B细胞活化因子在ITP中的机制研究
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摘要
第Ⅰ部分BAFF及BR3-Fc融合蛋白在ITP中的机制研究
免疫性血小板减少症(immune thrombocytopenia, ITP)是由于机体免疫失耐受,产生的抗血小板自身抗体与血小板表面特异性抗原结合,导致血小板在网状内皮系统过度破坏引起血小板减少。由自身反应性B细胞产生的自身抗体,主要是针对自身血小板膜糖蛋白Ⅱb／Ⅲa和／或Ⅰb／Ⅸ抗原的自身抗体,尤其是IgG抗体,在ITP的发病中起到重要的作用。此外,多种细胞免疫机制异常,如Th1极化、调节性T细胞数目降低或者抑制功能缺陷、细胞毒性T细胞(cytotoxic T lymphocyte, CTL)介导的血小板破坏等在ITP的发病中发挥重要作用。到目前为止,出现上述异常的原因尚未明确。ITP的主要治疗包括糖皮质激素、免疫球蛋白、抗-D和脾切除等,但仅有1／3的患者能够获得长期缓解。
B细胞活化因子(B-cell activating factor, BAFF,亦称为BlyS,TALL-1,THANK, zTNF4 and TNFSF13B)是近年来发现的肿瘤坏死因子超家族(tumor necrosis factor super family, TNFSF)的新成员,它在B细胞发育、稳定及其自身反应性和T细胞共刺激等方面起到重要的作用。此外,BAFF在Th1介导的炎症应答中也起到重要的作用。目前研究发现BAFF主要与三个受体结合,B细胞成熟抗原(B-cell maturation antigen, BCMA,亦称TNFRSF17)、转膜激活剂、钙调节剂和亲环素配体相互作用物(transmembrane activator and calcium-modulating cyclophilin ligand interactor, TACI,亦称TNFRSF13B)和BAFF受体(BAFF receptor, BAFF-R,亦称BR3, TNFRSF13C)。BR3是B细胞存活最主要的受体,它广泛表达于包括未成熟B、过渡期B、成熟B、记忆B、生发中心B以及浆细胞在内的B细胞亚群。此外,体内和体外研究表明,表达于T细胞上的BR3与BAFF结合后能够刺激T细胞的增殖。
许多研究表明,BAFF在自身免疫中发挥重要的作用。自身抗原结合的B细胞对BAFF介导的存活信号依赖增强。在许多自身免疫性疾病患者中,如风湿性关节炎(rheumatoid arthritis, RA)、系统性红斑狼疮(systemic lupus erythematosus, SLE)、干燥综合征(Sjogren's syndrome, S S)、多发性硬化(multiple sclerosis,MS)等,血浆BAFF表达水平异常升高。抑制BAFF信号通路可能是B细胞介导的自身反应性疾病一种有效的治疗方法。动物实验和临床试验研究表明应用BAFF阻断剂(如TACI-Ig,BAFF-R-Ig,BR3-Fc)阻断BAFF在某些自身免疫性疾病中是一种有效的治疗手段。
目的：
检测ITP患者BAFF表达以及rhBAFF和BR3-Fc对B细胞、T细胞、血小板、干扰素-γ(interferon-gamma,IFN-γ)、白介素-4(interleukin-4,IL-4)的作用,探讨BAFF和BR3-Fc在ITP发病中的可能机制
方法：
◆BAFF检测部分抽取25例活动性ITP患者,20例稳定期患者和24例对照的外周血,分离血浆和单个核细胞
◆酶联免疫吸附实验(enzyme-linked immunoadsorbent assay, ELISA)法和实时定量聚合酶链反应(real-time polymerase chain reaction, RT-PCR检测BAFF蛋白和mRNA水平；改良单克隆抗体特异性俘获血小板抗原(immobilization of platelet antigens magnetic activated cell separation, MAIPA)检测抗GPIIb/Ⅲa和GPIb/IX自身抗体
◆体外部分抽取18例活动性ITP患者和15例对照的外周血15ml,分离单个核细胞和血小板,加入不同的细胞因子共同培养,分为3组：1640组(Ⅰ组)、rhBAFF 20ng／ml组(Ⅱ组)和rhBAFF+BR3-Fc组(Ⅲ组)。流式细胞术检测CD19+B细胞、CD4+T细胞、CD8+T细胞及血小板的凋亡；ELISA法检测培养上清中IFN-γ、IL-4和自身抗体的表达
结果：
◆BAFF在活动性ITP中表达升高,稳定期患者与对照无显著性差异
在活动性ITP患者中BAFF蛋白水平(均值±标准差,593.1±219.0 pg/ml)明显高于稳定期患者(432.5±121.4pg/ml, P<0.05)和正常对照(454.4±132.5pg/ml,P<0.05)。活动性ITP患者BAFF mRNA水平是稳定期患者和对照的3.1倍(P<0.01)和2.5倍(P<0.01)。而在稳定期患者无论BAFF蛋白还是mRNA水平均与正常对照无明显差异(P>0.05)。BAFF水平与GPIIb/IIIa和GPIb/Ⅸ无显著性相关。
◆RhBAFF促进CD19+存活,BR3-Fc纠正rhBAFF对其影响
RhBAFF降低ITP患者CD19+细胞的凋亡(Ⅱ组：7.0±4.3％vsⅠ组：13.2±7.4％,P<0.05),加入BR3-Fc后CD19+细胞的凋亡升高,但与1640组无显著性差异(Ⅲ组：11.2±4.1％vsⅠ组：13.2±7.4%,P>0.05)。
◆RhBAFF促进CD8+存活,BR3-Fc纠正rhBAFF对其影响
ITP患者中Ⅰ、Ⅱ、Ⅲ组CD8+细胞的凋亡率分别为6.5±3.2％,4.4±2.2％和6.3±2.9%。RhBAFF促进CD8+存活,BR3-Fc纠正rhBAFF对其影响。
◆RhBAFF和BR3-Fc对CD4+细胞凋亡无显著影响
RhBAFF对CD4+细胞凋亡无显著影响。ITP患者中Ⅰ、Ⅱ、Ⅲ组CD8+细胞的凋亡率分别为8.6±4.5％,5.3±1.8％和8.2±3.8％。
◆RhBAFF促进血小板凋亡,BR3-Fc抑制其凋亡
RhBAFF促进ITP患者血小板的凋亡(Ⅱ组：8.4±4.9％vsⅠ组：3.2±1.0％,P<0.051,加入BR3-Fc后血小板的凋亡降低(Ⅲ组：5.0±3.0％vsⅡ组：8.4±4.9％,P<0.05)。
此外,我们应用线粒体膜电位检测试剂盒JC-1检测血小板凋亡进一步验证了我们的结果。
◆在ITP患者中各组IFN-γ均显著高于对照组,rhBAFF促进ITP患者IFN-γ分泌,而BR3-Fc抑制其表达
ITP患者中Ⅰ、Ⅱ、Ⅲ组IFN-γ分别为74.0±12.5pg／ml、95.1±25.7pg／ml和82.4±17.4pg/ml, rhBAFF 20ng／ml组IFN-γ水平显著高于1640组,加入BR3-Fc后IFN-γ表达下降。IL-4低于最低检测值。
结论：
◆BAFF在活动性ITP的表达升高,而稳定期患者无异常,与病情变化相关。
◆BAFF可能通过促进CD19+B和CD8+T细胞存活、促进血小板的凋亡、促进Th1类细胞因子分泌等机制在ITP的发病中起到重要的作用；BR3-Fc纠正了rhBAFF对上述细胞和因子的作用。
◆阻断ITP中过高的BAFF可能成为ITP治疗的一个新的靶点。
第Ⅱ部分大剂量地塞米松疗法抑制ITP患者BAFF的表达
ITP的治疗主要有糖皮质激素、免疫球蛋白、脾切除、达那唑及免疫抑制剂等,其中糖皮质激素是ITP的一线治疗药物。在过去50多年里,泼尼松一直是治疗ITP的首选,开始剂量1.0-1.5mg／kg/d,分次口服4-6周,此后逐渐减量至血小板在安全水平以上(血小板>30x109／L)的最低用量时给予维持治疗,约70％患者可获得缓解,而30％患者治疗无效,属于难治性ITP。该方法治疗有效率与剂量相关,长期应用副作用较大,并且减量过程中容易复发,此时给予重复治疗很难再取得持续缓解,应考虑换用其他治疗。脾切除是泼尼松治疗失败患者的第一选择,其长期缓解率为50％-70％,仍然有30％左右患者治疗无效,另外还有部分泼尼松治疗失败的患者由于年龄、基础疾病等因素而不能行脾切除手术治疗,这些难治性ITP患者的治疗一直是临床上非常棘手的问题。
近年来大剂量地塞米松(High dose dexamethasone, HD-DXM)疗法较传统的强的松治疗取得了更好的疗效。与泼尼松相比,HD-DXM冲击治疗起效快、副反应轻微,效果相当于免疫球蛋白,但是花费明显减少。对于初诊的ITP患者,HD-DXM冲击治疗效果显著,80％以上有治疗反应,已经作为一线治疗选择。对于复发／难治性ITP患者HD-DXM冲击治疗总体有效率不高,只有部分患者有治疗反应。HD-DXM冲击治疗尚不能代替或避免脾切除,不过可以作为脾切除前的应急准备。
