免疫性血小板减少症患者骨髓间质细胞及树突状细胞免疫功能异常的研究
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摘要
第一部分
沙利度胺纠正ITP患者骨髓间质细胞诱导调节性树突状细胞功能的研究
研究背景：
免疫性血小板减少症(Immune thrombocytopenia, ITP)是临床上最为常见的一种自身免疫性出血性疾病(外周血血小板计数<100×109/L)。ITP患者临床症状主要表现为皮肤黏膜出血,内脏出血,以及致死性颅内出血。目前ITP的发病机制研究热点主要分为血小板破坏增多以及血小板生成不足两部分。经典的体液免疫机制提出,ITP患者血小板糖蛋白(Glycoprotein, GP)特异性的自身抗体的产生不但加速了血小板破坏同时还减少了血小板的生成。细胞免疫方面的研究发现,ITP患者T辅助细胞(T helper lymphocyte, Th)中Thl/Th2细胞亚群平衡失调,调节性T细胞(Regulatory T cells, Treg)功能障碍数量异常,细胞毒性T细胞(Cytotoxic T lymphocyte, CTL)直接介导的血小板破坏以及Th17细胞过度增殖活化均直接参与了ITP患者的病理生理过程。近期研究发现ITP患者骨髓间质细胞的增殖及免疫功能异常为ITP发病机制研究及治疗提供了新的思路。
骨髓间质细胞(Mesenchymal stem cells, MSC)发现与40年前,是一种非造血并具有多向分化功能的干细胞。体外培养的MSC主要表达一些非特异性表面标志如：CD105, CD73, CD166, CD44, CD90, CD271以及CD29等,并且具有分化为脂肪细胞,成骨细胞,软骨细胞的能力。MSC在固有免疫以及适应性免疫中的作用备受关注。MSCs可以直接干预固有免疫中的补体、Toll样受体(Toll-like receptor).巨噬细胞(Macrophage, MO)、树突状细胞(Dendritic cell, DC)、肥大细胞以及自然杀伤细胞的功能。在适应性免疫中,MSC可以直接抑制T细胞的功能、调节Th细胞的平衡、诱导Treg,并且可以调控B细胞以及抗原提呈细胞(Antigen presenting cell, APC)的功能。MSC在维持免疫耐受中具有显著地作用,其可以阻断APC与T细胞之间的抗原递呈并可以诱导APC向调节性DC方向分化,进而抑制免疫效应细胞的激活增殖。ITP患者的MSC是否依然具有诱导APC向调节性方向分化的能力尚不清楚。
目前ITP患者一线治疗药物主要以糖皮质激素、静脉注射用免疫球蛋白(Intravenous immune globulin G, IVIg)为主,然而有近1/3的患者无效或反复发作。二线治疗药物主要有美罗华(Rituximab)、促血小板生成素(Thrombopoietin, TPO)、罗米司亭(Romiplostim)以及艾曲波帕(Eltrombopag)等新生药物组成,用于治疗经糖皮质激素类药物、免疫球蛋白治疗无效的患者,但是治疗费用昂贵。沙利度胺(Thalidomide, THD)曾用作非巴比妥酸盐药物镇静催眠药,作为一种免疫抑制剂,目前沙利度胺广泛用于：类风湿关节炎、系统性硬化症、干燥综合征、移植物抗宿主病(GvHD)、克罗恩病等免疫相关性病。2004年Falco等利用沙利度胺成功的治愈了一位伴发多发性骨髓瘤的ITP患者。迄今为止,没有与ITP相关的MSC诱导调节性DC研究的报道以及THD对ITP患者MSC免疫功能的影响的报道。
研究目的：
检测正常对照与ITP患者MSC的增殖能力,抑制淋巴细胞激活的能力以及诱导调节性DC的能力差异。检测THD干预前后,ITP患者MSC增殖能力的变化及其分子生物学机制以及对淋巴细胞激活的能力和诱导调节性DC能力的变化。检测TIEG基因在MSC诱导调节性DC中的作用。探讨THD纠正ITP患者MSC功能异常的可能机制。
研究方法：
1.细胞增殖实验(CCK8)：健康对照以及ITP患者的MSC种板后加入不同浓的THDC(0,0.05,0.1,0.5,1,5,10,50mg/mL)培养3天,应用Cell Counting Kit-8(CCK8)试剂盒检测并比较健康对照以及ITP患者MSC细胞增殖情况的变化。
2.细胞凋亡实验(Annexin V/7AAD):健康对照以及ITP患者的MSC种板后加入THD培养3天,应用Annexin V fluorescein isothiocyanate/7-amino-actinomycin D (AnnexinV/7-AAD)试剂盒检测并比较健康对照以及ITP患者MSC细胞凋亡情况的变化。
3.细胞周期实验(PI)：健康对照以及ITP患者的MSC种板后加入THD培养3天,应用碘化丙啶(propidium iodide, PI)试剂盒检测并比较健康对照以及ITP患者MSC细胞周期情况的变化。
4.表达芯片：ITP患者的MSC种板后加入THD培养12小时,应用Human Genome Array (CapitalBio Corp)检测并比较ITP患者MSC在THD干预前后表达基因谱的变化,寻找THD干预前后的差异基因。
5.差异性表达基因验证实验：健康对照以及ITP患者的MSC种板后加入THD培养12小时,实时荧光定量法(real-time reverse-transcription polymerase chain reaction, real-time RT-PCR)鉴证表达芯片中筛选的差异基因。
6.MSC抑制试验：健康对照以及ITP患者的MSC经过THD干预前后,分别与同种异基因的佛波酯(phorbolmyristate acetate, PMA)激活的琥珀酰亚胺酯(Carboxyfluorescein diacetate succinimidyl ester, CFSE)标记的CD4+T细胞、同种同基因的DC共培养。流式细胞术检测T细胞的增殖情况用以检测健康对照以及ITP患者的MSC抑制功能的差异以及THD对MSC抑制功能的影响。
7.调节性DC抑制功能试验：mDC与健康对照以及ITP患者的MSC以及THD干预后的MSC共培养后,经过免疫磁珠分选,再与同种异基因的PMA激活的CFSE标记的CD4+T细胞、同种同基因的DC共培养。流式细胞术检测T细胞的增殖情况用以检测健康对照以及ITP患者的MSC诱导调节性DC功能的差异以及THD对MSC诱导调节性DC功能的影响。
8.培养上清细胞因子检测：健康对照以及ITP患者的MSC经过THD干预前后分别与同种异基因的CD4+T细胞、同种同基因的DC共培养,细胞培养上清中的IL-4、IFN-r, IL-12、IDO、TGF-β、IL-10蛋白应用酶联免疫吸附实验试(enzyme-linked immunoabsorbent assay, ELISA)法检测。
9.DC成熟度检测：mDC与健康对照以及ITP患者的MSC以及THD干预后的MSC共培养后,标记CD80,CD86流式抗体,上机检测THD干预MSC前后对共培养体系中DC细胞的成熟度的影响。
10.慢病毒干扰试验：健康对照MSC分别与TIEG1病毒干扰前后的DC共培养。然后经过免疫磁珠法分选出nDC、MSC-DC、MSC-DC、(TIEGl shRNA)、 MSC-DC (control shRNA)分别与同种异基因的CD4+T细胞共培养。流式细胞术检测T细胞的增殖情况用以验证TIEG1基因在MSC诱导调节性DC中的作用。
研究结果：
1.THD可以显著地促进ITP患者MSC的增殖能力,并且在0.1μg/mL到10μg/mL浓度区间呈现浓度依赖性递增。但当THD浓度大于10μg/mL时对MSC具有抑制性作用。ITP患者MSC处于S+G2期的细胞比例较正常人明显减低,经过THD干预之后其比例显著上升。正常人与ITP患者的MSC凋亡率无明显差别,THD干预之后可以明显的降低二者的细胞凋亡率。
2.THD干预前后表达芯片的结果显示P27、P16、caspase-8、caspase-10、klf4、 c-myc、oct3/4以及TGF-β与’THD的干预密切相关。THD干预后ITP患者MSC的caspase-8、caspase-10基因明显下调,而正常人的无明显变化。THD干预后正常人和ITP患者的c-myc基因均显著下调。THD干预后,ITP患者的oct3/4、TGF-β基因显著上调,而正常人无明显变化。在正常人中klf4基因上调、p27基因下调而在ITP患者中无明显差别。p16基因在THD干预前后在正常人与ITP患者中均无明显变化。
3.MSC抑制试验结果显示：正常人与ITP患者的MSC均不能独自抑制PMA激活的CD4+T细胞增殖。正常人MSC能够明显抑制mDC激活的CD4+T细胞增殖但ITP患者MSC失去了抑制mDC激活的CD4+T细胞增殖能力,THD干预后的MSC恢复了抑制功能。正常人及THD干预后的ITP患者MSC均可以通过DC部分的抑制PMA激活的CD4+T细胞。
4.调节性DC抑制性试验显示：正常人MSC诱导的调节性DC以及THD干预后的ITP患者MSC诱导的调节性DC均失去了激活CD4+T的能力,但是ITP患者的MSC诱导的DC细胞例外。虽然所有条件下诱导的调节性DC均无法单独的显著的抑制PMA激活的CD4+T的增殖。但是正常人MSC诱导的调节性DC以及THD干预后的ITP患者MSC诱导的调节性DC均可以显著的抑制同种异基因mDC激活的CD4+T细胞的增殖,然而ITP患者的MSC诱导的DC失去了这种功能。
5.正常人MSC与THD干预后的ITP患者的MSC和mDC共培养后均可以下调CD80、CD86的表达,而与ITP患者共培养的DC无明显变化。
6.细胞培养上清中,THD干预后组上清中的IL-4、IDO表达量较干预前明显增多；IL-12、IFN-γ表达量明显较少。另外与单独的DC培养上清以及THD干预前MSC/DC共培养上清相比,THD干预后MSC与DC共培养后可以明显提高上清中IL-10、TGF-β的表达量。
7.慢病毒干扰实验结果显示,MSC与干扰TIEG1基因后的DC共培养,无法诱导其向至耐受性方向分化。MSC诱导调节性DC是TIEG1依赖性的。
结论：
1.ITP患者MSC的增殖能力较正常人明显减低。THD干预后可以明显的恢复ITP患者MSC的增殖能力,促使其向S+G2其转化并减低其凋亡率。
2.ITP患者的MSC的免疫抑制功能障碍,THD干预后部分的恢复ITP患者MSC直接的免疫抑制功能,并能纠正ITP患者MSC诱导调节性DC的能力。
3.MSC诱导调节性DC的是TIEG1依赖性的。
第二部分
CD205在ITP患者树突状细胞中的表达及大剂量地塞米松对其调控作用的研究
研究背景：
树突状细胞(Dendritic cell, DC)在1973年首次被Steinman发现并报道。作为抗原递呈细胞,DC广泛分布在全身各组织器官,捕捉内吞处理异常抗原并与主要组织相容性复合体一类分子和二类分子结合分别递呈给CD4+T细胞和CD8+T细胞,在维持机体自身免疫平衡及阻止病理性自身免疫性疾病中发挥着至关重要的作用。DC通过自身表达的不同受体来区分自体抗原以及异体抗原,其可以通过C型选择素受体(C-type lectin receptors, CLRs)识别自身抗原,如：糖蛋白或者通过Toll样受体(Toll-like receptors, TLRs)识别异己抗原,如：微生物抗原。免疫稳态下,抗原与特定的CLRs结合后经由DC递呈,通常会诱导特异性的免疫耐受。CD205主要表达在DC细胞,是目前知之甚少的CLRs家族成员之一,CD205在维持机体对自体抗原的免疫耐受中发挥着重要作用。免疫稳态情况下,自身抗原在CD205介导下诱导T细胞的特异性耐受。并且CD205+DC细胞可以诱导Foxp3-CD4+T向Foxp3+调节性T细胞(regulatory T cell, Treg)方向分化。相关研究表明免疫性血小板减少症患者(Immune thrombocytopenia, ITP)的DC激活同种异基因T细胞的能力较正常人明显增高。但是CD205在ITP患者DC中表达的情况尚未有报道。
