【作者】 罗心静；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 类风湿关节炎(rheumatoid arthritis,RA)是一种以累及周围关节为主要表现的系统性炎症性自身免疫病。其病理特征表现为关节滑膜面的炎症及滑膜组织的增生导致关节腔的损伤和关节的破坏。尽管RA的发病机制迄今仍未完全阐明,但细胞因子网络在RA的滑膜炎症及骨质破坏中起到重要作用,是使RA病变持续存在、迁延进展的关键因素。热休克蛋白(heat shock protein,HSP)是一高度保守的应激蛋白家族,广泛存在于原核细胞和真核细胞中。HSP70是热休克蛋白家族中最重要的成员,其基本功能是参与新生蛋白质的折叠、解聚、移位等,故有“分子伴侣”(molecular chaperone)之称。长期以来,热休克蛋白被认为是细胞内的蛋白质,只能在细胞内发挥功能。近年研究发现,HSP70能被释放到细胞外环境中,称为细胞外HSP70(extracellular HSP70);而且细胞外HSP70可能作为免疫系统的“危险信号”,调节免疫细胞的功能,参与免疫性疾病的病理过程。近年来研究发现,RA患者关节液中HSP70水平明显升高。然而,RA关节液和血清中HSP70与RA活动性之间相关性如何?关节液中HSP70的来源和释放机制是什么?细胞外HSP70对RA炎症是否有影响?目前尚无相关研究。本研究首先观察了RA患者血清和关节液中HSP70水平的改变及其临床意义;然后探讨热应激和TNF-α对细胞外HSP70释放的影响及其机制;并从整体动物水平和细胞水平探讨细胞外HSP70对RA的作用及其机制。主要的实验结果如下:1、RA患者血清和关节液中HSP70的改变及临床意义。(1)类风湿关节炎(RA)患者血清中HSP70含量变化:RA患者血清中HSP70含量(2.84±1.16 ng/ml)明显高于骨关节炎(OA)(0.74±0.34 ng/ml)和正常对照组(0.84±0.42 ng/ml),并且活动期RA血清中HSP70含量(3.31±1.09 ng/ml)明显高于非活动期RA(1.89±0.06 ng/ml);RA患者血清中HSP70水平与肿瘤坏死因子(TNF)-α、白介素(IL)-6水平呈正相关;RA患者血清HSP70的水平与血沉(ESR)、C-反应蛋白(CRP)、类风湿因子(RF)呈正相关。(2)类风湿关节炎(RA)患者关节液中HSP70含量变化:RA患者关节液中HSP70含量(88.97±21.49 ng/ml)明显高于OA组(33.13±17.27 ng/ml),并且活动期RA(102.55±19.61 ng/ml)明显高于非活动期RA(80.36±10.88 ng/ml);RA患者血清HSP70水平与TNF-α、IL-6、IL-10水平呈正相关。上述结果提示细胞外HSP70产生可能与RA炎症刺激有关,也提示细胞外HSP70可能参与RA的炎症过程,细胞外HSP70水平可以考虑作为RA患者的病情活动指标。2、应激对成纤维样滑膜细胞HSP70释放的影响。(1)热应激对HSP70释放的影响:成纤维样滑膜细胞(fibroblast-like synoviocytes,FLSs)遭受不同温度的热休克(heat shock,HS)时,细胞内HSP70表达和细胞外HSP70释放都明显增加;重度HS引起HSP70释放增多同时伴有乳酸脱氢酶(LDH)释放增多;轻度HS也能诱导HSP70释放,却没有明显LDH释放。(2)炎症应激对HSP70释放的影响:TNF-α能剂量和时间依赖性诱导成纤维样滑膜细胞HSP70的表达和释放增多,但是LDH释放并没有明显增多。这提示多种应激可导致HSP70的主动释放。(3)脂筏在HSP70主动释放中的作用:脂筏结构的破坏剂β甲基环状糊精(methyl-β-cyclodextrin,MβD)处理成纤维样滑膜细胞后能上调热休克和TNF-α诱导的细胞内HSP70表达水平,同时MβD能剂量依赖性抑制热休克或TNF-α所致成纤维样滑膜细胞HSP70的主动释放。以上研究表明,各种应激刺激,包括TNF-α能促进成纤维样滑膜细胞HSP70的表达和主动释放,而脂筏与成纤维样滑膜细胞HSP70的主动释放有关。3、HSP70对成纤维样滑膜细胞炎症细胞因子分泌的影响及机制。(1)细胞外HSP70对成纤维样滑膜细胞炎症细胞因子分泌的影响:本研究用重组的人HSP70处理成纤维样滑膜细胞,结果发现HSP70单独处理不能影响促炎因子IL-6、IL-8和巨噬细胞趋化蛋白(MCP)-1的分泌,但能剂量依赖性抑制TNF-α所致成纤维样滑膜细胞中IL-6、IL-8和MCP-1的分泌;HSP70能剂量和时间依赖性直接诱导抗炎细胞因子IL-10的生成;煮沸(100℃)能消除HSP70对细胞因子分泌的影响,而多粘菌素B(polymyxin B,PMB)不能消除HSP70对细胞因子分泌的影响。以上结果表明细胞外HSP70通过抑制成纤维样滑膜细胞促炎因子分泌和促进抗炎因子分泌而发挥抗炎作用,而且细胞外HSP70对细胞因子分泌的影响与内毒素污染无关。(2)HSP70作用于成纤维样滑膜细胞的受体类型:本研究首先通过RT-PCR和Western blotting实验证实了Toll样受体(toll-likereceptor,TLR)2和TLR4在成纤维样滑膜细胞中的表达;采用专一针对抗TLR2或TLR4的单克隆抗体封闭成纤维样滑膜细胞膜上TLR2或TLR4,发现抗TLR4抗体预处理废除了HSP70对促炎细胞因子IL-6、IL-8、MCP-1和抗炎细胞因子IL-10分泌的影响,而抗TLR2抗体预处理却不能废除HSP70对上述细胞因子分泌的影响。以上结果表明细胞外HSP70对成纤维样滑膜细胞炎症因子分泌的影响是TLR4依赖性的,即成纤维样滑膜细胞表面TLR4可能是细胞外HSP70的受体。(3)HSP70对炎症信号通路的影响:重组的人HSP70能够显著抑制TNF-α所致JNK、ERK、p38 MAPK的活化;HSP70能够抑制TNF-α所致IκB的降解和NF-κB p65的核移位;HSP70也能够直接诱导STAT3的活化和SOCS3的表达。上述研究表明,细胞外HSP70通过抑制MAPK、NF-κB通路的活化及激活JAK-STAT3-SOCS3通路来调节炎症细胞因子分泌。4、HSP70对胶原性关节炎(collagen induced arthritis,CIA)动物模型的影响。本研究首先应用基因工程技术制备重组的人HSP70,然后在小鼠初次免疫胶原后的第28天出现关节炎时给予重组HSP70腹腔注射。结果显示,HSP70能够减轻CIA小鼠关节肿胀和降低关节炎评分;组织病理学检查显示HSP70能够减少滑膜组织炎症细胞渗出和骨质破坏。此外,HSP70明显降低CIA小鼠血清中TNF-α和IL-6水平。上述研究表明细胞外HSP70能够缓解小鼠胶原性关节炎的炎症,对RA具有治疗作用。综上所述,RA患者血清和关节液中HSP70水平明显升高,并且与RA疾病活动性和炎症细胞因子之间存在相关性,提示细胞外HSP70产生可能与RA炎症刺激有关,细胞外HSP70水平可作为RA疾病活动性的参考指标;热休克或TNF-α诱导了成纤维样滑膜细胞HSP70的主动释放,而且HSP70的主动释放与成纤维样滑膜细胞膜上的脂筏结构有关,提示RA患者关节液中高水平的HSP70可能来源于成纤维样滑膜细胞受应激刺激后的主动分泌;重组人HSP70通过抑制成纤维样滑膜细胞促炎因子IL-6、IL-8和MCP-1的分泌和促进抗炎细胞因子IL-10分泌来发挥抗炎作用;HSP70对细胞因子分泌的影响具有TLR4依赖性,提示成纤维样滑膜细胞表面的TLR4可能是HSP70作用的受体;HSP70通过抑制MAPK、NF-κB通路和激活JAK-STAT3-SOCS3通路来调节成纤维样滑膜细胞炎症因子的分泌;重组HSP70减轻CIA小鼠关节肿胀,抑制滑膜组织炎症细胞渗出和骨质破坏,降低血清促炎因子的水平,从而能有效缓解CIA小鼠病情恶化,对RA具有治疗作用。更多还原

