特发性血小板减少性紫癜免疫发病机制的临床与基础研究
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摘要
特发性血小板减少性紫癜(idiopathic thrombocytopenic purpura,ITP)属于自身免疫性疾病,又称免疫性血小板减少性紫癜(autoimmune thrombocytopenic purpura ITP),其免疫机制是ITP患者体内产生抗血小板膜糖蛋白的血小板相关抗体,使血小板被网状内皮系统或补体系统过度破坏,而引起血小板减少,出现出血的临床表现。随着研究的深入,发现除体液免疫外,细胞免疫在ITP发病机制中的作用不容忽视,其中针对血小板膜糖蛋白的抗原特异性自身反应性T细胞的异常活化起着关键性作用,一旦这种血小板抗原特异性自身反应性T细胞的免疫耐受机制被打破,可辅助同种自身抗原特异性B细胞产生自身抗体导致自身免疫病ITP的发生。研究证实,CD4+CD25+调节性T细胞(regulatory T cell,Treg)及树突状细胞(DC)在维持机体自身免疫耐受中均发挥着及其重要的作用,CD4+CD25+ Treg细胞和/或DC的免疫缺陷均可导致自身免疫性疾病的发生。其中CD4+CD25+ Treg细胞由胸腺髓质中的T细胞分化发育而来,通过主动抑制自身反应性T细胞的活化、维持自身免疫耐受;而DC作为已知功能最强的抗原递呈细胞(APC),能唯一激活初始型T细胞致敏,产生初次免疫应答,成熟DC联合抗原作用可加强免疫应答,而不成熟DC又可促进机体产生免疫抑制或免疫耐受,因此,DC亦被视为机体内重要的免疫调节细胞。T细胞异常活化后辅助B细胞活化,分泌抗体;同时活化的B细胞可作为APC直接活化T细胞,因此B细胞在自身反应性T细胞的活化效应中亦发挥一定的作用。本课题旨从临床和基础两方面来探讨CD4+CD25+ Treg细胞、DC及B细胞在ITP免疫发病机制中的作用,为ITP的免疫治疗开辟新的思路。通过对ITP患者外周血循环中CD4+CD25+ Treg细胞数量及体外免疫抑制功能的检测,大剂量地塞米松(HD-DXM)治疗前后CD4+CD25+ Treg细胞数量及相关抗炎细胞因子IL-10、TGF-β1的变化来阐明CD4+CD25+ Treg在ITP患者中的免疫异常并探讨HD-DXM免疫抑制作用的机理;检测了ITP患者外周血循环中DC细胞表面协同刺激分子的表达、HD-DXM治疗前后不同DC亚型数量的变化、体外CD4+ T淋巴细胞增殖实验等来初步探讨DC在ITP发病机制中的意义;针对ITP患者外周血循环中B细胞数量及其细胞协同刺激分子的表达探讨其临床意义。
1.特发性血小板减少性紫癜(ITP)患者中CD4+CD25+调节性T细胞的免疫异常为探讨CD4+CD25+ Treg细胞在ITP发病机制中的作用,本研究运用了流式细胞术检测32例ITP患者外周血中CD4+CD25+ T细胞、CD4+CD25high T细胞、CD4+ FOXP3+T细胞及CD4+CD25+FOXP3+ T细胞的数量;实时荧光定量PCR检测ITP患者外周血单个核细胞(PBMC)Foxp3 mRNA的表达水平;将ITP患者CD4+CD25high T细胞与自身CD4+CD25- T细胞混合培养,检测CD4+CD25high T细胞的免疫抑制功能。结果发现,ITP患者外周血中CD4+CD25+T细胞约占CD4+T细胞(15.64±5.82)%,明显高于正常对照组(9.30±3.95)%(P﹤0.001),CD4+CD25high T细胞比例为(1.53±0.66)%,与对照组比较(1.36±0.55)%无显著性差异(P>0.05);CD4+ FOXP3+T细胞和CD4+CD25+ FOXP3+ T细胞分别为(1.82±1.42)%和(1.25±0.94)%,均明显低于对照组(3.90±1.37)%(P﹤0.001)和(2.65±0.92)%(P﹤0.001)。ITP患者外周血PBMC中FOXP3 mRNA表达水平较对照组明显下调(P﹤0.001),CD4+CD25high T细胞的免疫抑制活性较对照组明显减弱(p<0.001)。
由此表明,在ITP的免疫发病机制中,存在CD4+CD25+ Treg细胞数量的减少和免疫抑制功能的减弱,可能致使机体内血小板自身反应性T细胞的活化不能被有效的抑制而诱发、维持和加重了患者的自身免疫。随着CD4+CD25+ Treg细胞免疫调节(抑制)功能的深入研究,将会为临床治疗ITP特别是难治性ITP提供一个新的免疫治疗途径。
2.慢性ITP患者大剂量地塞米松治疗前后CD4+CD25+调节性T的变化及与抗炎细胞因子的关系
前期研究已证实在ITP患者中存在CD4+CD25+ Treg细胞的免疫异常,为证实在ITP患者中CD4+CD25+ Treg细胞与抗炎细胞因子IL-10、TGF-β1是否有一定的关联及大剂量地塞米松(HD-DXM)对CD4+CD25+ Treg细胞及IL-10、TGF-β1的影响,本研究采用流式细胞术检测了26例慢性ITP患者在HD-DXM治疗前后CD4+CD25+ T细胞、CD4+CD25high T细胞、CD4+ FOXP3+ T细胞的数量变化;酶联免疫吸附实验(ELISA)检测ITP患者在HD-DXM治疗前后血清中IL-10及TGF-β1的浓度变化;实时荧光定量PCR检测ITP患者HD-DXM治疗前后外周血CD4+ T细胞中FOXP3、IL-10、TGF-βmRNA的表达水平;免疫组化检测DXM治疗后难治性ITP患者脾脏组织中FOXP3基因的表达。结果发现,26例慢性ITP患者在确诊后予HD-DXM治疗,初始反应率达92.3%。所有患者在初始治疗后一周平均血小板数达(84.9±30.4)×109/L,范围(20～150)×109/L。