Activation of EP4 receptors prevents endotoxin-induced neutrophil infiltration into the airways and enhances microvascular barrier function

Abstract

Background and purpose: Pulmonary vascular dysfunction is a key event in acute lung injury. We recently demonstrated that PGE₂ , via activation of E-prostanoid (EP)₄ receptors, strongly enhances microvascular barrier function in vitro. The aim of this study was to investigate the beneficial effects of concomitant EP₄ receptor activation in murine models of acute pulmonary inflammation.

Experimental approach: Pulmonary inflammation in male BALB/c mice was induced by LPS (20 μg per mouse intranasally) or oleic acid (0.15 μL·g^-1 , i.v^. ). In-vitro, endothelial barrier function was determined by measuring electrical impedance.

Key results: PGE₂ activation of EP₄ receptors reduced neutrophil infiltration, pulmonary vascular leakage and TNF-α concentration in bronchoalveolar lavage fluid from LPS-induced pulmonary inflammation. Similarly, pulmonary vascular hyperpermeability induced by oleic acid was counteracted by EP₄ receptor activation. In lung function assays, the EP₄ agonist ONO AE1-329 restored the increased resistance and reduced compliance upon methacholine challenge in mice treated with LPS or oleic acid. In agreement with these findings, EP₄ receptor activation increased the in vitro vascular barrier function of human and mouse pulmonary microvascular endothelial cells and diminished the barrier disruption induced by LPS. The EP₂ agonist ONO AE1-259 likewise reversed LPS-induced lung dysfunction without enhancing vascular barrier function.

Conclusion and implications: Our results show that activation of the EP₄ receptor strengthens the microvascular barrier function and thereby ameliorates the pathology of acute lung inflammation, including neutrophil infiltration, vascular oedema formation and airway dysfunction. This suggests a potential benefit for EP₄ agonists in acute pulmonary inflammation.

Figure 1

Neutrophil counts in BAL fluid of (A) mice treated intranasally with vehicle (veh; n = 18), LPS (20 μg per mouse; n = 17), PGE₂ (20 μg per mouse) concomitantly with LPS (n = 22) or pretreated with the EP₄ receptor antagonists GW627368X (GW; 10 mg·kg⁻¹ s.c.; n = 8) or ONO AE3-208 (10 mg·kg⁻¹ s.c.; n = 5) 30 min before intranasal LPS or LPS concomitantly applied with PGE₂ (n = 6 or 10 respectively). (B) Intranasal application of vehicle (veh, n = 8) or LPS (20 μg per mouse, n = 11) alone or in combination with the EP₄ receptor agonist ONO AE1-329 (n = 3–10). Each point shows the neutrophil count in BAL fluid from one animal. Data were analysed using one-way anova and multiple comparisons were calculated with Tukey’s post test. *P < 0.05, **P < 0.01, ***P < 0.001. (C–F) Haematoxylin/eosin staining of lungs with intranasal application of (C) vehicle, (D) LPS (20 μg per mouse), (E) PGE₂ (20 μg per mouse) given concomitantly with LPS and (F) ONO AE1-329 (20 μg per mouse) concomitantly applied with LPS. Lung histology micrographs are representative of three independent experiments; scale bar = 50 μm.

Figure 2

Evans blue extravasation in the lung of mice intranasally treated (A) with vehicle (veh, n = 7), LPS (20 μg per mouse, n = 22) or LPS in combination with PGE₂ (20 μg per mouse, n = 20). (B) Intranasal treatment with vehicle (veh, n = 5), LPS (20 μg per mouse, n = 7) or in combination with the EP₄ receptor agonist ONO AE1-329 (20 μg per mouse, n = 6). Each point shows μg Evans blue (EB) per lung from one animal. Data were analysed using one-way anova and multiple comparisons were calculated with Tukey’s post test. **P < 0.01, ***P < 0.001.

Figure 3

TNF-α (A,C) and IL-6 (B,D) levels in the BAL fluid of (A,B) mice treated intranasally with vehicle (veh; n = 18), LPS (20 μg per mouse; n = 17), PGE₂ (20 μg per mouse) concomitantly with LPS (n = 22) or pretreated with the EP₄ receptor antagonists GW627368X (GW; 10 mg·kg⁻¹ s.c.; n = 8) or ONO AE3-208 (10 mg·kg⁻¹ s.c.; n = 5) 30 min before intranasal LPS or LPS concomitantly applied with PGE₂ (n = 6 or 10 respectively). (C,D) Intranasal application of vehicle (veh, n = 8) or LPS (20 μg per mouse, n = 11) alone or in combination with the EP₄ receptor agonist ONO AE1-329 (n = 3–10). Each point shows the cytokine concentration (ng mL⁻¹) from one animal. Data were analysed using one-way anova and multiple comparisons were calculated with Tukey’s post test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

