天然免疫分子TLR4在肾移植受者中的表达及其临床意义
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摘要
遗传基因不同的个体之间的器官移植,由于供、受体移植抗原的不同,可引起宿主的免疫反应,即排斥反应(rejection response)。随着肾移植技术的不断成熟和新型免疫抑制的应用,肾移植的成功率较以往大大改善,在防治肾移植术后排斥反应及各种并发症方面取得了较大进展,国内外的移植中心也积累了丰富的治疗经验。但是,排斥反应仍然是影响肾移植长期效果的重要因素。虽然近年关于肾移植术后排斥反应的基础和临床研究取得较大进展,但其发生的具体机制目前仍不是十分清楚。
目前的研究结果表明,器官移植后排异反应的发生机制和发生过程包括:移植物损伤、抗原的处理与递呈、抗原的识别与T细胞活化、免疫应答的生成与效应。在移植免疫中,T淋巴细胞在双信号刺激下才能被激活和发生增殖,从而启动由淋巴细胞、细胞因子、补体等参与的错综复杂的移植排斥反应。如果抗原提呈细胞(antigen present cell,APC)只提供MHC-抗原肽复合物而不提供CD80、CD86等共刺激信号,将使淋巴细胞无能,诱导免疫耐受。
当前,国内外关于移植免疫的研究主要集中在以T/B淋巴细胞及其下游细胞因子为中心的获得性免疫(adaptive immunity)过程,并取得了较大进展。基于相关的研究结果,大量的针对获得性免疫系统的抗排斥药物如钙调神经蛋白抑制剂(Calcineurin inhibitors,CNIs)如环孢素(CsA)、他克莫司(FK506),抗增殖类如硫唑嘌呤(Aza)、吗替麦考酚酯(MMF),抗淋巴细胞单/多克隆抗体如ATG、OKT3等相继面世,大大降低了肾移植术后急性排斥反应的发生率,肾移植术后人/肾存活率也较以往大大提高。但是,由于T/B淋巴细胞活化后移植免疫网络的复杂性,以该免疫网络某一个环节为靶点的治疗措施往往事倍功半,仍难以完全避免排斥反应的发生。
显然,针对淋巴细胞活化的上游信号传导者APC的干预措施,可以从源头上找到抑制移植免疫活化的靶点,在防治急性排斥反应方面将具有显而易见的优势。
天然免疫(innate immunity)是机体抵御外来入侵的第一道屏障,不仅可以直接杀伤外来入侵者,还可以将外来抗原提呈给T淋巴细胞,启动获得性免疫应答。APC表面共刺激分子(costimulatory molecule)的表达是决定T细胞对异基因免疫反应性的关键因素。APC表面CD80、CD86等共刺激分子的下调可以使APC转变为致耐受性细胞,使T细胞失去发生异基因反应的能力。但问题是,CD80、CD86的表达是如何调控的?由谁调控?
1997年,人类天然免疫模式识别受体Toll样受体(toll like receptors,TLRs)的发现使得以APC为主要研究对象的移植免疫研究成为可能。早期关于TLRs的研究主要集中在抗感染免疫方面,TLRs是天然免疫用以识别各种病原微生物保守序列中共有结构的“工具”,TLR与各种病原微生物共有的保守结构也即病原相关分子模式(pathogen-associated molecular patterns,PAMPs)结合后,激活相应的信号传导通路,促进单核/巨噬细胞、树突状细胞等抗原提呈细胞的成熟与活化,并通过核因子κB(nuclear factor-kappa B,NF-κb)和MAP激酶等通路上调CD80、CD86等共刺激分子的表达,将外来抗原提呈给下游淋巴细胞,启动天然免疫,消灭入侵的病原体。随着研究的进一步深入,发现TLRs还在移植免疫、动脉粥样硬化形成中起重要作用。其中,TLR4与移植免疫的关系似乎更为密切,其在器官移植中的研究也显示出极具诱人的前景。
目前国、内外关于TLR4在临床器官移植中的作用的研究刚刚起步,临床证据较少。已有的研究表明,TLR4和肝移植、肺移植等的急性排斥反应关系密切。TLR4的基因多态性影响肾移植急性排斥反应的发生率。具有TLR4变异体(Asp299Gly或Thr399Ile)的肺移植和肾移植患者,手术后急性排斥反应的发生次数和发生率显著低于TLR4正常者;在慢性排斥的心脏移植患者中,外周血单核细胞TLR4的表达水平升高。基于TLR4在免疫及炎症反应中的重要作用,国外有学者通过对TLR4和IL-6等炎症因子进行联合测定,以评估机体对病原体的防御能力。TLR4除在抗原提呈细胞(antigen present cell,APC)单核/巨噬细胞、树突状细胞表达以外,在肾小管上皮细胞、肾足细胞、系膜细胞表面也有表达,并可能参与免疫复合物相关性肾小球肾炎及移植物的排斥反应。
我们的研究首先以人外周血CD14阳性单核细胞为靶细胞,应用三色标记流式细胞技术,对天然免疫分子TLR4及其下游分子CD80在肾移植围手术期表达的变化规律进行监测,并研究TLR4与急性排斥反应的关系:考虑到TLR4的表达在围手术期可能被移植手术、应激反应、围手术期用药等影响,因此我们选取肾移植手术1年以后的患者为研究对象,以观察TLR4在此类患者发生急性排斥反应时的表达情况及其对激素冲击治疗的反应;最后,我们应用免疫组织化学方法,检测TLR4在发生排斥反应的移植肾组织的表达情况,以进一步探讨TLR4参与急性排斥反应的证据。
通过研究TLR4在肾移植受者中的表达情况,本文旨在探讨如下问题:1、TLR4水平的高低是否可以预测肾移植术后急性排斥反应的发生风险?2、TLR4在临床肾移植围手术期的动态变化规律及其与急性排斥反应的关系?3、肾移植远期发生急性排斥反应时TLR4的表达情况及抗排斥治疗对其影响;4、TLR4水平是否可以评估移植肾功能的长期预后?5、TLR4在发生急、慢性排斥反应的移植肾组织中的表达情况。
第一章肾移植围手术期患者Toll样受体4/CD80测定及其意义
目的为研究围手术期TLR4在移植免疫中的作用,我们以CD14阳性单核细胞为靶细胞,观察TLR4/CD80在肾移植围手术期的变化规律及其与急性排斥反应的关系,探讨TLR4参与围手术期急性排斥反应的可能机制。
方法选取初次肾移植受者32例,所有受者术前群体反应抗体检测均阴性。流式细胞术测定受者肾移植术前及术后第1、4、7、14、21、28、35d外周血单核细胞TLR4/CD80表达的百分率;根据肾移植术后2周内发生排斥与否将患者分为排斥组(7例)和无排斥组(25例)。选取10例PRA阴性的健康成年志愿者为正常对照组。急性排斥反应的诊断标准根据临床表现、实验室检查、移植肾多普勒超声检查、必要时行移植肾穿刺活检等综合判断。实验组测量值用均数±标准差((?)±s)表示,应用SPSS13.0统计分析软件对数据进行分析,各组间比较应用单因素方差分析(One-Way ANOVA)、不同时间点的比较应用重复测量的方差分析,P＜0.05为差异有统计学意义。
结果肾移植术前,外周血单核细胞TLR4、CD80的表达:排斥组为8.03±0.84%、0.85±0.3 1%(n＝7),无排斥组为6.14±0.85%、0.84±0.39%(n＝25),正常对照组为6.37±0.56%、0.85±0.35%(n＝10);排斥组TLR4的表达显著高于无排斥组和正常对照组(P＜0.01),无排斥组与正常对照组比较无显著差异(P＝0.448);三组之间CD80的表达无显著差异(P＝0.995)。肾移植术后,患者单核细胞TLR4/CD80的表达在第4d开始出现升高趋势,排斥组为16.5±1.02%、7.82±1.66%,无排斥组为11.6±0.98%、2.26±0.96%,较术前均显著升高(P＜0.01);第7天左右达高峰,排斥组为36.4±4.86%、9.53±1.97%,无排斥组为22.7±3.45%、1.87±0.72%,并随着肾功能的恢复逐渐回落,排斥组于第35天降至7.10±0.82%、0.87±0.57%,无排斥组第21天降至7.24±0.76%、0.81±0.37%;与无排斥组相比较,排斥组TLR4/CD80表达的峰值较高(P＜0.01),且持续时间较长。
结论术前单核细胞表面TLR4的表达水平可以预测肾移植术后急性排斥反应的发生风险,高TLR4的肾移植受者易于发生急性排斥反应;TLR4在肾移植术后早期上调,并随着肾功能的恢复逐渐回落至术前水平,围手术期发生急性排斥反应的患者,TLR4/CD80表达的峰值较高,持续时间较长;天然免疫分子TLR4通过刺激下游分子CD80的表达,启动获得性免疫反应,参与急性排斥反应的发生。
第二章Toll样受体4在肾移植术后急性排斥反应中的检测及意义
目的研究外周血单核细胞Toll样受体4(TLR4)在肾移植术后远期急性排斥反应发生时的表达情况,及甲基强的松龙(MP)治疗对TLR4的表达的影响。探讨TLR4在肾移植术后急性排斥反应监测中的临床应用价值。
方法选取13例肾移植手术1年后发生急性排斥反应的患者,应用流式细胞术测定甲基强的松龙(MP)冲击治疗前、后外周血CD14~+单核细胞TLR4表达的百分率,并与20例未发生排斥反应的移植肾功能正常患者相比较,20例健康志愿者作为正常对照。肾移植术后远期急性排斥反应的诊断标准:(1)临床表现为发热、尿量减少、血压升高、移植肾区胀痛、体重增加、尿蛋白增多、血肌酐升高,排除感染、外伤、严重心、脑血管疾病等。(2)彩色多普勒超声检查显示肾血管阻力指数升高,排除其他原因如梗阻、肾动脉狭窄等引起的肾功能损害。(3)肾活检病理符合移植肾急性排斥反应病理表现(Banff标准),排除慢性排斥反应、急性环孢素A肾中毒等原因导致的肾功能损害。实验组测量值用均数±标准差((?)±s)表示,应用SPSS13.0统计分析软件对数据进行分析,统计学方法采用单因素方差分析(One-Way ANOVA)和重复测量的方差分析进行统计学分析。P＜0.05为差异有统计学意义。
结果发生急性排斥反应的患者外周血CD14~+单核细胞TLR4表达的百分率(30.45±5.17%)显著高于移植肾功能正常组(6.01±0.93%,P＜0.01)及正常对照组(7.15±0.85%,P＜0.01);MP冲击治疗两周后,发生急性排斥反应的患者TLR4的表达显著下降(P＜0.01),其下降程度与激素治疗敏感与否有关。
结论TLR4在肾移植远期的急性排斥反中表达上调,MP冲击治疗显著降低TLR4的表达水平;激素治疗不敏感的患者,TLR4表达的水平较高;TLR4水平的监测似乎可以作为糖皮质激素治疗敏感性的判断指标;高TLR4水平可能提示移植肾功能预后不良。
第三章Toll样受体4在肾移植术后移植肾组织中的表达
目的研究Toll样受体4(TLR4)在肾移植术后急、慢性排斥反应移植肾组织中的表达情况,探讨TLR4参与移植免疫的相关机制。
方法选取2007年3月～2008年12月我院肾移植术后发生急、慢性排斥反应的移植肾组织标本各10例,正常肾组织标本10例。应用免疫组织化学方法并结合移植肾病理分级(Banff标准),分析TLR4在移植肾组织中的表达情况。采用SPSS13.0统计软件建立数据库。两组间的均数比较用独立样本f检验;多组间的均数比较采用单因素方差分析,组间两两比较用LSD法。P＜0.05为差异有显著性意义。
结果三组移植肾组织标本均有TLR4的表达。急性排斥反应组与慢性排斥反应组的表达强度均高于正常组,且差异有统计学意义(P＝0.000),而急性排斥反应组与慢性排斥反应组之间无显著差异(P＝0.889)。在急性排斥反应组中,TLR4的表达随移植肾病理分级升高而增强,病理分级Ⅰ级明显高于Ⅱ级(t＝6.894,P＝0.030);慢性排斥反应组TLR4的表达与病理分级无明显相关性(F＝0.026,P＝0.975)。移植肾组织中TLR4的来源主要是肾小管上皮细胞及侵入肾组织的单核细胞。
