP (100 μM) with or without PGE₂ (1 μM) for 5 min. Data are shown as the mean ± SEM (n = 3). * p < 0.05 indicates a significant difference from the control.

Figure 3

Involvement of P2X4 receptor (P2X4R) activation in the synergistic effect of prostaglandin (PG) E₂ and ATP on mast cell (MC) degranulation. (A) Bone marrow-derived MCs (BMMCs) were stimulated concurrently with PGE₂ (1 μM) and a vehicle, ATP, ADP, or UTP (100 μM) for 5 min. (B) BMMCs were preincubated with a vehicle, the P2Y₁ antagonist MRS2179 (10 μM), or the P2Y₂ antagonist AR-C118925 (10 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE₂ (1 μM) for 5 min. (C) BMMCs were preincubated with a vehicle, the P2X1 antagonist NF449 (10 μM), or the P2X7 antagonist AZ10606120 (1 μM) for 3 min and then stimulated with ATP (100 μM) with or without PGE₂ (1 μM) for 5 min. (D) BMMCs were preincubated with a vehicle, the P2X4 antagonist 5-BDBD (10 μM), or the P2X4R positive allosteric modulator ivermectin (10 μg/mL) for 5 min and then stimulated with ATP (100 μM) with or without PGE₂ (1 μM) for 5 min. (E) BMMCs prepared from wild type and P2X4R-deficient mice (P2X4R KO) were stimulated with ATP (100 μM) with or without PGE₂ (1 μM) for 5 min. Data are shown as the mean ± SEM (n = 3). N.S. no significant difference, * p < 0.05 and ** p < 0.01 indicate significant differences.

Figure 4

Effect of tyrosine kinase and phosphoinositide 3-kinase (PI3K)/Akt signaling pathway inhibitors on mast cell (MC) degranulation induced by co-stimulation with ATP and prostaglandin (PG)E₂. (A) Bone marrow-derived MCs (BMMCs) were preincubated with a vehicle or the Src tyrosine kinase inhibitor PP2 (1 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE₂ (1 μM) or 2,4-dinitrophenyl human serum albumin (DNP-HSA, 10 ng/mL). (B) BMMCs were preincubated with a vehicle or the Syk inhibitor R406 (2 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE₂ (1 μM) (n = 3). (C) BMMCs were preincubated with a vehicle, the PI3K inhibitor LY294002 (10 μM), or the control compound LY303511 (10 μM) for 5 min and then stimulated with ATP (0.1 mM) with or without PGE₂ (1 μM) (n = 3). (D) BMMCs were preincubated with a vehicle or the PI3Kγ inhibitor AS605240 (1 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE₂ (1 μM) (n = 3). (E) BMMCs were preincubated with a vehicle or the Akt inhibitor triciribin (10 μM) for 5 min, and then stimulated with ATP (100 μM) with or without PGE₂ (1 μM) (n = 3). Data are shown as the mean ± SEM. N.S. no significant difference, ** p < 0.01 indicates a significant difference.

Figure 5

Effect of co-stimulation with ATP and prostaglandin (PG)E₂ on Syk, extracellular signal-regulated kinase (ERK)1/2, and Akt phosphorylation in bone marrow-derived mast cells (BMMCs). (A) BMMCs were stimulated with ATP (100 μM) with or without PGE₂ (1 μM, upper) or 2,4-dinitrophenyl human serum albumin (DNP-HAS,10 ng/mL, lower) for 1 min. Cell lysates were subjected to western blot analysis for phospho-Syk and total-Syk. (B) BMMCs were stimulated with ATP (100 μM) with or without PGE₂ (1 μM) for 1 (left) or 3 (right) min. Cell lysates were subjected to western blot analysis for phospho-Akt and total Akt (upper) or phospho-ERK 1/2 and total-ERK 1/2 (lower). The numbers below each image indicate normalized relative phosphorylated protein intensity; the results for no stimulation are set to one. Blots are representative of three independent experiments.

Figure 6

Effects of co-stimulating bone marrow-derived mast cells (BMMCs) with ATP and prostaglandin (PG)E₂ on intracellular Ca²⁺ concentration ([Ca²⁺]i) levels. (A) BMMCs prepared from widg type (WT) or P2X4 receptor deficient (P2X4RKO) mice were loaded with Fura-2 acetoxymethyl ester and changes in [Ca²⁺]i were monitored after stimulating with ATP (black line), PGE₂ (gray line), or ATP plus PGE₂ (dotted line) at the time indicated by the arrow. The Ca²⁺ data are representative of four independent experiments. (B)Summary of the data obtained in A. Results are indicated as fold of ATP-induced response (WT; 205 ± 38 nM, n = 4, P2X4RKO; 115 ± 16 nM, n = 4). Data are shown as the mean ± SEM (n = 4). N.S.; no significant difference, * p < 0.05 indicates a significant difference. (C) BMMCs prepared from WT or P2X4RKO were preincubated with or without the PI3Kγ inhibitor AS605240 (1 μM) for 5 min and then stimulated with ATP (100 μM) plus PGE₂ (1 μM). The superimposed [Ca²⁺]i changes are representative of at least four different BMMC preparations obtained from different animals. (D) Summary of the data obtained in C. Results are indicated as fold of control response (WT; 342 ± 45 nM, n = 3, P2X4RKO; 158 ± 29 nM, n = 4). Data are shown as the mean ± SEM (n = 3–4). N.S.; no significant difference, * p < 0.05 indicates a significant difference.

Figure 7

Comparison of prostaglandin (PG) E₂-induced hyperpermeability in wild type, P2X4 receptor-deficient (P2X4RKO), and mast cell-deficient Kit ^Wsh/Wsh mice. PGE₂ (1.5 nmol) was intradermally injected into the ear and vascular permeability measured 30 min later. Data are shown as the mean ± SEM (n = 5–6). N.S. no significant difference, * p < 0.05 indicates a significant difference.

Figure 8

Proposed mechanism of interaction between P2X4 receptor (P2X4R) and EP3 receptor (EP3R) signals for the synergistic degranulation in mast cells(MCs). Extracellular ATP released from damaged cells stimulates MC P2X4R ①, leading to Ca²⁺ influx. Such conditions are accompanied by an inflammation with increased production of prostaglandin (PG)E₂. In MCs, PGE₂ stimulates Gi-coupled EP3R ②, leading to activation of phosphoinositide 3-kinase (PI3K)γ and G protein-coupled inwardly-rectifying K⁺ channel (GIRK) via βγ-complex of the G protein ③. Activation of PI3Kγincreases phosphoinositide-3,4,5-trisphosphate (PIP3) levels in plasma membrane ④, thereby promoting P2X4R channel activity ⑤. GIRK activation may cause hyperpolarization of the membrane potential ⑥, which would increase the driving force of Ca²⁺ inflow through P2X4R ⑦. These interactions between P2X4R and EP3R signals lead to the observed synergy in MC degranulation ⑧. P2X4R signal also promotes IgE-dependent tyrosine kinase-mediated signals to induce facilitated degranulation, as described previously ⑨ [12].