研究表明在某些自身免疫性疾病中如SLE、大疱性类天疱疮中糖皮质激素能够显著降低患者BAFF表达水平。另一项研究表明DXM在体外能够抑制风湿性关节炎患者纤维母样滑膜细胞BAFF的表达,并且呈剂量和时间依赖性。而关于HD-DXM对ITP患者BAFF表达的影响目前尚未见报道。
目的：
研究大剂量地塞米松(HD-DXM)对ITP患者BAFF的作用,进一步探讨HD-DXM在ITP治疗中的可能机制
方法：
◆留取20例ITP患者(应用激素治疗前2周各抽血一次)和24例对照的外周血3ml。分离血浆和单个核细胞。ELISA检测血浆BAFF蛋白水平,实时定量PCR检测PBMCs中BAFF mRNA水平。
◆其中10例ITP患者和14例对照抽血15ml,分离单个核细胞。加入不同浓度DXM,培养后分离上清检测BAFF、IFN-γ和IL-4,分离细胞实时定量PCR检测BAFF mRNA水平。CCK-8法检测淋巴细胞增殖。
结果：
◆HD-DXM临床疗效
80％患者(16／20)获得长期反应,15％患者(3／20)获得部分反应,5％患者(1／20)无反应。HD-DXM治疗2周后平均血小板数目是165×109／L(范围23-332×109/L)。
◆BAFF在活动性ITP(应用HD-DXM治疗前)明显升高,应用HD-DXM后,ITP患者BAFF水平明显降低
活动性ITP患者中BAFF蛋白水平及其mRNA水平显著升高。应用HD-DXM后,ITP患者BAFF蛋白水平(均值297pg／ml,范围184-545pg/ml)明显低于用药前(均值597 pg／ml,范围300-1026pg/ml)和正常对照(均值454 pg／ml,208-804pg/ml, P<0.001); BAFF mRNA水平亦明显低于激素前患者(P<0.001)和对照组(P<0.001)。
◆BAFF变化与HD-DXM治疗反应密切相关
应用HD-DXM后,20例患者中有18例BAFF水平明显低于治疗前水平,1例患者BAFF水平与治疗前相比无明显降低,1例BAFF水平略微升高。这两例患者均对HD-DXM治疗无反应,后来随访发现为难治性ITP,并进行脾切除。
◆DXM体外抑制BAFF的表达
我们体外培养10例未治疗的ITP患者和11例对照PBMCs,加入不同浓度的DXM后体外培养72小时,我们发现DXM明显抑制BAFF的表达,并且呈剂量依赖性。
◆DXM体外抑制IFN-γ的表达
我们体外培养ITP患者和对照PBMCs,加入不同浓度的DXM(OnM,7.8nM, 15.6nM,31.3nM,62.5nM,125nM,250nM1和10μg/ml PHA,于37℃含5％CO2孵箱孵育72小时后,收集上清,ELISA检测IFN-γ和IL-4表达,DXM能够显著抑制IFN-γ表达,并且呈剂量依赖性,而IL-4水平低于最低检测值。
◆DXM体外抑制淋巴细胞的增殖
CCK-8法检测DXM对淋巴细胞增殖的影响,我们发现DXM明显抑制淋巴细胞增殖,并且呈剂量依赖性。
◆BAFF与ITP患者临床实验室指标相关性分析
BAFF水平和血小板数目无显著性相关,且BAFF水平的变化和血小板数目的改变无显著性相关。
结论：
◆HD-DXM抑制ITP患者血浆BAFF及其mRNA水平的表达；
◆DXM体外抑制BAFF、IFN-γ的表达和淋巴细胞的增殖。
第Ⅲ部分白介素-21在ITP中的作用及其机制研究
白介素-2家族主要包括IL-2、IL-4、IL-7、IL-9和IL-15,在促进和维持T淋巴细胞群中起主要作用。由这些细胞因子介导的详细的分子信号传递途径尚未完全澄清.然而JAK/STAT,MAPK和P13这三个主要的途径已经清楚。
IL-21是IL-2家族的新成员,主要由活化的CD4+T细胞和自然杀伤T细胞表达,除此以外,近来发现IL-21还可以由Th17细胞表达。IL-21／IL-21R信号在天然免疫和获得性免疫应答中均起到重要的作用,它能够促进T细胞介导的体液免疫应答与抗体生成、增强NK细胞的自然杀伤能力、促进Th细胞向Th17细胞的分化、影响Th细胞向Th1／Th2细胞的分化等。在许多自身免疫性疾病中(如SLE,RA,SS等)IL-21和／或IL-21R表达异常。目前,未见关于IL-21／IL-21R在ITP患者中的报道。
目的：
通过对ITP患者外周血中表达IL-21的细胞亚群和Th17、Th1、Tc1细胞亚群的检测,探讨IL-21在ITP中的作用及可能机制
材料与方法：
◆病例及正常对照的选择：活动性ITP患者26例,均符合ITP的诊断标准。正常对照21例,均为健康志愿者,无感染、无病毒性肝炎、无免疫性疾病及半年内未使用免疫抑制剂、抗HIV抗体阴性、肝肾功能正常,其年龄和性别均与ITP患者相匹配。
◆应用三色流式细胞术检测ITP患者和正常对照外周血中各细胞亚群的比例。三种细胞亚群的界定分别为：表达IL-21的细胞为CD3+CD8-IL-21+和CD3+CD8+IL-21+,Th17细胞为CD3+CD8-IL-17A+,Th1细胞为CD3+CD8-IFN-γ+,Tc1细胞为CD3+CD8+IFN-γ+
◆应用ELISA技术检测ITP患者和正常对照血浆中IL-21的表达水平。
◆应用改良的MAIPA技术(单克隆抗体特异性俘获血小板抗原技术)测定ITP患者体内的抗血小板膜GPⅡb／Ⅲa和Ib／Ⅸ自身抗体。
结果：
◆IL-21在ITP中表达升高
我们应用流式细胞术检测ITP患者外周血中IL-21的表达,发现IL-21表达于CD3+CD8-细胞和CD3+CD8+细胞上。ITP患者中CD3+CD8-IL-21+细胞亚群(4.4±0.8％)比例显著高于对照组(2.3±0.8％,P<0.001),相似的,ITP患者中CD3+CD8+IL-21+细胞亚群(0.8±0.5％)比例显著高于对照组(0.5±0.3％,P<0.05)。
我们应用ELISA法检测ITP患者和对照血浆中IL-21的表达,我们发现ITP血浆IL-21水平(148.3±124.2pg／ml)高于对照组(78.0±32.0pg／ml,P<0.05)。
◆ITP患者中Th17、Th1、Tc1细胞亚群表达升高
ITP患者外周血Th17细胞的比例(2.1±0.8％)明显高于正常对照组(1.2±0.5％,P<0.05)。ITP患者外周血Thl细胞的比例(18.6±2.3％)明显高于正常对照组(10.9±2.6％,P<0.001),并且ITP患者Tc1细胞的比例(21.4±8.4％)较正常对照组(11.4±3.8％)亦显著升高(P<0.001)。
◆各细胞亚群的相关性分析
在ITP患者中,CD3+CD8-IL-21+细胞与Th17细胞(P<0.01)和Thl细胞(P<0.01)存在明显的正相关,而CD3+CD8+IL-21+细胞与Tc1细胞不存在相关性(P>0.05)。
◆抗血小板自身抗体与各细胞亚群的相关性分析
应用MAIPA法检测抗血小板自身抗体,并与各细胞亚群进行相关性分析,我们发现自身抗体与各个细胞亚群均不存在相关性(P>0.05)。
结论：
◆ITP患者中IL-21表达升高；
◆在ITP患者中,CD3+CD8-IL-21+细胞与Th17细胞和Th1细胞存在明显的正相关,提示CD3+CD8-IL-21+细胞、Th17细胞和Th1细胞可能通过相互作用而在ITP的发病中起到协同作用。
英文摘要ⅠThe effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia
Immune thrombocytopenia (ITP) is an autoimmune disorder in which the patients'immune system is activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. In addition, several abnormalities involving the cellular mechanisms of immune modulation such as Th1 bias, the decreased number or defective function of regulatory T cells and the platelet destruction by cytotoxic T cells (CTL) have been described.
