【作者】 田莉；

【导师】 龚非力； 谭政；

【作者基本信息】 华中科技大学 ， 免疫学， 2011， 博士

【摘要】 IL-16最初作为CD4分子的配体而被发现,其可趋化所有CD4+细胞,尤其是CD4+T细胞。IL-16在不同的病理过程中发挥不同的作用,在有些疾病中是趋化因子,在有些疾病中为促炎症因子,在其他疾病中还可能作为抑制因子发挥作用。、结核性胸腔积液(TBPE)内活性IL-16水平增高前期研究发现：结核性胸腔积液(TBPE)和恶性胸腔积液(MPE)中均含高水平IL-16,且TBPE内活性IL-16水平明显高于MPE。本文借助ELISA检测胸水上清活性IL-16水平,结果显示：与MPE相比,TBPE内活性IL-16水平增高。此结果提示,结核性胸水内活性IL-16来源可能与恶性胸水内活性IL-16来源不同,或二者IL-16的来源(产生细胞)相同,但表达水平各异。二、胸腔积液内活性IL-16的细胞来源正常情况下,IL-16前体蛋白组成性表达于各种免疫细胞和非免疫细胞内。文献已报道：与恶性胸水相比,结核性胸水淋巴细胞中CD4+T细胞数量及比例均明显升高,而CD8+T细胞比例降低。为探讨TBPE内IL-16水平升高是否由于CD4+T细胞数量增加所致,借助流式细胞术检测两种胸腔积液中T细胞亚群(CD3+CD8+T细胞和CD3+CD8-T细胞)和B细胞胞内IL-16(包括IL-16前体)表达。1.T细胞和B细胞组成性表达IL-16流式细胞术检测结核病和肿瘤患者外周血、胸腔积液T/B细胞中IL-16+细胞比例。结果显示,两组患者外周血和胸水内几乎全部T细胞(包括CD3+CD8-和CD3+CD8+T细胞)均组成性表达IL-16(包括IL-16前体蛋白),绝大多数B细胞也表达IL-16。2.IL-16+细胞中CD3+CD8-T细胞占绝对优势进一步分析IL-16+细胞中淋巴细胞亚群比例(包括T细胞和B细胞)。结果表明,两组患者外周血和胸水内IL-16+(包括IL-16前体蛋白)单个核细胞中,CD3+CD8-T细胞占绝对优势,其次是CD3+CD8+T细胞。3.T细胞内IL-16表达水平(平均荧光强度MFI)高于B细胞两组患者胸腔积液IL-16+T细胞(包括CD3+CD8+IL-16+T细胞和CD3+CD8-IL-16+T细胞)内IL-16表达水平(MFI)明显高于IL-16+B细胞。两组患者外周血CD3+CD8-IL-16+T细胞IL-16表达水平(MFI)显著高于IL-16+B细胞。结论：就单个细胞而言,T细胞内IL-16表达水平较高。4.TBPE和MPE内CD4+T和CD8+T细胞IL-16分泌水平存在差异ELISA检测结果显示：结核性胸水来源的CD4+T细胞培养上清活性IL-16水平(193.5±43.1ng/L)明显低于CD8+T细胞培养上清活性IL-16水平(377±100.2ng/L)；恶性胸水来源CD4+T细胞培养上清活性IL-16水平(352.8±100.2ng/L)显著高于CD8+T细胞培养上清活性IL-16水平(261.5±k62.2ng/L)。三、对结核性胸腔积液内IL-16水平增高的初步机制研究胸水CD4+T细胞和CD8+T细胞胞内IL-16蛋白表达水平无明显差异,但两类细胞所分泌活性IL-16水平有明显差异。作者猜测,上述IL-16分泌水平的差异可能与细胞内Caspase-3活化相关。文献已报道,活化的Caspase-3在IL-16前体剪切和活性IL-16分泌中发挥重要作用。活化的Caspase-3可剪切IL-16前体蛋白,所产生的羧基端121个氨基酸肽段可被分泌至细胞外,此肽段即活性IL-16。本文借助流式细胞术检测结核性及肿瘤PB和PE中CD3+CD8-/CD3+CD8+T细胞内Caspase-3活化水平,结果发现：结核性胸腔积液CD3+CD8-T细胞内Caspase-3活化水平高于CD3+CD8+T细胞。而ELISA检测结果显示,CD3+CD8+T细胞IL-16分泌水平比CD3+CD8-T细胞高。这一看似矛盾的实验结果提示：CD4+T细胞和CD8+T细胞分泌IL-16的机制可能各异,可能还存在其他途径参与IL-16剪切。四.结核性胸腔积液T细胞分泌IL-16的病理生理意义文献已报道：结核性胸腔积液内IFN-γ、IL-12p40和IL-18水平显著高于恶性胸腔积液,提示结核性胸腔积液以Th1细胞应答占优势,而恶性胸腔积液内Th2细胞应答较强；Elizabeth等发现,CCR5存在的条件下,IL-16与CD4结合明显增强,导致IL-16趋化细胞的能力增强,而CCR5优势表达于Thl细胞表面。因此,IL-16对Th1细胞具有强的趋化作用,此与结核性胸膜发生、发展相关。我们基于上述实验结果推测：结核性胸水内CD8+T细胞可能通过某种机制而快速释放活性IL-16,后者通过与CD4结合,可趋化、募集CD4+T细胞(主要是Th1细胞)浸润至胸水内。从胸膜外其他组织浸润至胸水内的CD4+T细胞(主要是Th1细胞)继而分泌更多IL-16,介导更多细胞趋化、募集和活化,形成正反馈机制,从而参与结核性胸膜炎发生和发展。结论1.结核性胸水内IL-16水平增高2.胸水T细胞高表达IL-16,是IL-16的主要来源,结核性胸水T细胞增多与IL-16水平增高呈正相关3.Caspase-3并非唯一参与IL-16剪切和分泌的蛋白,可能还有其他酶或蛋白影响IL-16分泌4.不同类型细胞中,IL-16分泌机制可能各异5.IL-16可通过选择性趋化Th1细胞而参与结核性胸膜炎发生、发展更多还原

