ITP患者骨髓CD8~+T细胞对巨核细胞生长和血小板生成的影响及机制探讨
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摘要
特发性血小板减少性紫癜(ITP)是临床最为常见的出血性、自身免疫性疾病,约占出血性疾病总数的30%。以外周血小板减少、骨髓巨核细胞正常或增多伴成熟障碍为主要表现。目前,治疗以长期使用肾上腺皮质激素、免疫抑制剂及脾切除等方法为主,容易引发感染、骨髓抑制等,并且约有1/3的患者上述治疗无效,迁延不愈,甚至危及生命。其发病机制复杂,至今尚未完全阐明。随着对ITP发病机制的深入了解,将会为临床提供新的治疗靶点。
长期以来由自身抗体介导的血小板破坏增多被看成是ITP的主要病因,即血小板膜表面糖蛋白(GP)作为自身抗原,特别是GPⅡb/Ⅲa和GPIb/Ⅸ复合物,刺激机体免疫系统产生特异性自身抗体,通过其Fab段与自身抗原结合。这些被自身抗体结合的血小板容易被机体的网状内皮系统所清除,从而导致血小板减少。
由于GP在巨核祖细胞阶段就开始表达在细胞膜上,并且在巨核细胞成熟过程中膜表面的表达量逐步升高,因此ITP患者的巨核细胞生长和血小板生成也应该受到影响。目前普遍认为,除血小板破坏增多外,血小板生成减少也参与ITP的发病。血小板动力学研究及骨髓巨核细胞形态学检查均证实ITP患者的血小板生成减少。最近,Chang等和McMillan等发现ITP患者血浆和纯化的血小板自身抗体可抑制体外巨核细胞生长,导致巨核细胞数量明显降低。充分证明血小板特异性抗原的自身抗体不仅介导外周血小板破坏,还影响骨髓中巨核细胞的生长和发育成熟。
曾认为体液免疫异常在ITP的发病过程中起决定性作用,但随着研究的深入,这一观点已日益受到挑战。近几年细胞免疫功能失调在ITP发病中的作用越来越受重视,尤其是T细胞在其中所起的作用。研究表明ITP的发病机制是以细胞免疫紊乱为中心的,其中Fas/FasL途径介导的T淋巴细胞凋亡障碍是重要原因。Yoshimura等研究发现ITP患者血清中sFas水平明显高于正常人,且其活化的CD8~+T细胞比例显著升高,提示高浓度的sFas抑制了免疫细胞发生Fas/FasL途径介导的激活诱导的细胞死亡(AICD),导致激活的T淋巴细胞因凋亡不足而大量积累于体内,不仅介导体液免疫异常、产生自身抗体,而且体外实验已证实细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL,CD8~+T)能直接参与对ITP患者血小板的杀伤,导致血小板持续破坏。
血小板生成与成熟巨核细胞的凋亡密切相关,巨核细胞凋亡异常即会引起血小板生成障碍。对ITP患者骨髓巨核细胞超微结构的研究表明,患者的巨核细胞存在广泛的异常,这些异常的巨核细胞发生与凋亡相似但不同于凋亡的程序性细胞死亡,是导致血小板生成减少的重要原因。ITP患者骨髓巨核细胞发生异常凋亡的机制还不清楚。
ITP患者骨髓中活化的CD8~+T细胞是否影响巨核系造血,目前尚不清楚。我们通过体外培养巨核细胞观察了ITP患者骨髓CD8~+T细胞对巨核细胞生成数量和质量的影响以及地塞米松对这种影响的逆转并探讨了相关机制,为临床治疗提供依据。
第一部分ITP患者骨髓CD8~+T细胞对体外巨核细胞生成数量的影响
研究目的:研究ITP患者骨髓中活化的CD8~+T细胞对自体巨核细胞生成数量的影响。
研究方法:*抽取15例慢性ITP患者和13例对照10mL骨髓以及30mL外周血。
*分离骨髓和外周血中的单个核细胞。
*检测单个核中CD8~+T细胞的比例。
*用免疫磁珠分离技术分离骨髓单个核细胞中CD8~+T细胞。
*分离外周血中的血小板。
*检测骨髓中CD8~+T细胞对自身血小板的增殖反应。
*将骨髓单个核细胞(MNC),分为MNC组(单个核细胞直接培养)、CD8~-组(单个核细胞去除CD8~+T细胞后培养)、CD8~+组(纯化的CD8~+T细胞1:1加入自体去CD8~+T细胞后的单个核细胞中共同培养)和DEX组(在CD8~+组加入地塞米松)。
*体外无血清半固体培养和液体培养定向扩增巨核细胞。
*流式细胞仪检测CD41a的表达。
*巨核祖细胞集落分析。
研究结果:*ITP患者骨髓中CD8~+T/CD3~+T比例为59.94%±5.15%,明显高于ITP患者外周血中的比例49.33%±3.70%(P＜0.05),也明显高于正常骨髓中CD8~+T/CD3~+T比例38.70%±3.43%(P＜0.05)。
*正常骨髓中的CD8~+T细胞和自身血小板在一起体外培养7天后,不发生增殖反应(cpm:1934±319),而ITP骨髓中的CD8~+T细胞和自身血小板在一起培养后,发生明显的增殖反应(cpm:13526±1037,P＜0.05)。
*正常骨髓CD8~-组形成的CFU-MK和CD41a~+细胞数(61.00±16.00和4.61±0.53)与MNC组(59.23±16.20和4.57±0.58)和CD8~+组(60.77±16.48和4.60±0.57)相比,差异无统计学意义(均P＞0.05),提示正常骨髓中的CD8~+T细胞不影响CFU-MK和CD41a~+细胞数。
*ITP患者骨髓CD8~-组形成的CFU-MK(59.40±12.76)与MNC组(55.87±18.40)和CD8~+组(54.47±21.48)相比,差异无统计学意义(均P＞0.05);CD8~-组CD41a+细胞数(5.01±0.58)低于MNC组(5.75±0.70,P＜0.05),CD8~+组与CD8~-组相比,CD41a~+细胞(6.18±0.79,P＜0.05)增加,提示ITP骨髓中的CD8~+T细胞不影响自身CFU-MK数量,但CD41a~+细胞数随培养体系中CD8~+T细胞数的增多而增多。
