【作者】 施维维；

【导师】 袁发焕；

【作者基本信息】 第三军医大学 ， 内科学， 2013， 博士

【摘要】 研究背景与目的抗中性粒细胞胞浆抗体(ANCA)相关性血管炎(ANCA associated vasculitis, AAV)是一组累及多个器官系统的全身性自身免疫性疾病，主要包括韦格纳氏肉芽肿(Wegener’s granulomatosis,WG)、显微镜下多血管炎(microscopic polyangiitis, MPA)、变应性肉芽肿性血管炎(Churg-Strauss syndrome,CSS)和肾脏局限性血管炎(Renal-limitedvasculitis,RLV)。当肾脏受累时，通常表现为寡免疫复合物局灶节段坏死型肾小球肾炎(PiFSGN)，该病进展迅猛、死亡率高，严重威胁人类健康。ANCA是针对中性粒细胞胞浆中抗原物质而产生的一组异质性自身抗体，是目前诊断ANCA相关性血管炎(AAV)的敏感血清学指标。ANCA特异性经典靶抗原主要包括：蛋白酶3(Proteinase3，PR3)和髓过氧化物酶(Myeloperoxidase，MPO)。目前的研究已经证实，ANCA对ANCA相关性血管炎(AAV)的发生有致病作用，它与中性粒细胞表面相对应靶抗原的结合是诱发该病的核心机制。在正常情况下，静息状态的中性粒细胞膜表面并不表达与ANCA相对应的靶抗原，而仅仅在中性粒细胞质内才有表达。只有经过炎性因子预处理或者刺激的中性粒细胞，这些靶抗原才会由胞质内转移并暴露到细胞膜表面，ANCA才能通过其Fab2段与中性粒细胞表面相应的靶抗原结合，从而激活中性粒细胞，产生局部炎症反应。邻近吞噬细胞对凋亡中性粒细胞及其细胞碎片的及时清除是消除炎性反应的必要条件，起着维持机体稳态的作用。如果机体对凋亡中性粒细胞的清除出现障碍，则会导致炎性疾病的发生。在ANCA相关性血管炎(AAV)患者的病变微血管周围及肾组织节段坏死区，聚集着大量未清除的激活状态中性粒细胞，以及位于胞外的溶酶体酶及其核碎片，这些中性粒细胞浸润及其细胞成分沉积的程度与肾脏损伤程度密切相关。有研究指出，中性粒细胞凋亡延迟,存活时间延长和过度激活,致使大量蛋白酶、炎性介质和氧自由基释放和分泌,可能是造成炎性反应过度和组织损害的关键原因，同时也增强了与血管内皮细胞黏附能力，破坏内皮细胞完整性，诱发并加重炎症反应。中性粒细胞胞外捕网(NETs)是新近发现的一种不同于凋亡、坏死以及自噬的死亡方式。通过将释放自身线粒体或核内染色质dsDNA骨架到胞外，形成网状结构，粘附、阻碍并杀死入侵病原体，以此起到防御病原体入侵的功能。然而，与此同时在释放到细胞外的染色质dsDNA骨架上也负载着多种抗微生物肽和自身抗原，如：髓过氧化酶(MPO)，蛋白酶3(PR3)，弹性蛋白酶(Elastase)，组织蛋白酶G(Cathepsin G)以及乳铁蛋白(Lactoferrin，LF)等。这说明，发生中性粒细胞胞外捕网(NETs)为自身免疫性疾病的发生提供了危险的自身抗原，是造成自身免疫性疾病的重要原因之一。树突状细胞是机体免疫系统功能最强的抗原递呈细胞(Antigen-presenting cells，APC)，在诱导机体免疫反应和维持耐受性过程中起着至关重要的作用，也是唯一能活化CD4~+T细胞的递呈抗原细胞。经树突状细胞激活后的CD4~+T细胞朝着Th17细胞分化，会打破免疫耐受。已证实，在ANCA相关性血管炎患者肾组织病理标本中，有大量不成熟的树突状细胞浸润，并且参与激活CD4~+Th17细胞增殖分化，促发了ANCA相关性血管炎(AAV)。溶酶体膜蛋白-2(LAMP-2)抗体是近年来发现的ANCA新亚型，其靶抗原与MPO、PR3的最大不同在于：LAMP-2不仅在静息的中性粒细胞胞内及细胞表面均有表达，并且能够自由地穿梭于细胞内外，为循环抗体的穿梭提供条件。LAMP-2抗体能直接通过与正常中性粒细胞表面靶抗原结合，激活中性粒细胞。目前已经证实，LAMP-2抗体普遍存在于寡免疫复合物局灶节段坏死性肾小球肾炎(PiFSGN)患者血清中，而且它与该型肾炎病变的相关性为PR3抗体和MPO抗体的两倍。但是LAMP-2抗体诱发ANCA相关血管炎的具体机制尚不清楚。本课题拟采用免疫磁珠分选法、流式细胞术、ELISA、免疫组织化学法、激光共聚焦等技术，观察LAMP-2抗体对中性粒细胞(中性粒细胞凋亡、中性粒细胞胞外捕网(NETs)形成)的影响，LAMP-2抗体作用的中性粒细胞对不成熟树突状细胞活化程度、细胞分泌炎性因子微环境对初始CD4~+T细胞向Th17分化的影响，以探讨LAMP-2抗体介导的中性粒细胞凋亡异常和胞外捕网在ANCA相关性血管炎发生中的免疫机制，为临床诊断、治疗ANCA相关性血管炎提供新的靶点和思路。实验方法第一部分1.通过免疫磁珠法，分选健康志愿者外周血CD14~+单核细胞，体外培养获得不成熟树突状细胞，并通过光学显微镜和流式细胞术检测对其进行鉴定。2.采用梯度密度离心法，分离出健康志愿者外周血中性粒细胞，并通过免疫荧光法和流式细胞术检测对其进行鉴定。3.通过流式细胞术检测， LAMP-2抗体直接刺激中性粒细胞24h后，中性粒细胞凋亡率的变化。4. LAMP-2抗体直接刺激中性粒细胞24h后，经过0.01M PBS缓冲液多次洗涤得到凋亡中性粒细胞，随后通过FITC-Annexin V标记这些凋亡中性粒细胞，将它们与树突状细胞共培养。采用激光共聚焦技术检测，树突状细胞能否吞噬凋亡中性粒细胞，建立树突状细胞-凋亡中性粒细胞荧光示踪吞噬体系。5.在树突状细胞-凋亡中性粒细胞荧光示踪吞噬体系中，结合树突状细胞表面活化标志(CD80、CD83、CD86和HLA-DR)，通过流式细胞术检测，树突状细胞吞噬凋亡中性粒细胞后的表型变化。6.通过ELISA检测，LAMP-2抗体作用的凋亡中性粒细胞对树突状细胞分泌的细胞因子谱(IL-1β、IL-6、IL-23)的变化。7.通过免疫磁珠法，分选健康志愿者外周血初始CD4~+T细胞，并通过流式细胞术检测对其纯度进行鉴定。8.在上述处理组中性粒细胞与树突状细胞共培养体系中加入健康志愿者外周血来源初始CD4~+T细胞，通过流式细胞术检测以确定初始CD4~+T细胞的分化方向。第二部分1.