IDO在新月体肾炎中的表达、意义及其体内干预的实验研究
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摘要
一、研究背景和目的
新月体肾炎是肾脏病理变化最严重进展最快的一类肾小球肾炎,是由大量炎性细胞浸润肾组织和肾小球包曼氏囊导致的严重肾小球疾病。虽然新月体肾炎发病率相对较低,但其病变迅速,疾病总的预后不佳,多数患者在数周或数月内发展至终末期肾功能衰竭而进入透析、移植阶段。以往认为新月体肾炎主要是由体液免疫所介导,但越来越多的研究证据表明自身抗体介导的体液免疫并非新月体性肾炎发病的关键机制,而CD4+ T细胞介导的细胞免疫在疾病的发生发展中发挥更为关键的作用。因此进一步研究新月体肾炎发病机制中效应性T细胞增殖和凋亡的调控机理,对新月体肾炎发病机制的阐明及寻找新的治疗策略具有重要的学术意义和实用前景。
色氨酸是哺乳动物体内的必需氨基酸。即使是能合成色氨酸的细菌在适宜的条件下也会从周围环境中摄取色氨酸,由此可见色氨酸在生物代谢中的重要作用。吲哚胺2,3-双加氧酶(Indoleamine2,3-dioxygenase,IDO)是催化色氨酸代谢通路中的裂解吡咯环关键步骤的限速酶。近年研究发现无论是抗原递呈细胞如树突状细胞、单核-巨噬细胞还是其它组织固有细胞表达的IDO均可显著抑制T细胞介导的细胞免疫。由于色氨酸是代谢旺盛T细胞增殖活化中所必需的氨基酸,而犬尿氨酸对T细胞具有细胞毒性,可促使T细胞凋亡,因此IDO通过降解色氨酸形成色氨酸耗竭和高犬尿氨酸微环境能高效诱导T细胞免疫耐受的形成。目前IDO已成为T细胞免疫相关疾病如母胎耐受、肿瘤免疫逃逸、移植物抗排异以及自身免疫性疾病自身耐受机制的研究热点。新近研究表明内源性IFN-γ诱导抗原递呈细胞IDO表达上调可能是内源性IFN-γ在多个自身免疫性动物模型和移植物抗排异中发挥免疫耐受作用的重要机制,既往研究表明IFN-γ基因敲除鼠可诱导更为严重的新月体肾炎,这些研究资料提示IDO可能在新月体肾炎的发病机制中具有重要作用。表达MHCⅡ类分子的肾小管上皮细胞可作为抗原递呈细胞对T细胞的功能活性进行调控,而IDO最显著的特征就是表达于多种抗原递呈细胞, IDO在肾炎病变组织尤其肾小管上皮细胞表达目前尚未见报道,因此我们拟研究IDO在新月体肾炎病变中的表达情况及其在新月体肾炎发病中可能的作用机制。
目前最具特征性和最广泛采用的新月体肾炎动物模型是肾毒血清性肾炎(NTN)模型,其是通过过继异种抗-GBM抗血清作为肾小球植入抗原而诱导的新月体肾炎。基于以上分析,我们拟首先检测新月体肾炎患者肾组织中IDO的表达并探讨其意义,之后在体外模拟病理条件下探讨肾小管上皮细胞IDO的表达变化规律及其免疫学效应。通过构建重组腺病毒Ad-IDO,在新月体肾炎模型鼠体内分别给予IDO特异性阻断剂1-甲基色氨酸和Ad-IDO腺病毒体内基因转染干预NTN大鼠体内IDO的生物活性,以IDO未干预鼠为对照组,通过检测脾脏淋巴细胞凋亡率、24h尿蛋白量、肾组织炎细胞浸润、增殖以及肾组织病理损害程度以揭示IDO在新月体肾炎发病机制中的作用,从而为寻找新月体肾炎治疗的新策略提供新思路。
二、方法
1.采用免疫组织化学染色法检测正常肾组织和新月体肾炎患者肾组织中IDO和PCNA的表达情况,同时对肾小管间质病变程度进行分级,分析肾组织IDO表达变化与肾组织浸润PCNA阳性细胞数和肾小管间质病变程度的相关性。
2.为了进一步明确肾小管上皮细胞IDO的表达变化规律,我们在体外培养人肾小管上皮细胞株HK2细胞,模拟病理条件给予IFN-γ刺激培养,通过RT-PCR、免疫细胞化学染色、高效液相色谱(HPLC)等方法检测不同培养条件下HK2细胞IDO的诱导表达情况。为观察HK2细胞诱导表达IDO的生物学效应,我们将IFN-γ刺激培养后的HK2细胞与Jurkat T淋巴细胞共培养,并设立IDO阻断剂共培养组和未予IFN-γ刺激HK2共培养对照组。通过细胞计数法检测共培养Jurkat细胞的生长增殖情况并计算其倍增时间,流式细胞法检测各共培养组Jurkat细胞的细胞周期及凋亡情况。
3.构建Ad-IDO重组腺病毒。首先酶切pOCUS-IDO质粒获得小鼠全长IDOcDNA片段,再将其亚克隆至穿梭质粒pAdTrack-CMV上,在BJ5183细菌中和AdEasy-1进行同源重组,获得Ad-IDO重组子,酶切、测序鉴定,并通过脂质体法将其转染293细胞进行病毒包装、扩增。对转染Ad-IDO腺病毒重组质粒的293细胞进行PCR、生物活性和安全性检测,同时纯化Ad-IDO腺病毒及并测定其病毒滴度。
4.建立NTN大鼠新月体肾炎模型。首先检测NTN大鼠和正常大鼠血清中色氨酸和犬尿氨酸浓度,之后分别给予NTN大鼠注射Ad-IDO腺病毒或腹腔内注射IDO特异性阻断剂——1-甲基色氨酸以干预其体内IDO活性。以IDO未干预的NTN大鼠和正常大鼠为对照组,检测各组大鼠血清色氨酸、犬尿氨酸浓度,并以犬尿氨酸/色氨酸浓度比值反应体内的IDO活性变化,通过检测脾脏淋巴细胞凋亡率、24h蛋白尿量、肾组织病理变化以及肾组织内浸润CD4+T细胞、PCNA阳性细胞情况,以揭示体内干预IDO活性对新月体肾炎病情的影响,从而为IDO在新月体肾炎发病机制中作用的阐明提供实验依据。
三、结果
1.在正常肾组织未检测到IDO表达,PCNA阳性细胞也非常罕见;而在新月体肾炎标本肾小管上皮细胞IDO的表达显著上调,PCNA阳性细胞数也明显增高。通过分析新月体肾炎患者肾小管上皮细胞IDO的表达强度与肾小管间质PCNA阳性细胞数及肾小管间质病理损害程度之间的相关性,发现肾小管上皮细胞IDO的表达强度和肾小管-间质PCNA阳性细胞数呈显著负相关,与肾小管-间质病理损害程度之间亦呈显著负相关。
2.正常培养条件下的HK2细胞不表达IDOmRNA及IDO,而在IFN-γ刺激培养条件下HK2细胞IDOmRNA和IDO的表达显著上调,并在一定范围内IFN-γ刺激IDO的表达上调呈时间依赖性和剂量依赖性地增高,高效液相色谱检测显示HK2细胞诱导表达的IDO具有降解色氨酸的生物活性。与对照组相比,和诱导表达IDO的HK2细胞共培养的Jurkat细胞生长增殖受抑、细胞周期阻滞于G1期,而S期减少,同时细胞凋亡率明显增高,而加1-MT阻断剂共培养的Jurkat细胞其生长增殖、细胞周期和凋亡率与对照组相比无显著差异。
