【作者】 单宁宁；

【导师】 侯明；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 免疫性血小板减少性紫癜(immune thrombocytopenia, ITP),亦称特发性血小板减少性紫癜(idiopathic thrombocytopenic purpura, ITP),是一种自身免疫性出血性疾病,该病的特点是患者体内抗血小板抗体增多,血小板破坏过多而出血。其发病机制目前尚未完全明了,但确认与免疫应答异常有关。近年来,国内外学者在ITP方面进行了大量的基础和临床研究,虽取得阶段性成果,但其发病机制、早期诊断和治疗措施仍处于探索之中。深入研究ITP发病免疫异常机制,探讨阻断ITP发病途径无疑对于ITP的诊治具有重要意义。大量实验研究表明,ITP的发病与细胞免疫功能异常关系密切。尽管B细胞过度增殖、活化和成熟产生多种针对血小板的自身抗体(包括抗血小板膜糖蛋白(glycoprotein,GP)Ⅱb/Ⅲa、GP Ib抗体)是ITP发病的直接原因,但T细胞亚群功能和比例失调导致B细胞激活可能才是ITP主要的发病机制,部分情况下,细胞毒性T细胞可直接导致ITP患者的血小板溶解。许多研究表明在ITP中,T辅助细胞(Th)1和Th2处于失衡状态,Th1／Th2比例明显上调。Th可以分化为Th1、Th2和Th17亚群。目前研究资料表明,Th1／Th2细胞的平衡,是体内体液免疫及细胞免疫间重要的调节枢纽,在自身免疫病的发生发展过程中起重要作用。Th1细胞主要分泌IL-2,IFN-γ,TNF-α等细胞因子,介导迟发型超敏反应(DTH)和巨噬细胞活化等细胞免疫反应；Th2细胞主要分泌IL-4,IL-5,IL-10和IL-13等细胞因子,促进B细胞增殖并分化成浆细胞,分泌特异性抗体,提高黏膜免疫力,介导体液免疫和Ⅰ型超敏反应。近年来,研究发现CD8+T至少也可分为两群,一群与Th1细胞相似,分泌IL-2,IFN-γ,TNF-α等细胞因子,称为Tc1；另一群与Th2细胞相似分泌IL-4,IL-5,IL-10等细胞因子,称为Tc2。人们将Th1和Tc1,Th2和Tc2分别统称为Th1类、Th2类细胞。Th细胞之间的失衡与许多疾病的发生、发展密切相关,多数活动性器官自身免疫病倾向于Th1细胞反应,而Th2细胞反应往往与自身免疫病保护有关。本研究证实ITP患者存在Th1极化现象,与Panitsas FP等人研究相符。作为T细胞炎症介质的强有力的诱导者,IL-18通过刺激Th1反应、诱导严重免疫紊乱,从而参与一些免疫疾病的发生发展。IL-18是1996年正式命名的细胞因子,主要由单核巨噬细胞产生,是一种强有力的免疫调节因子,其结构与IL-1相似,功能与IL-12相似,但发挥作用却不依赖于两者。人在正常状态下,产生于细胞内的IL-18是以没有生物活性的前体形式存在,由193个氨基酸组成,无N糖基化位点和疏水信号肽序列,N-端有一36个氨基酸的前导序列。经IL-1β转化酶(IL-1βconverting enzyme, ICE)切除其前导序列后,IL-18才以单体的形式表现出生物学活性。与IL-18受体(interleukin 18 receptor, IL-18R)结合,通过IL-1受体相关激酶(interleukin-1 receptor-associated kinase, IRAK)途径活化肿瘤坏死因子受体相关因子6(TNF receptor-associated factor 6, TRAF-6)诱导NF-κB结合DNA信息,促进INF-γ等Th1细胞相关因子的释放而导致Th1／Th2失衡的发生。IL-18还可增强Th1细胞的FasL表达,增强FasL介导细胞毒效应,促使T、B细胞的凋亡发生,而凋亡细胞数量与自身免疫疾病活动性相关,因而IL-18在免疫系统和疾病的自我调节过程中发挥重要作用。IL-18结合蛋白(interleukin 18 binding protein, IL-18BP)为Novick等新近发现的一种糖蛋白,属于免疫超家族成员,能抑制IL-18诱导Th1细胞产生INF-γ,降低INF-γ对NF-κB的激活作用,是IL-18的天然拮抗剂,与IL-18协调作用参与细胞免疫反应。大量研究证实IL-18和IL-18BP在自身免疫性疾病的发生、发展中发挥重要的作用：在系统性红斑狼疮(systemic lupus erythematosus, SLE)、风湿性关节炎(rheumatoid arthritis,RA)等自身免疫性疾病的患者和动物模型中已经检测出上述因子mRNA和蛋白质的表达失衡,而通过内外源性IL-18BP抑制IL-18的作用已成为抗自身免疫性疾病的重要着眼点。但是,IL-18体系在ITP的疾病进程中的作用还没有得到清楚的了解。近年来,T细胞免疫球蛋白粘蛋白分子-3(T cell immunoglobulin-and mucin domain-containing molecules, Tim-3)在自身免疫性疾病中的作用日益受到人们的重视。Tim-3又称HAVcr-2(hepatitis A vires cell receptor 2),只选择性表达在分化的Th1细胞而不是Th2细胞上,通过与Tim-3配体(Tim-3 ligant, Tim-3L)即半乳糖凝集素-9(Galectin-9, Gal-9)结合,给T细胞提供一种负性共刺激信号,下调Th1及Th17细胞的反应,在自身和异体免疫性疾病以及免疫耐受中起着重要作用。既然ITP是一种Th1极化模式的自身免疫性疾病,而Tim-3特异性的表达于Th1细胞中,那么Tim-3是否在ITP发病中发挥作用呢?本研究以IL-18,IL-18BP,Tim-3等细胞因子的mRNA和蛋白质表达为指标,从细胞和分子水平探讨IL-18体系及Tim-3在ITP的发生和发展的作用。第一部分IL-18／IL-18BP在ITP发病及地塞米松治疗中的意义研究目的：探讨ITP患者体内IL-18,IL-18BP,IFN-γ,IL-4；转录因子T-bet,GATA-3的表达及地塞米松的作用,分析IL-18／IL-18BP在ITP患者中Th1类细胞优势应答中的作用。方法：(1)抽取37例ITP急性期患者,21例ITP缓解期患者和24例正常人的外周血15mL,其中17例初诊患者标本分别在大剂量地塞米松治疗前和治疗后两周采样。(2)从外周血中分离单个核细胞,提取mRNA。(3)应用ELISA检测ITP急性期,缓解期患者和正常对照外周血IL-18,IL-18BP.IFN-γ和IL-4的含量。(4)应用RT-PCR检测ITP急性期,缓解期患者和正常对照IL-18,IL-18BP,IFN-γ,IL-4和T-bet,GATA-3的表达。结果：(1)经ELISA检测,37例ITP急性期患者血浆中IL-18及IFN-γ的含量明显高于对照组和缓解期患者；IL-4的含量明显低于对照组；而IL-18BP的含量没有明显变化,IL-18／IL-18BP升高。