【作者】 李盼；

【导师】 龚非力； 郑芳；

【作者基本信息】 华中科技大学 ， 免疫学， 2011， 博士

【副题名】IL-33与EAE发病的相关注及其机制

【摘要】 NK细胞是一类重要的固有免疫细胞,在机体抗肿瘤、抗病毒中发挥重要作用。NK细胞最重要的生物学作用是杀伤靶细胞,其主要机制之一是：NK细胞与靶细胞接触而形成免疫突触,NK细胞脱颗粒并释放效应分子,其中穿孔素可在靶细胞表面形成微孔道,颗粒酶循该孔道进入靶细胞内而诱导凋亡。靶细胞内,颗粒酶诱导凋亡相关的caspase级联反应,通过直接作用pro-caspase切掉天冬氨酸残基激活下游caspase而发挥作用。免疫突触内部分胞毒效应分子未进入靶细胞,其转归及功能尚不清楚,待阐明的问题之一是：滞留免疫突触内的穿孔素和颗粒酶是否会伤害NK细胞本身；其代谢与去向如何；等等。文献已报道,细胞毒性细胞可通过独特机制处理胞内错位的颗粒酶B (GzmB)和穿孔素(PFN)。例如,NK细胞内表达GzmB特异性抑制剂—丝氨酸蛋白酶抑制剂9(PI-9),后者可及时灭活胞内渗漏的GzmB。鉴于NK细胞具有连续杀伤靶细胞的功能,我们提出如下推测：释放并滞留于免疫突触的效应分子可能被回收而重新利用。本课题以非何杰金淋巴瘤来源的NK92细胞系为模型,在靶细胞刺激下,探讨NK92细胞所释放GzmB的转归。一、NK92细胞在靶细胞刺激下出现继发性胞吞现象1.应用苯乙烯染料FM1-43观察NK92细胞继发性胞吞现象苯乙烯染料FM1-43可与细胞膜磷脂双分子层可逆性结合,发出荧光,而其在溶液中则几乎检测不到荧光。细胞发生内吞后,苯乙烯染料内吞入细胞内,细胞膜上荧光可用磷酸盐缓冲液洗掉。借此原理,本实验首先将NK92细胞用FM1-43预染,然后用猪内皮细胞(PEC)刺激1h,激光共聚焦显微镜(LCM)观察NK92细胞内吞情况。结果显示,未用PEC刺激的NK92细胞膜上显示荧光,PEC刺激的NK92细胞用磷酸盐缓冲液(PBS)冲洗细胞膜后,细胞内显示荧光,提示NK92细胞在胞吐后细胞膜发生内化。2.靶细胞刺激的细胞膜内吞过程依赖于clathrin细胞膜的内吞常见形式为依赖于clathrin的经典途径,也可依赖于巨胞饮(macropinocytosis)、小窝蛋白(caveola)和脂筏(lipid raft)等途径。我们采用clathrin特异性抑制剂CPZ预处理NK92细胞,再用FM1-43标记细胞膜,使用靶细胞刺激后激光共聚焦显微镜观察细胞内荧光。实验结果显示,CPZ处理后NK92细胞胞内荧光强度显著降低,提示NK92细胞膜内吞依赖于clathrin内吞途径。同时用流式细胞术检测细胞内荧光强度,结果显示使用CPZ后细胞内荧光强度约为对照组的48%。我们将NK92细胞用FM1-43标记,分为两组,分别用PEC在4℃和37℃刺激1h,结果显示：在4℃细胞并不发生内吞,提示NK92细胞内吞作用与温度相关,是一种主动内吞。二、靶细胞刺激下,NK92细胞继发性胞吞伴随GzmB回收1.NK92细胞内吞的GzmB进入早期内体NK92细胞膜内吞过程为经典的clathrin依赖途径,CPZ预处理的NK92细胞经靶细胞(PEC)刺激,收集未刺激组、刺激后15min、30min的细胞,免疫荧光检测GzmB和早期内体EEA-1在胞内共定位情况,结果显示：未刺激组EEA-1与GzmB均有表达,二者未出现共定位；靶细胞刺激15min后,GzmB含量明显增多,GzmB与EEA-1有明显共定位,用CPZ处理后共定位显著减少。靶细胞刺激30min后,GzmB与EEA-1共定位比15min组减少。提示NK92细胞继发性胞吞伴随GzmB回收。为进一步验证上述结果,本实验设计针对clathrin蛋白重链的siRNA,采用核转染方法瞬时转染NK92细胞。转染后的细胞用靶细胞刺激,免疫荧光法在15min检测GzmB和EEA-1共定位情况,结果显示干扰组EEA-1与GzmB共定位比对照组明显降低。2.NK92细胞内吞的GzmB可进入溶酶体Clathrin依赖的内吞途径包括从早期内体→晚期内体→溶酶体的全过程。免疫荧光检测靶细胞刺激60min后的NK92细胞GzmB与lamp1共定位情况。结果显示,与未刺激组相比,GzmB与lamp1出现明显共定位；使用CPZ或siRNA后,共定位明显减少。GzmB与lamp1共定位提示GzmB进入溶酶体,且进入后并未被降解。为此,使用溶酶体酶抑制剂leupeptin处理NK92细胞,免疫荧光检测GzmB和lamp1共定位,结果显示共定位并未增加,提示GzmB在溶酶体内未被降解,可供分选重复利用。三、抑制NK92细胞内吞能显著降低杀伤靶细胞活性采用K562作为NK92细胞的靶细胞,CFSE-PI法在不同的效靶比(2.5：1,5：1,10：1)下流式细胞术检测其杀伤率。结果显示：相对于对照组,不同效靶比下CPZ预处理过的NK92细胞杀伤活性明显下降(16.93% vs 1.45%,20.44%vs 1.69%,30.22% vs 2.79%)。当采用siRNA干扰clathrin后,重复以上细胞毒性实验,得到相似的结果,相比于对照组,不同效靶比下干扰组的杀伤活性明显下降(23.18% vs 10.63%,25.31%vs 13.02%,38.69% vs 15.51%)。四、NK92细胞杀伤靶细胞后上清及胞内GzmB含量检测为检测CPZ对NK92细胞胞吐后上清和细胞内颗粒酶B含量的影响,本实验在10：1效靶比条件下,NK92细胞与K562作用1h、3h、5h,ELISA检测上清GzmB含量,Western-blot检测胞内GzmB含量。结果显示,随杀伤时间延长,对照组和CPZ处理组上清GzmB含量逐渐上升,与对照组相比,1h和5h组CPZ处理组上清GzmB含量略有上升(1.8ng/ml vs 2.0ng/ml,2.45ng/ml vs 2.8 ng/ml),3h组GzmB含量有所下降(2.7ng/ml vs 2.25ng/ml)。相应各个时间点Western-blot检测胞内GzmB,杀伤后1h、3h、5h, CPZ预处理的NK92细胞内GzmB含量与对照组相比下降。五、结论1.靶细胞刺激NK92细胞,胞吐后发生继发性内吞,且这种内吞依赖于clathrin。2.NK92细胞继发性胞吞伴随效应分子GzmB回收。3. GzmB被NK92细胞回收,依此进入早期内体→晚期内体→lamp1+溶酶体→分泌型溶酶体,供再利用。4.抑制NK细胞内吞可显著降低NK细胞杀伤活性。多发性硬化症(multiple sclerosis, MS)是以神经脱髓鞘为主要病理改变的中枢神经系统(CNS)炎症性疾病。用于研究MS的经典动物模型是实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)。此模型通过主动免疫神经髓鞘来源的抗原或过继转移髓鞘特异性T细胞,在实验动物中枢神经系统诱导炎症,其脱髓鞘和炎性细胞浸润等病理表现及疾病表现类似于人MS,故被广泛应用于MS发生、发展、转归、调控等细胞和分子机制研究。