3.经酶切和测序鉴定表明成功构建了Ad-IDO腺病毒重组质粒。PCR检测显示AD293细胞中包装扩增的腺病毒重组基因组中有目的基因IDO的表达,而AdΦ腺病毒空载体转染的293细胞无IDO表达。HPLC检测显示表达的IDO具有降解色氨酸的生物学活性,因此构建的Ad-IDO腺病毒具有感染并表达有生物活性IDO的能力。氯化铯密度梯度离心法纯化了Ad-IDO腺病毒,并达到病毒体内转染所需滴度。重组腺病毒对非包装细胞Hela细胞无毒性,说明无野生型病毒的存在。
4.实验显示在NTN大鼠血清中色氨酸代谢率较正常大鼠显著升高。与IDO未干预NTN鼠相比,Ad-IDO转染NTN鼠的血清色氨酸浓度进一步降低,而犬尿氨酸浓度进一步升高,同时IDO有效转染了肾组织肾小管上皮细胞,表明Ad-IDO体内注射显著提高了体内IDO的生物学活性。Ad-IDO腺病毒转染NTN大鼠的脾脏淋巴细胞凋亡率明显增加,24h尿蛋白显著减少,而肾小球新月体比率明显降低,肾小球和肾小管-间质浸润的CD4+ T细胞和PCNA阳性细胞数也显著减少,各指标与IDO未干预NTN鼠相比差异显著;相反,腹腔注射1-MT的NTN大鼠其血清色氨酸降解明显减弱,24h尿蛋白量显著升高,肾组织病理损伤进一步加重,同时肾小球和肾小管-间质浸润CD4+ T细胞和PCNA阳性细胞数也明显增高,与IDO未干预NTN鼠相比差异显著。
四、结论
1.新月体肾炎患者肾标本中肾小管上皮细胞IDO的表达显著上调,并且IDO的表达强度与肾小管间质PCNA阳性细胞数和肾小管间质病变程度显著负关性,结合体外IFN-γ刺激培养条件下HK2细胞IDO的表达变化及其免疫学效应,说明病理条件下肾小管上皮细胞诱导表达的IDO可通过调控肾小管间质淋巴细胞的功能活性而减缓新月体肾炎肾小管间质的免疫病理损伤。
2.成功构建了具有转染并可表达生物活性IDO的Ad-IDO重组腺病毒。
3.与正常大鼠相比,NTN大鼠血清中色氨酸降解明显增强,而用1-MT阻断IDO活性后新月体肾炎的病情显著加重,说明NTN大鼠体内升高的IDO活性在保持新月体肾炎大鼠自身耐受机制而减缓肾炎病情中具有重要作用。采用Ad-IDO体内转染NTN大鼠可进一步提高大鼠体内IDO的生物活性,其通过抑制体内T淋巴细胞增殖、促进其凋亡而发挥减缓新月体肾炎病情的生物学效应。因此体内靶向加强病变肾组织局部IDO活性可望为新月体肾炎的治疗提供新的策略。
Background and objectives
Glomerulonephritis (GN) is an inflammatory disease of the renal glomeruli and is a major cause of end-stage renal failure. The most severe and rapidly progressive type of GN results from infiltration of inflammatory cells into the glomerular tuft and accumulation of cells and fibrin in Bowman’s space, these forms, known as crescentic GN. Increasing evidence has suggested that T cell-mediated cellular immunity may be a more important mediator than the function of self-antibody, CD4+ T helper 1(Th1) cell-mediated response has been suspected as the culprit for glomerular injury in crescentic GN. The glomerular influx of CD4+ T cells and macrophages results in local cell proliferation, and formation of glomerular crescents and severe glomerular injury. It is enforced by interrupting B7/CD28 co-stimulation pathway, and attenuated by Th2 cytokines or transfer of CD4+ CD25+ T cell, and independent of autologous antibodies(Ab). Further studies about the exact regulated mechanism of activation and effection of CD4+ T cell in pathogenic mechanisms of crescentic GN are needed, and these researches may lead to the development of novel therapies for crescentic glomerulonephritis.
