【作者】 王晶；

【导师】 李卓娅；

【作者基本信息】 华中科技大学 ， 免疫学， 2006， 博士

【摘要】 TNF-α(tumor necrosis factor，TNF-α)是由激活的单核／巨噬细胞，淋巴细胞等分泌的具有多种生物学活性的细胞因子，它能直接介导肿瘤细胞的凋亡和坏死，参与机体局部及全身炎症反应，介导免疫细胞的增殖分化，发挥免疫调节作用。在体内以两种形式存在：即26kD的跨膜型TNF-α(membrane associated TNF-α，mTNF-α)和17kD的分泌型TNF-α(secreted TNF-α，sTNF-α)，后者由mTNF-α酶解而来。两型TNF-α通过与两型TNF受体(TNFR1与TNFR2)结合而发挥广泛的生物学作用。成熟T细胞的AICD是机体清除免疫应答中过量的效应T细胞，调节免疫反应，维持免疫稳态和建立外周免疫耐受的重要机制。目前，已证实FasL／Fas是介导T细胞AICD重要的、但非唯一的死亡分子。关于TNF-α在AICD的作用主要限制于对sTNF-α的研究，mTNF-α正向信号在AICD中的作用则未见报道。近来研究发现，mTNF-α除了作为配体可与TNFR结合，向靶细胞传递正向信号而发挥生物学效应外，还可同时作为受体向效应细胞传递反向信号(reverse signaling)。有学者报道Infliximab可以诱发Crohn’s病患者肠相关淋巴组织固有层(lamina propria)T细胞凋亡及外周血活化的T细胞凋亡。提示mTNF-α的反向信号可以参与AICD。本课题以PHA活化白血病CD4~+淋巴细胞系Jurkat建立体外的AICD模型，研究和比较mTNF-α和sTNF-α对T细胞AICD的影响，以期阐明两型TNF-α正向信号及mTNF-α的反向信号在AICD中的生物学特征及生物学作用，从而深入探讨AICD的分子机制，为临床治疗自身免疫病提供新的线索。一、AICD实验模型的建立1．PHA诱导Jurkat细胞AICD的剂量依赖性体外以不同剂量的PHA刺激Jurkat细胞24h，借助Annexin／PI法观察AICD。结果显示，PHA可诱导Jurkat细胞发生凋亡，且呈剂量依赖性，即凋亡率随PHA的浓度的递增而升高，当PHA为1μg／ml时其凋亡率已达50％，5μg／ml为63％，20μg／ml则超过80％。2．PHA诱导Jurkat细胞AICD的时间动力学选取5μg／ml的PHA，观察其诱导Jurkat细胞AICD的时间动力学。凋亡在2h时已达22％，6h已升高至56％，随着PHA刺激时间的延长，其凋亡率缓慢上升，24h时凋亡率为63％，而72h时则达82％。选取PHA既可有效诱导AICD，凋亡值又适中的剂量(5μg／ml)和作用时间点(24h)作为诱导体外AICD模型的条件。3．TNF-α参与PHA诱导Jurkat细胞的AICD用本实验室制备的对mTNF-α无反向信号作用的抗TNF-α单克隆抗体中和内源性TNF-α，可部分抑制PHA诱导的AICD(抑制率14.01％，p＜0.05)，提示TNF-α参与PHA诱导Jurkat细胞的AICD。二、mTNF-α和sTNF-α正向信号在AICD中的作用1．mTNF-α正向信号在AICD中的作用1．1 AICD中Jurkat细胞表达内源性mTNF-α实验证实未刺激的Jurkat表达少量mTNF-α(4.5％)，PHA活化的T细胞表达mTNF-α明显增加，并随着刺激时间的延长而逐渐增高。24h时mTNF-α的表达率为45.8％。1．2 mTNF-α对未活化Jurkat的杀伤作用用1％多聚甲醛固定PHA活化24h的Jurkat细胞，以不同的效靶比与未活化的Jurkat细胞共同孵育24h，借助WST-1法检测活化T细胞的杀伤活性。结果显示活化T细胞对未活化细胞具有明显的杀伤作用，且随效靶比的升高而增强，5：1时杀伤率为71％。应用兔抗人TNF多克隆抗体(TNFpAb)及鼠抗人sTNF-α单克隆抗体(TNFmAb)，可部分阻断其杀伤作用(抑制率分别为53.86％和43.37％，p＜0.01)，提示活化T细胞的杀伤作用部分是由mTNF-α介导的。1．3 mTNF-α对活化Jurkat的杀伤作用用多聚赖氨酸使将PHA活化24h的Jurkat细胞贴壁，以效靶比5∶1加入PHA活化2h的Jurkat细胞(作为靶细胞)共同孵育48h。结果显示，活化Jurkat细胞对活化Jurkat细胞有明显致凋亡作用(44.37％，p＜0.01)，且抗TNF pAb和mAb可以部分阻断AICD(p＜0.01)。提示mTNF-α可能是AICD的效应分子之一。2．sTNF-α正向信号在AICD中的作用2．1活化Jurkat细胞分泌内源性sTNF-α收集PHA作用24小时的Jurkat细胞培养上清，以ELISA法未能检出sTNF-α的分泌。2．2 PHA诱导Jurkat细胞表达TACE进一步观察TNF转换酶(TACE)的表达是否受到抑制，流式细胞术结果显示PHA可以明显刺激Jurkat细胞表达TACE(表达率44.39％)。推测TACE可能导致大量可溶性TNFR的产生，从而影响sTNF-α的免疫学检测。2．3外源性sTNF-α在AICD中的作用为探索sTNF-α对T细胞是否具有致凋亡作用，用外源性sTNF-α(50U／ml)作用Jurkat细胞24h，结果显示sTNF-α可杀伤未活化(凋亡率23.81％，p＜0.01)和活化Jurkat细胞(凋亡率78.13％，p＜0.01)。三、mTNF-α反向信号在AICD中的作用1．mTNF-α反向信号促进PHA诱导的AICD1．1抗TNF-α多克隆抗体可促进PHA诱导的AICD用抗TNF-α多克隆抗体激活mTNF-α反向信号，结果显示，其不能诱导未活化Jurkat细胞发生凋亡，但可明显提高活化Jurkat细胞的凋亡率，增强率为20.11％(p＜0.01)。提示mTNF-α反向信号可增强AICD效应。1．2 sTNFR1也可促进AICD为进一步确认mTNF-α反向信号促进AICD的效应，我们用可溶性TNFRI(sTNFR1 20μg／ml)刺激mTNF-α反向信号，结果sTNFR1不能诱导未活化的Jurkat细胞凋亡，但可明显提高活化Jurkat细胞的凋亡率(凋亡率86.82％)，且呈剂量依赖性(p＜0.01)。提示mTNF-α反向信号确可促进AICD。2．mTNF-α反向信号增强AICD的机制2．1 mTNF-α反向信号促进PHA诱导的Fas和FasL的表达实验证实未经活化的Jurkat细胞低表达Fas／FasL；PHA可诱导Jurkat细胞表达Fas(33.28％)和FasL(34.98％)。用抗TNF-α多克隆抗体激活mTNF-α反向信号，可明显促进PHA诱导的Fas(52.19％)和FasL(47.74％)的表达(p＜0.01)，其增强率分别为56.82％，36.47％。2．2 mTNF-α反向信号促进PHA诱导的mTNF-α表达实验证实未活化Jurkat细胞低表达mTNF-α，PHA诱导其表达增加(45.51％)，而mTNF-α反向信号则可促进PHA诱导的mTNF-α表达率增至61.15％，与PHA刺激组相比，增强率为34.36％(p＜0.01)。2．3 mTNF-α反向信号促凋亡中TNFR1和TNFR2的变化实验证实未活化Jurkat细胞低表达TNFR1和TNFR2，PHA主要诱导Jurkat细胞表达TNFR1(45.29％)，而TNFR2表达则轻度上升(仅6.54％)，提示TNF-α在PHA诱导的AICD中主要通过含有死亡结构域的TNFR1介导凋亡。mTNF-α反向信号可明显上调TNFR1(59.32％％)和TNFR2的表达(55.66％，p＜0.01)。结论：上述结果提示①mTNF-α可能为AICD的效应分子之一，其正向和反向信号均参与AICD。②PHA主要诱导Jurkat细胞表达TNFR1，提示TNF-α主要通过含有死亡结构域的TNFR1介导凋亡。③mTNF-α反向信号促进PHA诱导AICD的主要分子机制：上调Fas和FasL、mTNF-α、TNFR1和TNFR2的表达。更多还原