IL-33是近年新发现的IL-1家族成员,其基因序列和结构与IL-1家族成员IL-18和IL-1β相似。IL-33受体是孤儿受体ST2,也称为IL-1 RL1。ST2基因主要编码两种形式ST2蛋白：膜结合型ST2和可溶性ST2。IL-33表达于人和小鼠多种组织,主要是上皮细胞、内皮细胞和成纤维细胞。IL-33具有重要免疫调节作用,并参与多种免疫相关疾病(如哮喘、类风湿关节炎、内毒性休克等)发生。IL-33主要介导Th2细胞应答,诱导Th2型细胞因子产生。有文献报道,中枢神经系统高表达IL-33。本课题主要观察正常小鼠和EAE小鼠中枢神经系统内IL-33的表达、来源及其参与EAE发病的作用及机制。一、IL-33在小鼠中枢神经系统的表达1.IL-33在正常小鼠中枢神经系统表达逆转录PCR、实时定量PCR、Western-blot检测正常C57BL/6小鼠脊髓、脑、皮肤、肺、肾脏、心脏、脾脏和肝脏组织IL-33表达；免疫荧光、免疫组织化学检测小鼠脊髓中IL-33表达。结果发现,在mRNA和蛋白水平,正常小鼠脑和脊髓IL-33表达水平远高于肝脏、脾脏等组织(约为12.5倍,P<0.01)。免疫荧光显示,正常小鼠脊髓高表达IL-33,主要定位于灰质,表达在细胞核内。免疫组织化学同样证明IL-33表达于细胞核内。Western-blot证明细胞核和细胞浆内均表达IL-33。2.脊髓中IL-33的细胞来源借助冰冻切片免疫荧光检测IL-33在正常小鼠脊髓的细胞定位,通过研究IL-33与星形胶质细胞标志物、神经胶质原纤维酸性蛋白(GFAP)、小胶质细胞标志物(CD68)、神经元特异性蛋白(NeuN)的组织共定位情况,确定IL-33在正常小鼠中的来源。结果显示,IL-33在体内与3种细胞标志物均未出现明显共定位,故暂时不能确定IL-33的细胞来源。3.EAE小鼠脊髓内IL-33表达PCR、Western-blot和免疫组织化学检测正常小鼠和EAE小鼠脊髓IL-33表达,分别提取正常小鼠和EAE模型小鼠脊髓胞核、胞浆蛋白,Western-blot检测IL-33表达改变。结果显示,与正常小鼠相比,在mRNA和蛋白水平上,EAE动物体内IL-33表达量降低；免疫组织化学显示正常小鼠IL-33表达于细胞核内,而EAE动物IL-33表达于胞浆,提示EAE动物体内IL-33发生从胞核向胞浆的转位；western-blot结果显示,正常小鼠IL-33表达于胞浆和胞核,EAE小鼠胞核不表达IL-33,胞浆中有表达。4.IL-33受体ST2在正常小鼠和EAE小鼠脊髓中表达免疫组化显示,正常小鼠ST2表达水平低,EAE小鼠ST2表达水平明显增高水平。二、IL-33参与小鼠EAE发生及其机制1.IL-33对小鼠EAE疾病进程的影响EAE小鼠于免疫前腹腔注射rIL-33(1μg/只)或发病后注射rIL-33(每天1μg/只,连续注射1周)。结果显示,注射rIL-33不影响小鼠EAE发病时间和疾病进程。2.抗IL-33多克隆抗体对EAE疾病的影响EAE小鼠于免疫前腹腔注射抗IL-33抗体(150μg/只)或发病后注射抗IL-33抗体(每隔3天150μg/只,连续注射5次),结果发现：抗IL-33抗体可明显加重EAE疾病症状,EAE发病率升高,临床评分提高；两者发病时间相似；EAE小鼠脊髓冰冻切片免疫荧光检测显示,CD4+T细胞浸润增加。3.抗IL-33多克隆抗体可增强Th1和Th17细胞应答从注射抗IL-33抗体的EAE小鼠采集并分离脾脏细胞,体外用20μg/mlMOG35-55抗原肽刺激淋巴细胞,72h后ELISA检测上清IFN-γ、IL-17、TNF-α水平,及脊髓组织IFN-γ、IL-17、TNF-αmRNA水平。结果显示,与对照组相比,上清内3种细胞因子水平和脊髓中3种细胞因子mRNA水平均明显升高。3.抗IL-33多抗明显降低脾脏、淋巴结CD11c+CD8a+DC数量,但不影响Treg数量体外取注射IL-33抗体的EAE小鼠的脾脏和淋巴结细胞,流式检测CD11c+CD8a+DC的数量,结果提示注射抗体后CD11c+CD8a+DC数量显著下降,CD4+CD25+Foxp3+Treg的数量并没有受到影响。更多还原

【Abstract】 Natural kiler cell is critical in the defence against viral infections and tumors, the most important role of NK cell is the cytotoxicity. They exert their cytotoxicity through forming’immunological synapse’ with their target cells, where they can recognize target cells and release lytic molecules (eg, Granzymes, Perforin etc). Granzyme B enter the target to induce apotosis by the micropore made by perforin. Granzyme B induced cell apotosis is closely related to caspase that cleaves after aspartic acid residues of pro-caspase. The perforin and granzyme in the target cells will be hydrolyzed by protease after exerting their cytotoxic fuction, on the other hand, if NK cell will be destroyed by perforin and granzymes in immunological synapse? The turnover of released lytic molecules are still largely unknown. There are mechanism of cytotoxic lymphocytes to deal with misdirected graznyme B and perforin. For example, serine proteinase inhibitor-9, the specific inhibitor of human granzyme B, could inactivate granzyme B leaked from vesiciles. Moreover, it is well known that NK cell could kill target cells serially, so we can speculate that realeased lytic