Tryptophan(Trp) is an essential amino acid for mammals. Bacteria which can synthesize tryptophan also obtain it from their environment when available. These presumably tryptophan is a very important consumed amino acid to synthesize. Indoleamine 2,3-dioxygenase (EC 1.13.11.42, IDO) is a heme-containing dioxygenase which catalyzes the first and rate-limiting step in the major pathway of L-tryptophan catabolism in mammals. Either antigen presenting cells (APC) such as dendritic cells (DCs) or tissue cells which express IDO, were shown to suppress the T-cell responses by depriving T-cells of Trp in vitro. The immunosuppressive activity of IDO was then applied to inhibit the T-cell-mediated reject on allografted pancreas islets in mice and such IDO-expressing islets have also been demonstrated to show a significant prolongation of graft survival. Similarly, it has recently been shown that the T-cell-mediated experimental asthma and experimental autoimmune encephalomyelitis were inhibited by the IDO which was up-regulated in pulmonary and microglial cells. Based on these findings in vitro and in experimental animal systems, the concept that cells expressing IDO can suppress the T-cell responses and induce tolerance has thus emerged as a new paradigm in immunology. Richard found IFN-γ–/– mice developed significant crescent formation and an increased cellular infiltrate in both the glomerulus and the interstitium compared with normal mice. But the mechanisms behind the protective effect of IFN-γin this model and in other models of organ-specific autoimmunity are complex and remain incompletely understood. Recently research showed IFN-γstimulation of APC could result in increased IDO expression, and the activity of IDO might be the important mechanisms of IFN-γin tolerance induction in many autoimmunity disease. Those investigation suggested that IDO may play an important role in crescentic GN. MHC class II-expressing renal tubular epithelial cells can function as APC for T cells,and no reports about whether the TEC express the IDO and its role in the crescentic GN. So we study the expression and function of IDO in crescentic GN. So we study the expression and function of IDO in crescentic GN. The best characterized and most widely used model of crescentic GN is nephrotoxic serum nephritis(NTN), in which heterologous anti-GBM antibody(Ab) acts as a planted antigen in glomeruli. We want to first examine the expression and function of IDO on crescentic GN kidney samples as well as on human renal tubular epithelial cells (RTEC) line HK-2 cells. Second, construct a recombinant adenovirus that expresses IDO for gene therapy in vivo. Last, we test the interference effects of enforceing IDO by gene transfection and interrupting IDO by 1-methyl-DL-tryptophan (1-MT) in crescentic GN in a rat model of NTN.