*与ITP患者的CD8~+组相比,DEX组产生的CFU-MK为56.93±14.54,P＞0.05,差异无统计学意义;DEX组产生的CD41a~+细胞数减少为(5.26±0.70)×10~5,P＜0.05,差异显著。
结论:ITP患者骨髓中活化的CD8~+T细胞可使巨核细胞数增加,巨核细胞数量增加不是由于巨核祖细胞增殖加速引起的。
第二部分ITP患者骨髓CD8~+T细胞对体外巨核细胞生成质量的影响
研究目的:初步探讨ITP患者骨髓中活化的CD8~+T细胞引起的巨核细胞数量增多与巨核细胞凋亡和血小板生成减少之间的关系。
研究方法:*检测培养体系中生成的血小板。
*CD41a~+细胞DNA倍体分析。
*CD41a~+细胞凋亡检测。
*检测CD41a~+细胞内Bcl-xl的表达。
*检测CD41a~+细胞膜表面Fas的表达。
*检测培养细胞上清液中sFas和sTRAIL水平。
研究结果:*正常骨髓中的CD8~+T细胞不影响血小板生成和巨核细胞凋亡。
*与ITP患者MNC组相比,CD8~-组产生的血小板明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,血小板减少,P＜0.05,差异显著。在ITP患者的三组中,CD8~+组培养体系中含有的CD8~+T细胞最多,所产生的CD41a~+细胞数最多,但产生的血小板最少。ITP患者MNC组血小板产生量与正常MNC组相比,明显减少,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞产生血小板的能力,血小板数随CD8~+T细胞数的增多而减少。
*与ITP患者MNC组相比,CD8~+组产生的CD41a~+细胞中多倍体细胞比例明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,多倍体细胞比例下降,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞的成熟,多倍体巨核细胞数随CD8~+T细胞数的增多而减少。
*与ITP患者MNC组相比,CD8~-组产生的CD41a~+细胞中凋亡细胞比例明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,凋亡细胞比例下降,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞的凋亡,凋亡的巨核细胞数随CD8~+T细胞数的增多而减少。
*与ITP患者MNC组相比,CD8~-组产生的CD41a~+细胞Bcl-xl表达强度明显下降,P＜0.05,差异显著;CD8~+组与CD8~-组相比,Bcl-xl表达强度明显增加,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞内Bcl-xl的表达,Bcl-xl的表达强度随CD8~+T细胞数的增多而增高。
*与ITP患者MNC组相比,CD8~-组产生的CD41a~+细胞Fas表达强度明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,Fas表达强度明显下降,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞膜表面Fas的表达,Fas的表达强度随CD8~+T细胞数的增多而下降。
*与ITP患者MNC直接培养相比,CD8~-组sFas水平明显下降,P＜0.05,差异显著;CD8~+组与CD8~-组相比,sFas水平明显升高,P＜0.05,差异显著;DEX组与CD8~+组相比,sFas水平明显下降,P＜0.05,差异显著。而各组细胞上清液中的sTRAIL水平太低,基本检测不到。提示ITP骨髓中的CD8~+T细胞影响自身骨髓细胞培养上清液中sFas水平,sFas水平随CD8~+T细胞数的增多而升高。
*与ITP患者的CD8~+组相比,DEX组产生的血小板增多为(16.36±6.86)×10~3,P＜0.05,差异显著;多倍体细胞数比例升高为21.37%±4.23%,P＜0.05,差异显著;CD41a~+细胞凋亡比例升高为19.04%±3.41%,P＜0.05,差异显著;Bcl-xl表达强度下降为30.70±5.88,P＜0.05,差异显著;Fas表达强度升高为26.57±4.63,P＜0.05,差异显著。
结论:ITP患者骨髓中活化的CD8~+T细胞抑制自体巨核细胞凋亡,导致巨核细胞数量增多但血小板生成减少,干预巨核细胞凋亡可能成为ITP治疗的一个新靶点。
Idiopathic thrombocytopenic purpura (ITP) is one of the most common forms of autoimmune disease affecting both adults and children, characterized by a low platelet count and normal or increased number of megakaryocytes in bone marrow. It is usually a persisting disease, which relapses frequently and requires a long-term treatment. In some cases, it progresses rapidly and even threatens the patients' survival. The severe side effects of routine treatment lead to a poor prognosis.
