Haemophilic arthropathy (HA), which shares some clinical and biological injury characteristics with rheumatoid arthritis (RA), is characterized by chronic proliferative synovitis and cartilage destruction. Anti-Fas mAb specifically targets the Fas molecule, which is expressed and activated on the cell surface of inflammatory synovial cells and plays a key role for induction of apoptosis. Caspases are the final executioners of apoptosis and their activation requires proteolytic processing of inactive zymogen into activated fragments.
To evaluate the effects of anti-Fas mAb on HA synoviocytes and its capacity of inducing apoptosis analysing caspase 3 activity.
HA synoviocytes were incubated with IgM 1000 ng/ml (control), TNFalpha 10 ng/ml, FGF 10 ng/ml, CH11 100 ng/ml (positive control of apoptosis) with or without anti-Fas mAb at different concentrations (from 0,1 to 1000 ng/ml) for 24 h. RA and healthy synoviocytes were used as controls. To measure cell proliferation/citotoxicity, the WST-1 assay has been performed. Caspase 3 activity has been evaluated with ELISA kit and western blot.
Anti-Fas mAb induced a citotoxic effect in HA (p < 0,001 for any dose), healthy (p < 0,001 at 100 and 1000 ng/ml) and RA synoviocytes (p < 0,05 for any dose) reaching a maximum effect at 1000 ng/ml. After stimulation with anti-Fas mAb combined with TNFalpha, there was a citotoxic effect on healthy (p < 0,001 at 10, 100, 1000 ng/ml anti-Fas mAb), RA (p < 0,001 for any dose) and HA synoviocytes (p < 0,005 at 1, 10, 100 and 1000 ng/ml anti-Fas mAb). After stimulation with anti-Fas mAb combined with FGF, there was a citotoxic effect on healthy, RA and HA synoviocytes (p < 0,001 for any dose). Caspase 3 levels were increased in HA synoviocytes after anti-Fas mAb treatment in a dose-dependent manner, even after co-stimulation with TNFalpha (p < 0,001 for any stimulus). CH11 induced an increase of caspase 3 levels in HA synoviocytes more than RA synoviocytes. Western blot showed that HA synoviocytes had higher levels of activated caspase 3 compared to RA synoviocytes after stimulation with Anti-Fas mAb, CH11 and co-stimulation with TNFalpha.
Anti-Fas mAb has a dose-dependent citotoxic effect on HA synoviocytes, even when associated with TNFalpha and FGF. Anti-Fas mAb is effective in increasing caspase 3 levels in HA synoviocytes in a dose-dependent manner. HA synoviocytes show higher levels of activated caspase 3 compared to RA synoviocytes. Our results suggest that anti-Fas IgM mAb may favour the induction of apoptosis in HA synoviocytes.