结果TLR4在发生急、慢性排斥反应的移植肾组织中均表达上调,其来源主要是肾小管上皮细胞及侵入肾组织的单核细胞。在急性排斥反应时,TLR4的表达随移植肾病理分级升高而增强。
In alloimmunity,T lymphocytes are activated by double signals,and then priming the complicated rejection procedure include lymphocytes,cytokins and complements.If antigen present cells(APCs) provide MHC-peptides only,do not present costimulatory signals such as CD80 and CD86 to T lymphocytes at the same time,T lymphocytes will become anergy,immune tolerance will be induced.
At present,research for alloimmunity is mainly focused on the immune procedure of T/B lymphocytes and their downstream cytokines.Because of the complexity of the immune procedure after T/B lymphocytes activation,treatments target on one point of the complicated procedure always obtain half the result with twice the effort.
Obviously,research focused on APCs-the transmitter of upstream signals for T/B lymphocytes are more superior.The expression of costimulatory molecules on APCs is crucial in determining T-cell immune responses to alloantigens.Decrease of costimulatory molecules such as CD80 and CD86 can converts APCs into tolerogenic cells.These APCs can deprive T lymphocytes the capacity of response to alloantigens.
But the question is,who is in charge of the regulatory of CD80 and CD86 expression? How the regulatory happened? In 1997,the discovery of toll like receptor(TLR)-human innate immunity pattern recognition receptor made it possible for the research focused on APCs in alloimmunity.In early time,the research for TLRs was mostly focused on anti-infection immunity.TLRs are‘tools' through which innate immunity identify various pathogenic microorganisms.When bind with conservative structure of pathogenic microorganism i.e pathogen-associated molecules,TLRs trigger natural immune response by including the production of inflammatory cytokines,that subsequently signal resulted in the induction of maturation of APCs such as mononuclear/macrophage cells and dendritic cells with up-regulated.Further research implied that TLRs played an important role in alloimmunity and atherogenesis.Among the total,the research of relationship between TLR4 and alloimmunity revealed attracting perspective.
Under the help of soluble CD14,TLR4 on APCs binding with LPS,the consequent signals activate NF-kappaB,induce the secretion of proinflammatory cytokine,up-regulation of CD80 and CD86,promote inflammatory process.Research implied,TLR4 may take part in alloimmunity procedure according to analogic pathway,the mechanism is still unclear.
At present,research for the role of TLR4 in clinical organ transplantation has just begin,clinical evidence were fewer.A recent clinical study suggests that recipients with a mutant TLR4 genotype(Asp299Gly or The399Ile) manifest reduced lung allograft rejection.Another research shows significantly elevated levels of TLR4 on circulating monocytes in hear-transpant recipients with chronic rejection when compare with control group.
Therefore,we investigate the development rule of innate immune molecules TLR4 in clinical renal transplantationand their roles in acute and chronic rejection by immune histochemistry and three-color fluorescent staining flow cytometry.Our study provided clinical and laboratory evidence for the role and clinical application value of TLR4 in organ transplantation,explored the mechanism of TLR4 involved in alloimmunity;We tried our best to offer new ideas for inducing immune tolerance in the future,developing new immunosuppressive drugs aimed at new target,the prevention and treatment of allograft rejection.
Chapter 1 Perioperative determination of toll-like receptor 4 and CD80 and their clinical significance in recipients of renal transplantation
Objective
To investigate the dynamic expression and clinical significance of TLR4/CD80 in patients before and early after renal transplantation,explore the relationship between the expression of TLR4/CD80 and acute rejection.
Method
32 patients who received renal transplantation in our hospital were involved in our research.All of them were primary recipients,and the level of panal reactive antibody(PRA) less than 1%.The expression of TLR4 and CD80 in CD14 positive monocyte of peripheral blood from patients on the 1~(st),4~(th),7~(th),14~(th),21~(th),28~(th) and 35 ~(th) days after transplantation were measured by three-color fluorescent staining flow cytometry.The patients were divided into rejection group(7 cases) and non-rejection group(25 cases) according to rejection episode record within two weeks.Normal control group(10 cases) was healthy adult volunteers.Diagnosis of acute rejection depended on clinical symptoms,lab test,color doppler sonography and renal biopsy. Results are expressed as mean±SD.A value of P＜0.05 was considered significant. Statistical analysis were performed using SPSS13.0 for windows.TLR4 values were expressed as percentage of positive CD 14+ monocytes.