Methods and results:
We fist examined the expression of IDO on crescentic GN and normal human kidney samples. In immunohistochemical staining revealed no expression of IDO in the normal kidney tissues, but much high expression in renal tubular epithelial cells of crescentic GN. The IDO expression had a negative correlation with the number of PCNA+ cells in tubulointerstitial and with the tubulointerstitial injury. The expression of IDO in RTEC line HK-2 cells also was examined by RT-PCR, immunocytochemistry and high performance liquid chromatography (HPLC),and rusults showed that the IDO is no expression on HK-2 cells in normal culture conditions, but its expression and the activity is dramatically increased by stimulation of IFN-γin a dose and time dependent manner compared with control groups. In vitro, pretreatment of IFN-γ-stimulated HK-2 cells cocultured with Jurkat cells can significantly inhibite the proliferation and promote the apoptosis of Jurkat cells. These results suggest that the activity of IDO negatively regulates T cell activation by TEC, and may play an important inhibitory role in TEC-mediated immune activation and immuno-pathology in the kidney.
Next, we construct a recombinant adenovirus that expresses IDO for gene therapy in vivo. Full-length mouse IDO cDNA was subcloned into pAdTrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with pAdEasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombined adenovirus DNA was transfected into 293 cells with LipofectamineTM2000 for packaging and amplification of Ad-IDO virus and the high-titer adenovirus supernatants were harvested. The expression of IDO is monitored by EGFP fluorescence in infected cells. After transfection with adenovirus DNA, infectious virus was only produced to cause cytopathic effect in the permissive cell line AD293 but not in the non-permissive cell line Hela, confirming only replication-defective but not wild type virus was generated. The specific expression of mouse IDO was verified by PCR in AD 293 cells after infection with Ad-IDO, but not Ad-EGFP, a similarly constructed control virus. Ad-IDO, but not Ad-EGFP, can degradate of tryptophan in culture medium. The results showed that we have successfully constructed a recombinant adenovirus Ad-IDO. The virus will be useful to enforce the activity of IDO in animal models of crescentic GN in vivo.
The last, we test the inteference effect of enforceing IDO by gene transfection and interrupting IDO by 1-MT in the development of crescentic GN in a rat model of NTN. It was demonstrated that the activity of IDO was increased significantly in NTN rat than in the normal rat, when administration of intravenous injection of Ad-IDO into NTN rat, not only had the IDO transfected on renal tubular epithelial cells, but also the tryptophan catabolism in serum was increased more higher than the control NTN rat, and ameliorated renal injury with decreased glomerular crescent formation, CD4+T cells and PCNA+ cells accumulation in kidney tissue and proteinuria compared with the control NTN rat, and prominent splenocyte cells apotosis(p<0.01).While the administration of 1-MT by intraperitoneally injection resulted in the decereased of IDO activity in NTN rat and accelerates crescentic GN renal pathology injured with significant increased glomerular crescent formation, CD4+T cells and PCNA+ cells accumulation in kidney tissue and proteinuria compared with the control NTN rat. Our results demonstrated the involvement of the role of IDO in the pathogenesis of crescentic GN, and enhancing the activity of IDO in vivo may be beneficial for the treatment of human crescentic GN.
Conclusions:
Our results suggest:
1.The upregulated IDO of renal tubular epithelial cells can suppresse T cell activation, and this might play an inhibited role in T cell mediated immune activation and immunopathology in crescentic GN.
2. The activity of IDO is increased in vivo in NTN rat, and which may play an important role in pathogenesis of crescentic GN. While enforced the activity of IDO in vivo might provided a novel therapy method for crescentic GN.
