[Effects of leflunomide and its active metabolite on the production and mRNA expression of TNF-alpha in peritoneal macrophages and synovial cells with adjuvant arthritis in rats].
OBJECTIVE
To observe the effects of leflunomide (LEF), an isoxazole immunomodulatory agent and its active metabolite, A771726, on the production and mRNA expression of TNF-alpha in peritoneal macrophages and synovial cells with adjuvant arthritis rats and to further investigate the immunosuppression effects of leflunomide and its mechanisms.


METHODS
ELISA methods were used for assaying the levels of TNF-alpha. The RT-PCR methods were used for measuring the expression of TNF-alpha.


RESULTS
The production of TNF-alpha was increased in the supernatant of PM psi in adjuvant arthritis (AA) model rat. LEF (5, 10, 25 mg.kg-1, ig) was shown to inhibit the release of TNF-alpha from peritoneal macrophages induced by LPS and the inhibitory effects were in a dose-effect relevance manner. A parallel investigation of cytokine mRNA expression was undertaken using semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR) to follow the kinetics of cytokine appearance in PM psi and synovial membrane tissue cells obtained from AA/normal rats treated with A771726. The results of RT-PCR from macrophages and synovial membrane tissue cells of AA rats at the peak of inflammatory phase showed that TNF-alpha mRNA expression levels were higher than those of normal rats, while the expression of TNF-alpha mRNA was reduced by treating with A771726 in vitro. On the other hand, the TNF-alpha mRNA expression showed kinetics very similar to those obtained by ELISA technique which measured protein expression.


CONCLUSIONS
Leflunomide and its active metabolite, A771726, was found to inhibit the production and mRNA expression of TNF-alpha in peritoneal macrophages and synovial cells with adjuvant arthritis rats model. It might be involved in the mechanisms of its anti-inflammation and immunosupression.
Li
Wei-dong
WD
Department of Pharmacology, School of Basic Medical Sciences, Medical Center of Peking University, Beijing 100083, China.
Lin
Zhi-bin
ZB
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism / Q000494:pharmacology; D001169:Arthritis, Experimental / Q000473:pathology*; D002478:Cells, Cultured; D005260:Female; D007555:Isoxazoles / Q000378:metabolism / Q000494:pharmacology*; D000077339:Leflunomide; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D013583:Synovial Membrane / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D000276:Adjuvants, Immunologic; D000894:Anti-Inflammatory Agents, Non-Steroidal; D007555:Isoxazoles; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000077339:Leflunomide