molecules could be recovered and used again.Human malignant non-Hodgkin’s lymphoma NK92 cell was used as a model to study the fate of released lytic molecule granzyme B under target cell stimulation.1. The study of secondary endocytosis of NK92 cell under target cell stimulation.1.1 Observation of secondary endocytosis of NK92 cell by FM1-43FM1-43 dyes reversibly partition into cell membranes. They have almost no fluorescent properties in aqueous solution, but they fluorescence intensely upon membrane binding. FM1-43 has the added advantage of being easily removed with PBS from the surface but not from the insides of cell membrane. FM1-43 prestained NK92 cell were stimulated by PEC for 1h, then endocytosis of NK92 cell were observed under confocal microscope. NK cell labeled with FM1-43 but neither stimulated by PEC nor washed showed fluroscence in cell membrane; NK cell stimulated by PEC and washed with PBS showed flurescence inside the cell, suggested that NK cell exerted membrane internalization after endocytosis.1.2 Membrane internalization under target cell stimulation was clathrin dependentEndocytic pathways are known to at least include both a clathrin-dependent and a caveolin-dependent pathway. NK92 cell were pretreated with CPZ (clathrin specific inhibitor) and labeled with FM1-43 dye, then cell were subjected to confocal microscope to observe fluroscence inside the cell. Our results showed that fluroscence inside NK92 cell was significantly decreased compared with control. It suggests that NK92 cell membrane internalization is calthrin-dependent. At the same time, fluorescence pretreated with CPZ observed by flow cytometry was 48% of control. NK92 cell were labeled with FM1-43 and separated to two groups:cells were stimulated by PEC cell at 4℃or 37℃, data showed that cells at 4℃cannot exert membrane internalization. It suggests that endocytosis of NK92 cell were dependent temperature.2. Endocytosis stimulated by target cell was associated with the recovery of granzyme B2.1 Recovered granzyme B is sorted to early endosomeNK92 cells were pretreated with CPZ and stimulated with target cells, then cells were harvested at 15min、30min and viewed under confocal microscope to observe colocalizaton of EEA-1 and GzmB. Our results showed that without PEC stimulation, an initial endosomal marker EEA-1 and granzyme B were localized in different compartments of NK cells. After 15 min of stimulation with PEC, most of the EEA-1 had colocalized with some of the granzyme B in NK cells. At 30 min, colocalization of EEA-1 and granzyme B became less prominent than before at 15 min.Further, the clathrin heavy chain targeting siRNA was transfected to NK92 using nucleofection. Transfected cell were viewed under confocal microscope to observe The clathrin heavy chain targeting siRNA, CHC siRNA treatment obtained similar results with CPZ treatment.2.2 Recovered granzyme B was sorted to LAMP-1-positive lysosomeClassical