【Abstract】 Tumor necrosis factor-α is a pleiotropic cytokine with a variety of biological activities produced chiefly by activated monocytes/macrophages, and lymphocytes. TNF-α has a wide range of biologieal effects such as tumoricidal activity, promotion of inflammation, regulation of immune response etc. TNF-α exerts biological effects with two forms, namely a 26kD transmembrane TNF-α (mTNF-α) and a 17kD secreted TNF-α ( sTNF-α) . mTNF-α is anchored on the plasma membrane with the hydrophobic region of its signal peptide. The extracellular segment of mTNF-α can be proteolytically cleaved by TNF-α converted enzyme with the release of sTNF. The two forms of TNF-α elicit a particularly broad spectrum of biological responses by binding two receptors, TNFR1 and TNFR2.The activation-induced cell death (AICD) of mature T lymphocytes represents a major mechanism of the body in the elimination excessive effector cells, modulation of the immune response, maintenance of immunological homeostasis and induction of peripheral immunological tolerance. It has been demonstrated that AICD is mediated mainly but not exclusively by the expression of the death molecule, Fas/FasL. No final conclusion has been reached so far about the role of TNF-α in AICD. Besides, the relevant studies carried out are focused only on sTNF-α, little has been reported concerning the role of mTNF-α in AICD.It was shown by recent studies that the binding of mTNF-α to TNF receptor. resulted in not only the transduction of a signal from TNFR to target cell (positivesignaling) but also the transmission of a signal from mTNF-α, like a receptor, toeffector cell (reverse signaling). It was reported that Infliximab treatment inducesapoptosis of lamina propria T lymphocytes and activated peripheral blood Tlymphocytes in Crohn’s disease.The aim of the present study is to compare the role of mTNF-a and sTNF-a in AICD of CD4~+ leukemia Jurkat T cells induced by PHA in an attempt to clarify the biological characteristics and regularities of TNF-a, particularly of positive and reverse signaling mTNF-a, so as to provide experimental basis for further study of the molecular mechanisms of AICD, and provide clues for treatment of autoimmune diseases.1. The experimental model of AICD(1) Dose dependent of AICD induced by PHA in Jurkat cellsTo induce AICD, we stimulated Jurkat cells with various concentrations of PHA.Jurkat cells can be induced to be apoptosis by PHA in a dose dependent manner, the apoptosis rate are proportional to the concentrations of PHA. The apoptosis are 50%, 63% and 80% at PHA 1μg/ml, 5μg/ml, 20μg/ml separately, suggesting that an experimental model of AICD was successfully established.