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

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.

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

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

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

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

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

DP₁ receptor-driven PKA activation cooperates with DP₂-driven Gα_i 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

PKA activation, but not Gα_i 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

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.