重症肌无力患者外周血调节性T细胞及相关可溶性因子在其发病机制中的研究
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摘要
重症肌无力(myasthenia gravis，MG)是乙酰胆碱受体抗体(acetylcholinereceptor antibodies，AChR-ab)介导的、细胞免疫依赖及补体参与的神经肌肉突触传递障碍的自身免疫性疾病。目前认为，致病性自身抗体的产生主要依赖于T辅助(Th)细胞产生的细胞因子，并且疾病进程中同时会伴有补体水平下降，存在大量的补体消耗。总之，MG是一种多种免疫相关因素参与的自身免疫性疾病，免疫平衡的破坏在其发病机制起到重要作用。近来外周免疫机制重要组成成分，调节性T细胞尤其是CD4~+CD25~+Treg细胞在各类自身免疫性疾病中的作用越来越受到重视。很多研究证实MG患者外周血中存在异常比例的CD4~+CD25~+Treg细胞并RMG患者胸腺CD4~+CD25~+Treg细胞功能异常，这些都可能参与疾病的发生与发展。我们着眼于外周血CD4~+CD25~+Treg细胞，特别是高表达CD25的Treg细胞，采用四色流式细胞仪分析这群细胞在MG患者外周血中的水平及其重要标志，特别是与Treg细胞生成和功能密切相关的重要的分子(如Foxp3)的表达情况；此外，我们还分析了MG患者其他各类调节性T细胞的表达以及B细胞的激活情况，及其与CD4~+CD25~(high)Treg细胞之间的关系；采用磁珠分选及氚标胸腺嘧啶脱氧核苷(~3H-TdR)掺入法检测MG患者外周血CD4~+CD25~+Treg细胞的功能情况。鉴于体液免疫MG在其发病机制中所起的重要作用，而调节性T细胞的生成与功能的发挥也离不开细胞因子的作用，我们还采用了酶联免疫吸附(ELISA)法检测了MG患者及正常对照体内的各类细胞因子及粘附分子的分泌，分析其与调节性T细胞之间的联系。
A：重症肌无力患者外周血CD_4~+CD_(25)~(high)调节性T细胞及其动态观察
目的研究重症肌无力(myasthenia gravis，MG)患者外周血CD4~+CD25~(high)T细胞的水平，以及各种治疗方法对其的影响。方法应用四色流式细胞仪检测55例MG患者与33名健康对照外周血CD4~+CD25~(high)T细胞百分率，对其中26例MG患者进行了治疗前后的动态检测。结果MG患者与健康对照外周血CD4~+CD25~(high)T细胞百分率分别为6．2％±3．4与5．2％±1．9，差异无统计学意义(p=0．061)，而21例非手术治疗患者其外周血CD4~+CD25~(high)T细胞百分率的变化与病情评分的变化呈负相关(r=-0．563，p=0．008)。结论这种短期非手术治疗前后的CD4~+CD25~(high)T细胞的变化情况可能与病情相关，但胸腺切除后早期观察的情况有所不同。
B：重症肌无力患者外周血调节性T细胞及B细胞激活因子受体的分析
目的基于外周血CD4~+CD25~(high)T细胞在重症肌无力(MG)患者中没有区别，本文进一步研究MG患者外周血CD4~+CD25~(high)T细胞Fxop3的表达及CD8~+调节性T细胞的水平及B细胞激活因子受体(B cell-activating factor receptor，BAFF-R)在B细胞上的表达情况。方法应用四色流式细胞仪检测61例MG患者与23名健康对照外周血调节性T细胞(CD4~+CD25~(high)Foxp3~+、CD8~+CD28~-、CD8~+CD122~+)以及CD19~+BAFF-R~+细胞的百分率。结果MG组与健康对照组外周血CD4~+CD25~(high)Foxp3~+T细胞的百分率分别为32．1％±16．1与65．2％±14．7，MG组该群调节性T细胞的水平明显低于健康对照(p<0．01)；两组CD8~+CD28~-及CD8~+CD122~+T细胞的水平差异无统计学意义(p>0．05)。此外，MG组外周血CD19~+BAFF-R~+细胞的水平(10．6％±5．6)显著高于健康对照组(5．4％±3．9，p<0．01)。大剂量激素或大剂量激素加丙种球蛋白治疗短期内可使MG组外周血CD4~+CD25~(high)Foxp3~+调节性T细胞的百分率增加(p<0．05)。结论MG患者Foxp3~+的CD4~+CD25~(high)调节性T细胞的减少提示MG患者存在免疫和耐受的失衡，显示了T细胞的自身免疫性。在B细胞方面，MG患者外周血CD19~+B细胞上BAFF-R表达增高，提示其体内B细胞已处于易激活状态。
C：重症肌无力患者外周血调节性CD4~+CD25~+T细胞的功能研究
目的：研究MG患者CD4~+CD25~+T细胞的功能。方法：流式细胞仪分析CD4~+CD25~(high)T细胞表面可能与功能相关的分子，GITR、Neuropilin-1、CTLA-4(CD152)及CD103，与凋亡相关的分子(CD95)；我们还分离出MG患者及正常对照的CD4~+CD25-~T细胞与CD4~+CD25~+T细胞，分组如下：A：CD4~+CD25~-T；B：CD4~+CD25~-T与CD4~+CD25~+T细胞；C：CD4~+CD25~+T细胞。各组单独培养或共培养后，氚标胸腺嘧啶脱氧核苷(~3H-TdR)掺入法检测各组增殖情况，ELISA检测细胞因子的分泌。结果：1)MG患者外周血CD4~+CD25~(high)Neuropilin-1~+T及CD4~+CD25~(high)CD103~+T细胞百分率明显低于正常对照；2)细胞增殖情况：MG患者B组CPM计数高于A组(p=0．0158)与C组(p=0．0676)，而正常对照各组之间未见差异。3)培养后细胞因子TGF-β、IL-10及sICAM-1的分泌结果：sICAM-1：A、B组：MG患者高于正常对照；C组两者未见显著差异。IL-10：A组、B组未见差异；而C组正常对照水平高于MG患者。TGF-β各组均未见显著差异。结论：与正常对照相比，MG患者外周血CD4~+CD25~+T细胞表面部分功能相关分子的表达降低，其抑制功能及IL-10的分泌也低于正常；此外，MG患者外周血CD4~+CD25~-T细胞可能分泌更多的sICAM-1。
A：重症肌无力患者血清及外周单个核细胞培养上清液各类细胞因子、sCTLA-4及sICAM-1的研究