TNF-α (A–C) and IL-6 (D) concentrations in the supernatants of isolated murine alveolar macrophages. Cells were treated with LPS (10 ng·mL⁻¹) for 4 h in the presence of (A) vehicle (veh) or PGE₂ (concentrations as indicated), n = 5, (B) vehicle (veh) or PGE₂ (30 nmol·L⁻¹) in the absence or presence of the EP₄ receptor antagonists ONO AE3-208 (300 nmol·L⁻¹) or GW627368X (GW, 1 μmol·L⁻¹) for 30 min, n = 6, (C) vehicle (veh) or the EP₄ receptor agonist ONO AE1-329 (concentrations as indicated), n = 8, (D) vehicle (veh) or PGE₂ (30 nmol·L⁻¹) or ONO AE1-329 (100 nmol·L⁻¹) in the absence or presence of the EP₄ receptor antagonist ONO AE3-208 (300 nmol·L⁻¹), n = 7. Data are shown as mean + SEM of n independent experiments performed in triplicate. Data were analysed using one-way anova for repeated measurements and multiple comparisons were calculated with Tukey’s post test. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with LPS treatment, #P < 0.05 as compared with PGE₂ or EP₄ agonist treatment.

Figure 5

Electrical resistance of murine endothelial cell monolayers treated with either vehicle or (A) PGE₂ or the (B) EP₄ agonist ONO AE1-329 at the indicated concentrations. (C). Cells were treated with PGE₂ (30 nmol·L⁻¹) or (D) the EP₄ agonist ONO AE1-329 (30 nmol·L⁻¹) alone or in the presence of the EP₄ antagonist ONO AE3-208 (1 μmol·L⁻¹, 15 min pretreatment). Data are shown as mean normalized resistance + SEM of four independent experiments performed in duplicate. Data were analysed by two-way anova for repeated measurements and multiple comparisons were calculated with Tukey’s post test. ***P < 0.001 as compared with vehicle; ###P < 0.001 as compared with PGE₂ or the EP₄ agonist. (E) EP₄ receptor expression on murine pulmonary microvascular cells using flow cytometry (typical example of n = 3). AB, antibody.

Figure 6

Electrical resistance of human endothelial cell monolayers treated with vehicle or LPS (1 μg·mL⁻¹) in the absence or presence of (A,C) PGE₂ (30 nmol·L⁻¹), (B,D) the EP₄ receptor agonist ONO AE1-329 (100 nmol·L⁻¹). (C,D) Cells were pretreated with the EP₄ receptor antagonist ONO AE3-208 (300 nmol·L⁻¹) for 15 min. Data are shown as mean normalized resistance + SEM of 4–7 independent experiments performed in duplicate. Data were analysed by two-way anova for repeated measurements and multiple comparisons were calculated with Tukey’s post test. ***P < 0.001 as compared with vehicle; ###P < 0.001 as compared with LPS.

Figure 7

Immunohistochemical staining of EP₄ receptors in murine lung tissue. (A) The endothelial layer of a lung vessel as indicated by the arrow is stained brown with the EP₄ receptor antibody. (B) Haematoxylin–eosin staining of a consecutive section which shows the same vessel. Scale bar = 50 μm. Micrographs are representative for five lungs.

Figure 8

Evans blue (EB) extravasation in the lung of mice that received vehicle (veh, n = 5) or oleic acid (0.15 μL·g⁻¹, n = 17) injected into the tail vein. The EP₄ receptor agonist ONO AE1-329 (20 μg per mouse, n = 15) was given intranasally. Each point shows microgram EB per lung of one animal 90 min after oleic acid injection. Data were analysed by one-way anova and multiple comparisons were calculated with Tukey’s post test. *P < 0.05.

Figure 9

Lung function measured by the flexiVent system. Resistance (A,C) and compliance (B,D) induced by inhalation of increasing concentrations of methacholine was recorded 4 h after intranasal application of LPS (20 μg per mouse; A,B) and 90 min after oleic acid (0.15 μL·g⁻¹ body weight; C,D) injection into the tail vein. Concomitant intranasal application of (A,B) PGE₂ (20 μg per mouse) or (A–D) the EP₄ receptor agonist ONO AE1-329 (20 μg per mouse). Data are shown as mean + SEM (n = 6 individual experiments per group). Data were analysed by two-way anova for repeated measurements and multiple comparisons were calculated with Tukey’s post test *P < 0.05, **P < 0.01, ***P < 0.001, compared with control; #P < 0.05, ##P < 0.01, compared with LPS or oleic acid.