B-cell activating factor (BAFF) (also known as BlyS, TALL-1, THANK, zTNF4 and TNFSF13B) belonging to the family of tumor necrosis factor (TNF) ligands is critical for the maintain of normal B-cell development, homeostasis, autoreactivity and T cell costimulation. In addition, it also augments certain Th1-associated inflammatory responses. BAFF binds to three receptors:B-cell maturation antigen (BCMA, TNFRSF17), transmembrane activator and calcium-modulating cyclophilin ligand (CAML) interactor (TACI, TNFRSF13B) and BAFF receptor (BR3/BAFF-R, TNFRSF13C). BR3, identified as the crucial receptor for B-cell survival, is expressed on a wide range of B-cell subsets, including immature, transitional, mature, memory and germinal center B cells, as well as on plasma cells. Furthermore, BAFF binding to BR3 on T cells has been shown to costimulate T-cell proliferation both in vitro and in vivo.
Several lines of evidence suggested that BAFF may play an important role in autoimmunity. Autoantigen-binding B cells may have an increased dependence on the BAFF survival signal. In addition, elevated BAFF plasma level was observed in many patients with autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogren's syndrome (SS), and multiple sclerosis (MS). Inhibition of BAFF signaling is a potentially therapeutic option for treatment of B cell-mediated autoimmune conditions. Data from animal tests and clinical trials had proved that blockade of BAFF by blocking reagents (TACI-Ig, BAFF-R-Ig, BR3-Fc) was effective therapeutic approach for some autoimmune diseases.
Objective:
To investigate the possible mechanism of BAFF and therapeutic effect of BR3-Fc in ITP, we measured the expression of plasma BAFF and BAFF mRNA, and the effects of rhBAFF and BR3-Fc on B cells, T cells, platelets, secretion of IFN-γand IL-4 were measured by flow cytometry and ELISA.
Methods:
◆Forty-five patients diagnosed with ITP were selected for detection of plasma BAFF and BAFF mRNA. Of these patients, twenty-five patients were active ITP patients who had not been treated with GCs for at least one month prior to sampling whereas twenty patients were in remission. Twenty-four healthy controls matched for sex and age with the study population were voluntary blood donors.
◆The expression of plasma BAFF and BAFF mRNA in ITP patients and controls were measured by ELISA and RT-PCR, the levels of plasma anti-platelet autoantibodies (GPIIb/IIIa and GPIb/IX) were measured by MAIPA.
◆Peripheral blood mononuclear cells (PBMCs) and platelets from additional eighteen ITP patients with active disease and fifteen controls were selected for detection of apoptosis on CD19+, CD4+, CD8+ cells, platelets, secretion of cytokines (IFN-y and IL-4) and autoantibodies by flow cytometry and ELISA.
Results:
◆Elevated levels of plasma BAFF and BAFF mRNA in active ITP patients
The level of plasma BAFF in ITP patients with active disease was significantly higher (mean±SD,593.1±219.0 pg/ml) than that in patients in remission (432.5±121.4pg/ml, P＜0.05) and controls (454.4±132.5pg/ml, P＜0.05). The relative amount of BAFF mRNA in patients with active disease was increased 3.1 and 2.5-fold compared to patients in remission (P＜0.01) and healthy controls (P<0.01), respectively. No significant difference of plasma BAFF or BAFF mRNA between patients in remission and healthy controls was found (P>0.05).
In addition, no association was found between the levels of BAFF and anti-platelet autoantibodies (P>0.05).
◆RhBAFF promotes the survival of CD19+ cells, BR3-Fc corrects its effect
RhBAFF significantly decreased the annexin V% of CD19+ cells in ITP patients (groupⅡ:7.0±4.3% vs groupⅠ:13.2±7.4%, P＜0.05) but not in controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of CD19+ cells (groupIII:11.2±4.1% vs groupⅠ:13.2±7.4%, P>0.05).
◆RhBAFF promotes the survival of CD8+ cells, BR3-Fc corrects its effect
RhBAFF significantly decreased the annexin V% of CD8+ cells in both ITP patients and controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of CD8+ cells only in ITP patients. The annexin V% on CD8+ cells in ITP patients in group I, group II and group III were 6.5±3.2%,4.4±2.2%and 6.3±2.9%, respectively, The annexin V% on CD8+ cells in controls in group I, group II and groupIII were 10.5±2.7%,8.3±3.2%and 8.9±4.0%, respectively.
◆No significant effect of rhBAFF and BR3-Fc on apoptosis of CD4+ cells
There was no significant effect of rhBAFF on annexin V% of CD4+ cells in both ITP patients and controls (P>0.05). The annexin V% of CD4+ cells in ITP patients in group I, group II and group III were 8.6±4.5%,5.3±1.8% and 8.2±3.8%, respectively.
◆RhBAFF promotes the apoptosis of platelets, BR3-Fc corrects its effect
RhBAFF significantly increased apoptosis of platelets in ITP patients (group II: 8.4±4.9% vs group I:3.2±1.0%, P＜0.05) but not in controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of platelets(group III:5.0±3.0% vs group II:8.4± 4.9%, P＜0.05). In order to further confirm the results, we also measured the apoptosis of platelets by mitochondrial membrane potential assay kit with JC-1 which was a more precise method for detection of platelet apoptosis. Similar results were found.
◆Effects of rhBAFF and/or BR3-Fc on secretion of cytokines by PBMCs
The levels of IFN-γand IL-4 in supernatant were measured by ELISA. RhBAFF promoted the secretion of IFN-γin the presence of PHA (10μg/ml) (P<0.05) in ITP patients but not controls, and combination of BR3-Fc and rhBAFF reduced the level of IFN-γcompared with group rhBAFF (20ng/ml) (P<0.05). The mean±SD of group I was 74.0±12.5pg/ml, and it increased to 95.1±25.7pg/ml in group II, and reduced to 82.4±17.4pg/ml in groupⅢ, similar to that in group I. There was no detectable level of IFN-y when incubating cells without PHA. The level of IL-4 was below the detectable limit of the assay used.
Conclusions:
◆The expression of BAFF is elevated in active ITP patients, and the expression of BAFF correlates to disease activity.
◆BAFF may play a pathogenic role in ITP by promoting the survival of CD19+ and CD8+ cells, increasing the apoptosis of platelets and the secretion of IFN-γ.
◆Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.
英文摘要ⅡHigh dose dexamethasone inhibit BAFF expression in patients with immune thrombocytopenia
The treatment regimens for ITP include glucorticosteroids (GCs), intravenous immunoglobulin (IVIg), intravenous anti-D immunoglobulin, splenectomy, danazol and other immunosuppressive drugs in which GCs have been widely recognized as the most appropriate first line treatment. Recently, HD-DXM has been used as the first-line therapy for adult patients with ITP, which has a higher response rate than conventional prednisone doses Some studies have showed that the treatment with GCs induces a marked decrease in BAFF levels in patients with SLE and bullous pemphigoid. Recently, a study about the inhibition of DXM on BAFF in fibroblast-like synoviocytes from patients with RA has been reported, which showed DXM inhibited the expression of BAFF in a dose-and time-dependent manner. Up to date, there is no data about the effect of HD-DXM on BAFF expression.
Objective:
To investigate the effects of HD-DXM on BAFF expression and its possible mechanism
Methods:
◆20 acitive ITP patients were enrolled in this study. Blood sampling was performed before and after treatment at the end of the second week with HD-DXM. Twenty-four healthy controls were voluntary blood donors.
◆The expression of plasma BAFF and BAFF mRNA in ITP patients and controls were measured by ELISA and RT-PCR.
