警报素IL-33介导狼疮肾炎炎症损伤的探究

硕士学位论文研究生:黄灿辉学科专业:内科学(风湿免疫方向)年级：2011级学号：2011212718学位类型:医学硕士(临床型)本课题受到国家自然科学基金（81300585）项目的资助广州医科大学•广州2014年5月目录中文摘要 (1)英文摘要 (6)主要英文缩写及中英文对照表 (11)第一章前言 (12)第二章实验材料及方法 (18)第三章实验结果 (33)第四章讨论 (41)第五章结论 (46)问题与展望 (47)参考文献 (48)综述 (54)攻读学位期间的研究成果 (63)致谢 (64)警报素IL-33介导狼疮肾炎炎症损伤的研究学位申请人：黄灿辉导师：陶怡教授专业：内科学（风湿免疫方向）广州医科大学附属第二医院风湿免疫科中文摘要研究背景及目的：系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种遗传、性激素、环境、感染等多因素参与其中的特异性自身免疫性疾病，其特征性表现为全身多系统和脏器受累，血清中存在大量自身抗体。

肾脏是SLE最容易受累的靶器官，狼疮肾炎（lupus nephritis，LN）是SLE最重要的临床表现之一。

LN对患者预后影响极大，肾功能衰竭是SLE的三大主要死亡原因之一。

目前LN的致病机制仍未完全清楚，治疗仍以传统抗慢性风湿病药物（Diseases Modifying Antirheumatic Drugs,DMARDS）为主，生物制剂的出现虽然带来了更多的治疗选择，但效果仍不尽人意。

因此，针对LN疾病机制的深入研究以及新药开发，依然任重道远。

固有免疫系统异常在SLE发病早期即出现，甚至早于临床表现，而且贯穿整个疾病过程。

病原相关分子模式（pathogen associated molecular patterns，PAMPs）和损伤相关分子模式（damage associated molecular patterns，DAMPs）是固有免疫系统识别外源性和内源性危险信号的方式。

他们可以被表达于免疫细胞上的模式识别受体（pattern recognition receptors，PRRs）所识别，从而启动免疫应答。

警报素是机体组织细胞损伤时所释放的内源性危险分子。

当机体细胞坏死或免疫细胞受到炎症刺激时，警报素通过被动或主动的方式释放到细胞间质中，与免疫细胞特异性受体结合，激活胞内信号通路，诱导炎症反应，募集趋化单核/巨噬细胞、中性粒细胞和特异性T细胞，从而启动固有免疫和调节特异性免疫反应。

在自身免疫性疾病中，由于自身免疫紊乱，警报素往往作为炎症因子参与疾病的损伤过程，从而加重组织损伤并形成恶性循环。

目前警报素在SLE、类风湿关节炎(Rheumatoid arthritis，RA)、系统性硬化、干燥综合征、炎症性肠病等自身免疫性疾病疾病被广泛研究。

不同的警报素之间具有类似的免疫激活模式。

白介素-33（interleukin-33,IL-33)是警报素的最新成员，其受体包括表达在免疫细胞表面的特异性受体ST2，以及被分泌到细胞外可以导致IL-33失活的诱骗受体可溶性ST2(Soluble ST2，sST2)。

IL-33/ST2信号通路的激活，可激活受体细胞下游有丝分裂原激活蛋白激酶（MAPK）及结合核因子κB（NF-κB），从而介导大量炎症因子产生，造成自身免疫性组织受损。

动物体内实验证明，予sST2治疗可以减轻RA、药物诱导急性肾损伤、哮喘等疾病的炎症受损。

临床研究发现, SLE患者血清IL33/sST2显著升高，而且升高水平与狼疮疾病活动度指数评分（SLEDAI）呈正相关。

SLE动物模型予以抗IL-33抗体治疗可以减轻狼疮肾炎病情。

以上研究提示IL-33/ST2通路可能以肾脏为靶点而参与SLE免疫激活，而拮抗该通路的激活有望成为治疗LN的新途径。

但目前关于IL-33/ST2通路如何参与狼疮肾炎炎症性损伤的机制仍不甚明了。

本研究旨在阐明：IL-33是否来源于肾脏，储备释放细胞是什么细胞，其表达水平是否可反应肾脏受损程度；外周血表面表达ST2受体是什么免疫细胞，以及IL-33与表达ST2受体的免疫细胞是如何通过相互作用导致肾脏炎症损伤等问题。

