Effects of cigarette smoke and alcohol on ciliated tracheal epithelium and inflammatory cell recruitment

Abstract

Ciliated epithelium represents the first line of host defense against lung infection. Most alcoholics smoke and are at high risk for developing lung infections. We reported that cigarette smoke activates protein kinase C (PKC) and alcohol desensitizes ciliary beat frequency (CBF) to beta-agonists in bovine bronchial epithelial cells in vitro. The combined effect of smoke and alcohol exposure on mouse ciliated tracheal epithelium has not been studied in vivo. We hypothesized that previously observed in vitro effects of smoke and alcohol exposure could be replicated in vivo. Female C57BL/6 mice were exposed to whole body cigarette smoke only, 20% alcohol ad libitum in drinking water only, or the combination of cigarette smoke plus alcohol for 6 wk. Bronchoalveolar lavage (BAL) cell populations, CBF, and airway kinase activity were assessed. Total BAL cells were decreased in animals exposed to alcohol alone and increased in animals exposed to smoke alone. Mice receiving smoke and alcohol had cell levels similar to smoke alone. Baseline CBF was not affected in any group; however, isoproterenol stimulation of CBF was blunted by alcohol exposure and actually slowed below baseline in the smoke plus alcohol group. Isoproterenol-induced PKA activity was inhibited in mice receiving alcohol independent of smoke exposure. Smoke activated PKC independent of alcohol. The isoproterenol-induced slowing below baseline of CBF after combined smoke and alcohol exposure demonstrates a novel ciliary impairment likely related to the combination of alcohol-mediated PKA desensitization and smoke-stimulated PKC activation, possibly through acetaldehyde present in the vapor phase of cigarette smoke.

Figure 1

Overall weight gain among the mice exposure groups is comparable. The vertical axis represents the mean (± SEM) of the weights of the mice within each exposure group. The horizontal axis represents the exposure to time in weeks (see Table 2 for details of the exposure protocol). Weight gain was roughly comparable among the four groups over the 6-wk experiment with no significant difference in weight between the groups. Open circles, air; open squares, air + alcohol; solid circles, smoke; solid squares, smoke + alcohol.

Figure 2

Total and differential BAL cell populations are reduced in alcohol-fed mice. The vertical axis represents the mean (± SEM) of the total or differential BAL cell counts within each exposure group. The horizontal axis represents the exposure (* P < 0.05 by ANOVA). (A) BAL total cell counts. Mice in the Air + Alcohol group had a mean decrease in total BAL cells compared with all other groups. Combined exposure to smoke and alcohol did not result in total BAL cell numbers significantly different from that of mice receiving smoke alone. (B) BAL total macrophage cell counts: The number of BAL macrophages was similarly decreased in the Air + Alcohol group compared with the other groups. Combined exposure to smoke and alcohol did not result in BAL macrophage cell numbers significantly different from that of mice receiving smoke alone. (C) BAL total PMN cell counts. The number of neutrophils in BAL fluid was significantly decreased in the Air + Alcohol group compared with the Air group. Smoke-exposed mice had a variable increase in the number of neutrophils. Total neutrophil counts from mice exposed to Smoke + Alcohol were comparable to those of the Air group. (D) BAL total lymphocyte cell counts. BAL lymphocytes were decreased in the Air + Alcohol group. This decrease not present when alcohol was combined with smoke exposure.

Figure 3

Alcohol desensitizes stimulated, but not baseline CBF. (A) Baseline CBF. The vertical axis represents the mean (± SEM) of the CBF of tracheal rings within each exposure group. The horizontal axis represents the exposure. Baseline (unstimulated) CBF is not altered by exposure to alcohol, smoke, or a combination of both. (B) β-agonist–stimulated CBF. The vertical axis represents the mean (± SEM) of the change in CBF in Hz after a 40-min treatment of tracheal rings with isoproterenol (100 µM) for each exposure group. The horizontal axis represents the exposure. Stimulation with isoproterenol induced a normal (> 1 Hz) increase in the Air and Smoke groups. The response to isoproterenol stimulation was blunted in the alcohol group and resulted in a significant decrease from baseline CBF in the group with Smoke + Alcohol exposure (* P < 0.05 by ANOVA).

Figure 4

Alcohol exposure, independent of smoke exposure, blunts β-agonist–induced PKA enzyme activity in ciliated tracheal epithelium. The vertical axis represents the mean (± SEM) of the PKA activity in pmol/min/mg of ciliated tracheal epithelial tissue within each exposure group. The horizontal axis represents the exposure. The open bars indicate the baseline (unstimulated) PKA activity. The solid bars indicate the isoproterenol (stimulated) PKA activity after treatment with 100 µM isoproterenol for 40 min. Isoproterenol induced a significant (2-fold) increase in PKA activity in the Air and Smoke groups. In contrast, mice exposed to alcohol, independent of smoke exposure, had either a blunted or no response to isoproterenol consistent with PKA desensitization (*P < 0.05 for + iso compared with baseline by paired Student’s t test).

Figure 5

Cigarette smoke, independent of alcohol exposure, activates baseline PKC activity in the ciliated tracheal epithelium. The vertical axis represents the mean (± SEM) of the PKC activity in pmol/min/mg of ciliated tracheal epithelial tissue within each exposure group. The horizontal axis represents the exposure. Cigarette smoke induced a significant (2-fold) increase PKC activity in the ciliated tracheal epithelium regardless of alcohol exposure (*P < 0.05 by ANOVA).