In order to establish the possible gender influence on the activity of leflunomide (LEF) in rheumatoid arthritis (RA), we evaluated the proapoptotic activity of the active LEF metabolite A77 1726 (LEF-M), in combination with sex hormones, on cultures of human macrophages. In particular, we focussed our investigation on the triggering phase of the apoptosis. Cultures of macrophages from activated THP-1 cells and from RA synovial tissues were treated with LEF-M alone [30muM] or in presence of 17beta-estradiol (E2) (10(-9)M) or testosterone (T) (10-(8)M) for 24 hours. FAS, FAS-L, FADD (Fas-Associated via Death Domain) and FLICE (FADD-Like Interleukin-1 beta Converting Enzyme) were evaluated by immunocytochemistry (ICC), Western blot (WB) and reverse transcriptase-multiplex polymerase chain reaction (RT-MPCR). Regarding macrophages from THP-1 cells (M), the ICC showed that LEF-M exerted a significant up-regulation on all investigated apoptotic proteins, when compared to untreated cells (control) (p<0.001). On the contrary, E2 significantly increased FAS-L positivity (p<0.05) and down-regulated FADD (p<0.01), while others apoptotic proteins were not modulated when compared to control. Regarding RA synovial macrophages (SM), the ICC showed that LEF-M exerted a significant up-regulation on FAS and FAS-L when compared to control (p<0.001). On the contrary, E2 down-regulated significantly FAS-L (p<0.001), while FAS was not modulated respect to control. T significantly increased the apoptotic proteins in all conditions. The results of the present study suggests a less efficient therapeutic effect of LEF in female patients, due to the contrasting action of estrogens on LEF- induced apoptosis.
Keywords: Rheumatoid arthritis; apoptosis; leflunomide; macrophages; sex hormones.