免疫复合物对人血管内皮细胞和单核细胞的活化作用及其机制研究
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摘要
第一部分
免疫复合物对人血管内皮细胞的活化作用及其机制研究
背景：
系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种以免疫复合物(immune complexes, ICs)形成为特征的慢性自身免疫性疾病,其中有文献报道免疫复合物包括核酸,核酸相关蛋白和相应的自身抗体三种成分。在系统性红斑狼疮患者外周血中,自然凋亡的细胞不能被单核巨噬细胞清除,结果导致了这些凋亡细胞的二次凋亡坏死,自身抗原成分核酸和核酸相关蛋白就从这些二次凋亡细胞的细胞核中释放出来,然后便产生了针对这些核自身抗原成分的相应的自身抗体,例如抗双链DNA抗体,导致了免疫复合物的形成。免疫复合物沉积在患者的靶器官如皮肤和肾脏中,导致了这些器官的炎症和损伤。
血管病变和血管炎,是系统性红斑狼疮的典型的并发症,有报道称其发生在10%至40%的系统性红斑狼疮患者,多见于患者的皮肤血管,肾小球,冠状动脉和脑血管。有文献报道系统性红斑狼疮血管病变和血管炎与免疫复合物沉积,内皮细胞活化以及炎症细胞的浸润有关。然而,免疫复合物对血管内皮细胞的作用及其机制尚不明确。
高迁移率族蛋白1(high-mobility group box1protein, HMGB1),也叫做两性霉素,属于核酸相关蛋白,是免疫复合物中的一种重要成分。它几乎在所有的真核细胞中表达,主要位于细胞核中,在细胞核中,它与核酸成分结合稳定核小体的结构,并且诱导DNA弯曲来调节转录。在系统性红斑狼疮患者中,HMGB1-核酸复合物从二次凋亡的细胞核中释放出来,与相应的自身抗体形成免疫复合物,在血浆中被检测到显著升高。在靶器官例如皮肤和肾脏中,包含HMGB1的免疫复合物大量沉积,导致了靶器官的慢性炎症和组织损伤。HMGB1的促炎症活性主要由其受体晚期糖基化终末产物受体(the receptor for advanced glycation end products, RAGE)来介导。RAGE是一种跨膜蛋白,是免疫球蛋白超家族的成员,在HMGB1介导的信号通路中,它是一个主要的信号转导受体。RAGE主要在血管内皮细胞,单核和巨噬细胞的细胞膜上表达,能够经过经典的信号激活途径来激活转录因子NF-κB家族成员p65,而且可以通过激活p65来调节自身的表达。
了解了HMGB1是免疫复合物的重要成分,并且HMGB1的促炎症活性主要由其受体RAGE来承载。那么,HMGB1-RAGE细胞信号途径有可能在免疫复合物对内皮细胞的作用中发挥重要的作用。然而目前为止,这一作用尚没有文献报道。因而,评价HMGB1-RAGE细胞信号通路在免疫复合物对内皮细胞的作用中发挥的作用来探讨HMGB1-RAGE细胞信号通路是不是免疫复合物对内皮细胞的作用中的一种可能的机制将会很有意义和价值。
HMGB1的作用能被HMGB1A-box特异性的阻断,RAGE能被可溶性RAGE (soluble RAGE, sRAGE)阻断。HMGB1A-box, sRAGE, Bay117082以及这些阻断剂的组合分别被用来阻断HMGB1-RAGE细胞信号途径上的免疫复合物中HMGB成分,内皮细胞表面的RAGE受体,内皮细胞的转录因子NF-κB p65以及它们的组合来检测免疫复合物对内皮细胞作用的发生是不是通过HMGB1-RAGE细胞信号途径。有报道称p38细胞分裂素活化蛋白激酶(mitogen-activated protein kinases, MAPK)和细胞外信号相关激酶1/2(extracellular signal-related kinases1and2, ERK1/2)信号途径位于RAGE介导的转录因子NF-κB活化的通路上,在本研究中,p38MAPK和ERK1/2的特异性阻断剂SB203580和PD98059分别用来阻断p38MAPK和ERK1/2,以评价这两个细胞信号途径在免疫复合物诱导的血管内皮细胞细胞因子分泌反应中所发挥的作用。
目的：
探索免疫复合物对人血管内皮细胞的作用,评价HMGB1-RAGE细胞信号通路在免疫复合物对人血管内皮细胞活化中所发挥的作用,来探讨人系统性红斑狼疮血管炎中免疫复合物对血管内皮细胞的活化作用及其其中一种可能的机制。
方法：
1.在免疫复合物刺激和阻断剂阻断实验中,把人脐静脉内皮细胞株CRL-1730细胞接种到直径为6cm的细胞培养皿中,在细胞达到80%单层细胞融合的时候,对细胞分组并且加药干预。先分别加入阻断剂HMGB1A-box (10μg/ml), sRAGE (20μg/ml), SB203580(10μM), PD98059(25μM), Bay117082(1μM或者它们的组合干预1小时,然后加入免疫复合物。孵育2小时后,NF-κB家族成员p65在细胞核中的水平的变化分别用免疫荧光,免疫组化和Western Blot的方法来检测。孵育6小时后,基因RAGE, ICAM-1, VCAM-1, IL-8, MCP-1, IL-6, TNF-a和IL-lβmRNA水平的变化用荧光实时定量PCR的方法来检测。孵育16小时后,细胞表面蛋白RAGE, ICAM-1, VCAM-1表达水平的变化用免疫荧光,免疫组化,流式以及细胞ELISA的方法来检测分析。孵育24小时后,细胞上清液中细胞因子IL-8, MCP-1, IL-6, TNF-α和IL-1β浓度的变化用ELISA的方法来检测。
2.在单核细胞跨血管内皮细胞迁移的实验中,先把人脐静脉内皮细胞株CRL-1730细胞接种在明胶包被的具有通透性的Transwell小室的滤膜上,形成(?)单血管内皮细胞层后,对该层血管内皮细胞分组并且用方法1中描述的方法用阻断剂和免疫复合物进行干预24小时。然后将单核细胞加入到上室中进行跨血管内皮细胞迁移,20小时后,将迁移至下室的单核细胞收集起来用细胞计数仪计数,并且比较不同干预组之间单核细胞跨内皮细胞迁移的差异。
3.在细胞活性测定实验中,把人脐静脉内皮细胞株CRL-1730以每孔105个细胞的密度接种到24孔板中,孵育过夜至细胞贴壁融合。将细胞分组,然后分别加入HMGB1A-box (10μg/ml), sRAGE (20μg/ml), SB203580(10μM), PD98059(25μM), Bay117082(1μM或者它们的组合进行干预。24小时后,将细胞收集起来,用细胞计数仪计数不同干预组细胞总数的变化,用台盼蓝染色的方法计数活细胞数目的差异,并且用CCK-8的方法检测细胞活性的差异。
4.实验中,用SPSS17.0统计软件进行统计分析,实验数据用均数±标准误来表示。不同实验组之间的差异用单因素方差分析进行分析,两组数据之间的比较用t检验进行分析,P值<0.05被认为具有统计学意义。
结果：
1.免疫复合物通过HMGB1-RAGE细胞信号通路上调人血管内皮细胞表面RAGE受体的表达。
2.免疫复合物通过HMGB1-RAGE细胞信号通路上调人血管内皮细胞表面粘附分子ICAM-1和VCAM-1的表达。
3.免疫复合物通过HMGB1-RAGE细胞信号途径增加了人血管内皮细胞趋化因子IL-8,MCP-1和促炎症细胞因子IL-6,TNF-α的分泌。
4. p38MAPK和ERK1/2信号途径在免疫复合物通过HMGB1-RAGE细胞信号途径诱导人血管内皮细胞活化中发挥重要作用。
5.免疫复合物通过HMGB1-RAGE细胞信号途径导致了人血管内皮细胞中转录因子NF-κB p65的活化。
6.免疫复合物通过HMGB1-RAGE细胞信号通路增强了单核细胞的跨血管内皮细胞迁移。
7. HMGB1-RAGE细胞信号途径上的阻断剂HMGB1-A-box, sRAGE, Bay117082, SB203580, PD98059以及它们的组合本身对人血管内皮细胞没有细胞毒性作用。
结论：
本研究证明免疫复合物通过HMGB1-RAGE细胞信号通路引发了人血管内皮细胞的促炎症反应,而且导致了血管内皮细胞功能的变化。这为免疫复合物对人血管内皮细胞的活化作用及其其中一种潜在的机制提供了证明,在理解系统性红斑狼疮血管炎的发病机制中发挥重要的作用,并且为将来系统性红斑狼疮血管炎的治疗提供了有力的证据。
第二部分
免疫复合物对人单核细胞的活化作用及其机制和治疗方法研究背景：
系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种以免疫复合物(immune complexes, ICs)形成为特征的慢性自身免疫性疾病,其中有文献报道免疫复合物包括核酸,核酸相关蛋白和相应的自身抗体三种成分。在系统性红斑狼疮患者中,自然凋亡的细胞不能被单核巨噬细胞清除,结果导致了这些凋亡细胞的二次凋亡坏死,自身抗原成分核酸和核酸相关蛋白就从这些二次凋亡细胞的细胞核中释放出来。然后,针对这些核自身抗原成分的相应的自身抗体,例如抗双链DNA抗体便产生了,导致了免疫复合物的形成。免疫复合物沉积在人体的靶器官如皮肤和肾脏中,导致了这些器官的炎症和损伤。
