Differential in vivo effects of whole cigarette smoke exposure versus cigarette smoke extract on mouse ciliated tracheal epithelium

Abstract

In this study the authors compared the affect of vapor phase cigarette smoke (CS) versus cigarette smoke extract (CSE) on the lungs and upper airway of C57BL/6 mice. The authors found that CSE treatment significantly increased neutrophil influx (P < .001), baseline ciliary beat frequency (CBF) (P < .05), and protein kinase C activity compared to CS and controls. Isoproterenol increased CBF with CS exposure, but decreased CBF with CSE (P < .01). Isoproterenol increased protein kinase A (PKA) activity in all groups except CSE. CSE exposure induced inflammatory cell bronchiolitis. These data indicate that CSE exposure has differential effects on the lungs and tracheal epithelium compared to CS exposure.

FIGURE 1

(A) Weight gain in mice exposed to air or whole cigarette smoke (CS) by inhalation, or intranasal administration of phosphate-buffered saline (PBS), or cigarette smoke extract (CSE). There were no significant differences in the weight or rate of weight gain for any of the treatment groups. (B) Serum cotinine levels in the CSE exposed mice were significantly higher than those in mice exposed to CS (P < .05).

FIGURE 2

Total BAL cell numbers. Mice were treated with whole body exposure to air or whole cigarette smoke (CS) or received intranasal administration of phosphate-buffered saline (PBS) or cigarette smoke extract (CSE). Mice treated with CSE had the greatest total number of BAL cells, significantly greater than all other groups (*P < .05, CS versus air; **P < .05 PBS versus air; ***P < .05, CSE versus CS and PBS; ***P < .001 CSE versus air).

FIGURE 3

Macrophages and neutrophils (PMNs) in the BAL. (A) CSE treatment reduced the percentage of macrophages in the BAL compared to all other groups (***P < .001 CSE versus CS, air; ***P < .01, CSE versus PBS). PBS treatment also decreased the percentage of macrophages recovered in the BAL (**P < .01) over CS- and air-treated groups. (B) Despite the decrease in the percentage of macrophages, treatment with CSE, PBS, and CS significantly increased the total number of macrophages (***P < .05, CSE versus CS; P < .01, CSE versus Air; **P < .05 PBS versus air; P < .05 CS versus Air). (C) The percentage of PMNs was increased in CSE treated mice compared to PBS (***P < .05), CS and air (***P < .001). PBS treatment also induced a significant increase in the percentage of neutrophils (**P < .001 PBS versus CS and air). (D) The number of PMNs was significantly higher in the CSE treated group compared to all other treatment groups (***P < .001). PBS also had a significant increase in the number of PMNs compared to the CS and air treatment groups (**P < .05).

FIGURE 4

Baseline (unstimulated) ciliary beat frequency. CBF was measured in all samples prior to stimulation with isoproterenol. Baseline CBF was significantly higher in the mice treated with CSE compared to all other groups (*P < .05).

FIGURE 5

Isoproterenol induced change in CBF. Following baseline CBF measurement, tracheal rings from treated mice were stimulated with 100 μM isoproterenol for 40 minutes. CBF increased normally in tracheal rings from mice treated with air or smoke. CBF did not increase in tracheal rings from mice treated with PBS. CBF decreased significantly in response to isoproterenol stimulation in the tracheal rings from mice treated with CSE (**P < .01).

FIGURE 6

Isoproterenol stimulated PKA activity. PKA activity was measured in tracheal tissue from treated mice with (filled bars) or without (open bars) 100 μM isoproterenol. Isoproterenol stimulation significantly increased the amount of PKA activity in the ciliated tracheal epithelium of mice treated with air, CS, and PBS (*P < .05). No significant increase in PKA activity was measured in response to isoproterenol in the ciliated tracheal epithelium of mice treated with CSE.

FIGURE 7

PKC activity in the ciliated tracheal epithelium. PKC activity in the ciliated tracheal epithelium of treated mice was measured. PKC activity was greatly increased and significantly higher in the tissue from CSE treated mice in comparison to all other groups (***P < .001). Tissues from CS treated mice had an increase in PKA activity significantly higher than that measured in Air treated mice (*P < .05), but not PBS treated mice.

FIGURE 8

Lung pathology in smoke exposed mice. H&E staining of a section of lung from mice exposed to air or to whole cigarette smoke (CS) by inhalation, intranasal administration of phosphate buffered saline (PBS), or cigarette smoke extract (CSE). Increased mild bronchiolitis is observed in the lungs of mice exposed to CS or PBS compared to air-exposed control mice. CSE-exposed mice lungs demonstrate a significant increase in inflammatory cell bronchiolitis compared to PBS-treated mice.