ITP是一种常见的自身免疫性疾病,其主要特征之一是抗自身血小板糖蛋白抗原(platelet glycoproteins, GPs)抗体介导的血小板破坏增多。作为CLRs家族的成员,CD205可能参与了DC识别并捕捉自身GPs的过程。目前,糖皮质激素作为ITP的一线用药,大剂量地塞米松(highdose dexamethasone, HD-dexa)冲击疗法的治疗效果已备受认可。HD-dexa可以通过调节FcγR的平衡减低单核细胞的吞噬功能以及调节患者Th1/Th2细胞平衡纠正ITP患者的异常免疫状态。但是应用HD-dexa治疗方案对ITP患者DC细胞的抗原递呈功能、对DC成熟状态的影响以及对DC表达CD205的作用仍未明确。
脾脏是人类重要的免疫器官,免疫应答及免疫耐受均离不开脾脏的功能。在ITP患者中,脾脏是ITP患者血小板破坏的主要器官之一。近期研究发现脾脏中淋巴小结正是ITP患者自身抗原与T细胞接触激活之处。ITP患者脾脏中DC细胞的分布情况以及DC细胞表达CD205的情况尚未清楚。
研究目的：
检测并比较CD205在ITP患者与正常人成熟与未成熟DC细胞中表达的差别。检测并比较HD.dexa对ITP患者与正常人DC成熟状态的影响以及CD205表达的影响。检测CD205表达量与糖皮质激素之间的关系。检测并比较ITP患者与正常人脾脏CD205+DC细胞的组织分布。
研究方法：
1.DC细胞的培养：抽取正常人与ITP患者外周血,Ficoll法分选外周血单个核细胞(Peripheral blood mononuclear cells,PBMC).用CD14+免疫磁珠筛选CD14+细胞后,用含有10%胎牛血清(fetal calf serum,FCS)的RPMI1640培养基重悬细胞并种板。每孔加入终浓度为1000u/mL的白细胞介素4(interleukin-4, IL.4)以及1000u/mL粒细胞生长因子的(granulocyte.macrophage Colony stimulating factor,GM-CSF).培养5天后加入1μg/mL脂多糖(Lipopolysaccharides,LPS)诱导DC成熟。继续培养7天后备用。
2.流式细胞术：ITP患者与正常人DC分别用FITC-或者PE-结合的CD80,CD86,以及CD205抗体标记,同时标记同型对照。应用FACScalibur flow cytometer(BD Biosciences)上机检测并应用Cell Quest Pro software(BDBiosciences)分析数据。
3.实时定量聚合酶链反应：分别收集ITP患者与正常人诱导的DC,提取总RNA用TAKARA逆转录试剂盒合成cDNA:用TOYOBO试剂盒,罗氏480PCR仪器,定量扩增目的基因。
4.地塞米松干预试验：诱导正常人成熟DC与未成熟DC,加入不同浓度地塞米松(0nm0l/L,10nmol/L,25nmol/L,50nmol/L,100nmol/L)继续培养3天。实时定量聚合酶链反应检测CD205.CD80.CD83.CD86的相对表达量,分析CD205.CD80.CD83.CD86与糖皮质激素治疗的相关性。
5.免疫荧光组织化学实验：取ITP患者与正常人脾脏组织,包埋、冰冻、切片-20℃保存备用。取组织切片,风干后丙酮固定,用含1%胎牛蛋白(bovine serum albumin, BSA)的磷酸盐缓冲液(hosphate-buffered saline,PBS)水化,一抗染色过夜,PBS缓冲液洗3次后荧光二抗染色1小时。用Molecular Devices Olympus AX70显微镜观测结果,并用METAMORPH Meta图像分析软件获取组织图像。
研究结果：
1.正常人与ITP患者树突状细胞细胞CD205、CD80、CD83、CD86表达的情况：
1.1在正常人中,随着树突状细胞的趋于成熟,CD205、CD80、CD83、CD86的表达明显增高。
1.2ITP患者未成熟树突状细胞CD205表达较正常人明显减少并且随着DC的趋于成熟,CD205的表达量没有显著增加。ITP患者未成熟树突状细胞CD80、CD83、CD86较正常人明显增高,并且伴随着树突状细胞的成熟CD80、CD83、CD86的表达量较正常人明显升高。
2. HD-dexa治疗前后ITP患者DC细胞CD205、CD80、CD83、CD86表达的情况变化：HD-dexa治疗后的患者成熟树突状细胞CD205表达量明显升高,未成熟树突状细胞CD205表达量没有明显的变化。与此相似,成熟及不成熟树突状细胞CD80、CD86的表达量明显降低。虽然未成熟树突状细胞CD83的表达量在HD-dexa治疗后显著降低,但成熟树突状细胞CD83的表达量无明显变化。
3. HD-dexa对DC表达CD205的影响：mDC的CD205表达量随着地塞米松的浓度递增,imDC的CD205表达量无明显变化。CD80、CD83、CD86的表达与地塞米松的浓度无明显相关性。
4.脾脏免疫组织荧光化学结果：
4.1HE染色结果显示：ITP患者脾脏淋巴小结数量较正常人明显增多,淋巴小结中B细胞区和T细胞区明显增大。
4.2正常人脾脏DC细胞表型呈现CD205+CD80-CD86-HLA-DR+状态,分布于红髓与白髓交界的淋巴小结周围带。ITP患者脾脏DC细胞表型呈现CD205low/-CD80+CD86+HLA-DR+状态,聚集在T细胞区以及B细胞区。
结论：
1.ITP患者DC细胞CD205表达量较正常人明显减低。并且ITP患者DC细胞趋于成熟状态。
2. HD-dexa治疗后的患者CD205表达量明显增加并且DC的成熟度明显降低
3.地塞米松与mDC细胞CD205表达成浓度依赖性递增。
4.ITP患者脾脏淋巴小结数量较正常人明显增多,淋巴小结明显增大。DC细胞聚集在T细胞区以及B细胞区呈CD205low/-CD80+CD86+表型。
Part Ⅰ
Thalidomide corrects impaired mesenchymal stem cell function in inducing tolerogenic DCs in patients with immune thrombocytopenia
Background:
Immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disease in clinical work (peripheral blood platelet count<100×109/L). Mucocutaneous hemorrhage, visceral hemorrhage, menorrhagia as well as fatal intracranial hemorrhage are main manifestations of patients. At present, the research focus of the pathogenesis of ITP is mainly divided into two parts, increased platelet destruction and decreased platelet production. As the pathogenesis of humoral immunology showed, the production of platelet specific autoantibodies against glycoproteins abated thrombocytopoiesis and escalated platelet destruction. As the pathogenesis of cellular immunology showed, the disorder of Thl/Th2balance, the defect regulator T cells (Treg), the directly mediated platelet destruction by Cytotoxic T cells (CTL) and the excessive activation and proliferaton of Th17are all directly involved in the pathophysiological process of ITP. Recent, the dysfunction of bone marrow stromal cell was found which was provided a new insight into the nosogenesis and treatment of ITP.
Bone marrow stromal cells (MSC) were found in40years ago. MSC is a kind of non-hematopoietic stem cells and has the potence of multi-directional differentiation. MSC expanded in vitro, express numerous of nonspecific surface marks including CD29, CD44, CD73, CD90, CD105, CD166and CD271, and possess the capacity to differentiate into osteogenesis, chondrogenesis and adipogenesis. The role of MSC in innate immunity and adaptive immunity have attracted much attention. MSC could directly intervene with the function of complement, toll-like receptors (toll-like receptor), macrophages (macrophage, M0), dendritic cells (dendritic cells, DC), mast cells and natural killer cells (NK) in the innate immune. In adaptive immunity, the MSC can directly inhibit T cell function, adjust the balance of Th cells, induce Treg, delect B cells and modulate antigen presenting cells (APC) function. MSC has a significantly role in maintaining immune tolerance. It can block the connection between T cells and APC and induce APC to differentiate into regulatory population, thereby inhibiting the activation and proliferation of immune effector cells. It isn't clear that MSC of ITP can yet have the ability to induce APC differention into regulatory population.