有文献报道系统性红斑狼疮患者的单核细胞异常活化,并且通过产生一系列的细胞因子和功能异常来启动和维持自身免疫反应。其中,IL-6, TNF-α和MCP-1是由单核细胞产生的,在系统性红斑狼疮患者的单核细胞中异常地表达升高,并且在系统性红斑狼疮的发病过程中发挥了重要作用的细胞因子。单核细胞是人外周血中IL-6的主要来源,IL-6在系统性红斑狼疮中促进B细胞分化成为产生自身抗体的浆细胞,并且促进自身抗体的产生。TNF-α是一个重要的促炎症因子,它的过表达导致了炎症的持续。MCP-1是一种作用非常强的单核细胞趋化蛋白,它促使单核细胞聚集在炎症发生的部位。最近对狼疮肾炎的研究表明,异常活化的单核巨噬细胞在狼疮肾炎中大量浸润,促进了狼疮肾炎的炎症和损伤,并且影响狼疮肾炎的最终结果。
包含核酸,核酸相关蛋白和相应自身抗体三种成分的免疫复合物是具有致病性的。有文献报道,从系统性红斑狼疮患者提取的免疫复合物中的核酸成分与从凋亡细胞中释放的染色质片段的大小差不多,这些片段含有丰富的寡聚核苷酸A类CpG,可以特异性的被表达于单核细胞,类浆细胞样树突状细胞和B细胞上的模式识别受体TLR9识别。TLR9的活化可以被其拮抗剂抑制性的寡聚脱氧核苷酸ODN2088特异性的阻断。HMGB1,高迁移率族蛋白1,也叫做两性霉素,属于免疫复合物中的核酸相关蛋白成分,是免疫复合物中另外一个重要的成分。它表达于几乎所有的有核细胞,并且主要位于细胞的细胞核中。在细胞核中,它与核苷酸结合来稳定核小体的结构,并且诱导DNA弯曲来调节转录。在系统性红斑狼疮患者中,HMGB1从二次凋亡的细胞核中释放出来,并且在血浆中被检测到显著升高。一旦释放到细胞外的环境中,HMGB1就作为一种炎症刺激因子,刺激炎症细胞进一步产生更多的细胞因子。HMGB1的促炎症作用主要通过与其细胞表面受体RAGE来发生。RAGE是第一个确定的HMGB1的受体,主要表达在单核细胞,血管平滑肌细胞,内皮细胞等细胞的表面,可被可溶性RAGE阻断。有文献报道免疫复合物能活化类浆细胞样树突状细胞和自身反应性B细胞来诱导IFN-α和其它促炎症因子的产生,促进疾病的发展,通过免疫复合物中的核酸和HMGB1成分活化细胞受体TLR9和RAGE。然而,免疫复合物对单核细胞的作用及其潜在的机制尚不明确。
细胞表面受体RAGE和细胞浆受体TLR9都能够通过经典的活化途径活化NF-κB家族成员p65,p65能调节IL-6, TNF-α和MCP-1的表达。另外,RAGE受体被激活后,通过信号通路活化NF-κB家族成员p65,p65还能调节RAGE自身的表达。在本研究中,免疫复合物被用来检测其对单核细胞的作用,可溶性RAGE, ODN2088和Bay117082被用来分别阻断RAGE受体,TLR9和NF-κB p65来检测免疫复合物对单核细胞作用的可能的机制。
虽然近来在抗炎症治疗方面有所进展,但是当前对系统性红斑狼疮治疗的选择还存在很多分歧和不确定。前面已有研究报道过氧化物酶体增值物受体γ(PPAR-γ)激动剂在系统性红斑狼疮小鼠模型中是一种很有效的治疗方法。它能够减少自身抗体的产生,而且能够减少肾脏炎症和损伤。研究报道,PPAR-γ激动剂包括噻畔烷二酮类(thiazolidinediones, TZDs),前列腺素类和非甾体类抗炎药物(NSAIDs)三种,激活了表达于单核细胞,角质化细胞等细胞核内的核受体PPAR-γ以后,通过与转录因子NF-κB的相互作用来调节促炎症因子的表达。也有文献报道,PPAR-γ激动剂通过抑制NF-κB p65的核转移和通过形成PPAR-γ-p65复合物,来减少NF-κB p65与其靶核苷酸调节序列的结合。在本研究中,我们选择噻唑烷二酮类药物匹格列酮(pioglitazone),它是PPAR-γ受体的一种药理性激动剂,作为PPAR-γ激动剂的代表,在体外实验检测PPAR-γ激动剂对系统性红斑狼疮单核细胞异常活化的作用及其机制,在前面报道的对狼疮鼠的有效的治疗作用的基础上,进一步探测它的对系统性红斑狼疮的治疗作用。
目的：
在本研究中,我们探索免疫复合物对系统性红斑狼疮患者单核细胞异常活化所起的作用及其潜在的机制,并且为系统性红斑狼疮患者单核细胞异常活化提出一种可能的治疗方法。
方法：
1.把人单核细胞白血病细胞株U937以每孔106个细胞的密度接种到6孔板中,将细胞分组,分别与sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM或者PPAR-γ激动剂匹格列酮(10μM)孵育1小时,然后加入免疫复合物。孵育2小时后,用Western Blot的方法来检测NF-κB家族成员p65在细胞核中水平的变化,用免疫沉淀的方法检测PPAR-γ-p65复合物的形成。孵育6小时后,用荧光实时定量RT-PCR的方法来检测基因RAGE, IL-6, TNF-α和MCP-1mRNA水平的变化。孵育16小时后,用流式和Western Blot的方法来检测RAGE蛋白表达的变化。孵育24小时后,用ELISA的方法来检测细胞上清液中细胞因子IL-6, TNF-α和MCP-1的浓度的变化,用Transwell的方法来检测单核细胞跨血管内皮细胞迁移的变化。
2.在检测细胞活性的实验中,把人单核细胞白血病细胞株U937以每孔5×105个细胞的密度接种到12孔板中,将细胞分组,分别与sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM)或者PPAR-γ激动剂匹格列酮(10μM)孵育24小时。分别用细胞计数仪计数细胞总数,台盼蓝染色活细胞数以及CCK-8的方法来检测细胞活性的变化。
3.实验中,用SPSS17.0统计软件进行统计分析,实验数据用均数士标准误来表示。不同实验组之间的差异用单因素方差分析进行分析,两组数据之间的比较用t检验进行分析,P值<0.05被认为具有统计学意义。
结果：
1.免疫复合物通过TLR9介导的NF-κB的活化上调人单核细胞表面受体RAGE的表达,这一作用可被PPAR-γ激动剂抑制。
2.免疫复合物通过RAGE/TLR9介导的NF-κB的活化增加人单核细胞细胞因子IL-6, MCP-1和TNF-α的分泌,这一作用可能被PPAR-γ激动剂抑制。
3.免疫复合物通过RAGE/TLR9介导的NF-κB的活化增强单核细胞跨血管内皮细胞迁移,这一作用可被PPAR-γ激动剂抑制。
4.免疫复合物通过RAGE/TLR9介导的NF-κB的活化增加人单核细胞NF-κB家族成员p65的核转移,这一作用可被PPAR-γ激动剂抑制。
5. PPAR-γ激动剂匹格列酮通过以剂量依赖的形式形成PPAR-γ-p65复合物抑制免疫复合物诱导的人单核细胞NF-κB p65的活化。
6.实验中用到的sRAGE, ODN2088, Bay117982和匹格列酮对单核细胞没有细胞毒性。
结论：
本研究证实了免疫复合物对系统性红斑狼疮单核细胞异常活化所起的作用并且对免疫复合物如何导致单核细胞异常活化提供了一种可能的机制,这为系统性红斑狼疮发病机制的研究提供了重要证据。另外,本研究对系统性红斑狼疮单核细胞异常活化提出了一种可能的治疗方法,这种方法将很有可能成为系统性红斑狼疮治疗的新的选择。
Part Ⅰ
The activating effects and potential mechanisms of immune complexes on human endothelial cells
Backgrounds:
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by formation of immune complexes (ICs), which contain autoantigens nucleic acids, nucleic acids-associated protein and corresponding antibodies. The apoptotic cells which are impared cleared by monocytes/macrophages result in secondary necrosis, autoantigens nucleic acids and nucleic acids-associated protein are released by the nuclei of these secondary apoptotic cells in patients with SLE. The corresponding autoantibodies against these nuclear autoantigens, such as the anti-double-stranded DNA (dsDNA) antibodies are generated and lead to the formation of ICs. Deposition of ICs on target organs, such as skin and kidney, results in end organs damage.