材料和方法：1、SPF级8周龄雌性MRL/lpr狼疮小鼠以及同龄雌性C57BL/6J健康小鼠各5只，动态观察2组小鼠一般情况以及检测尿蛋白水平。

18周龄时处死，检测血清ds-DNA、血清肌酐、尿素水平，行肾组织HE、PASM、MASSON染色观察肾脏病变。

2、Real time PCR方法检查2组小鼠肾组织IL-33mRNA表达水平。

western blot 检测肾组织IL-33蛋白表达水平。

分析IL-33表达水平与尿蛋白、血清ds-DNA 的相关性。

3、通过免疫组化技术，定位IL-33在肾组织的表达位置，进一步通过免疫荧光双染定位IL-33的表达细胞来源。

4、流式细胞术检测SLE患者及健康体检者外周血CD4+T细胞和CD14+单核细胞表面ST2受体的表达水平。

使用软件Image J进行图像分析，所有数据用SPSS17.0统计软件处理，正态分布并且方差齐同的定量资料以均数±标准差表示，偏态分布资料以中位数表示，2组间均数比较采用t检验或秩和检验。

等级资料组间比较采用秩和检验，相关性分析采用spearman相关系数。

p<0.05表示差异有统计学意义。

结果：1、小鼠一般情况及尿蛋白水平2组动物在处死前均存活，C57组精神状态良好，食欲良好，活跃，皮毛有光泽，未见脱毛，四肢关节无肿胀。

MRL/lpr组精神状态一般，食量较对照组减少，懒动,小鼠全身不同程度浮肿。

浅表处可触及数量不等肿大淋巴结。

皮毛颜色缺少光泽，但未见明显脱毛。

部分小鼠后膝关节轻度-中度肿胀。

尿蛋白从14周龄开始检测，每隔2周一次，MRL/lpr组所有小鼠均出现了不同程度的蛋白尿，但整体上没有出现尿蛋白进行性增多趋势。

C57组所有小鼠尿蛋白均为阴性。

2、小鼠血清尿素氮（BUN）、肌酐（Scr）及ds-DNA的测定MRL/lpr组血清BUN为10.04±1.79mmol/L，C57组为7.42±1.00 mmol/L，2组之间差别无统计学意义（P=0.300）；MRL/lpr组血清Scr为25.60±7.30 ummol/L，C57组为25.75±8.54ummol/L，同样2组之间差别无统计学意义（P=0.878）。

血清ds-DNA，MRL/lpr组均为阳性（2+～4+），而C57组则未见阳性表达，2组之间差异有统计学意义（p=0.003）。

3、普通及特殊病理切片结果HE染色：C57小鼠肾组织可见肾小球结构显示清晰，小球内可见血管袢；MRL/lpr狼疮小鼠则见肾小球明显肥大，结构紊乱，小球及肾间质可见大量淋巴细胞浸润。

PASM染色：MRL/lpr狼疮小鼠可见肾小球基底膜明显增厚，内皮及上皮细胞增生明显；而C57小鼠基底膜基本正常，未见明显内皮及上皮细胞增生。

MASSON染色：MRL/lpr狼疮小鼠可见肾小球内大量嗜红免疫复合物沉积，肾小管内可见大量蛋白管型；而C57小鼠肾小球结构清晰，未见免疫复合物沉积，肾小管管腔未见蛋白管型。