The first-line treatment drugs for ITP patients mainly include glucocorticoid and intravenous immunoglobulin G (IVIg). However more than one-third of patients has no response to these drugs or relapse. The second-line drugs, mainly of rituximab, thrombopoietin (TPO), romiplostim and eltrombopag, were offered in the treatment of patients failed to respond to first-line treatment drugs but costly. Thalidomide (THD) have been used as a barbiturate sedative hypnotics drugs. As a kind of immunosuppressant, THD were widely used in rheumatoid arthritis, systemic sclerosis, sjogren's syndrome, graft versus host disease (GvHD), crohn's disease and other immune related diseases.2004, Falco et al successfully managed an ITP patient with multiple myeloma with THD. By far, it wasn't reported that the study on MSC induction of regulatory DC in ITP and the influence of THD on MSC immune function of patients with ITP.
Objective:
To compare the proliferation capacity, the inhibition of lymphocyte activation ability and the induction of induced regulatory DC function of MSC in newly diagnosed ITP patients and healthy controls. After THD modulated, to detect the changes of MSC proliferation in newly diagnosed ITP patients and healthy controls. As well as, to investigate the molecular mechanisms before and after modulated with THD and to test the changes of induction of regulatory DC function. To detect the role of TIEG1gene in the process of MSC induction of regulatory DC.
Materials and methods:
1. Cell proliferation assay:At passage4, MSCs were cultured in triplicate in a96-well plate and incubated in humidified air in5%CO2at37℃. After adherence, these cells were treated with THD at concentrations of0(commensurable DMSO),0.05,0.1,0.5,1,5,10, and50mg/mL. The proliferation assay was assessed on day3using the Cell Counting Kit-8(CCK8) according to the manufacturer's recommendations.
2. Cell Apoptosis assay:Briefly, MSCs (ITP/healthy controls, cultured in commensurable DMSO) or THD-MSCs (ITP/healthy controls, cultured in0.5mg/mL THD) were harvested and stained with Annexin V-fluorescein isothiocyanate/7-amino-actinomycin D (Annexin V/7-AAD) according to the manufacturer's recommendations. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
3. Cell-cycle analysis:Briefly, MSCs (ITP/healthy controls, cultured in commensurable DMSO) or THD-MSCs (ITP/healthy controls, cultured in0.5mg/mL THD) were harvested and stained with propidium iodide (PI) according to the manufacturer's recommendations. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
4. Microarray analysis:microarray analysis was conducted on THD-MSCs (ITP, cultured in0.5mg/mL THD) and MSCs (ITP, cultured in commensurable DMSO) for12hours from3independent patients. The abundance of messenger RNA (mRNA) transcripts was measured using the35k human Genome Array (CapitalBio Corp) representing39600transcripts and～25100genes. The data were analyzed using the Molecule Annotation System (MAS)
5. Differential expression gene validation test:Total RNA was extracted using TRIzol reagent (Invitrogen) from MSCs (ITP/control) and THD-MSCs (ITP/control), cultured in DMSO or in0.5mg/mL THD for12hours. Real-time reverse-transcription polymerase chain reaction (qRT-PCR) were used to verify the differential expression gene.
6. MSC inhibition assays:After labeling with CFSE, CD4+T cells or phorbol myristate acetate (PMA) preactivated CD4+T cells were cultured alone or cocultured with MSCs (control), MSCs (ITP), or THD-MSCs (ITP), respectively, in the presence or absence of mDCs in triplicate. All cells were incubated for3days and then collected for fluorescence-activated cell sorter (FACS) analysis.
7. Tolerogenic DC inhibition assays:After coculture with MSCs (control), MSCs (ITP), or THD-MSCs (ITP), the MSC-DCs were sorted using anti-HLA-DR immunomagnetic beads (Miltenyi Biotec). CFSE-labeled allogeneic CD41T cells were cocultured with PMA, mDCs, MSC (control)-DCs, MSC (ITP)-DCs, THD-MSC (ITP)-DCs, MSC (control)-DCs+PMA, MSC (ITP)-DCs+PMA, THD-MSC (ITP)-DCs+PMA, MSC (control)-DCs+mDCs, MSC (ITP)-DCs+mDCs, or THD-MSC (ITP)-DCs+mDCs, respectively. All cells were cultured in triplicate and incubated for3days and then collected for FACS analysis.
8. Cytokine production:CD4+T cells, mDCs and MSCs were cultured alone or in combination. The levels of IL-4, IFN-y, IL-12, IDO, TGF-p, and IL-10in cultural supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) after3days.
9. Dendritic cells maturity test:After co-cultured with MSC (ITP), MSC (THD-ITP) and MSC (congtrol). The DC staining was performed using fluorescein isothiocyanate-or phycoerythrin-conjugated anti-CD80and anti-CD86(BD Pharmingen) monoclonal antibodies. The isotype IgG of same species origin was used as a control. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
10. Lentivirus interference:TIEG1small hairpin RNA (shRNA)(h) lentiviral particles and control shRNA lentiviral particles were purchased from Santa Cruz Biotechnology. The mDCs were preinfected with prepared virus and then co-cultured with MSC. After sorted using anti-HLA-DR immunomagnetic beads, the CD4+T cells were co-cultured with mDC, MSC-DC, MSC-DC (TIEG1shRNA), MSC-DC (control shRNA), respectively. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
Results:
1. THD with concentration radients fromO.1μg/mL to10μg/mL could stimulate the proliferationof MSCs (ITP) in a dose-dependent manner. Yet for all patients, higher THD concentrations (>10μg/mL) would lead to an opposite result. The proliferation of MSCs from both ITP patients and healthy controls could be promoted after0.5mg/mL THD modulation. There were less cells entered into S+G2phases in the MSC (ITP) group compared with those in MSCs (control). After THD modulation, the frequency of cells in S and G2phases was increased in THD-MSCs (ITP). The apoptosis analysis of MSCs showed there was no significant difference of Annexin-V+/7-AAD+cells between patients and healthy controls, and THD modulation could decrease the apoptosis rate both in patients and in controls.
2. Differentially expressed genes indexed by Gene Ontology (Go) biological process of MSCs were shown in the Gene Expression Omnibus database under accession number GSE44948; enrichment for variants was associated with DNA-dependent regulation of transcription, cell cycle, mitosis, DNA replication, cell proliferation, apoptosis, regulation of cell growth, and regulation of apoptosis, etc. We selected several genes associated with cell proliferation, apoptosis and cell cycle, including p27, p16, caspase-8, caspase-10, klf4, c-myc, oct3/4, and TGF-β which may be associated with THD modulation. The expression of caspase-8and caspase-10was downregulated in THD-MSCs (ITP), while there was no significant difference between MSCs (control) and THD-MSCs (control). The expression of c-myc was downregulated in both THD-MSCs (ITP) and THD-MSCs (control). The expression of oct3/4and TGF-β was upregulated in THD-MSCs (ITP), while there was no obvious change between MSCs (control) and THD-MSCs (control). The expression of klf4and p27was upregulated or downregulated, respectively, while there was no obvious change between MSCs (ITP) and THD-MSCs (ITP). There was no obvious change of p16either in MSCs (control) or in MSCs (ITP) before and after modulated with THD. We confirmed the downregulation of caspase-8and caspase-10, and upregulation of oct3/4and TGF-β, after THD modulation was specific for MSC (ITP) and explained the THD induced increase in MSC proliferation, as well as their potentially ability to induce tolerogenic DCs.
3. The data demonstrated that mDCs and PMA significantly stimulated T-cell proliferation. When MSCs (control) or THD-MSCs (ITP) were added to mDC cocultures in the absence of PMA, they inhibited T-cell proliferation, whereas the addition of MSCs (ITP) had no inhibitory effect. Notwithstanding, THD-MSCs (ITP) were unable to prevent T-cell proliferation activated in the presence of PMA. The data also showed that addition of mDCs to the coculture system alleviated T-cell proliferation.
4. The mDCs regulated by MSCs from healthy controls suppressed the proliferation of T lymphocytes, whereas the mDCs regulated by MSCs from ITP patients did not. However, the inhibitory capacity of mDCs was increased when they were incubated with THD modulated ITP-MSCs. We also investigated whether the inhibitory effect of these tolerogenic DCs was strong enough to silence the lymphocytes activated by PMA. The data demonstrated that mDCs regulated by MSCs from ITP patients, healthy controls, or THD modulated MSCs were unable to inhibit T-cell proliferation. These results further confirmed that the MSCs from ITP patients had lost the ability to induce tolerogenicity in mDCs. Although the tolerogenic mDCs could not prevent T-cell proliferation activated by PMA, they did block the allogeneic mDC-activated T-cell proliferation.
5. Furthermore, we conducted phenotypic analysis of the mDCs cocultured with MSCs from healthy controls, from ITP patients or THD-modulated MSCs. MSCs from ITP patients failed to downregulate CD80and CD86expression in mDCs, while MSCs from healthy controls significantly inhibited the expression of these costimulatory factors. Accordingly, THD-MSCs also downregulated the expression of CD80and CD86in mDCs.