The pathogenetic mechanism of ITP is not completely clear yet. It has long been believed that thrombocytopenia is mediated by autoantibodies that are directed against various platelet membrane receptors, including platelet glycoproteins such as glycoprotein II b/IIIa (GPIIb/IIIa) or GP I b/IX complexes. Binding of autoantibodies to these target antigens eventually results in platelet destruction by the reticuloendothelial system.
Since the target antigens are present on both platelets and their precursors, megakaryocytes, it is possible that megakaryocytopoiesis and thrombopoiesis are also impaired during ITP, which could further aggravate the thrombocytopenia caused initially by increased peripheral destruction of platelets. This hypothesis that megakaryocytopoiesis and thrombopoiesis may be disrupted in ITP has been supported by platelet kinetic studies and morphologic alterations of ITP marrow megakaryocytes. Recently, Chang et al reported that plasma from patients with childhood ITP suppressed in vitro megakaryocyte production. Similarly, McMillan et al studied the effect of plasma from adult patients with chronic ITP on in vitro megakaryocyte production. Their further study proved the suppression was mediated by plasma autoantibodies. Taken together, these data indicate that autoantibodies not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.
However, these mechanisms cannot account for all observations made in this disorder. Some ITP patients' platelets are absent of detectable antigen-specific autoantibodies and remission in ITP can occur despite the presence of platelet autoantibodies. It is difficult to interpret all the impairment of platelet with antibody-mediated immunity. These phenomena indicate the presence of other mechanisms in ITP.
It has been well known that T-lymphocyte abnormalities may have pathogenetic importance in ITP patients. Mature reactive T-cell clones have also been shown to be deleted peripherally through activation-induced cell death (AICD). AICD is induced in T cells via different death pathways, of which Fas/FasL is the best characterized. Yoshimura et al found a higher level of soluble Fas which protects cells from undergoing FasL induced apoptosis in patients with chronic ITP. Furthermore, Olsson et al reported that apoptotic resistance of T cells in patients with active ITP may lead to defective clearance of potentially pathogenic reactive T cells through AICD and consequently, may allow continuing autoimmune platelet destruction, i.e. platelet antibody production and cell-mediated cytotoxicity. Most recently, in vitro studies suggested that cytotoxic T-lymphocyte (CTL) (CD8~+) may be involved in the pathogenesis of chronic ITP through cell-mediated destruction of autologous platelets
In normal physiology platelet production and mature megakaryocyte apoptosis are closely related events. In disease, however, the decreased megakaryocyte apoptosis might disrupt platelet formation. Growing evidence suggests that ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like programmed cell death which contribute to thrombocytopenia. Triggers for the different cell death pattern are largely unknown.
To understand the contribution of CD8~+ T cells in bone marrow to the pathogenesis of ITP, we investigated the effect and mechanism of the CD8~+ T cells of patients with ITP on autologous megakaryocytopoiesis.
I Effects of CD8~+ T cells from patients with ITP on quantity of in vitro megakaryocytopoiesis
Objective: To investigate the effects of CD8~+ T lymphocytes in bone marrow from patients with chronic ITP on quantity of in vitro autologous megakaryocytopoiesis.
Methods: * 10 mL bone marrow and 30 mL blood from 15 chronic ITP patients and 13 controls were collected respectively.
* Mononuclear cells (MNCs) were prepared.
* CD8~+T/CD3~+ T ratio in MNCs was detected.
* CD8~+ T lymphocytes were positively selected using CD8~+ magnetic microbeads, according to the manufacture's recommendations.
* Platelets were prepared from peripheral blood.
* Proliferative responses by CD8~+ T cells in bone marrow to autologous platelets were performed.
* The prepared bone marrow cells were divided into four groups: MNCs were cultured directly (group MNC); CD8~+T depleted MNCs were cultured (group CD8~+T-dep); purified CD8~+ T cells were 1:1 added to autologous CD8~+T-dep MNCs in coculture (group coculture); and dexamethasone was added to coculture (group DEX) and adjusted final concentration to 1.0×10~(-6) mol/L.
*The prepared cells were planted in semi-solid and liquid culture systems.
*Megakaryocytes were recognized as CD41a~+ events by fluorescence -activated cell sorter (FACS).
* Megakaryocyte colony forming units (CFU-MK) were quantitated.
Results: * The ratio of CD8~+T/CD3~+T in ITP bone marrow was 59.94%±5.15%, which was significantly higher than that in ITP peripheral blood (49.33%±3.70%; p<0.05) and in normal bone marrow (38.70%±3.43%; p<0.05).