目的：重症肌无力(MG)是一种T细胞介导的抗体依赖的神经肌肉接头障碍的自身免疫性疾病。许多研究表明T辅助细胞及细胞因子可能参与了MG的发生发展。为此，我们研究了MG患者及正常对照血清及外周单个核细胞(PBMC)培养上清液Th1、Th2、Th3、炎症性细胞因子、sCTLA-4及sICAM-1的分泌情况，分析其在MG中的作用。方法：主要采用应用酶联免疫吸附实验(ELISA)方法检测75例MG患者与50例正常对照血清及其中40例MG患者与20例正常对照PBMC培养48小时后上清液各类可溶性因子的分泌水平。结果：血清及细胞培养上清液中MG患者IL-2，IL-4，IL-10，IL-13，IFN-γ，TNF-β，TGF-β及sCTLA-4的水平与正常对照相比未见显著差异(P>0．05)，MG患者血清中IL-12的水平有所下降(P<0．05)，而sICAM-1水平在血清(p=0．054)及上清液(P<0．01)中均高于正常对照。结论：MG患者血清及培养上清液中各类细胞因子水平与正常对照相比
B：检测重症肌无力患者体内sICAM-1水平的有效方法
目的细胞间粘附分子(ICAM-1)，尤其是可溶性ICAM-1(sICAM-1)是很多疾病的重要相关指标。本实验旨在研究sICAM-1与重症肌无力(MG)的关系。方法应用酶联免疫吸附技术(ELISA)定量检测MG患者及正常对照血清及外周血单个核细胞(PBMC)培养上清液中sICAM-1的水平。结果血清中MG患者与正常对照之间sICAM-1的水平无差异，而PBMC培养上清液中MG患者显著高于正常对照；10例正常对照PBMC培养液中加自身血清的sICAM-1水平显著高于加胎牛血清(FBS)的培养上清液，而IL-10、IL-12、TNF-α的水平均为加FBS高于加自身血清，IL-4则未见变化；加入激素共培养48小时后，7例MG患者sICAM-1水平显著下降，而大剂量激素治疗一月后患者血清水平却未见变化。结论MG患者的PBMC分泌更多的sICAM-1；与血清相比，检测PBMC培养上清液中sICAM-1的水平是一种较好的研究方法；激素可以减少PBMC中sICAM-1的分泌，但是这种改变未见于高剂量激素治疗后MG患者的血清水平。
结论：
1．MG患者存在CD4~+CD25~+Treg细胞的缺陷，并且B细胞处于更易激活的状态。这种T细胞的失衡和B细胞的活化可能有助于MG的发生和发展。
2．MG患者体内血清IL-2、IL-4、IL-10、IL-13、IFN-γ、TNF-α、TGF-β以及sCTLA-4未见显著差异，但其sICAM-1的水平高于正常，并且升高的sICAM-1可能主要由MG患者外周血反应性CD4~+CD25~-T细胞分泌。
3．动态观测MG患者外周血CD4~+CD25~(high)T细胞的表达及其外周血单个核细胞sICAM-1的分泌有助于进一步了解疾病的变化情况。
Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction of skeletal muscles. Acetylcholine receptor (AChR) antibodies are present in sera from 80% to 90% of patients with generalized MG. Both AChR-specific Th1, Th2 cells and cyokines are documented in MG patients, and the involvement of the complement system should cause an increased consumption of complement components such as C3 and C4. But it is not yet clear what are essential for driving the pathogenesis of MG patients. The pathogenesis of MG is involved by many of the relative immune factors. The imbalance of immune system plays an important role in it. As impaired regulatory T cell activity can result in autoimmune diseases, regulatory T cells play a central role in the maintenance of peripheral tolerance. Current evidence proves the disfunction of regulatory T cells (Tregs), especially CD4~+CD25~+Tregs in both circulation and thymus maybe contribute to the development of MG. We used flowcytometry to detect the percentage of circulating CD4~+CD25~(high) Treg and the expression of some molecules which are important for the development and function of the cell, such as Foxp3 and so on; We also detected the level of other regulatory T cells and the activation of B cell in peripheral circulation of MG patients; And we used MACS and ~3H-TdR to isolate the circulating CD4~+CD25~+Treg and CD4~+CD25~-T cell for co-culture and to analyse the function of CD4~+CD25~+Treg in MG patients. Because of the important role of humoral immunity in MG, we used ELISA to detecte the soluble molecules in sera and PBMC cutured supernatants of MG patients and the healthy controls. We also aimed to find the relationship between the CD4~+CD25~(high) Treg and the other regulatory T cells, BAFFR~+ B cells, the soluble molecules in MG.
