【作者】 王燕；

【导师】 陈政良；

【作者基本信息】 南方医科大学 ， 免疫学， 2012， 博士

【摘要】 20世纪90年代后,“天然免疫重新崛起”,发现天然免疫系统通过多种模式识别受体(pattern recognition receptor,PRR)识别病原相关分子模式(pathogen associated molecular pattern,PAMP),既作为抵抗病原体感染的第一道防线,又可指导获得性免疫以适当的方式(应答类型)和强度对之应答,有效地清除病原体。天然免疫的这种指导作用,首先是识别非己的外源性(如病原体)配体和异常(如感染、损伤等)情况下产生的内源性配体,然后通过抗原提呈、提供共刺激信号和产生不同谱系细胞因子而实现的。该过程中,起关键作用的是抗原提呈细胞(antigen presenting cell,APC)。专职APC包括巨噬细胞(macrophage,MΦ)、树突状细胞(dendritic cell, DC)和B淋巴细胞。迄今,该领域一直是免疫学研究的前沿和热点。然而,上述多数仅为表象性描述,天然免疫对获得性免疫应答的指导作用特别是其机制,尚需更深入的研究予以阐明。甘露聚糖结合凝集素(mannan-binding lectin, MBL)系由肝细胞合成、分泌的血浆蛋白,是天然免疫系统中一个关键的可溶性PRR。MBL属于C型凝集素超家族中胶凝素家族成员,具有“郁金香”样结构,类似补体C1q。成熟MBL肽链有4个结构域,自N端至C端依次为：富含半胱氨酸(Cys)的N端区、胶原样区(collagen-like region,CLR)、颈区和C端糖识别域(carbohydrate-recognition domain,CRD)。完整的人MBL分子是同质三肽链结构亚单位的寡聚体,多至六聚体。只有高聚体MBL分子才具有生物学活性。MBL可选择性识别多种病原体表面的糖结构,包括D-甘露糖、L-盐藻糖、N-乙酰葡萄糖胺、N-乙酰甘露糖胺等,通过凝集素途径以不依赖抗体和Clq的方式激活补体系统,从而发挥溶破和间接调理功能,并能与吞噬细胞表面胶凝素受体结合而介导调理吞噬作用,还能清除免疫复合物和凋亡细胞。而且,MBL也是一个重要的黏膜表面防御分子。MBL结构基因的3个点突变(CGT52TGT、GGC54GAC和GGA57GAA)以及启动子和5’非翻译区突变所致血浆MBL低下而引起的调理吞噬缺损为迄今所发现的最常见的遗传性免疫缺损病,MBL缺损者终生处于高度的感染风险之中,随时可患各种感染甚至是威胁生命的感染,也易患自身免疫性疾病如系统性红斑狼疮(systemic lupus erythematosus,SLE)、类风湿性关节炎(rheumatoid arthritis,RA)、Sjogren’s综合征等；而凝集素途径活性过强时可能导致炎症性损伤。MBL是一种急性期反应蛋白,在应激(如病原体感染、外科手术等)时,其浓度可升高2-3倍。目前有关MBL在天然免疫中作用的研究很多,主要集中在补体激活和调理吞噬方面,但对其与获得性免疫的关系尚未涉及,其免疫调节作用的研究也极少,所见的唯一报道是Fraser等发现MBL可抑制细菌脂多糖(lipopolysaccharide,LPS)刺激的单核细胞(monocyte,Mo)分泌白细胞介素(interleukin,IL)-1而促进其产生IL-10、IL-6等细胞因子。因此,研究MBL与获得性免疫的关系,特别是其对单核巨噬细胞分化发育的调节作用,具有重要的科学意义和潜在的医学实践意义。已发现,胶凝素家族的其他成员如肺表面活性物质蛋白A和D (surfactant proteins A and D, SP-A and SP-D)及补体Clq具有多种免疫调节功能,如SP-A能抑制DC的分化成熟,通过与CD14相互作用调节细菌LPS诱导的细胞免疫应答,抑制LPS诱导的单核巨噬细胞分泌肿瘤坏死因子-α (Tumor necrosis factor-α,TNF-α)等细胞因子,还可以直接与Toll样受体4(Toll-like receptor4, TLR4)、MD-2分子相互作用调节炎性细胞反应；SP-D可增强DC的抗原提呈能力,增强抗感染免疫,亦可通过与CD14结合,改变CD14-LPS相互作用等；SP-A和SP-D还可直接抑制T淋巴细胞增殖及功能,避免由于免疫应答过强造成的肺部损伤。Clq也可以直接调节DC的分化成熟并抑制LPS诱导的细胞因子产生。因为MBL与C1q、SP-A及SP-D结构的同源性、且均能与胶凝素受体结合,及MΦ和DC均表达胶凝素受体并介导多种免疫调节功能,所以我们推论MBL必定参与获得性免疫应答。