Mechanisms Of The Transcription Of IL-8 In Human Bronchial Epithelial Cells Treated By CSE

Cigarette smoke mediated oxidative stress and inflammatory events in the bronchial epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Lots of harmful substances generated by smoking including free radicals and oxides can trigger pro-inflammatory cytokines, which are increased in the lungs of smokers. The airway epithelium is the primary target for any inhaled environmental agents and plays a critical role in the release of pro-inflammatory mediators. Previous in vivo findings have supported that cigarette smoke can induce pro-inflammatory cytokine (Interleukin-8, IL-8) release in smokers and in rodent lungs. However, the precise molecular mechanisms as how cigarette smoke generates signals for pro-inflammatory cytokine release, particularly in bronchial epithelium are not yet clearly understood.ObjectiveHere we examined the acute effect of aqueous cigarette smoke extracts (CSE) in cultured normal human bronchial epithelial cell line (BEAS-2B), and detected the changes of the transcription of Interleukin-8. IL-8 is a multifunctional cytokine that has significant neutrophil chemoattractant and activating properties. It was reported that NF-κB and C/EBPβinvolve in IL-8 promoter transactivation as well as in its generation. Herein, we used three kinds of mutant BEAS-2B cells to determine effects of NF-κB and C/EBPβon the transcription of Interleukin-8 in cigarette smoke extract treated BEAS-2B cells. These three kinds of cells were transfected by luciferase plasmid and EGFP plasmid. Luciferase constructs under the control of the IL-8 promoter (IL-8 WT BEAS-2B cells), as well as mutants in the NF-κB site (IL-8mNFκB-BEAS-2B cells) or in the C/EBPβsite (IL-8mC/EBPβ-BEAS-2B cells).Methods1 Preparation of cigarette smoke extracts solution CSE was prepared by using an improved modification of the method described by Su Y, and diluted by RPMI1640 medium without FBS.2 Using MTT colorimetric methods to get IC50BEAS-2B cells were treated by 0%,2.5%,5.0%,7.5%,10.0%,12.5%. CSE for 24h and added in MTT to get the inhibitions of BEAS-2B cells under the pression of different CSE concentrations. And then we got the Inhibitory Concentration 50(IC50).3 CSE of different concentrations treated BEAS-2B cells, and we detected the transcription of IL-8 mRNA by fluorescent quantitative PCRBased on the results of the MTT colorimetric methods, BEAS-2B cells were treated with 0%,2.5%,5.0%,7.5%,10.0%CSE for 24h for each experimental condition, total RNA was extracted and 1μg of the resulting RNA was then reverse transcribed to cDNA. And fluorescent quantitative PCR assay was used to detect the transcription of IL-8 mRNA.4 7.5%CSE stimulated three kinds of mutant BEAS-2B cells, and we detected the transcription of fLCF mRNA by fluorescent quantitative PCRThree kinds of mutant BEAS-2B cells were treated by the 7.5%CSE for 24h for each experimental condition, total RNA was extracted and 1μg of the resulting RNA was then reverse transcribed to cDNA. And fluorescent quantitative PCR assay was used to detect the transcription of fLCF mRNA.5 Luciferase assays7.5%CSE treated normal BEAS-2B cells and three kinds of mutant BEAS-2B cells for 24h. And then we measured the luciferase activity.Results1 Morphologic change of the BEAS-2B cells treated with CSEAfter the stimulated by CSE, the shapes of BEAS-2B cells became irregular. There were more floating cells compared with cells without CSE. We observed a CSE dose dependant increase in cellular damage. And the cell damage became more seriously with the increase of CSE.2 Changes of green fluorescent protein (EGFP) in three kinds of mutant BEAS-2B cells before and after CSE treated CSE had an obvious cyto-inhibition. Cells with green fluorescent protein (EGFP) became fewer and round observed under the fluorescence microscope.3 Results of the MTT colorimetric methodCSE had an obvious cyto-inhibition (F=25.06, P<0.001). With the increase of the concentration of CSE, cyto-inhibition became higher. There was a dose-effect relationship between CSE and the cyto-inhibition. And Inhibitory Concentration 50 of CSE was 7.5%.4 Transcription of IL-8 mRNA in BEAS-2B cells treated with different concentrations of CSE detected by fluorescent quantitative PCRBEAS-2B cells were stimulated with 2.5%,5.0%,7.5%and 10.0%CSE for 24h. IL-8 mRNA levels were up-regulated by CSE (2.5%,5.0%and 7.5%), (P<0.001)and had a CSE dose dependent increase. However CSE at a higher concentration (10.0%) made lots of cells dead and did not increase IL-8 mRNA level (P>0.05).5 Transcription of fLCF mRNA in three kinds of mutant BEAS-2B cells treated with 7.5%CSE detected by fluorescent quantitative PCRThe transcriptions of fLCF mRNA of IL-8mNF-κB-and IL-8mC/EBPβ-BEAS-2B cells were lower than that of IL-8 WT BEAS-2B cells. The transcription of fLCF mRNA of IL-8 WT BEAS-2B cells was 1.32 times than that of IL-8mNF-κB-BEAS-2B cells and 1.54 times than that of IL-8mC/EBPβ-BEAS-2B cells.6 Luciferase assays7.5%CSE treated BEAS-2B cells and three kinds of mutant BEAS-2B cells for 24h. We found that the luciferase activities in IL-8mNF-κB-BEAS-2B cells and in IL-8mC/EBPp-BEAS-2B cells were lower than that in IL-8 WT BEAS-2B cells. And the discrepancy had a statistical significance (P< 0.05).ConclusionsNF-κB regulated the release of Interleukin-8 in BEAS-2B cells treated by cigarette smoke extract. And this study showed for the first time that C/EBPβtranscription factor involved in the precise molecular mechanisms of the release of Interleukin-8 in BEAS-2B cells treated by cigarette smoke extract.