OBJECTIVE: To investigate if type II alveolar epithelial cells express Toll-like receptor 4 (TLR4) and to investigate the role of TLR4 in airway inflammation of chronic obstructive pulmonary diseases (COPD). METHODS: A549, the line of human type II alveolar epithelial cells were cultured and divided into 3 groups: normal control group, E1A(+) group transfected with adenovirus E1A plasmid, E1A(-) group transferred with blank plasmid without adenovirus E1A. Lipopolysaccharide (LPS) of the concentrations of 0, 0.1, 1, and 10 microg/ml, IL-1 beta of the concentrations of 0, and 0.1 ng/ml, and cigarette smoking extract (CSE) of the concentrations of 0, 10%, 20%, and 40% were used to stimulated the A549 cells for 12 and 24 h. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of IL-8 and TLR4. Western blotting was used to detect the protein expression of nuclear factor kappaB (NF-kappaB) subunit P65. RESULTS: Twenty-four hours after the stimulation of 10 microg/ml LPS, 0.1 ng/ml IL-1beta, and 20% CSE, the IL-8 mRNA expression of the E1A(+) group was 2.82, 1.87, and 4.70 respectively, all significantly higher than those of the normal control group (0.95, 0.78, and 1.02 respectively, all P < 0.05) and those of the E1A(-) group (0.97, 0.81, and 1.12 respectively, all P < 0.05). Twelve and twenty-four hours after the stimulation of 10 microg/ml of LPS, the TLR4 mRNA expression of the E1A+ group were 4.52 and 7.99, both significantly higher than those of the normal control group (1.91 and 3.81 respectively, both P < 0.05) and those of the E1A(-) group (2.00 and 3.88 respectively, both P < 0.05). IL-1beta increased the expression of TLR4 mRNA too, but CSE did not change the expression of TLR4 mRNA in all these groups. LPS, IL-1beta, and CSE all increased the expression levels of NF-kappaB subunit P65 protein. CONCLUSIONS: Pulmonary type II epithelial cells express TLR4. LPS and IL-1beta up-regulate the release of IL-8 which may be mediated via the activation of NF-kappaB induced by TLR4.
OBJECTIVE: To investigate if type II alveolar epithelial cells express Toll-like receptor 4 (TLR4) and to investigate the role of TLR4 in airway inflammation of chronic obstructive pulmonary diseases (COPD). METHODS: A549, the line of human type II alveolar epithelial cells were cultured and divided into 3 groups: normal control group, E1A(+) group transfected with adenovirus E1A plasmid, E1A(-) group transferred with blank plasmid without adenovirus E1A. Lipopolysaccharide (LPS) of the concentrations of 0, 0.1, 1, and 10 microg/ml, IL-1 beta of the concentrations of 0, and 0.1 ng/ml, and cigarette smoking extract (CSE) of the concentrations of 0, 10%, 20%, and 40% were used to stimulated the A549 cells for 12 and 24 h. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of IL-8 and TLR4. Western blotting was used to detect the protein expression of nuclear factor kappaB (NF-kappaB) subunit P65. RESULTS: Twenty-four hours after the stimulation of 10 microg/ml LPS, 0.1 ng/ml IL-1beta, and 20% CSE, the IL-8 mRNA expression of the E1A(+) group was 2.82, 1.87, and 4.70 respectively, all significantly higher than those of the normal control group (0.95, 0.78, and 1.02 respectively, all P < 0.05) and those of the E1A(-) group (0.97, 0.81, and 1.12 respectively, all P < 0.05). Twelve and twenty-four hours after the stimulation of 10 microg/ml of LPS, the TLR4 mRNA expression of the E1A+ group were 4.52 and 7.99, both significantly higher than those of the normal control group (1.91 and 3.81 respectively, both P < 0.05) and those of the E1A(-) group (2.00 and 3.88 respectively, both P < 0.05). IL-1beta increased the expression of TLR4 mRNA too, but CSE did not change the expression of TLR4 mRNA in all these groups. LPS, IL-1beta, and CSE all increased the expression levels of NF-kappaB subunit P65 protein. CONCLUSIONS: Pulmonary type II epithelial cells express TLR4. LPS and IL-1beta up-regulate the release of IL-8 which may be mediated via the activation of NF-kappaB induced by TLR4.
Authors: Urvashi Bhan; Megan N Ballinger; Xianying Zeng; Michael J Newstead; Matthew D Cornicelli; Theodore J Standiford Journal: PLoS One Date: 2010-03-26 Impact factor: 3.240