(2)经RT-PCR检测,ITP急性期患者外周血单个核细胞的IL-18,IFN-γ和T-bet的表达明显升高；IL-4和GATA-3的表达明显降低,IL-18BP的含量没有明显变化。(3)大剂量地塞米松治疗后,IL-18,IFN-γ和T-bet含量明显降低,IL-18BP含量明显升高。结论：(1)证实了IL-18,IL-18BP参与了ITP发病；(2)大剂量地塞米松能够促进IL-18BP的生成,拮抗IL-18的作用而缓解ITP的发病；(3)应用外源性IL-18BP干预IL-18的作用可能成为ITP治疗的一个新靶点。第二部分IL-18BP对ITP患者外周血单个核细胞免疫干预机制探讨目的：研究外源性IL-18BP对ITP外周血单个核细胞(PBMCs)中IFN-γ,IL-4,IL-10等细胞因子的表达及细胞凋亡的影响,初步探讨IL-18BP在ITP免疫治疗中的作用和意义。方法：(1)收集我院2007年12月—2008年12月10例初诊ITP急性期患者及10例正常对照；(2)从外周血中分离单个核细胞,取不同浓度的IL-18BP与PBMCs细胞共同培养48h；(3)采用ELISA和RT-PCR的方法检测IFN-γ,IL-4,IL-10,IL-2及TNF-α蛋白和mRNA的表达；(4)CCK-8法检测IL-18BP作用后淋巴细胞的增殖情况；(5)AnnexinV-FITC/PI流式细胞技术检测IL-18BP作用后淋巴细胞凋亡的情况。结果：(1)IL-18BP明显抑制ITP患者和正常对照组PBMCs中IFN-γ蛋白和mRNA的表达(P<0.01),与对照组相比,ITP组抑制更为明显(P<0.01)；同时IL-18BP能促进ITP患者和正常对照组PBMCs中IL-10的表达；(2)IL-18BP对ITP患者和正常对照组的淋巴细胞增殖没有影响(P>0.05)；(3)AnnexinV-FITC-FCM分析见,ITP急性期PBMCs加入IL-18BP后凋亡细胞增加,而正常对照组无差异(P>0.05)。结论：IL-18／IL-18BP在ITP的免疫紊乱和发生发展中起着一定的作用,外源性IL-18BP能够抑制ITP患者体外PBMCs中IFN-γ的表达,促进IL-10的生成；还可通过调节淋巴细胞凋亡对ITP免疫失调起调节作用。也提示我们通过增加IL-18BP含量拮抗IL-18产生和活性可能是ITP治疗的一个策略和方向。第三部分IL-18／IL-18BP及Tim-3在ITP患者脾脏的表达目的：探讨ITP患者脾脏IL-18,IL-18BP,IFN-γ,IL-4,Tim-3及转录因子T-bet,GATA-3的表达,分析IL-18,IL-18BP,Tim-3在ITP患者中Th1类细胞优势应答中的作用。方法：(1)收集我院2007年12月—2008年12月ITP患者行外科手术治疗脾切除标本12例；同时另选2001年1月—2008年12月因创伤性脾破裂而手术切除的正常脾标本10例作对照研究；(2)采用免疫组化检测ITP及对照组脾脏IL-18,IL-18BP,Tim-3的表达；(3)采用免疫荧光检测ITP及对照组脾脏IL-18,IL-18BP,Tim-3的表达;(4)从脾中分离单个核细胞,置于含PHA 10μg／mL,10%FCS的RPMI1640完全培养基中；在37℃,5%CO2培养箱中培养48小时后收集细胞悬液及PBMCs备用；(5)采用ELISA的方法检测IFN-γ,IL-4,IL-18及IL-18BP蛋白的表达;(6)采用RT-PCR的方法检测IFN-γ,IL-4,IL-18及IL-18BP及转录因子T-bet,GATA-3的表达。结果：(1)免疫组化及免疫荧光结果显示：IL-18及IL-18BP在ITP患者脾脏中的表达明显高于正常对照组；而Tim-3在ITP患者脾脏中的表达和阳性率均低于正常对照组：(2)脾PBMCs培养48小时后,ITP患者IL-18和IFN-γ的含量较正常对照明显升高；IL-18BP含量两组间无统计学意义(P=0.646)；IL-4低于检测限；(3)RT-PCR检测脾PBMCs中细胞因子mRNA结果显示ITP患者IL-18的表达是正常对照的3倍,IL-18BP的含量较正常对照明显升高。Th1细胞因子IFN-γ及转录T-bet表达升高,而Th2细胞因子IL-4和转录因子GATA-3的含量较正常对照明显下降,比较具有统计学意义(P<0.05)。脾PBMCs在PHA的刺激下培养48h后ITP患者IL-18和IFN-γ的表达较正常对照明显升高。结论：(1)ITP患者脾脏中存在Th1／Th2细胞因子极化；(2)ITP患者脾脏中Tim-3-Gal-9通路失衡,可能参与ITP的发病；(3)ITP患者脾脏CD4+T细胞分化紊乱,分泌Thl细胞因子(IL-18,IFN-γ)能力异常升高。更多还原

【Abstract】 Immune thrombocytopenia (ITP) is an acquired chronic autoimmune-mediated bleeding disorder in which platelets are opsonized by platelet autoantibodies that target several platelet glycoproteins including GPIIb/IIIa and GPIb/IX and prematurely destroyed in the reticuloendothelial system of the spleen, liver or bone marrow. Apart from phagocytosis, destruction mechanisms include complement activation and T cell abnormalities.It has become evident that T helper 1-(Th1) and Th2 cells might have pathogenetic importance in ITP.T-box expressed in T cells (T-bet) and GATA-binding protein 3 (GATA-3) are two major T transcription factors that regulate the expression of Thl or Th2 cytokine genes respectively and play a crucial role in T-cell differentiation. We, hereby, evaluated the relevance of Th1 type (i.e., IFN-γand T-bet) versus Th2 type (i.e., IL-4 and GATA-3) in the pathogenesis of ITP focusing on the