Vasculopathy and vasculitis, which are typical complications of SLE, are reported in10%to40%of SLE patients and are usually seen in cutaneous vessels, renal glomeruli, coronary and brain vessels. Vasculopathy and vasculitis are reported to be associated with the deposition of ICs on endothelium, endothelial cells activation and inflammatory cells infiltration into the inflammatory sites. However, the effects of ICs on the endothelial cells and the potential mechanisms remain unclear.
HMGB1, also known as amphotericin, which belongs to the nucleic acids-associated protein, is an essential component of ICs. HMGB1is expressed in almost all eukaryotic cell types and is located mainly in the cell nucleus, where it binds to nucleotides to stabilize the structure of nucleosomes and to induce DNA binding to regulate transcription. In SLE patients, HMGB1-nucleic acids complexes are released from the secondary necrotic cells, form ICs with the corresponding antibodies and are found to be significantly elevated in the sera. Depositon of HMGB1-containing ICs are also found in target tissues, such as the skin and kidney, contributing to the chronic inflammation and tissue injury. The proinflammatory activity of HMGB1is mostly attributed to its ligation with RAGE. RAGE, a member of the immunoglobulin superfamily of cell surface molecules, is a central signal transduction receptor for HMGB1-modified adducts. It is expressed on cells such as endothelial cells, monocytes and macrophages and can signal to transcription factor NF-κB family member p65through the classical activation pathway. Furthermore, RAGE signaling to the NF-κB p65regulates the expression of RAGE itself.
Knowing that HMGB1is one important component of ICs and the proinflammatory activity of HMGB1is mostly attributed to its ligation with RAGE. The cell signaling HMGB1-RAGE axis may play important roles in the effects of ICs on the endothelial cells. However, the roles of cell signaling HMGB1-RAGE axis in the effects of ICs on the endothelial cells has not been evaluated so far. It would be attracted and worthy to evaluate the involvement of the cell signaling HMGB1-RAGE axis in the effects of ICs on endothelial cells to examine whether it is one potential mechanism of ICs'effects on human endothelial cells.
The effects of HMGB1can be specially blocked by HMGB1A-box, RAGE can be blocked by sRAGE. HMGB1A-box, sRAGE, Bay117082and combination of these blockers were used to block the effects of the HMGB1component in ICs, RAGE, NF-κB p65and combine effects of them in human endothelial cells, respectively to detect whether the effects of ICs on human endothelial cells occur involving in the cell signaling HMGB1-RAGE axis. p38 mitogen-activated protein kinases (MAPK) and extracellular signal-related kinases1and2(ERK1/2) pathways are reported to be involved in the signaling pathway of RAGE-mediated NF-κB activation. Specific inhibitors of p38MAPK (SB203580) and ERK1/2(PD98059) were also used to assess the contribution of p38MAPK and ERK1/2pathways to ICs-stimulated cytokine responses in human endothelial cells.
Objectives:
To explored the effects of ICs on the endothelial cells and evaluate the involvement of the cell signaling HMGB1-RAGE axis in these effects to examine one potential mechanism of the effects of ICs on human endothelial cells in the pathogenesis SLE vasculitis.
Methods:
1. For ICs stimulation and blocking assays, human CRL-1730cells were seeded into6cm-dishes at80%confluent monolayers and pretreated with HMGB1A-box (10μg/ml), sRAGE (20μg/ml) SB203580(10μM), PD98059(25μM), Bay117082(1μM) or combination of them for1hour before the addition of ICs. After incubation for2hours, immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses of NF-κB family member p65levels in the nucleus were performed. After incubation for6hours, RAGE, ICAM-1, VCAM-1, IL-8, MCP-1, IL-6, TNF-α and IL-1β mRNA levels were detected by Quantitative real-time-PCR. After incubation for16hours, cell surface RAGE, ICAM-1, VCAM-1proteins expression levels were analyzed by ICC, IF, flow cytometry (FCM) or cellular enzyme-linked immunosorbent assay (cellular ELISA) analysis. After incubation for24hours, the concentration of cytokines IL-8, MCP-1, IL-6, TNF-a and IL-1β in the cell-free supernatants were detected by ELISA.
2. For monocytes transendothelial migration experiments, human CRL-1730cells were seeded on gelatin-coated permeable Transwell filters to form a monolayer and pretreated by blockers and ICs as described above for24hours before monocytes migration. After incubation for20hours, the cells that had transmigrated to the lower chamber were harvested and counted using a TC10Automated Cell Counter. And the difference of monocytes transendothelial migration between different endothelial cells groups are analysed.
3. For cell viability assay, human CRL-1730cells were seeded onto24-well plates at105cells/well and allowed to attach and grow overnight to confluence, and then they were incubated with control medium alone, HMGB1A-box (10μg/ml), sRAGE (20μg/ml), SB203580(10μM), PD98059(25μM), Bay117082(1μM) or combination of them. After treatment for24hours, cells were harvested and overall cell counts were measured and the number of viable cells was determined by hemocytometer counts of Trypan Blue-impermeable cells. In another test, cell viability was determined using the CCK-8kit.
4. Statistical analyses were performed using SPSS statistics software version17.0. Unless otherwise indicated, data are presented as mean±SE. Differences of group comparisons were analyzed using one-way ANOVA and subsequent appropriate post-hoc analysis by unpaired Student's t-tests. A P value<0.05was considered statistically significant.
Results:
1. ICs up-regulate RAGE expression involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
2. ICs up-rcgulate the cell surface adhesion molecules ICAM-1and VCAM-1expression involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
3. ICs increase the secretion of chemokines IL-8, MCP-1and proinflammatory cytokines IL-6, TNF-a involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
4. Activation of p38MAPK and ERK1/2pathways in the cell siganling HMGB1-RAGE axis induced by ICs in human endothelial cells
5. ICs lead to activation of NF-κB p65involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
6. Monocytes transendothelial migration is enhanced by ICs pretreated human endothelial cells which occurs involving in the cell signaling HMGB1-RAGE axis of human endothelial cells
7. Blockers HMGB1-A-box, sRAGE, Bay117082, SB203580, PD98059or combination of them used in the experiments were not toxic to human endothelial cells.
Conclusions:
This study demonstrates that ICs elicit proinflammatory responses in human endothelial cells and contribute to alterations in human endothelial cells function involving in the cell signaling HMGB1-RAGE axis, it provides a demonstration for the effects and a potential mechanism of the effects of ICs on human endothelial cells, which is important in understanding the pathogenesis of lupus vasculitis and may provide important evidence for the therapy of SLE vasculitis in the future.
Part II
The activating effects and potential mechanisms of immune complexes on human monocytes and its possible therapeutic methods
Backgrounds:
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by formation of immune complexes (ICs), which contain autoantigens nucleic acids, nucleic acids-associated protein and corresponding antibodies. In SLE patients, the apoptotic cells which are impaired cleared by monocytes/macrophages result in secondary necrosis, autoantigens nucleic acids and nucleic acids-associated protein are released by the nuclei of these secondary apoptotic cells. The corresponding autoantibodies against these nuclear autoantigens, such as the anti-double-stranded DNA (dsDNA) antibodies are generated and lead to the formation of ICs. Deposition of ICs on target organs, such as the kidney, results in end organs damage.
Monocytes that are aberrantly activated in the patients of SLE initiate and maintain autoimmune responses via production of several cytokines and functional alteration. IL-6, TNF-a and MCP-1are cytokines secreted by monocytes, which are aberrantly up-regulated in the monocytes of SLE patients and play very important roles in the pathogenesis of SLE. Monocytes are the primary source of IL-6in the peripheral blood, IL-6promotes the production of autoantibodies and is required for the differentiation of B cells into antibodies secreting plasma cells. TNF-a is a pro-inflammatory cytokine and its over-expression leads to perpetuation of inflammation. MCP-1, which is known for its ability as a potent chemoattractant of monocytes, facilitates the recruitment of monocytes into the inflammatory sites. Recent studies on lupus nephritis demonstrated that activated monocytes/macrophages vigorously participate in and amplify renal inflammation and injury, which influence the outcome of lupus nephritis.
