B-232. Development of a Novel In vivo Assay to Examine Smoke- and Alcohol-Induced Alterations in Bacterial Uptake by Alveolar Macrophages

A. M. Pitz, M. J. Gentry-Nielsen;
Creighton Univ., Omaha, NE.

Using a rat model that mimics the human alcohol-abusing smoker, our laboratory has shown ethanol (EtOH) ingestion significantly decreases non-neutrophil-mediated pulmonary killing of Streptococcus pneumoniae. Unexpectedly, concomitant exposure to cigarette smoke reversed this EtOH-induced defect. Although alveolar macrophages (MΦ) phagocytose S. pneumoniae poorly, we hypothesized that EtOH decreases MΦ uptake of pneumococci, and smoke exposure reverses this detrimental EtOH-induced effect by activating the MΦs. To test our hypothesis, we developed a novel flow cytometric assay to quantify in vivo uptake of bacteria by MΦs within rats’ lungs. Male rats (n = 7-8/group) were exposed for 1 h twice daily for 12 wks to either cigarette smoke (smoke-exposed) or room air (sham-exposed). During the final 5 wks, the rats were pair-fed liquid diets containing either 36% or 0% EtOH calories. They then were infected transtracheally with 1 x 107 cfu of live S. pneumoniae labeled with the newly available fluorochrome allophycocyanin-Cy7. Exactly 15 min later, each rat was euthanized and bronchoalveolar lavage was performed. MΦs in the lavage fluid were collected by differential centrifugation, purified by magnetic cell sorting, and analyzed in a FACSAria flow cytometer for uptake of the fluorescent bacteria. As hypothesized, EtOH ingestion significantly reduced the mean fluorescence of MΦs from sham-exposed rats (624 vs. 1218; p<0.001), but not smoke-exposed rats (2935 vs. 2071; p=0.8). Smoke exposure greatly increased the fluorescence of MΦs from rats in both feeding groups. However, based on the fluorescence of MΦs from an uninfected smoke-exposed rat, we determined that the increase was due to enhanced autofluorescence rather than augmented bacterial uptake. This precluded an accurate comparison of uptake by MΦs from smoke-exposed vs. sham-exposed rats. Based on these results, we believe we have developed a workable assay to quantify bacterial uptake by MΦs within the lungs of experimental animals. Applications would include comparison of bacterial strains, opsonic factors, and innate MΦ defects, but are limited to experimental conditions that do not result in across-the-board MΦ activation.