Activated prostaglandin D2 receptors on macrophages enhance neutrophil recruitment into the lung

Abstract

Background: Prostaglandin (PG) D2 is an early-phase mediator in inflammation, but its action and the roles of the 2 D-type prostanoid receptors (DPs) DP1 and DP2 (also called chemoattractant receptor-homologous molecule expressed on T(H)2 cells) in regulating macrophages have not been elucidated to date.

Objective: We investigated the role of PGD2 receptors on primary human macrophages, as well as primary murine lung macrophages, and their ability to influence neutrophil action in vitro and in vivo.

Methods: In vitro studies, including migration, Ca(2+) flux, and cytokine secretion, were conducted with primary human monocyte-derived macrophages and neutrophils and freshly isolated murine alveolar and pulmonary interstitial macrophages. In vivo pulmonary inflammation was assessed in male BALB/c mice.

Results: Activation of DP1, DP2, or both receptors on human macrophages induced strong intracellular Ca(2+) flux, cytokine release, and migration of macrophages. In a murine model of LPS-induced pulmonary inflammation, activation of each PGD2 receptor resulted in aggravated airway neutrophilia, tissue myeloperoxidase activity, cytokine contents, and decreased lung compliance. Selective depletion of alveolar macrophages abolished the PGD2-enhanced inflammatory response. Activation of PGD2 receptors on human macrophages enhanced the migratory capacity and prolonged the survival of neutrophils in vitro. In human lung tissue specimens both DP1 and DP2 receptors were located on alveolar macrophages along with hematopoietic PGD synthase, the rate-limiting enzyme of PGD2 synthesis.

Conclusion: For the first time, our results show that PGD2 markedly augments disease activity through its ability to enhance the proinflammatory actions of macrophages and subsequent neutrophil activation.

Keywords: D-type prostanoid receptor 1; D-type prostanoid receptor 2/chemoattractant receptor–homologous molecule expressed on T(H)2 cells; hematopoietic prostaglandin D synthase; macrophages; neutrophils; prostaglandin D(2); pulmonary inflammation.

Fig 1

PGD₂ receptors DP₁ and DP₂ are expressed on macrophages and induce Ca²⁺ flux and migration. A, Flow cytometric histograms of DP₂ and DP₁ staining (filled histograms) on MDMs, respectively. B, Immunohistochemistry of healthy human lung tissue showing DP₂- and DP₁-positive alveolar macrophages (arrows). C, Representative Ca²⁺ responses of MDMs over time. D, MDM migration toward PGD₂ is blocked by DP₁- and DP₂-specific antagonists (n = 4-5). *P < .05 and ***P < .001.

Fig 2

PGD₂ acting through DP₁ and DP₂ promotes neutrophil influx into lungs and aggravates airway hyperreactivity. A and B, Neutrophil infiltration is increased in animals pretreated with PGD₂ (Fig 2, A) or DP₁- and DP₂-selective agonists (Fig 2, B). C-F, PGD₂ pretreatment increases Ly6G-positive neutrophil infiltration in the peribronchial and alveolar space (representative pictures, scale bar = 50µm; Fig 2, C), MPO activity (Fig 2, D), Evans blue dye extravasation (Fig 2, E), and airway hyperreactivity (vs vehicle/LPS; Fig 2, F). *P < .05, **P < .01, and ***P < .001.

Fig 3

Blocking of endogenous PGD₂ ameliorates LPS-induced neutrophil influx into the alveolar space and pulmonary tissue. A and B, DP₁ antagonist reduced neutrophil counts in the bronchoalveolar space (Fig 3, A), whereas DP₂ antagonist reduced MPO activity (Fig 3, B; n = 6-9). *P < .05 versus vehicle. C, Lipid mediators in BAL fluid 4 hours after vehicle or LPS treatment were quantified by using liquid chromatography– tandem mass spectrometry (n = 4). HHTrE, 12S-hydroxy-5Z,8E,10E-heptoadectrienoic acid. *P < .05 versus vehicle.

Fig 4

Macrophage depletion prevents the proinflammatory effect of PGD₂ on neutrophil recruitment. A, Neutrophil numbers in BAL fluid. B, MPO activity in lung tissue. *P < .05, **P < .01, and ***P < .001. C, Macrophage depletion prevents reduction in lung function induced by PGD₂ (n = 6-8). *P < .05, **P < .01, and ***P < .001 versus vehicle/LPS.

Fig 5

PGD₂ receptor activation on MDMs increases neutrophil migration toward IL-8 (A and B) and prolongs neutrophil survival (C and D) in vitro. Fig 5, A, C, and D: n = 5-10; Fig 5, B: n = 3-5. *P < .05 and **P < .01 versus vehicle. PI, Propidium iodide.

Fig 6

Increased numbers of HPGDS-expressing cells in lungs of patients with ARDS. Representative immunohistochemical staining of human lung samples showing positive cells for HPGDS (brown) in a control subject (A) and a patient with ARDS (B). Stainings are representative pictures of 5 patients and control subjects. Note the high amount of HPGDS found in alveolar macrophages. Bar = 100 µm.