新月体肾炎是肾脏病理变化最严重进展最快的一类肾小球肾炎,是由大量炎性细胞浸润肾组织和肾小球包曼氏囊导致的严重肾小球疾病。虽然新月体肾炎发病率相对较低,但其病变迅速,疾病总的预后不佳,多数患者在数周或数月内发展至终末期肾功能衰竭而进入透析、移植阶段。以往认为新月体肾炎主要是由体液免疫所介导,但越来越多的研究证据表明自身抗体介导的体液免疫并非新月体性肾炎发病的关键机制,而CD4+ T细胞介导的细胞免疫在疾病的发生发展中发挥更为关键的作用。因此进一步研究新月体肾炎发病机制中效应性T细胞增殖和凋亡的调控机理,对新月体肾炎发病机制的阐明及寻找新的治疗策略具有重要的学术意义和实用前景。
色氨酸是哺乳动物体内的必需氨基酸。即使是能合成色氨酸的细菌在适宜的条件下也会从周围环境中摄取色氨酸,由此可见色氨酸在生物代谢中的重要作用。吲哚胺2,3-双加氧酶(Indoleamine2,3-dioxygenase,IDO)是催化色氨酸代谢通路中的裂解吡咯环关键步骤的限速酶。近年研究发现无论是抗原递呈细胞如树突状细胞、单核-巨噬细胞还是其它组织固有细胞表达的IDO均可显著抑制T细胞介导的细胞免疫。由于色氨酸是代谢旺盛T细胞增殖活化中所必需的氨基酸,而犬尿氨酸对T细胞具有细胞毒性,可促使T细胞凋亡,因此IDO通过降解色氨酸形成色氨酸耗竭和高犬尿氨酸微环境能高效诱导T细胞免疫耐受的形成。目前IDO已成为T细胞免疫相关疾病如母胎耐受、肿瘤免疫逃逸、移植物抗排异以及自身免疫性疾病自身耐受机制的研究热点。新近研究表明内源性IFN-γ诱导抗原递呈细胞IDO表达上调可能是内源性IFN-γ在多个自身免疫性动物模型和移植物抗排异中发挥免疫耐受作用的重要机制,既往研究表明IFN-γ基因敲除鼠可诱导更为严重的新月体肾炎,这些研究资料提示IDO可能在新月体肾炎的发病机制中具有重要作用。表达MHCⅡ类分子的肾小管上皮细胞可作为抗原递呈细胞对T细胞的功能活性进行调控,而IDO最显著的特征就是表达于多种抗原递呈细胞, IDO在肾炎病变组织尤其肾小管上皮细胞表达目前尚未见报道,因此我们拟研究IDO在新月体肾炎病变中的表达情况及其在新月体肾炎发病中可能的作用机制。
目前最具特征性和最广泛采用的新月体肾炎动物模型是肾毒血清性肾炎(NTN)模型,其是通过过继异种抗-GBM抗血清作为肾小球植入抗原而诱导的新月体肾炎。基于以上分析,我们拟首先检测新月体肾炎患者肾组织中IDO的表达并探讨其意义,之后在体外模拟病理条件下探讨肾小管上皮细胞IDO的表达变化规律及其免疫学效应。通过构建重组腺病毒Ad-IDO,在新月体肾炎模型鼠体内分别给予IDO特异性阻断剂1-甲基色氨酸和Ad-IDO腺病毒体内基因转染干预NTN大鼠体内IDO的生物活性,以IDO未干预鼠为对照组,通过检测脾脏淋巴细胞凋亡率、24h尿蛋白量、肾组织炎细胞浸润、增殖以及肾组织病理损害程度以揭示IDO在新月体肾炎发病机制中的作用,从而为寻找新月体肾炎治疗的新策略提供新思路。
二、方法
1.采用免疫组织化学染色法检测正常肾组织和新月体肾炎患者肾组织中IDO和PCNA的表达情况,同时对肾小管间质病变程度进行分级,分析肾组织IDO表达变化与肾组织浸润PCNA阳性细胞数和肾小管间质病变程度的相关性。
2.为了进一步明确肾小管上皮细胞IDO的表达变化规律,我们在体外培养人肾小管上皮细胞株HK2细胞,模拟病理条件给予IFN-γ刺激培养,通过RT-PCR、免疫细胞化学染色、高效液相色谱(HPLC)等方法检测不同培养条件下HK2细胞IDO的诱导表达情况。为观察HK2细胞诱导表达IDO的生物学效应,我们将IFN-γ刺激培养后的HK2细胞与Jurkat T淋巴细胞共培养,并设立IDO阻断剂共培养组和未予IFN-γ刺激HK2共培养对照组。通过细胞计数法检测共培养Jurkat细胞的生长增殖情况并计算其倍增时间,流式细胞法检测各共培养组Jurkat细胞的细胞周期及凋亡情况。
3.构建Ad-IDO重组腺病毒。首先酶切pOCUS-IDO质粒获得小鼠全长IDOcDNA片段,再将其亚克隆至穿梭质粒pAdTrack-CMV上,在BJ5183细菌中和AdEasy-1进行同源重组,获得Ad-IDO重组子,酶切、测序鉴定,并通过脂质体法将其转染293细胞进行病毒包装、扩增。对转染Ad-IDO腺病毒重组质粒的293细胞进行PCR、生物活性和安全性检测,同时纯化Ad-IDO腺病毒及并测定其病毒滴度。
4.建立NTN大鼠新月体肾炎模型。首先检测NTN大鼠和正常大鼠血清中色氨酸和犬尿氨酸浓度,之后分别给予NTN大鼠注射Ad-IDO腺病毒或腹腔内注射IDO特异性阻断剂——1-甲基色氨酸以干预其体内IDO活性。以IDO未干预的NTN大鼠和正常大鼠为对照组,检测各组大鼠血清色氨酸、犬尿氨酸浓度,并以犬尿氨酸/色氨酸浓度比值反应体内的IDO活性变化,通过检测脾脏淋巴细胞凋亡率、24h蛋白尿量、肾组织病理变化以及肾组织内浸润CD4+T细胞、PCNA阳性细胞情况,以揭示体内干预IDO活性对新月体肾炎病情的影响,从而为IDO在新月体肾炎发病机制中作用的阐明提供实验依据。
三、结果
1.在正常肾组织未检测到IDO表达,PCNA阳性细胞也非常罕见;而在新月体肾炎标本肾小管上皮细胞IDO的表达显著上调,PCNA阳性细胞数也明显增高。通过分析新月体肾炎患者肾小管上皮细胞IDO的表达强度与肾小管间质PCNA阳性细胞数及肾小管间质病理损害程度之间的相关性,发现肾小管上皮细胞IDO的表达强度和肾小管-间质PCNA阳性细胞数呈显著负相关,与肾小管-间质病理损害程度之间亦呈显著负相关。
2.正常培养条件下的HK2细胞不表达IDOmRNA及IDO,而在IFN-γ刺激培养条件下HK2细胞IDOmRNA和IDO的表达显著上调,并在一定范围内IFN-γ刺激IDO的表达上调呈时间依赖性和剂量依赖性地增高,高效液相色谱检测显示HK2细胞诱导表达的IDO具有降解色氨酸的生物活性。与对照组相比,和诱导表达IDO的HK2细胞共培养的Jurkat细胞生长增殖受抑、细胞周期阻滞于G1期,而S期减少,同时细胞凋亡率明显增高,而加1-MT阻断剂共培养的Jurkat细胞其生长增殖、细胞周期和凋亡率与对照组相比无显著差异。
