Background We have previously described that cyclosporin A inhibits CD69 expression induced by IL-15 on human peripheral blood lymphocytes (PBL).1 The expression of CD69 has a putative role in the intercellular contact-mediated TNF-alpha production by macrophages and could be a potential target in the treatment of Rheumatoid Arthritis. In addition, Janus Kinase 3 (JAK3) has been involved in the signal transmission of the IL-2 and IL-15 receptors. Furthermore, it has been described that A77 1726 (the active metabolite of leflunomide) may inhibit the effect of some tyrosine kinases, including the JAK3.
Objectives To analyse the effect of A77 1726 upon the CD69 expression induced by different cytokines on PBL.
Methods Purified PBL from healthy donors were incubated with IL-2 (50 IU/ml), IL-6 (5 ng/ml), IL-15 (5 ng/ml), TNF-alpha (20 ng/ml), or medium, separately and in the presence of cyclosporin A (0.1 ? 10 μg/ml) and/or A77 1726 (0.1 ? 100 μM). After 24 h of culture, cells were washed and CD69 expression assessed through indirect immunofluorescence and flow cytometry. Data are shown as mean ± SD. Paired Student t test was used for statistical analysis.
Results Cells incubated with IL-15 in the presence of A77 1726 showed a significant lower CD69 expression than those incubated with IL-15 alone in a dose dependent manner (p < 0.05 for all concentrations). The highest inhibitory effect, without evidence of toxicity, was observed at 100 μM with a reduction in CD69 expression of 47.3 ± 20.3% (n = 6). A similar effect was achieved with a specific JAK3 inhibitor, 4-(4?-Hydroxyphenyl) amino-6,7-dimethoxyquinazoline. A77 1726 also significantly inhibited the effect of IL-2 and IL-6, but not the CD69 up-regulation mediated by TNF-alpha. Treatment of PBL with a combination of CsA (0.1 μg/ml) and A77 1726 (0.1–1 μM) induced a similar inhibition of the CD69 expression achieved with each of the drugs at the maximal dose assayed (10 μg/ml and 100μM, respectively).
Conclusion The inhibition of IL-15 induced CD69 expression on PBL by A77 1726 might be also responsible for the therapeutic effect of leflunomide in RA. Furthermore, the combination of cyclosporin A and leflunomide might be an interesting approach to the treatment of this disease, as demonstrated in an antigen induced arthritis model in rats.2
Ortiz AM, García-Vicuña R, Sancho D, Laffon A, Sánchez-Madrid F, González-Álvaro I. Cyclosporin A inhibits CD69 expression induced on synovial fluid and peripheral blood lymphocytes by IL-15. J Rheumatol. 2000;21:2329–38
Thoss K, Henzgen S, Petrow PK, Katenkamp D, Brauer R. Immunomodulation of rat antigen-induced arthritis by leflunomide alone and in combination with cyclosporin A. Inflamm Res. 1996;45:103–7
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