6. Since IL-10and TGF-β are potent immunosuppressive cytokines tolerogenic DC-mediated inhibition. The levels of IL-10and TGF-β in the culture supernatants of THD-MSCs (ITP) and mDCs were higher than those of mDCs alone or mDCs plus MSCs (ITP). These results indicate that the MSC-elicited inhibitory property of DCs is related to DC maturation state and TGF-β or IL-10cytokine production. We also assayed the levels of IL-4, IFN-y, IDO, and IL-12in culture supernatants. When CD4+T cells and mDCs were cocultured with THD-MSCs (ITP), the level of IL-4and IDO was higher while the IFN-y level was lower than that cocultured with MSCs (ITP) or without MSCs. There was no significant difference in IL-12level between cultures with THD-MSCs (ITP) or with MSCs (ITP), though in both cases it was lower than that without any MSCs.
7. The mDCs infected with TIEG1shRNA (h) lentiviral particles lost the ability to suppress T-cell proliferation. The results indicate that the acquisition of tolerance by mDCs upon incubation with MSCs depends on TIEG1.
Conclusions:
1. The MSCs from ITP patients had lost their regulatory capacity, which could be increased by THD modulation. THD with concentration radients could stimulate the proliferationof MSCs (ITP) in a dose-dependent manner. After THD modulation, the frequency of cells in S and G2phases was increased in THD-MSCs (ITP). THD modulation could decrease the apoptosis rate both in patients and in controls. After coculture with MSCs, the mDCs may have tolerogenic rather than stimulatory effects and play an important role in mediating the inhibitory activity of MSCs.
2. The study demonstrated that the regulatory ability of MSCs from ITP patients could be regained upon THD modulation. After coculture with MSCs, the mDCs differentiated into a novel tolerogenic DC population.
3. The acquisition of tolerance by mDCs upon incubation with MSCs depends on TIEG1.
Part Ⅱ
Expression of CD205on dendritic cells and its regulation by high dose dexamethasone in immune thrombocytopenia
Background:
Dendritic cells (DCs) were found in1973by Steinman. As antigen presenting cells, DCs are located throughout the body to capture and internalize invading pathogens, and subsequently process and present antigens on MHC class Ⅰ and class Ⅱ molecules to CD8+and CD4+T cells, respectively. DCs play a crucial role in maintaining the balance of immune system and preventing pathological autoimmune diseases. DCs were well equipped to distinguish self to non-self-antigens by the variable expression of cell-surface receptors. Such as C-type lectin receptors (CLRs) for glycoproteins and Toll-like receptors (TLRs) for microbial antigens. In steady state, uptake of antigen by CLRs may induce antigen-specific tolerance. CD205is one of the least understood member of the family of CLRs which is predominantly expressed by the DCs. Recent study showed CD205+DCs are much more effective inducers of functional Foxp3+Treg from Foxp3-CD4+T cells. In addition, ITP DCs have increased capacity to present apoptotic platelets to T lymphocytes. But the expression of CD205on DCs of patients with ITP has not been reported.
Immune thrombocytopenia (ITP) is a common autoimmune diseases. It is characterized with anti-platelet glycoproteins (GPs) antibody mediated increased destruction of platelets. As a member of the family of CLRs, CD205may participate in the process of DC to identify and capture self-GPs. Corticosteroids is the most appropriate first-line treatment for patients with ITP and highdose dexamethasone (HD-dexa) in4-day cycles has proven its clinical efficacy in the treatment of ITP. The mechanism study of the HD-dexa treatment proved that by regulating the balance of the Fc gamma receptor (FcR) and regulating the polarization of Th1/Th2 cells, the abnormal immune status in patients with ITP was restored. But the expression of CD205on dendritic cells and its regulation by high dose dexamethasone in ITP patients was still not clear.
The spleen is of particular interest because it is the major site of immune response, where the immune response is triggered and regulated, especially for platelets destruction in ITP. Recently, Daridon et al. reported that proliferative lymphoid nodules are the sites of auto-antigen stimulation in ITP potentially related to a lack of control by T cells and/or the present auto-antigen. The distribution of DC in spleen and the expression of CD205of DC of patients with in spleen is not yet clear.
Objective:
The aim of this study was to determine the expression status of CD205in mature and immature DC in ITP patients and healthy controls. To detect the effect of high dose dexamethasone to mature conditions and the expression of CD205of DC in patients with ITP. To investigate the correlation between the effect of dexamethasone and the expression of CD205of DC. To compare the distribution of CD205+DC and the mature conditions of CD205+DC in patients with ITP and healthy controls.
Materials and methods:
1. Preparetion of DCs:Peripheral blood mononuclear cells (PBMCs) were prepared from peripheral blood of ITP patients and healthy controls. CD14+cells were negatively selected from the PBMCs using micromagnetic beads, according to the manufacturer's instructions. The purified CD14+cells were then incubated in RPMI1640culture medium with10%fetal calf serum,1000u/mL granulocyte-macrophage colony stimulating factor and1000u/mL interleukin-4for5days. Lipopolysaccharides (LPS,1ug/mL) were added to the cells for an additional2days. At day7the DCs were collected for flow cytometry and quantitative real time-PCR (qRT-PCR) analysis.
2. Cell-surface staining for flow cytometric determination of CD205, CD80, CD83, CD86:Cell (ITP and healthy nomal) staining was performed using FITC-or PE-conjugated anti-CD80, anti-CD86and anti-CD205monoclonal antibodies. The same-species-isotype IgG was used as an isotype control. Data acquisition was performed using a FACScalibur flow cytometer (BD Biosciences), and analyzed with Cell Quest Pro software (BD Biosciences).
3. Real-time reverse transcription-PCR (RT-PCR) analysis:Total RNA was extracted from cells (ITP and healthy nomal) by TRIzol (Invitrogen). cDNA was synthesized using the PrimeScript RT reagent kit (Takara) according to the manufacturer's instructions. qPCR was performed with Power SYBR Green PCR Master Mix (Takara) under the following parameters:denaturation95℃,5minutes; followed by45cycles of95℃,10seconds;60℃,10seconds;72℃,10seconds. Melt-curve analysis was performed following amplification. The delta Ct calculation method using arithmetic formulas was used for relative quantification of target gene.
4. Dexamethasone intervention trial:In order to further observe the relation between the expression of CD205, CD80, CD83, CD86and the effect of dexamethasone treatment, we put the follow concentration of dexamethasone (0nmol/L,10nmol/L,25nmol/L,50nmol/L,100nmol/L) in use. After modulated with dexamethasone for3days, the DCs were collected for quantitative real time-PCR analysis.
5. Immunofluorescence:Spleen tissue was frozen in OCT Compound and stored at-80℃. Frozen sections (5μm) prepared using a Microm cryostat were air-dried and stored at-20℃. Upon thawing, sections were airdried, fixed for10min in acetone, rehydrated in phosphate-buffered saline (PBS)/1%bovine serum albumin (BSA) and stained overnight in4℃with purified mouse anti-human CD86(mouse IgG2b, k) or purified mouse anti-human CD80(mouse IgGl, k), as well as rabbit anti human CD205(IgG, R18-D). After washing3times in PBS, sections were stained with rhodamine (TRITC)-conjugated affinipure goat anti-rabbit IgG (H+L) secondary antibodies or fluorescein-conjugated affinipure goat anti-mouse IgG (H+L) secondary antibodies (Zhongshanjinqiao, Beijing) diluted in PBS/1%BSA to2μg/ml. Slides were viewed on a Molecular Devices Olympus AX70 deconvolution microscope (Olympus America Inc, Lake Success, NJ) running METAMORPH Meta Imaging series software (Universal Imaging Corporation, West Chester, PA).
Results:
1. The expression of CD205, CD80, CD83and CD86of mature and immature DC of patients with ITP and healthy controls.
1.1With DCs mature process, the frequency of CD205, CD80, CD83and CD86positive DCs were all rised in control.
1.2The frequency of CD205failed up-regulation along with DCs maturation in ITP patients. Further more, we observed an increased frequency of CD80and CD86of imDC in ITP patients compared with that in controls, In addition, with DCs maturation the frequency of CD80, CD83and CD86significantly up-regulated in patients with ITP than that in controls.
2. Low expression of CD205of DCs in patients with ITP can be up-regulated by HD-dexa treatment:After administration of HD-dexa treatment to ITP patients results in elevation of CD205mRNA levels of mDC but not in imDC. In biological accordance with this marked augmentation, CD80and CD86mRNA levels steadily declined over the study period but CD83mRNA levels had no obvious change before and after HD-dexa therapy although the CD83mRNA levels reduced in imDC.
3. Administration of dexamethasone results in up-regulation of CD205expression in a dose-dependent manner in mDCs:In mDCs, the expression amount of CD205had positive relevance to the dose of dexamethasone but not in imDCs, while there is no causal link between the relative quantity of CD80, Cd83, and CD86and the dosageof dexamethasone in the mature and immature DCs.
4. The DCs in ITP spleen were typically CD205low and in a mature functional state.
4.1The HE staining (hematoxylin-eosin staining) of the tissues showed that the spleens in ITP patients had far more white pulps than control in the field. In the meanwhile, the filtration of lymphocytes in the white pulp area was much bigger than the control group.
4.2The CD205could be found in the border region between the red and white pulp in control spleen and can be found in the T-cell areas and the B-cell areas of ITP spleen. We verified that costimulatory (CD80and CD86) molecules were expressed in both T-cell and B-cell areas, no matter in control spleen or in ITP spleen. But the frequency of CD80and CD86positive cells were lower in the control human spleen than that in ITP spleen.
Conclusions:
1. The expression of CD205was significantly reduced in patients with ITP. In addition, the mature condition of DC in patients in a semi-mature state.
2. After HD-dexa treatment, the expression of CD205was markedly up-regulated while the expression of CD80, CD86was dramatically down-regulated.
3. Administration of dexamethasone results in up-regulation of CD205expression in a dose-dependent manner in mDCs.
4. Our study indicate that most CD205+DCs of the control human spleen in an immature functional state. But in ITP spleen, the DCs were typically CD205low or negative and in a mature functional state.