ICs which contain nucleic acids, nucleic acids-associated protein and corresponding antibodies are pathogenic. It has been reported that the nucleic acids component in ICs isolated from SLE patients are of similar size as the cleaved chromatin fragments released from apoptotic cells, these fragments are rich in oligonucleotide class A CpG (CpG-A) and can be specifically recognized by TLR9, which is a pattern recognition receptor expressed on monocytes, pDCs, B cells and so on. TLR9activation can be specifically blocked by its antagonist inhibitory oligodeoxynucleotide ODN2088. High Mobility Group Box1(HMGB1; also known as amphotericin) which belongs to the nucleic acids-associated protein in ICs, is another essential component of ICs. It is expressed in eukaryotic cells and is located mainly in the cell nucleus, where it binds to nucleotides to stabilize the structure of nucleosomes and to induce DNA binding to regulate transcription. In SLE patients, HMGB1released from the secondary necrotic cells are found to be significantly elevated in the sera. Once released into the extracellular milieu, it functions as a cytokine that activates inflammatory cells to the further production of cytokines. The pro-inflammatory cytokine activity of HMGB1is mostly attributed to its association with RAGE. RAGE is the first described receptor for HMGB1and is expressed on the surface of monocytes, vascular smooth muscle cells, neurons, endothelial cells and so on. RAGE can be blocked by soluble RAGE (sRAGE). It has been reported that ICs can stimulate pDCs and autoreactive B cells to induce IFN-a and other pro-inflammatory cytokines production and disease development through RAGE and TLR9due to the nucleic acids-associated protein HMGB1and nucleic acids component in the ICs. However, the effects of ICs on monocytes and the potential mechanisms remain unclear.
Both cell surface receptor RAGE and cytosolic receptor TLR9can signal to transcription factor NF-κB family member p65through the classical activation pathway, which can regulate the expression of IL-6, TNF-α and MCP-1. Furthermore, RAGE signal to NF-κB p65can regulate the expression of RAGE itself. In this paper, ICs were used to detect its effects on monocytes, sRAGE, ODN2088, and Bay117082were used to block RAGE, TLR9and NF-κB p65, respectively, to detect the possible mechanisms of ICs'effects on monocytes.
Despite the recent advances in anti-inflammatory therapy, current treatment options for SLE are diverse and poorly defined. It has been reported that PPAR-y agonist is a very useful therapeutic strategy in mouse models of SLE, it can reduce autoantibodies production, it can also reduce renal inflammation and injury. It is reported that the PPAR-y agonists which contains thiazolidinediones (TZDs), prostanoids and non-steroidal anti-inflammatory drugs (NSAIDs) activate the nuclear hormone receptor PPAR-y, which is expressed in monocytes, keratinocytes and so on, and regulate the expression of pro-inflammatory proteins by interacting with transcription factors NF-κB, it is also reported that PPAR-y agonists can inhibit the nuclear translocation of NF-κB p65and promote PPAR-y to form complexes with p65in the nucleus, which reduces the binding of p65to its target sequences. Here we chose pioglitazone, which belongs to TZDs and pharmacological agonist of PPAR-y as a representative of the PPAR-y agonists to examine the effects of PPAR-y agonists on monocytes aberrant activation and their mechanisms in SLE in vitro to further detect its therapeutic effects in SLE in addition to its effective therapeutic effects in mouse models of SLE reported before.
Objectives:
In this study, we try to explore the effects of ICs on monocytes aberrant activation in SLE and the potential mechanisms, and to propose a potential therapeutic strategy to the monocytes aberrant activation in SLE.
Methods:
1. Human U937cells were seeded onto6-well plates at106cells/well and pretreated with sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM) or PPAR-y agonist pioglitazone (10μM) for one hour before treating with ICs. After incubation for two hours, the level of NF-κB family member p65in the nucleus was analyzed by western blot and immunoprecipitation (IP) assays were performed to detect the formation of complexes PPAR-y-p65. After incubation for six hours, RAGE, IL-6, TNF-a and MCP-1mRNA expression levels were determined using real-time RT-PCT. After incubation for16hours, RAGE protein expression level was determined by flow cytometry and western blot analysis. After incubation for24hours, the concentrations of cytokines IL-6, TNF-a and MCP-1in the cell-free supernatants were examined and transendothelial migration assays were performed to detect the changes of monocytes transendothelial migration.
2. For the cell viability assay, human U937cells were seeded onto12-well plates at5×105cells/well and incubated with control medium alone or sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM) or PPAR-y agonist pioglitazone (10μM) for24hours. Cell viability was determined by measuring overall cell counts, hemocytometer counts of Trypan Blue-impermeable cells and CCK-8.
3. Statistical analyses were performed using SPSS Statistics software (version17.0). Unless otherwise indicated, the data were presented as mean±SE. Differences of group comparisons were analyzed using one-way ANOVA and subsequent appropriate post hoc analysis by unpaired Student's t test. A P value<0.05was considered statistically significant.
Results:
1. Cell surface receptor RAGE expression are up-regulated by ICs through TLR9mediated NF-κB activation and can be inhibited by PPAR-y agonist in human monocytes
2. The secretion of monocytic cytokines IL-6, MCP-1and TNF-a is increased by ICs through RAGE/TLR9mediated NF-κB activation and can be inhibited by PPAR-y agonist in human monocytes
3. Increased nuclear import of NF-κB family member p65is induced by ICs through RAGE/TLR9and can be inhibited by PPAR-y agonist in human monocytes