critical role of IL-18.This latter cytokine was originally termed as IFN-y inducing factor that belongs to the IL-1 cytokine superfamily. IL-18 is an inflammatory cytokine, not only predominantly produced by Kupffer cells in the liver, but also expressed in pancreas, kidney, skeletal muscle, lung, osteoblasts, and keratinocytes. Up-regulation of IL-18 has previously been detected in multiple sclerosis (MS), lupus nephritis (LN), atopic dermatitis.Our data strongly support the notion that up-regulation of IL-18 may be an important determinant in the evolution of ITP. The biologic effects of IL-18 by promoting a cytokine imbalance toward a Th1-type immune response may induce ITP. IL-18BP is a constitutively secreted protein able to bind IL-18 with high-affinity, providing a potential mechanism whereby IL-18 activity is regulated. IL-18BP is induced by IFN-y, thus establishing a negative feedback loop that can be observed in vitro and in vivo. Human IL-18BPa and IL-18BPc isoforms are capable of binding to and neutralizing IL-18. The affinity of IL-18BPc isoform is 10 fold less than IL-18BPa isoform. IL-18BPa which was measured in this study is the main IL-18BP in humans. Data showed IL-18BP inhibits IL-18-induced IFN-y and IL-8 production and NF-κB activation in vitro and LPS-induced IFN-y production in vivo. We hypothesized both the onset and aggravation of ITP are promoted by an imbalance of immune response towards a Thl cytokine predominance due to IL-18 and related cytokine up-regulation.Up-to-date, the balance between IL-18 and IL-18BP in patients with ITP is still unknown. In the present study, we have explored the hypothesis that the imbalance of IL-18 and IL-18BP may be of importance in ITP. Plasma levels as well as mRNA expression in peripheral blood mononuclear cells (PBMCs) of IL-18, IL-18BP and other cytokines were measured in patients with active ITP, patients in remission and in healthy volunteers to investigate whether IL-18BP could act as a possible therapeutic approach against ITP.I Effects of IL-18/IL-18BP on the onset of ITP and mechanism of action of dexamethasoneObjective:To evaluate the balance of interleukin (IL)-18 and its endogenous antagonist IL-18 binding protein (IL-18BP) and the effects of high dose dexamethasone (HD-DXM) in patients with immune thrombocytopenia (ITP), plasma IL-18,IL-18BP, IFN-γand IL-4 levels, as well as platelets counts were measured in patients with active ITP (n=37), ITP in remission (n=21),ITP receiving HD-DXM(n=17) and in healthy subjects (n=24) were measured.Methods:(1)37 ITP patients were enrolled in this study. Blood sampling was performed before and after treatment at the end of the second week with high dose dexamethasone (HD-DXM);(2)Plasma IL-18, IL-18BP, IFN-y and IL-4 levels, as well as platelets counts were measured in patients with active ITP (n=23), ITP in remission (n=21), and in healthy subjects (n=24) by enzyme linked immunosorbent assay (ELISA);(3) IL-18, IL-18BP levels as well as IFN-y, IL-4 plasma levels and platelets counts were determined in 17 ITP patients before and after HD-DXM treatment and in 24 healthy subjects;(4) Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IL-18, IL-18BP,IFN-y,IL-4,T-box (T-bet) and GATA-binding protein 3(GATA-3) were studied in all subjects.Results:(1) IL-18 and IFN-γprotein and mRNA levels were significantly increased in patients with active ITP than in control subjects, but the IL-18BP were not significantly elevated in ITP patients, which resulted in an elevated ratio of IL-18/IL-18BP in patients with active disease.(2) HD-DXM administration increased IL-18BP levels significantly (P＜0.01) and reduced IL-18 expression (P＜0.05), which resulting in a downregulation of IL-18/IL-18BP ratio (P＜0.05), IFN-γand T-bet were decreased (P＜0.05) whereas IL-4 and GATA were increased (P＜0.05) after HD-DXM treatment.Conclusions:(1)Persistent overproduction of IL-18 in ITP is associated with disease progression and IL-18BP might inhibit this activity. (2)The HD-DXM-mediated Thl/Th2 cytokine profile alterations observed in this study could be the results of a downregulation of IL-18 and other Thl cytokines by induction of IL-18BP while permitting the production of Th2 cytokines.(3)These findings suggest blockage of IL-18 bioactivities by IL-18BP is likely a promising therapeutic concept.ⅡRecovery of Thl/Th2 balance in immune thrombocytopenia pateints through the actions of the cytokine IL-18BPObjective:To investigate the effects of IL-18BPa/Fc on the production of cytokines in the peripheral blood mononuclear cells (PBMCs) from patients with immune thrombocytopenia (ITP),10 ITP patients and 10 health controls were enrolled in this study.Methods:(1) 10 ITP patients and 10 health controls were enrolled in this study.(2) IFN-γ, IL-2, TNF-α, IL-4, IL-5 and IL-10 levels in the supernatants were determined after 48h of PBMCs culture with or without addition of IL-18BPa/Fc by ELISA.(3) Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IFN-γ, IL-4 and IL-18R were studied in all subjects.(4) Meanwhile, proliferation of PBMCs was examined by CCK-8 assay.(5) Flow cytometry (FCM) was applied to detect the apoptosis of cells by staining with annexinV-FITC/PI.Results:(1) IL-18BPa/Fc (200 ng/mL) had a significant effect on secretion of IL-10 from healthy and ITP patients PBMCs, while diminishing IFN-γrelease from cultures of PBMCs. Notably, IL-18BPa/Fc further reinforced dexamethasone (5 nM)-mediated reduction of PHA-induced IFN-γproduction by an additional 49.9%.(2) Compared with healthy controls, the annexin V% was significantly decreased in ITP patients (ITP:7.1±3.2%, controls:10.9±2.1%, P=0.02). IL-18BPa/Fc significantly increased the annexin V% in ITP patients(p=0.04) but not in controls(p=0.96). The annexin V% in ITP patients in groupⅠ(IL-18BPa/Fc Ong/mL), groupⅡ(IL-18BPa/Fc lOng/mL) were 7.1±3.2% and 9.1±5.1% respectively, The annexin V% in controls in groupⅠand groupⅡwere 10.9±2.1% and 10.8±2.6% respectively.