Smoke exposure exacerbates an ethanol-induced defect in mucociliary clearance of Streptococcus pneumoniae

Abstract

Background: Alcoholics and smokers are particularly susceptible to pulmonary infections caused by Streptococcus pneumoniae, the pneumococcus. Infection begins when pneumococci colonizing the nasopharynx are aspirated into the lower respiratory tract. The major host defense against this movement is the mucociliary clearance apparatus. Both cigarette smoke and ethanol (EtOH) exposure alter ciliary beating and protein kinase activity in the respiratory mucosa in vitro, but their effects on bacterial clearance in the intact animal have not been determined.

Methods: Male Sprague Dawley rats were exposed twice daily for 12 weeks to either the smoke generated from 30 cigarettes (smoke-exposed) or room air (sham-exposed). For the last five weeks of smoke exposure, the rats were fed Lieber-DeCarli liquid diets containing 0%, 16%, 26%, or 36% EtOH calories. The rats then were infected intranasally with S. pneumoniae, and movement of the organisms into the lower respiratory tract was quantified by plate counts of the tracheas and lungs 4 hr later. Ciliary beat frequency (CBF) analysis was performed on tracheal ring explants from each animal before and after stimulation with the beta-agonist isoproterenol, and tracheal epithelial cell protein kinase C (PKC) activity was measured.

Results: Ingestion of any of the EtOH-containing diets resulted in a dose-dependent increase in movement of S. pneumoniae into the rats' lungs. This EtOH-induced defect was augmented further by concurrent smoke exposure, although smoke exposure alone had little effect on S. pneumoniae movement. Smoke, but not EtOH exposure, activated tracheal epithelial cell PKC. Increased movement of organisms into lungs correlated with a decrease in CBF and loss of the ciliary response to isoproterenol.

Conclusion: EtOH ingestion in our model facilitated movement of S. pneumoniae into rats' lungs, a phenomenon exacerbated by concurrent smoke exposure. Furthermore, the organism's movement into the lungs correlated with a blunting of the rats' ciliary response to an established stimulus. Defects in mucociliary clearance thus may be one cause of the increased risk of pneumococcal infections in people who abuse alcohol, particularly if they also smoke.

Figure 1

Log of cfu/ml of S. pneumoniae isolated from lung tissue of individual smoke-exposed [A] and sham-exposed [B] rats (n = 8–9). Rats were sacrificed 4 h after intranasal infection with 1 × 10⁸ cfu of type 3 S. pneumoniae. The lungs were removed and homogenized in 10 ml of PBS, and bacteria were enumerated by standard plate counts. Horizontal bars represent mean of the log values with samples having 0 cfu being counted as 0 for graphing and statistical purposes.

Figure 1

Log of cfu/ml of S. pneumoniae isolated from lung tissue of individual smoke-exposed [A] and sham-exposed [B] rats (n = 8–9). Rats were sacrificed 4 h after intranasal infection with 1 × 10⁸ cfu of type 3 S. pneumoniae. The lungs were removed and homogenized in 10 ml of PBS, and bacteria were enumerated by standard plate counts. Horizontal bars represent mean of the log values with samples having 0 cfu being counted as 0 for graphing and statistical purposes.

Figure 2

Computerized spectrum analysis of CBF performed on tracheal ring explants (n = 8–9). Unstimulated (baseline) measurements were recorded immediately on a thermostatically controlled microscope. Samples then were treated with the β-agonist isoproterenol and stimulated CBF was determined. * Stimulated value for 0% diet group significantly greater than 26% and 36% diet groups within the same exposure group (p<0.001 by ANOVA). † Sham stimulated value significantly greater than sham unstimulated value (p<0.001 by t-test). ‡ Sham stimulated value greater than smoke stimulated value (p=0.007 by t-test).

Figure 3

Baseline PKC activity in cigarette smoke-exposed (black bars) or sham-exposed (white bars) rats consuming different concentrations of EtOH. Tracheal epithelial cells were extracted and assayed for PKC activity in the particulate fraction of the cells. Bars represent SEM of tracheal epithelial cell PKC activity from six different rats normalized against total cellular protein (*p<0.05 for smoke-exposed vs. sham-exposed rats).