Endothelial microparticles induce inflammation in acute lung injury

Abstract

Background: Previously, we have shown that endothelial microparticles (EMPs) injected into mice induce acute lung injury (ALI) [1]. In this study, we hypothesize that EMPs induce ALI by initiating cytokine release in the lung, leading to recruitment and activation of neutrophils.

Materials and methods: C57BL/6J male mice (8-10 wk old) were intravenously injected with EMPs (200,000/mL), LPS (2 mg/kg), or both. Bronchoalveolar lavage (BAL) and serum levels of IL-1β and TNF-α were analyzed by enzyme-linked immunoassay (ELISA). Morphometric analysis was performed on H and E stained lung sections. Myeloperoxidase (MPO) levels were determined via an enzymatic assay and immunofluorescence of stained sections.

Results: EMPs led to significantly increased pulmonary and systemic IL-1β and TNF-α levels, which correlated with increased neutrophil recruitment to the lung. MPO levels in the lungs were increased significantly following injection of EMPs or LPS, compared to PBS. In mice treated with EMPs and LPS either simultaneously or successively, the cytokine and MPO levels were significantly increased over that of either treatment alone.

Conclusion: EMPs contribute to lung injury through the initiation of a cytokine cascade that increases recruitment of neutrophils and subsequent release of MPO. Furthermore, treatment of mice with both EMPs and LPS induced greater lung injury than either treatment alone, suggesting that EMPs prime the lung for increased injury by other pathogens. Therapies aimed at reducing or blocking EMPs may be a useful strategy for attenuating lung injury.

FIG. 1

Effects of EMPs on BAL (A) and serum (B) IL-1β after treatment with phosphate-buffered saline (PBS), endothelial microparticles (EMP), lipopolysaccharide (LPS), and simultaneous (EL), or sequential (E/L) treatment with EMPs and LPS. Error bars represent standard deviation (SD); *P ≤ 0.05; n = 3 per group.

FIG. 2

Effects of EMPs on BAL (A) and serum (B) TNF-α after treatment with phosphate-buffered saline (PBS), endothelial microparticles (EMP), lipopolysaccharide (LPS), and simultaneous (EL), or successive (E/L) treatment with EMPs and LPS (EMP/LPS). Error bars represent standard deviation (SD); *P ≤ 0.05; n = 3 per group.

FIG. 3

H and E stained lung sections (20× magnification) from mice treated with PBS (A), EMPs (B), LPS (C), and simultaneous (EL), or sequential (E/L) treatment with EMPs plus LPS (D) and (E), respectively. Perivascular edema is notable in the four experimental treatment groups compared with PBS control (black arrow heads). (Color version of figure is available online.)

FIG. 4

Representative H and E stained sections of lung tissue (20× magnification) from mice treated with PBS (A), EMPs (B), LPS (C), and simultaneous (EL), or successive (E/L) injection of EMPs plus LPS (D) and (E), respectively, demonstrating increasing degrees of pleuritis (black arrow heads), edema, and alveolar hemorrhage. (Color version of figure is available online.)

FIG. 5

Myeloperoxidase activity (A) and immunofluorescent staining (B) in lung tissue. Increasing amounts of MPO staining (B; white arrows) can be seen in the vascular wall and parenchyma after injection with EMPs, LPS, co-administration of EMPs and LPS (EL), and EMPs followed in 6 h by LPS (E/L) compared with PBS. (Color version of figure is available online.)