HD-DXM治疗后,慢性ITP患者外周血中CD4+FOXP3+ T细胞(P﹤0.0001)、CD4+CD25+ T细胞(P﹤0.0001)、CD4+CD25high T细胞(P﹤0.0001)数量较治疗前均显著增加。慢性ITP患者治疗前血清IL-10浓度为(441.10±239.26) pg/ml,正常对照组为(349.88±21.27) pg/ml,两者比较无显著性差异(P﹥0.05);HD-DXM治疗后,IL-10浓度为(395.95±149.95) pg/ml,与治疗前比较无显著性差异(P﹥0.05);慢性ITP患者治疗前血清TGF-β浓度为(305.90±202.35) pg/ml,显著低于正常对照组(1479.00±182.70) pg/ml,(P﹤0.0001);HD-DXM治疗后,TGF-β浓度较治疗前明显上升(1414.70±270.35) pg/ml,(P﹤0.0001)。HD-DXM治疗后,慢性ITP患者外周血CD4+ T细胞FOXP3+基因mRNA表达水平较治疗前明显上升(P﹤0.05),并高于对照组(P﹤0.05);IL-10基因在HD-DXM治疗前后mRNA表达水平无变化,(P﹥0.05),与对照组比较亦均无显著性差异(P﹥0.05);治疗前, TGF-β1基因mRNA表达水平明显低于正常对照组(P﹤0.05),HD-DXM治疗后TGF-β1基因mRNA表达水平明显上升(P﹤0.0001);并高于对照组(P﹤0.05);HD-DXM治疗后,TGF-β1基因mRNA表达水平与FOXP3+基因呈正相关(r =0.403, P=0.041)。蛋白Foxp3在术前应用DXM治疗的慢性难治性ITP患者脾脏组织淋巴细胞内的表达明显强于正常脾破裂对照组但弱于乙肝患者组。
本实验结果表明在ITP免疫机制异常中,CD4+CD25+ Treg及细胞因子TGF-β1发挥着一定的作用;而糖皮质激素可通过促进CD4+CD25+ Treg及TGF-β1的表达发挥免疫抑制效应。
3.慢性ITP患者中树突状细胞(DC)免疫异常及大剂量地塞米松治疗前后DC亚群数量的变化
DC被视为机体内重要的免疫调节细胞,在维持自身免疫耐受中发挥重要作用。为探讨DC在ITP发病机制中的作用,本研究采用流式细胞术检测26例慢性ITP患者在HD-DXM治疗前后外周血循环中不同DC亚群数量的变化及DC表面协同刺激分子的表达;体外诱导分化外周血单核细胞来源的DC,观察CD4+ T细胞、DC、自体血小板或同源异基因正常人血小板混合培养时的CD4+ T淋巴细胞增殖程度。结果发现,慢性ITP患者外周血循环中pDC和mDC的绝对数量与正常对照组比较均没有显著性差异(P﹥0.05和P﹥0.05);HD-DXM冲击治疗4天后,pDC绝对数较治疗前减少达75.5%( P﹤0.0001);而mDC绝对数较治疗前虽增加了24.3%( P<0.05),但mDC上CD11c表达的平均荧光强度(MFI)从治疗前340±30降至199±21(P﹤0.0001)。慢性ITP患者外周血循环中DC表面协同刺激分子CD40 (P﹥0.05)、CD80(P﹥0.05)、CD86(P﹥0.05)表达阳性率与正常对照组均无显著性差异;其中CD86表达的MFI在慢性ITP患者中明显高于对照组(P﹤0.05),而CD40 (P﹥0.05)及CD80 (P﹥0.05)表达的MFI均无差异。当正常人外周血单核细胞来源的DC、自身血小板及CD4+ T淋巴细胞混合培养6天后, CD4+ T淋巴细胞不发生增殖反应;而慢性ITP患者的CD4+ T淋巴细胞则发生明显的增殖(P﹤0.05);同时当外周血单核细胞来源的DC及自身CD4+ T淋巴细胞与同源异基因血小板混合培养6天后,慢性ITP患者的CD4+ T淋巴细胞的增殖反应强度显著高于正常对照组(P﹤0.05)。
本实验结果表明DC参与了ITP的免疫发病机制,减少外周血循环中pDC数量、干预mDC的成熟可能是HD-DXM有效发挥其免疫抑制作用的机制之一。有文献报道,在一些自身免疫病中,能通过耐受性DC诱导自身反应性T细胞免疫耐受达到阻止疾病发展的目的,在难治性ITP的治疗中,是否也能通过应用耐受性DC达到治疗ITP的目的呢?值得进一步深入研究。
4.ITP患者外周血B细胞数量及表面协同刺激分子的表达。
在自身免疫过程中,B细胞的活化大多需要T细胞(Th)的辅助,同时B细胞特别是活化的B细胞亦可作为抗原递呈细胞(APC)直接活化T细胞。为探讨B细胞在ITP发病机制中的作用,本研究采用流式细胞术检测ITP患者外周血循环中CD19+ B细胞数量、CD19+ B细胞表面协同刺激分子的表达及血小板相关免疫球蛋白G(PAIgG)。结果发现,ITP患者包括未治疗组及激素治疗组与正常对照组比较,CD19+ B细胞比例、CD19+ CD40+/CD19+阳性率及MFI,均无显著性差异(P﹥0.05);未治疗组与激素治疗组间亦无明显差异(P﹥0.05)。未治疗组及激素治疗组分别与正常对照组比较,CD19+ CD80+/CD19+阳性率及MFI均无显著性差异(P﹥0.05和P﹥0.05),而激素治疗组CD19+ CD80+/CD19+阳性率低于未治疗组(P﹤0.05),MFI无差异(P﹥0.05)。ITP未治疗组与正常对照组比较,CD19+ CD86+/CD19+阳性率高于对照组(P﹤0.05),MFI无差异;ITP激素治疗组CD19+ CD86+/CD19+阳性率及MFI与对照组比较无差异(P﹥0.05);而激素治疗组CD19+ CD86+/CD19+阳性率低于未治疗组(P﹤0.05),MFI无差异(P﹥0.05)。未治疗组18例ITP患者中有12例PAIgG异常增高(PAIgG正常值为35.6±7.4%),均值为(57.3±12.1)%;在激素治疗组15例ITP患者中仅有4例PAIgG异常增高,均值为(46.1±9.6)%;异常增高的PAIgG的表达与外周血B细胞数量及其表面协同刺激分子的表达均无相关性(P﹥0.05)。
