20-HETE mediates ozone-induced, neutrophil-independent airway hyper-responsiveness in mice

Abstract

Background: Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways.

Methodology/principal findings: Male BALB/c mice were exposed to ozone (3 or 6 ppm) or filtered air (controls) for 2 h. Precision cut lung slices (PCLS; 250 microm thickness) containing an intrapulmonary airway ( approximately 0.01 mm(2) lumen area) were prepared immediately after exposure or 16 h later. After 24 h, airways were contracted to carbachol (CCh). Log EC(50) and E(max) values were then calculated by measuring the airway lumen area with respect to baseline. In parallel studies, dexamethasone (2.5 mg/kg), or 1-aminobenzotriazol (ABT) (50 mg/kg) were given intraperitoneal injection to naïve mice 18 h prior to ozone exposure. Indomethacin (10 mg/kg) was administered 2 h prior. Cell counts, cytokine levels and liquid chromatography-mass spectrometry (LC-MS) for lipid analysis were assessed in bronchoalveolar lavage (BAL) fluid from ozone exposed and control mice. Ozone acutely induced AHR to CCh. Dexamethasone or indomethacin had little effect on the ozone-induced AHR; while, ABT, a cytochrome P450 inhibitor, markedly attenuated airway sensitivity. BAL fluid from ozone exposed animals, which did not contain an increase in neutrophils or interleukin (IL)-6 levels, increased airway sensitivity following in vitro incubation with a naïve PCLS. In parallel, significant increases in oxidized lipids were also identified using LC-MS with increases of 20-HETE that were decreased following ABT treatment.

Conclusions/significance: These data show that ozone acutely induces AHR to CCh independent of inflammation and is insensitive to steroid treatment or cyclooxygenase (COX) inhibition. BAL fluid from ozone exposed mice mimicked the effects of in vivo ozone exposure that were associated with marked increases in oxidized lipids. 20-HETE plays a pivotal role in mediating acute ozone-induced AHR.

Figure 1. Ozone increases murine airway sensitivity to carbachol.

(A) A representive contraction of a murine airway to carbachol (CCh) where A is the airway and V is the blood vessel; and a mean concentration-response curve calculated by changes in the airway lumen area with respect to the baseline image. Slices (n = 13) were prepared as described in the methods. A CCh EC₅₀ value of 0.66 µM and E_max values of 67.1±5.0% were obtained. Airways from mice exposed to ozone at concentrations of (B) 3 ppm or (C) 6 ppm for 2 h, were also contracted to CCh. Mice were sacrificed either immediately (T = 0 h), or the next day (T = 16 h). (D) Area under the curve (AUC) units were used to statistically analyze data. Mean ± SEM shown. NS  =  Non-significant; * P≤0.05; *** P≤0.001 vs filtered air control. At least 6 animals were used in each group, and never more than 8.

Figure 2. BAL fluid cell counts and KC levels following ozone exposure.

Broncho-alveolar lavage (BAL) fluid was collected from mice following exposure to ozone (6 ppm; 2 h) either immediately following exposure (T = 0 h) or the following day (T = +16 h). (A) Macrophage and neutrophil cell counts; (B) mouse (m)KC levels. Data expressed as mean ± SEM shown; *** P≤0.001 vs. filtered air control.

Figure 3. BAL fluid from ozone-exposed mice induces AHR in slices from naïve mice.

Lung slices from naïve mice were incubated with BAL fluid from mice exposed to ozone (6 ppm; 2 h; T = 0 h), or filtered air. (A) Airways were contracted to carbachol (CCh) to create a concentration-response curve. BAL fluid from ozone-exposed mice (B) decreased log EC₅₀ values; and, (C) increased maximum contraction compared to slices incubated with BAL fluid from air-exposed mice. Data expressed as mean ± SEM shown; * P≤0.05 vs. filtered air control. At least 14 airways were used in each group, and never more than 19.

Figure 4. Ozone increases oxidized lipids levels in the BAL fluid.

Mice were administered either dexamethasone (2.5 mg/kg) or ABT (50 mg/kg) 18 h prior or indomethacin (10 mg/kg) 2 hr prior to ozone (6 ppm, 2 h; T = 0 h) exposure, with control mice exposed to filtered air. BAL fluid was retrieved and analyzed for (A) PGE₂, (B) PGD₂, (C) 20-HETE and (D) AA as previously described. Data is expressed as mean ± SEM shown. **** P<0.001 vs. control; ** P≤0.01 vs. control; # P≤0.05 vs. ozone. At least 3 animals were used in each group, and never more than 7.

Figure 5. Ozone did not increase CYP enzymes measured.

mRNA was extracted from whole lungs of mice exposed to ozone (6 ppm; 2 h; T = 0 h) and CYP isotypes were quantified using Taq Man qPCR, as described in the methods. Data was run in triplicate and expressed as mean ± SEM (n = 5 in each group).

Figure 6. CYP inhibition abrogates ozone-induced increases in airway sensitivity.

Mice were administered either (A) dexamethasone (2.5 mg/kg; 18 h prior); (B) indomethacin (10 mg/kg; 2 hr prior); or (C) ABT (50 mg/kg; 18 hr prior) to ozone (6 ppm, 2 h; T = 0 h) exposure, with control mice exposed to filtered air. Lung slices were prepared, and airways were contracted to carbachol (CCh) to create concentration response curves, from which (D) log EC₅₀ values and (E) maximum contractions were calculated. Data is expressed as Mean ± SEM shown. NS  =  Non-significant; **** P<0.001 vs. control; # # P≤0.01 vs. ozone. At least 6 animals were used in each group, and never more than 10.

Figure 7. 20-HETE increases murine airway sensitivity to carbachol.

Lung slices from naïve mice were incubated over-night with 20-HETE (10 and 1 ng/mL). (A) Airways were contracted to carbachol (CCh) to create concentration-response curves. Incubation with 20-HETE (B) decreased log EC₅₀ values; but, (C) had no effect on maximum contraction compared to control slices. Data expressed as mean ± SEM shown; * P≤0.05 and ** P≤0.01 vs control (exact P values shown). At least 8 airways were used in each group, and never more than 12.