Results
Before ransplanstation,the expression of TLR4 and CD80:8.03±0.84%and 0.85±0.31%(n=7) in rejection group,6.14±0.85%and 0.84±0.39%(n=25) in non-rejection group,6.37±0.56%and 0.85±0.35%(n=10) in normal control group. The expression of TLR4 of rejection group was higher than non-rejection group and normal control group(P<0.01).The expression of CD80 of three groups had no significant difference(P=0.995).After ransplanstation,the expression of TLR4 and CD80 in the two groups increased to 16.5±1.02%and 7.82±1.66%in rejection group, 11.6±0.98%and 2.26±0.96%in non-rejection group at the 4~(th) day,and got to peak at the 7~(th) day:36.4±4.86%and 9.53±1.97%in rejection group,22.7±3.45%and 1.87±0.72%in non-rejection group,then displayed downtrend,rejection group decreased to 7.10±0.82%and 0.87±0.57%at the 35~(th) day,non-rejection group decreased to 7.2±0.76%and 0.81±0.37%at the 21~(th) day.Compared with non-rejection group,rejection group showed higher peak value of expression of TLR4/CD80(P<0.01) and longer consistant time.
Conclusions
The high expression of TLR4 increased the risk of acute rejection.Early after renal transplantation,TLR4 was up-regulated and contributed to the happeness of acute rejection.
Chapter 2 Determination of toll-like receptor 4 in acute rejection after renal transplantation.
Objective
To investigate the relationship between the expression of TLR4 in CD14 positive monocyte of peripheral blood and acute rejection happened 1 year after transplantation,explore the change of TLR4 after methylprednisolone(MP) pulse therapy.
Method
13 recipients who developed acute rejection,20 recipients with normal graft function and 20 healthy volunteers were involved in our research.All of the recipients were performed renal transplantation in our hospital at least one year ago.We used flow cytometry to assess TLR4 expression among circulating CD14 positive monocytes before and after methylprednisolone(MP) pulse therapy in recipients with acute rejection.Results are expressed as mean±SD.A value of P＜0.05 was considered significant.Statistical analysis were performed using SPSS13.0 for windows.
Results
We demonstrated significantly higher TLR 4 expression on circulating monocytes among renal transplantation recipients with acute rejection compared with those in clinically stable with normal renal function and healthy volunteers.MP pulse therapy significantly inhibited the expression of TLR 4 on the monocytes of recipients with acute rejection.The degree of inhibition was related with the reactivity of MP pulse therapy.
Conclusions
The expression of TLR4 in acute rejection after renal transplantation was up-regulated.Monitoring of the level of TLR4 can be used to evaluate the reactivity of MP pulse therapy.
Chapter 3 Expression of toll-like receptor 4 in renal allograft tissue
Objective
To investigate the expression of TLR4 in the renal tissue of recipients with acute rejection and chronic rejection.
Method
20 recipients who underwent kidney transplantation in our hospital were included in this study:acute-rejection group(n＝10),chronic-rejection group(n＝10). 10 control group were included.We used immune histochemistry combined with Banff 97 classification to investigate the expression of TLR4 in graft tissue.Results are expressed as mean±SD.A value of P＜0.05 was considered significant. Statistical analysis were performed using SPSS13.0 for windows.
Results
The expression of TLR4 were found in all of three groups'graft tissue. Acute-rejection group and chronic-rejection group were higher than control group. No significance was found in acute-rejection group and chronic-rejection group.In acute-rejection group,the expression of TLR4 were in those patients who had higher classification of pathology.The main source of TLR4 were renal tubular epithelial cells and monocytes.
Conclusions
The expression of TLR4 were increased in renal allograft patients with acute rejection or chronic rejection.
目前的研究结果表明,器官移植后排异反应的发生机制和发生过程包括:移植物损伤、抗原的处理与递呈、抗原的识别与T细胞活化、免疫应答的生成与效应。在移植免疫中,T淋巴细胞在双信号刺激下才能被激活和发生增殖,从而启动由淋巴细胞、细胞因子、补体等参与的错综复杂的移植排斥反应。如果抗原提呈细胞(antigen present cell,APC)只提供MHC-抗原肽复合物而不提供CD80、CD86等共刺激信号,将使淋巴细胞无能,诱导免疫耐受。
当前,国内外关于移植免疫的研究主要集中在以T/B淋巴细胞及其下游细胞因子为中心的获得性免疫(adaptive immunity)过程,并取得了较大进展。基于相关的研究结果,大量的针对获得性免疫系统的抗排斥药物如钙调神经蛋白抑制剂(Calcineurin inhibitors,CNIs)如环孢素(CsA)、他克莫司(FK506),抗增殖类如硫唑嘌呤(Aza)、吗替麦考酚酯(MMF),抗淋巴细胞单/多克隆抗体如ATG、OKT3等相继面世,大大降低了肾移植术后急性排斥反应的发生率,肾移植术后人/肾存活率也较以往大大提高。但是,由于T/B淋巴细胞活化后移植免疫网络的复杂性,以该免疫网络某一个环节为靶点的治疗措施往往事倍功半,仍难以完全避免排斥反应的发生。
显然,针对淋巴细胞活化的上游信号传导者APC的干预措施,可以从源头上找到抑制移植免疫活化的靶点,在防治急性排斥反应方面将具有显而易见的优势。
天然免疫(innate immunity)是机体抵御外来入侵的第一道屏障,不仅可以直接杀伤外来入侵者,还可以将外来抗原提呈给T淋巴细胞,启动获得性免疫应答。APC表面共刺激分子(costimulatory molecule)的表达是决定T细胞对异基因免疫反应性的关键因素。APC表面CD80、CD86等共刺激分子的下调可以使APC转变为致耐受性细胞,使T细胞失去发生异基因反应的能力。但问题是,CD80、CD86的表达是如何调控的?由谁调控?