通过免疫荧光法，鉴定LAMP-2抗体刺激中性粒细胞3h后，中性粒细胞胞外捕网（NETs）的形成，以及自身抗原(LAMP-2、MPO、PR3)的暴露情况。2.将PKH-26标记的树突状细胞，与SYTOX green标记的LAMP-2抗体诱发了胞外捕网的中性粒细胞共培养4h。采用激光共聚焦技术观察，经胞外捕网释放出的染色质丝状物与树突状细胞的接触情况。3．将PKH-26标记的树突状细胞与LAMP-2抗体诱发了胞外捕网的中性粒细胞共培养18h，采用FITC-PR3、MPO和LAMP-2单克隆抗体标记经胞外捕网暴露的自身抗原。采用激光共聚焦技术观察，树突状细胞能否摄入暴露在细胞外的自身抗原(LAMP-2、MPO、PR3)，建立树突状细胞-自身抗原荧光示踪吞噬体系。4.在树突状细胞-自身抗原荧光示踪吞噬体系中，结合树突状细胞表面活化标志(CD80、CD83、CD86和HLA-DR)，通过流式细胞术检测，树突状细胞摄入中性粒细胞胞外捕网（NETs）暴露出的自身抗原后表型的变化。5.通过ELISA，检测LAMP-2抗体介导的中性粒细胞胞外捕网对树突状细胞分泌的细胞因子谱(IL-1β、IL-6、IL-23)的变化。6.在上述处理组中性粒细胞与树突状细胞共培养体系中，加入健康志愿者外周血来源初始CD4~+T细胞，通过流式细胞术检测以确定初始CD4~+T细胞的分化方向。结果第一部分1.经LAMP-2抗体作用24h后，中性粒细胞凋亡率较自然凋亡对照组显著降低。2.不成熟树突状细胞吞噬了FITC-Annexin V标记的LAMP-2抗体作用的凋亡中性粒细胞。建立了树突状细胞-凋亡中性粒细胞荧光示踪吞噬体系。3.不成熟树突状细胞吞噬了LAMP-2抗体作用的凋亡中性粒细胞后，其表面标志(CD80、CD83、CD86和HLA-DR)升高。4. LAMP-2抗体作用的凋亡中性粒细胞促进树突状细胞分泌IL-1β、IL-6，未检测到IL-23。5. LAMP-2抗体作用的凋亡中性粒细胞与树突状细胞共培养体系，未检测到初始CD4~+T细胞向Th17细胞分化。第二部分1. LAMP-2抗体作用于健康志愿者外周血中性粒细胞3h，诱发中性粒细胞胞外捕网(NETs)的发生，释放的染色质dsDNA，并暴露自身抗原(MPO、PR3和LAMP-2)。2.不同时相点显示树突状细胞对自身抗原摄入的过程：4h时，LAMP-2抗体诱发中性粒细胞胞外捕网(NETs)“吐出”的染色质dsDNA丝状物，紧紧与不成熟树突状细胞相接触。18h时，不成熟树突状细胞已经摄入了经中性粒细胞胞外捕网(NETs)暴露在胞质外的自身抗原PR3、MPO和LAMP-2。建立了树突状细胞-自身抗原荧光示踪吞噬体系。3.树突状细胞摄入了中性粒细胞胞外捕网(NETs)暴露在胞质外的自身抗原后，其表面活化标志(CD80、CD83、CD86和HLA-DR)表达升高。4. LAMP-2抗体诱发的中性粒细胞胞外捕网(NETs)，促进树突状细胞分泌IL-1β、IL-6、IL-23。5. LAMP-2抗体诱发胞外捕网的中性粒细胞(NETs)与树突状细胞共培养体系，引起初始CD4~+T细胞向Th17细胞分化。此外，在细胞上清中发现Th17细胞分泌的特异指标IL-17a。6.在加入DNase I破坏胞外捕网释放出的dsDNA染色质结构后，原本暴露在细胞外的自身抗原(LAMP-2、MPO、PR3)局限在细胞内；原本对树突状细胞表面标志活化作用和促进IL-1β、IL-6、IL-23分泌的作用和对初始CD4~+T细胞向Th17细胞分化的影响均消失。结论1. LAMP-2抗体刺激中性粒细胞后降低中性粒细胞的凋亡率。2.树突状细胞吞噬了LAMP-2抗体作用的凋亡异常中性粒细胞，建立了树突状细胞-凋亡中性粒细胞荧光示踪吞噬体系。3. LAMP-2抗体诱导的凋亡中性粒细胞，部分活化了树突状细胞，促进了IL-1β、IL-6分泌，但未检测到与Th17细胞增值存活相关的IL-23。未发现初始CD4~+T细胞向Th17细胞分化。4. LAMP-2抗体诱发中性粒细胞胞外捕网(NETs)形成，促使中性粒细胞胞浆抗原(MPO，PR3和LAMP-2)的释放和暴露。5.树突状细胞摄入了经中性粒细胞胞外捕网(NETs)暴露在外的自身抗原(MPO，PR3和LAMP-2)，建立了树突状细胞-自身抗原荧光示踪体系。6.LAMP-2抗体诱发的中性粒细胞胞外捕网向树突状细胞呈递自身抗原，活化树突状细胞，体系中细胞因子谱发生变化，并导致初始CD4~+T细胞向Th17细胞分化。更多还原

【Abstract】 BackgroundAnti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (ANCA associatedvasculitis, AAV) represents a group of systemic autoimmune diseases, including Wegener ’sgranulomatosis (WG), microscopic polyangiitis (MPA), Churg-Strauss syndrome (CSS) andrenal-limited vasculitis(RLV). When idneys are involved, they usually presentpauci-immune focal segmental necrotising glomerulonephritis (PiFSGN), which leads torapid and irreversible renal failure. It is acknowledged as a local clinical manifestation ofanti-neutrophil cytoplasmic autoantibody-associated system vasculitis (AAV).ANCAs are a heterogeneous group of autoantibodies produced by stiumulatingneutrophil cytoplasmic antigens, which are sensitive serological