Activated Platelets Induce an Anti-Inflammatory Response of Monocytes/Macrophages through Cross-Regulation of PGE₂and Cytokines

Figures

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  Figure 1. Collagen-activated platelets selectively regulate the synthesis of IL-10 and TNFα in cocultures with murine macrophages. (a), (b) Murine bone marrow-derived macrophages (BMDM) were incubated alone, with untreated platelets (untreated PLTs) or with platelets prestimulated with collagen (1 μg/ml; act. PLTs) for 3 hours and the concentrations of IL-10 and TNFα were determined by ELISA. (c) Murine BMDM were incubated with untreated platelets with or without murine IL-10 (125 pg/ml). TNFα levels were determined in the medium. Data are presented as mean ± SEM from 4 experiments. One-way ANOVA/Bonferroni ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.002, ∗∗∗∗P < 0.0004.

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  Figure 2. Cross-regulation of PGE2 and TNFα through an induction of IL-10 in murine macrophages by platelet derived PGE2. (a) Murine BMDM were incubated alone, with untreated platelets, or with collagen-activated platelets for 3 hours. (b), (c) Murine BMDM were incubated with untreated platelets with or without PGE2 (1 μM) for 3 hours. (d) Murine platelets were incubated with or without collagen (1 μg/ml) for 30 minutes. (e), (f) BMDM from wild-type mice (b) or COX-1-deficient mice (c) were coincubated with BAPTA-AM-inactivated platelets (30 μM) or with collagen-activated platelets isolated from wild-type and COX-1-deficient mice for 3 hours. The concentrations of PGE2 in the medium were determined by LC-MS/MS. IL-10 levels and TNFα levels in the medium were determined by ELISA. Data are presented as mean ± SEM from 4 experiments. One-way ANOVA/Bonferroni ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.002, ∗∗∗∗P < 0.0004.

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  Figure 3. Influence of EP receptor agonists on the release of TNFα and IL-10 from platelet-stimulated murine macrophages. Murine BMDM were incubated alone or with untreated platelets and the respective EP agonist concentrations for 3 hours (400 nM ONO-Di-004, 170 nM butaprost, 10 nM sulprostone, 5 nM L-902,688, 1 μM PGE2). TNFα (a) or IL-10 (b) were determined by ELISA. Data are presented as mean ± SEM from 4 experiments. One-way ANOVA/Bonferroni ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.002, ∗∗∗∗P < 0.0004.

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  Figure 4. EP2 and EP4 decrease TNFα through IL-10 and its receptor IL-10 R. Murine BMDM were incubated alone or with untreated platelets ± IL-10 Rα antibody (9 μg/ml) 30 minutes before the application of the respective EP agonist concentrations for 3 hours (40 nM ONO-Di-004, 100 nM butaprost, 5 nM L-902,688, 30 nM PGE2). TNFα was determined by ELISA. Data are presented as mean ± SEM from 4 preparations. One-way ANOVA/Bonferroni ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.002.

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  Figure 5. Ex vivo validation of the cross-regulation between platelet PGE2 and IL-10 and TNFα released from EP2- and EP4-deficient macrophages. Murine BMDM from wild-type, EP2-deficient, and EP4-deficient mice were incubated alone, with untreated platelets (untreated PLTs) or with platelets prestimulated with collagen (1 μg/ml; act. PLTs) for 3 hours. The concentrations of PGE2 (a) were determined by LC-MS/MS. IL-10 levels (b) and TNFα levels (c) were determined by ELISA. One-way ANOVA/Bonferroni ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.002, ∗∗∗∗P < 0.0004, #< 0.00008.

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  Figure 6. PGE2 mediates inhibition of TNFα release from platelet-stimulated human monocytes. (a), (b), (c) Human monocytes were incubated alone, with untreated human platelets or with collagen-activated human platelets for 3 hours and PGE2 (a), TNFα (b), or IL-10 (c) were determined. (d), (e) Human monocytes were incubated alone or with untreated platelets with or without PGE2 (1 μM). (f) Human monocytes were incubated with untreated platelets with or without human IL-10 (125 pg/ml). The concentrations of PGE2, TNFα, and IL-10 within the cell-free supernatants were determined by LC-MS/MS or rather ELISA within cell-free supernatants. Data are presented as mean ± SEM from 4 experiments. One-way ANOVA/Bonferroni (a)–(e) or two-tailed t-test (f) ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.002.

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  Figure 7. Influence of EP receptor agonists on the release of TNFα and IL-10 from platelet-stimulated human monocytes. Human monocytes were incubated alone or with untreated platelets and the respective EP agonist concentrations for 3 hours (400 nM ONO-Di-004, 170 nM butaprost, 10 nM sulprostone, 5 nM L-902,688, 1 μM PGE2). TNFα (a) or IL-10 (b) were determined by ELISA. Data are presented as mean ± SEM from 4 experiments. One-way ANOVA/Bonferroni ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.002.

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  Figure 8. EP2 decreases TNFα through IL-10 and its receptor IL-10 R. Human monocytes were incubated with untreated platelets ± IL-10 Rα antibody (22.5 μg/ml) 30 minutes before the application of the respective EP agonist concentrations for 3 hours (40 nM ONO-Di-004, 75 nM butaprost). TNFα was determined by ELISA. Data are presented as mean ± SEM from 4 preparations. One-way ANOVA/Bonferroni ∗P < 0.05.

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  Figure 9. Summary of the platelet-induced crosstalk between PGE2, IL-10, and TNFα in murine macrophages and human monocytes. Activated platelets produce and release PGE2 in cocultures with macrophages. PGE2 binds to EP1, EP2 (orange), and in case of murine macrophages additionally to EP4 (green). The binding of PGE2 to EP1 decreased synthesis and secretion of TNFα independently of IL-10. The activation of EP2 and EP4 in murine macrophages and EP2 in human monocytes increased the secretion of IL-10 which binds to its receptor IL-10 R, leading to an inhibition of the production and release of TNFα.