Figure 3

Detection of apoptotic cells and Fas/Fas ligand (FasL) protein expression in grafted rheumatoid arthritis (RA) synovium. The RA synovium in severe combined immunodeficiency (SCID) mice was harvested approximately two weeks after engraftment without or with three day treatment with 10¹¹ particles of recombinant FasL adenovirus (Ad-FasL), LacZ adenovirus (Ad-LacZ), or PBS before it was collected. (a-c) Fas and FasL protein expression in RA synovium in vivo were examined using an immunohistochemistry staining system. (a) Fas protein is highly expressed on synoviocytes in RA synovium (original magnification 400×). (b) FasL protein is lacking in RA synoviocytes treated with Ad-LacZ (original magnification 400×); (c) but a significant increase in FasL protein expression in RA synoviocytes appears three days after Ad-FasL injection (original magnification 400×). (d-i) Apoptotic cells in RA synovium were examined using the ApopTag fluorescein in situ apoptosis detection kit (Intergen). The red propidium iodide counter-staining indicates the nucleus of all synovial cells in RA synovium. The green fluorescein stains the fragmented DNA in the nucleus. (d) Apoptotic cells in PBS treated RA synovium are almost undetectable (original magnification 200×). (e) A slightly increased apoptotic cell population was seen in Ad-LacZ treated RA synovium (original magnification 200×), (f) while around a 15-fold increased frequency of apoptotic cells in RA synovium was observed in Ad-FasL treated RA synovium (original magnification 200×; arrows point to TUNEL-positive cells). (g-i) The location of the fragmented DNA was observed using a laser scanning confocal microscope. (g) The nucleus of synovial cells in RA synovium was stained with propidium iodide (red; bar = 15 μm). (h) Fragmented DNA was stained with fluorescein (green; bar = 15 μm). (i) The localization of the fragmented DNA was determined by the overlap of the fluorescein staining and propidium iodide counter-staining (bar = 15 μm), which characterizes cell death.