Construction of a recombinant adenovirus vector expressing IL-18BP/IL-4 fusion gene and the anti-inflammatory effect induced by this gene on lipopolysaccharide-stimulated synovial fibroblasts.
OBJECTIVE
To construct a recombinant adenovirus vector for expressing the IL-18 binding protein (IL-18BP)/IL-4 fusion gene and confirm the anti-inflammatory effect of this gene.


METHODS
The recombinant virus expressing IL-18BP/IL-4 fusion protein (AD-IL-18BP/IL-4) was constructed. AD-IL-18BP/IL-4 was used to infect synovial fibroblasts (SF). ELISA and Western blot analysis were used to determine the expressions of the proteins IL-4 and IL-18BP. To investigate the protective effects of this vector on rheumatoid arthritis, SF were infected with AD-IL-18BP/IL-4 and stimulated by LPS (1 microg/ml) 4 h later. The expression levels of TNF-alpha, IL-6, IL-8, and IL-18 in the culture supernatant were detected by ELISA and production of PGE2 and NO was estimated. The protein expression of COX-2, iNOS, and NF-kappaB p50 in treated SF was analyzed by Western blot.


RESULTS
AD-IL-18BP/IL-4 can effectively express the IL-18BP/IL-4 fusion protein. The expressions of TNF-alpha, IL-6, IL-8, and IL-18 were significantly inhibited in LPS-stimulated SF after treatment with AD-IL-18BP/IL-4. The production of PGE2 and NO was significantly decreased. Moreover, NF-kappaB p50, COX-2, and iNOS levels in SF were markedly suppressed by AD-IL-18BP/IL-4.


CONCLUSIONS
AD-IL-18BP/IL-4 can suppress the production and expression of inflammatory cytokines such as COX-2, iNOS, and NF-kappaB in LPS-stimulated SF.
Yao
Hang-Ping
HP
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Institute of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, 310003 Hangzhou, China. yaohangping@zju.edu.cn
Qian
Yun
Y
Shao
Xue-Ting
XT
Xu
Zhe-Rong
ZR
Cheng
Lin-Fang
LF
Feng
Lei
L
Wu
Nan-Ping
NP
Yang
Yun-Mei
YM
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D015847:Interleukin-4 / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D036341:Intercellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; C118370:interleukin-18 binding protein; D015847:Interleukin-4; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D015232:Dinoprostone