(2) Time-course of AICD induced by PHA in Jurkat cellsTo observe the apoptosis development of AICD, the apoptosis rates were determined at PHA 5μg/ml. The apoptosis activity was already detectable (22%) at 2h, and increased rapidly to 56% at 6h, after that it increased slowly at 24h(63%), 72h(83%). Jurkat cells were incubated for 24h with 5μg/ml of PHA as a model of AICD in the present work to induce the apoptosis effectively and also moderately in order to observe the promoting and inhibitory effect.(3) TNF-α was involved in AICD of Jurkat cells induced by PHAAICD could be partially blocked by anti-TNF-α mAb which cannot exhibit the reverse signaling to neutralize the endogenous TNF-α, (inhibitory rate 14.01%, p<0.05), indicating that TNF-α was involved in AICD of Jurkat cells induced by PHA.2. Positive signal of mTNF-α and sTNF-α in AICD of Jurkat cells (1) Positive signal of mTNF-α in AICDA. Expression of endogenous mTNF-α in AICDThe mTNF-α expression level on the surface of Jurkat cells was analyzed by flow cytometry before and after PHA stimulation. The mTNF-α expression was significantly increased in the PHA-activated Jurkat T cells compared with the control Jurkat cells (4.5% versus 45.8% at 24h). The expression rate of mTNF-α increased gradually in a time dependent manner.B. Cytotoxicyty of mTNF-α against non-activated Jurkat cellsJurkat cells were stimulated with PHA for 24h, then washed and fixed with 1% polyformaldehyde, washed with acidified buffer (glycine HC1 buffer), were used as effectors which express mTNF-α. The effector cells were mixed with non-activated Jurkat cells as targets at different ratios and the extent of cell death was determined by the WST-1 assay. Prestimulated Jurkat cells induced death of their non-activated counterparts. The percentage of cell death increased with the effector to target ratios, reaching 71% at ratio 5:1. Anti-TNF rabbit pAb and anti-TNF mouse mAb partly block the cytotoxicity (inhibitory rate at 53.86%, 43.37%, respectively, p<0.01). These results indicate that the cytotoxicity induced by activated Jurkat cells were partly mediated by mTNF-α.C. Cytotoxicity of mTNF-α on PHA activated Jurkat cellsThe PHA-activated Jurkat cells (24h) expressing mTNF-α were used as effectors and mixed with PHA-activated cells (2h) as targets for 48h. mTNF-α on PHA activated Jurkat cells induced significant apoptotic cell death (44.37%) against activated Jurkat target cells. Anti-TNF pAb and anti-TNF mAb partly block apoptosis (p<0.01). mTNF-α could therefore account for the AICD of activated Jurkat cells.(2) Positive signal of sTNF-α in AICDA. Secretion of endogenous sTNF-α by PHA activated Jurkat cellsJurkat cells were stimulated with PHA for 24h, and detect the sTNF-α release by the ELISA assay. Endogenous sTNF-α was non-detectable.B. PHA induce TACE on Jurkat cellsWe next investigated the expression of TNF converting enzyme (TACE), FACS analysis showed that TACE expression on Jurkat cells was greatly increased stimulated by PHA (44.39%). It was possible that the TACE-mediated cleavage of mTNF-α generates a large amount of soluble TNFR that affects the detection of sTNF-α.C. Cytotoxicity of exogenous sTNF-α on PHA activated and non-activated Jurkat cellsTo observe the apoptosis effect- induced by sTNF-α, Jurkat cells were treated with exogenous sTNF-α(50U/ml) for 24h. sTNF-α are cytotoxic against both non-activated Jurkat cells and PHA-activated cells, with the apoptosis rate of 23.81% and 78.13%, respectively(p<0.01). 