本实验结果表明,在ITP患者中存在B细胞协同刺激分子CD86的表达异常,糖皮质激素能通过有效降低B细胞表面CD80和CD86的表达而发挥免疫抑制作用。
Idiopathic thrombocytopenic purpura (ITP) belongs to an autoimmune disease. Its immunologic abnormality is related to platelet-associated immunoglobulin (PAIg), which results in the over-destruction of platelet in reticuloendothelial system (RES) or complement system. Patients with ITP manifest thrombocytopenia and clinical bleeding symptom. With the development of research, it has been discovered that the cellular immune dysfunction especially activated self-reactive T cells in response to glucoprotein on platelet membrane might play critical roles in the pathogenesis of ITP, which results in autoimmune intolerance. Both a new type of regulatory T cells-CD4+CD25+ T cells that was firstly reported by Sakaguchi et al. in 1956 and dendritic cells (DCs) play important roles in the maintenance of immunological self-tolerance. Natural CD4+CD25+ regulatory T cells (Treg) are generated in the thymus, which suppress the activation of self-reactive T cells and maintain autoimmune tolerance. Known as the strongest antigen-presenting cell (APC), DCs stimulate immune response. Mature DCs present antigen and have an increased T-cell stimulatory capacity, whereas immature DCs are considered to induce immune tolerance. Therefore, DCs also regulate immune response. The activation of B cells depends on T cells help. Meanwhile, B cells can directly activate T cells as APC. B cells also play vital roles in the activation of self-reactive T cells.
In our study, basic and clinical study was carried out to explore CD4+CD25+ Treg cells, DC, and B cells effects on the immune pathogenesis of ITP. We seek to clarify the immune mechanism of CD4+CD25+ Treg cells in ITP and the relevantly immune effect of high dose dexamethasome (HD-DXM) by investigating the circulating number and the immunologic suppression of CD4+CD25+ Treg cells, the changes of CD4+CD25+ Treg cells number and anti-inflammatory cytokine IL-10 or TGF-β1 after the treatment with HD-DXM in ITP patients; To clarify the immune mechanism of DCs by investigating the expression of co-stimulatory molecules on circulating DCs, the changes of DCs subsets number after treatment with HD-DXM in ITP patients, and the proliferation of CD4+ T cells; To clarify the immune mechanism of B cells by investigating the circulating number of B cells and the expression of co-stimulatory molecules on B cells.
