Eliminating or blocking 12/15-lipoxygenase reduces neutrophil recruitment in mouse models of acute lung injury

Abstract

Introduction: Acute lung injury (ALI) is a common disease in critically ill patients with a high morbidity and mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme generating 12-hydroxy-eicosatetraenoic acid (12-HETE) and 15-HETE from arachidonic acid. It has been shown that 12/15-LO is involved in the regulation of vascular permeability during ALI.

Methods: To test whether 12/15-LO participates in leukocyte recruitment into the lung, we investigated the role of 12/15-LO in mouse models of lipopolysaccharide (LPS)-induced pulmonary inflammation and acid-induced ALI, a clinically relevant model of acute lung injury.

Results: The increase in neutrophil recruitment following LPS inhalation was reduced in 12/15-LO-deficient (Alox15(-/-)) mice and in wild-type (WT) mice after the blocking of 12/15-LO with a pharmacological inhibitor. Bone marrow chimeras revealed that 12/15-LO in hematopoietic cells regulates neutrophil accumulation in the interstitial and alveolar compartments, whereas the accumulation of neutrophils in the intravascular compartment is regulated by 12/15-LO in non-hematopoietic and hematopoietic cells. Mechanistically, the increased plasma levels of the chemokine CXCL1 in Alox15(-/-) mice led to a reduced response of the neutrophil chemokine receptor CXCR2 to stimulation with CXCL1, which in turn abrogated neutrophil recruitment. Alox15(-/-) mice also showed decreased edema formation, reduced neutrophil recruitment and improved gas exchange in an acid-induced ALI model.

Conclusions: Our findings suggest that 12/15-LO modulates neutrophil recruitment into the lung by regulating chemokine/chemokine receptor homeostasis.

Figure 1

12/15-lipoxygenase is involved in LPS-induced pulmonary inflammation. Neutrophil recruitment into the interstitial (A) and BAL (B) compartments of the lung and the wet/dry ratio (C) of WT mice and Alox15^-/- mice with (white bars) or without (black bars) LPS inhalation (24 h) was measured by flow cytometry (n = 3 to 4). Exemplary histological images of lungs from WT mice and Alox15^-/- mice under baseline conditions and after LPS exposure (D-G). Bar indicates 50 µm. ^#P < 0.05, *P < 0.05 versus WT LPS.

Figure 2

Pharmacological inhibition of 12/15-LO reduces neutrophil recruitment. (A-C) After pharmacological blockade of 12/15-LO by the inhibitor CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate) or in vehicle control mice, neutrophil recruitment (24 h) into the lung was investigated by flow cytometry. Number of neutrophils in the intravascular (A), interstitial (B), and alveolar compartments (C) (n = 4). Wet/dry ratio under baseline conditions and after LPS exposure (D) (n = 3). Exemplary histological images of lungs from WT mice and Alox15^-/- mice under baseline conditions and after LPS exposure (E-H). Bar indicates 50 µm. ^#P < 0.05

Figure 3

Hematopoietic 12/15-LO is responsible for the regulation of neutrophil recruitment into the lung. Neutrophil recruitment into the lung in response to LPS (white bars) was tested in BM chimeras (WT into WT; WT into Alox15^-/-; Alox15^-/- into WT; Alox15^-/- into Alox15^-/-) (n = 4 to 5 mice per group). Mice lacking hematopoietic and non-hematopoietic Alox15 showed a reduced neutrophil accumulation in the intravascular compartment 24 h after LPS inhalation (A). Reduced neutrophil recruitment into the interstitial (B) and alveolar (C) compartment was observed when either hematopoietic or both hematopoietic and non-hematopoietic Alox15 was eliminated. n = 4 to 5. ^#P < 0.05.

Figure 4

Elimination of 12/15-LO activity alters chemokine homeostasis. CXCL1 (A) and CXCL2 (B) concentrations in the plasma was measured in WT mice (black bars) and Alox15^-/- mice (white bars) under baseline conditions and 3 h after LPS inhalation (n = 3 to 4). CXCL1 (C) and CXCL2 (D) concentrations in the lung were measured in WT mice (black bars) and Alox15^-/- mice (white bars) under baseline conditions and 3 h after LPS inhalation (n = 3 to 4). CXCL1 concentration (E) was measured in the plasma of vehicle- or CDC-treated WT mice and CXCR2 expression (F) on circulating neutrophils under baseline conditions and 3 h after LPS inhalation was quantified by using flow cytometry (n = 3). (G-J) CXCL1 protein expression shown by immunohistochemistry in WT and Alox15^-/- mice under baseline conditions and 3 h after LPS inhalation. Black arrows indicate CXCL1-producing cells (n = 3). Bar indicates 100 µm. ^#P < 0.05

Figure 5

Elimination of 12/15-LO leads to a reduced response of neutrophils to CXCL1. (A) Flow cytometry analysis of CXCR2 expression was performed on peripheral blood neutrophils from WT and Alox15^-/- mice under baseline conditions. Whole blood was stained for CD45, 7/4, GR-1 and CXCR2. CXCR2 expression on the surface of peripheral blood neutrophils was determined by using flow cytometry. (n = 3). (B) Flow cytometry analysis of CXCR2 expression on isolated neutrophils treated with or without CXCL1 (10 pg/ml) for one hour (n = 6). Migration of labeled neutrophils from WT mice (circles) and Alox15^-/- mice (triangles) toward different concentrations of CXCL1 (C) or fMLP (D) was investigated in an in vitro chemotaxis assay. Neutrophils were applied on the top of the filter and allowed to migrate toward the chemoattractant for one hour at 37°C. Migrated cells were measured by fluorescence intensity. Chemotactic index was calculated as the ratio of migrated cells with chemoattractant to migrated cells without chemoattractant and ED₅₀ was determined (n = 3).

Figure 6

12/15-LO-deficiency influences acid-induced acute lung injury. Acid-induced ALI was initiated by intratracheal instillation of 2 µl/g HCL (pH 1.5) in WT and Alox15^-/- mice. (A) After two hours, the lungs were lavaged and the neutrophil count in the BAL fluid was determined (n ≥3 mice). (B) Wet/dry ratio of lungs from WT and Alox15^-/- mice after saline (SAL) or HCL instillation (n = 3). (C) Arterial blood was withdrawn from WT mice and Alox15^-/- mice two hours after saline (SAL) or HCL instillation and the paO₂/FiO₂ ratio was calculated (n = 3). Exemplary histological images of lungs from WT mice and Alox15^-/- mice after saline (SAL) or HCL instillation (D-G). Bar indicates 50 µm. ^#P < 0.05

Figure 7

Pharmacological blockade of 12/15-LO influences acid-induced acute lung injury. Acid-induced ALI was initiated by intratracheal instillation of 2 µl/g HCL (pH 1.5) in vehicle- and CDC-treated WT mice. (A) Wet/dry ratio of lungs from vehicle- and CDC-treated WT mice after saline (SAL) or HCL instillation (n = 3). (B) Arterial blood was withdrawn from vehicle- and CDC-treated WT mice two hours after saline (SAL) or HCL instillation and the paO₂/FiO₂ ratio was calculated (n = 3). Exemplary histological images of lungs from vehicle- and CDC-treated WT mice after saline (SAL) or HCL instillation (C-F). Bar indicates 50 µm. ^#P < 0.05