4. Monocytes transendothelial migration is increased by ICs stimulation through RAGE/TLR-9mediated NF-κB activation and can be inhibited by PPAR-y agonist
5. The PPAR-y agonist pioglitazone inhibits ICs-induced NF-κB p65activation via formation of PPAR-y-p65complexes in a dose dependent manner in human monocytes
6. sRAGE, ODN2088, Bay117982and pioglitazone used in the experiements are no cytotoxic to human monocytes.
Conclusions:
This study provides a demonstration of ICs'activating effects on the aberrant activation of monocytes and provides one possible mechanism of how ICs activate monocytes in SLE, which plays important roles in the pathogenesis of SLE. In addition, this study suggests a possible way to inhibit the monocytic activation in SLE, which has the potential to become one of the therapeutic options for SLE.
免疫复合物对人血管内皮细胞的活化作用及其机制研究
背景：
系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种以免疫复合物(immune complexes, ICs)形成为特征的慢性自身免疫性疾病,其中有文献报道免疫复合物包括核酸,核酸相关蛋白和相应的自身抗体三种成分。在系统性红斑狼疮患者外周血中,自然凋亡的细胞不能被单核巨噬细胞清除,结果导致了这些凋亡细胞的二次凋亡坏死,自身抗原成分核酸和核酸相关蛋白就从这些二次凋亡细胞的细胞核中释放出来,然后便产生了针对这些核自身抗原成分的相应的自身抗体,例如抗双链DNA抗体,导致了免疫复合物的形成。免疫复合物沉积在患者的靶器官如皮肤和肾脏中,导致了这些器官的炎症和损伤。
血管病变和血管炎,是系统性红斑狼疮的典型的并发症,有报道称其发生在10%至40%的系统性红斑狼疮患者,多见于患者的皮肤血管,肾小球,冠状动脉和脑血管。有文献报道系统性红斑狼疮血管病变和血管炎与免疫复合物沉积,内皮细胞活化以及炎症细胞的浸润有关。然而,免疫复合物对血管内皮细胞的作用及其机制尚不明确。
高迁移率族蛋白1(high-mobility group box1protein, HMGB1),也叫做两性霉素,属于核酸相关蛋白,是免疫复合物中的一种重要成分。它几乎在所有的真核细胞中表达,主要位于细胞核中,在细胞核中,它与核酸成分结合稳定核小体的结构,并且诱导DNA弯曲来调节转录。在系统性红斑狼疮患者中,HMGB1-核酸复合物从二次凋亡的细胞核中释放出来,与相应的自身抗体形成免疫复合物,在血浆中被检测到显著升高。在靶器官例如皮肤和肾脏中,包含HMGB1的免疫复合物大量沉积,导致了靶器官的慢性炎症和组织损伤。HMGB1的促炎症活性主要由其受体晚期糖基化终末产物受体(the receptor for advanced glycation end products, RAGE)来介导。RAGE是一种跨膜蛋白,是免疫球蛋白超家族的成员,在HMGB1介导的信号通路中,它是一个主要的信号转导受体。RAGE主要在血管内皮细胞,单核和巨噬细胞的细胞膜上表达,能够经过经典的信号激活途径来激活转录因子NF-κB家族成员p65,而且可以通过激活p65来调节自身的表达。
了解了HMGB1是免疫复合物的重要成分,并且HMGB1的促炎症活性主要由其受体RAGE来承载。那么,HMGB1-RAGE细胞信号途径有可能在免疫复合物对内皮细胞的作用中发挥重要的作用。然而目前为止,这一作用尚没有文献报道。因而,评价HMGB1-RAGE细胞信号通路在免疫复合物对内皮细胞的作用中发挥的作用来探讨HMGB1-RAGE细胞信号通路是不是免疫复合物对内皮细胞的作用中的一种可能的机制将会很有意义和价值。
HMGB1的作用能被HMGB1A-box特异性的阻断,RAGE能被可溶性RAGE (soluble RAGE, sRAGE)阻断。HMGB1A-box, sRAGE, Bay117082以及这些阻断剂的组合分别被用来阻断HMGB1-RAGE细胞信号途径上的免疫复合物中HMGB成分,内皮细胞表面的RAGE受体,内皮细胞的转录因子NF-κB p65以及它们的组合来检测免疫复合物对内皮细胞作用的发生是不是通过HMGB1-RAGE细胞信号途径。有报道称p38细胞分裂素活化蛋白激酶(mitogen-activated protein kinases, MAPK)和细胞外信号相关激酶1/2(extracellular signal-related kinases1and2, ERK1/2)信号途径位于RAGE介导的转录因子NF-κB活化的通路上,在本研究中,p38MAPK和ERK1/2的特异性阻断剂SB203580和PD98059分别用来阻断p38MAPK和ERK1/2,以评价这两个细胞信号途径在免疫复合物诱导的血管内皮细胞细胞因子分泌反应中所发挥的作用。
目的：
探索免疫复合物对人血管内皮细胞的作用,评价HMGB1-RAGE细胞信号通路在免疫复合物对人血管内皮细胞活化中所发挥的作用,来探讨人系统性红斑狼疮血管炎中免疫复合物对血管内皮细胞的活化作用及其其中一种可能的机制。
方法：
1.在免疫复合物刺激和阻断剂阻断实验中,把人脐静脉内皮细胞株CRL-1730细胞接种到直径为6cm的细胞培养皿中,在细胞达到80%单层细胞融合的时候,对细胞分组并且加药干预。先分别加入阻断剂HMGB1A-box (10μg/ml), sRAGE (20μg/ml), SB203580(10μM), PD98059(25μM), Bay117082(1μM或者它们的组合干预1小时,然后加入免疫复合物。孵育2小时后,NF-κB家族成员p65在细胞核中的水平的变化分别用免疫荧光,免疫组化和Western Blot的方法来检测。孵育6小时后,基因RAGE, ICAM-1, VCAM-1, IL-8, MCP-1, IL-6, TNF-a和IL-lβmRNA水平的变化用荧光实时定量PCR的方法来检测。孵育16小时后,细胞表面蛋白RAGE, ICAM-1, VCAM-1表达水平的变化用免疫荧光,免疫组化,流式以及细胞ELISA的方法来检测分析。孵育24小时后,细胞上清液中细胞因子IL-8, MCP-1, IL-6, TNF-α和IL-1β浓度的变化用ELISA的方法来检测。
2.在单核细胞跨血管内皮细胞迁移的实验中,先把人脐静脉内皮细胞株CRL-1730细胞接种在明胶包被的具有通透性的Transwell小室的滤膜上,形成(?)单血管内皮细胞层后,对该层血管内皮细胞分组并且用方法1中描述的方法用阻断剂和免疫复合物进行干预24小时。然后将单核细胞加入到上室中进行跨血管内皮细胞迁移,20小时后,将迁移至下室的单核细胞收集起来用细胞计数仪计数,并且比较不同干预组之间单核细胞跨内皮细胞迁移的差异。
3.在细胞活性测定实验中,把人脐静脉内皮细胞株CRL-1730以每孔105个细胞的密度接种到24孔板中,孵育过夜至细胞贴壁融合。将细胞分组,然后分别加入HMGB1A-box (10μg/ml), sRAGE (20μg/ml), SB203580(10μM), PD98059(25μM), Bay117082(1μM或者它们的组合进行干预。24小时后,将细胞收集起来,用细胞计数仪计数不同干预组细胞总数的变化,用台盼蓝染色的方法计数活细胞数目的差异,并且用CCK-8的方法检测细胞活性的差异。
4.实验中,用SPSS17.0统计软件进行统计分析,实验数据用均数±标准误来表示。不同实验组之间的差异用单因素方差分析进行分析,两组数据之间的比较用t检验进行分析,P值<0.05被认为具有统计学意义。
结果：
1.免疫复合物通过HMGB1-RAGE细胞信号通路上调人血管内皮细胞表面RAGE受体的表达。
2.免疫复合物通过HMGB1-RAGE细胞信号通路上调人血管内皮细胞表面粘附分子ICAM-1和VCAM-1的表达。
3.免疫复合物通过HMGB1-RAGE细胞信号途径增加了人血管内皮细胞趋化因子IL-8,MCP-1和促炎症细胞因子IL-6,TNF-α的分泌。
4. p38MAPK和ERK1/2信号途径在免疫复合物通过HMGB1-RAGE细胞信号途径诱导人血管内皮细胞活化中发挥重要作用。
5.免疫复合物通过HMGB1-RAGE细胞信号途径导致了人血管内皮细胞中转录因子NF-κB p65的活化。
6.免疫复合物通过HMGB1-RAGE细胞信号通路增强了单核细胞的跨血管内皮细胞迁移。
7. HMGB1-RAGE细胞信号途径上的阻断剂HMGB1-A-box, sRAGE, Bay117082, SB203580, PD98059以及它们的组合本身对人血管内皮细胞没有细胞毒性作用。
结论：
本研究证明免疫复合物通过HMGB1-RAGE细胞信号通路引发了人血管内皮细胞的促炎症反应,而且导致了血管内皮细胞功能的变化。这为免疫复合物对人血管内皮细胞的活化作用及其其中一种潜在的机制提供了证明,在理解系统性红斑狼疮血管炎的发病机制中发挥重要的作用,并且为将来系统性红斑狼疮血管炎的治疗提供了有力的证据。
第二部分
免疫复合物对人单核细胞的活化作用及其机制和治疗方法研究背景：
系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种以免疫复合物(immune complexes, ICs)形成为特征的慢性自身免疫性疾病,其中有文献报道免疫复合物包括核酸,核酸相关蛋白和相应的自身抗体三种成分。在系统性红斑狼疮患者中,自然凋亡的细胞不能被单核巨噬细胞清除,结果导致了这些凋亡细胞的二次凋亡坏死,自身抗原成分核酸和核酸相关蛋白就从这些二次凋亡细胞的细胞核中释放出来。然后,针对这些核自身抗原成分的相应的自身抗体,例如抗双链DNA抗体便产生了,导致了免疫复合物的形成。免疫复合物沉积在人体的靶器官如皮肤和肾脏中,导致了这些器官的炎症和损伤。
有文献报道系统性红斑狼疮患者的单核细胞异常活化,并且通过产生一系列的细胞因子和功能异常来启动和维持自身免疫反应。其中,IL-6, TNF-α和MCP-1是由单核细胞产生的,在系统性红斑狼疮患者的单核细胞中异常地表达升高,并且在系统性红斑狼疮的发病过程中发挥了重要作用的细胞因子。单核细胞是人外周血中IL-6的主要来源,IL-6在系统性红斑狼疮中促进B细胞分化成为产生自身抗体的浆细胞,并且促进自身抗体的产生。TNF-α是一个重要的促炎症因子,它的过表达导致了炎症的持续。MCP-1是一种作用非常强的单核细胞趋化蛋白,它促使单核细胞聚集在炎症发生的部位。最近对狼疮肾炎的研究表明,异常活化的单核巨噬细胞在狼疮肾炎中大量浸润,促进了狼疮肾炎的炎症和损伤,并且影响狼疮肾炎的最终结果。
包含核酸,核酸相关蛋白和相应自身抗体三种成分的免疫复合物是具有致病性的。有文献报道,从系统性红斑狼疮患者提取的免疫复合物中的核酸成分与从凋亡细胞中释放的染色质片段的大小差不多,这些片段含有丰富的寡聚核苷酸A类CpG,可以特异性的被表达于单核细胞,类浆细胞样树突状细胞和B细胞上的模式识别受体TLR9识别。TLR9的活化可以被其拮抗剂抑制性的寡聚脱氧核苷酸ODN2088特异性的阻断。HMGB1,高迁移率族蛋白1,也叫做两性霉素,属于免疫复合物中的核酸相关蛋白成分,是免疫复合物中另外一个重要的成分。它表达于几乎所有的有核细胞,并且主要位于细胞的细胞核中。在细胞核中,它与核苷酸结合来稳定核小体的结构,并且诱导DNA弯曲来调节转录。在系统性红斑狼疮患者中,HMGB1从二次凋亡的细胞核中释放出来,并且在血浆中被检测到显著升高。一旦释放到细胞外的环境中,HMGB1就作为一种炎症刺激因子,刺激炎症细胞进一步产生更多的细胞因子。HMGB1的促炎症作用主要通过与其细胞表面受体RAGE来发生。RAGE是第一个确定的HMGB1的受体,主要表达在单核细胞,血管平滑肌细胞,内皮细胞等细胞的表面,可被可溶性RAGE阻断。有文献报道免疫复合物能活化类浆细胞样树突状细胞和自身反应性B细胞来诱导IFN-α和其它促炎症因子的产生,促进疾病的发展,通过免疫复合物中的核酸和HMGB1成分活化细胞受体TLR9和RAGE。然而,免疫复合物对单核细胞的作用及其潜在的机制尚不明确。
细胞表面受体RAGE和细胞浆受体TLR9都能够通过经典的活化途径活化NF-κB家族成员p65,p65能调节IL-6, TNF-α和MCP-1的表达。另外,RAGE受体被激活后,通过信号通路活化NF-κB家族成员p65,p65还能调节RAGE自身的表达。在本研究中,免疫复合物被用来检测其对单核细胞的作用,可溶性RAGE, ODN2088和Bay117082被用来分别阻断RAGE受体,TLR9和NF-κB p65来检测免疫复合物对单核细胞作用的可能的机制。