◆Moreover, we evaluated the effects of DXM on BAFF expression, secretion of IFN-γand IL-4, and proliferation of lymphocytes by ELISA, real time quantitative PCR and cell proliferation assay [cell counting kit-8 (CCK-8)] respectively in in vitro experiment.
Results:
◆Clinical therapeutic effect of HD-DXM
Responses were reached in patients:CR in 16 (80%), R in 3 (15%), and NR in 1 (5%). The mean platelet count was 165×109/L (range 23 to 332×109/L) two weeks after the initiation of treatment.
◆Decreased expression of BAFF in active ITP patients with HD-DXM treatment
Both plasma BAFF and its mRNA expression are elevated in active ITP patients before HD-DXM treatment. And plasma BAFF correlated with its mRNA levels in active ITP patients (r=0.45, P＜0.01).
After administration of HD-DXM, plasma BAFF levels in ITP patients were significantly decreased (297±120 pg/ml) compared with pretherapy (597±197 pg/ml, P＜0.001), and lower than that of the normal controls (454±132 pg/ml, P＜0.001).
Similar results were found on BAFF mRNA levels. After administration of HD-DXM, the relative amount of BAFF mRNA was 0.32 fold and 0.13 fold of that of healthy controls (P<0.001) and untreated patients respectively (P<0.001).
◆Changes of BAFF correlated with clinical responses
After administration of HD-DXM, eighteen of twenty patients had reduced plasma BAFF level, with two patients who relapsed shortly after discontinuation of the therapy and later received splenectomy had similar or even higher expression.
◆Expression of BAFF is inhibited by DXM in vitro
Because BAFF is elevated in untreated ITP patients, and after administration of HD-DXM, it reduced significantly, we investigated whether DXM, a most used glucocorticoids for ITP therapy, is able to inhibit the expression of BAFF. We cultured PBMCs from 10 untreated ITP patients with active disease and 11 controls were adjusted to 1×106/ml in RPMI-1640 culture medium, cultured at a density of 1×106 cells/well in a 24-well culture plate and incubated with different concentrations of DXM at 37℃with 5% CO2, after 72h, cells were harvested for RT-PCR.The results showed that DXM suppressed the expression of BAFF in a dose-dependent manner.
◆Effects of DXM on the expression of IFN-γand IL-4
PBMCs from active ITP patients and healthy controls were cultured with different concentrations of DXM (OnM,7.8nM,15.6nM,31.3nM,62.5nM,125nM, 250nM) at 37℃with 5% CO2, with 10μg/ml phytohemagglutinin (PHA). After 72h, supernatants were collected and levels of IFN-y and IL-4 were measured. When PHA was added, levels of IFN-y were available in all samples. A dose-dependent inhibition of IFN-y by DXM was obvious. The levels of plasma IL-4 were lower than the detection limits.
◆Effects of DXM on the proliferation of PBMCs
After cultured with different concentrations of DXM (OuM,0.125uM,0.25uM, 0.5uM, 1uM,2uM and 4uM) for 72h, proliferation in active ITP patients and controls was assayed by CCK-8. The results showed DXM inhibited the proliferation of PBMCs of both patients and controls, reaching about 50% of inhibition with 0.5uM DXM, and about 75% inhibition with 2uM DXM in active ITP patients. Similar results in healthy controls were observed.
◆Correlation of BAFF with clinical and laboratory parameters in ITP patients
Correlations between levels of BAFF and platelet counts were analyzed in ITP patients, and no significant associations were found (P>0.05). There was no significant correlation between changes of BAFF and platelets.
Conclusions:
◆HD-DXM inhibits the expression of plasma BAFF and its mRNA levels in ITP patients;
◆DXM inhibits the expression of BAFF, IFN-y and proliferation of lymphocytes in vitro.
英文摘要ⅢElevated interleukin-21 correlated to Th17 and Th1 cells in patients with immune thrombocytopenia
IL-21, a recently discovered cytokine, was originally thought to be restricted to CD4+ T cells (Th1 and Th2 cells) and NKT cells, but it is now clear that IL-21 is also produced by Th17 cells. IL-21/IL-21R system is involved in the regulation of central functions of the immune system, including promoting T-cell-mediated humoral immune responses and antibody production, increasing the cytolytic potential of NK cells, promoting the differentiation of naive Th cells into Th17 cells. Data from mouse models have suggested that IL-21 promotes autoimmunity and blocking the action of IL-21 holds promises in a number of disease settings. IL-21 and/or IL-21 R are also implicated in the pathogenesis of some autoimmune diseases such as SLE, RA, SS in humans.
Up to date, there is no data about IL-21 in patients with ITP. To further investigate the possible role of IL-21 in the pathogenesis of ITP, we measured the levels of IL-21 and correlated its levels to Th17 cells, Th1 cells and Tel cells. Objective:
To investigate the possible role of IL-21, Th17, Th1 and Tc1 cells, as well as their relationship in the pathogenesis of ITP, and evaluated their clinical relevance
Methods:
◆Patients and controls:Twenty-six adult chronic ITP patients were enrolled by diagnostic criteria for ITP. The control group consisted of twenty-one adult healthy volunteers matched for sex and age with the study population.
◆We examined the levels of CD3+CD8-IL-21+, CD3+CD8+IL-21+,Th17,Th1 and Tc1 cells in peripheral blood which was activated in vitro by PMA/ionomycin in short-term cultures in ITP patients and controls by flow cytometry through intracellular cytokines analysis. Th17 cells and Th1 cells were identified as those that were CD3+CD8-IL-17A+and CD3+CD8-IFN-γ+, and Tc1 cells were those that were CD3+CD8+IFN-γ+.
◆ELISA was used to detect the expression of IL-21 in plasma.
◆Anti-platelet autoantibodies were detected using the modified MAIPA assay.
Results:
◆Elevated IL-21 in active ITP patients
IL-21 was expressed on both CD3+CD8-T cells and CD3+CD8+T cells. The percentage of CD3+CD8-IL-21+T cells was significantly elevated in active ITP patients (mean±SD,4.4±0.8%) compared to healthy controls (2.3±0.8%, P＜0.001). Similarly, elevated percentage of CD3+CD8+IL-21+T cells was found in active ITP patients (0.8±0.5%) compared to healthy controls (0.5±0.3%, P＜0.05)
We investigated plasma IL-21 by ELISA. The level of IL-21 was significantly increased in active ITP patients (148.3±124.2pg/ml) compared to controls (78.0±32.0pg/ml, P＜0.05).
◆Increased expression of Th17, Th1 and Tc1 cells in active ITP patients treatment
IL-17 was expressed on CD3+CD8-T cells and IFN-y was expressed on both CD3+CD8-T cells and CD3+CD8+T cells. ITP patients had significantly increased percentage of Th17 cells, Thl cells and Tc1 cells. The percentage of Th17 cells was significantly increased in ITP patients (2.1±0.8%) compared to controls (1.2±0.5%, P＜0.05). Similarly, we found significantly increased percentage of Thl cells and Tc1 cells in ITP patients (Th1 cells:18.6±2.3%, Tc1 cells:21.4±8.4%) compared to controls (Th1 cells:10.9±2.6%, Tc1 cells:11.4±3.8%).
◆Correlation between CD3+CD8-IL-21+T cells, CD3+CD8+IL-21+T cells, Th17 cells, Th1 cells and Tc1 cells in active ITP patients
We observed that the percentage of CD3+CD8-IL-21+T cells positively correlated to Th17 cells (P＜0.01) and Thl cells (P＜0.01). No significant correlation between CD3+CD8+IL-21+T cells and Tc1 cells was found (P>0.05).
◆Correlation of cells with autoantibodies in ITP patients
The levels of CD3+CD8-IL-21+T cells, CD3+CD8+IL-21+T cells, Th17 cells, Th1 cells and Tc1 cells were not significantly different for patients who had positive MAIPA test compared to those with negative MAIPA test (P>0.05).
Conclusions:
◆ITP patients have elevated IL-21 level;
◆Our results indicated a possible role of IL-21 in ITP patients correlated to Th17 and Th1 cells and blockade of IL-21 may be a reasonable therapeutic strategy for ITP especially those with active disease.