沙利度胺纠正ITP患者骨髓间质细胞诱导调节性树突状细胞功能的研究
研究背景：
免疫性血小板减少症(Immune thrombocytopenia, ITP)是临床上最为常见的一种自身免疫性出血性疾病(外周血血小板计数<100×109/L)。ITP患者临床症状主要表现为皮肤黏膜出血,内脏出血,以及致死性颅内出血。目前ITP的发病机制研究热点主要分为血小板破坏增多以及血小板生成不足两部分。经典的体液免疫机制提出,ITP患者血小板糖蛋白(Glycoprotein, GP)特异性的自身抗体的产生不但加速了血小板破坏同时还减少了血小板的生成。细胞免疫方面的研究发现,ITP患者T辅助细胞(T helper lymphocyte, Th)中Thl/Th2细胞亚群平衡失调,调节性T细胞(Regulatory T cells, Treg)功能障碍数量异常,细胞毒性T细胞(Cytotoxic T lymphocyte, CTL)直接介导的血小板破坏以及Th17细胞过度增殖活化均直接参与了ITP患者的病理生理过程。近期研究发现ITP患者骨髓间质细胞的增殖及免疫功能异常为ITP发病机制研究及治疗提供了新的思路。
骨髓间质细胞(Mesenchymal stem cells, MSC)发现与40年前,是一种非造血并具有多向分化功能的干细胞。体外培养的MSC主要表达一些非特异性表面标志如：CD105, CD73, CD166, CD44, CD90, CD271以及CD29等,并且具有分化为脂肪细胞,成骨细胞,软骨细胞的能力。MSC在固有免疫以及适应性免疫中的作用备受关注。MSCs可以直接干预固有免疫中的补体、Toll样受体(Toll-like receptor).巨噬细胞(Macrophage, MO)、树突状细胞(Dendritic cell, DC)、肥大细胞以及自然杀伤细胞的功能。在适应性免疫中,MSC可以直接抑制T细胞的功能、调节Th细胞的平衡、诱导Treg,并且可以调控B细胞以及抗原提呈细胞(Antigen presenting cell, APC)的功能。MSC在维持免疫耐受中具有显著地作用,其可以阻断APC与T细胞之间的抗原递呈并可以诱导APC向调节性DC方向分化,进而抑制免疫效应细胞的激活增殖。ITP患者的MSC是否依然具有诱导APC向调节性方向分化的能力尚不清楚。
目前ITP患者一线治疗药物主要以糖皮质激素、静脉注射用免疫球蛋白(Intravenous immune globulin G, IVIg)为主,然而有近1/3的患者无效或反复发作。二线治疗药物主要有美罗华(Rituximab)、促血小板生成素(Thrombopoietin, TPO)、罗米司亭(Romiplostim)以及艾曲波帕(Eltrombopag)等新生药物组成,用于治疗经糖皮质激素类药物、免疫球蛋白治疗无效的患者,但是治疗费用昂贵。沙利度胺(Thalidomide, THD)曾用作非巴比妥酸盐药物镇静催眠药,作为一种免疫抑制剂,目前沙利度胺广泛用于：类风湿关节炎、系统性硬化症、干燥综合征、移植物抗宿主病(GvHD)、克罗恩病等免疫相关性病。2004年Falco等利用沙利度胺成功的治愈了一位伴发多发性骨髓瘤的ITP患者。迄今为止,没有与ITP相关的MSC诱导调节性DC研究的报道以及THD对ITP患者MSC免疫功能的影响的报道。
研究目的：
检测正常对照与ITP患者MSC的增殖能力,抑制淋巴细胞激活的能力以及诱导调节性DC的能力差异。检测THD干预前后,ITP患者MSC增殖能力的变化及其分子生物学机制以及对淋巴细胞激活的能力和诱导调节性DC能力的变化。检测TIEG基因在MSC诱导调节性DC中的作用。探讨THD纠正ITP患者MSC功能异常的可能机制。
研究方法：
1.细胞增殖实验(CCK8)：健康对照以及ITP患者的MSC种板后加入不同浓的THDC(0,0.05,0.1,0.5,1,5,10,50mg/mL)培养3天,应用Cell Counting Kit-8(CCK8)试剂盒检测并比较健康对照以及ITP患者MSC细胞增殖情况的变化。
2.细胞凋亡实验(Annexin V/7AAD):健康对照以及ITP患者的MSC种板后加入THD培养3天,应用Annexin V fluorescein isothiocyanate/7-amino-actinomycin D (AnnexinV/7-AAD)试剂盒检测并比较健康对照以及ITP患者MSC细胞凋亡情况的变化。
3.细胞周期实验(PI)：健康对照以及ITP患者的MSC种板后加入THD培养3天,应用碘化丙啶(propidium iodide, PI)试剂盒检测并比较健康对照以及ITP患者MSC细胞周期情况的变化。
4.表达芯片：ITP患者的MSC种板后加入THD培养12小时,应用Human Genome Array (CapitalBio Corp)检测并比较ITP患者MSC在THD干预前后表达基因谱的变化,寻找THD干预前后的差异基因。
5.差异性表达基因验证实验：健康对照以及ITP患者的MSC种板后加入THD培养12小时,实时荧光定量法(real-time reverse-transcription polymerase chain reaction, real-time RT-PCR)鉴证表达芯片中筛选的差异基因。
6.MSC抑制试验：健康对照以及ITP患者的MSC经过THD干预前后,分别与同种异基因的佛波酯(phorbolmyristate acetate, PMA)激活的琥珀酰亚胺酯(Carboxyfluorescein diacetate succinimidyl ester, CFSE)标记的CD4+T细胞、同种同基因的DC共培养。流式细胞术检测T细胞的增殖情况用以检测健康对照以及ITP患者的MSC抑制功能的差异以及THD对MSC抑制功能的影响。
7.调节性DC抑制功能试验：mDC与健康对照以及ITP患者的MSC以及THD干预后的MSC共培养后,经过免疫磁珠分选,再与同种异基因的PMA激活的CFSE标记的CD4+T细胞、同种同基因的DC共培养。流式细胞术检测T细胞的增殖情况用以检测健康对照以及ITP患者的MSC诱导调节性DC功能的差异以及THD对MSC诱导调节性DC功能的影响。
8.培养上清细胞因子检测：健康对照以及ITP患者的MSC经过THD干预前后分别与同种异基因的CD4+T细胞、同种同基因的DC共培养,细胞培养上清中的IL-4、IFN-r, IL-12、IDO、TGF-β、IL-10蛋白应用酶联免疫吸附实验试(enzyme-linked immunoabsorbent assay, ELISA)法检测。
9.DC成熟度检测：mDC与健康对照以及ITP患者的MSC以及THD干预后的MSC共培养后,标记CD80,CD86流式抗体,上机检测THD干预MSC前后对共培养体系中DC细胞的成熟度的影响。
10.慢病毒干扰试验：健康对照MSC分别与TIEG1病毒干扰前后的DC共培养。然后经过免疫磁珠法分选出nDC、MSC-DC、MSC-DC、(TIEGl shRNA)、 MSC-DC (control shRNA)分别与同种异基因的CD4+T细胞共培养。流式细胞术检测T细胞的增殖情况用以验证TIEG1基因在MSC诱导调节性DC中的作用。
研究结果：
1.THD可以显著地促进ITP患者MSC的增殖能力,并且在0.1μg/mL到10μg/mL浓度区间呈现浓度依赖性递增。但当THD浓度大于10μg/mL时对MSC具有抑制性作用。ITP患者MSC处于S+G2期的细胞比例较正常人明显减低,经过THD干预之后其比例显著上升。正常人与ITP患者的MSC凋亡率无明显差别,THD干预之后可以明显的降低二者的细胞凋亡率。
2.THD干预前后表达芯片的结果显示P27、P16、caspase-8、caspase-10、klf4、 c-myc、oct3/4以及TGF-β与’THD的干预密切相关。THD干预后ITP患者MSC的caspase-8、caspase-10基因明显下调,而正常人的无明显变化。THD干预后正常人和ITP患者的c-myc基因均显著下调。THD干预后,ITP患者的oct3/4、TGF-β基因显著上调,而正常人无明显变化。在正常人中klf4基因上调、p27基因下调而在ITP患者中无明显差别。p16基因在THD干预前后在正常人与ITP患者中均无明显变化。
3.MSC抑制试验结果显示：正常人与ITP患者的MSC均不能独自抑制PMA激活的CD4+T细胞增殖。正常人MSC能够明显抑制mDC激活的CD4+T细胞增殖但ITP患者MSC失去了抑制mDC激活的CD4+T细胞增殖能力,THD干预后的MSC恢复了抑制功能。正常人及THD干预后的ITP患者MSC均可以通过DC部分的抑制PMA激活的CD4+T细胞。
4.调节性DC抑制性试验显示：正常人MSC诱导的调节性DC以及THD干预后的ITP患者MSC诱导的调节性DC均失去了激活CD4+T的能力,但是ITP患者的MSC诱导的DC细胞例外。虽然所有条件下诱导的调节性DC均无法单独的显著的抑制PMA激活的CD4+T的增殖。但是正常人MSC诱导的调节性DC以及THD干预后的ITP患者MSC诱导的调节性DC均可以显著的抑制同种异基因mDC激活的CD4+T细胞的增殖,然而ITP患者的MSC诱导的DC失去了这种功能。
5.正常人MSC与THD干预后的ITP患者的MSC和mDC共培养后均可以下调CD80、CD86的表达,而与ITP患者共培养的DC无明显变化。
6.细胞培养上清中,THD干预后组上清中的IL-4、IDO表达量较干预前明显增多；IL-12、IFN-γ表达量明显较少。另外与单独的DC培养上清以及THD干预前MSC/DC共培养上清相比,THD干预后MSC与DC共培养后可以明显提高上清中IL-10、TGF-β的表达量。
7.慢病毒干扰实验结果显示,MSC与干扰TIEG1基因后的DC共培养,无法诱导其向至耐受性方向分化。MSC诱导调节性DC是TIEG1依赖性的。
结论：
1.ITP患者MSC的增殖能力较正常人明显减低。THD干预后可以明显的恢复ITP患者MSC的增殖能力,促使其向S+G2其转化并减低其凋亡率。
2.ITP患者的MSC的免疫抑制功能障碍,THD干预后部分的恢复ITP患者MSC直接的免疫抑制功能,并能纠正ITP患者MSC诱导调节性DC的能力。
3.MSC诱导调节性DC的是TIEG1依赖性的。
第二部分
CD205在ITP患者树突状细胞中的表达及大剂量地塞米松对其调控作用的研究
研究背景：
树突状细胞(Dendritic cell, DC)在1973年首次被Steinman发现并报道。作为抗原递呈细胞,DC广泛分布在全身各组织器官,捕捉内吞处理异常抗原并与主要组织相容性复合体一类分子和二类分子结合分别递呈给CD4+T细胞和CD8+T细胞,在维持机体自身免疫平衡及阻止病理性自身免疫性疾病中发挥着至关重要的作用。DC通过自身表达的不同受体来区分自体抗原以及异体抗原,其可以通过C型选择素受体(C-type lectin receptors, CLRs)识别自身抗原,如：糖蛋白或者通过Toll样受体(Toll-like receptors, TLRs)识别异己抗原,如：微生物抗原。免疫稳态下,抗原与特定的CLRs结合后经由DC递呈,通常会诱导特异性的免疫耐受。CD205主要表达在DC细胞,是目前知之甚少的CLRs家族成员之一,CD205在维持机体对自体抗原的免疫耐受中发挥着重要作用。免疫稳态情况下,自身抗原在CD205介导下诱导T细胞的特异性耐受。并且CD205+DC细胞可以诱导Foxp3-CD4+T向Foxp3+调节性T细胞(regulatory T cell, Treg)方向分化。相关研究表明免疫性血小板减少症患者(Immune thrombocytopenia, ITP)的DC激活同种异基因T细胞的能力较正常人明显增高。但是CD205在ITP患者DC中表达的情况尚未有报道。