* CD8~+ T cells derived from patients with chronic ITP showed vigorous proliferation after being incubated with autologous platelets for 7 days, but CD8~+ T cells from controls did not show reactivity (p<0.05).
* The counts of CFU-MK and megakaryocytes were very similar among group MNC, group CD8~+T-dep and group coculture, which were not influenced by the depletion or addition of autologous CD_8~+T cells from normal bone marrow.
* When numbers of CFU-MK of ITP were compared, there was no statistical difference between group MNC and group CD8~+T-dep (55.87±18.40 vs. 59.40±12.76; p>0.05), between group CD8~+T-dep and group coculture (59.40±12.76 vs. 54.47±21.48; p>0.05),nor between group MNC and group coculture(55.87±18.40 vs. 54.47±21.48; p>0.05). CFU-MK formation of chronic ITP patients was not influenced by the depletion or addition of autologous CD8~+ T cells. When megakaryocyte counts were compared, the depletion of CD8~+ T cells caused a significant reduction in megakaryocytes when compared with group MNC, the addition of CD8~+ T cells increased megakaryocytes when compared with group CD8~+T-dep, and DEX decreased megakaryocytes when compared with group coculture. Also, megakaryocyte count of group coculture was maximal in coculture containing the most numerous CD8~+T cells.
Conclusion: ITP CD8~+ T cells increased megakaryocytes, and the increased megakaryocyte count was not mediated by accelerated proliferation of megakaryocyte progenitors.
II Effects of CD8~+ T cells from patients with ITP on quality of in vitro megakaryocytopoiesis
Objective: To investigate the connection between the increased megakaryocytes mediated by ITP CD8~+ T cells and the suppressed apoptotic megakaryocytes and platelet production.
Methods: * Platelet count was analyzed in cultured cells.
* Megakaryocyte ploidy was measured. CD41a~+ cells were gated and ploidy distribution was assessed by the intensity of the PI fluorescence.
* Apoptosis in megakaryocytes was measured using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturers instructions..
* Bcl-xl expression in megakaryocytes was performed by first incubating the cells with PEcy5-conjugated CD41a mAb. After staining, cells were fixed in 1% paraformaldehyde, permeabilized with 0.1% saponin, and incubated with FITC-conjugated Bcl-xl. CD41a~+ cells were gated and Bcl-xl expression was shown as mean fluorescence intensity (MFI) within that population.
* Fas expression in megakaryocytes was performed by sequentially incubating with PEcy5-conjugated CD41a mAb and FITC-conjugated Fas mAb. CD41a~+ cells were gated and Fas expression was reported as MFI within that population.
* The levels of soluble Fas (sFas) and soluble TRAIL (sTRAIL) of cell-free supernatants of megakaryocytic cultures were quantified by the respectiveenzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.
Results: * The megakaryocytic characteristics were very similar among group MNC, group CD8~+T-dep and group coculture in controls. The depletion or addition of autologous CD8~+ T cells from normal bone marrow did not affect apoptotic megakaryocytes and platelet production.
* A significant reduction in platelets was noted in cocultures when compared with CD8~+T-dep cultures and MNC cultures. The count of platelets in group CD8~+T-dep was remarkably higher than that of group MNC. When the yield of megakaryocytes was maximal in cocultures, in contrast, the platelet production reached the minimum, indicating the ability of platelet production by megakaryocytes dropped, though the quantity of megakaryocytes increased.
* The percentage of polyploidy (N≥4) megakaryocytes in group CD8~+T-dep was significantly higher than that in group MNC (p<0.05). The addition of CD8~+ T cells decreased the percentage of polyploidy megakaryocytes when compared with group CD8~+T-dep (p<0.05).
* The percentage of apoptotic megakaryocytes was significantly higher in group CD8~+T-dep than that in group MNC (p<0.05). The addition of CD8~+T cells decreased the percentage of apoptotic megakaryocytes when compared with group CD8~+T-dep (p<0.05).
* The expression of Bcl-xl was significantly lower in group CD8~+T-dep than that in group MNC (p<0.05). The addition of CD8~+T cells increased the expression of Bcl-xl when compared with group CD8~+T-dep (p<0.05).
* The expression of Fas was significantly higher in group CD8~+T-dep than that in group MNC (p<0.05). The addition of CD8~+ T cells decreased the expression of Fas when compared with group CD8~+T-dep (p<0.05).
* The sFas level of the group CD8~+T-dep (415.01±178.47) was significantly lower than that of the group MNC (977.46±287.31) and the group coculture (1424.88±469.93, both P<0.05). sTRAIL level was too low to be detected by ELISA.
* Compared with the ITP group coculture, the platelet numbers and the percentage of polyploid nuclei of the group DEX increased largely(16.36±6.86 and 21.37±4.23 respectively, both P<0.05), the percentage of apoptotic CD41a~+ cells and the Fas expression level increased(19.04±3.41 and 26.57±4.63 respectively, both P<0.05), the Bcl-xl expression level reduced(30.70±5.88, P<0.05), and the sFas level reduced(719.13±220.53, P<0.05).
