【作者】 冯晓明；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2008， 博士

【摘要】 白介素27(IL-27)是一个IL-12相关的多功能的细胞因子,它在免疫和炎症发生的过程中表现出似乎相互矛盾的作用。IL-27包含p28和EBB两个亚基,作用于WSX-1亚基(也叫作TCCR)和gp130亚基构成的异二聚体受体(IL-27R)传递信号。很多类型细胞如T细胞、自然杀伤细胞、B细胞、单核细胞、肥大细胞和中性粒细胞都表达IL-27R。IL-27R与配体的结合诱导了胞内的信号途径,包括Janus kinases(JAK)和STAT转录因子。起初IL-27被认为通过诱导Ⅰ型辅助性T细胞(TH1 cells)的分化而促进炎症的发生。很多研究表明,在TH1分化的早期,IL-27诱导转录因子T-bet的表达,T-bet可上调初始CD4+T细胞IL-12受体的β2亚单位(IL-12Rβ2)的表达,使其获得对IL-12的反应性;也有研究表明,IL-27可以不依赖于T-bet而促进TH1分化。然而,在许多感染和自身免疫疾病动物模型中,缺乏IL-27受体的小鼠在TH1和TH2型反应中都发展成为过度的病理性炎症反应,这表明IL-27有抗炎作用。就IL-27的抗炎机制而言,IL-27可通过抑制活化的CD4+T细胞IL-2的产生而抑制其增殖。IL-27还能在很多动物模型中如实验性自身免疫性脑脊髓炎(EAE,多发性硬化的动物模型),抑制产IL-17的辅助性T细胞(TH17细胞)介导的炎性反应。最近的研究还发现了IL-27的另一个新功能,即IL-27能诱导产生IL-10的调节性T细胞的产生,IL-10参与IL-27对IL-17的抑制作用,但不为IL-27抑制Th1细胞IL-2的产生所必需。在继发性转移而形成的EAE模型中,IL-10介导了IL-27对脑膜自身反应性T细胞的抑制作用。IL-27的抗炎和促炎作用有待于进一步研究。MHCⅡ类分子和它们的辅助分子,如恒定链(Ii)和非经典的MHCⅡ类分子(DM和DO)在胸腺分化(thymic education)的过程中限制机体对自身和外源性抗原的免疫反应有重要作用,他们的表达也对外周T细胞激活至关重要。MHCⅡ的表达被很多细胞因子包括干扰素-γ(IFN-γ)调控,IFN-γ能通过上调免疫细胞MHCⅡ的表达而增强免疫反应。MHCⅡ基因的转录主要由一个关键的的调节子,Ⅱ类转录活化子(classⅡtransactivator,CIITA)控制,CIITA的基因至少包含3种不同的启动子(CIITApⅠ、CIITApⅢ和CIITApⅣ),这些启动子表现出组织细胞特异性的激活及对细胞因子的不同反应性。我们的研究主要集中在探讨IL-27促炎的机制。首先我们观察了IL-27对内皮细胞的免疫调控作用。IL-27刺激人脐静脉内皮细胞(HUVECs)可快速诱导干扰素调节因子1(IFN regulatory factor-1,IRF-1)的表达,并显著升高CIITA亚型Ⅳ的表达。与CIITAⅣ的表达相一致,MHCⅡ基因的表答也升高。IL-27也能增强MHCⅠ类分子的表达。另外,在IL-27的作用下,β2-m和TAP-1 mRNA的表达水平也升高。基因芯片分析表明IL-27可以显著上调内皮细胞很多和免疫调节相关的基因,包括趋化因子CXCL9、CXCL10、CX3CL1的表达。这些结果首次表明IL-27能在内皮细胞上调MHCⅡ类和Ⅰ类分子的表达,提示IL-27对血管内皮的免疫功能有重要的调控作用。我们进一步观察IL-27对THP-1单核细胞系的作用。THP-1细胞是研究单核细胞的很好的模型。与在内皮细胞相似,IL-27可上调THP-1细胞CIITAⅢ和CIITAⅣ的mRNA表达水平,导致细胞表面MHCⅡ类分子的表达增加,以及HLA-DM和DO的表达水平升高。另外,IL-27可上调beta-2m、TAP-1和MHCⅠ类分子的表达。进一步的研究表明,IL-27可增强THP-1细胞表面共刺激分子CD80和CD86及粘附分子CD54的表达。这些结果表明IL-27可促进单核巨噬细胞的抗原提呈功能。为了进一步分析IL-27通过何种信号机制参与调节CIITA和MHCⅡ的表达,我们用ERK抑制剂U0126、p38MAPK抑制剂SB203580和PI3K抑制剂LY294002来观察各条信号通路在IL-27诱导CIITA和MHCⅡ表达中的作用。我们的结果表明p38MAPK和PI3K信号通路都为IL-27作用下最优化的MHCⅡ的表达所必需,而ERK信号通路对IL-27诱导的MHCⅡ表达有抑制作用。另外,PI3K信号通路也为IL-27诱导最优化的MHCⅠ表达所必需。这些结果表明PI3K信号通路参与正调控IL-27介导的效应,而ERK信号通路对IL-27诱导的MHCⅡ类分子的表达有抑制作用。进一步的研究表明,TLR2激动剂Pam3CSK4和TLR4激动剂LPS介导的TLR信号能部分阻断IL-27诱导得THP-1细胞MHCⅡ的表达。LPS的抑制效应部分通过ERK和p38MAPK信号途径介导。研究表明IL-27对很多实体瘤有很强的抗肿瘤效应。IL-27的抗肿瘤机制主要包括诱导CD4+T细胞、CD8+T细胞和NK细胞的活化。IL-27还能通过诱导IP-10和MIG的表达而抑制肿瘤的血管新生。我们的另外一项研究是构建IL-27的表达载体而评估其对小鼠白血病模型的抗肿瘤效应。我们用重叠延伸PCR来扩增信号肽和单链IL-27融合基因,然后把这些基因插入许多真核表达载体中,包括pIRES2-EGFP、pcDNA3.1(-)、p3XFLAG-CMV-9和PCAGEN。用免疫沉淀和western blot检测IL-27表达载体转染的293T细胞上清中IL-27的分泌和表达。我们用液压基因转移法(hydrodynamic gene transfer,HGT)将IL-27表达载体导入小鼠体内后,在小鼠血清中也能检测到IL-27的表达。我们用能否诱导HUVECsCIITAmRNA的表达来鉴定IL-27的生物学活性。总之,我们的研究首次表明IL-27能够诱导单核细胞和内皮细胞CIITA和MHCⅡ的表达。IL-27还能上调MHCⅠ类分子、共刺激分子和粘附分子的表达。另外,IL-27能激活一个重要的转录因子IRF-1。ERK、p38MAPK和PI3K信号通路可被IL-27直接激活并在调节IL-27效应的过程中有关键作用。这些结果表明IL-27有调节抗原提呈的新功能,并揭示了一种IL-27促进T细胞的激活和获得性免疫发生的新机制。更多还原

【Abstract】 Interleukin-27(IL-27) is IL-12-related pleiotropic cytokine that has seemingly paradoxical functions in inflammation.IL-27 contains two subunits,p28 and EBI3 and signals through a heterodimeric receptor(IL-27R) consisting of the WSX-1 subunit(also called TCCR) and the gp130 subunit.Several cell types,including T cells,natural killer cells,B cells,monocytes,mast cells and neutrophils,express IL-27R.Ligation of IL-27R induces intracellular signaling involving Janus kinases and STAT transcription factors.Originally,IL-27 was thought to promote inflammation by inducing