BRD 7552 receptor br Materials and methods br Results
Materials and methods
Results To examine the apoptosis in FLS in vitro, we measured the number of BRD 7552 receptor in early apoptosis (Annexin V+, 7-AAD−) and late apoptosis/necrosis (Annexin V+, 7-AAD+) using flow cytometry after incubation with or without stimulating Fas antibody. In the absence of Fas activation, the frequency of apoptotic cells in FLS from RA patients was lower compared to that from OA patients (Fig. 1A). However, after stimulation with Fas agonist antibody, FLS from RA patients underwent apoptosis more readily than those from OA patients. The proportion of total apoptotic cells (Annexin V+) was significantly increased by Fas stimulation in FLS from RA patients (Fig. 1B). Thus, we observed that RA synovial cells were sensitive to Fas-mediated apoptotic signaling, consistent with previous studies , , .
Discussion Cellular hyperplasia is one of the main pathological findings in the inflamed synovium of RA patients , . Increased proliferation and/or impaired apoptosis might contribute to the expansion of synovial cells , . It is well known that there is contradictory evidence in the evaluation of the level of apoptosis in FLS, showing that RA FLS have an even greater susceptibility to Fas-mediated apoptosis in vitro in contrast to their apparent lack of apoptosis in vivo , . However, the mechanisms responsible for this discrepancy remain under investigation. In the present study, our findings suggest that the presence of sFas induced by TNF-α could account for the resistance to Fas-mediated apoptosis in synovial cells of RA patients, although FLS exhibited high expression of mFas in RA. Synovial cells from patients with RA exhibited a unique phenotype of resistance to apoptotic signals. However, there are several lines of in vitro evidence demonstrating that RA FLS have an increased susceptibility to apoptosis in response to Fas stimulation , , . Recent study showed that Fas-mediated signaling can exert pro-apoptotic effects in FLS from patients with hemophilic arthropathy as well as from patients with RA . In the present study, we also observed enhanced apoptosis induction in RA FLS after stimulation with Fas activating antibody (Fig. 1). Thus, there should be mechanisms for lack of apoptosis in the synovial tissue of RA to explain the cellular hyperplasia of an inflamed joint . Cellular and molecular mechanisms of decreased apoptosis in RA include high levels of anti-apoptotic molecules such as bcl-2 (B cell lymphoma gene 2) and FLIP (also known as CFLAR, CASP8 and FADD-like apoptosis regulator) , . In addition, certain factors present in the inflammatory milieu of the synovium appear to play a role in inhibition of apoptosis. TNF-α sensitizes FLS to Fas-mediated apoptosis as well as proliferation , . Besides the direct effects of TNF-α on FLS, TNF-α may exert an indirect influence on cellular proliferation. In this context, interestingly, we found that levels of sFas were strikingly elevated in the synovial fluid of RA patients relative to their peripheral blood. Further, we found in our current analyses that sFas was markedly induced by TNF-α, which was abundantly present in the synovial cavity of RA patients (Fig. 3C, D). A recent paper, however, showed that therapy with TNF-α blockers induced elevation of serum sFas levels with significant improvement in disease activity, suggesting that circulating sFas levels may negatively correlate with disease activity in RA upon anti-TNF-α treatment . On the other hand, previous several studies have shown that increases in sFas levels were positively correlated with disease activity of RA, including an accumulation of sFas in the joints of active RA patients , . Thus we need to consider various factors affecting the effects of sFas as well as TNF-α on FLS apoptosis in RA, including different sample sources and types of therapy used for RA. Indeed, our and previous reports indicated that an alteration of sFas levels was more prominent in the synovial fluid than in the peripheral blood of patients with RA (Fig. 2B) .