1. The immune dysfunction of CD4+CD25+ Treg cells in patients with ITP.
To study the effect of CD4+CD25+ Treg cells in the pathogenesis of ITP, we used flow cytometry and found the proportion of CD4+CD25+ T cells in the peripheral blood of patients with ITP [(15.64±5.82)%] was significantly higher than that in normal control group ([9.30±3.95)%(]P﹤0.001); There was no significantly difference in the percentage of CD4+CD25high T cells between ITP patients and control [(1.53±0.66)% versus (1.36±0.55)%( P=0.317)]; But the number of CD4+ FOXP3+ T cells ([1.82±1.42)% versus (3.90±1.37)% (P﹤0.001)]and CD4+CD25+ FOXP3+ T cells[(1.25±0.94)% versus (2.65±0.92)% ( P﹤0.001)] in patients was significantly lower than that in control group. The expression of Foxp3 mRNA in patients was reduced (P﹤0.001) by using real-time PCR. The suppressive activity of CD4+CD25 high T cells was lower than that of healthy controls (P﹤0.001).
The results implied that patients with ITP show a decreased circulating number and reduced suppressive activity of CD4+CD25+ Treg cells.
2. The changes of CD4+CD25+ Treg cells after the treatment with HD-DXM in ITP patients and the relationship between CD4+CD25+ Treg cells and anti-inflammatory cytokine IL-10 or TGF-β1.
To study the relationship between CD4+CD25+ Treg cells and anti-inflammatory cytokine IL-10 or TGF-β1 and the effects of high dose dexamethasome (HD-DXM) on them contained CD4+CD25+ Treg cells, IL-10 and TGF-β1. A good initial response to HD-DXM occurred in 24 of the 26 patients with chronic ITP (92.3%): the mean platelet count was (84.9±30.4)×109/L [range, (20～150)×109/L]] one week after the initiation of treatment with HD-DXM. We used flow cytometry and found the number of CD4+ CD25+ T cells (P﹤0.0001), CD4+CD25high T cells ( P﹤0.0001) , and CD4+FOXP3+ T cells ( P﹤0.0001) in patients with chronic ITP was significantly increased after the treatment with HD-DXM. Meanwhile the TGF-β1 and IL-10 in the serum of chronic ITP patients were also measured by Enzyme-linked immunosorbent assay (ELISA), the results showed the expression level of TGF-β1 was lower than that of healthy controls([305.90±202.35)pg/ml versus (1479.00±182.70)pg/ml (P﹤0.0001)] and HD-DXM could significantly increase TGF-β1 levels in chronic ITP patients(1414.70±270.35) pg/ml,(P﹤0.0001). There was no significantly difference in the expression level of IL-10 between chronic ITP patients and control [(441.10±239.26)pg/ml versus (349.88±21.27)pg/ml ( P﹥0.05)] and there was no remarkably changes of IL-10 in patients after HD-DXM treatment (395.95±149.95) pg/ml,(P﹥0.05). Using the methods of real-time PCR examine the expression of gene Foxp3, IL-10, TGF-β1 in the CD4+ T cells . Both expressions of Foxp3 and TGF-β1 gene were lower than that of controls (P﹤0.05 and P﹤0.05); HD-DXM administration significantly increased the expressions of Foxp3 and TGF-β1 gene (P﹤0.05 and P﹤0.0001)that were even higher than that of controls(P﹤0.05 and P﹤0.05); There was a positive correlation between the expressions of Foxp3 and TGF-β1 gene after treatment with HD-DXM (r =0.403, P=0.041). The expression rate of Foxp3 protein in spleen T cells was observed using immunohistochemistry. The Foxp3 protein level in refractory ITP patients who received DXM therapy before spleen surgery was higher as compared with that in splenic rupture group; whereas lower than that in HBV hepatits group. Together, the results suggested that both CD4+CD25+ Treg cells and TGF-β1 play roles in the pathogenesis of ITP; Glucocorticosteroid may inhibit immune response by affecting CD4+CD25+ Treg cells and TGF-β1.
3. The changes of circulating DC number after the treatment with HD-DXM and the expression level of co-stimulatory molecule on DC in ITP patients.
To study the effect of DCs in the pathogenesis of ITP, flow cytometry was used to analyze the absolute number of circulating DCs subsets including mDCs and pDCs in 26 chronic ITP patients, meanwhile the co-stimulatory molecule on total DC. No statistically significant difference was found between patients and controls in the absolute number of circulating mDCs or pDCs (P﹥0.05 and P﹥0.05). HD-DXM therapy suppressed pDCs by 75.5% (P<0.0001), whereas stimulated mDCs by 24.3% (P<0.05); Interestingly, the median fluorescence intensity (MFI) of CD11c in mDCs was remarkable reduced from 340±30 to 199±21 (P<0.0001). The MFI of CD86 on DCs was higher in ITP patients than in healthy controls (P<0.05), while the proportion of CD86, CD40, CD80 and the MFI of CD40, CD80 in ITP patients were normal (P﹥0.05). Monocyte-derived DCs were propagated in granulocyte-macrophage colony stimulating factor (GM-CSF) combined with IL-4. Briefly, autologous CD4+ T cells and monocyte-derived DCs were cultured with autologous or allogeneic normal fresh platelets in 96-well plates. After 6 days of incubation, 3H-TdR was used to assay the proliferation of CD4+ T cells. In ITP patients DCs, cultured with autologous or allogeneic platelets, were highly efficient in stimulating autologous CD4+ T cells proliferaton as compared to DCs derived from health donors(P﹤0.05 and P﹤0.05). Our results indicated that DCs also play an important role in the pathogenesis of ITP , and DXM may affect immune response by decreasing the number of circulating pDCs and inhibiting the maturation of mDCs.
4.The circulating number of B cells and the expression level of co-stimulatory molecule on B cells in ITP patients.
To study the effect of B cells in the pathogenesis of ITP, flow cytometry was used to analyze the proportion of circulating CD19+ B cells, the expression level of co-stimulatory molecule on CD19+ B cells in ITP patients, the expression of PAIgG. The proportion of CD86 on CD19+ B cells was higher in untreated group of ITP patients than in healthy controls (P<0.05); while the proportion of CD19+ cells , CD19+ CD40+ cells, CD19+CD80+ cells and the MFI of CD86, CD40, CD80 in untreated group of ITP patients were normal (P﹥0.05). The proportion of CD80 and CD86 on CD19+ B cells in treated group of ITP patients was lower than that in untreated group (P<0.05 and P<0.05). The high level of PAIgG in 12 of the 18 untreated patients with ITP and 4 of the 15 treated patients with ITP was detected. No association was observed between high level PAIgG and the expression level of CD40, CD80, CD86 on CD19+ B cells.
1.特发性血小板减少性紫癜(ITP)患者中CD4+CD25+调节性T细胞的免疫异常为探讨CD4+CD25+ Treg细胞在ITP发病机制中的作用,本研究运用了流式细胞术检测32例ITP患者外周血中CD4+CD25+ T细胞、CD4+CD25high T细胞、CD4+ FOXP3+T细胞及CD4+CD25+FOXP3+ T细胞的数量;实时荧光定量PCR检测ITP患者外周血单个核细胞(PBMC)Foxp3 mRNA的表达水平;将ITP患者CD4+CD25high T细胞与自身CD4+CD25- T细胞混合培养,检测CD4+CD25high T细胞的免疫抑制功能。结果发现,ITP患者外周血中CD4+CD25+T细胞约占CD4+T细胞(15.64±5.82)%,明显高于正常对照组(9.30±3.95)%(P﹤0.001),CD4+CD25high T细胞比例为(1.53±0.66)%,与对照组比较(1.36±0.55)%无显著性差异(P>0.05);CD4+ FOXP3+T细胞和CD4+CD25+ FOXP3+ T细胞分别为(1.82±1.42)%和(1.25±0.94)%,均明显低于对照组(3.90±1.37)%(P﹤0.001)和(2.65±0.92)%(P﹤0.001)。ITP患者外周血PBMC中FOXP3 mRNA表达水平较对照组明显下调(P﹤0.001),CD4+CD25high T细胞的免疫抑制活性较对照组明显减弱(p<0.001)。
由此表明,在ITP的免疫发病机制中,存在CD4+CD25+ Treg细胞数量的减少和免疫抑制功能的减弱,可能致使机体内血小板自身反应性T细胞的活化不能被有效的抑制而诱发、维持和加重了患者的自身免疫。随着CD4+CD25+ Treg细胞免疫调节(抑制)功能的深入研究,将会为临床治疗ITP特别是难治性ITP提供一个新的免疫治疗途径。
2.慢性ITP患者大剂量地塞米松治疗前后CD4+CD25+调节性T的变化及与抗炎细胞因子的关系
前期研究已证实在ITP患者中存在CD4+CD25+ Treg细胞的免疫异常,为证实在ITP患者中CD4+CD25+ Treg细胞与抗炎细胞因子IL-10、TGF-β1是否有一定的关联及大剂量地塞米松(HD-DXM)对CD4+CD25+ Treg细胞及IL-10、TGF-β1的影响,本研究采用流式细胞术检测了26例慢性ITP患者在HD-DXM治疗前后CD4+CD25+ T细胞、CD4+CD25high T细胞、CD4+ FOXP3+ T细胞的数量变化;酶联免疫吸附实验(ELISA)检测ITP患者在HD-DXM治疗前后血清中IL-10及TGF-β1的浓度变化;实时荧光定量PCR检测ITP患者HD-DXM治疗前后外周血CD4+ T细胞中FOXP3、IL-10、TGF-βmRNA的表达水平;免疫组化检测DXM治疗后难治性ITP患者脾脏组织中FOXP3基因的表达。结果发现,26例慢性ITP患者在确诊后予HD-DXM治疗,初始反应率达92.3%。所有患者在初始治疗后一周平均血小板数达(84.9±30.4)×109/L,范围(20～150)×109/L。HD-DXM治疗后,慢性ITP患者外周血中CD4+FOXP3+ T细胞(P﹤0.0001)、CD4+CD25+ T细胞(P﹤0.0001)、CD4+CD25high T细胞(P﹤0.0001)数量较治疗前均显著增加。慢性ITP患者治疗前血清IL-10浓度为(441.10±239.26) pg/ml,正常对照组为(349.88±21.27) pg/ml,两者比较无显著性差异(P﹥0.05);HD-DXM治疗后,IL-10浓度为(395.95±149.95) pg/ml,与治疗前比较无显著性差异(P﹥0.05);慢性ITP患者治疗前血清TGF-β浓度为(305.90±202.35) pg/ml,显著低于正常对照组(1479.00±182.70) pg/ml,(P﹤0.0001);HD-DXM治疗后,TGF-β浓度较治疗前明显上升(1414.70±270.35) pg/ml,(P﹤0.0001)。HD-DXM治疗后,慢性ITP患者外周血CD4+ T细胞FOXP3+基因mRNA表达水平较治疗前明显上升(P﹤0.05),并高于对照组(P﹤0.05);IL-10基因在HD-DXM治疗前后mRNA表达水平无变化,(P﹥0.05),与对照组比较亦均无显著性差异(P﹥0.05);治疗前, TGF-β1基因mRNA表达水平明显低于正常对照组(P﹤0.05),HD-DXM治疗后TGF-β1基因mRNA表达水平明显上升(P﹤0.0001);并高于对照组(P﹤0.05);HD-DXM治疗后,TGF-β1基因mRNA表达水平与FOXP3+基因呈正相关(r =0.403, P=0.041)。蛋白Foxp3在术前应用DXM治疗的慢性难治性ITP患者脾脏组织淋巴细胞内的表达明显强于正常脾破裂对照组但弱于乙肝患者组。
