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, 162 (8), 1674-85

Co-operative Signalling Through DP(1) and DP(2) Prostanoid Receptors Is Required to Enhance Leukotriene C(4) Synthesis Induced by Prostaglandin D(2) in Eosinophils

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Co-operative Signalling Through DP(1) and DP(2) Prostanoid Receptors Is Required to Enhance Leukotriene C(4) Synthesis Induced by Prostaglandin D(2) in Eosinophils

F P Mesquita-Santos et al. Br J Pharmacol.

Abstract

Background and purpose: Prostaglandin (PG) D(2) has emerged as a key mediator of allergic inflammatory pathologies and, particularly, PGD(2) induces leukotriene (LT) C(4) secretion from eosinophils. Here, we have characterized how PGD(2) signals to induce LTC(4) synthesis in eosinophils.

Experimental approach: Antagonists and agonists of DP(1) and DP(2) prostanoid receptors were used in a model of PGD(2) -induced eosinophilic inflammation in vivo and with PGD(2) -stimulated human eosinophils in vitro, to identify PGD(2) receptor(s) mediating LTC(4) secretion. The signalling pathways involved were also investigated.

Key results: In vivo and in vitro assays with receptor antagonists showed that PGD(2) -triggered cysteinyl-LT (cysLT) secretion depends on the activation of both DP(1) and DP(2) receptors. DP(1) and DP(2) receptor agonists elicited cysLTs production only after simultaneous activation of both receptors. In eosinophils, LTC(4) synthesis, but not LTC(4) transport/export, was activated by PGD(2) receptor stimulation, and lipid bodies (lipid droplets) were the intracellular compartments of DP(1) /DP(2) receptor-driven LTC(4) synthesis. Although not sufficient to trigger LTC(4) synthesis by itself, DP(1) receptor activation, signalling through protein kinase A, did activate the biogenesis of eosinophil lipid bodies, a process crucial for PGD(2) -induced LTC(4) synthesis. Similarly, concurrent DP(2) receptor activation used Pertussis toxin-sensitive and calcium-dependent signalling pathways to achieve effective PGD(2) -induced LTC(4) synthesis.

Conclusions and implications: Based on pivotal roles of cysLTs in allergic inflammatory pathogenesis and the collaborative interaction between PGD(2) receptors described here, our data suggest that both DP(1) and DP(2) receptor antagonists might be attractive candidates for anti-allergic therapies.