免疫性血小板减少症(immune thrombocytopenia, ITP)是由于机体免疫失耐受,产生的抗血小板自身抗体与血小板表面特异性抗原结合,导致血小板在网状内皮系统过度破坏引起血小板减少。由自身反应性B细胞产生的自身抗体,主要是针对自身血小板膜糖蛋白Ⅱb／Ⅲa和／或Ⅰb／Ⅸ抗原的自身抗体,尤其是IgG抗体,在ITP的发病中起到重要的作用。此外,多种细胞免疫机制异常,如Th1极化、调节性T细胞数目降低或者抑制功能缺陷、细胞毒性T细胞(cytotoxic T lymphocyte, CTL)介导的血小板破坏等在ITP的发病中发挥重要作用。到目前为止,出现上述异常的原因尚未明确。ITP的主要治疗包括糖皮质激素、免疫球蛋白、抗-D和脾切除等,但仅有1／3的患者能够获得长期缓解。
B细胞活化因子(B-cell activating factor, BAFF,亦称为BlyS,TALL-1,THANK, zTNF4 and TNFSF13B)是近年来发现的肿瘤坏死因子超家族(tumor necrosis factor super family, TNFSF)的新成员,它在B细胞发育、稳定及其自身反应性和T细胞共刺激等方面起到重要的作用。此外,BAFF在Th1介导的炎症应答中也起到重要的作用。目前研究发现BAFF主要与三个受体结合,B细胞成熟抗原(B-cell maturation antigen, BCMA,亦称TNFRSF17)、转膜激活剂、钙调节剂和亲环素配体相互作用物(transmembrane activator and calcium-modulating cyclophilin ligand interactor, TACI,亦称TNFRSF13B)和BAFF受体(BAFF receptor, BAFF-R,亦称BR3, TNFRSF13C)。BR3是B细胞存活最主要的受体,它广泛表达于包括未成熟B、过渡期B、成熟B、记忆B、生发中心B以及浆细胞在内的B细胞亚群。此外,体内和体外研究表明,表达于T细胞上的BR3与BAFF结合后能够刺激T细胞的增殖。
许多研究表明,BAFF在自身免疫中发挥重要的作用。自身抗原结合的B细胞对BAFF介导的存活信号依赖增强。在许多自身免疫性疾病患者中,如风湿性关节炎(rheumatoid arthritis, RA)、系统性红斑狼疮(systemic lupus erythematosus, SLE)、干燥综合征(Sjogren's syndrome, S S)、多发性硬化(multiple sclerosis,MS)等,血浆BAFF表达水平异常升高。抑制BAFF信号通路可能是B细胞介导的自身反应性疾病一种有效的治疗方法。动物实验和临床试验研究表明应用BAFF阻断剂(如TACI-Ig,BAFF-R-Ig,BR3-Fc)阻断BAFF在某些自身免疫性疾病中是一种有效的治疗手段。
目的：
检测ITP患者BAFF表达以及rhBAFF和BR3-Fc对B细胞、T细胞、血小板、干扰素-γ(interferon-gamma,IFN-γ)、白介素-4(interleukin-4,IL-4)的作用,探讨BAFF和BR3-Fc在ITP发病中的可能机制
方法：
◆BAFF检测部分抽取25例活动性ITP患者,20例稳定期患者和24例对照的外周血,分离血浆和单个核细胞
◆酶联免疫吸附实验(enzyme-linked immunoadsorbent assay, ELISA)法和实时定量聚合酶链反应(real-time polymerase chain reaction, RT-PCR检测BAFF蛋白和mRNA水平；改良单克隆抗体特异性俘获血小板抗原(immobilization of platelet antigens magnetic activated cell separation, MAIPA)检测抗GPIIb/Ⅲa和GPIb/IX自身抗体
◆体外部分抽取18例活动性ITP患者和15例对照的外周血15ml,分离单个核细胞和血小板,加入不同的细胞因子共同培养,分为3组：1640组(Ⅰ组)、rhBAFF 20ng／ml组(Ⅱ组)和rhBAFF+BR3-Fc组(Ⅲ组)。流式细胞术检测CD19+B细胞、CD4+T细胞、CD8+T细胞及血小板的凋亡；ELISA法检测培养上清中IFN-γ、IL-4和自身抗体的表达
结果：
◆BAFF在活动性ITP中表达升高,稳定期患者与对照无显著性差异
在活动性ITP患者中BAFF蛋白水平(均值±标准差,593.1±219.0 pg/ml)明显高于稳定期患者(432.5±121.4pg/ml, P<0.05)和正常对照(454.4±132.5pg/ml,P<0.05)。活动性ITP患者BAFF mRNA水平是稳定期患者和对照的3.1倍(P<0.01)和2.5倍(P<0.01)。而在稳定期患者无论BAFF蛋白还是mRNA水平均与正常对照无明显差异(P>0.05)。BAFF水平与GPIIb/IIIa和GPIb/Ⅸ无显著性相关。
◆RhBAFF促进CD19+存活,BR3-Fc纠正rhBAFF对其影响
RhBAFF降低ITP患者CD19+细胞的凋亡(Ⅱ组：7.0±4.3％vsⅠ组：13.2±7.4％,P<0.05),加入BR3-Fc后CD19+细胞的凋亡升高,但与1640组无显著性差异(Ⅲ组：11.2±4.1％vsⅠ组：13.2±7.4%,P>0.05)。
◆RhBAFF促进CD8+存活,BR3-Fc纠正rhBAFF对其影响
ITP患者中Ⅰ、Ⅱ、Ⅲ组CD8+细胞的凋亡率分别为6.5±3.2％,4.4±2.2％和6.3±2.9%。RhBAFF促进CD8+存活,BR3-Fc纠正rhBAFF对其影响。
◆RhBAFF和BR3-Fc对CD4+细胞凋亡无显著影响
RhBAFF对CD4+细胞凋亡无显著影响。ITP患者中Ⅰ、Ⅱ、Ⅲ组CD8+细胞的凋亡率分别为8.6±4.5％,5.3±1.8％和8.2±3.8％。
◆RhBAFF促进血小板凋亡,BR3-Fc抑制其凋亡
RhBAFF促进ITP患者血小板的凋亡(Ⅱ组：8.4±4.9％vsⅠ组：3.2±1.0％,P<0.051,加入BR3-Fc后血小板的凋亡降低(Ⅲ组：5.0±3.0％vsⅡ组：8.4±4.9％,P<0.05)。
此外,我们应用线粒体膜电位检测试剂盒JC-1检测血小板凋亡进一步验证了我们的结果。
◆在ITP患者中各组IFN-γ均显著高于对照组,rhBAFF促进ITP患者IFN-γ分泌,而BR3-Fc抑制其表达
ITP患者中Ⅰ、Ⅱ、Ⅲ组IFN-γ分别为74.0±12.5pg／ml、95.1±25.7pg／ml和82.4±17.4pg/ml, rhBAFF 20ng／ml组IFN-γ水平显著高于1640组,加入BR3-Fc后IFN-γ表达下降。IL-4低于最低检测值。
结论：
◆BAFF在活动性ITP的表达升高,而稳定期患者无异常,与病情变化相关。
◆BAFF可能通过促进CD19+B和CD8+T细胞存活、促进血小板的凋亡、促进Th1类细胞因子分泌等机制在ITP的发病中起到重要的作用；BR3-Fc纠正了rhBAFF对上述细胞和因子的作用。
◆阻断ITP中过高的BAFF可能成为ITP治疗的一个新的靶点。
第Ⅱ部分大剂量地塞米松疗法抑制ITP患者BAFF的表达
ITP的治疗主要有糖皮质激素、免疫球蛋白、脾切除、达那唑及免疫抑制剂等,其中糖皮质激素是ITP的一线治疗药物。在过去50多年里,泼尼松一直是治疗ITP的首选,开始剂量1.0-1.5mg／kg/d,分次口服4-6周,此后逐渐减量至血小板在安全水平以上(血小板>30x109／L)的最低用量时给予维持治疗,约70％患者可获得缓解,而30％患者治疗无效,属于难治性ITP。该方法治疗有效率与剂量相关,长期应用副作用较大,并且减量过程中容易复发,此时给予重复治疗很难再取得持续缓解,应考虑换用其他治疗。脾切除是泼尼松治疗失败患者的第一选择,其长期缓解率为50％-70％,仍然有30％左右患者治疗无效,另外还有部分泼尼松治疗失败的患者由于年龄、基础疾病等因素而不能行脾切除手术治疗,这些难治性ITP患者的治疗一直是临床上非常棘手的问题。
近年来大剂量地塞米松(High dose dexamethasone, HD-DXM)疗法较传统的强的松治疗取得了更好的疗效。与泼尼松相比,HD-DXM冲击治疗起效快、副反应轻微,效果相当于免疫球蛋白,但是花费明显减少。对于初诊的ITP患者,HD-DXM冲击治疗效果显著,80％以上有治疗反应,已经作为一线治疗选择。对于复发／难治性ITP患者HD-DXM冲击治疗总体有效率不高,只有部分患者有治疗反应。HD-DXM冲击治疗尚不能代替或避免脾切除,不过可以作为脾切除前的应急准备。
