T-bet expression in the iris and spleen parallels disease expression during endotoxin-induced uveitis.
BACKGROUND
T lymphocytes have been implicated in the development of endotoxin-induced uveitis (EIU). T-bet is a Th1 cell-specific transcription factor that is involved in differentiation and effector functions. The aim of this study was to investigate kinetics of T-bet expression at the mRNA and protein levels during EIU using real-time PCR and whole-mount immunohistochemistry.


METHODS
A single footpad injection of 200 mug of lipopolysaccharide (LPS) was administered to male Wistar rats in order to induce EIU. Clinical changes were followed by slit-lamp examination. The expression of T-bet mRNA in the spleen was evaluated 0, 8, 16, 24, 48, and 96 h after LPS injection using real-time PCR. Immunohistochemistry was performed on the iris whole-mounts as well as on frozen sections of the spleen to evaluate T-bet protein expression. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed on the iris whole-mounts to assay apoptotic cells.


RESULTS
Uveitis was observed in all rats that received LPS. T-bet(+) cells and TUNEL(+) cells in the iris whole-mounts showed a similar pattern in cell number and distribution and both types of cells were observed at 8 h, significantly increased 24 h, and decreased 48 h after LPS injection. T-bet expression at both the mRNA and protein levels in spleen also paralleled ocular inflammation. It was weakly detectable after 0 h, increased after 8 h (index 1.3, T-bet(+) cells OD 17.43+/-2.15), reached its peak after 24 h (index 4.00, OD 53.52+/-4.00), and decreased 48 h following LPS injection (index 1.38, OD 25.75+/-2.45).


CONCLUSIONS
The results show that T-bet expression in both the iris and the spleen, and in apoptotic cells in the iris parallel the severity of intraocular inflammation after systemic LPS administration. These results suggest that T-bet may play a significant role in the dynamics of EIU.
Li
Bing
B
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, People's Republic of China.
Yang
Peizeng
P
Chu
Liqun
L
Zhou
Hongyan
H
Huang
Xiangkun
X
Zhu
Lianxiang
L
Kijlstra
Aize
A
D000818:Animals; D017209:Apoptosis; D005786:Gene Expression Regulation / Q000502:physiology*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007498:Iris / Q000378:metabolism* / Q000473:pathology; D008070:Lipopolysaccharides; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012486:Salmonella typhimurium; D013154:Spleen / Q000378:metabolism* / Q000473:pathology; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism; D014605:Uveitis / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology; D000098815:T-bet Transcription Factor
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor