Objective: To 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.
Methods: Three 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.
Results: A549 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.
Conclusion: Hyperoxia 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.