Conclusion: CD8~+ T cells in bone marrow of patients with chronic ITP might suppress megakaryocyte apoptosis leading to the increased megakaryocytes and the impaired platelet production. Megakaryocyte apoptosis would be a novel target for the management of ITP.
长期以来由自身抗体介导的血小板破坏增多被看成是ITP的主要病因,即血小板膜表面糖蛋白(GP)作为自身抗原,特别是GPⅡb/Ⅲa和GPIb/Ⅸ复合物,刺激机体免疫系统产生特异性自身抗体,通过其Fab段与自身抗原结合。这些被自身抗体结合的血小板容易被机体的网状内皮系统所清除,从而导致血小板减少。
由于GP在巨核祖细胞阶段就开始表达在细胞膜上,并且在巨核细胞成熟过程中膜表面的表达量逐步升高,因此ITP患者的巨核细胞生长和血小板生成也应该受到影响。目前普遍认为,除血小板破坏增多外,血小板生成减少也参与ITP的发病。血小板动力学研究及骨髓巨核细胞形态学检查均证实ITP患者的血小板生成减少。最近,Chang等和McMillan等发现ITP患者血浆和纯化的血小板自身抗体可抑制体外巨核细胞生长,导致巨核细胞数量明显降低。充分证明血小板特异性抗原的自身抗体不仅介导外周血小板破坏,还影响骨髓中巨核细胞的生长和发育成熟。
曾认为体液免疫异常在ITP的发病过程中起决定性作用,但随着研究的深入,这一观点已日益受到挑战。近几年细胞免疫功能失调在ITP发病中的作用越来越受重视,尤其是T细胞在其中所起的作用。研究表明ITP的发病机制是以细胞免疫紊乱为中心的,其中Fas/FasL途径介导的T淋巴细胞凋亡障碍是重要原因。Yoshimura等研究发现ITP患者血清中sFas水平明显高于正常人,且其活化的CD8~+T细胞比例显著升高,提示高浓度的sFas抑制了免疫细胞发生Fas/FasL途径介导的激活诱导的细胞死亡(AICD),导致激活的T淋巴细胞因凋亡不足而大量积累于体内,不仅介导体液免疫异常、产生自身抗体,而且体外实验已证实细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL,CD8~+T)能直接参与对ITP患者血小板的杀伤,导致血小板持续破坏。
血小板生成与成熟巨核细胞的凋亡密切相关,巨核细胞凋亡异常即会引起血小板生成障碍。对ITP患者骨髓巨核细胞超微结构的研究表明,患者的巨核细胞存在广泛的异常,这些异常的巨核细胞发生与凋亡相似但不同于凋亡的程序性细胞死亡,是导致血小板生成减少的重要原因。ITP患者骨髓巨核细胞发生异常凋亡的机制还不清楚。
ITP患者骨髓中活化的CD8~+T细胞是否影响巨核系造血,目前尚不清楚。我们通过体外培养巨核细胞观察了ITP患者骨髓CD8~+T细胞对巨核细胞生成数量和质量的影响以及地塞米松对这种影响的逆转并探讨了相关机制,为临床治疗提供依据。
第一部分ITP患者骨髓CD8~+T细胞对体外巨核细胞生成数量的影响
研究目的:研究ITP患者骨髓中活化的CD8~+T细胞对自体巨核细胞生成数量的影响。
研究方法:*抽取15例慢性ITP患者和13例对照10mL骨髓以及30mL外周血。
*分离骨髓和外周血中的单个核细胞。
*检测单个核中CD8~+T细胞的比例。
*用免疫磁珠分离技术分离骨髓单个核细胞中CD8~+T细胞。
*分离外周血中的血小板。
*检测骨髓中CD8~+T细胞对自身血小板的增殖反应。
*将骨髓单个核细胞(MNC),分为MNC组(单个核细胞直接培养)、CD8~-组(单个核细胞去除CD8~+T细胞后培养)、CD8~+组(纯化的CD8~+T细胞1:1加入自体去CD8~+T细胞后的单个核细胞中共同培养)和DEX组(在CD8~+组加入地塞米松)。
*体外无血清半固体培养和液体培养定向扩增巨核细胞。
*流式细胞仪检测CD41a的表达。
*巨核祖细胞集落分析。
研究结果:*ITP患者骨髓中CD8~+T/CD3~+T比例为59.94%±5.15%,明显高于ITP患者外周血中的比例49.33%±3.70%(P＜0.05),也明显高于正常骨髓中CD8~+T/CD3~+T比例38.70%±3.43%(P＜0.05)。
*正常骨髓中的CD8~+T细胞和自身血小板在一起体外培养7天后,不发生增殖反应(cpm:1934±319),而ITP骨髓中的CD8~+T细胞和自身血小板在一起培养后,发生明显的增殖反应(cpm:13526±1037,P＜0.05)。
*正常骨髓CD8~-组形成的CFU-MK和CD41a~+细胞数(61.00±16.00和4.61±0.53)与MNC组(59.23±16.20和4.57±0.58)和CD8~+组(60.77±16.48和4.60±0.57)相比,差异无统计学意义(均P＞0.05),提示正常骨髓中的CD8~+T细胞不影响CFU-MK和CD41a~+细胞数。
*ITP患者骨髓CD8~-组形成的CFU-MK(59.40±12.76)与MNC组(55.87±18.40)和CD8~+组(54.47±21.48)相比,差异无统计学意义(均P＞0.05);CD8~-组CD41a+细胞数(5.01±0.58)低于MNC组(5.75±0.70,P＜0.05),CD8~+组与CD8~-组相比,CD41a~+细胞(6.18±0.79,P＜0.05)增加,提示ITP骨髓中的CD8~+T细胞不影响自身CFU-MK数量,但CD41a~+细胞数随培养体系中CD8~+T细胞数的增多而增多。
