Caramés B, López-Armada M J, Cillero-Pastor B, Lires-Dean M, Vaamonde C, Galdo F, Blanco F J
Osteoarticular and Aging Research Laboratory, Biomedical Research Center, Spain.
Osteoarthritis Cartilage. 2008 Jun;16(6):715-22. doi: 10.1016/j.joca.2007.10.006. Epub 2007 Nov 28.
The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1beta on cell death in normal human chondrocytes.
Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 microM), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay.
At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha+Ro was higher than the level induced by Ro alone. The combination of IL-1beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1beta+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha+Ro. As quantified by flow cytometry, TNF-alpha+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1beta+Ro. Pre-incubation for 2h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha+Ro at 24 h. Indomethacin increased the cell death induced by IL-1beta+Ro; however, apoptosis induced by TNF-alpha+Ro was not modified by indomethacin.
These results confirm that TNF-alpha and IL-1beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction.
软骨细胞凋亡导致的死亡是退行性关节疾病（如骨关节炎，OA）的特征。肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）已被证明在OA的发展中起重要作用。在本研究中，我们分析了TNF-α和IL-1β对正常人软骨细胞死亡的影响。
从30名成年尸体供体尸检获得的膝关节软骨中分离出正常人软骨细胞。在存在或不存在Ro 31-8220（Ro：双吲哚马来酰亚胺的结构相关类似物，可抑制丝裂原活化蛋白激酶磷酸酶1 [MKP-1]）（Ro；10 microM）的情况下，用TNF-α（10 ng/ml）或IL-1β（5 ng/ml）刺激细胞，Ro是一种MKP-1抑制剂，可诱导软骨细胞凋亡。通过流式细胞术（碘化丙啶）评估细胞凋亡，并用4',6'-二脒基-2-苯基吲哚二盐酸盐评估核形态。通过蛋白质印迹分析caspase-8、-7和-3以及Bcl-2的表达，并通过流式细胞术测量caspase-3和-8的活化。通过酶联免疫吸附测定评估前列腺素E2（PGE2）。
在24小时时，TNF-α+Ro诱导的凋亡（亚二倍体）核百分比高于单独Ro诱导的水平。IL-1β（5 ng/ml）与Ro的组合未显示协同作用。形态学分析表明，TNF-α+Ro处理导致大量细胞核浓缩和DNA片段化的细胞。蛋白质印迹研究表明，IL-1β+Ro没有像TNF-α+Ro那样诱导caspase-8、-7和-3的时间依赖性活化。通过流式细胞术定量分析，TNF-α+Ro诱导的caspase-3和-8活化水平高于IL-1β+Ro。用caspase-3、-7、-8和泛caspase抑制剂预孵育2小时可显著降低TNF-α+Ro处理在24小时时诱导的亚二倍体DNA峰。吲哚美辛增加了IL-1β+Ro诱导的细胞死亡；然而，吲哚美辛并未改变TNF-α+Ro诱导的细胞凋亡。
这些结果证实，在该人软骨细胞模型中，TNF-α和IL-1β对细胞凋亡的调节方式不同，并且这些细胞因子的不同作用与PGE2无关。吲哚美辛增强了IL-1对细胞死亡的作用，这可能解释了吲哚美辛对关节破坏进展的报道作用。