clathrin-dependent endocytosis pathway includes form early endosome, late endosome and lysosome. NK92 cell stimulated by target cell for 60min, cells were harvested, viewed under confocal microscope to observe colocalization of GzmB and lamp-1. Our results showed that colocalization was significant decreased treated with CPZ or CHC siRNA.Proteins sorted for degeneration will be cleaved by lysosomal enzymes and cannot be detected by lysosome surface maker co-localization assay, so the observed colocalization of granzyme B and LAMP-1 means the granzyme B was not recovered for degeneration. The colocalization was not increased treated by lysosomal inhibitor leupepetin, it would appear reasonable to suggest that most of, if not all, the recovered granzyme B was sorted to LAMP-1+lysosomes, re-cycled, and used again.3. Inhibition of clathrin-dependent endocytosis attenuated the cytotoxicity of NK92 cells.The cytotoxicity of NK92 cell against K562 at different E/T ratio (2.5:1,5:1, 10:1) was analysed by FCM. Results showed that cytotoxicity of NK92 cell pretreated with CPZ decreased compared with control (16.93% vs 1.45%, 20.44% vs 1.69%,30.22% vs 2.79%)Similar results obtained from NK92 cell transfected with CHC siRNA, Results showed that cytotoxicity of NK92 cell pretreated with CHC siRNA decreased compared with control (23.18% vs 10.63%,25.31% vs 13.02%, 38.69% vs15.51%)。4. Quantity of granzyme B in the supernatants and NK92 cellsTo examine the effect of CPZ on exocytosis of NK92 cell, at the indicated time(1h,3h,5h) of NK92 cell incubated with K562, supernatants were collected for granzyme B measurement. At the same time, NK92 cell were collected and lysed for western-blot. Granzyme B in the supernatant of NK92 and incubated PEC cells increased in the CPZ pre-treatment group as time went on. The quantity of granzyme B had risen slightly at the 1h and 5h junctions compared with control (1.8 ng/ml vs 2.0 ng/ml,2.45 ng/ml vs 2.8 ng/ml), but decreased compared at 3h with control (2.7 ng/ml vs 2.25 ng/ml). In contrast, CPZ pre-treatment caused a decrease of intracellular granzyme B.5. Conclusion1. NK92 cell could undergo clathrin-dependent endocytosis under target cell stimulation.2. Endocytosis stimulated by target cell was associated with the recovery of granzyme B.3. Granzyme B is recovered into early endosome、late endosome and lamp1+lysosome to be used again.4. Inhibition of clathrin-dependent endocytosis attenuated the cytotoxicity of NK92 cell. Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that is both clinically and pathologically heterogeneous. Mechanistic studies of the complex pathogenesis of MS have relied extensively on animal models of CNS demyelination. The most commonly used animal model for MS is EAE. EAE is induced in multiple species either by immunization of animals with myelin antigens in adjuvant or by adoptive transfer of activated myelin-specific T cells into animals.Interleukin (IL)-33 is a