针对此设想,本课题组已经做了大量研究,并得出结论：(1)MBL能够以Ca2+依赖、糖敏感形式与自身的DC、T细胞和B细胞系Raji细胞结合。(2)MBL对DC的分化成熟具有双向调节作用,它既可以直接促进DC的成熟,又可以抑制LPS诱导的DC成熟,初步证实了MBL可以通过调节DC的分化成熟和功能而参与获得性免疫应答的调节。(3)高浓度MBL可以抑制Raji细胞的增殖,MBL还能直接调控T细胞和B细胞的功能,由此我们取得了MBL与获得性免疫直接联系的初步证据。综上所述,MBL不仅在天然免疫中发挥重要作用,而且在获得性免疫中也扮演独特的角色。目前,有关MBL调节获得性免疫应答的报道罕见,虽然其作用机理还有待于进一步的研究予以阐明,但这些发现为进一步探讨MBL调节获得性免疫应答的作用及其机制提供了切入点,后续MBL免疫调节作用的研究及其分子机制的阐明,将进一步揭示MBL这种重要的天然免疫分子的新的生物学意义。在现有研究基础之上,本课题继续探讨MBL在调节获得性免疫应答中所扮演的角色。单核巨噬细胞作为连接天然免疫与获得性免疫的桥梁,在免疫应答中发挥重要的作用。本课题以单核巨噬细胞为切入点,初步探索MBL对单核巨噬细胞分化发育和增殖的影响及其分子机制。我们首先联合应用配体与抗体亲和层析柱,从人冰冻血浆中提取高纯度的具有生物学活性的天然MBL。在此基础上,探讨了MBL对MΦ分化发育的影响：包括MBL对MΦ的形态、表面标志、细胞因子释放、吞噬功能及刺激同种异体T细胞增殖能力的调节作用,为进一步阐明MBL调节单核巨噬细胞分化发育提供依据,丰富天然免疫指导获得性免疫应答的基础理论。同时还研究了MBL对Mo增殖,周期和凋亡的影响及其作用机制。本课题分为两部分,兹分述如下。第一章MBL调节单核巨噬细胞的分化发育Mo作为髓系的前体细胞之一,同时具有向DC和MΦ两个方向分化的潜能。在不同的细胞因子刺激下,Mo可以在两者之间择一而行。目前已知可以调控Mo发育方向的细胞因子包括IL-10、IL-6和TNF-a等。MBL作为天然免疫系统中一个关键的可溶性PRR,是由肝细胞分泌的血浆蛋白,属于C型凝集素超家族中胶凝素家族成员,本课题组前期研究发现,MBL可抑制Mo向DC分化,而诱导Mo分化为MΦ。MΦ既可作为效应细胞直接清除病原体,又可提呈抗原给T细胞并启动特异性免疫应答,在调控机体免疫反应中起着重要作用。若证实MBL对Mo分化为不同亚型MΦ有调节作用,则有理由推测,不同亚型MΦ可能诱导不同类型的获得性免疫应答,提示MBL参与调节获得性免疫应答。因此,本研究通过体外培养人外周血Mo,进一步探讨MBL调节单核巨噬细胞分化发育的作用。首先用传统方法从人冰冻血浆中提取MBL。在提取过程中会污染LPS,为排除LPS对研究MBL调节功能的干扰作用,应用多粘菌素B凝胶柱去除LPS,经配体结合实验鉴定,MBL仍保持良好的甘露聚糖(mannan)结合活性。取人新鲜抗凝血,先用Ficoll-Hypaque密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),再用磁式细胞分选器(magnetic activated cell sortor,MACS)负选法纯化Mo,用含10%胎牛血清(fetal bovine serum, FBS)的RPMI-1640培养基体外培养,同时加入不同浓度MBL和或巨噬细胞集落刺激因子(macrophage colony stimulatory factor,M-CSF),于37℃,5%CO2培养。在Mo向MΦ分化的过程中,激光共聚焦显微镜观察发现细胞表面同时表达MΦ表面标志MAC-1(CDllb)和CD14,充分证实Mo己分化为MΦ。接下来,我们深入探讨了MBL对MΦ表型及功能的影响。(1)流式细胞术(flow cytometry,FCM)检测MBL与Mo结合；(2)相差显微镜下观察MΦ形态；(3)FCM检测MΦ表面分子表达；(4)酶联免疫吸附试验(enzyme-linked immunosorbence assay,ELISA)检测MΦ分泌的细胞因子,real-time PCR检测MΦ分泌的细胞因子mRNA水平；(5)激光共聚焦显微镜和FCM检测MΦ吞噬功能；(6) Cell Counting Kit-8(CCK-8)法检测MΦ刺激同种异体T细胞增殖的能力。实验结果显示：(1)MBL直接结合Mo,且结合量与MBL有浓度依赖关系,提示Mo表面可能存在MBL受体或结合蛋白。