• Zhongguo Wei Zhong Bing Ji Jiu Yi Xue · Nov 2011

    [Hyperoxia-induced up-regulation of Toll-like receptors expression in alveolar epithelial cells].

    • Dong Huang, Fang Fang, and Feng Xu.
    • Department of Pediatrics, Guizhou Province People's Hospital,Guiyang, Guizhou, China.
    • Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2011 Nov 1;23(11):645-9.

    ObjectiveTo examine the correlation between the hyperoxia-induced reactive oxygen species (ROS) production and Toll-like receptors (TLRs) expression in cultured alveolar epithelial cells in order to understand the role of TLRs signaling in inflammatory response during acute lung injury.MethodsThree groups of cultured human lung adenocarcinoma cell line A549 were exposed to: ()air, )hyperoxia(95%O2 + 5%COz) and ® N-acetylcysteine (NAC) pretreatment + hyperoxia. The level of intracellular ROS, TLR2/4 mRNA, TLR2/4 protein and cytokine interleukin-6/8 (IL-6/8) concentrations in the culture supernatant were measured by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR)and enzyme linked immunosorbent assay (ELISA) in cells harvested at different time points into the treatment.ResultsA549 cells were found to have a baseline (mainly intracellular) TLR2/4 expression.Continued exposure to hyperoxia caused () a progressive increase in intracellular ROS, which became significantly higher than the air treated cells after 2 hours of exposure (11. 820±3. 123 vs. 7. 223 ± 1. 170,P < 0.01), and reached a peak after 48 hours of exposure (113. 837 ± 5. 247, P < 0. 01); © an increase in intracellular TLR2/4 mRNA which peaked after 2 hours of exposure (TLR2 mRNA: 1. 820 ± 0. 056 vs.1. 263 ± 0. 023; TLR4 mRNA: 2. 080 ± 0. 220 vs. 1. 317 ± 0. 107, both P < 0.01); © significant increase inTLR2/4 protein expression (mainly intracellular), both peaked after exposure for 6 hours (TLR2 protein:8. 370 ± 1. 548 vs. 3. 930 ± 0.277; TLR4 protein: 25. 803 ± 5. 783 vs. 8. 867+2. 230, both P < 0.01); a progressive increase in culture supernatant IL-6 (ng/L), IL-8 (ng/L) concentration, both peaked at 48 hours (IL-6: 2 213.41 69.99 vs. 9. 76 ± i1. 47; IL-8: 11 520. 38 ± 429. 93 vs. 159. 56 ± 20. 80, both P < 0.01). NAC pre-treatment significantly suppressed the hyperoxia induced intracellular ROS (14. 050 ± 1. 257 vs. 31.180+2.336, P < 0.01) production, the hyperoxia induced expression of TLR2/4 (TLR2 mRNA: 1. 270±0. 061 vs. 1. 683+0. 025; TLR4 mRNA: 1. 5870. 058 vs. 1. 650 ± 0. 139; TLR2 protein: 3. 458 ± 0.258 vs. 8.371 + 1.548; TLR4 protein: 11.611 ± 3.403 vs. 25. 803 ± 5.783, all P < 0.05), and the hyperoxia induced increase in IL-6 and IL-8 in supernatant level (IL-6: 8.42+0.70 vs. 73.51+16.70; IL-8: 134.94 ± 5.19 vs. 772.82 ± 96.05, both P < 0.05), as seen in the hyperoxia treated groups.ConclusionHyperoxia induced intracellular ROS production can up-regulate TLR2/4 expression in A549 cells, leading to the release pro-inflammatory cytokines IL-6 and IL-8 from these cells.

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