A: Dynamic study of circulating CD4~+CD25~(high) regulatory T cells inmyasthenia gravis
Objective The aim of this study is to observe the difference of blood CD_4~+CD_(25)~(high) T cells between the MG patients and healthy controls and to investigate the dynamic changes of the population in MG patients with various treatments. Methods The peripheral blood CD_4~+CD_(25)~(high) T cells of 55 MG patients and 33 healthy controls were detected by four-color flow cytometry. And 26 MG patients were detected before and after various treatments. Results There was no significant difference in the percentages of CD_4~+CD_(25)~(high) T cells between MG patients (6.2%±3.4) and the healthy control (5.2%±1.9, p=0.061). After short-term non-operated treatment, the percentages of CD_4~+CD_(25)~(high) T cells were elevated, and had a negative correlation with the decrease of MGFA in 21 MG patients (r=-0.563, P=0.008). Conclusions Our results indicated that changes of the percentage of CD_4~+CD_(25)~(high) T cells in MG patients after short-term treatment might be related to the disease. Except thymectomy, other treatments contributed to the increase of peripheral CD_4~+CD_(25)~(high) regulatory T cells in MG patients after short term treatment.
B: Circulating regulatory T cells and BAFF receptor on B cells in patients withmyasthenia gravis
Objectives To investigate the levels of circulating regulatory T cells and B cell-activating factor receptor (BAFF-R) on B cells from patients with myasthenia gravis (MG). Methods Using flow cytometry, we tested the levels of circulating regulatory T cells ( CD4~+CD25~(high)Foxp3~+, CD8~+CD28~-, CD8~+CD122~+ ) and CD19~+BAFF-R~+ B cells in 61 MG patients and 23 healthy controls. Results The percentages of the circulating CD4~+CD25~(high)Foxp3~+ T cells in MG patients ( 32.1%±16.1 ) were significantly declined compared with healthy controls (65.2%±14.7, p<0.01). The expression of BAFF-R on CD19~+ B cells in MG patients (10.6%±5.6) was higher than that in healthy controls (5.4%±3.9, p<0.01). There were no significant differences in the subpopulations of CD8~+CD28~- and CD8~+CD122~+T cells between MG patients and healthy controls (p>0.05) . Treatment of high-dose corticosteroid with or without additional intravenous IgG elevated the percentages of CD4~+CD25~(high)Foxp3~+ T cells in 20 MG patients (p<0.05). Conclusions These results revealed that decrease of circulating CD4~+CD25~(high)Foxp3~+ regulatory T cells in MG patients might be related to the pathogenesis of MG. An elevated BAFF-R on CD19~+ B cells demonstrated that B cells were more active in MG patients.