1997年,人类天然免疫模式识别受体Toll样受体(toll like receptors,TLRs)的发现使得以APC为主要研究对象的移植免疫研究成为可能。早期关于TLRs的研究主要集中在抗感染免疫方面,TLRs是天然免疫用以识别各种病原微生物保守序列中共有结构的“工具”,TLR与各种病原微生物共有的保守结构也即病原相关分子模式(pathogen-associated molecular patterns,PAMPs)结合后,激活相应的信号传导通路,促进单核/巨噬细胞、树突状细胞等抗原提呈细胞的成熟与活化,并通过核因子κB(nuclear factor-kappa B,NF-κb)和MAP激酶等通路上调CD80、CD86等共刺激分子的表达,将外来抗原提呈给下游淋巴细胞,启动天然免疫,消灭入侵的病原体。随着研究的进一步深入,发现TLRs还在移植免疫、动脉粥样硬化形成中起重要作用。其中,TLR4与移植免疫的关系似乎更为密切,其在器官移植中的研究也显示出极具诱人的前景。
目前国、内外关于TLR4在临床器官移植中的作用的研究刚刚起步,临床证据较少。已有的研究表明,TLR4和肝移植、肺移植等的急性排斥反应关系密切。TLR4的基因多态性影响肾移植急性排斥反应的发生率。具有TLR4变异体(Asp299Gly或Thr399Ile)的肺移植和肾移植患者,手术后急性排斥反应的发生次数和发生率显著低于TLR4正常者;在慢性排斥的心脏移植患者中,外周血单核细胞TLR4的表达水平升高。基于TLR4在免疫及炎症反应中的重要作用,国外有学者通过对TLR4和IL-6等炎症因子进行联合测定,以评估机体对病原体的防御能力。TLR4除在抗原提呈细胞(antigen present cell,APC)单核/巨噬细胞、树突状细胞表达以外,在肾小管上皮细胞、肾足细胞、系膜细胞表面也有表达,并可能参与免疫复合物相关性肾小球肾炎及移植物的排斥反应。
我们的研究首先以人外周血CD14阳性单核细胞为靶细胞,应用三色标记流式细胞技术,对天然免疫分子TLR4及其下游分子CD80在肾移植围手术期表达的变化规律进行监测,并研究TLR4与急性排斥反应的关系:考虑到TLR4的表达在围手术期可能被移植手术、应激反应、围手术期用药等影响,因此我们选取肾移植手术1年以后的患者为研究对象,以观察TLR4在此类患者发生急性排斥反应时的表达情况及其对激素冲击治疗的反应;最后,我们应用免疫组织化学方法,检测TLR4在发生排斥反应的移植肾组织的表达情况,以进一步探讨TLR4参与急性排斥反应的证据。
通过研究TLR4在肾移植受者中的表达情况,本文旨在探讨如下问题:1、TLR4水平的高低是否可以预测肾移植术后急性排斥反应的发生风险?2、TLR4在临床肾移植围手术期的动态变化规律及其与急性排斥反应的关系?3、肾移植远期发生急性排斥反应时TLR4的表达情况及抗排斥治疗对其影响;4、TLR4水平是否可以评估移植肾功能的长期预后?5、TLR4在发生急、慢性排斥反应的移植肾组织中的表达情况。
第一章肾移植围手术期患者Toll样受体4/CD80测定及其意义
目的为研究围手术期TLR4在移植免疫中的作用,我们以CD14阳性单核细胞为靶细胞,观察TLR4/CD80在肾移植围手术期的变化规律及其与急性排斥反应的关系,探讨TLR4参与围手术期急性排斥反应的可能机制。
方法选取初次肾移植受者32例,所有受者术前群体反应抗体检测均阴性。流式细胞术测定受者肾移植术前及术后第1、4、7、14、21、28、35d外周血单核细胞TLR4/CD80表达的百分率;根据肾移植术后2周内发生排斥与否将患者分为排斥组(7例)和无排斥组(25例)。选取10例PRA阴性的健康成年志愿者为正常对照组。急性排斥反应的诊断标准根据临床表现、实验室检查、移植肾多普勒超声检查、必要时行移植肾穿刺活检等综合判断。实验组测量值用均数±标准差((?)±s)表示,应用SPSS13.0统计分析软件对数据进行分析,各组间比较应用单因素方差分析(One-Way ANOVA)、不同时间点的比较应用重复测量的方差分析,P＜0.05为差异有统计学意义。
结果肾移植术前,外周血单核细胞TLR4、CD80的表达:排斥组为8.03±0.84%、0.85±0.3 1%(n＝7),无排斥组为6.14±0.85%、0.84±0.39%(n＝25),正常对照组为6.37±0.56%、0.85±0.35%(n＝10);排斥组TLR4的表达显著高于无排斥组和正常对照组(P＜0.01),无排斥组与正常对照组比较无显著差异(P＝0.448);三组之间CD80的表达无显著差异(P＝0.995)。肾移植术后,患者单核细胞TLR4/CD80的表达在第4d开始出现升高趋势,排斥组为16.5±1.02%、7.82±1.66%,无排斥组为11.6±0.98%、2.26±0.96%,较术前均显著升高(P＜0.01);第7天左右达高峰,排斥组为36.4±4.86%、9.53±1.97%,无排斥组为22.7±3.45%、1.87±0.72%,并随着肾功能的恢复逐渐回落,排斥组于第35天降至7.10±0.82%、0.87±0.57%,无排斥组第21天降至7.24±0.76%、0.81±0.37%;与无排斥组相比较,排斥组TLR4/CD80表达的峰值较高(P＜0.01),且持续时间较长。
结论术前单核细胞表面TLR4的表达水平可以预测肾移植术后急性排斥反应的发生风险,高TLR4的肾移植受者易于发生急性排斥反应;TLR4在肾移植术后早期上调,并随着肾功能的恢复逐渐回落至术前水平,围手术期发生急性排斥反应的患者,TLR4/CD80表达的峰值较高,持续时间较长;天然免疫分子TLR4通过刺激下游分子CD80的表达,启动获得性免疫反应,参与急性排斥反应的发生。
第二章Toll样受体4在肾移植术后急性排斥反应中的检测及意义
目的研究外周血单核细胞Toll样受体4(TLR4)在肾移植术后远期急性排斥反应发生时的表达情况,及甲基强的松龙(MP)治疗对TLR4的表达的影响。探讨TLR4在肾移植术后急性排斥反应监测中的临床应用价值。
方法选取13例肾移植手术1年后发生急性排斥反应的患者,应用流式细胞术测定甲基强的松龙(MP)冲击治疗前、后外周血CD14~+单核细胞TLR4表达的百分率,并与20例未发生排斥反应的移植肾功能正常患者相比较,20例健康志愿者作为正常对照。肾移植术后远期急性排斥反应的诊断标准:(1)临床表现为发热、尿量减少、血压升高、移植肾区胀痛、体重增加、尿蛋白增多、血肌酐升高,排除感染、外伤、严重心、脑血管疾病等。(2)彩色多普勒超声检查显示肾血管阻力指数升高,排除其他原因如梗阻、肾动脉狭窄等引起的肾功能损害。(3)肾活检病理符合移植肾急性排斥反应病理表现(Banff标准),排除慢性排斥反应、急性环孢素A肾中毒等原因导致的肾功能损害。实验组测量值用均数±标准差((?)±s)表示,应用SPSS13.0统计分析软件对数据进行分析,统计学方法采用单因素方差分析(One-Way ANOVA)和重复测量的方差分析进行统计学分析。P＜0.05为差异有统计学意义。
结果发生急性排斥反应的患者外周血CD14~+单核细胞TLR4表达的百分率(30.45±5.17%)显著高于移植肾功能正常组(6.01±0.93%,P＜0.01)及正常对照组(7.15±0.85%,P＜0.01);MP冲击治疗两周后,发生急性排斥反应的患者TLR4的表达显著下降(P＜0.01),其下降程度与激素治疗敏感与否有关。
结论TLR4在肾移植远期的急性排斥反中表达上调,MP冲击治疗显著降低TLR4的表达水平;激素治疗不敏感的患者,TLR4表达的水平较高;TLR4水平的监测似乎可以作为糖皮质激素治疗敏感性的判断指标;高TLR4水平可能提示移植肾功能预后不良。
第三章Toll样受体4在肾移植术后移植肾组织中的表达
目的研究Toll样受体4(TLR4)在肾移植术后急、慢性排斥反应移植肾组织中的表达情况,探讨TLR4参与移植免疫的相关机制。
方法选取2007年3月～2008年12月我院肾移植术后发生急、慢性排斥反应的移植肾组织标本各10例,正常肾组织标本10例。应用免疫组织化学方法并结合移植肾病理分级(Banff标准),分析TLR4在移植肾组织中的表达情况。采用SPSS13.0统计软件建立数据库。两组间的均数比较用独立样本f检验;多组间的均数比较采用单因素方差分析,组间两两比较用LSD法。P＜0.05为差异有显著性意义。
结果三组移植肾组织标本均有TLR4的表达。急性排斥反应组与慢性排斥反应组的表达强度均高于正常组,且差异有统计学意义(P＝0.000),而急性排斥反应组与慢性排斥反应组之间无显著差异(P＝0.889)。在急性排斥反应组中,TLR4的表达随移植肾病理分级升高而增强,病理分级Ⅰ级明显高于Ⅱ级(t＝6.894,P＝0.030);慢性排斥反应组TLR4的表达与病理分级无明显相关性(F＝0.026,P＝0.975)。移植肾组织中TLR4的来源主要是肾小管上皮细胞及侵入肾组织的单核细胞。
结果TLR4在发生急、慢性排斥反应的移植肾组织中均表达上调,其来源主要是肾小管上皮细胞及侵入肾组织的单核细胞。在急性排斥反应时,TLR4的表达随移植肾病理分级升高而增强。
In alloimmunity,T lymphocytes are activated by double signals,and then priming the complicated rejection procedure include lymphocytes,cytokins and complements.If antigen present cells(APCs) provide MHC-peptides only,do not present costimulatory signals such as CD80 and CD86 to T lymphocytes at the same time,T lymphocytes will become anergy,immune tolerance will be induced.