ITP是一种常见的自身免疫性疾病,其主要特征之一是抗自身血小板糖蛋白抗原(platelet glycoproteins, GPs)抗体介导的血小板破坏增多。作为CLRs家族的成员,CD205可能参与了DC识别并捕捉自身GPs的过程。目前,糖皮质激素作为ITP的一线用药,大剂量地塞米松(highdose dexamethasone, HD-dexa)冲击疗法的治疗效果已备受认可。HD-dexa可以通过调节FcγR的平衡减低单核细胞的吞噬功能以及调节患者Th1/Th2细胞平衡纠正ITP患者的异常免疫状态。但是应用HD-dexa治疗方案对ITP患者DC细胞的抗原递呈功能、对DC成熟状态的影响以及对DC表达CD205的作用仍未明确。
脾脏是人类重要的免疫器官,免疫应答及免疫耐受均离不开脾脏的功能。在ITP患者中,脾脏是ITP患者血小板破坏的主要器官之一。近期研究发现脾脏中淋巴小结正是ITP患者自身抗原与T细胞接触激活之处。ITP患者脾脏中DC细胞的分布情况以及DC细胞表达CD205的情况尚未清楚。
研究目的：
检测并比较CD205在ITP患者与正常人成熟与未成熟DC细胞中表达的差别。检测并比较HD.dexa对ITP患者与正常人DC成熟状态的影响以及CD205表达的影响。检测CD205表达量与糖皮质激素之间的关系。检测并比较ITP患者与正常人脾脏CD205+DC细胞的组织分布。
研究方法：
1.DC细胞的培养：抽取正常人与ITP患者外周血,Ficoll法分选外周血单个核细胞(Peripheral blood mononuclear cells,PBMC).用CD14+免疫磁珠筛选CD14+细胞后,用含有10%胎牛血清(fetal calf serum,FCS)的RPMI1640培养基重悬细胞并种板。每孔加入终浓度为1000u/mL的白细胞介素4(interleukin-4, IL.4)以及1000u/mL粒细胞生长因子的(granulocyte.macrophage Colony stimulating factor,GM-CSF).培养5天后加入1μg/mL脂多糖(Lipopolysaccharides,LPS)诱导DC成熟。继续培养7天后备用。
2.流式细胞术：ITP患者与正常人DC分别用FITC-或者PE-结合的CD80,CD86,以及CD205抗体标记,同时标记同型对照。应用FACScalibur flow cytometer(BD Biosciences)上机检测并应用Cell Quest Pro software(BDBiosciences)分析数据。
3.实时定量聚合酶链反应：分别收集ITP患者与正常人诱导的DC,提取总RNA用TAKARA逆转录试剂盒合成cDNA:用TOYOBO试剂盒,罗氏480PCR仪器,定量扩增目的基因。
4.地塞米松干预试验：诱导正常人成熟DC与未成熟DC,加入不同浓度地塞米松(0nm0l/L,10nmol/L,25nmol/L,50nmol/L,100nmol/L)继续培养3天。实时定量聚合酶链反应检测CD205.CD80.CD83.CD86的相对表达量,分析CD205.CD80.CD83.CD86与糖皮质激素治疗的相关性。
5.免疫荧光组织化学实验：取ITP患者与正常人脾脏组织,包埋、冰冻、切片-20℃保存备用。取组织切片,风干后丙酮固定,用含1%胎牛蛋白(bovine serum albumin, BSA)的磷酸盐缓冲液(hosphate-buffered saline,PBS)水化,一抗染色过夜,PBS缓冲液洗3次后荧光二抗染色1小时。用Molecular Devices Olympus AX70显微镜观测结果,并用METAMORPH Meta图像分析软件获取组织图像。
研究结果：
1.正常人与ITP患者树突状细胞细胞CD205、CD80、CD83、CD86表达的情况：
1.1在正常人中,随着树突状细胞的趋于成熟,CD205、CD80、CD83、CD86的表达明显增高。
1.2ITP患者未成熟树突状细胞CD205表达较正常人明显减少并且随着DC的趋于成熟,CD205的表达量没有显著增加。ITP患者未成熟树突状细胞CD80、CD83、CD86较正常人明显增高,并且伴随着树突状细胞的成熟CD80、CD83、CD86的表达量较正常人明显升高。
2. HD-dexa治疗前后ITP患者DC细胞CD205、CD80、CD83、CD86表达的情况变化：HD-dexa治疗后的患者成熟树突状细胞CD205表达量明显升高,未成熟树突状细胞CD205表达量没有明显的变化。与此相似,成熟及不成熟树突状细胞CD80、CD86的表达量明显降低。虽然未成熟树突状细胞CD83的表达量在HD-dexa治疗后显著降低,但成熟树突状细胞CD83的表达量无明显变化。
3. HD-dexa对DC表达CD205的影响：mDC的CD205表达量随着地塞米松的浓度递增,imDC的CD205表达量无明显变化。CD80、CD83、CD86的表达与地塞米松的浓度无明显相关性。
4.脾脏免疫组织荧光化学结果：
4.1HE染色结果显示：ITP患者脾脏淋巴小结数量较正常人明显增多,淋巴小结中B细胞区和T细胞区明显增大。
4.2正常人脾脏DC细胞表型呈现CD205+CD80-CD86-HLA-DR+状态,分布于红髓与白髓交界的淋巴小结周围带。ITP患者脾脏DC细胞表型呈现CD205low/-CD80+CD86+HLA-DR+状态,聚集在T细胞区以及B细胞区。
结论：
1.ITP患者DC细胞CD205表达量较正常人明显减低。并且ITP患者DC细胞趋于成熟状态。
2. HD-dexa治疗后的患者CD205表达量明显增加并且DC的成熟度明显降低
3.地塞米松与mDC细胞CD205表达成浓度依赖性递增。
4.ITP患者脾脏淋巴小结数量较正常人明显增多,淋巴小结明显增大。DC细胞聚集在T细胞区以及B细胞区呈CD205low/-CD80+CD86+表型。
Part Ⅰ
Thalidomide corrects impaired mesenchymal stem cell function in inducing tolerogenic DCs in patients with immune thrombocytopenia
Background:
Immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disease in clinical work (peripheral blood platelet count<100×109/L). Mucocutaneous hemorrhage, visceral hemorrhage, menorrhagia as well as fatal intracranial hemorrhage are main manifestations of patients. At present, the research focus of the pathogenesis of ITP is mainly divided into two parts, increased platelet destruction and decreased platelet production. As the pathogenesis of humoral immunology showed, the production of platelet specific autoantibodies against glycoproteins abated thrombocytopoiesis and escalated platelet destruction. As the pathogenesis of cellular immunology showed, the disorder of Thl/Th2balance, the defect regulator T cells (Treg), the directly mediated platelet destruction by Cytotoxic T cells (CTL) and the excessive activation and proliferaton of Th17are all directly involved in the pathophysiological process of ITP. Recent, the dysfunction of bone marrow stromal cell was found which was provided a new insight into the nosogenesis and treatment of ITP.
Bone marrow stromal cells (MSC) were found in40years ago. MSC is a kind of non-hematopoietic stem cells and has the potence of multi-directional differentiation. MSC expanded in vitro, express numerous of nonspecific surface marks including CD29, CD44, CD73, CD90, CD105, CD166and CD271, and possess the capacity to differentiate into osteogenesis, chondrogenesis and adipogenesis. The role of MSC in innate immunity and adaptive immunity have attracted much attention. MSC could directly intervene with the function of complement, toll-like receptors (toll-like receptor), macrophages (macrophage, M0), dendritic cells (dendritic cells, DC), mast cells and natural killer cells (NK) in the innate immune. In adaptive immunity, the MSC can directly inhibit T cell function, adjust the balance of Th cells, induce Treg, delect B cells and modulate antigen presenting cells (APC) function. MSC has a significantly role in maintaining immune tolerance. It can block the connection between T cells and APC and induce APC to differentiate into regulatory population, thereby inhibiting the activation and proliferation of immune effector cells. It isn't clear that MSC of ITP can yet have the ability to induce APC differention into regulatory population.