【Abstract】 Rheumatoid arthritis (RA) is an autoimmune disease, characterized by chronic inflammation of synovial membranes and proliferation of the synovial lining, leading to cartilage damage and ultimately joint destruction. Although the pathogenesis of RA remains unclear, a variety of cytokines have been implicated in the development of the disease. Heat shock proteins (HSPs) are a superfamily of highly conserved proteins found in all eukaryotes and prokaryotes. Among them HSP70 is the most important subfamily which contains multiple members such as constitutive HSP70 (HSC70) and stress-inducible HSP70 (HSP70). Although HSP70 are traditionally regarded as intracellular proteins and their primary functions appear to be as molecular chaperones, involved in protein folding and transport, accumulating data suggest that HSP70 are also actively released and have important extracellular functions. It has been shown that HSP70 are released from a variety of cells in response to cellular stress. HSP70 may serve as a "danger signal" to the innate immune system and may also be relevant to the pathogenesis of autoimmune diseases. Although elevated HSP70 have been found in synovial fluid from RA patients, their sources and functions remain unclear.In present studies, the investigators observed the clinical significance of HSP70 alteration in serum or synovial fluid of RA patients, explored the effects of stress on HSP70 release and its mechanism, and further studied the effects of extracellular HSP70 on inflammatory response and its mechanisms from TNF-αstimulated fibroblast-like synoviocytes (FLSs) as well as a mouse model with collagen induced arthritis (CIA). The main results are as follows:1.The clinical significance of HSP70 alteration in serum or synovial fluid of RA patients. 1) Changes of HSP70 in serum of RA patients. The serum levels of HSP70 were significantly higher in RA patients than those in osteoarthritis (OA) or nomal control groups, and the serum levels of HSP70 were higher in active RA than those in inactive RA. The levels of HSP70 were correlated with the levels of TNF-αand IL-6 in serum of RA. Moreover, the levels of HSP70 in serum of RA were correlated with erythrocyte sedimentation rate (ESR), C-response protein (CRP) and rheumatoid factor (RF). 2) Changes of HSP70 in synovial fluid (SF) of RA patients. The SF levels of HSP70 were significantly higher in RA group than those in OA group, while the SF levels of HSP70 were higher in active RA than those in inactive RA. The levels of HSP70 were correlated with TNF-α, IL-6 and IL-10 in SF of RA. It suggests that extracellular HSP70 is correlated with the inflammatory response in RA, and the examination of HSP70 levels may be helpful in the evaluation of prognosis of RA.2. The effects of stress on HSP70 release in FLSs. Firtly, the effects of heat stress on HSP70 release were observed. Exposure of FLSs from RA patients to nonlethal HS resulted in a marked up-regulation of intracellular HSP70 and HSP70 release without significant cell death as indicated by LDH release. Then, the effects of inflammatory stress on HSP70 were studied. Treatment of FLSs with TNF-αdose- and time-dependently increased the expression of intracellular HSP70 and the release of HSP70 into the medium, without significant increase in cell death as indicated by LDH release. In addition, the investigators explored the role of lipid raft in HSP70 release. Treatment of FLSs with methyl-β-cyclodextrin (MβD), a lipid raft-disrupting reagent, inhibited nonlethal HS or TNF-αinduced HSP70 release from FLSs. It suggests that FLSs can actively release HSP70 when exposed to different kinds of stressors, and lipid raft may be involved in HSP70 release in FLSs.3. The effects of extracellular HSP70 on inflammatory cytokine secretion in FLSs and its mechanisms. 