C: The function of circulating CD4~+CD25~+ regulatory T cells in myasthenia gravis
Objectives To investigate the function of circulating CD4~+CD25~+ Treg cells in patients with Myasthenia Gravis (MG). Methods By using flow cytometry, we detected the fluorescence intensity of function-relative molecules on circulating CD4~+CD25~(high)T cells (GITR/Neuropilin-l/CTLA-4/CD103) and appotosis-relative molecule (CD95) in 20 MG patients and 16 healthy controls. And we sorted the CD4~+CD25~+T and CD4~+CD25~-T cells from 10 MG patients and 8 healthy controls and cultured in differents experimental groups: Group A: CD4~+CD25~+ T cell alone; Group B: CD4~+CD25~+T plus CD4~+CD25~-T cell; Group C: CD4~+CD25~-T cell alone). The proliferation was detected by ~3H-TdR incorperation and the cytokines were detected by ELISA. Results The pecentages of CD4~+CD25~(high)Neuropilin-1~+ and CD4~+CD25~(high)CD103~+T cells were lower in MG patients than in the healthy controls. The proliferation of the group B was higher than group A and C in MG patients, while there was no difference in healthy controls. The secretion of sICAM-1 in group A and B were higher in MG patients, and the secretion of IL-10 in group C was lower in MG patients. Conclusions Some function-relative molecules in circulating CD4~+CD25~+ T cells in MG patients were decreased. The immunosuppression and the secretion of IL-10 of CD4~+CD25~+ T cells in MG patients were abnormal as well.
A: The level of different kinds of cytokines, sCTLA-4 and sICAM-1from serum and cultured-cell supernatant in myasthenia gravis
Objectives Myasthenia gravis (MG) is caused by T-cell dependent autoantibodies against muscle acetylcholine receptors (AChR) at the neuromuscular junction. Methods Here, we adopted ELISA and flow cytometry techniques to measure the levels of Th1, Th2, Th3 cytokines, inflammatory cytokines, sCTLA-4 and sICAM-1 from 75 MG patients and 50 healthy controls. Results There were no differences in the levels of IL-2, IL-4, IL-10, IL-13, IFN-γ, TNF-α, TGF-βand sCTLA-4 between MG patients and healthy controls in both sera and culture supernatants. The level of IL-12 was decreased in culture supernatants from MG patients, and the level of sICAM-1 was increased in both sera and culture supernatants from MG patients. Conclusions It revealed that there were no significant differences in the levels of different soluble factors between MG patients and healthy controls. While the function of sICAM-1 should be studied further.
B: An effective method of detection sICAM-1 for patients withmyasthenia gravis
Objectives ICAM-1 is a member of the immunoglobulin supergene family and a reliable marker of disease, especially soluble ICAM-1. Methods In this study, we used ELISA to detect the level of sICAM-1 in the serum and PBMC culture supernatant from MG patients and healthy controls. Results There was no significant (p=0.20) differences of the level of sICAM-1 in serum between MG patients and healthy controls. The level of the sICAM-1 in culture supernatant was increased in MG patients (10.4±1.4) compared with healthy controls (4.5±0.1) (after cultured with FBS (p=0.0048) or with serum of themselves (p=0.024) for 48 hours). The level of IL-10, IL-12 and TNF-αwere all elevated cultured with FBS compared with the serum of themselves, except for IL-4 (no difference). The supernatant level of sICAM-1 from seven MG patients co-cultured with corticosteroid was decreased (p=0.0136) after 48 hours, while there was no significant difference of the level of sICAM-1 in serum before and after treated with high dosage of corticosteroid in fourteen MG patients (p=0.329). Conclusions So we drew the conclusion that PBMC secreted more sICAM-1 in MG patients than normal. It was a better method to analyze the supernatant level of sICAM-1 in PBMC cultured with FBS in MG patients. Corticosteroid can down-regulate the secretion of sICAM-1 but it was not seen in the level in serum after treated with high dosage of corticosteroid in MG.