T helper type 1(TH1) differentiation early during immune responses.Several studies have shown that during the early stage of TH1 differentiation,IL-27 induces expression of the transcription factor T-bet,which triggers upregulation of theβ2-subunit of the interleukin 12 receptor(IL-12Rβ2) on naive T cells,thus conferring responsiveness to IL-12.An IL-27-induced T-bet-independent pathway of TH1 differentiation has also been reported.However,in infectious and autoimmune animal models,mice deficient in IL-27R develop excessive,pathological inflammation during both TH1 and TH2 responses.Regarding the anti-inflammatory effects of IL-27,it has been shown that IL-27 inhibits the proliferation of activated CD4+T cells by suppressing IL-2 production.Furthermore, IL-27 suppresses the proinflammatory immune responses of IL-17-producing T helper cells(TH-17 cells) in several animal models,including experimental autoimmune encephalomyelitis(EAE),an autoimmune inflammatory demyelinating disease of the central nervous system and a model of human multiple sclerosis.Recent works also identified a novel role of IL-27 in the induction of CD4+ and CD8+ effector T cells positive for IL-10,and demonstrated IL-10 was involved in the IL-27-mediated suppression of IL-17 but was dispensable for the IL-27-induced suppression of IL-2. IL-10 mediated the suppressive effect of IL-27 on encephalitogenic T cells in adoptively transferred EAE.The mechanisms that orchestrate the pro-inflammatory and anti-inflammatory effects of IL-27 are incompletely understood.Major histocompatibility complex classⅡ(MHCⅡ) proteins and their accessory molecules,such as the invariant chain(Ii) and the nonclassical MHCⅡproteins(DM and DO),are crucial for restricting immune reactivity to self versus foreign antigens during thymic education,and their expression level is also crucial for determining T-cell activation in the periphery.MHCⅡexpression is regulated by multiple cytokines,including interferon-gamma(IFN-γ),which can enhance the immune response by upregulating MHCⅡexpression in immune cells.MHCⅡgene transcription is regulated centrally by the master regulator of its activation,the classⅡtransactivator(CⅡTA).The gene encoding CⅡTA uses at least three distinct promoters (CⅡTApI,CⅡTApⅢand CⅡTApⅣ),and display tissue-restricted expression and differential responses to cytokines.Our study focused on the the mechanisms by which IL-27 mediated its pro-inflammatory effects.Firstly we explored the immunoregulatory effects of IL-27 on endothelial cells(EC).We reveal a role for IL-27 in the induction of MHC expression in primary human umbilical vein endothelial cells(HUVECs).Stimulation of HUVECs by IL-27 rapidly induces IFN regulatory factor-1(IRF-1),and dramatically increases the expression of CⅡTA isoformⅣ.Expression of CⅡTAⅣcorrelates with increased MHC classⅡgene expression.IL-27 also enhances expression of MHC classⅠmolecules.Furthermore,expression ofβ2-m and TAP-1 transcripts increases in response to IL-27.Microarray analysis demonstrates that IL-27 significantly up-regulates a panel of genes that correlate with immune regulation,including the chemokines CXCL9,CXCL10 and CX3CL1 in HUVECs. This first demonstration that both MHCⅡandⅠexpression are increased in EC after IL-27 stimulation suggests that IL-27 may be important in conferring immune