In Vitro Analysis of the Mode of Inhibition of T-Cell Proliferation by Leflunomide, A Novel Immunomodulatory Agent
Hamzavi, Fasahat H.
Leflunomide (HW A 486) is a novel immunosuppressive drug that has shown promising results in preventing graft rejection and curing some autoimmune diseases in rats. The mechanism by which HWA 486 inhibits T-cell proliferation has not yet been characterized. This study utilized the human interleukin-2 (IL-2) dependent leukemia T-cell line, Kit 225 (Hori et al., 1987), to determine at which point of the signal transduction pathway of T-cell proliferation HWA 486 is taking its effect. Results of [3H] thymidine experiments demonstrated that HW A 486 was unable to inhibit proliferation of Kit 225 cells. The ineffectiveness of HW A 486 to inhibit proliferation of Kit 225 cells was unexpected, but subsequent research on this cell line (Sawami et al., 1992) indicated that the Kit 225 cell line does not express lck tyrosine kinase activity. Lck is a protein bound to the cytoplasmic end of the interleukin-2 receptor (IL-2R) and is thought to be necessary in the signal transduction pathway of T-cell proliferation (Horak et al., 1991). The effect of HW A 486 on general tyrosine kinase activity was then assessed by immunoblotting analysis of phosphotyrosine kinase activity of Kit 225 cells. Immunoblotting indicated that HWA 486 was ineffective in inhibiting the other tyrosine kinase activity involved in Kit 225 cells. On the other hand, genistein, a general tyrosine kinase inhibitor, was able to slightly inhibit this kinase activity. Since HWA 486 was ineffective in inhibiting the general tyrosine kinase activity within the lck deficient Kit 225 cells, it was possible to assume that HW A 486 was specific for lck tyrosine kinase activity. It is therefore possible to deduce that a hypothetical mechanism by which HWA 486 takes its effect is to inhibit lck tyrosine kinase activity of the IL-2R which in turn inhibits T-cell proliferation.
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