3.经酶切和测序鉴定表明成功构建了Ad-IDO腺病毒重组质粒。PCR检测显示AD293细胞中包装扩增的腺病毒重组基因组中有目的基因IDO的表达,而AdΦ腺病毒空载体转染的293细胞无IDO表达。HPLC检测显示表达的IDO具有降解色氨酸的生物学活性,因此构建的Ad-IDO腺病毒具有感染并表达有生物活性IDO的能力。氯化铯密度梯度离心法纯化了Ad-IDO腺病毒,并达到病毒体内转染所需滴度。重组腺病毒对非包装细胞Hela细胞无毒性,说明无野生型病毒的存在。
4.实验显示在NTN大鼠血清中色氨酸代谢率较正常大鼠显著升高。与IDO未干预NTN鼠相比,Ad-IDO转染NTN鼠的血清色氨酸浓度进一步降低,而犬尿氨酸浓度进一步升高,同时IDO有效转染了肾组织肾小管上皮细胞,表明Ad-IDO体内注射显著提高了体内IDO的生物学活性。Ad-IDO腺病毒转染NTN大鼠的脾脏淋巴细胞凋亡率明显增加,24h尿蛋白显著减少,而肾小球新月体比率明显降低,肾小球和肾小管-间质浸润的CD4+ T细胞和PCNA阳性细胞数也显著减少,各指标与IDO未干预NTN鼠相比差异显著;相反,腹腔注射1-MT的NTN大鼠其血清色氨酸降解明显减弱,24h尿蛋白量显著升高,肾组织病理损伤进一步加重,同时肾小球和肾小管-间质浸润CD4+ T细胞和PCNA阳性细胞数也明显增高,与IDO未干预NTN鼠相比差异显著。
四、结论
1.新月体肾炎患者肾标本中肾小管上皮细胞IDO的表达显著上调,并且IDO的表达强度与肾小管间质PCNA阳性细胞数和肾小管间质病变程度显著负关性,结合体外IFN-γ刺激培养条件下HK2细胞IDO的表达变化及其免疫学效应,说明病理条件下肾小管上皮细胞诱导表达的IDO可通过调控肾小管间质淋巴细胞的功能活性而减缓新月体肾炎肾小管间质的免疫病理损伤。
2.成功构建了具有转染并可表达生物活性IDO的Ad-IDO重组腺病毒。
3.与正常大鼠相比,NTN大鼠血清中色氨酸降解明显增强,而用1-MT阻断IDO活性后新月体肾炎的病情显著加重,说明NTN大鼠体内升高的IDO活性在保持新月体肾炎大鼠自身耐受机制而减缓肾炎病情中具有重要作用。采用Ad-IDO体内转染NTN大鼠可进一步提高大鼠体内IDO的生物活性,其通过抑制体内T淋巴细胞增殖、促进其凋亡而发挥减缓新月体肾炎病情的生物学效应。因此体内靶向加强病变肾组织局部IDO活性可望为新月体肾炎的治疗提供新的策略。
Background and objectives
Glomerulonephritis (GN) is an inflammatory disease of the renal glomeruli and is a major cause of end-stage renal failure. The most severe and rapidly progressive type of GN results from infiltration of inflammatory cells into the glomerular tuft and accumulation of cells and fibrin in Bowman’s space, these forms, known as crescentic GN. Increasing evidence has suggested that T cell-mediated cellular immunity may be a more important mediator than the function of self-antibody, CD4+ T helper 1(Th1) cell-mediated response has been suspected as the culprit for glomerular injury in crescentic GN. The glomerular influx of CD4+ T cells and macrophages results in local cell proliferation, and formation of glomerular crescents and severe glomerular injury. It is enforced by interrupting B7/CD28 co-stimulation pathway, and attenuated by Th2 cytokines or transfer of CD4+ CD25+ T cell, and independent of autologous antibodies(Ab). Further studies about the exact regulated mechanism of activation and effection of CD4+ T cell in pathogenic mechanisms of crescentic GN are needed, and these researches may lead to the development of novel therapies for crescentic glomerulonephritis.