*与ITP患者的CD8~+组相比,DEX组产生的CFU-MK为56.93±14.54,P＞0.05,差异无统计学意义;DEX组产生的CD41a~+细胞数减少为(5.26±0.70)×10~5,P＜0.05,差异显著。
结论:ITP患者骨髓中活化的CD8~+T细胞可使巨核细胞数增加,巨核细胞数量增加不是由于巨核祖细胞增殖加速引起的。
第二部分ITP患者骨髓CD8~+T细胞对体外巨核细胞生成质量的影响
研究目的:初步探讨ITP患者骨髓中活化的CD8~+T细胞引起的巨核细胞数量增多与巨核细胞凋亡和血小板生成减少之间的关系。
研究方法:*检测培养体系中生成的血小板。
*CD41a~+细胞DNA倍体分析。
*CD41a~+细胞凋亡检测。
*检测CD41a~+细胞内Bcl-xl的表达。
*检测CD41a~+细胞膜表面Fas的表达。
*检测培养细胞上清液中sFas和sTRAIL水平。
研究结果:*正常骨髓中的CD8~+T细胞不影响血小板生成和巨核细胞凋亡。
*与ITP患者MNC组相比,CD8~-组产生的血小板明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,血小板减少,P＜0.05,差异显著。在ITP患者的三组中,CD8~+组培养体系中含有的CD8~+T细胞最多,所产生的CD41a~+细胞数最多,但产生的血小板最少。ITP患者MNC组血小板产生量与正常MNC组相比,明显减少,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞产生血小板的能力,血小板数随CD8~+T细胞数的增多而减少。
*与ITP患者MNC组相比,CD8~+组产生的CD41a~+细胞中多倍体细胞比例明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,多倍体细胞比例下降,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞的成熟,多倍体巨核细胞数随CD8~+T细胞数的增多而减少。
*与ITP患者MNC组相比,CD8~-组产生的CD41a~+细胞中凋亡细胞比例明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,凋亡细胞比例下降,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞的凋亡,凋亡的巨核细胞数随CD8~+T细胞数的增多而减少。
*与ITP患者MNC组相比,CD8~-组产生的CD41a~+细胞Bcl-xl表达强度明显下降,P＜0.05,差异显著;CD8~+组与CD8~-组相比,Bcl-xl表达强度明显增加,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞内Bcl-xl的表达,Bcl-xl的表达强度随CD8~+T细胞数的增多而增高。
*与ITP患者MNC组相比,CD8~-组产生的CD41a~+细胞Fas表达强度明显增加,P＜0.05,差异显著;CD8~+组与CD8~-组相比,Fas表达强度明显下降,P＜0.05,差异显著。提示ITP骨髓中的CD8~+T细胞影响自身巨核细胞膜表面Fas的表达,Fas的表达强度随CD8~+T细胞数的增多而下降。
*与ITP患者MNC直接培养相比,CD8~-组sFas水平明显下降,P＜0.05,差异显著;CD8~+组与CD8~-组相比,sFas水平明显升高,P＜0.05,差异显著;DEX组与CD8~+组相比,sFas水平明显下降,P＜0.05,差异显著。而各组细胞上清液中的sTRAIL水平太低,基本检测不到。提示ITP骨髓中的CD8~+T细胞影响自身骨髓细胞培养上清液中sFas水平,sFas水平随CD8~+T细胞数的增多而升高。
*与ITP患者的CD8~+组相比,DEX组产生的血小板增多为(16.36±6.86)×10~3,P＜0.05,差异显著;多倍体细胞数比例升高为21.37%±4.23%,P＜0.05,差异显著;CD41a~+细胞凋亡比例升高为19.04%±3.41%,P＜0.05,差异显著;Bcl-xl表达强度下降为30.70±5.88,P＜0.05,差异显著;Fas表达强度升高为26.57±4.63,P＜0.05,差异显著。
结论:ITP患者骨髓中活化的CD8~+T细胞抑制自体巨核细胞凋亡,导致巨核细胞数量增多但血小板生成减少,干预巨核细胞凋亡可能成为ITP治疗的一个新靶点。
Idiopathic thrombocytopenic purpura (ITP) is one of the most common forms of autoimmune disease affecting both adults and children, characterized by a low platelet count and normal or increased number of megakaryocytes in bone marrow. It is usually a persisting disease, which relapses frequently and requires a long-term treatment. In some cases, it progresses rapidly and even threatens the patients' survival. The severe side effects of routine treatment lead to a poor prognosis.