研究表明在某些自身免疫性疾病中如SLE、大疱性类天疱疮中糖皮质激素能够显著降低患者BAFF表达水平。另一项研究表明DXM在体外能够抑制风湿性关节炎患者纤维母样滑膜细胞BAFF的表达,并且呈剂量和时间依赖性。而关于HD-DXM对ITP患者BAFF表达的影响目前尚未见报道。
目的：
研究大剂量地塞米松(HD-DXM)对ITP患者BAFF的作用,进一步探讨HD-DXM在ITP治疗中的可能机制
方法：
◆留取20例ITP患者(应用激素治疗前2周各抽血一次)和24例对照的外周血3ml。分离血浆和单个核细胞。ELISA检测血浆BAFF蛋白水平,实时定量PCR检测PBMCs中BAFF mRNA水平。
◆其中10例ITP患者和14例对照抽血15ml,分离单个核细胞。加入不同浓度DXM,培养后分离上清检测BAFF、IFN-γ和IL-4,分离细胞实时定量PCR检测BAFF mRNA水平。CCK-8法检测淋巴细胞增殖。
结果：
◆HD-DXM临床疗效
80％患者(16／20)获得长期反应,15％患者(3／20)获得部分反应,5％患者(1／20)无反应。HD-DXM治疗2周后平均血小板数目是165×109／L(范围23-332×109/L)。
◆BAFF在活动性ITP(应用HD-DXM治疗前)明显升高,应用HD-DXM后,ITP患者BAFF水平明显降低
活动性ITP患者中BAFF蛋白水平及其mRNA水平显著升高。应用HD-DXM后,ITP患者BAFF蛋白水平(均值297pg／ml,范围184-545pg/ml)明显低于用药前(均值597 pg／ml,范围300-1026pg/ml)和正常对照(均值454 pg／ml,208-804pg/ml, P<0.001); BAFF mRNA水平亦明显低于激素前患者(P<0.001)和对照组(P<0.001)。
◆BAFF变化与HD-DXM治疗反应密切相关
应用HD-DXM后,20例患者中有18例BAFF水平明显低于治疗前水平,1例患者BAFF水平与治疗前相比无明显降低,1例BAFF水平略微升高。这两例患者均对HD-DXM治疗无反应,后来随访发现为难治性ITP,并进行脾切除。
◆DXM体外抑制BAFF的表达
我们体外培养10例未治疗的ITP患者和11例对照PBMCs,加入不同浓度的DXM后体外培养72小时,我们发现DXM明显抑制BAFF的表达,并且呈剂量依赖性。
◆DXM体外抑制IFN-γ的表达
我们体外培养ITP患者和对照PBMCs,加入不同浓度的DXM(OnM,7.8nM, 15.6nM,31.3nM,62.5nM,125nM,250nM1和10μg/ml PHA,于37℃含5％CO2孵箱孵育72小时后,收集上清,ELISA检测IFN-γ和IL-4表达,DXM能够显著抑制IFN-γ表达,并且呈剂量依赖性,而IL-4水平低于最低检测值。
◆DXM体外抑制淋巴细胞的增殖
CCK-8法检测DXM对淋巴细胞增殖的影响,我们发现DXM明显抑制淋巴细胞增殖,并且呈剂量依赖性。
◆BAFF与ITP患者临床实验室指标相关性分析
BAFF水平和血小板数目无显著性相关,且BAFF水平的变化和血小板数目的改变无显著性相关。
结论：
◆HD-DXM抑制ITP患者血浆BAFF及其mRNA水平的表达；
◆DXM体外抑制BAFF、IFN-γ的表达和淋巴细胞的增殖。
第Ⅲ部分白介素-21在ITP中的作用及其机制研究
白介素-2家族主要包括IL-2、IL-4、IL-7、IL-9和IL-15,在促进和维持T淋巴细胞群中起主要作用。由这些细胞因子介导的详细的分子信号传递途径尚未完全澄清.然而JAK/STAT,MAPK和P13这三个主要的途径已经清楚。
IL-21是IL-2家族的新成员,主要由活化的CD4+T细胞和自然杀伤T细胞表达,除此以外,近来发现IL-21还可以由Th17细胞表达。IL-21／IL-21R信号在天然免疫和获得性免疫应答中均起到重要的作用,它能够促进T细胞介导的体液免疫应答与抗体生成、增强NK细胞的自然杀伤能力、促进Th细胞向Th17细胞的分化、影响Th细胞向Th1／Th2细胞的分化等。在许多自身免疫性疾病中(如SLE,RA,SS等)IL-21和／或IL-21R表达异常。目前,未见关于IL-21／IL-21R在ITP患者中的报道。
目的：
通过对ITP患者外周血中表达IL-21的细胞亚群和Th17、Th1、Tc1细胞亚群的检测,探讨IL-21在ITP中的作用及可能机制
材料与方法：
◆病例及正常对照的选择：活动性ITP患者26例,均符合ITP的诊断标准。正常对照21例,均为健康志愿者,无感染、无病毒性肝炎、无免疫性疾病及半年内未使用免疫抑制剂、抗HIV抗体阴性、肝肾功能正常,其年龄和性别均与ITP患者相匹配。
◆应用三色流式细胞术检测ITP患者和正常对照外周血中各细胞亚群的比例。三种细胞亚群的界定分别为：表达IL-21的细胞为CD3+CD8-IL-21+和CD3+CD8+IL-21+,Th17细胞为CD3+CD8-IL-17A+,Th1细胞为CD3+CD8-IFN-γ+,Tc1细胞为CD3+CD8+IFN-γ+
◆应用ELISA技术检测ITP患者和正常对照血浆中IL-21的表达水平。
◆应用改良的MAIPA技术(单克隆抗体特异性俘获血小板抗原技术)测定ITP患者体内的抗血小板膜GPⅡb／Ⅲa和Ib／Ⅸ自身抗体。
结果：
◆IL-21在ITP中表达升高
我们应用流式细胞术检测ITP患者外周血中IL-21的表达,发现IL-21表达于CD3+CD8-细胞和CD3+CD8+细胞上。ITP患者中CD3+CD8-IL-21+细胞亚群(4.4±0.8％)比例显著高于对照组(2.3±0.8％,P<0.001),相似的,ITP患者中CD3+CD8+IL-21+细胞亚群(0.8±0.5％)比例显著高于对照组(0.5±0.3％,P<0.05)。
我们应用ELISA法检测ITP患者和对照血浆中IL-21的表达,我们发现ITP血浆IL-21水平(148.3±124.2pg／ml)高于对照组(78.0±32.0pg／ml,P<0.05)。
◆ITP患者中Th17、Th1、Tc1细胞亚群表达升高
ITP患者外周血Th17细胞的比例(2.1±0.8％)明显高于正常对照组(1.2±0.5％,P<0.05)。ITP患者外周血Thl细胞的比例(18.6±2.3％)明显高于正常对照组(10.9±2.6％,P<0.001),并且ITP患者Tc1细胞的比例(21.4±8.4％)较正常对照组(11.4±3.8％)亦显著升高(P<0.001)。
◆各细胞亚群的相关性分析
在ITP患者中,CD3+CD8-IL-21+细胞与Th17细胞(P<0.01)和Thl细胞(P<0.01)存在明显的正相关,而CD3+CD8+IL-21+细胞与Tc1细胞不存在相关性(P>0.05)。
◆抗血小板自身抗体与各细胞亚群的相关性分析
应用MAIPA法检测抗血小板自身抗体,并与各细胞亚群进行相关性分析,我们发现自身抗体与各个细胞亚群均不存在相关性(P>0.05)。
结论：
◆ITP患者中IL-21表达升高；
◆在ITP患者中,CD3+CD8-IL-21+细胞与Th17细胞和Th1细胞存在明显的正相关,提示CD3+CD8-IL-21+细胞、Th17细胞和Th1细胞可能通过相互作用而在ITP的发病中起到协同作用。
英文摘要ⅠThe effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia
Immune thrombocytopenia (ITP) is an autoimmune disorder in which the patients'immune system is activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. In addition, several abnormalities involving the cellular mechanisms of immune modulation such as Th1 bias, the decreased number or defective function of regulatory T cells and the platelet destruction by cytotoxic T cells (CTL) have been described.