4、IL-33在肾组织的表达水平MRL/lpr组较C57组表达IL-33 mRNA水平平均升高2.73±1.34倍，两组比较有统计学差异（p=0.045）。

说明从mRNA水平看，MRL/lpr小鼠肾组织上调了IL-33的表达。

MRL/lpr组较C57组表达IL-33 蛋白水平显著升高（灰度比值为0.558±0.094 vs 0.034±0.002），C57组IL-33表达量极少，两组比较有统计学差异（p=0.0005）。

说明从蛋白水平看，MRL/lpr小鼠肾组织上调了IL-33的表达。

5、MRL/lpr小鼠肾组织IL-33蛋白与尿蛋白、血清ds-DNA的相关性分析spearman相关系数分析显示MRL/lpr小鼠肾组织IL-33灰度比值变化与半定量尿蛋白变化呈明显正相关（r2=0.72，p=0.037）。

而与血清ds-DNA半定量水平则无明显相关（r2=0.386，p=0.215），但也出现正相关趋势。

6、IL-33在小鼠肾组织的表达来源细胞MRL/lpr在肾小球基底膜区细胞有较多IL-33表达，肾间质少见IL-33表达。

C57只有极个别肾小球基底膜区细胞表达IL-33，较MRL/lpr显著减少。

进一步行免疫荧光双染检测（IL-33+VWF）发现，IL-33表达在肾小球内皮细胞。

7、LN患者与健康体检者外周血细胞ST2受体的表达与健康对照组相比，ST2 受体高表达于LN患者外周血 CD4+ T 细胞及 CD14+单核细胞表面，2组有显著差异。