The pathogenetic mechanism of ITP is not completely clear yet. It has long been believed that thrombocytopenia is mediated by autoantibodies that are directed against various platelet membrane receptors, including platelet glycoproteins such as glycoprotein II b/IIIa (GPIIb/IIIa) or GP I b/IX complexes. Binding of autoantibodies to these target antigens eventually results in platelet destruction by the reticuloendothelial system.
Since the target antigens are present on both platelets and their precursors, megakaryocytes, it is possible that megakaryocytopoiesis and thrombopoiesis are also impaired during ITP, which could further aggravate the thrombocytopenia caused initially by increased peripheral destruction of platelets. This hypothesis that megakaryocytopoiesis and thrombopoiesis may be disrupted in ITP has been supported by platelet kinetic studies and morphologic alterations of ITP marrow megakaryocytes. Recently, Chang et al reported that plasma from patients with childhood ITP suppressed in vitro megakaryocyte production. Similarly, McMillan et al studied the effect of plasma from adult patients with chronic ITP on in vitro megakaryocyte production. Their further study proved the suppression was mediated by plasma autoantibodies. Taken together, these data indicate that autoantibodies not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.
However, these mechanisms cannot account for all observations made in this disorder. Some ITP patients' platelets are absent of detectable antigen-specific autoantibodies and remission in ITP can occur despite the presence of platelet autoantibodies. It is difficult to interpret all the impairment of platelet with antibody-mediated immunity. These phenomena indicate the presence of other mechanisms in ITP.
It has been well known that T-lymphocyte abnormalities may have pathogenetic importance in ITP patients. Mature reactive T-cell clones have also been shown to be deleted peripherally through activation-induced cell death (AICD). AICD is induced in T cells via different death pathways, of which Fas/FasL is the best characterized. Yoshimura et al found a higher level of soluble Fas which protects cells from undergoing FasL induced apoptosis in patients with chronic ITP. Furthermore, Olsson et al reported that apoptotic resistance of T cells in patients with active ITP may lead to defective clearance of potentially pathogenic reactive T cells through AICD and consequently, may allow continuing autoimmune platelet destruction, i.e. platelet antibody production and cell-mediated cytotoxicity. Most recently, in vitro studies suggested that cytotoxic T-lymphocyte (CTL) (CD8~+) may be involved in the pathogenesis of chronic ITP through cell-mediated destruction of autologous platelets
In normal physiology platelet production and mature megakaryocyte apoptosis are closely related events. In disease, however, the decreased megakaryocyte apoptosis might disrupt platelet formation. Growing evidence suggests that ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like programmed cell death which contribute to thrombocytopenia. Triggers for the different cell death pattern are largely unknown.
To understand the contribution of CD8~+ T cells in bone marrow to the pathogenesis of ITP, we investigated the effect and mechanism of the CD8~+ T cells of patients with ITP on autologous megakaryocytopoiesis.
I Effects of CD8~+ T cells from patients with ITP on quantity of in vitro megakaryocytopoiesis
Objective: To investigate the effects of CD8~+ T lymphocytes in bone marrow from patients with chronic ITP on quantity of in vitro autologous megakaryocytopoiesis.
Methods: * 10 mL bone marrow and 30 mL blood from 15 chronic ITP patients and 13 controls were collected respectively.
* Mononuclear cells (MNCs) were prepared.
* CD8~+T/CD3~+ T ratio in MNCs was detected.
* CD8~+ T lymphocytes were positively selected using CD8~+ magnetic microbeads, according to the manufacture's recommendations.
* Platelets were prepared from peripheral blood.
* Proliferative responses by CD8~+ T cells in bone marrow to autologous platelets were performed.
* The prepared bone marrow cells were divided into four groups: MNCs were cultured directly (group MNC); CD8~+T depleted MNCs were cultured (group CD8~+T-dep); purified CD8~+ T cells were 1:1 added to autologous CD8~+T-dep MNCs in coculture (group coculture); and dexamethasone was added to coculture (group DEX) and adjusted final concentration to 1.0×10~(-6) mol/L.
*The prepared cells were planted in semi-solid and liquid culture systems.
*Megakaryocytes were recognized as CD41a~+ events by fluorescence -activated cell sorter (FACS).
* Megakaryocyte colony forming units (CFU-MK) were quantitated.
Results: * The ratio of CD8~+T/CD3~+T in ITP bone marrow was 59.94%±5.15%, which was significantly higher than that in ITP peripheral blood (49.33%±3.70%; p<0.05) and in normal bone marrow (38.70%±3.43%; p<0.05).
* CD8~+ T cells derived from patients with chronic ITP showed vigorous proliferation after being incubated with autologous platelets for 7 days, but CD8~+ T cells from controls did not show reactivity (p<0.05).