【Abstract】 IL-16 is a cytokine found as a ligand for CD4 and can chemoattract all CD4+ cells, especially CD4+T cells. Subsequent researches demonstrated cytokine IL-16 was not only chemokine, but also pro-inflammatory factor, in addition to inhibitory cytokine.Ⅰ. Increased IL-16 released in tuberculous pleural effusionIt was observed the concentration of IL-16 in tuberculous PE was significantly greater than that in malignant PE. ELISA was used to detect the concentration of secreted IL-16 in TBPE and MPE. It was found increased IL-16 released in tuberculous pleural effusion, in accordance with previous research. These results demonstrated that different or more pleural sources of IL-16 existed in tuberculous patients, or different expression levels despite same source.Ⅱ. The cell source of active IL-16 in pleural effusionPro-IL-16 is constitutively expressed in immune and non-immune cells. Lymphocytes, especially CD4+T cells, were dominant in pleural effusion. It was reported the absolute lymphocyte counts, especially CD4+T cells counts, displayed the highest values in tuberculous PE and showed a significant increase of CD4+T cells and decrease of CD8+T cells in camparison with that in malignant PE. To find out whether the increased CD4+T cells are the source of increased IL-16 in PE, FCM was used to evaluate IL-16(including pro-IL-16) protein expression in T cells(including CD3+CD8+T cells and CD3+CD8-T cells) and B cells.1. Cytokine IL-16 (including pro-IL-16) constitutively expressed in T cells and B cells both in blood and pleural effusion FCM was used to test the percentage of IL-16+cells in T cells and B cells from TB patients and Malignant patients. It was found IL-16 were almost constitutively expressed in all T cells (CD8+and CD8-) and a vast majority of B cells, not only in PB but also in PE.2. CD3+CD8- T cells were dominant in IL-16 positive cells in all situations.In the reversed orientation, the percentage of CD3+CD8-T cells, CD3+CD8+T cells and CD19+B cells in IL-16 positive cells were also analysed. Data demonstrated CD3+CD8-T cells were dominant in IL-16 positive cells in all situation, followed by CD3+CD8+T cells.3. The MFI of IL-16 in T cells is much higher than in B cellsThe average IL-16 protein expressions in IL-16+T cells (including CD3+CD8+T cells and CD3+CD8-T cells) showed obviously greater values in comparison with those in IL-16+B cells from tuberculous and malignant pleural effusion.The protein amounts of IL-16 were much greater in CD3+CD8-IL-16+T cells than those in CD19+IL-16+B cells in Tuberculous and malignant blood.For single cell, T cell expressed more IL-16 proteins.4. Different IL-16 secretion abilities of CD4+T cells and CD8+T cells in Tuberculous group and malignant groupThe mean bioactive IL-16 levels secreted by CD4+T cells (193.5±43.1 ng/L) were significant lower than those secreted by CD8+T cells (377±100.2 ng/L) in Tuberculous pleural effusion. In contrast, bioactive IL-16 concentrations secreted by CD4+T cells (352.8±100.2 ng/L) were significant higher