title: In vitro effects of water-pipe smoke condensate on the endocytic activity of Type II alveolar epithelial cells (A549) with bacillus Calmette-Guérin.
abstract: OBJECTIVE
  Tuberculosis (TB) is a major global health problem and poses immense threats to many populations. The association between tobacco smoke and TB has already been studied. Water-pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally as it is considered by users as being safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be predisposing factors that enhance the incidence of pulmonary disorders in water-pipe smokers. For example, uncontrolled macropinocytosis occurs in alveolar epithelial cells following exposure to water-pipe smoke, which may predispose individuals to pulmonary infection. In this work, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) by macropinocytosis in Type II alveolar epithelial cells (A549).
  
  
  METHODS
  A549 cells were treated by WPC (4mg/mL) for 24 h, 48 h, 72 h, and 96 h, respectively. The effect on cell proliferation was studied using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction assay. Cells were exposed to fluorescein isothiocyanate (FITC)-dextran (1mg/mL; control) and FITC-BCG (multiplicity of infection, 10) for 20min at 37°C before their collection and the uptake of BCG-FITC was determined by flow cytometry. Similar experiments were performed at 4°C as a control.
  
  
  RESULTS
  WPC (4mg/mL) after 72h (1.4±0.2-fold, p<0.05) and 96h (1.6±0.2-fold, p<0.05) hours increased the uptake of BCG-FITC. No effect on BCG-FITC uptake was observed at 24h or 48h. WPC also significantly increased the uptake of FITC-dextran (2.9±0.3-fold, p<0.05) after 96h. WPC also significantly decreased cell proliferation after 24h (84±2%), 48h (78±3%), 72h (64±2%, p<0.05), and 96h (45±2%, p<0.05).
  
  
  CONCLUSIONS
  WPC exposure increased epithelial cells' permeability and death and enhanced their capacity for macropinocytosis. Our in vitro data suggest possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacteria. Further studies will be conducted to understand the mechanism of action of WPC.
authors.affiliation: Cell and Molecular Biology Group, Airways Disease Section, National Heart and Lung Institute, Imperial College London, London, UK. Electronic address: ianmadcock@hotmail.com.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Adcock
authors.affiliation: Clinical Tuberculosis and Epidemiology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Esmaeil
authors.initials: E
authors.lastname: Mortaz
authors.affiliation: Clinical Tuberculosis and Epidemiology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Shamila D
authors.initials: SD
authors.lastname: Alipoor
authors.affiliation: Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands; Nutricia Research Centre for Specialized Nutrition, Utrecht, The Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: Garssen
authors.affiliation: Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Akbar Velayati
keywords: Endocytosis activity; Type II alveolar epithelial cells (A549); Water pipe