本实验结果表明在ITP免疫机制异常中,CD4+CD25+ Treg及细胞因子TGF-β1发挥着一定的作用;而糖皮质激素可通过促进CD4+CD25+ Treg及TGF-β1的表达发挥免疫抑制效应。
3.慢性ITP患者中树突状细胞(DC)免疫异常及大剂量地塞米松治疗前后DC亚群数量的变化
DC被视为机体内重要的免疫调节细胞,在维持自身免疫耐受中发挥重要作用。为探讨DC在ITP发病机制中的作用,本研究采用流式细胞术检测26例慢性ITP患者在HD-DXM治疗前后外周血循环中不同DC亚群数量的变化及DC表面协同刺激分子的表达;体外诱导分化外周血单核细胞来源的DC,观察CD4+ T细胞、DC、自体血小板或同源异基因正常人血小板混合培养时的CD4+ T淋巴细胞增殖程度。结果发现,慢性ITP患者外周血循环中pDC和mDC的绝对数量与正常对照组比较均没有显著性差异(P﹥0.05和P﹥0.05);HD-DXM冲击治疗4天后,pDC绝对数较治疗前减少达75.5%( P﹤0.0001);而mDC绝对数较治疗前虽增加了24.3%( P<0.05),但mDC上CD11c表达的平均荧光强度(MFI)从治疗前340±30降至199±21(P﹤0.0001)。慢性ITP患者外周血循环中DC表面协同刺激分子CD40 (P﹥0.05)、CD80(P﹥0.05)、CD86(P﹥0.05)表达阳性率与正常对照组均无显著性差异;其中CD86表达的MFI在慢性ITP患者中明显高于对照组(P﹤0.05),而CD40 (P﹥0.05)及CD80 (P﹥0.05)表达的MFI均无差异。当正常人外周血单核细胞来源的DC、自身血小板及CD4+ T淋巴细胞混合培养6天后, CD4+ T淋巴细胞不发生增殖反应;而慢性ITP患者的CD4+ T淋巴细胞则发生明显的增殖(P﹤0.05);同时当外周血单核细胞来源的DC及自身CD4+ T淋巴细胞与同源异基因血小板混合培养6天后,慢性ITP患者的CD4+ T淋巴细胞的增殖反应强度显著高于正常对照组(P﹤0.05)。
本实验结果表明DC参与了ITP的免疫发病机制,减少外周血循环中pDC数量、干预mDC的成熟可能是HD-DXM有效发挥其免疫抑制作用的机制之一。有文献报道,在一些自身免疫病中,能通过耐受性DC诱导自身反应性T细胞免疫耐受达到阻止疾病发展的目的,在难治性ITP的治疗中,是否也能通过应用耐受性DC达到治疗ITP的目的呢?值得进一步深入研究。
4.ITP患者外周血B细胞数量及表面协同刺激分子的表达。
在自身免疫过程中,B细胞的活化大多需要T细胞(Th)的辅助,同时B细胞特别是活化的B细胞亦可作为抗原递呈细胞(APC)直接活化T细胞。为探讨B细胞在ITP发病机制中的作用,本研究采用流式细胞术检测ITP患者外周血循环中CD19+ B细胞数量、CD19+ B细胞表面协同刺激分子的表达及血小板相关免疫球蛋白G(PAIgG)。结果发现,ITP患者包括未治疗组及激素治疗组与正常对照组比较,CD19+ B细胞比例、CD19+ CD40+/CD19+阳性率及MFI,均无显著性差异(P﹥0.05);未治疗组与激素治疗组间亦无明显差异(P﹥0.05)。未治疗组及激素治疗组分别与正常对照组比较,CD19+ CD80+/CD19+阳性率及MFI均无显著性差异(P﹥0.05和P﹥0.05),而激素治疗组CD19+ CD80+/CD19+阳性率低于未治疗组(P﹤0.05),MFI无差异(P﹥0.05)。ITP未治疗组与正常对照组比较,CD19+ CD86+/CD19+阳性率高于对照组(P﹤0.05),MFI无差异;ITP激素治疗组CD19+ CD86+/CD19+阳性率及MFI与对照组比较无差异(P﹥0.05);而激素治疗组CD19+ CD86+/CD19+阳性率低于未治疗组(P﹤0.05),MFI无差异(P﹥0.05)。未治疗组18例ITP患者中有12例PAIgG异常增高(PAIgG正常值为35.6±7.4%),均值为(57.3±12.1)%;在激素治疗组15例ITP患者中仅有4例PAIgG异常增高,均值为(46.1±9.6)%;异常增高的PAIgG的表达与外周血B细胞数量及其表面协同刺激分子的表达均无相关性(P﹥0.05)。
本实验结果表明,在ITP患者中存在B细胞协同刺激分子CD86的表达异常,糖皮质激素能通过有效降低B细胞表面CD80和CD86的表达而发挥免疫抑制作用。
Idiopathic thrombocytopenic purpura (ITP) belongs to an autoimmune disease. Its immunologic abnormality is related to platelet-associated immunoglobulin (PAIg), which results in the over-destruction of platelet in reticuloendothelial system (RES) or complement system. Patients with ITP manifest thrombocytopenia and clinical bleeding symptom. With the development of research, it has been discovered that the cellular immune dysfunction especially activated self-reactive T cells in response to glucoprotein on platelet membrane might play critical roles in the pathogenesis of ITP, which results in autoimmune intolerance. Both a new type of regulatory T cells-CD4+CD25+ T cells that was firstly reported by Sakaguchi et al. in 1956 and dendritic cells (DCs) play important roles in the maintenance of immunological self-tolerance. Natural CD4+CD25+ regulatory T cells (Treg) are generated in the thymus, which suppress the activation of self-reactive T cells and maintain autoimmune tolerance. Known as the strongest antigen-presenting cell (APC), DCs stimulate immune response. Mature DCs present antigen and have an increased T-cell stimulatory capacity, whereas immature DCs are considered to induce immune tolerance. Therefore, DCs also regulate immune response. The activation of B cells depends on T cells help. Meanwhile, B cells can directly activate T cells as APC. B cells also play vital roles in the activation of self-reactive T cells.
In our study, basic and clinical study was carried out to explore CD4+CD25+ Treg cells, DC, and B cells effects on the immune pathogenesis of ITP. We seek to clarify the immune mechanism of CD4+CD25+ Treg cells in ITP and the relevantly immune effect of high dose dexamethasome (HD-DXM) by investigating the circulating number and the immunologic suppression of CD4+CD25+ Treg cells, the changes of CD4+CD25+ Treg cells number and anti-inflammatory cytokine IL-10 or TGF-β1 after the treatment with HD-DXM in ITP patients; To clarify the immune mechanism of DCs by investigating the expression of co-stimulatory molecules on circulating DCs, the changes of DCs subsets number after treatment with HD-DXM in ITP patients, and the proliferation of CD4+ T cells; To clarify the immune mechanism of B cells by investigating the circulating number of B cells and the expression of co-stimulatory molecules on B cells.
1. The immune dysfunction of CD4+CD25+ Treg cells in patients with ITP.
To study the effect of CD4+CD25+ Treg cells in the pathogenesis of ITP, we used flow cytometry and found the proportion of CD4+CD25+ T cells in the peripheral blood of patients with ITP [(15.64±5.82)%] was significantly higher than that in normal control group ([9.30±3.95)%(]P﹤0.001); There was no significantly difference in the percentage of CD4+CD25high T cells between ITP patients and control [(1.53±0.66)% versus (1.36±0.55)%( P=0.317)]; But the number of CD4+ FOXP3+ T cells ([1.82±1.42)% versus (3.90±1.37)% (P﹤0.001)]and CD4+CD25+ FOXP3+ T cells[(1.25±0.94)% versus (2.65±0.92)% ( P﹤0.001)] in patients was significantly lower than that in control group. The expression of Foxp3 mRNA in patients was reduced (P﹤0.001) by using real-time PCR. The suppressive activity of CD4+CD25 high T cells was lower than that of healthy controls (P﹤0.001).
The results implied that patients with ITP show a decreased circulating number and reduced suppressive activity of CD4+CD25+ Treg cells.
2. The changes of CD4+CD25+ Treg cells after the treatment with HD-DXM in ITP patients and the relationship between CD4+CD25+ Treg cells and anti-inflammatory cytokine IL-10 or TGF-β1.