Tryptophan(Trp) is an essential amino acid for mammals. Bacteria which can synthesize tryptophan also obtain it from their environment when available. These presumably tryptophan is a very important consumed amino acid to synthesize. Indoleamine 2,3-dioxygenase (EC 1.13.11.42, IDO) is a heme-containing dioxygenase which catalyzes the first and rate-limiting step in the major pathway of L-tryptophan catabolism in mammals. Either antigen presenting cells (APC) such as dendritic cells (DCs) or tissue cells which express IDO, were shown to suppress the T-cell responses by depriving T-cells of Trp in vitro. The immunosuppressive activity of IDO was then applied to inhibit the T-cell-mediated reject on allografted pancreas islets in mice and such IDO-expressing islets have also been demonstrated to show a significant prolongation of graft survival. Similarly, it has recently been shown that the T-cell-mediated experimental asthma and experimental autoimmune encephalomyelitis were inhibited by the IDO which was up-regulated in pulmonary and microglial cells. Based on these findings in vitro and in experimental animal systems, the concept that cells expressing IDO can suppress the T-cell responses and induce tolerance has thus emerged as a new paradigm in immunology. Richard found IFN-γ–/– mice developed significant crescent formation and an increased cellular infiltrate in both the glomerulus and the interstitium compared with normal mice. But the mechanisms behind the protective effect of IFN-γin this model and in other models of organ-specific autoimmunity are complex and remain incompletely understood. Recently research showed IFN-γstimulation of APC could result in increased IDO expression, and the activity of IDO might be the important mechanisms of IFN-γin tolerance induction in many autoimmunity disease. Those investigation suggested that IDO may play an important role in crescentic GN. MHC class II-expressing renal tubular epithelial cells can function as APC for T cells,and no reports about whether the TEC express the IDO and its role in the crescentic GN. So we study the expression and function of IDO in crescentic GN. So we study the expression and function of IDO in crescentic GN. The best characterized and most widely used model of crescentic GN is nephrotoxic serum nephritis(NTN), in which heterologous anti-GBM antibody(Ab) acts as a planted antigen in glomeruli. We want to first examine the expression and function of IDO on crescentic GN kidney samples as well as on human renal tubular epithelial cells (RTEC) line HK-2 cells. Second, construct a recombinant adenovirus that expresses IDO for gene therapy in vivo. Last, we test the interference effects of enforceing IDO by gene transfection and interrupting IDO by 1-methyl-DL-tryptophan (1-MT) in crescentic GN in a rat model of NTN.
