Background Membrane bound FasL (mFasL) is able to induce fibroblast-like synoviocytes (FLS) cell death. In experimental arthritis mouse models, injection of agonistic antibody (Ab) anti-Fas decreased the symptoms. However, soluble FasL (sFasL) is increased in RA patients serum and correlated with disease activity. Recent data indicated that mFasL could be protective whereas sFasL could be deleterious (#1) suggesting that they could have different functions.
Objectives We therefore analyzed the effect of different FasL preparation mimicking sFasL or mFasL on RAFLS proliferation or apoptosis and on cytokines production.
Methods RAFLS were treated with different FasL preparations (FasL-Flag± Ab anti-Flag, FasL-Fc or sFasL) or with agonistic Ab anti-Fas. Apoptosis was then analyzed by FACS on basis of the annexin V-FITC binding and TOPRO-3 up-take. Proliferation was measured using tritiated [3H] thymidine. Activation of signaling pathways was analyzed by western blot and their influence was assessed using chemical inhibitors. VEGF, IL-6 and IL-8 were measured using commercially available ELISA.
Results FasL-Flag alone (mimicking sFasL) was not able to induced FLS apoptosis (8%±8 n=5) while proliferation was significantly activated (3.3±1 fold; n=5; p<0.05). Similarly, sFasL was only able to strongly induce RAFLS proliferation (8.1±3.3 fold; n=3). In an other hand, membrane bound FasL (FasL-Flag+Ab α-Flag) significantly induced RAFLS apoptosis (52%±18; n=5) but also a slighter but significant proliferation (2.2±0.3 fold; n=4). Duality of mFasL was confirmed using agonistic Ab anti-Fas. (mimicking mFasL) with pro-apoptotic (38%±18; n=2) and proliferative effect (2.5±0.15 fold). Finally, growing concentration of FasL-Fc leads to aggregation of the protein, mimicking mFas or sFasL at high and low concentration respectively. Dose responses confirmed mFasL and sFAsL effects. FasL activated Akt and ERK (n=5) but also activated caspase-8. A pan-caspases inhibitor (z-VAD-FMK) prevented mFasL-induced apoptosis, but also blocked mFasL and sFasL-induced proliferation (n=4). Using specific inhibitors for caspases 8, 3 and 9, we found that caspase 8 is involved in FasL proliferation. Because ERK and Akt pathways are involved in TRAIL induced proliferation, we tested whether caspases participate on MAPK and Akt activation after FasL stimulation. This was not confirmed, since caspases inhibition did not inhibit FasL-induced kinases activation (n=3). Moreover, sFasL but not FasL-Fc, induced significant production of VEGF, IL-6 and IL-8 in RA FLS
Conclusions mFasL induces preferentially RAFLS apoptosis, whereas sFasL only induces RAFLS proliferation and cytokines production. According to what we have already described for TRAIL, caspases are involved in FasL-induced apoptosis and proliferation. This is the first demonstration of sFasL and mFasL have different effects on RAFLS proliferation and cytokines production. sFasL by enhancing RAFLS proliferation and cytokines could have a deleterious role in RA. Therefore, its blockage could be a therapeutic tool to prevent RA.
O’Reilly et al. 2009, Nature Medecine
Disclosure of Interest None Declared