B-cell activating factor (BAFF) (also known as BlyS, TALL-1, THANK, zTNF4 and TNFSF13B) belonging to the family of tumor necrosis factor (TNF) ligands is critical for the maintain of normal B-cell development, homeostasis, autoreactivity and T cell costimulation. In addition, it also augments certain Th1-associated inflammatory responses. BAFF binds to three receptors:B-cell maturation antigen (BCMA, TNFRSF17), transmembrane activator and calcium-modulating cyclophilin ligand (CAML) interactor (TACI, TNFRSF13B) and BAFF receptor (BR3/BAFF-R, TNFRSF13C). BR3, identified as the crucial receptor for B-cell survival, is expressed on a wide range of B-cell subsets, including immature, transitional, mature, memory and germinal center B cells, as well as on plasma cells. Furthermore, BAFF binding to BR3 on T cells has been shown to costimulate T-cell proliferation both in vitro and in vivo.
Several lines of evidence suggested that BAFF may play an important role in autoimmunity. Autoantigen-binding B cells may have an increased dependence on the BAFF survival signal. In addition, elevated BAFF plasma level was observed in many patients with autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogren's syndrome (SS), and multiple sclerosis (MS). Inhibition of BAFF signaling is a potentially therapeutic option for treatment of B cell-mediated autoimmune conditions. Data from animal tests and clinical trials had proved that blockade of BAFF by blocking reagents (TACI-Ig, BAFF-R-Ig, BR3-Fc) was effective therapeutic approach for some autoimmune diseases.
Objective:
To investigate the possible mechanism of BAFF and therapeutic effect of BR3-Fc in ITP, we measured the expression of plasma BAFF and BAFF mRNA, and the effects of rhBAFF and BR3-Fc on B cells, T cells, platelets, secretion of IFN-γand IL-4 were measured by flow cytometry and ELISA.
Methods:
◆Forty-five patients diagnosed with ITP were selected for detection of plasma BAFF and BAFF mRNA. Of these patients, twenty-five patients were active ITP patients who had not been treated with GCs for at least one month prior to sampling whereas twenty patients were in remission. Twenty-four healthy controls matched for sex and age with the study population were voluntary blood donors.
◆The expression of plasma BAFF and BAFF mRNA in ITP patients and controls were measured by ELISA and RT-PCR, the levels of plasma anti-platelet autoantibodies (GPIIb/IIIa and GPIb/IX) were measured by MAIPA.
◆Peripheral blood mononuclear cells (PBMCs) and platelets from additional eighteen ITP patients with active disease and fifteen controls were selected for detection of apoptosis on CD19+, CD4+, CD8+ cells, platelets, secretion of cytokines (IFN-y and IL-4) and autoantibodies by flow cytometry and ELISA.
Results:
◆Elevated levels of plasma BAFF and BAFF mRNA in active ITP patients
The level of plasma BAFF in ITP patients with active disease was significantly higher (mean±SD,593.1±219.0 pg/ml) than that in patients in remission (432.5±121.4pg/ml, P＜0.05) and controls (454.4±132.5pg/ml, P＜0.05). The relative amount of BAFF mRNA in patients with active disease was increased 3.1 and 2.5-fold compared to patients in remission (P＜0.01) and healthy controls (P<0.01), respectively. No significant difference of plasma BAFF or BAFF mRNA between patients in remission and healthy controls was found (P>0.05).
In addition, no association was found between the levels of BAFF and anti-platelet autoantibodies (P>0.05).
◆RhBAFF promotes the survival of CD19+ cells, BR3-Fc corrects its effect
RhBAFF significantly decreased the annexin V% of CD19+ cells in ITP patients (groupⅡ:7.0±4.3% vs groupⅠ:13.2±7.4%, P＜0.05) but not in controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of CD19+ cells (groupIII:11.2±4.1% vs groupⅠ:13.2±7.4%, P>0.05).
◆RhBAFF promotes the survival of CD8+ cells, BR3-Fc corrects its effect
RhBAFF significantly decreased the annexin V% of CD8+ cells in both ITP patients and controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of CD8+ cells only in ITP patients. The annexin V% on CD8+ cells in ITP patients in group I, group II and group III were 6.5±3.2%,4.4±2.2%and 6.3±2.9%, respectively, The annexin V% on CD8+ cells in controls in group I, group II and groupIII were 10.5±2.7%,8.3±3.2%and 8.9±4.0%, respectively.
◆No significant effect of rhBAFF and BR3-Fc on apoptosis of CD4+ cells
There was no significant effect of rhBAFF on annexin V% of CD4+ cells in both ITP patients and controls (P>0.05). The annexin V% of CD4+ cells in ITP patients in group I, group II and group III were 8.6±4.5%,5.3±1.8% and 8.2±3.8%, respectively.
◆RhBAFF promotes the apoptosis of platelets, BR3-Fc corrects its effect
RhBAFF significantly increased apoptosis of platelets in ITP patients (group II: 8.4±4.9% vs group I:3.2±1.0%, P＜0.05) but not in controls. BR3-Fc corrected the effect of rhBAFF on apoptosis of platelets(group III:5.0±3.0% vs group II:8.4± 4.9%, P＜0.05). In order to further confirm the results, we also measured the apoptosis of platelets by mitochondrial membrane potential assay kit with JC-1 which was a more precise method for detection of platelet apoptosis. Similar results were found.
◆Effects of rhBAFF and/or BR3-Fc on secretion of cytokines by PBMCs
The levels of IFN-γand IL-4 in supernatant were measured by ELISA. RhBAFF promoted the secretion of IFN-γin the presence of PHA (10μg/ml) (P<0.05) in ITP patients but not controls, and combination of BR3-Fc and rhBAFF reduced the level of IFN-γcompared with group rhBAFF (20ng/ml) (P<0.05). The mean±SD of group I was 74.0±12.5pg/ml, and it increased to 95.1±25.7pg/ml in group II, and reduced to 82.4±17.4pg/ml in groupⅢ, similar to that in group I. There was no detectable level of IFN-y when incubating cells without PHA. The level of IL-4 was below the detectable limit of the assay used.
Conclusions:
◆The expression of BAFF is elevated in active ITP patients, and the expression of BAFF correlates to disease activity.
◆BAFF may play a pathogenic role in ITP by promoting the survival of CD19+ and CD8+ cells, increasing the apoptosis of platelets and the secretion of IFN-γ.
◆Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.
英文摘要ⅡHigh dose dexamethasone inhibit BAFF expression in patients with immune thrombocytopenia
The treatment regimens for ITP include glucorticosteroids (GCs), intravenous immunoglobulin (IVIg), intravenous anti-D immunoglobulin, splenectomy, danazol and other immunosuppressive drugs in which GCs have been widely recognized as the most appropriate first line treatment. Recently, HD-DXM has been used as the first-line therapy for adult patients with ITP, which has a higher response rate than conventional prednisone doses Some studies have showed that the treatment with GCs induces a marked decrease in BAFF levels in patients with SLE and bullous pemphigoid. Recently, a study about the inhibition of DXM on BAFF in fibroblast-like synoviocytes from patients with RA has been reported, which showed DXM inhibited the expression of BAFF in a dose-and time-dependent manner. Up to date, there is no data about the effect of HD-DXM on BAFF expression.
Objective:
To investigate the effects of HD-DXM on BAFF expression and its possible mechanism
Methods:
◆20 acitive ITP patients were enrolled in this study. Blood sampling was performed before and after treatment at the end of the second week with HD-DXM. Twenty-four healthy controls were voluntary blood donors.
◆The expression of plasma BAFF and BAFF mRNA in ITP patients and controls were measured by ELISA and RT-PCR.
◆Moreover, we evaluated the effects of DXM on BAFF expression, secretion of IFN-γand IL-4, and proliferation of lymphocytes by ELISA, real time quantitative PCR and cell proliferation assay [cell counting kit-8 (CCK-8)] respectively in in vitro experiment.