Conclusions:
1. The disfunction of circulating CD4~+CD25~(high) regulatory T cells and more active B cell in MG patients reveal the imbalance of T cells and the activation of B cell might be related to the pathogenesis of MG.
2. There was no difference in the levels of IL-2, IL-4, IL-10, IL-13, IFN-γ, TNF-α, TGF-βand sCTLA-4 in sera and culture supernatants between MG patients and healthy controls. But the level of sICAM-1 was increased in culture supernatants from MG patients and most of the increase of sICAM-1 was likely caused by effctive CD4~+CD25~-T cells in MG patients.
3. Dynamic observation of the circulating CD4~+CD25~(high) T cells and the secretion of sICAM-1 in cultured supernatant of PBMC in MG patients would be helpful for understanding the change of the disease.
A：重症肌无力患者外周血CD_4~+CD_(25)~(high)调节性T细胞及其动态观察
目的研究重症肌无力(myasthenia gravis，MG)患者外周血CD4~+CD25~(high)T细胞的水平，以及各种治疗方法对其的影响。方法应用四色流式细胞仪检测55例MG患者与33名健康对照外周血CD4~+CD25~(high)T细胞百分率，对其中26例MG患者进行了治疗前后的动态检测。结果MG患者与健康对照外周血CD4~+CD25~(high)T细胞百分率分别为6．2％±3．4与5．2％±1．9，差异无统计学意义(p=0．061)，而21例非手术治疗患者其外周血CD4~+CD25~(high)T细胞百分率的变化与病情评分的变化呈负相关(r=-0．563，p=0．008)。结论这种短期非手术治疗前后的CD4~+CD25~(high)T细胞的变化情况可能与病情相关，但胸腺切除后早期观察的情况有所不同。
B：重症肌无力患者外周血调节性T细胞及B细胞激活因子受体的分析
目的基于外周血CD4~+CD25~(high)T细胞在重症肌无力(MG)患者中没有区别，本文进一步研究MG患者外周血CD4~+CD25~(high)T细胞Fxop3的表达及CD8~+调节性T细胞的水平及B细胞激活因子受体(B cell-activating factor receptor，BAFF-R)在B细胞上的表达情况。方法应用四色流式细胞仪检测61例MG患者与23名健康对照外周血调节性T细胞(CD4~+CD25~(high)Foxp3~+、CD8~+CD28~-、CD8~+CD122~+)以及CD19~+BAFF-R~+细胞的百分率。结果MG组与健康对照组外周血CD4~+CD25~(high)Foxp3~+T细胞的百分率分别为32．1％±16．1与65．2％±14．7，MG组该群调节性T细胞的水平明显低于健康对照(p<0．01)；两组CD8~+CD28~-及CD8~+CD122~+T细胞的水平差异无统计学意义(p>0．05)。此外，MG组外周血CD19~+BAFF-R~+细胞的水平(10．6％±5．6)显著高于健康对照组(5．4％±3．9，p<0．01)。大剂量激素或大剂量激素加丙种球蛋白治疗短期内可使MG组外周血CD4~+CD25~(high)Foxp3~+调节性T细胞的百分率增加(p<0．05)。结论MG患者Foxp3~+的CD4~+CD25~(high)调节性T细胞的减少提示MG患者存在免疫和耐受的失衡，显示了T细胞的自身免疫性。在B细胞方面，MG患者外周血CD19~+B细胞上BAFF-R表达增高，提示其体内B细胞已处于易激活状态。
C：重症肌无力患者外周血调节性CD4~+CD25~+T细胞的功能研究
目的：研究MG患者CD4~+CD25~+T细胞的功能。方法：流式细胞仪分析CD4~+CD25~(high)T细胞表面可能与功能相关的分子，GITR、Neuropilin-1、CTLA-4(CD152)及CD103，与凋亡相关的分子(CD95)；我们还分离出MG患者及正常对照的CD4~+CD25-~T细胞与CD4~+CD25~+T细胞，分组如下：A：CD4~+CD25~-T；B：CD4~+CD25~-T与CD4~+CD25~+T细胞；C：CD4~+CD25~+T细胞。各组单独培养或共培养后，氚标胸腺嘧啶脱氧核苷(~3H-TdR)掺入法检测各组增殖情况，ELISA检测细胞因子的分泌。结果：1)MG患者外周血CD4~+CD25~(high)Neuropilin-1~+T及CD4~+CD25~(high)CD103~+T细胞百分率明显低于正常对照；2)细胞增殖情况：MG患者B组CPM计数高于A组(p=0．0158)与C组(p=0．0676)，而正常对照各组之间未见差异。3)培养后细胞因子TGF-β、IL-10及sICAM-1的分泌结果：sICAM-1：A、B组：MG患者高于正常对照；C组两者未见显著差异。IL-10：A组、B组未见差异；而C组正常对照水平高于MG患者。TGF-β各组均未见显著差异。结论：与正常对照相比，MG患者外周血CD4~+CD25~+T细胞表面部分功能相关分子的表达降低，其抑制功能及IL-10的分泌也低于正常；此外，MG患者外周血CD4~+CD25~-T细胞可能分泌更多的sICAM-1。
A：重症肌无力患者血清及外周单个核细胞培养上清液各类细胞因子、sCTLA-4及sICAM-1的研究