markers for diagnosis ofANCA-associated vasculitis (AAV). Myeloperoxidase (MPO) and Proteinase3(PR3) aretwo major ANCA antigens. The present study has confirmed that ANCAs play a pathogenicrole in ANCA-associated vasculitis (AAV), and it is the core pathogenic mechanism thatANCAs bind with target antigens on the surface of neutrophils. Neutrophils can beactivated when ANCAs recognize the conformational epitopes of MPO and PR3. Understeady circumstances, neutrophils do not express target antigens for ANCA on cell surfaces,but only in the cytoplasm of neutrophils. These target antigens would be transferred fromthe cytoplasm to the cell surface and exposed the binding antigens only when neutrophilswere primed or stimulated by inflammatory cytokines. In this case, ANCAs can bind withtarget antigens on neutrophil surface through their Fab2segment, and then neutrophilswould be activated, resulting in local inflammation. Apoptotic neutrophils and their cell debris which ingested by nearby phagocytoticcells and timely removed are imperative conditions to eliminate the inflammatory response,maintaining homeostasis. However, the obstacled clearance of apoptotic neutrophils wouldresult in inflammatory diseases. In patients with ANCA associated pauci-immune focalsegmental necrotising glomerulonephritis (PiFSGN), numbers of activated neutrophils andlysosomal enzymes, which released from its nuclear debris, are accumulated aroundinjuried capillaries. These neutrophils infiltration and cellular components deposit areclosely associated with kidney damages. Research indicates that many diseases incidence(such as SIRS, ARDS and MODS etc.) are due to neutrophil delayed apoptosis or apoptosisobstructions. A large number of adhesion molecules expressed on cell surface whenneutrophil apoptosis disorders or even delay, which enhance the ability of adhesion tovascular endothelial cells, destroy the integrity of endothelial cells, and induce theinflammatory response.Neutrophil extracellular traps (NETs) is a newly recognized mode of neutrophilcell-death, which extrudes its structures of decondensed chromatin decorated withantimicrobial peptides, such as myeloperoxidase (MPO), elastase, proteinase3(PR3),cathepsin G, lactoferrin and others. Recent studies have demonstrated that the formation ofneutrophil extracellular traps (NETs) also participate in pathogenesis of AAV. It couldtransfer cytoplasmic neutrophil autoantigens to dendritic cells, which induce ANCAassociated autoimmunity (AAV) in mice. And ANCA can be further effect on neutrophils bypromoting more neutrophil extracellular nets (NETs) formation, which