虽然近来在抗炎症治疗方面有所进展,但是当前对系统性红斑狼疮治疗的选择还存在很多分歧和不确定。前面已有研究报道过氧化物酶体增值物受体γ(PPAR-γ)激动剂在系统性红斑狼疮小鼠模型中是一种很有效的治疗方法。它能够减少自身抗体的产生,而且能够减少肾脏炎症和损伤。研究报道,PPAR-γ激动剂包括噻畔烷二酮类(thiazolidinediones, TZDs),前列腺素类和非甾体类抗炎药物(NSAIDs)三种,激活了表达于单核细胞,角质化细胞等细胞核内的核受体PPAR-γ以后,通过与转录因子NF-κB的相互作用来调节促炎症因子的表达。也有文献报道,PPAR-γ激动剂通过抑制NF-κB p65的核转移和通过形成PPAR-γ-p65复合物,来减少NF-κB p65与其靶核苷酸调节序列的结合。在本研究中,我们选择噻唑烷二酮类药物匹格列酮(pioglitazone),它是PPAR-γ受体的一种药理性激动剂,作为PPAR-γ激动剂的代表,在体外实验检测PPAR-γ激动剂对系统性红斑狼疮单核细胞异常活化的作用及其机制,在前面报道的对狼疮鼠的有效的治疗作用的基础上,进一步探测它的对系统性红斑狼疮的治疗作用。
目的：
在本研究中,我们探索免疫复合物对系统性红斑狼疮患者单核细胞异常活化所起的作用及其潜在的机制,并且为系统性红斑狼疮患者单核细胞异常活化提出一种可能的治疗方法。
方法：
1.把人单核细胞白血病细胞株U937以每孔106个细胞的密度接种到6孔板中,将细胞分组,分别与sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM或者PPAR-γ激动剂匹格列酮(10μM)孵育1小时,然后加入免疫复合物。孵育2小时后,用Western Blot的方法来检测NF-κB家族成员p65在细胞核中水平的变化,用免疫沉淀的方法检测PPAR-γ-p65复合物的形成。孵育6小时后,用荧光实时定量RT-PCR的方法来检测基因RAGE, IL-6, TNF-α和MCP-1mRNA水平的变化。孵育16小时后,用流式和Western Blot的方法来检测RAGE蛋白表达的变化。孵育24小时后,用ELISA的方法来检测细胞上清液中细胞因子IL-6, TNF-α和MCP-1的浓度的变化,用Transwell的方法来检测单核细胞跨血管内皮细胞迁移的变化。
2.在检测细胞活性的实验中,把人单核细胞白血病细胞株U937以每孔5×105个细胞的密度接种到12孔板中,将细胞分组,分别与sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM)或者PPAR-γ激动剂匹格列酮(10μM)孵育24小时。分别用细胞计数仪计数细胞总数,台盼蓝染色活细胞数以及CCK-8的方法来检测细胞活性的变化。
3.实验中,用SPSS17.0统计软件进行统计分析,实验数据用均数士标准误来表示。不同实验组之间的差异用单因素方差分析进行分析,两组数据之间的比较用t检验进行分析,P值<0.05被认为具有统计学意义。
结果：
1.免疫复合物通过TLR9介导的NF-κB的活化上调人单核细胞表面受体RAGE的表达,这一作用可被PPAR-γ激动剂抑制。
2.免疫复合物通过RAGE/TLR9介导的NF-κB的活化增加人单核细胞细胞因子IL-6, MCP-1和TNF-α的分泌,这一作用可能被PPAR-γ激动剂抑制。
3.免疫复合物通过RAGE/TLR9介导的NF-κB的活化增强单核细胞跨血管内皮细胞迁移,这一作用可被PPAR-γ激动剂抑制。
4.免疫复合物通过RAGE/TLR9介导的NF-κB的活化增加人单核细胞NF-κB家族成员p65的核转移,这一作用可被PPAR-γ激动剂抑制。
5. PPAR-γ激动剂匹格列酮通过以剂量依赖的形式形成PPAR-γ-p65复合物抑制免疫复合物诱导的人单核细胞NF-κB p65的活化。
6.实验中用到的sRAGE, ODN2088, Bay117982和匹格列酮对单核细胞没有细胞毒性。
结论：
本研究证实了免疫复合物对系统性红斑狼疮单核细胞异常活化所起的作用并且对免疫复合物如何导致单核细胞异常活化提供了一种可能的机制,这为系统性红斑狼疮发病机制的研究提供了重要证据。另外,本研究对系统性红斑狼疮单核细胞异常活化提出了一种可能的治疗方法,这种方法将很有可能成为系统性红斑狼疮治疗的新的选择。
Part Ⅰ
The activating effects and potential mechanisms of immune complexes on human endothelial cells
Backgrounds:
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by formation of immune complexes (ICs), which contain autoantigens nucleic acids, nucleic acids-associated protein and corresponding antibodies. The apoptotic cells which are impared cleared by monocytes/macrophages result in secondary necrosis, autoantigens nucleic acids and nucleic acids-associated protein are released by the nuclei of these secondary apoptotic cells in patients with SLE. The corresponding autoantibodies against these nuclear autoantigens, such as the anti-double-stranded DNA (dsDNA) antibodies are generated and lead to the formation of ICs. Deposition of ICs on target organs, such as skin and kidney, results in end organs damage.
Vasculopathy and vasculitis, which are typical complications of SLE, are reported in10%to40%of SLE patients and are usually seen in cutaneous vessels, renal glomeruli, coronary and brain vessels. Vasculopathy and vasculitis are reported to be associated with the deposition of ICs on endothelium, endothelial cells activation and inflammatory cells infiltration into the inflammatory sites. However, the effects of ICs on the endothelial cells and the potential mechanisms remain unclear.
HMGB1, also known as amphotericin, which belongs to the nucleic acids-associated protein, is an essential component of ICs. HMGB1is expressed in almost all eukaryotic cell types and is located mainly in the cell nucleus, where it binds to nucleotides to stabilize the structure of nucleosomes and to induce DNA binding to regulate transcription. In SLE patients, HMGB1-nucleic acids complexes are released from the secondary necrotic cells, form ICs with the corresponding antibodies and are found to be significantly elevated in the sera. Depositon of HMGB1-containing ICs are also found in target tissues, such as the skin and kidney, contributing to the chronic inflammation and tissue injury. The proinflammatory activity of HMGB1is mostly attributed to its ligation with RAGE. RAGE, a member of the immunoglobulin superfamily of cell surface molecules, is a central signal transduction receptor for HMGB1-modified adducts. It is expressed on cells such as endothelial cells, monocytes and macrophages and can signal to transcription factor NF-κB family member p65through the classical activation pathway. Furthermore, RAGE signaling to the NF-κB p65regulates the expression of RAGE itself.
Knowing that HMGB1is one important component of ICs and the proinflammatory activity of HMGB1is mostly attributed to its ligation with RAGE. The cell signaling HMGB1-RAGE axis may play important roles in the effects of ICs on the endothelial cells. However, the roles of cell signaling HMGB1-RAGE axis in the effects of ICs on the endothelial cells has not been evaluated so far. It would be attracted and worthy to evaluate the involvement of the cell signaling HMGB1-RAGE axis in the effects of ICs on endothelial cells to examine whether it is one potential mechanism of ICs'effects on human endothelial cells.
The effects of HMGB1can be specially blocked by HMGB1A-box, RAGE can be blocked by sRAGE. HMGB1A-box, sRAGE, Bay117082and combination of these blockers were used to block the effects of the HMGB1component in ICs, RAGE, NF-κB p65and combine effects of them in human endothelial cells, respectively to detect whether the effects of ICs on human endothelial cells occur involving in the cell signaling HMGB1-RAGE axis. p38 mitogen-activated protein kinases (MAPK) and extracellular signal-related kinases1and2(ERK1/2) pathways are reported to be involved in the signaling pathway of RAGE-mediated NF-κB activation. Specific inhibitors of p38MAPK (SB203580) and ERK1/2(PD98059) were also used to assess the contribution of p38MAPK and ERK1/2pathways to ICs-stimulated cytokine responses in human endothelial cells.
Objectives:
To explored the effects of ICs on the endothelial cells and evaluate the involvement of the cell signaling HMGB1-RAGE axis in these effects to examine one potential mechanism of the effects of ICs on human endothelial cells in the pathogenesis SLE vasculitis.