At present,research for alloimmunity is mainly focused on the immune procedure of T/B lymphocytes and their downstream cytokines.Because of the complexity of the immune procedure after T/B lymphocytes activation,treatments target on one point of the complicated procedure always obtain half the result with twice the effort.
Obviously,research focused on APCs-the transmitter of upstream signals for T/B lymphocytes are more superior.The expression of costimulatory molecules on APCs is crucial in determining T-cell immune responses to alloantigens.Decrease of costimulatory molecules such as CD80 and CD86 can converts APCs into tolerogenic cells.These APCs can deprive T lymphocytes the capacity of response to alloantigens.
But the question is,who is in charge of the regulatory of CD80 and CD86 expression? How the regulatory happened? In 1997,the discovery of toll like receptor(TLR)-human innate immunity pattern recognition receptor made it possible for the research focused on APCs in alloimmunity.In early time,the research for TLRs was mostly focused on anti-infection immunity.TLRs are‘tools' through which innate immunity identify various pathogenic microorganisms.When bind with conservative structure of pathogenic microorganism i.e pathogen-associated molecules,TLRs trigger natural immune response by including the production of inflammatory cytokines,that subsequently signal resulted in the induction of maturation of APCs such as mononuclear/macrophage cells and dendritic cells with up-regulated.Further research implied that TLRs played an important role in alloimmunity and atherogenesis.Among the total,the research of relationship between TLR4 and alloimmunity revealed attracting perspective.
Under the help of soluble CD14,TLR4 on APCs binding with LPS,the consequent signals activate NF-kappaB,induce the secretion of proinflammatory cytokine,up-regulation of CD80 and CD86,promote inflammatory process.Research implied,TLR4 may take part in alloimmunity procedure according to analogic pathway,the mechanism is still unclear.
At present,research for the role of TLR4 in clinical organ transplantation has just begin,clinical evidence were fewer.A recent clinical study suggests that recipients with a mutant TLR4 genotype(Asp299Gly or The399Ile) manifest reduced lung allograft rejection.Another research shows significantly elevated levels of TLR4 on circulating monocytes in hear-transpant recipients with chronic rejection when compare with control group.
Therefore,we investigate the development rule of innate immune molecules TLR4 in clinical renal transplantationand their roles in acute and chronic rejection by immune histochemistry and three-color fluorescent staining flow cytometry.Our study provided clinical and laboratory evidence for the role and clinical application value of TLR4 in organ transplantation,explored the mechanism of TLR4 involved in alloimmunity;We tried our best to offer new ideas for inducing immune tolerance in the future,developing new immunosuppressive drugs aimed at new target,the prevention and treatment of allograft rejection.
Chapter 1 Perioperative determination of toll-like receptor 4 and CD80 and their clinical significance in recipients of renal transplantation
Objective
To investigate the dynamic expression and clinical significance of TLR4/CD80 in patients before and early after renal transplantation,explore the relationship between the expression of TLR4/CD80 and acute rejection.
Method
32 patients who received renal transplantation in our hospital were involved in our research.All of them were primary recipients,and the level of panal reactive antibody(PRA) less than 1%.The expression of TLR4 and CD80 in CD14 positive monocyte of peripheral blood from patients on the 1~(st),4~(th),7~(th),14~(th),21~(th),28~(th) and 35 ~(th) days after transplantation were measured by three-color fluorescent staining flow cytometry.The patients were divided into rejection group(7 cases) and non-rejection group(25 cases) according to rejection episode record within two weeks.Normal control group(10 cases) was healthy adult volunteers.Diagnosis of acute rejection depended on clinical symptoms,lab test,color doppler sonography and renal biopsy. Results are expressed as mean±SD.A value of P＜0.05 was considered significant. Statistical analysis were performed using SPSS13.0 for windows.TLR4 values were expressed as percentage of positive CD 14+ monocytes.
Results
Before ransplanstation,the expression of TLR4 and CD80:8.03±0.84%and 0.85±0.31%(n=7) in rejection group,6.14±0.85%and 0.84±0.39%(n=25) in non-rejection group,6.37±0.56%and 0.85±0.35%(n=10) in normal control group. The expression of TLR4 of rejection group was higher than non-rejection group and normal control group(P<0.01).The expression of CD80 of three groups had no significant difference(P=0.995).After ransplanstation,the expression of TLR4 and CD80 in the two groups increased to 16.5±1.02%and 7.82±1.66%in rejection group, 11.6±0.98%and 2.26±0.96%in non-rejection group at the 4~(th) day,and got to peak at the 7~(th) day:36.4±4.86%and 9.53±1.97%in rejection group,22.7±3.45%and 1.87±0.72%in non-rejection group,then displayed downtrend,rejection group decreased to 7.10±0.82%and 0.87±0.57%at the 35~(th) day,non-rejection group decreased to 7.2±0.76%and 0.81±0.37%at the 21~(th) day.Compared with non-rejection group,rejection group showed higher peak value of expression of TLR4/CD80(P<0.01) and longer consistant time.
Conclusions
The high expression of TLR4 increased the risk of acute rejection.Early after renal transplantation,TLR4 was up-regulated and contributed to the happeness of acute rejection.
Chapter 2 Determination of toll-like receptor 4 in acute rejection after renal transplantation.
Objective
To investigate the relationship between the expression of TLR4 in CD14 positive monocyte of peripheral blood and acute rejection happened 1 year after transplantation,explore the change of TLR4 after methylprednisolone(MP) pulse therapy.
Method
13 recipients who developed acute rejection,20 recipients with normal graft function and 20 healthy volunteers were involved in our research.All of the recipients were performed renal transplantation in our hospital at least one year ago.We used flow cytometry to assess TLR4 expression among circulating CD14 positive monocytes before and after methylprednisolone(MP) pulse therapy in recipients with acute rejection.Results are expressed as mean±SD.A value of P＜0.05 was considered significant.Statistical analysis were performed using SPSS13.0 for windows.
Results
We demonstrated significantly higher TLR 4 expression on circulating monocytes among renal transplantation recipients with acute rejection compared with those in clinically stable with normal renal function and healthy volunteers.MP pulse therapy significantly inhibited the expression of TLR 4 on the monocytes of recipients with acute rejection.The degree of inhibition was related with the reactivity of MP pulse therapy.
Conclusions
The expression of TLR4 in acute rejection after renal transplantation was up-regulated.Monitoring of the level of TLR4 can be used to evaluate the reactivity of MP pulse therapy.
Chapter 3 Expression of toll-like receptor 4 in renal allograft tissue
Objective
To investigate the expression of TLR4 in the renal tissue of recipients with acute rejection and chronic rejection.
Method
20 recipients who underwent kidney transplantation in our hospital were included in this study:acute-rejection group(n＝10),chronic-rejection group(n＝10). 10 control group were included.We used immune histochemistry combined with Banff 97 classification to investigate the expression of TLR4 in graft tissue.Results are expressed as mean±SD.A value of P＜0.05 was considered significant. Statistical analysis were performed using SPSS13.0 for windows.
Results
The expression of TLR4 were found in all of three groups'graft tissue. Acute-rejection group and chronic-rejection group were higher than control group. No significance was found in acute-rejection group and chronic-rejection group.In acute-rejection group,the expression of TLR4 were in those patients who had higher classification of pathology.The main source of TLR4 were renal tubular epithelial cells and monocytes.
Conclusions
The expression of TLR4 were increased in renal allograft patients with acute rejection or chronic rejection.
引文
[1]李清芬.免疫应答的分子机制.见:龚非力主编.医学免疫学.第2版.北京:科学出版社.
[2]Appleman LJ,Boussiotis VA.T cell anergy and costimulation.Immunol Rev.2003 Apr;192:161-80.Review.
[3]Slavik JM,Hutchcroft JE,Bierer BE.CD28/CTLA-4 and CD80/CD86 families:signaling and function.Immunol Res.1999;19(1):1-24.Review.
[4]Flechner SM.Cyclosporine:a new and promising immunosuppressive agent.Urol Clin North Am.1983 May;10(2):263-75.Review.
[5]Halloran PF,Madrenas J.The mechanism of action of cyclosporine:a perspective for the 90's.Clin Biochem.1991 Feb;24(1):3-7.Review.
[6]Yin M,Ochs RS.Mechanism for the paradoxical inhibition and stimulation of calcineurin by the immunosuppresive drug tacrolimus(FK506).Arch Biochem Biophys.2003 Nov 15;419(2):207-13.
