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. 2003 May;62(5):440–443. doi: 10.1136/ard.62.5.440

Active leflunomide metabolite inhibits interleukin 1ß, tumour necrosis factor α, nitric oxide, and metalloproteinase-3 production in activated human synovial tissue cultures

O Elkayam 1, I Yaron 1, I Shirazi 1, R Judovitch 1, D Caspi 1, M Yaron 1
PMCID: PMC1754531  PMID: 12695157

Abstract

Background: Leflunomide is now an approved agent for the management of adult rheumatoid arthritis (RA). Its active metabolite A771726 inhibits de novo pyrimidine biosynthesis. Although considered to be an immunosuppressive agent, its mechanism of action remains obscure.

Objectives: Evaluation of the leflunomide active metabolite A771726 (LEF) effect on interleukin 1ß (IL1ß), tumour necrosis factor (TNFα), nitric oxide (NO), and stromelysin (metalloproteinase-3 (MMP-3)) production by activated human synovial tissue in culture.

Methods: Synovial tissue was obtained during surgery from patients undergoing total knee replacement owing to RA or osteoarthritis (OA), cut into small pieces, and cultured in Petri dishes with test materials as previously described. IL1ß, TNFα, NO, and MMP-3 were measured in the culture media after 48 hours incubation with different doses of LEF by methods previously described.

Results: LEF (0.3, 3, and 9 µg/ml) inhibited IL1ß production in the presence of lipopolysaccharide (LPS; 3 µg/ml) in a dose dependent manner (p<0.01) at LEF 0.3 µg/ml. TNFα production in the presence of IL1ß (1 ng/ml) was also inhibited in a dose dependent manner (p<0.05 at LEF 0.3 µg/ml). NO and MMP-3 production in the presence of LPS (3 µg/ml) was inhibited as well (p<0.01 at LEF 1 µg/ml and at LEF 0.3 µg/ml, respectively). Synovial cell viability evaluated by the tetrazolium salt XTT was unaffected by the LEF concentration used. There was no qualitative difference in the response of OA and RA synovial tissue.

Conclusion: Leflunomide may modulate the rheumatoid articular process by inhibition of local production of IL1ß, TNFα, NO, and MMP-3.

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Figure 1.

Figure 1

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Effect of the leflunomide active metabolite (LEF) on IL1ß levels in the medium of synovial tissue cultures in the presence of LPS (3 µg/ml). Bars show the mean (SEM) of four separate experiments (two RA and two OA, n=8). The absolute mean (SEM) levels of IL1ß in control and LPS stimulated cultures were 1.38 (0.012) and 5.16 (0.08) pg/mg synovia, respectively.

Figure 2.

Figure 2

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Effect of leflunomide active metabolite (LEF) on TNFα levels in levels in the medium of synovial tissue cultures in the presence of IL1ß (1 ng/ml). Bars show the mean (SEM) of four separate experiments (two RA and two OA, n=8). The absolute mean (SEM) levels of TNFα in control and IL1ß stimulated cultures were 2.33 (0.23) and 4.95 (0.49) pg/mg synovia, respectively.

Figure 3.

Figure 3

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Effect of leflunomide active metabolite (LEF) on nitric oxide (NO) levels in the medium of synovial tissue cultures in the presence of LPS (3 µg/ml). Bars show the mean (SEM) of four separate experiments (two RA, n=4). The absolute mean (SEM) levels of NO in control and LPS stimulated cultures were 1.08 (0.01) and 2.45 (0.245) µmol/mg synovia, respectively.

Figure 4.

Figure 4

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Effect of LEF on stromelysin (MMP-3) levels in the medium of synovial tissue cultures in the presence of LPS (3 µg/ml). Bars show the mean of four separate experiments (two RA, n=4). The absolute mean (SEM) levels of MMP-3 in controls and LPS stimulated cultures were 1929 (83) and 5394 (197) ng/mg synovia, respectively.

Figure 5.

Figure 5

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Effect of LEF on cell viability. Bars represent the percentage of viable cells incubated with LEF in comparison with control. LEF at a concentration of 0.3–9 µg/ml did not show any deleterious effect on cell viability, which remained >90% in comparison with controls.

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