结论：1、IL-33高表达于MRL/lpr小鼠肾组织，并且表达水平与可反应肾损害程度的尿蛋白水平呈正相关。

2、IL-33在肾组织的表达来源细胞是肾小球内皮细胞。

3、LN患者外周血单核细胞和CD4T细胞表面高表达ST2受体，提示IL-33可能通过介导外周血单核细胞和CD4T细胞趋化浸润至肾脏组织而导致肾脏炎症损伤。

关键词：系统性红斑狼疮警报素白介素-33 ST2受体Research on the role of alarmin IL-33 in thepathogenesis of lupus nephritisMajor: Internal medicineCandidate: Huang CanhuiSupervisor: professor.Tao YiAbstractBackground and ObjectivesSystemic lupus erythematosus (SLE) is a specific autoimmune disease that involves in genetics, hormones, environment, infection and other factors. It could be characterized by multi-system organ involvement and lots of autoantibodies in serum. The kidney is the easiest target organ involvement and lupus nephritis(LN) is the most important clinical manifestation of SLE.LN greatly impacts on the prognosis and renal failure is a major cause of death in SLE patients. The pathogenesis of LN is not absolute clear.Diseases modifying antirheumatic drugs (DMARDS) are still the main treatment of LN currently. Although the emergence of biologics brings more treatment options, but the results are still unsatisfactory.So,for deeper research on the pathogenesis of LN and development of new drugs remains to be done.Innate immune system abnormalities occur in the early onset of SLE, even before clinical manifestations, and throughout the course of the disease. Pathogen associated molecular patterns(PAMPs) and damage associated molecular patterns(DAMPs) are the ways to recognize exogenous and endogenous danger signals by innate immune system. They can be expressed on cells of the innate immune pattern recognition receptors (PRRs) identified, then initiate an immune response.'Alarmins' are a group of endogenous proteins or molecules that releasing from cells during cellular demise to alert the host innate immune system.Alarmins bind thespecific receptors expressing on the immune cells and then act the nuclear factor NF-KB axis, recruit monocyte / macrophages, neutrophils and special T cells, thereby initiate innate and specific immune response which involve SLE, rheumatoid arthritis (RA), atherosclerosis, hepatitis and cancer disease process. Different alarmins have a similar pattern of immune activation.Interleukin-33(IL-33) is the newest partner of alarmins. Its specific receptor is ST2. ST2 is known to exist in a transmembrane form (ST2L) and also alternatively spliced to produce a secreted soluble form (sST2). IL-33 stimulates target cells by binding to ST2, thereby activating nuclear factor (NF)-kB and mitogen-activated protein kinase pathways, releasing lots of inflammatory factors and inducing tissue damage.sST2 treatment can attenuate inflammatory tissue damage from the research of RA, acute kidney injury induced by drugs, asthma and etc in vivo.The clinical researches have found that serum of IL-33 or sST2 significantly increased in the SLE patients. The level of IL-33 or sST2 is positively correlated with SLE disease activity index (SLEDAI).The therapy with anti-IL-33 protein can attenuate severity of the LN in the animal model of SLE. The above results suggest that IL-33/ST2 pathway may participate in the kidney target SLE immune activation, and antagonized the activated pathway may become a new approach for the treatment of LN. But the mechanism about how the IL-33/ST2 pathway involving in lupus nephritis inflammation injury is still unclear. Our study aimed to clarify these questions: Whether IL-33 derived from the kidney of LN.Which cells may release the IL-33, and whether its level is associated with the severity of kidney damage. Further more, we will study which immune cells express ST2 receptor in peripheral blood, and how IL-33 and immune cells interact to injure the kidney.Material and Methods:1.Grouping Matched group:8 weeks old SPF level female C57BL/6j mice;LN modelgroup: 8 weeks old SPF level female MRL/1pr mice; Five for each group. Two groups were observed in general and tested the level of urinary protein. All of themice sacrificed in 18weeks,testing the level of serum ds-DNA,BUN and Scr and observing on renal lesion in renal tissue of HE, PASM, MASSON staining.2.Real time PCR examed the renal tissue IL-33 mRNA expression level in 2 groupsof mice. Western blot examed the renal tissue IL-33 protein expression level in 2 groups of mice and then analyzed the correlation between IL-33 expression levels and urinary protein, serum ds-DNA.3.Immunohistochemistry expressed IL-33 in renal tissue of position, further more,double immunofluorescence localized IL-33 in derived cells.4.ELISA tested serum levels of IL-33, sST2 in the LN patients with or without flare.5.Flow cytometry Detected the expression levels of surface ST2 receptors onperipheral blood CD4+T cells and CD14+ mononuclear cells in SLE patients and the healthy control.Results:1. The general situation and urinary protein levels in miceTwo groups of mice were survived before sacrificing. C57 group is a good mental state, good appetite, activity, fur shiny, no hair, no joint swelling of limbs. MRL/lpr group