Methods:
1. For ICs stimulation and blocking assays, human CRL-1730cells were seeded into6cm-dishes at80%confluent monolayers and pretreated with HMGB1A-box (10μg/ml), sRAGE (20μg/ml) SB203580(10μM), PD98059(25μM), Bay117082(1μM) or combination of them for1hour before the addition of ICs. After incubation for2hours, immunocytochemistry (ICC), immunofluorescence (IF) and Western blot analyses of NF-κB family member p65levels in the nucleus were performed. After incubation for6hours, RAGE, ICAM-1, VCAM-1, IL-8, MCP-1, IL-6, TNF-α and IL-1β mRNA levels were detected by Quantitative real-time-PCR. After incubation for16hours, cell surface RAGE, ICAM-1, VCAM-1proteins expression levels were analyzed by ICC, IF, flow cytometry (FCM) or cellular enzyme-linked immunosorbent assay (cellular ELISA) analysis. After incubation for24hours, the concentration of cytokines IL-8, MCP-1, IL-6, TNF-a and IL-1β in the cell-free supernatants were detected by ELISA.
2. For monocytes transendothelial migration experiments, human CRL-1730cells were seeded on gelatin-coated permeable Transwell filters to form a monolayer and pretreated by blockers and ICs as described above for24hours before monocytes migration. After incubation for20hours, the cells that had transmigrated to the lower chamber were harvested and counted using a TC10Automated Cell Counter. And the difference of monocytes transendothelial migration between different endothelial cells groups are analysed.
3. For cell viability assay, human CRL-1730cells were seeded onto24-well plates at105cells/well and allowed to attach and grow overnight to confluence, and then they were incubated with control medium alone, HMGB1A-box (10μg/ml), sRAGE (20μg/ml), SB203580(10μM), PD98059(25μM), Bay117082(1μM) or combination of them. After treatment for24hours, cells were harvested and overall cell counts were measured and the number of viable cells was determined by hemocytometer counts of Trypan Blue-impermeable cells. In another test, cell viability was determined using the CCK-8kit.
4. Statistical analyses were performed using SPSS statistics software version17.0. Unless otherwise indicated, data are presented as mean±SE. Differences of group comparisons were analyzed using one-way ANOVA and subsequent appropriate post-hoc analysis by unpaired Student's t-tests. A P value<0.05was considered statistically significant.
Results:
1. ICs up-regulate RAGE expression involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
2. ICs up-rcgulate the cell surface adhesion molecules ICAM-1and VCAM-1expression involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
3. ICs increase the secretion of chemokines IL-8, MCP-1and proinflammatory cytokines IL-6, TNF-a involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
4. Activation of p38MAPK and ERK1/2pathways in the cell siganling HMGB1-RAGE axis induced by ICs in human endothelial cells
5. ICs lead to activation of NF-κB p65involving in the cell signaling HMGB1-RAGE axis in human endothelial cells
6. Monocytes transendothelial migration is enhanced by ICs pretreated human endothelial cells which occurs involving in the cell signaling HMGB1-RAGE axis of human endothelial cells
7. Blockers HMGB1-A-box, sRAGE, Bay117082, SB203580, PD98059or combination of them used in the experiments were not toxic to human endothelial cells.
Conclusions:
This study demonstrates that ICs elicit proinflammatory responses in human endothelial cells and contribute to alterations in human endothelial cells function involving in the cell signaling HMGB1-RAGE axis, it provides a demonstration for the effects and a potential mechanism of the effects of ICs on human endothelial cells, which is important in understanding the pathogenesis of lupus vasculitis and may provide important evidence for the therapy of SLE vasculitis in the future.
Part II
The activating effects and potential mechanisms of immune complexes on human monocytes and its possible therapeutic methods
Backgrounds:
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by formation of immune complexes (ICs), which contain autoantigens nucleic acids, nucleic acids-associated protein and corresponding antibodies. In SLE patients, the apoptotic cells which are impaired cleared by monocytes/macrophages result in secondary necrosis, autoantigens nucleic acids and nucleic acids-associated protein are released by the nuclei of these secondary apoptotic cells. The corresponding autoantibodies against these nuclear autoantigens, such as the anti-double-stranded DNA (dsDNA) antibodies are generated and lead to the formation of ICs. Deposition of ICs on target organs, such as the kidney, results in end organs damage.
Monocytes that are aberrantly activated in the patients of SLE initiate and maintain autoimmune responses via production of several cytokines and functional alteration. IL-6, TNF-a and MCP-1are cytokines secreted by monocytes, which are aberrantly up-regulated in the monocytes of SLE patients and play very important roles in the pathogenesis of SLE. Monocytes are the primary source of IL-6in the peripheral blood, IL-6promotes the production of autoantibodies and is required for the differentiation of B cells into antibodies secreting plasma cells. TNF-a is a pro-inflammatory cytokine and its over-expression leads to perpetuation of inflammation. MCP-1, which is known for its ability as a potent chemoattractant of monocytes, facilitates the recruitment of monocytes into the inflammatory sites. Recent studies on lupus nephritis demonstrated that activated monocytes/macrophages vigorously participate in and amplify renal inflammation and injury, which influence the outcome of lupus nephritis.
ICs which contain nucleic acids, nucleic acids-associated protein and corresponding antibodies are pathogenic. It has been reported that the nucleic acids component in ICs isolated from SLE patients are of similar size as the cleaved chromatin fragments released from apoptotic cells, these fragments are rich in oligonucleotide class A CpG (CpG-A) and can be specifically recognized by TLR9, which is a pattern recognition receptor expressed on monocytes, pDCs, B cells and so on. TLR9activation can be specifically blocked by its antagonist inhibitory oligodeoxynucleotide ODN2088. High Mobility Group Box1(HMGB1; also known as amphotericin) which belongs to the nucleic acids-associated protein in ICs, is another essential component of ICs. It is expressed in eukaryotic cells and is located mainly in the cell nucleus, where it binds to nucleotides to stabilize the structure of nucleosomes and to induce DNA binding to regulate transcription. In SLE patients, HMGB1released from the secondary necrotic cells are found to be significantly elevated in the sera. Once released into the extracellular milieu, it functions as a cytokine that activates inflammatory cells to the further production of cytokines. The pro-inflammatory cytokine activity of HMGB1is mostly attributed to its association with RAGE. RAGE is the first described receptor for HMGB1and is expressed on the surface of monocytes, vascular smooth muscle cells, neurons, endothelial cells and so on. RAGE can be blocked by soluble RAGE (sRAGE). It has been reported that ICs can stimulate pDCs and autoreactive B cells to induce IFN-a and other pro-inflammatory cytokines production and disease development through RAGE and TLR9due to the nucleic acids-associated protein HMGB1and nucleic acids component in the ICs. However, the effects of ICs on monocytes and the potential mechanisms remain unclear.
Both cell surface receptor RAGE and cytosolic receptor TLR9can signal to transcription factor NF-κB family member p65through the classical activation pathway, which can regulate the expression of IL-6, TNF-α and MCP-1. Furthermore, RAGE signal to NF-κB p65can regulate the expression of RAGE itself. In this paper, ICs were used to detect its effects on monocytes, sRAGE, ODN2088, and Bay117082were used to block RAGE, TLR9and NF-κB p65, respectively, to detect the possible mechanisms of ICs'effects on monocytes.
Despite the recent advances in anti-inflammatory therapy, current treatment options for SLE are diverse and poorly defined. It has been reported that PPAR-y agonist is a very useful therapeutic strategy in mouse models of SLE, it can reduce autoantibodies production, it can also reduce renal inflammation and injury. It is reported that the PPAR-y agonists which contains thiazolidinediones (TZDs), prostanoids and non-steroidal anti-inflammatory drugs (NSAIDs) activate the nuclear hormone receptor PPAR-y, which is expressed in monocytes, keratinocytes and so on, and regulate the expression of pro-inflammatory proteins by interacting with transcription factors NF-κB, it is also reported that PPAR-y agonists can inhibit the nuclear translocation of NF-κB p65and promote PPAR-y to form complexes with p65in the nucleus, which reduces the binding of p65to its target sequences. Here we chose pioglitazone, which belongs to TZDs and pharmacological agonist of PPAR-y as a representative of the PPAR-y agonists to examine the effects of PPAR-y agonists on monocytes aberrant activation and their mechanisms in SLE in vitro to further detect its therapeutic effects in SLE in addition to its effective therapeutic effects in mouse models of SLE reported before.
Objectives:
In this study, we try to explore the effects of ICs on monocytes aberrant activation in SLE and the potential mechanisms, and to propose a potential therapeutic strategy to the monocytes aberrant activation in SLE.