The first-line treatment drugs for ITP patients mainly include glucocorticoid and intravenous immunoglobulin G (IVIg). However more than one-third of patients has no response to these drugs or relapse. The second-line drugs, mainly of rituximab, thrombopoietin (TPO), romiplostim and eltrombopag, were offered in the treatment of patients failed to respond to first-line treatment drugs but costly. Thalidomide (THD) have been used as a barbiturate sedative hypnotics drugs. As a kind of immunosuppressant, THD were widely used in rheumatoid arthritis, systemic sclerosis, sjogren's syndrome, graft versus host disease (GvHD), crohn's disease and other immune related diseases.2004, Falco et al successfully managed an ITP patient with multiple myeloma with THD. By far, it wasn't reported that the study on MSC induction of regulatory DC in ITP and the influence of THD on MSC immune function of patients with ITP.
Objective:
To compare the proliferation capacity, the inhibition of lymphocyte activation ability and the induction of induced regulatory DC function of MSC in newly diagnosed ITP patients and healthy controls. After THD modulated, to detect the changes of MSC proliferation in newly diagnosed ITP patients and healthy controls. As well as, to investigate the molecular mechanisms before and after modulated with THD and to test the changes of induction of regulatory DC function. To detect the role of TIEG1gene in the process of MSC induction of regulatory DC.
Materials and methods:
1. Cell proliferation assay:At passage4, MSCs were cultured in triplicate in a96-well plate and incubated in humidified air in5%CO2at37℃. After adherence, these cells were treated with THD at concentrations of0(commensurable DMSO),0.05,0.1,0.5,1,5,10, and50mg/mL. The proliferation assay was assessed on day3using the Cell Counting Kit-8(CCK8) according to the manufacturer's recommendations.
2. Cell Apoptosis assay:Briefly, MSCs (ITP/healthy controls, cultured in commensurable DMSO) or THD-MSCs (ITP/healthy controls, cultured in0.5mg/mL THD) were harvested and stained with Annexin V-fluorescein isothiocyanate/7-amino-actinomycin D (Annexin V/7-AAD) according to the manufacturer's recommendations. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
3. Cell-cycle analysis:Briefly, MSCs (ITP/healthy controls, cultured in commensurable DMSO) or THD-MSCs (ITP/healthy controls, cultured in0.5mg/mL THD) were harvested and stained with propidium iodide (PI) according to the manufacturer's recommendations. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
4. Microarray analysis:microarray analysis was conducted on THD-MSCs (ITP, cultured in0.5mg/mL THD) and MSCs (ITP, cultured in commensurable DMSO) for12hours from3independent patients. The abundance of messenger RNA (mRNA) transcripts was measured using the35k human Genome Array (CapitalBio Corp) representing39600transcripts and～25100genes. The data were analyzed using the Molecule Annotation System (MAS)
5. Differential expression gene validation test:Total RNA was extracted using TRIzol reagent (Invitrogen) from MSCs (ITP/control) and THD-MSCs (ITP/control), cultured in DMSO or in0.5mg/mL THD for12hours. Real-time reverse-transcription polymerase chain reaction (qRT-PCR) were used to verify the differential expression gene.
6. MSC inhibition assays:After labeling with CFSE, CD4+T cells or phorbol myristate acetate (PMA) preactivated CD4+T cells were cultured alone or cocultured with MSCs (control), MSCs (ITP), or THD-MSCs (ITP), respectively, in the presence or absence of mDCs in triplicate. All cells were incubated for3days and then collected for fluorescence-activated cell sorter (FACS) analysis.
7. Tolerogenic DC inhibition assays:After coculture with MSCs (control), MSCs (ITP), or THD-MSCs (ITP), the MSC-DCs were sorted using anti-HLA-DR immunomagnetic beads (Miltenyi Biotec). CFSE-labeled allogeneic CD41T cells were cocultured with PMA, mDCs, MSC (control)-DCs, MSC (ITP)-DCs, THD-MSC (ITP)-DCs, MSC (control)-DCs+PMA, MSC (ITP)-DCs+PMA, THD-MSC (ITP)-DCs+PMA, MSC (control)-DCs+mDCs, MSC (ITP)-DCs+mDCs, or THD-MSC (ITP)-DCs+mDCs, respectively. All cells were cultured in triplicate and incubated for3days and then collected for FACS analysis.
8. Cytokine production:CD4+T cells, mDCs and MSCs were cultured alone or in combination. The levels of IL-4, IFN-y, IL-12, IDO, TGF-p, and IL-10in cultural supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) after3days.
9. Dendritic cells maturity test:After co-cultured with MSC (ITP), MSC (THD-ITP) and MSC (congtrol). The DC staining was performed using fluorescein isothiocyanate-or phycoerythrin-conjugated anti-CD80and anti-CD86(BD Pharmingen) monoclonal antibodies. The isotype IgG of same species origin was used as a control. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
10. Lentivirus interference:TIEG1small hairpin RNA (shRNA)(h) lentiviral particles and control shRNA lentiviral particles were purchased from Santa Cruz Biotechnology. The mDCs were preinfected with prepared virus and then co-cultured with MSC. After sorted using anti-HLA-DR immunomagnetic beads, the CD4+T cells were co-cultured with mDC, MSC-DC, MSC-DC (TIEG1shRNA), MSC-DC (control shRNA), respectively. Data acquisition was performed on a flow cytometer (FACSCalibur; Becton Dickinson; or Gallios; Beckman Coulter).
Results:
1. THD with concentration radients fromO.1μg/mL to10μg/mL could stimulate the proliferationof MSCs (ITP) in a dose-dependent manner. Yet for all patients, higher THD concentrations (>10μg/mL) would lead to an opposite result. The proliferation of MSCs from both ITP patients and healthy controls could be promoted after0.5mg/mL THD modulation. There were less cells entered into S+G2phases in the MSC (ITP) group compared with those in MSCs (control). After THD modulation, the frequency of cells in S and G2phases was increased in THD-MSCs (ITP). The apoptosis analysis of MSCs showed there was no significant difference of Annexin-V+/7-AAD+cells between patients and healthy controls, and THD modulation could decrease the apoptosis rate both in patients and in controls.
2. Differentially expressed genes indexed by Gene Ontology (Go) biological process of MSCs were shown in the Gene Expression Omnibus database under accession number GSE44948; enrichment for variants was associated with DNA-dependent regulation of transcription, cell cycle, mitosis, DNA replication, cell proliferation, apoptosis, regulation of cell growth, and regulation of apoptosis, etc. We selected several genes associated with cell proliferation, apoptosis and cell cycle, including p27, p16, caspase-8, caspase-10, klf4, c-myc, oct3/4, and TGF-β which may be associated with THD modulation. The expression of caspase-8and caspase-10was downregulated in THD-MSCs (ITP), while there was no significant difference between MSCs (control) and THD-MSCs (control). The expression of c-myc was downregulated in both THD-MSCs (ITP) and THD-MSCs (control). The expression of oct3/4and TGF-β was upregulated in THD-MSCs (ITP), while there was no obvious change between MSCs (control) and THD-MSCs (control). The expression of klf4and p27was upregulated or downregulated, respectively, while there was no obvious change between MSCs (ITP) and THD-MSCs (ITP). There was no obvious change of p16either in MSCs (control) or in MSCs (ITP) before and after modulated with THD. We confirmed the downregulation of caspase-8and caspase-10, and upregulation of oct3/4and TGF-β, after THD modulation was specific for MSC (ITP) and explained the THD induced increase in MSC proliferation, as well as their potentially ability to induce tolerogenic DCs.
3. The data demonstrated that mDCs and PMA significantly stimulated T-cell proliferation. When MSCs (control) or THD-MSCs (ITP) were added to mDC cocultures in the absence of PMA, they inhibited T-cell proliferation, whereas the addition of MSCs (ITP) had no inhibitory effect. Notwithstanding, THD-MSCs (ITP) were unable to prevent T-cell proliferation activated in the presence of PMA. The data also showed that addition of mDCs to the coculture system alleviated T-cell proliferation.
4. The mDCs regulated by MSCs from healthy controls suppressed the proliferation of T lymphocytes, whereas the mDCs regulated by MSCs from ITP patients did not. However, the inhibitory capacity of mDCs was increased when they were incubated with THD modulated ITP-MSCs. We also investigated whether the inhibitory effect of these tolerogenic DCs was strong enough to silence the lymphocytes activated by PMA. The data demonstrated that mDCs regulated by MSCs from ITP patients, healthy controls, or THD modulated MSCs were unable to inhibit T-cell proliferation. These results further confirmed that the MSCs from ITP patients had lost the ability to induce tolerogenicity in mDCs. Although the tolerogenic mDCs could not prevent T-cell proliferation activated by PMA, they did block the allogeneic mDC-activated T-cell proliferation.
5. Furthermore, we conducted phenotypic analysis of the mDCs cocultured with MSCs from healthy controls, from ITP patients or THD-modulated MSCs. MSCs from ITP patients failed to downregulate CD80and CD86expression in mDCs, while MSCs from healthy controls significantly inhibited the expression of these costimulatory factors. Accordingly, THD-MSCs also downregulated the expression of CD80and CD86in mDCs.