decreased appetite, lazy in different degree edema, color of hair lacks luster, but no obvious epilation. Some mice had mild to moderate swelling of knee joint. The testing of urine protein began form 14 weeks old every two weeks. All MRL/lpr mice detected in deferent degree urine protein but did not increase overall. The urine protein of C57 mice always was negative.2. The level of serum BUN, Scr and ds-DNA in miceMRL/lpr mice serum BUN level is 10.04±1.79mmol/L,while C57 mice’s is 7.42±1.00 mmol/L.There is no statistical significance between two groups(p=0.300). MRL/lpr mice serum Scr level is 25.60±7.30 ummol/L,while C57 mice’s is 25.75±8.54ummol/L.There is also no statistical significance between two groups(p=0.878).The serum ds-DNA is positive in all MRL/lpr mice(from 2+ to 4+) ,but negative in C57 mice. There is statistical significance between two groups(p=0.003)3. General and special pathology staining in miceHE staining:Renal glomerular structure clearly displays and the ball can be seen in the vascular loop in C57 mice. Disordered glomerular hypertrophy and large lymphocytes infiltrate in renal interstitium in MRL/lpr mice. PASM staining: MRL/lpr mice’s Glomerular basement membrane is thick, endothelial and epithelial cells hyperplasia, while the basement membrane in C57 mice is normal. Masson staining: lots of eosinophilic immune complex deposit in glomerular and protein tube s in the renal tubules in MRL/lpr mice. There is neither eosinophilic immune complex nor protein tubes in the kidney of C57 mice.4. IL-33 expression in the mice kidney tissueMRL/lpr group increase the expression of IL-33 mRNA by an average of 2.73 ±1.34 times comparing with C57 group, a significant difference between two groups (p=0.045). It indicates that MRL/lpr mice has up-regulated the expression of IL-33 in gene levels. By western blot, MRL/lpr group also significantly increased the expression of IL-33 protein (OD value 0.558±0.094 vs. 0.034±0.002).The expression of C57 is little, a significant difference between two groups (p=0.0005). It indicates that MRL/lpr mice have up-regulated the expression of IL-33 in protein levels.5. Correlation analysis between IL-33 and urine protein/serum ds-DNA in renal tissue of MRL/lpr miceThe spearman correlation coefficient indicates that a significant positive correlation between the changes of MRL/lpr mice in renal tissue of IL-33 gray ratio and semi quantitative urine protein (r2=0.72, p=0.037).Although there is no significant relation between il-33 and Semi quantitative ds-DNA level (r2=0.386, p=0.215), but positive correlation trend between them.6. The expression of IL-33 in renal tissue cells of miceBy immunochemistry, IL-33 expresses in cells of the glomerular basement membrane zone, but rare in renal interstitial in MRL/lpr. C57 significantly reduces the expression of IL-33.Further, double Immunofluorescence shows that the expression of IL-33 by Glomerular endothelial cells.7. The expression of ST2 receptor in Peripheral blood cells of LN and healthy controlST2 receptor is up-regulates on the surface of CD4+T cells and CD14+ mononuclear cells in Peripheral blood of LN patients, compared with the healthy control. There is significant different between them.Conclusions:1. MRL/lpr renal tissue up-regulates IL-33 expression. The level positively relates to urinary protein level which could reflect the degree of kidney damage.2. IL-33 comes from Glomerular endothelial cells in the renal tissue when the tissue suffers injury.3. LN patients up-regulate ST2 receptor on the surface of CD4+T cells and CD14+ mononuclear cells in Peripheral blood. It may indicate that IL-33 induces inflammatory injury by Mediating peripheral blood mononuclear cells and CD4+T cells infiltrating into the renal tissue.Key words: systemic lupus erythematosus；alarmin；interleukin-33；ST2主要英文缩写及中英文对照表英文缩略词英文全称中文全称IL-33 interleukin-33 白介素-33SLE systemic lupus erythematosus 系统性红斑狼疮LN lupus nephritis 狼疮肾炎DMARDS Diseases Modifying Antirheumatic Drugs 缓解病情抗风湿药RA rheumatoid arthritis 类风湿关节炎APC antigen presenting cells 抗原递呈细胞T-reg cell T- regulatory cell 调节性T细胞PAMPs pathogen associated molecular patterns 病原相关分子模式DAMPs damage associated molecular patterns 损伤相关分子模式PRRs pattern recognition receptors 模式识别受体HMGB1 high mobility group protein B1 高迁移速率组蛋白B1 HDGF hepatomaderived growth factor 肝癌来源的生长因子IL-1RAcP interleukin-1 receptor accessory protein IL-1受体辅助蛋白MAPK mitogen-activated protein kinase 丝分裂原激活蛋白激酶NF-κB nuclear factor-κB 核因子κBsST2 Soluble ST2 可溶性ST2PBMC peripheral blood mononucleated cell 外周血单个核细胞Scr serum creatinine 血清肌酐BUN blood urea nitrogen 血液尿素氮ds-DNA Double stranded-DNA 双链DNAGAPDH glyceraldehyde-3-phosphate dehydrogenase 磷酸甘油醛脱氢酶IFN-γ interferon-γγ干扰素TNF-a tumor necrosis factor-a 肿瘤坏死因子-a NKT cell natural killer cell 自然杀伤细胞MDSCs myeloid-derived suppressor cells 骨髓来源的抑制性细胞SLEDAI SLE Disease Activity Index SLE疾病活动性指数第一章前言一、系统性红斑狼疮概述系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种遗传、性激素、环境、感染等多因素参与其中的特异性自身免疫性疾病，其特征性表现为全身多系统和脏器受累，血清中存在大量自身抗体。