recently described member of the IL-1 family that also includes IL-1p and IL-18. Like IL-1 and IL-18, IL-33 was found to have strong immunomodulatory functions. However, unlike IL-1 and IL-18, which mainly promote T helper (Th) type 1 responses, IL-33 has been shown to induce the production of Th2 cytokines (IL-5 and IL-13). IL-33 was identified as the ligand for the orphan receptor ST2 (also known as IL-1RL1). The ST2 gene encodes two isoforms of ST2 protein:ST2L, a membrane-bound form; and soluble ST2 (sST2). IL-33 mRNA is expressed by multiple organs and cells types in human and mice. IL-33 is expressed by fibroblasts, epithelial cells, endothelials, activated macrophages, particularly in high endothelial venules. IL-33 appears to play an important role in several inflammatory disorders, including asthma, rheumatoid arthritis and anaphylactic shock.IL-33 is highly expressed in CNS, so in this study, we examine the expression and sourse of IL-33 in normal mice and mice with EAE, to further investigate the contribution of IL-33 to the induction and augmentation of EAE.1. Expression of IL-33 in the CNS of mice1.1 Expression of IL-33 in the CNS of normal miceWe investigated the expression of IL-33 in various tissues (including spinal cord, brain, skin, lung, kidney, heart, spleen, liver) of normal C57BL/6 mice by PCR and western-blot. IL-33 mRNA transcripts of the central nerve system tissues (including spinal cord and brain) were more abundant compared with liver and spleen (12.5-fold increase, P<0.01). Immunofluroscence demonstrates that IL-33 is also highly expressed in gray matter of spinal cord and colocolize with nucleus. Immunohistochemistry also demonstrates that IL-33 is expressed in nucleus.1.2 The cell sourse of IL-33 in the spinal cordThe distributions of IL-33 in the spinal cord were examined by double immunostaining of spinal cord frozen sections with specific antibodies to glial fibrillary acidic protein (GFAP), a marker for astrocytes; CD68, a marker for microglia; neuron specific protein, a marker for neurons. Results showed that IL-33 did not colocolize with markers of glial cells and neurons.1.3 The expression of IL-33 in the mice of EAEWe investigated the expression of IL-33 in the spinal cord of mice of EAE by PCR, western-blot and immunohistochemistry. Cytoplasmic and nuclear proteins were prepared from tissues of spinal cords of normal mice and mice with EAE. IL-33 expression is decreased in mice with EAE compared with normal mice. Nuclear fractionation experiment demonstrated that IL-33 was located in the nucleus and cytoplasm, while under EAE, IL-33 was undetectable in the nuclear fraction. This was further confirmed by immunohistochemistry, as shown in staining of parallel sections showed that IL-33 was expressed in cytoplasm and nuclear in normal mice, but under inflammation in EAE, cells were swelling and IL-33 was mainly expressed in cytoplasm.1.4 The expression of ST2 in the spinal