目的：重症肌无力(MG)是一种T细胞介导的抗体依赖的神经肌肉接头障碍的自身免疫性疾病。许多研究表明T辅助细胞及细胞因子可能参与了MG的发生发展。为此，我们研究了MG患者及正常对照血清及外周单个核细胞(PBMC)培养上清液Th1、Th2、Th3、炎症性细胞因子、sCTLA-4及sICAM-1的分泌情况，分析其在MG中的作用。方法：主要采用应用酶联免疫吸附实验(ELISA)方法检测75例MG患者与50例正常对照血清及其中40例MG患者与20例正常对照PBMC培养48小时后上清液各类可溶性因子的分泌水平。结果：血清及细胞培养上清液中MG患者IL-2，IL-4，IL-10，IL-13，IFN-γ，TNF-β，TGF-β及sCTLA-4的水平与正常对照相比未见显著差异(P>0．05)，MG患者血清中IL-12的水平有所下降(P<0．05)，而sICAM-1水平在血清(p=0．054)及上清液(P<0．01)中均高于正常对照。结论：MG患者血清及培养上清液中各类细胞因子水平与正常对照相比
B：检测重症肌无力患者体内sICAM-1水平的有效方法
目的细胞间粘附分子(ICAM-1)，尤其是可溶性ICAM-1(sICAM-1)是很多疾病的重要相关指标。本实验旨在研究sICAM-1与重症肌无力(MG)的关系。方法应用酶联免疫吸附技术(ELISA)定量检测MG患者及正常对照血清及外周血单个核细胞(PBMC)培养上清液中sICAM-1的水平。结果血清中MG患者与正常对照之间sICAM-1的水平无差异，而PBMC培养上清液中MG患者显著高于正常对照；10例正常对照PBMC培养液中加自身血清的sICAM-1水平显著高于加胎牛血清(FBS)的培养上清液，而IL-10、IL-12、TNF-α的水平均为加FBS高于加自身血清，IL-4则未见变化；加入激素共培养48小时后，7例MG患者sICAM-1水平显著下降，而大剂量激素治疗一月后患者血清水平却未见变化。结论MG患者的PBMC分泌更多的sICAM-1；与血清相比，检测PBMC培养上清液中sICAM-1的水平是一种较好的研究方法；激素可以减少PBMC中sICAM-1的分泌，但是这种改变未见于高剂量激素治疗后MG患者的血清水平。
结论：
1．MG患者存在CD4~+CD25~+Treg细胞的缺陷，并且B细胞处于更易激活的状态。这种T细胞的失衡和B细胞的活化可能有助于MG的发生和发展。
2．MG患者体内血清IL-2、IL-4、IL-10、IL-13、IFN-γ、TNF-α、TGF-β以及sCTLA-4未见显著差异，但其sICAM-1的水平高于正常，并且升高的sICAM-1可能主要由MG患者外周血反应性CD4~+CD25~-T细胞分泌。
3．动态观测MG患者外周血CD4~+CD25~(high)T细胞的表达及其外周血单个核细胞sICAM-1的分泌有助于进一步了解疾病的变化情况。
Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction of skeletal muscles. Acetylcholine receptor (AChR) antibodies are present in sera from 80% to 90% of patients with generalized MG. Both AChR-specific Th1, Th2 cells and cyokines are documented in MG patients, and the involvement of the complement system should cause an increased consumption of complement components such as C3 and C4. But it is not yet clear what are essential for driving the pathogenesis of MG patients. The pathogenesis of MG is involved by many of the relative immune factors. The imbalance of immune system plays an important role in it. As impaired regulatory T cell activity can result in autoimmune diseases, regulatory T cells play a central role in the maintenance of peripheral tolerance. Current evidence proves the disfunction of regulatory T cells (Tregs), especially CD4~+CD25~+Tregs in both circulation and thymus maybe contribute to the development of MG. We used flowcytometry to detect the percentage of circulating CD4~+CD25~(high) Treg and the expression of some molecules which are important for the development and function of the cell, such as Foxp3 and so on; We also detected the level of other regulatory T cells and the activation of B cell in peripheral circulation of MG patients; And we used MACS and ~3H-TdR to isolate the circulating CD4~+CD25~+Treg and CD4~+CD25~-T cell for co-culture and to analyse the function of CD4~+CD25~+Treg in MG patients. Because of the important role of humoral immunity in MG, we used ELISA to detecte the soluble molecules in sera and PBMC cutured supernatants of MG patients and the healthy controls. We also aimed to find the relationship between the CD4~+CD25~(high) Treg and the other regulatory T cells, BAFFR~+ B cells, the soluble molecules in MG.