function on vascular endothelium.We further examined the effects of IL-27 on THP-1 monocytic cells,a well characterized model of monocytes.Similarly,we revealed that IL-27 increased both CⅡTAⅢand CⅡTAⅣmRNA levels,leading to an enhanced suface expression of classⅡmolecules and elevated mRNA levels of HLA-DM and DO.In addition,IL-27 up-regulates beta-2m,TAP-1 and classⅠMHC expression.Furthermore IL-27 enhances co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54 expression in THP-1 cells.These observations indicate a crucial role of IL-27 in regulating the antigen presentation function of monocytes/macrophages.To delineate the signaling mechanisms by which IL-27 regulate the expression of CⅡTA and classⅡMHC in THP-1 cells,we use ERK inhibitor U0126,p38MAPK inhibitor SB203580 and PI3K inhibitor LY294002 to examine the roles of various signaling pathway in IL-27 induced CⅡTA and classⅡMHC expression.Our results demonstrate that both p38MAPK and PI3K signalings are essential for optimal expression of classⅡMHC in THP-1 cells in response to IL-27,whereas ERK activation plays an inhibitory role in IL-27 induced classⅡMHC expression.PI3K is also required for maximal expression of classⅠMHC expression in THP-1 cells. These data suggest that PI3K signal playways are critically involved in IL-27-mediated effects,yet ERK act as negative regulator of classⅡexpression. Further work using TLR2 agonist Pam3CSK4 and TLR4 agonist LPS shows that these TLR signalings partially prevent IL-27 induced CⅡTA and MHC expression in THP-1 cells.The inhibitory effect of LPS on IL-27 effects is partially mediated through ERK and p38MAPK signaling.IL-27 has been reported to have potent anti-tumor activity against a varity of solid tumors through mechamis involving the induction of CD4+ and CD8+ T cells,and NK cells.IL-27 can also inhibit tumor angiogenesis through induction of chemokines IP-10 and MIG.Another work we have done is construction of IL-27 expression vectors to assess the anti-leukemic activity of IL-27 against murine leukemia models. We used overlap PCR to amplify the signal peptide and the single chain IL-27 gene, and inserted the gene into a variety of eukaryiotic expression vectors including pIRES2-EGFP,pcDNA3.1,p3×FLAG-CMV-9 and PCAGEN.Immunoprecipitation and western blot have been performed to confirm the expression and secrection of IL-27 from the supernatant of 293T cells transfected with IL-27 expressing vectors. Expression of IL-27 was also detected in the serum of mice treated with IL-27 expressing vectors by hydrodynamic gene transfer(HGT).The bioactivity of IL-27 was confirmed by the induction of CⅡTA expression in HUVECs.In conclusion,our work for the first time demonstrates that IL-27 increases CⅡTA and classⅡMHC expression in both monocytes and endothelial cells.IL-27 also up-regulates the expression classⅠMHC,co-stimulatory molecules and adhesion molecules.In addition,IL-27 could activate the critcal transcription factor IRF-1. ERK,p38MAPK and PI3K signaling pathways can be directly activated by IL-27,and they play cucial roles in modulating IL-27 mediated effects.Our results demonstrate novel roles of IL-27 in the regulation of antigen presentation and uncover a new mechanism by which IL-27 promote the activation of T cells and the generation of effective adaptive immune responses.更多还原