[7]Borel JF,Baumann G.Molecular mechanisms of immunosuppressive agents.Bull Mem Acad R Med Belg.1994;149(1-2):97-101.
[8]Taylor AL,Watson C J,Bradley JA.Immunosuppressive agents in solid organ transplantation:Mechanisms of action and therapeutic efficacy.Crit Rev OncoI Hematol.2005 Oct;56(1):23-46.Review.
[9]Superdock KR,Helderman JH.Immunosuppressive drugs and their effects.Semin Respir Infect.1993 Sep;8(3):152-9.Review.
[10]Khanna AK.Mechanism of the combination immunosuppressive effects of rapamycin with either cyclosporine or tacrolimus.Transplantation.2000 Aug 27;70(4):690-4.
[11]Dilioglou S,Cruse JM,Lewis RE.Function of CD80 and CD86 on monocyte-and stem cell-derived dendritic cells.Exp Mol Pathol.2003 Dec;75(3):217-27.
[12]Yamashita K, Masunaga T, Yanagida N, et al.Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies.Transplantation.2003 Oct 15;76(7):1089-96.
[13]Montgomery SP, Xu H, Tadaki DK, et al.Combination induction therapy with monoclonal antibodies specific for CD80, CD86, and CD 154 in nonhuman primate renal transplantation.Transplantation.2002 Nov 27;74(10): 1365-9.
[14]Jiga LP, Bauer TM, Chuang JJ,et al.Generation of tolerogenic dendritic cells by treatment with mitomycin C: inhibition of allogeneic T-cell response is mediated by downregulation of ICAM-1, CD80, and CD86.Transplantation.2004 Jun15;77(11): 1761-4.
[15]Medzhitov R, Preston-Hurlburt P, Janeway CA Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature.1997Jul 24;388(6640):394-7.
[16]Janeway CA Jr.Approaching the asymptote? Evolution and revolution in immunology.Cold Spring Harb Symp Quant Biol.1989;54 Pt 1:1-13.
[17]Takeda K, Akira S.Toll-like receptors in innate immunity.Int Immunol.2005 Jan;17(1):l-14.Review.
[18]Tesar BM, Goldstein DR.Toll-like receptors and their role in transplantation.Front Biosci, 2007, 12(5): 4221-38.
[19]Brandl K, Gluck T, Hartmann P, et al.A designed TLR4/MD-2 complex to capture LPS.J Endotoxin Res.2005;11(4):197-206.
[20]Philpott DJ, Girardin SE.The role of Toll-like receptors and Nod proteins in bacterial infection.Mol Immunol.2004 Nov;41(l 1):1099-108.Review.
[21]Means TK, Lien E, Yoshimura A, et al.The CD 14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors.J Immunol.1999 Dec 15;163(12):6748-55.
[22]Modlin RL.Activation of toll-like receptors by microbial lipoproteins: role in host defense.J Allergy Clin Immunol.2001 Oct;108(4 Suppl):S 104-6.Review.
[23]Schroder NW, Morath S, Alexander C, et al.Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), andCD14, whereas TLR-4 and MD-2 are not involved.J Biol Chem.2003 May 2;278(18):15587-94.
[24]Sivori S, Falco M, Delia Chiesa M, et al.CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells.Proc Natl Acad Sci U S A.2004 Jul 6;101(27):10116-21.
[25]Ishii KJ, Akira S.Toll-like Receptors and Sepsis.Curr Infect Dis Rep.2004 Oct;6(5):361-366.
[26]Lorenz E, Schwartz DA, Martin PJ, et al.Association of TLR4 mutations and the risk for acute GVHD after HLA-matched-sibling hematopoietic stem cell transplantation.Biol Blood Marrow Transplant.2001 ;7(7):384-7.
[27]Schmausser B, Andrulis M, Endrich S, et al.Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori.Int J Med Microbiol.2005 Jun;295(3): 179-85.
[28]Pespeni M, Mackersie RC, Lee H,et al.Serum levels of Hsp60 correlate with the development of acute lung injury after trauma.J Surg Res.2005 Jun l;126(1):41-7.
[29]Johnson GB, Brunn GJ, Platt JL.Cutting edge: an endogenous pathway to systemic inflammatory response syndrome (SIRS)-like reactions through Toll-like receptor 4.J Immunol.2004 Jan l;172(1):20-4.
[30]Liu L, Zhou X, Shi J,et al.Toll-like receptor-9 induced by physical trauma mediates release of cytokines following exposure to CpG motif in mouse skin.Immunology.2003 Nov;110(3):341-7.
[31]Kazemi MR, McDonald CM, Shigenaga JK,et al.Adipocyte fatty acid-binding protein expression and lipid accumulation are increased during activation of murine macrophages by toll-like receptor agonists.Arterioscler Thromb Vasc Biol.2005 Jun;25(6): 1220-4.
[32]Pasterkamp G, Van Keulen JK, De Kleijn DP.Role of Toll-like receptor 4 in the initiation and progression of atherosclerotic disease.Eur J Clin Invest.2004 May;34(5):328-34.Review.
[33]Bjorkbacka H, Kunjathoor VV, Moore KJ, et al。 Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways.Nat Med.2004 Apr; 10(4) :416-21.
[34]Hart AL, Al-Hassi HO, Rigby RJ, et ah Characteristics of intestinal dendritic cells in inflammatory bowel diseases.Gastroenterology.2005 Jul;129(1):50-65.
[35]Vives-Pi M, Somoza N, Fernandez-Alvarez J, et al.Evidence of expression of endotoxin receptors CD 14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and of sensitivity to endotoxin (LPS) in islet beta cells.Clin Exp Immunol.2003 Aug;133(2):208-18.
[36]Cario E, Podolsky DK.Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease.Infect Immun.2000 Dec;68(12):7010-7.
[37]Miyake K.Endotoxin recognition molecules, Toll-like receptor 4-MD-2.Semin Immunol.2004 Feb;16(1):l 1-6.Review.
[38]Espevik T, Latz E, Lien E, et al.Cell distributions and functions of Toll-like receptor 4 studied by fluorescent gene constructs.Scand J Infect Dis.2003;35(9):660-4.Review.
[39]Wang PL, Ohura K.Porphyromonas gingivalis lipopolysaccharide signaling in gingival fibroblasts-CD14 and Toll-like receptors.Crit Rev Oral Biol Med.2002;13(2):132-42.Review.
[40]Muzio M, Polentarutti N, Bosisio D, et al.Toll-like receptor family and signalling pathway.Biochem Soc Trans.2000 Oct;28(5):563-6.Review.
[41]Ellingsen E, Morath S, Flo T, et al.Induction of cytokine production in human T cells and monocytes by highly purified lipoteichoic acid: involvement of Toll-like receptors and CD14.Med Sci Monit.2002 May;8(5):BR149-56.
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[44]Schnare M, Barton GM, Holt AC, et al.Toll-like receptors control activation of adaptive immune responses.Nat Immunol.2001 Oct;2(10):947-50.
[45]Varadaradjalou S, Feger F, Thieblemont N, et al.Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells.Eur J Immunol.2003 Apr;33(4):899-906.
[46]Miller YI, Viriyakosol S, Binder CJ, et al.Minimally modified LDL binds to CD 14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells.J Biol Chem.2003 Jan 17;278(3):1561-8.
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[49]Yamamoto M, Sato S, Hemmi H, et al.TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway.Nat Immunol.2003 Nov;4(l 1):1144-50.
[50]Seya T, Oshiumi H, Sasai M, et al.TICAM-1 and TICAM-2: toll-like receptor adapters that participate in induction of type 1 interferons.Int J Biochem Cell Biol.2005 Mar;37(3):524-9.Review.
[51]Oshiumi H, Matsumoto M, Funami K, et al.TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction.Nat Immunol.2003 Feb;4(2):161-7.
[52]Akira S, Takeda K, Kaisho T.Toll-like receptors: critical proteins linking innate and acquired immunity.Nat Immunol.2001 Aug;2(8):675-80.Review.
[53]Pasare C, Medzhitov R.Toll-like receptors: linking innate and adaptive immunity.Microbes Infect.2004 Dec;6(15):1382-7.Review.