results in a viciouscycle of autoimmune reactions.DCs act as the most powerful potent antigen-presenting cells and the only antigenspresentation cells to CD4~+T cells in human immune system, which play a vital role inmediation of immune response and maintaining tolerance. Th17cells are recognized assubsets of CD4~+T cell relevanting to autoimmune diseasese. It is proved that highercontents of Th17cells in serum of ANCA associated vasculitis patients than that in ANCAnegative vasculitis and healthy people. Th17cells participate in the occurrence of ANCAassociated vasculitis and tissue damage. Research has been confirmed that ithere are a lot of immature dendritic cell infiltrationn renal pathological specimens of patients withANCA-associated vasculitis, and involved in activation of Th17cell proliferation anddifferentiation.Human anti-lysosome-associated membrane protein2(LAMP-2) antibody, a newsubtype of ANCA family, is universal prevalence in patients with active disease of PiFSGNand capable to lead to PiFSGN in WKY rats, as well as activating human neutrophils orprovoking endothelium apoptosis in vitro. The results have clearly demonstrated thatanti-LAMP-2antibody plays a pathogenic role in the development of AAV. However, howanti-LAMP-2antibody leads to AAV remains unknown.This project intends to adopt magnetic activated cell sorting, flow cytometry, ELISA,immunohistochemistry and confocal laser scanning techniques to observe the influence ofanti-LAMP-2antibody on neutrophil (neutrophil apoptosis process, neutrophil extracellularnets (NETs) formation), and the effects of neutrophil treated by anti-LAMP-2antibody onactivation of immature dendritic cells, and the secretion of inflammatory cytokinesmicroenvironment on initial CD4~+T cells into Th17differentiation. Based on these findings,we aim to test the feasibility that anti-LAMP-2antibody induce apoptosis disturbance orneutrophils extracelluar traps formation in AAV.Materials and MethodsPart11. CD14~+monocytes were separated and purityed by immunomagnetic beads, werecultured to become immature dendritic cells in vitro, and were detected by opticalmicroscopy and flow cytometry.2. Density gradient centrifugation method was used to isolate neutrophils fromperipheral bloods of healthy volunteers and identification the neutrophils throughimmunofluorescence and flow cytometry.3. Neutrophils were in presense of Anti-LAMP-2antibody for24h, and then apoptosisrate changes of neutrophils were detected by flow cytometry.4. Apoptotic neutrophils labeled by FITC-Annexin V were cultured with immaturedendritic cells (DCs), and then the state