[Effect of T-bet on biological functions of mouse macrophage Raw264.7].
OBJECTIVE
T-bet (T box expressed in T cells), a Th1-specific T box transcription factor, controls many kinds of immune cells, such as Th1, NK, CD8+, dendritic cells, and B cells. This study was to explore potential effects of T-bet gene on biological functions of mouse macrophage Raw264.7 cells in vitro.


METHODS
The eukaryotic expression vector carrying mouse T-bet (pcDNA3.0-mT-bet) was constructed and identified by consequence analysis, double restrictive endonucleases digestion and polymerase chain reaction (PCR). The gene expression in Raw264.7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. PBS, pcDNA3.0, and pcDNA3.0-mT-bet were transiently transfected into Raw264.7 cells respectively; cell cycle, MHC I/II expression levels, phagocytic activity of FITC-dextran, nitric oxide (NO) secretion level, and the cytotoxicity of Raw264.7 cells to mouse leukemia cell line L1210 were evaluated 48 hours after transfection.


RESULTS
Eukaryotic expression vector which could express T-bet protein in Raw264.7 cells was successfully constructed. There was no difference in cell cycle between pcDNA3.0 group and pcDNA3.0-mT-bet group. There was significant difference in MHC I expression level between pcDNA3.0 group (20.8+/-0.7) and pcDNA3.0-mT-bet group (24.8+/-0.6, P<0.05), but not in MHC II expression level; there was also difference in mean fluorescence intensity of phagocytized dextran between pcDNA3.0 group (28.2+/-0.4) and pcDNA3.0-mT-bet group (32.8+/-0.8, P<0.05); there was also significant difference in NO secretion level between pcDNA3 group (0 pmol) and pcDNA3.0-mT-bet group [(1.7+/-0.6) pmol, P<0.05] without lipopolysaccharide (LPS) stimulation; meanwhile, significant difference was also observed between pcDNA3.0 group [(10.5 +/-1.3) pmol] and pcDNA3.0-mT-bet group [(15.6+/-1.6) pmol, P<0.05] under the stimulation of LPS (10 microg/ml) for 20 h; there was also difference in cytotoxicity of Raw264.7 cells to L1210 cells in vitro between pcDNA3 group [(35.6+/-2.1)%] and pcDNA3.0-mT-bet group [(51.9+/-3.5)%, P<0.05].


CONCLUSIONS
T-bet up-regulates MHC I expression and NO secretion level in Raw264.7 cells, increases their cytotoxicity to L1210 cells, but has no influences on the cell cycle and MHC II expression.
Xiao
Lin
L
State Key Laboratory of Oncology in South China, Guangzhou, Guangdong, 510060, P. R. China.
Tan
Wei-Ping
WP
Xiao
Xia
X
Liang
Zhi-Hui
ZH
Wu
Jiang-Xue
JX
Li
Hong-Li
HL
Liu
Ran-Yi
RY
Huang
Bi-Jun
BJ
Huang
Wen-Lin
WL
D000818:Animals; D002453:Cell Cycle; D045744:Cell Line, Tumor; D005805:Genes, MHC Class I; D005822:Genetic Vectors; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D007939:Leukemia L1210 / Q000473:pathology; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010587:Phagocytosis; D010957:Plasmids; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation; D000098815:T-bet Transcription Factor
D015395:Histocompatibility Antigens Class I; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor; D015534:Trans-Activators; D009569:Nitric Oxide