View Abstract

A3809 - Cigarette Smoke Alters Endocytosis in Primary Human Small Airway Epithelial Cells
Author Block: P. Duffney1, T. H. Thatcher2, R. P. Phipps3, P. J. Sime4; 1University of Rochester, Rochester, NY, United States, 2Division of Pulmonary and Critical Care Medicine, University of Rochester, Rochester, NY, United States, 3Univ of Rochester, Rochester, NY, United States, 4Univ of RochesterMed Ctr, Rochester, NY, United States.
Rationale: Exposure to cigarette smoke is a risk factor for many pulmonary diseases. Those with diseases caused by smoking, such as chronic obstructive pulmonary disease, are at increased risk for lung infections, which can trigger exacerbations of COPD symptoms. Many pulmonary pathogens utilize endocytosis to infect host cells. Endocytosis is also vital for proper cell signaling and nutrient uptake. Lung epithelial cells are particularly susceptible to infection as they are the interface between the environment and the lung interstitium. We have previously shown that cigarette smoke alters innate immune signaling in response to viral infection. However, the effects of cigarette smoke exposure on lung epithelial cell endocytosis are largely unknown. Methods: Primary human small airway epithelial cells (SAECs) were grown on culture well inserts and exposed to either air or whole tobacco smoke at the air-liquid interface for 60 min. Cells were treated with fluorescently labeled polyinosinic-polycytidylic acid (FITC-poly I:C), bovine serum albumin (AF488-BSA), Cholera toxin B subunit (AF488-CtxB), or 3K molecular weight Dextran (FITC-Dextran) to investigate clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis respectively. Uptake of fluorescent ligands was determined by fluorescent microscopy and flow cytometry. For inhibitor experiments, cells were pretreated with either chlorprozamine or filipin to inhibit clathrin-mediated or caveolin-mediated endocytosis respectively. Results: Smoke-exposed cells had increased uptake of FITC-poly I:C starting 6 hours after smoke exposure. Uptake of AF488-CtxB was similarly increased in smoke exposed cells suggesting an upregulation of caveolin-mediated endocytosis. Interestingly, uptake of AF488-BSA and FITC-dextran was reduced in smoke exposed cells. Conclusion: These data show that cigarette smoke exposure leads to alteration of endocytosis in smoke exposed SAEC. This alteration favors an increase in caveolin-mediated endocytosis and down regulation of both macropinocytosis and clathrin-mediated endocytosis. Targeting alterations in endocytosis could be a new treatment paradigm to restore proper signaling in smoke-exposed epithelial cells.