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Link to original content: https://pubmed.ncbi.nlm.nih.gov/18957484/
Inhibition of tumour necrosis factor and IL-17 production by leflunomide involves the JAK/STAT pathway - PubMed Skip to main page content
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. 2009 Oct;68(10):1644-50.
doi: 10.1136/ard.2008.096743. Epub 2008 Oct 28.

Inhibition of tumour necrosis factor and IL-17 production by leflunomide involves the JAK/STAT pathway

I González-Alvaro  1 , A M Ortiz, C Domínguez-Jiménez, A Aragón-Bodi, B Díaz Sánchez, F Sánchez-Madrid
Affiliations

Inhibition of tumour necrosis factor and IL-17 production by leflunomide involves the JAK/STAT pathway

I González-Alvaro et al. Ann Rheum Dis. 2009 Oct.

Abstract

Objective: To study the effects of different disease-modifying antirheumatic drugs (DMARD) on different events mediated by IL-15-activated lymphocytes.

Methods: Peripheral blood lymphocytes (PBL) were isolated from healthy donors and activated with IL-15 after exposure to different DMARD: leflunomide, cyclosporin A, methotrexate, mycophenolic acid, FK-506, sulphasalazine and sodium aurothiomalate. The expression of different surface molecules on the PBL was then determined by flow cytometry. Cells were also co-cultured with the monocytic cell line THP-1 and the tumour necrosis factor (TNF) concentration in the supernatant was measured after 24 h using an immunoenzyme assay. The effect of the aforementioned drugs on IL-17 production by IL-15-activated PBL was also studied.

Results: Treatment of PBL with leflunomide, cyclosporin A and FK-506 inhibited the IL-15-induced expression of both CD54 and CD69 by PBL, as well as TNF production in co-cultures of activated PBL and THP-1 cells. The downregulation of CD54 and CD69 in PBL was correlated with the inhibition of TNF production. Likewise, leflunomide, cyclosporin A and FK-506 all inhibited IL-17 production in IL-15-activated PBL. Interestingly, the effect of leflunomide was not reverted by the presence of uridine in the medium. In addition, leflunomide inhibited the phosphorylation of STAT6 in vitro.

Conclusion: Inhibition of the JAK/STAT pathway may represent an additional effect of leflunomide in chronic polyarthritis because it impairs certain events that control proinflammatory TNF and IL-17 cytokine production.

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Conflict of interest statement

Competing interests: Declared. IG-A has received unrestricted grants from Abbott Laboratories and Bristol-Myers Squibb. CD-J was supported by grants from Sanofi-Aventis and Fundación Española de Reumatología. AA-B was supported by a DIB-SER grant from the Fundación Española de Reumatología.

