The role of cyclooxygenase-2 in mechanical ventilation-induced lung injury

Abstract

Mechanical ventilation is necessary for patients with acute respiratory failure, but can cause or propagate lung injury. We previously identified cyclooxygenase-2 as a candidate gene in mechanical ventilation-induced lung injury. Our objective was to determine the role of cyclooxygenase-2 in mechanical ventilation-induced lung injury and the effects of cyclooxygenase-2 inhibition on lung inflammation and barrier disruption. Mice were mechanically ventilated at low and high tidal volumes, in the presence or absence of pharmacologic cyclooxygenase-2-specific inhibition with 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404). Lung injury was assessed using markers of alveolar-capillary leakage and lung inflammation. Cyclooxygenase-2 expression and activity were measured by Western blotting, real-time PCR, and lung/plasma prostanoid analysis, and tissue sections were analyzed for cyclooxygenase-2 staining by immunohistochemistry. High tidal volume ventilation induced lung injury, significantly increasing both lung leakage and lung inflammation relative to control and low tidal volume ventilation. High tidal volume mechanical ventilation significantly induced cyclooxygenase-2 expression and activity, both in the lungs and systemically, compared with control mice and low tidal volume mice. The immunohistochemical analysis of lung sections localized cyclooxygenase-2 expression to monocytes and macrophages in the alveoli. The pharmacologic inhibition of cyclooxygenase-2 with CAY10404 significantly decreased cyclooxygenase activity and attenuated lung injury in mice ventilated at high tidal volume, attenuating barrier disruption, tissue inflammation, and inflammatory cell signaling. This study demonstrates the induction of cyclooxygenase-2 by mechanical ventilation, and suggests that the therapeutic inhibition of cyclooxygenase-2 may attenuate ventilator-induced acute lung injury.

Figure 1.

Mechanical ventilation induces lung leakage. High tidal volume mechanical ventilation significantly increased Evans blue dye deposition in lung tissue, compared with control and low tidal volume mice (A, light bars). Similarly, mechanical ventilation caused a stepwise increase in bronchoalveolar lavage protein compared with spontaneously breathing control mice (B, light bars). These effects were significantly attenuated in high tidal volume mice by the pharmacologic inhibition of cyclooxygenase-2 with 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404; hatched bars) (n = 4–7 mice per condition). LTV, low tidal volume ventilation; HTV, high tidal volume ventilation; COX-2, cyclooxygenase-2; BAL, bronchoalveolar lavage; EBD, Evans blue dye. *P < 0.01 versus control values. **P < 0.05 versus HTV alone.

Figure 2.

Mechanical ventilation induces lung inflammation. HTV ventilation induced a mild, but statistically significant, increase in alveolar leukocytes (A, light bars) that was significantly attenuated by the pharmacologic inhibition of COX-2 with CAY10404 (A, dark bars). Both LTV and HTV ventilation increased tissue polymorphonuclear leukocytes (PMNs) and tissue myeloperoxidase (MPO) activity compared with spontaneously breathing control mice (B and C, light bars), indicative of acute tissue inflammation. COX-2 inhibition with CAY10404 significantly decreased both tissue PMNs and tissue MPO activity in HTV mice, with a trend toward decreased MPO in control and LTV mice (B and C, dark bars). (D) Finally, mechanical ventilation induced a tidal volume–dependent increase in BAL IL-6, which was significantly attenuated in HTV mice treated with CAY10404. Lung homogenates were stained for ICAM-1 and VCAM-1 as representative inflammatory cell adhesion molecules. (Results were repeated in triplicate, and representative images are shown). (E, top left) Mechanical ventilation induced a stepwise increase in ICAM-1 expression compared with control mice. (E, top right) In contrast, whereas HTV ventilation increased VCAM-1 expression, LTV ventilation was associated with a decrease in VCAM-1 expression compared with control mice. (E, bottom) The pharmacologic inhibition of COX-2 significantly decreased ICAM-1 expression in both the LTV and HTV groups, but increased VCAM-1 in the LTV and HTV groups. LTV, low tidal volume ventilation; HTV, high tidal volume ventilation; BAL, bronchoalveolar lavage; ICAM-1, intercellular adhesion molecule–1; VCAM-1, vascular cell adhesion protein–1. *P < 0.05 versus control values. **P < 0.05 versus HTV alone.

Figure 3.

Mechanical ventilation induces COX-2 expression and activity. Lung expression of COX-2 was determined by Western blotting and quantitative real-time PCR analysis. (A) HTV mechanical ventilation significantly increased COX-2 protein expression, compared with control and LTV mice. No significant differences in COX-1 expression were evident between the three groups. (Results were repeated in triplicate, and representative images are shown). (B) No differences were evident in COX-2 mRNA expression between the three groups. BAL and plasma prostanoids were measured as markers for cyclooxygenase activity. HTV mechanical ventilation significantly increased lavage concentrations of both prostaglandin E2 (PGE2) (C) and 6-keto prostaglandin F1α (D), a stable prostacyclin metabolite, compared with control samples. Likewise, HTV mechanical ventilation increased plasma PGE2 (E) compared with control and LTV mice, but exerted no effect on plasma 2,3-dinor-6-keto prostaglandin F1α (2,3-dinor-6-keto PGF1α) (F), a stable prostacyclin metabolite. The pharmacologic inhibition of COX-2 with CAY10404 significantly decreased BAL PGE2 and 6-keto PGF1α as well as plasma PGE2 in HTV mice. LTV, low tidal volume ventilation; HTV, high tidal volume ventilation; BAL, bronchoalveolar lavage. *P < 0.05 versus control values. **P < 0.05 versus HTV alone.

Figure 4.

COX-2 expression in murine lungs. Murine lungs were formalin-fixed in situ and immunohistochemically stained with antibodies directed against COX-2. In addition to the expected prominent staining of bronchiolar epithelium in all groups, significant staining of mononuclear cells was evident, showing granular cytoplasmic positivity to COX-2. These cells were confirmed to be of the monocyte/macrophage lineage by costaining for CD45 and CD68 (not shown). Note the presence of COX-2–positive alveolar macrophages in the HTV group (dashed arrow), in addition to interstitial monocytes and macrophages located in close proximity to the alveolar space (solid arrows). Images represent 5-μm histologic sections. Boxed areas in left panels are presented at ×500 magnification in right panels.