[54]Andrade CF, Waddell TK, Keshavjee S, et al.Innate immunity and organ transplantation: the potential role of toll-like receptors.Am J Transplant.2005 May;5(5):969-75.Review.
[55]Tsoulfas G, Takahashi Y, Ganster RW, et al.Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury.Transplantation.2002 Jul 15;74(1):7-13.
[56]Asea A, Rehli M, Kabingu E, et al.Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4.J Biol Chem.2002 Apr 26;277(17): 15028-34.
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[58]Ohashi K, Burkart V, Flohe S, et al.Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex.J Immunol.2000 Jan15;164(2):558-61.
[59]Termeer C, Benedix F, Sleeman J, et al.Oligosaccharides of Hyaluronan ?activate dendritic cells via toll-like receptor 4.J Exp Med.2002 Jan 7;195(1):99-111.
[60]Goldstein DR, Tesar BM, Akira S, et al.Critical role of the Toll-like receptor signal adaptor protein MyD88 in acute allograft rejection.J Clin Invest.2003 May;lll(10):1571-8.
[61]Tsoulfas G, Takahashi Y, Ganster RW, et al.Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury.Transplantation.2002 Jul 15;74(1):7-13.
[62]Palmer SM, Burch LH, Trindade AJ, et al.Innate immunity influences long-term outcomes after human lung transplant.Am J Respir Crit Care Med.2005 Apr l;171(7):780-5.
[63]Palmer SM, Burch LH, Davis RD, et al.The role of innate immunity in acute allograft rejection after lung transplantation.Am J Respir Crit Care Med.2003 Sep 15;168(6):628-32.
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[65]Ducloux D, Deschamps M, Yannaraki M, et al.Relevance of Toll-like receptor-4 polymorphisms in renal transplantation.Kidney Int.2005 Jun;67(6):2454-61.
[66]Methe H, Zimmer E, Grimm C, et al.Evidence for a role of toll-like receptor 4 in development of chronic allograft rejection after cardiac transplantation.Transplantation.2004 Nov 15;78(9): 1324-31.
[1]Obhrai J,Goldstein DR.The role of toll-like receptors in solid organ transplantation.Transplantation,2006,81(4):497-502.
[2]Tesar BM,Goldstein DR.Toll-like receptors and their role in transplantation.Front Biosci,2007,12(5):4221-38.
[3]Palmer SM,Burch LH,Mir S,et al.Donor polymorphisms in Toll-like receptor-4 influence the development of rejection after renal transplantation.Clin Transplant,2006,20(1):30-6.
[4]Deng JF,Geng L,Qian YG,et al.The role of toll-like receptors 2 and 4 in acute allograft rejection after liver transplantation.Transplant Proc,2007,39(10):3222-4.
[5]李晓北,管德林,韩志友等.彩色多普勒超声诊断早期移植肾急性排斥.中华泌尿外科杂志,2001,22(12):723-5.
[6]于立新,余玉明;邓文锋等.三色和双色荧光标记流式细胞术对肾移植免疫反应新指标TLR4的监测.南方医科大学学报,2006(3):294-296.
[7]于立新,叶俊生,邓文锋,等.高度致敏受者肾移植的临床处理.中华泌尿外科杂志,2008(3):185-187
[8]Goldstein DR.Toll like receptors and acute allograft rejection.Transpl Immunol,2006,17(1):11-5.
[9]余玉明;于立新.TOLL样受体与移植免疫.国外医学.移植与血液净化分册,2005,3(6):34-37.
[10]Palmer SM,Burch LH,Trindade AJ,et al.Innate immunity influences long-term outcomes after human lung transplant.Am J Respir Crit Care Med.2005,171(7):780-785.
[11]Dilioglou S,Cruse JM,Lewis RE.Function of CD80 and CD86 on monocyte-and stem cell-derived dendritic cells.Exp Mol Pathol.2003 Dec;75(3):217-27.
[12]Liang X, Lu L, Chen Z, et al.Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival.Transplantation.2003 Aug 27;76(4):721-9.
[13]Birsan T, Hausen B, Higgins JP, et al.Treatment with humanized monoclonal antibodies against CD80 and CD86 combined with sirolimus prolongs renal allograft survival in cynomolgus monkeys.Transplantation.2003 Jun 27;75(12):2106-13.
[14]Kagaya F, Hori J, Kamiya K, et al.Inhibition of murine corneal allograft rejection by treatment with antibodies to CD80 and CD86.Exp Eye Res.2002 Jan;74(1):131-9.
[15]Nicod LP, Joudrier S, Isler P,et al.Upregulation of CD40, CD80, CD83 or CD86 on alveolar macrophages after lung transplantation.J Heart Lung Transplant.2005 Aug;24(8): 1067-75.
[16]Kaczorowski DJ, Nakao A, Mollen KP, et al.Toll-like receptor 4 mediates the early inflammatory response after cold ischemia/reperfusion.Transplantation, 2007,84(10):1279-87.
[17]Pulskens WP, Teske GJ, Butter LM, et al.Toll-like receptor-4 coordinates the innate immune response of the kidney to renal ischemia/reperfusion injury.PLoS ONE.2008;3(10):e3596.
[18]Kr(u|¨)ger B, Krick S, Dhillon N, et al.Donor Toll-like receptor 4 contributes to ischemia and reperfusion injury following human kidney transplantation.Proc Natl Acad Sci U S A.2009 Mar 3;106(9):3390-5.
[19]Wu H, Chen G, Wyburn KR,et al.TLR4 activation mediates kidney ischemia/reperfusion injury.J Clin Invest.2007 Oct;117(10):2847-59.
[20]Kim BS, Lim SW, Li C, et al.Ischemia-reperfusion injury activates innate immunity in rat kidneys.Transplantation.2005 May 27;79(10):1370-7.
[21]Flynn MG, McFarlin BK, Phillips MD, et al.Toll-like receptor 4 and CD 14 mRNA expression are lower in resistive exercise-trained elderly women.J Appl Physiol.2003, 95(5): 1833-42.
[22]Krauss-Etschmann S, Hartl D, Heinrich J, et al.Association between levels of Toll-like receptors 2 and 4 and CD 14 mRNA and allergy in pregnant women and their offspring.Clin Immunol.2006,118(2-3):292-9.
[1]Medzhitov R,Preston-Hurlburt P,Janeway CA Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J].Nature,1997,388(6640):394-397.
[2]Skinner NA,MacIsaac CM,Hamilton JA,Regulation of Toll-like receptor(TLR)2and TLR4 on CD 14dimCD 16+ monocytes in response to sepsis-related antigens.Clin Exp Immunol.2005 Aug;141(2):270-8.
[3]Obhral J,Goldstein DR.The role of toll-like receptors in solid organ transplantation[J].Transplantation,2006,81(4):497-502.
[4]Tesar BM,Goldstein DR.Toll-like receptors and their role in transplantation[J].Front Biosci,2007,12(5):4221-38.
[5]Goldstein DR.Toll-like receptors and other links between innate and acquired alloimmunity.Curr Opin Immunol.2004 Oct;16(5):538-44.Review.
[6]Deng JF,Geng L,Qian YG,et al.The role of toll-like receptors 2 and 4 in acute allograft rejection after liver transplantation[J].Transplant Proc,2007,39(10):3222-4.
[7]Palmer SM,Burch LH,Trindade A J,et al.Innate immunity influences long-term outcomes after human lung transplant[J].Am J Respir Crit Care Med,2005,171(7):780-785.
[8]Palmer SM,Burch LH,Mir S,et al.Donor polymorphisms in Toll-like receptor-4influence the development of rejection after renal transplantation[J].Clin Transplant.2006,20(1):30-36.
[9]Land WG.The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation.Transplantation.2005 Mar 15;79(5):505-14.
[1]Racusen LC, Solez K, Colvin RB, et al.The Banff 97 working classification of renal allograft pathology.Kidney Int ,1999 , 55 :713-23.
[2]Schroppel B, He JC.Expression of Toll-like receptors in the kidney: their potential role beyond infection.Kidney Int.2006 Mar;69(5):785-7.
[3]Tesar BM,Goldstein DR.Toll-like receptors and their role in transplantation[J].Front Biosci, 2007,12(5):4221-38.