3. mTNF-α reverse signaling in AICD of Jurkat cells(1) Enhancement of AICD via mTNF-α reverse signalingA. mTNF-α reverse signaling triggered by pAb against TNF-α accelerate AICD PHA-activated Jurkat cells were treated with anti-TNF pAb simultaneously. As acontrol, anti-TNF alone did not induce apoptosis of non-activated Jurkat cells, whereas the apoptosis rate of activated Jurkat cells was greatly induced by 20.11%, indicating that mTNF-α reverse signaling could enhance the AICD of Jurkat cells.B. Augment of AICD via mTNF-α reverse signaling triggered by sTNFR1To confirm the direct involvement of mTNF-α reverse signaling, 20μg/ml soluble TNF receptor (sTNFR1) were used to stimulate the reverse signaling of mTNF-α. sTNFR1 was able to increase the apoptosis rate of activated Jurkat cells instead of non-activated cell (86.82%, p<0.01). The apoptosis rate increased in sTNFR1 dose dependent manner.(2) Mechanism mediated by mTNF-α reverse signaling in AICD of Jurkat cells A. mTNF-α reverse signaling upregulate the expression of Fas/FasLAlthough non-activated Jurkat cells expressed almost undectable level of Fas and FasL, the expression of Fas and FasL was significantly induced by PHA. The treatmentof anti-TNF pAb further upregulate the expression of Fas/FasL (33.28% versus 52.19%, 34.98% versus 47.74%, respectively, p<0.01).B. Increase in PFLA induced mTNF-α expression by reverse signaling via mTNF-αUnstimulated Jurkat cells expressed low level of mTNF-α, after stimulated with PHA, the expression of mTNF-α was greatly induced. Anti-TNF pAb was able to significantly enhance the expression of mTNF-α on the surface of Jurkat cells compared to PHA alone (45.51%versus61.15%, p<0.01).C. Effect of mTNF-α reverse signaling on expression of TNFR1 and TNFR2 in PHA induced AICDNon-activated Jurkat cells express basal amount of TNFR1 and TNFR2. PHA predominantly induced the expression of TNFR1 (45.29%), whereas TNFR2 only slightly increased (6.54%), indicating that TNF-α induced the apoptosis in AICD of Jurkat cells mainly through TNFR1 which contains the death domain. The reverse signaling of mTNF-α induced by anti-TNF pAb obviously upregulate the expression of TNFR1 and TNFR2 (59.32% and 55.66%, separately, p<0.01).In summary, the results above suggest that the positive signaling and reverse signaling of mTNF-α might mediate the AICD of Jurkat cells induced by PHA. PHA predominantly induced the expression of TNFR1 compared with TNR2, indicating that TNF-α induced the apoptosis in AICD of Jurkat cells mainly through TNFR1 which contains the death domain. Upregulation of Fas/FasL, mTNF-α and TNFR1/TNFR2 may account for the mechanism of enhancement of AICD mediated by mTNF-α reverse signaling on Jurkat cells.更多还原