Methods and results:
We fist examined the expression of IDO on crescentic GN and normal human kidney samples. In immunohistochemical staining revealed no expression of IDO in the normal kidney tissues, but much high expression in renal tubular epithelial cells of crescentic GN. The IDO expression had a negative correlation with the number of PCNA+ cells in tubulointerstitial and with the tubulointerstitial injury. The expression of IDO in RTEC line HK-2 cells also was examined by RT-PCR, immunocytochemistry and high performance liquid chromatography (HPLC),and rusults showed that the IDO is no expression on HK-2 cells in normal culture conditions, but its expression and the activity is dramatically increased by stimulation of IFN-γin a dose and time dependent manner compared with control groups. In vitro, pretreatment of IFN-γ-stimulated HK-2 cells cocultured with Jurkat cells can significantly inhibite the proliferation and promote the apoptosis of Jurkat cells. These results suggest that the activity of IDO negatively regulates T cell activation by TEC, and may play an important inhibitory role in TEC-mediated immune activation and immuno-pathology in the kidney.
Next, we construct a recombinant adenovirus that expresses IDO for gene therapy in vivo. Full-length mouse IDO cDNA was subcloned into pAdTrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with pAdEasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombined adenovirus DNA was transfected into 293 cells with LipofectamineTM2000 for packaging and amplification of Ad-IDO virus and the high-titer adenovirus supernatants were harvested. The expression of IDO is monitored by EGFP fluorescence in infected cells. After transfection with adenovirus DNA, infectious virus was only produced to cause cytopathic effect in the permissive cell line AD293 but not in the non-permissive cell line Hela, confirming only replication-defective but not wild type virus was generated. The specific expression of mouse IDO was verified by PCR in AD 293 cells after infection with Ad-IDO, but not Ad-EGFP, a similarly constructed control virus. Ad-IDO, but not Ad-EGFP, can degradate of tryptophan in culture medium. The results showed that we have successfully constructed a recombinant adenovirus Ad-IDO. The virus will be useful to enforce the activity of IDO in animal models of crescentic GN in vivo.
The last, we test the inteference effect of enforceing IDO by gene transfection and interrupting IDO by 1-MT in the development of crescentic GN in a rat model of NTN. It was demonstrated that the activity of IDO was increased significantly in NTN rat than in the normal rat, when administration of intravenous injection of Ad-IDO into NTN rat, not only had the IDO transfected on renal tubular epithelial cells, but also the tryptophan catabolism in serum was increased more higher than the control NTN rat, and ameliorated renal injury with decreased glomerular crescent formation, CD4+T cells and PCNA+ cells accumulation in kidney tissue and proteinuria compared with the control NTN rat, and prominent splenocyte cells apotosis(p<0.01).While the administration of 1-MT by intraperitoneally injection resulted in the decereased of IDO activity in NTN rat and accelerates crescentic GN renal pathology injured with significant increased glomerular crescent formation, CD4+T cells and PCNA+ cells accumulation in kidney tissue and proteinuria compared with the control NTN rat. Our results demonstrated the involvement of the role of IDO in the pathogenesis of crescentic GN, and enhancing the activity of IDO in vivo may be beneficial for the treatment of human crescentic GN.
Conclusions:
Our results suggest:
1.The upregulated IDO of renal tubular epithelial cells can suppresse T cell activation, and this might play an inhibited role in T cell mediated immune activation and immunopathology in crescentic GN.
2. The activity of IDO is increased in vivo in NTN rat, and which may play an important role in pathogenesis of crescentic GN. While enforced the activity of IDO in vivo might provided a novel therapy method for crescentic GN.
引文
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