of dendritic cells phagocyting apoptotic neutrophils were assayed by confocal detection.5. Neutrophils were stimulated by anti-LAMP-2antibody for24h, and their influenceson MFI of immature dendritic cells (DCs) activation markers (CD80, CD83, CD86andHLA-DR) were detected by flow cytometry.6. Neutrophils were stimulated by anti-LAMP-2antibody for24h, and their influenceson the secretion of (IL-1β, IL-6, IL-23) from dendritic cells were detected by ELISA.7. CD4~+T cells were separated and purifyed by immunomagnetic beads and then wereidentificated them through flow cytometry.8. CD4~+T lymphocytes were added into the coculture system of neutrophils anddendritic cell.5days later, we use of flow cytometry to determine whether CD4~+T cells toTh17cells or not.Part21. Neutrophils were stimulated by anti-LAMP-2antibody for3h, and then neutrophilextracellular traps (NETs) formation, as well as the exposure of antigen (LAMP-2, MPO,PR3) was detected by immunofluorescence.2. Immature dendritic cells labeled with PKH-26were cultured with anti-LAMP-2antibody-induced neutrophilextracellular traps (NETs) stained with SYTOX green for4h,then the interaction of NETs with dendritic cells were observe by confocal detection.3. Immature dendritic cells labeled with PKH-26were cultured with anti-LAMP-2antibody-induced neutrophilextracellular traps(NETs) stained with FITC-PR3, MPO andLAMP-2monoclonal antibody for18h, then the stages of extracellular autoantigens(LAMP-2, MPO, PR3) transferred to dendritic cells were observe by confocal detection.4. Neutrophils were stimulated by anti-LAMP-2antibody for3h, and their influenceson MFI of immature dendritic cells (DCs) activation markers (CD80, CD83, CD86andHLA-DR) were detected by flow cytometry.5. Neutrophils were stimulated by anti-LAMP-2antibody for3h, and their influenceson the secretion of (IL-1β, IL-6, IL-23) from dendritic cells were detected by ELISA.6. CD4~+T lymphocytes were added into the coculture system of neutrophils anddendritic cell.5days later, we use of flow cytometry to determine whether CD4~+T cells toTh17cells or not. ResultsPart11. Neutrophil apoptosis rates were significantly decreased when they were in thepresence of anti-hLAMP-2antibody for24h.2. Apoptotic neutrophils labeled with FITC-Annexin V appeared in the cytoplasm ofimmature dendritic cells.3. Anti-LAMP-2antibody distrubed the process of neutrophil apoptosis, whichincreased the the surface activation markers (CD80, CD83, CD86and HLA-DR) ofdendritic cell; however, no significant changes of the activated phenotype (CD80, CD83,CD86and HLA-DR) were detected between anti-LAMP-2antibody