than those secreted by CD8+T cells (261.5±62.2 ng/L) in malignant PE.III. The mechanism involved in IL-16 increasment in pleural effusionThere is no difference of intracellular IL-16 protein levels but great difference of secreted active IL-16 levels between CD4+T cells and CD8+T cells in PE. We hypothesized that this critical difference might lie with Caspase-3 activation. It was reported active Caspase-3 was an important mechanism for pro-IL-16 processing and bioactive IL-16 releasing. FCM was used to detect the percentage of CD3+CD8+active Caspase-3+T cells, CD3+CD8-active Caspase-3+T cells and CD19+active Caspase-3+B cells in PE and PB from both TB patients and Malignant patients. Our data showed an obviously increase of the percentages of active Caspase-3 in CD3+CD8-T cells (4.4±1.1%) in comparison with CD3+CD8+T cells (2.7±0.6%) in PE from Tuberculous patients.IV. Active IL-16 released by T cells may participate in development of TB diseaseThe greater levels of IFN-gamma, IL-12p40 and IL-18 in tuberculous pleural effusion than in malignant pleural effusion demonstrated T cells in tuberculous pleural effusions are polarized to Thl, and Th2 may be dominant in malignant pleural effusion. Elizabeth has used T cells from CCR5null mice and determined CCR5 significantly increased IL-16 binding to CD4 and IL-16-induced migration. CCR5 was expressed predominantly in Thl cells, so IL-16 could preferentially chemoattract Thl cells. So IL-16 may participate in the induction and development of TB disease.Based on our data, one could hypothesize that CD8+T cells may represent a more rapidly release mechanism of IL-16 in tuberculous PE, and would contribute to recruitment of CD4+T cells by secretion of bioactive IL-16. Consequently, the CD4+T cells which migrating from other place into PE secrete more IL-16 and serve to function as a positive feedback mechanism for further cell recruitment and activation. It is the possible process for IL-16 involved in the development of TB disease.Conclusion1. Increased IL-16 released in tuberculous pleural effusion2. In comparion with other cells, T cells express higher levels of IL-16 protein, T cells are the main source of IL-16 in pleural effusion. The increasing of T cells amount is positively correlated with higher secreted IL-16 levels in TB pleural effusion.3. Caspase-3 may not be the unique enzyme for pro-IL-16 processing, some other proteins or enzymes have also involved in processing and secretion of IL-16.4. In different types of cells, there are different pathway for IL-16 secretion5. IL-16 may participate in development of TB disease, through preferentially chemoattracting Thl cells into pleural effusion更多还原