* The counts of CFU-MK and megakaryocytes were very similar among group MNC, group CD8~+T-dep and group coculture, which were not influenced by the depletion or addition of autologous CD_8~+T cells from normal bone marrow.
* When numbers of CFU-MK of ITP were compared, there was no statistical difference between group MNC and group CD8~+T-dep (55.87±18.40 vs. 59.40±12.76; p>0.05), between group CD8~+T-dep and group coculture (59.40±12.76 vs. 54.47±21.48; p>0.05),nor between group MNC and group coculture(55.87±18.40 vs. 54.47±21.48; p>0.05). CFU-MK formation of chronic ITP patients was not influenced by the depletion or addition of autologous CD8~+ T cells. When megakaryocyte counts were compared, the depletion of CD8~+ T cells caused a significant reduction in megakaryocytes when compared with group MNC, the addition of CD8~+ T cells increased megakaryocytes when compared with group CD8~+T-dep, and DEX decreased megakaryocytes when compared with group coculture. Also, megakaryocyte count of group coculture was maximal in coculture containing the most numerous CD8~+T cells.
Conclusion: ITP CD8~+ T cells increased megakaryocytes, and the increased megakaryocyte count was not mediated by accelerated proliferation of megakaryocyte progenitors.
II Effects of CD8~+ T cells from patients with ITP on quality of in vitro megakaryocytopoiesis
Objective: To investigate the connection between the increased megakaryocytes mediated by ITP CD8~+ T cells and the suppressed apoptotic megakaryocytes and platelet production.
Methods: * Platelet count was analyzed in cultured cells.
* Megakaryocyte ploidy was measured. CD41a~+ cells were gated and ploidy distribution was assessed by the intensity of the PI fluorescence.
* Apoptosis in megakaryocytes was measured using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturers instructions..
* Bcl-xl expression in megakaryocytes was performed by first incubating the cells with PEcy5-conjugated CD41a mAb. After staining, cells were fixed in 1% paraformaldehyde, permeabilized with 0.1% saponin, and incubated with FITC-conjugated Bcl-xl. CD41a~+ cells were gated and Bcl-xl expression was shown as mean fluorescence intensity (MFI) within that population.
* Fas expression in megakaryocytes was performed by sequentially incubating with PEcy5-conjugated CD41a mAb and FITC-conjugated Fas mAb. CD41a~+ cells were gated and Fas expression was reported as MFI within that population.
* The levels of soluble Fas (sFas) and soluble TRAIL (sTRAIL) of cell-free supernatants of megakaryocytic cultures were quantified by the respectiveenzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.
Results: * The megakaryocytic characteristics were very similar among group MNC, group CD8~+T-dep and group coculture in controls. The depletion or addition of autologous CD8~+ T cells from normal bone marrow did not affect apoptotic megakaryocytes and platelet production.
* A significant reduction in platelets was noted in cocultures when compared with CD8~+T-dep cultures and MNC cultures. The count of platelets in group CD8~+T-dep was remarkably higher than that of group MNC. When the yield of megakaryocytes was maximal in cocultures, in contrast, the platelet production reached the minimum, indicating the ability of platelet production by megakaryocytes dropped, though the quantity of megakaryocytes increased.
* The percentage of polyploidy (N≥4) megakaryocytes in group CD8~+T-dep was significantly higher than that in group MNC (p<0.05). The addition of CD8~+ T cells decreased the percentage of polyploidy megakaryocytes when compared with group CD8~+T-dep (p<0.05).
* The percentage of apoptotic megakaryocytes was significantly higher in group CD8~+T-dep than that in group MNC (p<0.05). The addition of CD8~+T cells decreased the percentage of apoptotic megakaryocytes when compared with group CD8~+T-dep (p<0.05).
* The expression of Bcl-xl was significantly lower in group CD8~+T-dep than that in group MNC (p<0.05). The addition of CD8~+T cells increased the expression of Bcl-xl when compared with group CD8~+T-dep (p<0.05).
* The expression of Fas was significantly higher in group CD8~+T-dep than that in group MNC (p<0.05). The addition of CD8~+ T cells decreased the expression of Fas when compared with group CD8~+T-dep (p<0.05).
* The sFas level of the group CD8~+T-dep (415.01±178.47) was significantly lower than that of the group MNC (977.46±287.31) and the group coculture (1424.88±469.93, both P<0.05). sTRAIL level was too low to be detected by ELISA.
* Compared with the ITP group coculture, the platelet numbers and the percentage of polyploid nuclei of the group DEX increased largely(16.36±6.86 and 21.37±4.23 respectively, both P<0.05), the percentage of apoptotic CD41a~+ cells and the Fas expression level increased(19.04±3.41 and 26.57±4.63 respectively, both P<0.05), the Bcl-xl expression level reduced(30.70±5.88, P<0.05), and the sFas level reduced(719.13±220.53, P<0.05).
Conclusion: CD8~+ T cells in bone marrow of patients with chronic ITP might suppress megakaryocyte apoptosis leading to the increased megakaryocytes and the impaired platelet production. Megakaryocyte apoptosis would be a novel target for the management of ITP.
引文
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