Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function

Abstract

Exposure to cigarette smoke is associated with airway epithelial mucus cell hyperplasia and a decrease in cilia and ciliated cells. Few models have addressed the long-term effects of chronic cigarette smoke exposure on ciliated epithelial cells. Our previous in vitro studies showed that cigarette smoke decreases ciliary beat frequency (CBF) via the activation of protein kinase C (PKC). We hypothesized that chronic cigarette smoke exposure in an in vivo model would decrease airway epithelial cell ciliary beating in a PKC-dependent manner. We exposed C57BL/6 mice to whole-body cigarette smoke 2 hours/day, 5 days/week for up to 1 year. Tracheal epithelial cell CBF and the number of motile cells were measured after necropsy in cut tracheal rings, using high-speed digital video microscopy. Tracheal epithelial PKC was assayed according to direct kinase activity. At 6 weeks and 3 months of smoke exposure, the baseline CBF was slightly elevated (~1 Hz) versus control mice, with no change in β-agonist-stimulated CBF between control mice and cigarette smoke-exposed mice. By 6 months of smoke exposure, the baseline CBF was significantly decreased (2-3 Hz) versus control mice, and a β-agonist failed to stimulate increased CBF. The loss of β-agonist-increased CBF continued at 9 months and 12 months of smoke exposure, and the baseline CBF was significantly decreased to less than one third of the control rate. In addition to CBF, ciliated cell numbers significantly decreased in response to smoke over time, with a significant loss of tracheal ciliated cells occurring between 6 and 12 months. In parallel with the slowing of CBF, significant PKC activation from cytosol to the membrane of tracheal epithelial cells was detected in mice exposed to smoke for 6-12 months.

Figure 1.

(A) Ciliary beat frequency (CBF) in tracheal epithelial cells of mice exposed to cigarette smoke. No change in baseline CBF was evident in mice exposed to cigarette smoke for 1.5 months and 3 months. Smoke exposure for 6 mo caused a small but significant (^#P < 0.05) decrease in baseline CBF of media-only cells. From 9 months onward, baseline CBF slowed significantly (*P < 0.01). Vertical axis represents the mean (±SEM) of CBF (n = 10). (B) Ciliary beat frequency (CBF) in tracheal epithelial cells stimulated with a β-agonist, isoproterenol (Iso; 10 μM), from mice exposed to cigarette smoke. Isoproterenol was able to stimulate an increase in CBF in mice exposed to smoke for 1.5 to 3 months. However, isoproterenol failed to stimulate an increase in CBF of mice exposed to smoke for 6 months or longer. Vertical axis represents the mean (±SEM) ΔCBF (n = 10).

Figure 2.

Quantification of numbers of motile cilia in mice exposed to cigarette smoke. Tracheal rings of mice were mapped using SAVA, and each whole-field analysis (WFA) was averaged for the total number of motile points. Ave #, average number. Smoke exposure for 6 to 12 months caused a significant (*P < 0.01) decrease in the numbers of motile cilia detected.

Figure 3.

Histologic sections represent mouse tracheae after exposure to cigarette smoke (magnifications, ×40 and ×100) for 1.5 to 12 months. Mice exposed to cigarette smoke for 1.5 to 3 months retained a pseudostratified columnar ciliated epithelial layer of cells similar to that in control mice (sham-exposed to room air) and harvested at 1 year. Mice exposed to smoke for 6 months reveal some areas of sparse or detaching ciliated cells. Mice exposed to smoke for 9 to 12 months have lost most of their ciliated cells. Control mice retain cilia throughout the 12-month study.

Figure 4.

Immunohistochemical staining for acetylated tubulin in tracheae of mice exposed or not exposed to smoke for 12 months. Cilia (green) are present at 12 months in mice not exposed to smoke (left column). After 12 months of smoke exposure, immunofluorescence images reveal a loss of cilia (right column). Lower images depict enlargements of boxed sections indicated in corresponding top images.

Figure 5.

Protein kinase C (PKC) activity in tracheal epithelial cells of mice exposed to cigarette smoke. The vertical axis represents the mean (±SEM) of PKA activity in pmol/min/mg of ciliated tracheal epithelial tissue within each exposure group (n = 10). Smoke exposure for 6 to 12 months caused a significant (^#P < 0.05 and *P < 0.01, vs. time-matched controls.) increase in the particulate fraction levels of PKC activity. Smoke exposure for 9 to 12 months caused a significant (^#P < 0.05) decrease in the cytosolic fraction levels of PKC activity. This result indicates the activated translocation of the enzyme to the particulate fraction.