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 Emax 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).
Table 1.
Level of eicosanoids in murine BAL fluid following ozone exposure.
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.