1) The effects of extracellular HSP70 on the secretion of inflammatory cytokine in FLSs. Recombinant human HSP70 inhibited the secretion of IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 induced by TNF-αstimulation in RA FLSs, although HSP70 alone had no effect on FLSs. HSP70 directly induced IL-10 secretion in dose-and time-dependent manners. Moreover, the effects of human HSP70 on cytokine secretion were completely abolished by the treatment of HSP70 with boiling but not with polymyxin B (PMB). 2) The analysis of receptor for extracellular HSP70 on cell surface of FLSs. By RT-PCR and Western blotting, it was shown that human RA FLSs could express TLR2 and TLR4. When the FLSs were preincubated with TLR2 or TLR4 antibody, and the effects of HSP70 on the secretion of IL-6, IL-8, MCP-1 and IL-10 were blocked by TLR4 antibody but not by TLR2 antibody. It suggests that the effects of human HSP70 on cytokine secretion in FLSs were mediated through TLR4, and TLR4 on cell surface of FLSs may be as the possible receptor of HSP70. 3) The effects of HSP70 on inflammatory signal pathways. Recombinant human HSP70 inhibited activation of MAPK signal pathways (p38, JNK, and ERK ) induced by TNF-α. HSP70 also inhibited the degradation of IκBαand nuclear translocation of NF-κB p65 induced by TNF-α. However, HSP70 directly induced the activation of STAT3 and expression of SOCS3. It suggests that the anti-inflammatory effect of extracellular HSP70 on human RA FLSs may be explained by its ability to suppress TNF-α-induced activation of MAPK and NF-κB, two vital pro-inflammatory signal pathways in RA, and promote activation of JAK-STAT3-SOCS3, a known anti-inflammatory signal pathway.4. The effect of HSP70 on collage induced arthritis (CIA). Recombinant human HSP70 significantly suppressed paw swelling of mice with CIA. Histological analysis confirmed the suppression of joint inflammation and bone destruction in mice with CIA after treatment with HSP70. Serum TNF-αand IL-6 levels were significantly reduced after treatment of HSP70. These results suggest that HSP70 is effective in suppressing inflammatory response and bone damage in CIA, and may have potential value in the treatment of rheumatoid arthritis.Taken together, it is suggested that 1) human HSP70 is up-regulated and actively released by RA FLSs exposed to stress; 2) Extracellular HSP70 inhibits the release of IL-6, IL-8 and MCP-1 induced by TNF-αand promotes the release of IL-10 in FLSs; 3) the anti-inflammatory role of extracellular HSP70 in human RA FLSs can be blocked by TLR4 antibody and recombinant human HSP70 inhibits the activation of MAPK and NF-κB signal pathways induced by TNF-α, but activates Jak-STAT3-SOCS3 pathway; 4) recombinant HSP70 suppresses systemic and joint inflammation and cartilage and bone destruction in mice with CIA, indicates a possible therapeutic effect of HSP70 on RA.更多还原