A: Dynamic study of circulating CD4~+CD25~(high) regulatory T cells inmyasthenia gravis
Objective The aim of this study is to observe the difference of blood CD_4~+CD_(25)~(high) T cells between the MG patients and healthy controls and to investigate the dynamic changes of the population in MG patients with various treatments. Methods The peripheral blood CD_4~+CD_(25)~(high) T cells of 55 MG patients and 33 healthy controls were detected by four-color flow cytometry. And 26 MG patients were detected before and after various treatments. Results There was no significant difference in the percentages of CD_4~+CD_(25)~(high) T cells between MG patients (6.2%±3.4) and the healthy control (5.2%±1.9, p=0.061). After short-term non-operated treatment, the percentages of CD_4~+CD_(25)~(high) T cells were elevated, and had a negative correlation with the decrease of MGFA in 21 MG patients (r=-0.563, P=0.008). Conclusions Our results indicated that changes of the percentage of CD_4~+CD_(25)~(high) T cells in MG patients after short-term treatment might be related to the disease. Except thymectomy, other treatments contributed to the increase of peripheral CD_4~+CD_(25)~(high) regulatory T cells in MG patients after short term treatment.
B: Circulating regulatory T cells and BAFF receptor on B cells in patients withmyasthenia gravis
Objectives To investigate the levels of circulating regulatory T cells and B cell-activating factor receptor (BAFF-R) on B cells from patients with myasthenia gravis (MG). Methods Using flow cytometry, we tested the levels of circulating regulatory T cells ( CD4~+CD25~(high)Foxp3~+, CD8~+CD28~-, CD8~+CD122~+ ) and CD19~+BAFF-R~+ B cells in 61 MG patients and 23 healthy controls. Results The percentages of the circulating CD4~+CD25~(high)Foxp3~+ T cells in MG patients ( 32.1%±16.1 ) were significantly declined compared with healthy controls (65.2%±14.7, p<0.01). The expression of BAFF-R on CD19~+ B cells in MG patients (10.6%±5.6) was higher than that in healthy controls (5.4%±3.9, p<0.01). There were no significant differences in the subpopulations of CD8~+CD28~- and CD8~+CD122~+T cells between MG patients and healthy controls (p>0.05) . Treatment of high-dose corticosteroid with or without additional intravenous IgG elevated the percentages of CD4~+CD25~(high)Foxp3~+ T cells in 20 MG patients (p<0.05). Conclusions These results revealed that decrease of circulating CD4~+CD25~(high)Foxp3~+ regulatory T cells in MG patients might be related to the pathogenesis of MG. An elevated BAFF-R on CD19~+ B cells demonstrated that B cells were more active in MG patients.