cord of normal mice and EAE miceST2 was expressed in in the white matter of normal, but in the mice of EAE, ST2 was expressed in the gray matter.2. Treatment of EAE mice with rIL-33 and IL-33 polyclonal antibody2.1 The effect of IL-33 in EAE miceWe examined the effect of recombinant IL-33 on the development of EAE. C57BL/6 mice were immunized with MOG35-55/CFA and were injected i.p. prior of immunization or daily with IL-33 (1μg per mouse) for 7 days from onset of the disease. Mice treated with IL-33 did not change the clinical score of the disease.2.2 Treatment of IL-33 polyclonal antibody in EAE miceMice received a neutralizing polyclonal antibody against murine IL-33. Anti-IL-33 antibody was given i.p.150μg per mouse every three days prior of immunization or from onset of disease. As control, mice were given the same amount of rabbit control IgG (purified normal rabbit IgG) by i.p. injection. To our surprise, mice developed significantly more severe disease as assessed by clinical score compared with control. Consistent with these observations, a massive infiltration of CD4+T cells were observed within the spinal cords of mice treated with anti-IL-33 antibody 21 days after the first immunization.2.3 Development of Th1-and Th17-type responses is enhanced in mice treated with anti-IL-33 antibodyWe harvested spleen cells from WT, IgG and anti-IL-33 antibody treated mice at the peak of the disease. Splenocytes were cultured in the presence of 20μg/ml of MOG35-55 peptide for 72 h. Supernatants of the cultures were harvested to measure T cell cytokine production by ELISA. Wild-type produced none or undetectable amounts of cytokines. Anti-IL-33 antibody treated group showed higher production of and IL-17A, IFN-γand TNF-αcompared with IgG treated group.We next examined the cytokine mRNA expression in the spinal cords of control (IgG treated) mice and anti-IL-33 antibody treated mice with EAE. Spinal cords were harvested at the peak of the disease. Total mRNA was extracted from the tissue in order to measure the cytokine mRNA by real-time RT-PCR analysis. At day 21 after immunization, we found that in the spinal cord of anti-IL-33 antibody treated mice, there was a significant higher expression of IL-17A, IFN-γ, TNF-α, compared with control mice. Anti-IL-33 treatment caused 2.5-fold increase of mRNA for IL-17A,3.2-fold increase for IFN-γ,3.8-fold increase for TNF-α.2.4 Anti-IL-33 polyantibody reduced the number of CD11c+CD8a+DC in spleen and draining lymph nodes but did not affect the number of regulatory T cells in spleenWe analyzed the draining lymph nodes and spleen of anti-IL-33 antibody treated mice and found significantly reduced numbers of CD8a+CD11c+DC by flow cytometry. However, no obvious changes in the frequency of CD4+CD25+Foxp3+ regulatory T cells was observed in spleen treated with anti-IL-33 antibody compared with IgG control. The regulatory T cells of draining lymph nodes were almost undetectable.更多还原