(3) As expected, significant difference was not found between the treated and the untreated group for the growth stimulating activity, indicating the addition of exogenous IL-18BPa/Fc exerted the marginal roles on cell proliferation. The results showed IL-18BPa/Fc indeed had no direct effect of stimulating cells proliferation.(4) After 48h culture with IL-18BPa/Fc, the expression of IL-18R protein and mRNA in PBMCs of ITP patients and controls was unaffected. Responsiveness of human IL-18R to IL-18BPa/Fc was obvious and reproducible. These observations revealed modulation of IL-18R expression was possibly irrelevant to IFN-y downregulation induced by IL-18BPa/Fc.Conclusions:The present data demonstrate that IL-18BPa/Fc may play a therapeutic role in ITP by downregulation of IFN-y and other Thl cytokines while permitting the production of Th2 cytokines (IL-10) via neutralizing the biologic activity of mature IL-18.ⅢAbnormal-expression of IL-18, IL-18BP and Tim-3 in the spleen of ITPObjective:To evaluate the balance of IL-18,IL-18BP and T cell immunoglobulin and mucin domain-3(Tim-3) in spleen of ITP, IL-18, IL-18BP and Tim-3 were measured. Methods:(1) 12 ITP patients spleens and 10 controls spleens were enrolled in this study.(2) IFN-y, IL-4, IL-18 and IL-18BP levels in the supernatants were determined after 48h of PBMCs culture.(3) Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IL-18, IL-18BP, IFN-γ, IL-4, T-box (T-bet) and GATA-binding protein 3(GATA-3) were studied in all subjects.(4) Using immunohistochemistry (IHC) and immunofluorescence, the expression of IL-18, IL-18BP and Tim-3 were studied in all subjects.Results:(1) The expression of IL-18, IL-18BP were much high in ITP patients than in normals using immunohistochemistry (IHC) and immunofluorescence; while the expressions of Tim-3 were declined in ITP than in normals.(2) IL-18 and IFN-y levels were significantly increased in patients with ITP than in control subjects after 48h of spleens lymphocytes culture, but the IL-18BP were not significantly elevated in ITP patients. The level of IL-4 was below the detectable limit of the assay used.(3)Using the REST software, the data are presented as the fold change in gene expression normalized to an endogenous reference gene and relative to healthy controls. The relative amount of mRNA gene expression of IL-18 and IFN-y were increased in active patients spleens compared to healthy controls (P＜0.01). IL-18 was up-regulated in active ITP patients compared to controls (P＜0.01).T-bet mRNA expression was significantly higher in active ITP patients compared to that of the control group.The decrease of IL-4 and GATA-3 was also observed.Conclusions:(1) We observed Thl polarization of the immune response in ITP spleens.(2) The abnormal pathway of Tim-3-galecti-9 may contribute to the ITP pathogenesis.更多还原