Shimizu Makoto, Yasuda Tadashi, Nakagawa Takefumi, Yamashita Eizaburo, Julovi Sohel M, Hiramitsu Teruko, Nakamura Takashi
Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, Japan.
J Rheumatol. 2003 Jun;30(6):1164-72.
To study the inhibitory effects of hyaluronan (HA) on the production of matrix metalloproteinase-1 (MMP-1) by rheumatoid synovial fibroblasts (RSF) stimulated by proinflammatory cytokines, tumor necrosis factor-a (TNF-a), and interleukin-1beta (IL-1beta).
HA of various sizes at various concentrations was added to monolayer cultures of RSF in the presence of TNF-a or IL-1beta, with or without pretreatment with a monoclonal antibody against CD44, OS/37. Concentrations of MMP-1 in cell lysates and conditioned media and of CD44 on RSF were assayed by immunoblotting. MMP-1 expression was analyzed by reverse transcriptase-polymerase chain reaction. Binding of HA to RSF was evaluated by confocal microscopy using fluorescein-conjugated HA and OS/37.
Treatment with HA (0.3 approximately 3.0 mg/ml) resulted in a significant decrease in the production of MMP-1 induced by TNF-a and IL-1beta, in a dose-dependent manner. HA of 250 approximately 2300 kDa at 3 mg/ml was found to suppress the induction of MMP-1 by TNF-a. HA decreased the cytokine-induced MMP-1 synthesis in RSF at mRNA and protein levels. The monoclonal antibody, which showed abundant expression of CD44 on RSF by immunofluorescein cytochemistry, partially blocked the binding of fluorescein-conjugated HA to RSF. Pretreatment with OS/37 reversed the inhibition of MMP-1 production in TNF-a or IL-1beta-stimulated RSF caused by HA.
HA suppresses the production of MMP-1 by TNF-a or IL-1beta-stimulated RSF. Based on data from anti-CD44 treatment, HA binding to CD44 is directly involved in the suppression of MMP-1 production. Those results provide the rationale for a therapeutic role of HA in treatment of rheumatoid joints.
研究透明质酸（HA）对促炎细胞因子肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）刺激的类风湿性滑膜成纤维细胞（RSF）产生基质金属蛋白酶-1（MMP-1）的抑制作用。
在存在TNF-α或IL-1β的情况下，将不同浓度的各种大小的HA添加到RSF的单层培养物中，有无抗CD44单克隆抗体OS/37预处理。通过免疫印迹法测定细胞裂解物和条件培养基中MMP-1的浓度以及RSF上CD44的浓度。通过逆转录聚合酶链反应分析MMP-1的表达。使用荧光素偶联的HA和OS/37通过共聚焦显微镜评估HA与RSF的结合。
用HA（0.3至3.0mg/ml）处理导致TNF-α和IL-1β诱导的MMP-1产生以剂量依赖性方式显著降低。发现3mg/ml的250至2300kDa的HA可抑制TNF-α诱导的MMP-1。HA在mRNA和蛋白质水平上降低了RSF中细胞因子诱导的MMP-1合成。通过免疫荧光细胞化学在RSF上显示CD44大量表达的单克隆抗体部分阻断了荧光素偶联的HA与RSF的结合。用OS/37预处理可逆转HA对TNF-α或IL-1β刺激的RSF中MMP-1产生的抑制作用。
HA抑制TNF-α或IL-1β刺激的RSF产生MMP-1。基于抗CD44治疗的数据，HA与CD44的结合直接参与MMP-1产生的抑制。这些结果为HA在类风湿关节治疗中的治疗作用提供了理论依据。