treated neutrophilsgroup and routinely apoptotic group.4. Anti-LAMP-2antibody-induced abnormal neutrophil apoptosis promoted dendriticcells to secretion of IL-1β, IL-6, but no IL-23has been detected.5. CD4~+T lymphocytes were added into the coculture system of neutrophils anddendritic cell.5days later, we found that part of CD4~+T cells differenticate into Treg cellsPart21. Anti-LAMP-2antibody induced neutrophil extracellular traps (NETs) formation,exposing LAMP-2, MPO and PR3along with NETs.2. Neutrophils were aged for3h in the presence of anti-LAMP-2antibody, and thenNETs stings were found to interact with imDCs after4hours coculture. On18hourscoculture, imDC to uptook of NETs autoantigens stained along with a mAb to PR3, MPOand LAMP-2, including LAMP-2, MPO and PR3directly conjugated with FITC dye.3. Anti-LAMP-2antibody-induced NETs significantly increased the markers indicativeof maturation on dendritic cells (CD80, CD83, CD86and HLA-DR).4. Anti-LAMP-2antibody-induced NETs significantly increased the secretion of IL-1β,IL-6,IL-23by dendritic cells.5. CD4~+T lymphocytes were added into neutrophils and dendritic cell coculturesystem.5days later, Th17cells and the secretion of IL-17a were detected.6. It is believed that NETs are adequately digested by DNase I. When DNase I addedinto Anti-LAMP-2antibody induced NETs, all chromatin fibers exposed along with NETs were all vanished. The increases of markers indicative of maturation on dendritic cells byanti-LAMP-2antibody–induced NETs were disappeared. And the differentiation of Th17cells and the secretion of IL-17a were prevented by using DNase I.Conclusions:1. Anti-LAMP-2antibody descreases the apoptotic rates of neutrophils.2.Dendritic cells engulfs LAMP-2antibody-induced apoptotic neutrophils. Thephagocytosis fluorescent tracer system of dendritic cells-apoptotic neutrophil wasestablished.3. Anti-LAMP-2antibody distrubs the process of neutrophil apoptosis, which partlyincreases the MFI of dendritic cell surface activation markers, and promotes the secretionof IL-1β,IL-6.In this case, no na ve CD4~+T cells differentiate into Th17cells are founded.4. Anti-LAMP-2antibody induces neutrophil extracellular traps (NETs) formation andexposes intracellular autoantigens (MPO, PR3, and LAMP-2).5. Dendritic cells engulfs autoantigens released from LAMP-2antibody-induced NETs.The phagocytosis fluorescent tracer system of dendritic cells-autoantigens was established.6. PBMC-derived DCs are actived by anti-hLAMP-2antibody induced-NETs.Moreover, coculture supernatants set up the microenviroment leading CD4~+T cellsdifferentiate into Th17cells.更多还原