[4]Banas MC, Banas B, Hudkins KL, et al.TLR4 links podocytes with the innate immune system to mediate glomerular injury.J Am Soc Nephrol.2008 Apr;19(4):704-13.
[5]Methe H, Zimmer E, Grimm C, et al.Evidence for a role of toll-like receptor 4 in development of chronic allograft rejection after cardiac transplantation.Transplantation.2004 Nov 15;78(9): 1324-31.
[6]Chowdhury P, Sacks SH, Sheerin NS.Toll-like receptors TLR2 and TLR4 initiate the innate immune response of the renal tubular epithelium to bacterial products.Clin Exp Immunol.2006 Aug;145(2):346-56.
[7]Zhang D, Zhang G, Hayden MS, et al.A toll-like receptor that prevents infection by uropathogenic bacteria.Science.2004 Mar 5;303(5663): 1522-6.
[8]Cunningham PN, Wang Y, Guo R, Role of Toll-like receptor 4 in endotoxin-induced acute renal failure.J Immunol.2004 Feb 15;172(4):2629-35.
[9]Lim SW, Li C, Ahn KO, et al.Cyclosporine-induced renal injury induces tolllike receptor and maturation of dendritic cells.Transplantation.2005 Sep 15;80(5):691-9.
[2]Appleman LJ,Boussiotis VA.T cell anergy and costimulation.Immunol Rev.2003 Apr;192:161-80.Review.
[3]Slavik JM,Hutchcroft JE,Bierer BE.CD28/CTLA-4 and CD80/CD86 families:signaling and function.Immunol Res.1999;19(1):1-24.Review.
[4]Flechner SM.Cyclosporine:a new and promising immunosuppressive agent.Urol Clin North Am.1983 May;10(2):263-75.Review.
[5]Halloran PF,Madrenas J.The mechanism of action of cyclosporine:a perspective for the 90's.Clin Biochem.1991 Feb;24(1):3-7.Review.
[6]Yin M,Ochs RS.Mechanism for the paradoxical inhibition and stimulation of calcineurin by the immunosuppresive drug tacrolimus(FK506).Arch Biochem Biophys.2003 Nov 15;419(2):207-13.
[7]Borel JF,Baumann G.Molecular mechanisms of immunosuppressive agents.Bull Mem Acad R Med Belg.1994;149(1-2):97-101.
[8]Taylor AL,Watson C J,Bradley JA.Immunosuppressive agents in solid organ transplantation:Mechanisms of action and therapeutic efficacy.Crit Rev OncoI Hematol.2005 Oct;56(1):23-46.Review.
[9]Superdock KR,Helderman JH.Immunosuppressive drugs and their effects.Semin Respir Infect.1993 Sep;8(3):152-9.Review.
[10]Khanna AK.Mechanism of the combination immunosuppressive effects of rapamycin with either cyclosporine or tacrolimus.Transplantation.2000 Aug 27;70(4):690-4.
[11]Dilioglou S,Cruse JM,Lewis RE.Function of CD80 and CD86 on monocyte-and stem cell-derived dendritic cells.Exp Mol Pathol.2003 Dec;75(3):217-27.
[12]Yamashita K, Masunaga T, Yanagida N, et al.Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies.Transplantation.2003 Oct 15;76(7):1089-96.
[13]Montgomery SP, Xu H, Tadaki DK, et al.Combination induction therapy with monoclonal antibodies specific for CD80, CD86, and CD 154 in nonhuman primate renal transplantation.Transplantation.2002 Nov 27;74(10): 1365-9.
[14]Jiga LP, Bauer TM, Chuang JJ,et al.Generation of tolerogenic dendritic cells by treatment with mitomycin C: inhibition of allogeneic T-cell response is mediated by downregulation of ICAM-1, CD80, and CD86.Transplantation.2004 Jun15;77(11): 1761-4.
[15]Medzhitov R, Preston-Hurlburt P, Janeway CA Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature.1997Jul 24;388(6640):394-7.
[16]Janeway CA Jr.Approaching the asymptote? Evolution and revolution in immunology.Cold Spring Harb Symp Quant Biol.1989;54 Pt 1:1-13.
[17]Takeda K, Akira S.Toll-like receptors in innate immunity.Int Immunol.2005 Jan;17(1):l-14.Review.
[18]Tesar BM, Goldstein DR.Toll-like receptors and their role in transplantation.Front Biosci, 2007, 12(5): 4221-38.
[19]Brandl K, Gluck T, Hartmann P, et al.A designed TLR4/MD-2 complex to capture LPS.J Endotoxin Res.2005;11(4):197-206.
[20]Philpott DJ, Girardin SE.The role of Toll-like receptors and Nod proteins in bacterial infection.Mol Immunol.2004 Nov;41(l 1):1099-108.Review.
[21]Means TK, Lien E, Yoshimura A, et al.The CD 14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors.J Immunol.1999 Dec 15;163(12):6748-55.
[22]Modlin RL.Activation of toll-like receptors by microbial lipoproteins: role in host defense.J Allergy Clin Immunol.2001 Oct;108(4 Suppl):S 104-6.Review.
[23]Schroder NW, Morath S, Alexander C, et al.Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), andCD14, whereas TLR-4 and MD-2 are not involved.J Biol Chem.2003 May 2;278(18):15587-94.
[24]Sivori S, Falco M, Delia Chiesa M, et al.CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells.Proc Natl Acad Sci U S A.2004 Jul 6;101(27):10116-21.
[25]Ishii KJ, Akira S.Toll-like Receptors and Sepsis.Curr Infect Dis Rep.2004 Oct;6(5):361-366.
[26]Lorenz E, Schwartz DA, Martin PJ, et al.Association of TLR4 mutations and the risk for acute GVHD after HLA-matched-sibling hematopoietic stem cell transplantation.Biol Blood Marrow Transplant.2001 ;7(7):384-7.
[27]Schmausser B, Andrulis M, Endrich S, et al.Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori.Int J Med Microbiol.2005 Jun;295(3): 179-85.
[28]Pespeni M, Mackersie RC, Lee H,et al.Serum levels of Hsp60 correlate with the development of acute lung injury after trauma.J Surg Res.2005 Jun l;126(1):41-7.
[29]Johnson GB, Brunn GJ, Platt JL.Cutting edge: an endogenous pathway to systemic inflammatory response syndrome (SIRS)-like reactions through Toll-like receptor 4.J Immunol.2004 Jan l;172(1):20-4.
[30]Liu L, Zhou X, Shi J,et al.Toll-like receptor-9 induced by physical trauma mediates release of cytokines following exposure to CpG motif in mouse skin.Immunology.2003 Nov;110(3):341-7.
[31]Kazemi MR, McDonald CM, Shigenaga JK,et al.Adipocyte fatty acid-binding protein expression and lipid accumulation are increased during activation of murine macrophages by toll-like receptor agonists.Arterioscler Thromb Vasc Biol.2005 Jun;25(6): 1220-4.
[32]Pasterkamp G, Van Keulen JK, De Kleijn DP.Role of Toll-like receptor 4 in the initiation and progression of atherosclerotic disease.Eur J Clin Invest.2004 May;34(5):328-34.Review.
[33]Bjorkbacka H, Kunjathoor VV, Moore KJ, et al。 Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways.Nat Med.2004 Apr; 10(4) :416-21.
[34]Hart AL, Al-Hassi HO, Rigby RJ, et ah Characteristics of intestinal dendritic cells in inflammatory bowel diseases.Gastroenterology.2005 Jul;129(1):50-65.
[35]Vives-Pi M, Somoza N, Fernandez-Alvarez J, et al.Evidence of expression of endotoxin receptors CD 14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and of sensitivity to endotoxin (LPS) in islet beta cells.Clin Exp Immunol.2003 Aug;133(2):208-18.
[36]Cario E, Podolsky DK.Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease.Infect Immun.2000 Dec;68(12):7010-7.
[37]Miyake K.Endotoxin recognition molecules, Toll-like receptor 4-MD-2.Semin Immunol.2004 Feb;16(1):l 1-6.Review.
[38]Espevik T, Latz E, Lien E, et al.Cell distributions and functions of Toll-like receptor 4 studied by fluorescent gene constructs.Scand J Infect Dis.2003;35(9):660-4.Review.
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