Figures

Figure 1
Figure 1
Effect of different disease-modifying antirheumatic drugs (DMARD) on the upregulation of CD69 induced by IL-15 in peripheral blood lymphocytes (PBL) and on tumour necrosis factor (TNF) production by THP-1 cells induced by IL-15-activated PBL. PBL were exposed to A77-1726 (the active metabolite of leflunomide; 100 μmol), cyclosporin A (10 μg/ml), methotrexate (50 μg/ml), mycophenolic acid (1 μmol), FK-506 (1 μg/ml), sulphasalazine (1000 μg/ml) or sodium aurothiomalate (1 μg/ml) for 30 minutes. The cells were then stimulated with IL-15 (50 μg/ml) for 24 h and afterwards they were used in different assays: (A) The expression of CD69 was assessed by flow cytometry. Data are shown as the median (top of histogram) and interquartile range (IQR; the 25th and 75th percentiles being the lower and upper ends of the bar, respectively) of the relative mean fluorescence intensity (rMFI) from 10 independent experiments. (B) The IL-15-stimulated PBL were washed and co-cultured with THP-1 cells for an additional 24 h. In some experiments, the PBL and THP-1 cells were separated with a 0.4 μm pore transwell to define the effects that were independent of cell–cell contact (Tw). TNF release was measured in cell-free supernatants by enzyme immunoassay. The data are shown as the median and IQR of TNF production (pg/ml) from 15 independent experiments. The statistical significance in these multiple experiments (A and B panels) was determined using the Kruskall–Wallis test, followed by comparison of each treatment with respect to the medium alone using a Mann–Whitney test with Bonferroni correction (significance level considered if p<0.01). (C) Effect of the combination of cyclosporin A and A77-1726 on TNF production. Cells were treated as described above, except in the combined condition in which the lymphocytes were incubated with cyclosporin A (0.1 μg/ml) and A77-1726 (3 μmol) for 10 minutes before IL-15 stimulation. The data are shown as the median and IQR of TNF production (pg/ml) from five independent experiments. (D and E) Correlation between the inhibitory effects of DMARD on lymphocyte CD69 (D) and CD54 (E) expression and TNF production. The data are the mean ± SEM of the percentage inhibition of CD69 or CD54 expression or TNF production, from eight independent experiments. Medium represents cells stimulated with IL-15 in the absence of DMARD. Statistical analysis was performed by using Spearman correlation test. CyA, cyclosporin A; GS, gold salts; MA, mycophenolic acid; MTX, methotrexate; SSZ, sulphasalazine.
Figure 2
Figure 2
The effect of disease-modifying antirheumatic drugs (DMARD) on IL-17 production induced by IL-15 in peripheral blood lymphocytes (PBL). (A) PBL were pre-treated with A77-1726 (100 μmol), cyclosporin A (10 μg/ml), methotrexate (50 μg/ml), mycophenolic acid (MA); 1 μmol), FK-506 (1 μg/ml), sulphasalazine (1000 μg/ml) and sodium aurothiomalate (1 μg/ml) for 30 minutes and then stimulated with IL-15 (50 μg/ml) for 72 h. The supernatants were then harvested and IL-17 release was measured in cell-free supernatants by enzyme immunoassay (EIA). Data are shown as the median (top of histogram) and interquartile range (IQR; the 25th and 75th percentiles being the lower and upper ends of the bar, respectively) of the percentage of IL-17 production from 11 independent experiments. IL-17 production in the condition without DMARD treatment was considered 100% and it corresponds to a median concentration of IL-17 of 216 pg/ml (IQR 144–233). The statistical significance (*) in these multiple condition experiments was determined using the Kruskall–Wallis test followed by comparison of each treatment with the results obtained with medium alone using the Mann–Whitney test with Bonferroni correction (significance level p<0.01). (B) PBL were pre-treated with A77-1726 (30 μmol) in the presence or absence of uridine (100 μmol), as well as with mycophenolic acid (1 μmol) in the presence or absence of guanosine (100 μmol) for 30 minutes and they were then stimulated with IL-15 (50 μg/ml) for 72 h. The supernatants were harvested and IL-17 release was measured in cell-free supernatants by EIA. The data are shown as the median and IQR of the percentage of IL-17 production from six independent experiments. IL-17 production in the condition without DMARD treatment was considered 100% and it corresponds to a median concentration of IL-17 of 382 pg/ml (IQR 249–624).
Figure 3
Figure 3
The inhibitory effect of A77-1726 on lymphocyte CD69 upregulation induced by IL-15 is mediated through the JAK/STAT pathway. (A) Peripheral blood lymphocytes (PBL) were treated with A77-1726 (30 μmol) for 30 minutes and then stimulated with IL-15 (50 μg/ml) for 24 h, in the absence (left panel) or presence (middle panel) of uridine (100 μmol). The effect of the JAK-3 inhibitor, 4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (10 μg/ml), is represented in the right panel. One representative experiment of four is shown and, in each panel, the following histograms are represented: negative control (dotted line), CD69 basal expression (green solid line), CD69 expression after IL-15 stimulation (black solid line), CD69 expression in PBL treated with A77-1726 or JAK-3 inhibitor and stimulated with IL-15 (red solid line). (B) PBL were pretreated with A77-1726 at different doses (1, 3, 10, 30 and 100 μmol) or with medium alone for 30 minutes, and they were then stimulated with IL-15 (50 ng/ml) for 5 minutes. The cells were lysed and the lysates processed to detect the phosphorylated form of STAT6 (upper line) or tubulin (lower line) as described in the Materials and methods section. The figure represents one of five independent experiments. (C) Band intensities were quantified from the nitrocellulose membranes using Image Gauge 3.4 software. Values were normalised considering 100% the intensity of the band obtained with lysates from PBL stimulated with IL-15 in absence of leflunomide pretreatment. Data are shown as the mean ± SEM of five independent experiments and the statistical significance was determined with an analysis of variance with the Bonferroni correction.
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