6. Since IL-10and TGF-β are potent immunosuppressive cytokines tolerogenic DC-mediated inhibition. The levels of IL-10and TGF-β in the culture supernatants of THD-MSCs (ITP) and mDCs were higher than those of mDCs alone or mDCs plus MSCs (ITP). These results indicate that the MSC-elicited inhibitory property of DCs is related to DC maturation state and TGF-β or IL-10cytokine production. We also assayed the levels of IL-4, IFN-y, IDO, and IL-12in culture supernatants. When CD4+T cells and mDCs were cocultured with THD-MSCs (ITP), the level of IL-4and IDO was higher while the IFN-y level was lower than that cocultured with MSCs (ITP) or without MSCs. There was no significant difference in IL-12level between cultures with THD-MSCs (ITP) or with MSCs (ITP), though in both cases it was lower than that without any MSCs.
7. The mDCs infected with TIEG1shRNA (h) lentiviral particles lost the ability to suppress T-cell proliferation. The results indicate that the acquisition of tolerance by mDCs upon incubation with MSCs depends on TIEG1.
Conclusions:
1. The MSCs from ITP patients had lost their regulatory capacity, which could be increased by THD modulation. THD with concentration radients could stimulate the proliferationof MSCs (ITP) in a dose-dependent manner. After THD modulation, the frequency of cells in S and G2phases was increased in THD-MSCs (ITP). THD modulation could decrease the apoptosis rate both in patients and in controls. After coculture with MSCs, the mDCs may have tolerogenic rather than stimulatory effects and play an important role in mediating the inhibitory activity of MSCs.
2. The study demonstrated that the regulatory ability of MSCs from ITP patients could be regained upon THD modulation. After coculture with MSCs, the mDCs differentiated into a novel tolerogenic DC population.
3. The acquisition of tolerance by mDCs upon incubation with MSCs depends on TIEG1.
Part Ⅱ
Expression of CD205on dendritic cells and its regulation by high dose dexamethasone in immune thrombocytopenia
Background:
Dendritic cells (DCs) were found in1973by Steinman. As antigen presenting cells, DCs are located throughout the body to capture and internalize invading pathogens, and subsequently process and present antigens on MHC class Ⅰ and class Ⅱ molecules to CD8+and CD4+T cells, respectively. DCs play a crucial role in maintaining the balance of immune system and preventing pathological autoimmune diseases. DCs were well equipped to distinguish self to non-self-antigens by the variable expression of cell-surface receptors. Such as C-type lectin receptors (CLRs) for glycoproteins and Toll-like receptors (TLRs) for microbial antigens. In steady state, uptake of antigen by CLRs may induce antigen-specific tolerance. CD205is one of the least understood member of the family of CLRs which is predominantly expressed by the DCs. Recent study showed CD205+DCs are much more effective inducers of functional Foxp3+Treg from Foxp3-CD4+T cells. In addition, ITP DCs have increased capacity to present apoptotic platelets to T lymphocytes. But the expression of CD205on DCs of patients with ITP has not been reported.
Immune thrombocytopenia (ITP) is a common autoimmune diseases. It is characterized with anti-platelet glycoproteins (GPs) antibody mediated increased destruction of platelets. As a member of the family of CLRs, CD205may participate in the process of DC to identify and capture self-GPs. Corticosteroids is the most appropriate first-line treatment for patients with ITP and highdose dexamethasone (HD-dexa) in4-day cycles has proven its clinical efficacy in the treatment of ITP. The mechanism study of the HD-dexa treatment proved that by regulating the balance of the Fc gamma receptor (FcR) and regulating the polarization of Th1/Th2 cells, the abnormal immune status in patients with ITP was restored. But the expression of CD205on dendritic cells and its regulation by high dose dexamethasone in ITP patients was still not clear.
The spleen is of particular interest because it is the major site of immune response, where the immune response is triggered and regulated, especially for platelets destruction in ITP. Recently, Daridon et al. reported that proliferative lymphoid nodules are the sites of auto-antigen stimulation in ITP potentially related to a lack of control by T cells and/or the present auto-antigen. The distribution of DC in spleen and the expression of CD205of DC of patients with in spleen is not yet clear.
Objective:
The aim of this study was to determine the expression status of CD205in mature and immature DC in ITP patients and healthy controls. To detect the effect of high dose dexamethasone to mature conditions and the expression of CD205of DC in patients with ITP. To investigate the correlation between the effect of dexamethasone and the expression of CD205of DC. To compare the distribution of CD205+DC and the mature conditions of CD205+DC in patients with ITP and healthy controls.
Materials and methods:
1. Preparetion of DCs:Peripheral blood mononuclear cells (PBMCs) were prepared from peripheral blood of ITP patients and healthy controls. CD14+cells were negatively selected from the PBMCs using micromagnetic beads, according to the manufacturer's instructions. The purified CD14+cells were then incubated in RPMI1640culture medium with10%fetal calf serum,1000u/mL granulocyte-macrophage colony stimulating factor and1000u/mL interleukin-4for5days. Lipopolysaccharides (LPS,1ug/mL) were added to the cells for an additional2days. At day7the DCs were collected for flow cytometry and quantitative real time-PCR (qRT-PCR) analysis.
2. Cell-surface staining for flow cytometric determination of CD205, CD80, CD83, CD86:Cell (ITP and healthy nomal) staining was performed using FITC-or PE-conjugated anti-CD80, anti-CD86and anti-CD205monoclonal antibodies. The same-species-isotype IgG was used as an isotype control. Data acquisition was performed using a FACScalibur flow cytometer (BD Biosciences), and analyzed with Cell Quest Pro software (BD Biosciences).
3. Real-time reverse transcription-PCR (RT-PCR) analysis:Total RNA was extracted from cells (ITP and healthy nomal) by TRIzol (Invitrogen). cDNA was synthesized using the PrimeScript RT reagent kit (Takara) according to the manufacturer's instructions. qPCR was performed with Power SYBR Green PCR Master Mix (Takara) under the following parameters:denaturation95℃,5minutes; followed by45cycles of95℃,10seconds;60℃,10seconds;72℃,10seconds. Melt-curve analysis was performed following amplification. The delta Ct calculation method using arithmetic formulas was used for relative quantification of target gene.
4. Dexamethasone intervention trial:In order to further observe the relation between the expression of CD205, CD80, CD83, CD86and the effect of dexamethasone treatment, we put the follow concentration of dexamethasone (0nmol/L,10nmol/L,25nmol/L,50nmol/L,100nmol/L) in use. After modulated with dexamethasone for3days, the DCs were collected for quantitative real time-PCR analysis.
5. Immunofluorescence:Spleen tissue was frozen in OCT Compound and stored at-80℃. Frozen sections (5μm) prepared using a Microm cryostat were air-dried and stored at-20℃. Upon thawing, sections were airdried, fixed for10min in acetone, rehydrated in phosphate-buffered saline (PBS)/1%bovine serum albumin (BSA) and stained overnight in4℃with purified mouse anti-human CD86(mouse IgG2b, k) or purified mouse anti-human CD80(mouse IgGl, k), as well as rabbit anti human CD205(IgG, R18-D). After washing3times in PBS, sections were stained with rhodamine (TRITC)-conjugated affinipure goat anti-rabbit IgG (H+L) secondary antibodies or fluorescein-conjugated affinipure goat anti-mouse IgG (H+L) secondary antibodies (Zhongshanjinqiao, Beijing) diluted in PBS/1%BSA to2μg/ml. Slides were viewed on a Molecular Devices Olympus AX70 deconvolution microscope (Olympus America Inc, Lake Success, NJ) running METAMORPH Meta Imaging series software (Universal Imaging Corporation, West Chester, PA).
Results:
1. The expression of CD205, CD80, CD83and CD86of mature and immature DC of patients with ITP and healthy controls.
1.1With DCs mature process, the frequency of CD205, CD80, CD83and CD86positive DCs were all rised in control.
1.2The frequency of CD205failed up-regulation along with DCs maturation in ITP patients. Further more, we observed an increased frequency of CD80and CD86of imDC in ITP patients compared with that in controls, In addition, with DCs maturation the frequency of CD80, CD83and CD86significantly up-regulated in patients with ITP than that in controls.
2. Low expression of CD205of DCs in patients with ITP can be up-regulated by HD-dexa treatment:After administration of HD-dexa treatment to ITP patients results in elevation of CD205mRNA levels of mDC but not in imDC. In biological accordance with this marked augmentation, CD80and CD86mRNA levels steadily declined over the study period but CD83mRNA levels had no obvious change before and after HD-dexa therapy although the CD83mRNA levels reduced in imDC.
3. Administration of dexamethasone results in up-regulation of CD205expression in a dose-dependent manner in mDCs:In mDCs, the expression amount of CD205had positive relevance to the dose of dexamethasone but not in imDCs, while there is no causal link between the relative quantity of CD80, Cd83, and CD86and the dosageof dexamethasone in the mature and immature DCs.
4. The DCs in ITP spleen were typically CD205low and in a mature functional state.
4.1The HE staining (hematoxylin-eosin staining) of the tissues showed that the spleens in ITP patients had far more white pulps than control in the field. In the meanwhile, the filtration of lymphocytes in the white pulp area was much bigger than the control group.
4.2The CD205could be found in the border region between the red and white pulp in control spleen and can be found in the T-cell areas and the B-cell areas of ITP spleen. We verified that costimulatory (CD80and CD86) molecules were expressed in both T-cell and B-cell areas, no matter in control spleen or in ITP spleen. But the frequency of CD80and CD86positive cells were lower in the control human spleen than that in ITP spleen.
Conclusions:
1. The expression of CD205was significantly reduced in patients with ITP. In addition, the mature condition of DC in patients in a semi-mature state.
2. After HD-dexa treatment, the expression of CD205was markedly up-regulated while the expression of CD80, CD86was dramatically down-regulated.
3. Administration of dexamethasone results in up-regulation of CD205expression in a dose-dependent manner in mDCs.
4. Our study indicate that most CD205+DCs of the control human spleen in an immature functional state. But in ITP spleen, the DCs were typically CD205low or negative and in a mature functional state.
引文
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