[Expression of recombinant mouse IL-1beta significantly down-regulates NK cell mediated cytolysis [corrected] against H22 hepatoma cells].
OBJECTIVE
To investigate the effect of the expression of recombinant IL-1beta in H22 hepatoma cells on its response to NK cell mediated cytotoxicity.


METHODS
BALB/c mouse was stimulated by 6% of starch. Total RNA was prepared from peripheral blood monocytes (PBMCs). IL-1beta gene (843 bp) was obtained by RT-PCR. The purified PCR product digested by Xho I and EcoR I was cloned into pIRES2-EGFP to construct the recombinant pIRES2-EGFP-mIL-1beta expression vector which was verified by PCR, restriction enzyme assay (Xho I and EcoR I) and DNA sequencing. Then the purified pIRES2-EGFP-mIL-1beta plasmid was transfected into H22 hepatoma cells by jetPEI. The expression level of recombinant IL-1beta was detected by RT-PCR and confocal microscopy. The cytotoxicity of wild-type spleenic NK cells against H22 cells was assessed by MTT assay.


RESULTS
After the total RNA isolated from the starch stimulated BALB/c mouse PBMC, 843 bp IL-1beta gene in length was prepared by RT-PCR. The purified PCR product digested by EcoR I and Xho I was ligated by pIRES2-EGFP to create pIRES2-EGFP-mIL-1beta expression plasmid which was verified by PCR, restriction enzyme assay and DNA sequencing. Then pIRES2-EGFP-mIL-1beta was transfected into H22 hepatoma cells by jetPEI. RT-PCR and confocal microscopy assay showed these cells expressed high level of recombinant IL-1beta expression vector. In a 4-hour based MTT assay, IL-1beta in H22 cells was more resistant to NK92 cell mediated cytotoxicity compared with the cells transfected with pIRES2-EGFP. Meanwhile, the cytolytic capacity of the spleenic NK cells separated from wild-type mouse decreased about 10% when the ratio of effector to target was 40:1.


CONCLUSIONS
The expression of proinflammatory cytokine IL-1beta can significantly down-regulate the cytolytic activity of NK cells against H22 hepatoma cells. It plays a crucial role in the immune escape of hepatoma from NK cell mediated innate immunity.
Xiao
Wei-Ling
WL
Key Laboratory of Molecular Immunology, Weifang Medical College, Weifang 261042, China. ixiaoxiaoyue@163.com
Liang
Shu-Juan
SJ
Mu
Dong-Zhen
DZ
Wang
Xue-Jing
XJ
Wu
Hui-Na
HN
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000473:pathology*; D045744:Cell Line, Tumor; D005260:Female; D053583:Interleukin-1beta / Q000235:genetics* / Q000276:immunology*; D007694:Killer Cells, Natural / Q000276:immunology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D053583:Interleukin-1beta; D011994:Recombinant Proteins