IL-1beta sensitizes rat intervertebral disc cells to Fas ligand mediated apoptosis in vitro.
OBJECTIVE
To determine the apoptotic effect of recombinant rat Fas Ligand on rat intervertebral disc cells pre-treated with IL-1beta in vitro, and the expression of Fas in cultured rat intervertebral disc cells.


METHODS
Cells were isolated from the inner annulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and divided in 5 treatment groups. IL-1beta (10 ng/mL), FasL (5, 20 ng/mL) with/without IL-1beta (10 ng/mL) pre-treatment was respectively added in Dulbeccoos modified Eagleos medium and Hamos F-12 medium with 1% fetal bovine serum. After 32 h, the cells were stained with annexin V-FITC and propidium iodide to evaluate apoptosis using flow cytometry and to analysis transcription of Fas using RT-PCR.


RESULTS
Compared with control group, FasL (20 ng/mL), IL-1beta (10 ng/mL)+FasL (5 ng/mL), and IL-1beta (10 ng/mL)+FasL (20 ng/mL) induced significant apoptosis of the disc cells (P<0.01). Apoptosis was also induced by FasL 5 ng/mL (P<0.05); whereas, apoptosis was not induced by IL-1beta (10 ng/mL) (P>0.05). IL-1beta (10 ng/mL) enhanced the apoptosis-inducing effects of FasL (5 ng/mL) and FasL (20 ng/mL) in disc cells. Fas gene transcription in all groups and Fas expression in the 5 treatment groups were approximately 1.2-2.1-fold greater than control group (respectively, P<0.05). Additionally, Fas expression in FasL with IL-1beta pre-treatment groups were significantly up-regulated than in FasL groups (P<0.01).


CONCLUSIONS
The results of this study showed disc cells pre-treated with IL-1beta increased apoptotic rate in response to FasL in vitro and provided insights to understand Fas/FasL system-mediated apoptosis in disc cells which would be enhanced due to inflammation factor in degenerative disc.
Cui
Li-Yang
LY
Department of Orthopaedic Surgery, the Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510120, China.
Liu
Shang-Li
SL
Ding
Yue
Y
Huang
Dong-Sheng
DS
Ma
Ruo-Fan
RF
Huang
Wei-Guo
WG
Hu
Bao-Shan
BS
Pan
Qiu-Hui
QH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D053222:Fas Ligand Protein / Q000494:pharmacology*; D005434:Flow Cytometry; D053583:Interleukin-1beta / Q000494:pharmacology*; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D008297:Male; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000187:drug effects; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
D053222:Fas Ligand Protein; D053583:Interleukin-1beta; D012333:RNA, Messenger; D019014:fas Receptor