Figures

Figure 1
Figure 1
Both DP1 and DP2 receptors control cysLTs production triggered by PGD2. In A, sensitized mice were pretreated with BWA868C (1 mg·kg−1) or ramatroban (1 mg·kg−1) and then stimulated with an i.pl. injection of PGD2 (35 pmol per cavity). Analysis of cysLTs synthesis was performed 24 h after PGD2 administration. Results are expressed as the means ± SEM from at least six animals. P≤ 0.05 compared with control animals and *P≤ 0.05 compared with PGD2-injected mice. In B, for in vitro analysis of LTC4 synthesis, human eosinophils were pretreated for 30 min with BWA868C (200 nM), ramatroban (200 nM), Cay10471 (200 nM) or SQ29548 (200 nM), and then stimulated for 1 h with PGD2 (25 nM). In vitro results are expressed as the means ± SEM from at least three independent experiments with eosinophils purified from different donors. P≤ 0.05 compared with control. *P≤ 0.05 compared with PGD2-stimulated eosinophils. cysLT, cysteinyl leukotriene; DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; i.pl., intrapleural; PGD2, prostaglandin D2.
Figure 2
Figure 2
DP1 and DP2 receptors cooperate to trigger cysLTs production. In A, sensitized mice received an i.pl. injection of PGD2 (35 pmol per cavity), BW245C (35 pmol per cavity), DK-PGD2 (35 pmol per cavity) or BW245C plus DK-PGD2 (both at 35 pmol per cavity). Analysis of cysLTs production within pleural fluids was performed 24 h after i.pl. administration. Results are expressed as the means ± SEM from at least six animals. P≤ 0.05 compared with control animals. In B, for in vitro analyses of LTC4 production in cell-free supernatants, human eosinophils were stimulated for 1 h with PGD2 (25 nM), BW245C (25 nM), DK-PGD2 (25 nM) or with a combination of BW245C plus DK-PGD2 (both at 25 nM). In vitro results are expressed as the means ± SEM from at least three independent experiments with eosinophils purified from different donors. P≤ 0.05 compared with control. P≤ 0.05 compared with PGD2-stimulated eosinophils. cysLT, cysteinyl leukotriene; DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; i.pl., intrapleural; PGD2, prostaglandin D2.
Figure 3
Figure 3
LTC4 synthesis is triggered within eosinophil cytoplasmic lipid bodies by simultaneous activation of DP1 and DP2 receptors by either PGD2 or the combination of BW245C and DK-PGD2 stimulation of human eosinophils in vitro. EicosaCell images illustrate intracellular immuno-detection of newly formed LTC4 (green) and of ADRP (red) in PGD2-stimulated, BW245-stimulated, DK-PGD2-stimulated or BW245C/DK-PGD2 co-stimulated human eosinophils (as indicated). Overlay images of identical fields are shown in the right column. Arrows indicate co-localization of immunolabelled synthesized LTC4 with ADRP-bearing lipid bodies. For EicosaCell analyses, cells were fixed and permeabilized with EDAC and sequentially incubated with anti-LTC4 and anti-ADRP antibodies and Alexa488-labelled anti-rabbit IgG plus Alexa546-labelled anti-guinea pig secondary antibodies. Images are representative of three independent experiments. ADRP, adipose-differentiation-related protein; DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; PGD2, prostaglandin D2.
Figure 4
Figure 4
DP1, but not DP2, activation triggers lipid body biogenesis within human eosinophils in vitro. In A, human eosinophils were stimulated with PGD2 (25 nM), BW245C (25 nM), DK-PGD2 (25 nM) or with a combination of BW245C plus DK-PGD2 (both at 25 nM). B shows a dose-response effect of PGD2 (25 nM), BW245C (25 nM) or DK-PGD2 (25 nM) on lipid body biogenesis after stimulation of human eosinophils. Analysis of lipid body biogenesis was performed 1 h after stimulation in osmium-stained cells. Results are expressed as means ± SEM from at least three different experiments with eosinophils purified from distinct donors. P≤ 0.05 compared with control. DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; PGD2, prostaglandin D2.
Figure 5
Figure 5
DP1, but not DP2 receptors, control eosinophil lipid body biogenesis triggered by PGD2 either in vivo or in vitro. In A, sensitized mice were pretreated with BW868c (1 mg·kg−1) or ramatroban (1 mg·kg−1), and then stimulated with an i.pl. injection of PGD2 (35 pmol/cavity). Analysis of lipid body biogenesis was performed 24 h after PGD2 administration in osmium-stained cells. Results are expressed as means ± SEM from at least six animals. P≤ 0.05 compared with control animals and *P≤ 0.05 compared with PGD2-injected mice. In B, for in vitro analysis of lipid body biogenesis, human eosinophils were pretreated for 30 min with BW868c (200 nM), ramatroban (200 nM), Cay10471 (200 nM) or SQ29548 (200 nM), stimulated for 1 h with PGD2 (25 nM) and subsequently stained with osmium. In vitro results are expressed as the means ± SEM from at least three different experiments with eosinophils purified from distinct donors. P≤ 0.05 compared with control. *P≤ 0.05 compared with PGD2-stimulated eosinophils. DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; i.pl., intrapleural; PGD2, prostaglandin D2.
Figure 6
Figure 6
DP1 receptor-driven PKA activation cooperates with DP2-driven Gαi protein activation and calcium influx to mediate lipid body-driven LTC4 sytnhesis within human eosinophils triggered by in vitro PGD2. Human eosinophils were pretreated for 30 min with H-89 (10 µM) and PKI (10 µM) in A, or with PTX (1 µg·mL−1) or BAPTA-AM (25 µg·mL−1) in B and then stimulated with PGD2 (25 nM). In vitro analysis of LTC4 production in cell-free supernatants and lipid body biogenesis were analysed 1 h after PGD2. Results are expressed as the means ± SEM from at least three different experiments with eosinophils purified from different donors. P≤ 0.05 compared with control group. *P≤ 0.05 compared with PGD2-stimulated eosinophils. DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; PGD2, prostaglandin D2; PTX, Pertussis toxin.
Figure 7
Figure 7
PKA activation, but not Gαi protein and calcium influx, mediates lipid body biogenesis within human eosinophils triggered by BW245C in vitro. Human eosinophils were pretreated for 30 min with PTX (1 µg·mL−1), BAPTA-AM (25 µg·mL−1), H-89 (10 µM) or PKI (10 µM), and then stimulated with BW245C (25 nM). Lipid body biogenesis was analysed 1 h after BW245C stimulation. Results are expressed as the means ± SEM from at least three different experiments with eosinophils purified from different donors. P≤ 0.05 compared with control group. *P≤ 0.05 compared with PGD2-stimulated eosinophils. PTX, Pertussis toxin.
Figure 8
Figure 8
Cooperation between DP1 and DP2 receptors to trigger lipid body-driven LTC4 synthesis within human eosinophils also takes place in allergic inflammatory response in vivo. Sensitized mice were pretreated with BWA868C (1 mg·kg−1), ramatroban (1 mg·kg−1) or Cay10471 (1 mg·kg−1), and then challenged with an i.pl. injection of ovalbumin (12 µg per cavity). Analyses of lipid body biogenesis and cysLTs production were performed 24 h after allergic challenge. Results are expressed as means ± SEM from at least six animals. P≤ 0.05 compared with saline-challenged mice and *P≤ 0.05 compared with ovalbumin-challenged mice. cysLT, cysteinyl leukotriene; DP1, D prostanoid receptor 1; DP2, D prostanoid receptor 2; i.pl., intrapleural.

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