Methods:
1. Human U937cells were seeded onto6-well plates at106cells/well and pretreated with sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM) or PPAR-y agonist pioglitazone (10μM) for one hour before treating with ICs. After incubation for two hours, the level of NF-κB family member p65in the nucleus was analyzed by western blot and immunoprecipitation (IP) assays were performed to detect the formation of complexes PPAR-y-p65. After incubation for six hours, RAGE, IL-6, TNF-a and MCP-1mRNA expression levels were determined using real-time RT-PCT. After incubation for16hours, RAGE protein expression level was determined by flow cytometry and western blot analysis. After incubation for24hours, the concentrations of cytokines IL-6, TNF-a and MCP-1in the cell-free supernatants were examined and transendothelial migration assays were performed to detect the changes of monocytes transendothelial migration.
2. For the cell viability assay, human U937cells were seeded onto12-well plates at5×105cells/well and incubated with control medium alone or sRAGE (20μg/ml), ODN2088(12μg/ml), Bay117082(1μM) or PPAR-y agonist pioglitazone (10μM) for24hours. Cell viability was determined by measuring overall cell counts, hemocytometer counts of Trypan Blue-impermeable cells and CCK-8.
3. Statistical analyses were performed using SPSS Statistics software (version17.0). Unless otherwise indicated, the data were presented as mean±SE. Differences of group comparisons were analyzed using one-way ANOVA and subsequent appropriate post hoc analysis by unpaired Student's t test. A P value<0.05was considered statistically significant.
Results:
1. Cell surface receptor RAGE expression are up-regulated by ICs through TLR9mediated NF-κB activation and can be inhibited by PPAR-y agonist in human monocytes
2. The secretion of monocytic cytokines IL-6, MCP-1and TNF-a is increased by ICs through RAGE/TLR9mediated NF-κB activation and can be inhibited by PPAR-y agonist in human monocytes
3. Increased nuclear import of NF-κB family member p65is induced by ICs through RAGE/TLR9and can be inhibited by PPAR-y agonist in human monocytes
4. Monocytes transendothelial migration is increased by ICs stimulation through RAGE/TLR-9mediated NF-κB activation and can be inhibited by PPAR-y agonist
5. The PPAR-y agonist pioglitazone inhibits ICs-induced NF-κB p65activation via formation of PPAR-y-p65complexes in a dose dependent manner in human monocytes
6. sRAGE, ODN2088, Bay117982and pioglitazone used in the experiements are no cytotoxic to human monocytes.
Conclusions:
This study provides a demonstration of ICs'activating effects on the aberrant activation of monocytes and provides one possible mechanism of how ICs activate monocytes in SLE, which plays important roles in the pathogenesis of SLE. In addition, this study suggests a possible way to inhibit the monocytic activation in SLE, which has the potential to become one of the therapeutic options for SLE.
引文
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[14]Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, et al. Understanding RAGE, the receptor for advanced glycation end products. Journal of molecular medicine (Berlin, Germany) 2005;83(11):876-886.
[15]Liang Y, Zhou Y, Shen P. NF-kappaB and its regulation on the immune system. Cellular& molecular immunology 2004;1(5):343-350.
[16]Yeh CH, Sturgis L, Haidacher J, Zhang XN, Sherwood SJ, Bjercke RJ, et al. Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion. Diabetes 2001;50(6):1495-1504.
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[18]Hoffmann A, Natoli G, Ghosh G. Transcriptional regulation via the NF-kappaB signaling module. Oncogene 2006;25(51):6706-6716.
[19]Yang H, Ochani M, Li J, Qiang X, Tanovic M, Harris HE, et al. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proceedings of the National Academy of Sciences of the United States of America 2004; 101(1):296-301.
[20]Kokkola R, Li J, Sundberg E, Aveberger AC, Palmblad K, Yang H, et al. Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity. Arthritis and rheumatism 2003;48(7):2052-2058.
[21]Li M, Shang DS, Zhao WD, Tian L, Li B, Fang WG, et al. Amyloid beta interaction with receptor for advanced glycation end products up-regulates brain endothelial CCR5 expression and promotes T cells crossing the blood-brain barrier. J Immunol 2009; 182(9):5778-5788.
[22]Hsieh HL, Schafer BW, Weigle B, Heizmann CW. S100 protein translocation in response to extracellular S100 is mediated by receptor for advanced glycation endproducts in human endothelial cells. Biochemical and biophysical research communications 2004;316(3):949-959.
[23]Skurk T, van Harmelen V, Hauner H. Angiotensin Ⅱ stimulates the release of interleukin-6 and interleukin-8 from cultured human adipocytes by activation of NF-kappaB. Arteriosclerosis, thrombosis, and vascular biology 2004;24(7):1199-1203.
[24]Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, et al. Advanced glycation end products activate endothelium through signal-transduction receptor RAGE:a mechanism for amplification of inflammatory responses. Circulation 2002; 105(7):816-822.
[25]Rasheed Z, Akhtar N, Haqqi TM. Advanced glycation end products induce the expression of interleukin-6 and interleukin-8 by receptor for advanced glycation end product-mediated activation of mitogen-activated protein kinases and nuclear factor-kappaB in human osteoarthritis chondrocytes. Rheumatology (Oxford, England);50(5):838-851.
[26]Shanmugam N, Kim YS, Lanting L, Natarajan R. Regulation of cyclooxygenase-2 expression in monocytes by ligation of the receptor for advanced glycation end products. The Journal of biological chemistry 2003;278(37):34834-34844.
[27]Bave U, Alm GV, Ronnblom L. The combination of apoptotic U937 cells and lupus IgG is a potent IFN-alpha inducer. J Immunol 2000;165(6):3519-3526.
[28]Bave U, Vallin H, Alm GV, Ronnblom L. Activation of natural interferon-alpha producing cells by apoptotic U937 cells combined with lupus IgG and its regulation by cytokines. Journal of autoimmunity 2001; 17(1):71-80.
[29]Bave U, Magnusson M, Eloranta ML, Perers A, Alm GV, Ronnblom L. Fc gamma RⅡa is expressed on natural IFN-alpha-producing cells (plasmacytoid dendritic cells) and is required for the IFN-alpha production induced by apoptotic cells combined with lupus IgG. J Immunol 2003;171(6):3296-3302.
[30]Vallin H, Perers A, Alm GV, Ronnblom L. Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus. J Immunol 1999; 163(11):6306-6313.
[31]Haraldsen G, Kvale D, Lien B, Farstad IN, Brandtzaeg P. Cytokine-regulated expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in human microvascular endothelial cells. J Immunol 1996;156(7):2558-2565.
[32]Smith CW. Leukocyte-endothelial cell interactions. Seminars in hematology 1993;30(4 Suppl 4):45-53; discussion 54-45.
[33]Akashi Y, Oshima S, Takeuchi A, Kubota T, Shimizu J, Shimizu E, et al. [Identification and analysis of immune cells infiltrating into the glomerulus and interstitium in lupus nephritis]. Nihon Rinsho Men'eki Gakkai kaishi = Japanese journal of clinical immunology 1995;18(5):545-551.
[34]Hill GS, Delahousse M, Nochy D, Remy P, Mignon F, Mery JP, et al. Predictive power of the second renal biopsy in lupus nephritis:significance of macrophages. Kidney international 2001;59(1):304-316.
[35]Isbel NM, Nikolic-Paterson DJ, Hill PA, Dowling J, Atkins RC. Local macrophage proliferation correlates with increased renal M-CSF expression in human glomerulonephritis. Nephrol Dial Transplant 2001; 16(8):1638-1647.
[36]Popovic M, Laumonnier Y, Burysek L, Syrovets T, Simmet T. Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration. Journal of leukocyte biology 2008;84(1):215-223.
[37]Zoja C, Angioletti S, Donadelli R, Zanchi C, Tomasoni S, Binda E, et al. Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-kappaB dependent up-regulation of IL-8 and MCP-1. Kidney international 2002;62(3):846-856.
[38]Hooper WC, Phillips DJ, Renshaw MA, Evatt BL, Benson JM. The up-regulation of IL-6 and IL-8 in human endothelial cells by activated protein C.J Immunol 1998;161(5):2567-2573.
[39]Bell CW, Jiang W, Reich CF,3rd, Pisetsky DS. The extracellular release of HMGB1 during apoptotic cell death. American journal of physiology 2006;291 (6):C 1318-1325.
[40]Fitzner N, Zahner L, Habich C, Kolb-Bachofen V. Stimulatory type A CpG-DNA induces a Th2-like response in human endothelial cells. International immunopharmacology;11(11):1789-1795.
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[42]Florey OJ, Johns M, Esho OO, Mason JC, Haskard DO. Antiendothelial cell antibodies mediate enhanced leukocyte adhesion to cytokine-activated endothelial cells through a novel mechanism requiring cooperation between Fc{gamma}RIIa and CXCR1/2.Blood 2007;109(9):3881-3889.
[43]Li Y, Lee PY, Reeves WH. Monocyte and macrophage abnormalities in systemic lupus erythematosus. Archivum immunologiae et therapiae experimentalis;58(5):355-364.
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