C: The function of circulating CD4~+CD25~+ regulatory T cells in myasthenia gravis
Objectives To investigate the function of circulating CD4~+CD25~+ Treg cells in patients with Myasthenia Gravis (MG). Methods By using flow cytometry, we detected the fluorescence intensity of function-relative molecules on circulating CD4~+CD25~(high)T cells (GITR/Neuropilin-l/CTLA-4/CD103) and appotosis-relative molecule (CD95) in 20 MG patients and 16 healthy controls. And we sorted the CD4~+CD25~+T and CD4~+CD25~-T cells from 10 MG patients and 8 healthy controls and cultured in differents experimental groups: Group A: CD4~+CD25~+ T cell alone; Group B: CD4~+CD25~+T plus CD4~+CD25~-T cell; Group C: CD4~+CD25~-T cell alone). The proliferation was detected by ~3H-TdR incorperation and the cytokines were detected by ELISA. Results The pecentages of CD4~+CD25~(high)Neuropilin-1~+ and CD4~+CD25~(high)CD103~+T cells were lower in MG patients than in the healthy controls. The proliferation of the group B was higher than group A and C in MG patients, while there was no difference in healthy controls. The secretion of sICAM-1 in group A and B were higher in MG patients, and the secretion of IL-10 in group C was lower in MG patients. Conclusions Some function-relative molecules in circulating CD4~+CD25~+ T cells in MG patients were decreased. The immunosuppression and the secretion of IL-10 of CD4~+CD25~+ T cells in MG patients were abnormal as well.
A: The level of different kinds of cytokines, sCTLA-4 and sICAM-1from serum and cultured-cell supernatant in myasthenia gravis
Objectives Myasthenia gravis (MG) is caused by T-cell dependent autoantibodies against muscle acetylcholine receptors (AChR) at the neuromuscular junction. Methods Here, we adopted ELISA and flow cytometry techniques to measure the levels of Th1, Th2, Th3 cytokines, inflammatory cytokines, sCTLA-4 and sICAM-1 from 75 MG patients and 50 healthy controls. Results There were no differences in the levels of IL-2, IL-4, IL-10, IL-13, IFN-γ, TNF-α, TGF-βand sCTLA-4 between MG patients and healthy controls in both sera and culture supernatants. The level of IL-12 was decreased in culture supernatants from MG patients, and the level of sICAM-1 was increased in both sera and culture supernatants from MG patients. Conclusions It revealed that there were no significant differences in the levels of different soluble factors between MG patients and healthy controls. While the function of sICAM-1 should be studied further.
B: An effective method of detection sICAM-1 for patients withmyasthenia gravis
Objectives ICAM-1 is a member of the immunoglobulin supergene family and a reliable marker of disease, especially soluble ICAM-1. Methods In this study, we used ELISA to detect the level of sICAM-1 in the serum and PBMC culture supernatant from MG patients and healthy controls. Results There was no significant (p=0.20) differences of the level of sICAM-1 in serum between MG patients and healthy controls. The level of the sICAM-1 in culture supernatant was increased in MG patients (10.4±1.4) compared with healthy controls (4.5±0.1) (after cultured with FBS (p=0.0048) or with serum of themselves (p=0.024) for 48 hours). The level of IL-10, IL-12 and TNF-αwere all elevated cultured with FBS compared with the serum of themselves, except for IL-4 (no difference). The supernatant level of sICAM-1 from seven MG patients co-cultured with corticosteroid was decreased (p=0.0136) after 48 hours, while there was no significant difference of the level of sICAM-1 in serum before and after treated with high dosage of corticosteroid in fourteen MG patients (p=0.329). Conclusions So we drew the conclusion that PBMC secreted more sICAM-1 in MG patients than normal. It was a better method to analyze the supernatant level of sICAM-1 in PBMC cultured with FBS in MG patients. Corticosteroid can down-regulate the secretion of sICAM-1 but it was not seen in the level in serum after treated with high dosage of corticosteroid in MG.
Conclusions:
1. The disfunction of circulating CD4~+CD25~(high) regulatory T cells and more active B cell in MG patients reveal the imbalance of T cells and the activation of B cell might be related to the pathogenesis of MG.
2. There was no difference in the levels of IL-2, IL-4, IL-10, IL-13, IFN-γ, TNF-α, TGF-βand sCTLA-4 in sera and culture supernatants between MG patients and healthy controls. But the level of sICAM-1 was increased in culture supernatants from MG patients and most of the increase of sICAM-1 was likely caused by effctive CD4~+CD25~-T cells in MG patients.
3. Dynamic observation of the circulating CD4~+CD25~(high) T cells and the secretion of sICAM-1 in cultured supernatant of PBMC in MG patients would be helpful for understanding the change of the disease.
引文
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25. Robert W, Arthur M, Hans L. Altered tumor growth factor β mRNA expression is associated with thymectomy-related clinical remission in myasthenia gravis. Neurol Sci, 1997,151:49-55.
26. Lan RY, Ansari AA, Lian ZX, et al. Regulatory T cells: development, function and role in autoimmunity. Autoimmun Rev, 2005, 4: 351- 363.
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