Pundt Noreen, Peters Marvin A, Wunrau Christina, Strietholt Simon, Fehrmann Carsten, Neugebauer Katja, Seyfert Christine, van Valen Frans, Pap Thomas, Meinecke Ingmar
Institute of Experimental Musculoskeletal Medicine, University Hospital Muenster, Domagkstr 3, Muenster 48149, Germany.
Arthritis Res Ther. 2009;11(1):R16. doi: 10.1186/ar2607. Epub 2009 Feb 5.
The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.
Synovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 microg/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISAPlus) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 microg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis.
Freshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G2/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G0/G1 phase.
Our data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.
类风湿性关节炎（RA）滑膜的特征是存在一群具有侵袭性的活化滑膜成纤维细胞（RASF），这些细胞在关节软骨和骨破坏中起主要作用。越来越多的证据表明，RASF对Fas配体（FasL）诱导的凋亡相对抵抗，但有关肿瘤坏死因子相关凋亡诱导配体（TRAIL）的数据一直存在矛盾。在此，我们假设RASF对受体介导的凋亡的敏感性取决于这些细胞的增殖状态，因此在体外分析了FasL和TRAIL诱导的RASF程序性细胞死亡的细胞周期依赖性。
通过酶消化从RA患者中分离滑膜成纤维细胞，并在标准条件下培养。使用流式细胞术和碘化丙啶染色进行细胞周期分析。用0.5 mM羟基脲或2.5 μg/ml诺考达唑以及无胎牛血清的胰岛素-转铁蛋白-亚硒酸钠补充培养基使RASF在细胞周期的各个阶段同步化或停滞。用100 ng/ml FasL或100 ng/ml TRAIL刺激18小时诱导凋亡。使用Apo-ONE均相半胱天冬酶-3/7检测法（德国曼海姆普洛麦格有限公司）和细胞死亡检测（酶联免疫吸附测定法）（德国曼海姆罗氏诊断有限公司）测量凋亡反应。用1 μg/ml星形孢菌素处理的细胞作为阳性对照。通过荧光激活细胞分选分析确定Fas和TRAIL受体（TRAILR1-4）的表达。
新鲜分离的RASF在体外仅显示低增殖，并且随着时间的推移增殖率进一步降低，特别是当RASF汇合时。RASF表达Fas、TRAIL受体-1和TRAIL受体-2，并且表达水平与细胞周期无关。然而，增殖率显著影响对FasL和TRAIL诱导的凋亡的敏感性。具体而言，增殖的RASF对FasL和TRAIL诱导的凋亡的敏感性低于增殖率降低的RASF。此外，与G0/G1期同步化的RASF相比，S期或G2/M期同步化的RASF对TRAIL诱导的凋亡更不敏感。
我们的数据表明，RASF对FasL和TRAIL诱导的凋亡的敏感性取决于细胞周期。这些结果可能解释了关于RASF经历FasL和TRAIL介导的细胞死亡能力的一些相互矛盾的数据，并表明使RASF对凋亡敏感的策略可能包括靶向细胞周期调节基因。
