Pivotal role of the 5-lipoxygenase pathway in lung injury after experimental sepsis

Abstract

Postsepsis lung injury is a common clinical problem associated with significant morbidity and mortality. Leukotrienes (LTs) are important lipid mediators of infection and inflammation derived from the 5-lipoxygenase (5-LO) metabolism of arachidonate with the potential to contribute to lung damage after sepsis. To test the hypothesis that LTs are mediators of lung injury after sepsis, we assessed lung structure, inflammatory mediators, and mechanical changes after cecal ligation and puncture surgery in wild-type (WT) and 5-LO knockout (5-LO(-/-)) mice and in WT mice treated with a pharmacologic LT synthesis inhibitor (MK886) and LT receptor antagonists (CP105,696 and montelukast). Sixteen hours after surgery, WT animals exhibited severe lung injury (by histological analysis), substantial mechanical impairment (i.e., an increase in static lung elastance), an increase in neutrophil infiltration, and high levels of LTB4, cysteinyl-LTs (cys-LTs), prostaglandin E2, IL-1β, IL-6, IL-10, IL-17, KC (CXCL1), and monocyte chemotactic protein-1 (CCL2) in lung tissue and plasma. 5-LO(-/-) mice and WT mice treated with a pharmacologic 5-LO inhibitor were significantly protected from lung inflammation and injury. Selective antagonists for BLT1 or cys-LT1, the high-affinity receptors for LTB4 and cys-LTs, respectively, were insufficient to provide protection when used alone. These results point to an important role for 5-LO products in sepsis-induced lung injury and suggest that the use of 5-LO inhibitors may be of therapeutic benefit clinically.

Figure 1.

Morphologic changes in sham and cecal ligation and puncture (CLP) wild-type (WT) (sv129 strain) and 5-lipoxygenase knockout (5-LO^−/−) mice. (A) Representative lung sections from sham/WT (upper left), CLP/WT (lower left), sham/5-LO^−/− (upper right), and CLP/5-LO^−/− mice (lower right) obtained 16 hours after surgery and stained with hematoxylin and eosin. (B) Morphometry of sham and CLP WT and 5-LO^−/− mice. Lungs were collected 16 hours after surgery and prepared for histology. Data are presented as the mean ± SEM. *P < 0.05 compared with sham group. ^#P < 0.05 compared with CLP WT mice.

Figure 2.

Leukotriene (LT) production and neutrophil influx into the lung after CLP. Concentrations of LTB₄ (A) and cys-LTs (B) in lung homogenates of C57Bl/6 mice were obtained 16 hours after sham, and CLP was determined by enzyme immunoassay. Neutrophil infiltration in WT and 5-LO^−/− mice (C) and MK886-, CP 105,696–, and montelukast-treated mice lungs (D) was evaluated 16 hours post-sham or CLP by MPO activity. Data are presented as the mean ± SEM. *P < 0.05 compared with sham group. ^#P < 0.05 compared with CLP/WT mice.

Figure 3.

Lung cytokine levels in WT (sv129 strain) and 5-LO^−/− mice subjected to sham or CLP. IL-1β, IL-6, IL-10, IL-17, KC, and MCP-1 levels were quantified in lungs 16 hours after sham or CLP of WT and 5-LO^−/− mice. Cytokines were measured by ELISA. Data are presented as the mean ± SEM. *P < 0.05 compared with sham group. ^#P < 0.05 compared with CLP/WT group.

Figure 4.

Effect of MK886, BLT1, and cys-LT1 receptor antagonists on lung cytokine levels after CLP. IL-1β, IL-6, IL-10, KC, and MCP-1 levels were quantified in lungs of C57Bl/6 mice 16 hours after sham or CLP in MK886-, CP105,696 (CP)-, or montelukast (Monte)-treated or nontreated mice. Cytokines were measured by ELISA. Data are presented as the mean ± SEM. *P < 0.05 compared with sham group. ^#P < 0.05 compared with CLP untreated mice.

Figure 5.

Plasma cytokine levels after sepsis in WT (sv129 strain) and 5-LO^−/− mice. IL-1β, IL-6, IL-10, IL-17, KC, and MCP-1 levels were quantified in plasma 16 hours after sham or CLP in WT and 5-LO^−/− mice by ELISA. Data are presented as the mean ± SEM. *P < 0.05 compared with sham group. ^#P < 0.05 compared with CLP/WT mice.

Figure 6.

Effects of MK886, BLT1, and cys-LT1 receptor antagonists on plasma cytokine levels after sepsis. IL-1β, IL-6, IL-10, KC, and MCP-1 levels were quantified in lungs 16 hours after sham or CLP in untreated or treated C57Bl/6 mice with MK886, CP105,696 (CP), or montelukast (Monte) by ELISA. Data are presented as the mean ± SEM. *P < 0.05 compared with sham group. ^#P < 0.05 compared with CLP untreated mice.

Figure 7.

Lung function in 5-LO^−/− mice and MK886-treated animals after CLP. Static elastance (Est) was evaluated 16 hours after sham or CLP in WT (sv129 strain) and 5-LO^−/− mice (A) and in untreated or treated C57Bl/6 mice with MK886, CP105,696 (CP), or montelukast (Monte) (B). Data are presented as the mean ± SEM of six animals per group. *P < 0.05 compared with sham group. ns = nonsignificant.