To study the relationship between CD4+CD25+ Treg cells and anti-inflammatory cytokine IL-10 or TGF-β1 and the effects of high dose dexamethasome (HD-DXM) on them contained CD4+CD25+ Treg cells, IL-10 and TGF-β1. A good initial response to HD-DXM occurred in 24 of the 26 patients with chronic ITP (92.3%): the mean platelet count was (84.9±30.4)×109/L [range, (20～150)×109/L]] one week after the initiation of treatment with HD-DXM. We used flow cytometry and found the number of CD4+ CD25+ T cells (P﹤0.0001), CD4+CD25high T cells ( P﹤0.0001) , and CD4+FOXP3+ T cells ( P﹤0.0001) in patients with chronic ITP was significantly increased after the treatment with HD-DXM. Meanwhile the TGF-β1 and IL-10 in the serum of chronic ITP patients were also measured by Enzyme-linked immunosorbent assay (ELISA), the results showed the expression level of TGF-β1 was lower than that of healthy controls([305.90±202.35)pg/ml versus (1479.00±182.70)pg/ml (P﹤0.0001)] and HD-DXM could significantly increase TGF-β1 levels in chronic ITP patients(1414.70±270.35) pg/ml,(P﹤0.0001). There was no significantly difference in the expression level of IL-10 between chronic ITP patients and control [(441.10±239.26)pg/ml versus (349.88±21.27)pg/ml ( P﹥0.05)] and there was no remarkably changes of IL-10 in patients after HD-DXM treatment (395.95±149.95) pg/ml,(P﹥0.05). Using the methods of real-time PCR examine the expression of gene Foxp3, IL-10, TGF-β1 in the CD4+ T cells . Both expressions of Foxp3 and TGF-β1 gene were lower than that of controls (P﹤0.05 and P﹤0.05); HD-DXM administration significantly increased the expressions of Foxp3 and TGF-β1 gene (P﹤0.05 and P﹤0.0001)that were even higher than that of controls(P﹤0.05 and P﹤0.05); There was a positive correlation between the expressions of Foxp3 and TGF-β1 gene after treatment with HD-DXM (r =0.403, P=0.041). The expression rate of Foxp3 protein in spleen T cells was observed using immunohistochemistry. The Foxp3 protein level in refractory ITP patients who received DXM therapy before spleen surgery was higher as compared with that in splenic rupture group; whereas lower than that in HBV hepatits group. Together, the results suggested that both CD4+CD25+ Treg cells and TGF-β1 play roles in the pathogenesis of ITP; Glucocorticosteroid may inhibit immune response by affecting CD4+CD25+ Treg cells and TGF-β1.
3. The changes of circulating DC number after the treatment with HD-DXM and the expression level of co-stimulatory molecule on DC in ITP patients.
To study the effect of DCs in the pathogenesis of ITP, flow cytometry was used to analyze the absolute number of circulating DCs subsets including mDCs and pDCs in 26 chronic ITP patients, meanwhile the co-stimulatory molecule on total DC. No statistically significant difference was found between patients and controls in the absolute number of circulating mDCs or pDCs (P﹥0.05 and P﹥0.05). HD-DXM therapy suppressed pDCs by 75.5% (P<0.0001), whereas stimulated mDCs by 24.3% (P<0.05); Interestingly, the median fluorescence intensity (MFI) of CD11c in mDCs was remarkable reduced from 340±30 to 199±21 (P<0.0001). The MFI of CD86 on DCs was higher in ITP patients than in healthy controls (P<0.05), while the proportion of CD86, CD40, CD80 and the MFI of CD40, CD80 in ITP patients were normal (P﹥0.05). Monocyte-derived DCs were propagated in granulocyte-macrophage colony stimulating factor (GM-CSF) combined with IL-4. Briefly, autologous CD4+ T cells and monocyte-derived DCs were cultured with autologous or allogeneic normal fresh platelets in 96-well plates. After 6 days of incubation, 3H-TdR was used to assay the proliferation of CD4+ T cells. In ITP patients DCs, cultured with autologous or allogeneic platelets, were highly efficient in stimulating autologous CD4+ T cells proliferaton as compared to DCs derived from health donors(P﹤0.05 and P﹤0.05). Our results indicated that DCs also play an important role in the pathogenesis of ITP , and DXM may affect immune response by decreasing the number of circulating pDCs and inhibiting the maturation of mDCs.
4.The circulating number of B cells and the expression level of co-stimulatory molecule on B cells in ITP patients.
To study the effect of B cells in the pathogenesis of ITP, flow cytometry was used to analyze the proportion of circulating CD19+ B cells, the expression level of co-stimulatory molecule on CD19+ B cells in ITP patients, the expression of PAIgG. The proportion of CD86 on CD19+ B cells was higher in untreated group of ITP patients than in healthy controls (P<0.05); while the proportion of CD19+ cells , CD19+ CD40+ cells, CD19+CD80+ cells and the MFI of CD86, CD40, CD80 in untreated group of ITP patients were normal (P﹥0.05). The proportion of CD80 and CD86 on CD19+ B cells in treated group of ITP patients was lower than that in untreated group (P<0.05 and P<0.05). The high level of PAIgG in 12 of the 18 untreated patients with ITP and 4 of the 15 treated patients with ITP was detected. No association was observed between high level PAIgG and the expression level of CD40, CD80, CD86 on CD19+ B cells.
引文
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