Results:
◆Clinical therapeutic effect of HD-DXM
Responses were reached in patients:CR in 16 (80%), R in 3 (15%), and NR in 1 (5%). The mean platelet count was 165×109/L (range 23 to 332×109/L) two weeks after the initiation of treatment.
◆Decreased expression of BAFF in active ITP patients with HD-DXM treatment
Both plasma BAFF and its mRNA expression are elevated in active ITP patients before HD-DXM treatment. And plasma BAFF correlated with its mRNA levels in active ITP patients (r=0.45, P＜0.01).
After administration of HD-DXM, plasma BAFF levels in ITP patients were significantly decreased (297±120 pg/ml) compared with pretherapy (597±197 pg/ml, P＜0.001), and lower than that of the normal controls (454±132 pg/ml, P＜0.001).
Similar results were found on BAFF mRNA levels. After administration of HD-DXM, the relative amount of BAFF mRNA was 0.32 fold and 0.13 fold of that of healthy controls (P<0.001) and untreated patients respectively (P<0.001).
◆Changes of BAFF correlated with clinical responses
After administration of HD-DXM, eighteen of twenty patients had reduced plasma BAFF level, with two patients who relapsed shortly after discontinuation of the therapy and later received splenectomy had similar or even higher expression.
◆Expression of BAFF is inhibited by DXM in vitro
Because BAFF is elevated in untreated ITP patients, and after administration of HD-DXM, it reduced significantly, we investigated whether DXM, a most used glucocorticoids for ITP therapy, is able to inhibit the expression of BAFF. We cultured PBMCs from 10 untreated ITP patients with active disease and 11 controls were adjusted to 1×106/ml in RPMI-1640 culture medium, cultured at a density of 1×106 cells/well in a 24-well culture plate and incubated with different concentrations of DXM at 37℃with 5% CO2, after 72h, cells were harvested for RT-PCR.The results showed that DXM suppressed the expression of BAFF in a dose-dependent manner.
◆Effects of DXM on the expression of IFN-γand IL-4
PBMCs from active ITP patients and healthy controls were cultured with different concentrations of DXM (OnM,7.8nM,15.6nM,31.3nM,62.5nM,125nM, 250nM) at 37℃with 5% CO2, with 10μg/ml phytohemagglutinin (PHA). After 72h, supernatants were collected and levels of IFN-y and IL-4 were measured. When PHA was added, levels of IFN-y were available in all samples. A dose-dependent inhibition of IFN-y by DXM was obvious. The levels of plasma IL-4 were lower than the detection limits.
◆Effects of DXM on the proliferation of PBMCs
After cultured with different concentrations of DXM (OuM,0.125uM,0.25uM, 0.5uM, 1uM,2uM and 4uM) for 72h, proliferation in active ITP patients and controls was assayed by CCK-8. The results showed DXM inhibited the proliferation of PBMCs of both patients and controls, reaching about 50% of inhibition with 0.5uM DXM, and about 75% inhibition with 2uM DXM in active ITP patients. Similar results in healthy controls were observed.
◆Correlation of BAFF with clinical and laboratory parameters in ITP patients
Correlations between levels of BAFF and platelet counts were analyzed in ITP patients, and no significant associations were found (P>0.05). There was no significant correlation between changes of BAFF and platelets.
Conclusions:
◆HD-DXM inhibits the expression of plasma BAFF and its mRNA levels in ITP patients;
◆DXM inhibits the expression of BAFF, IFN-y and proliferation of lymphocytes in vitro.
英文摘要ⅢElevated interleukin-21 correlated to Th17 and Th1 cells in patients with immune thrombocytopenia
IL-21, a recently discovered cytokine, was originally thought to be restricted to CD4+ T cells (Th1 and Th2 cells) and NKT cells, but it is now clear that IL-21 is also produced by Th17 cells. IL-21/IL-21R system is involved in the regulation of central functions of the immune system, including promoting T-cell-mediated humoral immune responses and antibody production, increasing the cytolytic potential of NK cells, promoting the differentiation of naive Th cells into Th17 cells. Data from mouse models have suggested that IL-21 promotes autoimmunity and blocking the action of IL-21 holds promises in a number of disease settings. IL-21 and/or IL-21 R are also implicated in the pathogenesis of some autoimmune diseases such as SLE, RA, SS in humans.
Up to date, there is no data about IL-21 in patients with ITP. To further investigate the possible role of IL-21 in the pathogenesis of ITP, we measured the levels of IL-21 and correlated its levels to Th17 cells, Th1 cells and Tel cells. Objective:
To investigate the possible role of IL-21, Th17, Th1 and Tc1 cells, as well as their relationship in the pathogenesis of ITP, and evaluated their clinical relevance
Methods:
◆Patients and controls:Twenty-six adult chronic ITP patients were enrolled by diagnostic criteria for ITP. The control group consisted of twenty-one adult healthy volunteers matched for sex and age with the study population.
◆We examined the levels of CD3+CD8-IL-21+, CD3+CD8+IL-21+,Th17,Th1 and Tc1 cells in peripheral blood which was activated in vitro by PMA/ionomycin in short-term cultures in ITP patients and controls by flow cytometry through intracellular cytokines analysis. Th17 cells and Th1 cells were identified as those that were CD3+CD8-IL-17A+and CD3+CD8-IFN-γ+, and Tc1 cells were those that were CD3+CD8+IFN-γ+.
◆ELISA was used to detect the expression of IL-21 in plasma.
◆Anti-platelet autoantibodies were detected using the modified MAIPA assay.
Results:
◆Elevated IL-21 in active ITP patients
IL-21 was expressed on both CD3+CD8-T cells and CD3+CD8+T cells. The percentage of CD3+CD8-IL-21+T cells was significantly elevated in active ITP patients (mean±SD,4.4±0.8%) compared to healthy controls (2.3±0.8%, P＜0.001). Similarly, elevated percentage of CD3+CD8+IL-21+T cells was found in active ITP patients (0.8±0.5%) compared to healthy controls (0.5±0.3%, P＜0.05)
We investigated plasma IL-21 by ELISA. The level of IL-21 was significantly increased in active ITP patients (148.3±124.2pg/ml) compared to controls (78.0±32.0pg/ml, P＜0.05).
◆Increased expression of Th17, Th1 and Tc1 cells in active ITP patients treatment
IL-17 was expressed on CD3+CD8-T cells and IFN-y was expressed on both CD3+CD8-T cells and CD3+CD8+T cells. ITP patients had significantly increased percentage of Th17 cells, Thl cells and Tc1 cells. The percentage of Th17 cells was significantly increased in ITP patients (2.1±0.8%) compared to controls (1.2±0.5%, P＜0.05). Similarly, we found significantly increased percentage of Thl cells and Tc1 cells in ITP patients (Th1 cells:18.6±2.3%, Tc1 cells:21.4±8.4%) compared to controls (Th1 cells:10.9±2.6%, Tc1 cells:11.4±3.8%).
◆Correlation between CD3+CD8-IL-21+T cells, CD3+CD8+IL-21+T cells, Th17 cells, Th1 cells and Tc1 cells in active ITP patients
We observed that the percentage of CD3+CD8-IL-21+T cells positively correlated to Th17 cells (P＜0.01) and Thl cells (P＜0.01). No significant correlation between CD3+CD8+IL-21+T cells and Tc1 cells was found (P>0.05).
◆Correlation of cells with autoantibodies in ITP patients
The levels of CD3+CD8-IL-21+T cells, CD3+CD8+IL-21+T cells, Th17 cells, Th1 cells and Tc1 cells were not significantly different for patients who had positive MAIPA test compared to those with negative MAIPA test (P>0.05).
Conclusions:
◆ITP patients have elevated IL-21 level;
◆Our results indicated a possible role of IL-21 in ITP patients correlated to Th17 and Th1 cells and blockade of IL-21 may be a reasonable therapeutic strategy for ITP especially those with active disease.
引文
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35. Becker-Merok A, Nikolaisen C and Nossent HC. B-lymphocyte activating factor in systemic lupus erythematosus and rheumatoid arthritis in relation to autoantibody levels, disease measures and time. Lupus 2006; 15:570-576.
36. Vugmeyster Y, Seshasayee D, Chang W, et al. A soluble BAFF antagonist, BR3-Fc, decreases peripheral blood B cells and lymphoid tissue marginal zone and follicular B cells in cynomolgus monkeys. Am J Pathol 2006; 168:476-489.
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