(2)在人外周血Mo分化为MΦ的过程中细胞贴壁生长,体积明显增大,出现两种不同形态的细胞,圆形细胞是常规巨噬细胞(standard macrophage, s-MΦ),梭形细胞是类成纤维细胞的巨噬细胞(fibroblast macrophage, f-MΦ),两者均表达MΦ表面标志MAC-1’和CD14。我们还发现低浓度MBL (2.5μg/ml)增加f-MΦ百分比(F=49.2P=0.000),而高浓度MBL (20μg/ml)减少f-MΦ百分比(F=49.2P=0.000),提示MBL调节MΦ的分化,即低浓度MBL促进Mo向f-MΦ分化,而高浓度MBL促进Mo向s-MΦ分化。(3)MBL下调MΦ表面分子HLA-DR. CD86和CD206的表达(F=3.831P=0.026;F=10.034P=0.001;F=4.524P=0.015),提示MBL可能通过抑制MΦ抗原提呈功能参与调节获得性免疫应答。(4)MBL促进MΦ分泌抑炎细胞因子IL-10(F=6.985P=0.001;F=25.481P=0.000)和转化生长因子-β (transforming growth factor-β,TGF-β)(F=341.411P=0.000; F=8.129P=0.001; F=5.486P=0.007),抑制MΦ分泌IL-12(F=114.179P=0.000; F=63.646P=0.000),同时抑制MΦ分泌促炎细胞因子IL-6(F=97.711P=0.000; F=212.528P=0.000; F=28.464P=0.000)和TNF-a (F=10.385P=0.000), MΦ分泌的细胞因子表达水平的变化提示MBL可能诱导人Mo向M2c型巨噬细胞分化。MBL还抑制MΦ分泌趋化性细胞因子macrophage chemoattractant protein-1(MCP-1)(F=471.826P=0.000)。(5)两种不同形态的MOΦ (s-MΦ和f-MΦ均可吞噬大肠杆菌或酵母菌,且吞噬百分率不受MBL诱导分化的影响(F=2.674P=0.076),提示MBL诱导分化不影响MΦ吞噬功能。(6)MBL降低MΦ刺激同种异体T细胞增殖的能力(F=24.343P=0.000; F=18.604P=0.000; F=6.463P=0.001),提示MBL通过下调MΦ功能来抑制获得性免疫应答,即MBL诱导分化的MΦ启动和介导获得性免疫应答的能力减弱。以上结果提示,MBL可以促进人外周血Mo向M2c型MΦ分化,高分泌IL-10和TGF-p,同时伴随抗原提呈功能减退,抑制免疫应答和炎症反应。由此可见,MBL既可参与天然免疫应答,也可参与获得性免疫应答的调节。研究机体内不同环境对MΦ分化发育的影响,不仅有助于我们更好的理解疾病的发生机制,更可为研究以MΦ为靶标的新型防治手段提供理论依据。第二章MBL调节单核细胞的增殖、周期和凋亡Mo是机体天然免疫和获得性免疫应答的重要组成部分,是单核吞噬系统(mononuclear phagocyte system,MPS)的主要成员之一。大量研究表明Mo是MO的前体细胞,他们共同构成了外来微生物入侵时机体的有效防御系统,同时对于生理状态下清除机体内衰老和凋亡细胞,维持机体的内环境稳态发挥了不可替代的作用,他们还具有识别和杀伤肿瘤细胞的能力。Mo来源的MΦ还具有较强的抗原处理和提呈能力,能够通过分泌多种细胞因子发挥免疫调节作用和介导炎症反应。MPS的异常发育必然导致机体免疫功能和内环境的紊乱,最终引发机体的各种疾病。本章工作主要评价MBL对人Mo增殖、周期和凋亡的影响,并对其作用机制进行初步探讨。无菌分离人外周血PBMC,MACS负选法纯化Mo,并复苏人原始单核细胞系U937细胞,用含10%FBS的RPMI-1640培养基体外培养,同时加入不同浓度MBL和或M-CSF,于37℃,5%CO2培养。(1)CCK-8法检测MBL对Mo和U937增殖的影响；(2)流式细胞仪Propidium Iodide(PI)单标法检测MBL对U937细胞周期进程的影响；(3) Real-time PCR检测Mo和U937细胞周期相关基因mRNA表达的变化；(4)流式细胞仪Annexin V-FITC/PI双标法检测U937细胞凋亡率;(5) Real-time PCR检测Mo和U937细胞凋亡相关基因mRNA表达的变化；(6) Caspase-3活性检测试剂盒检测U937细胞内caspase-3酶活性。实验结果显示：(1)MBL对体外培养的Mo生长具有双向作用,低浓度MBL(≤4μg/ml)促进Mo增殖；而高浓度MBL (4-20μg/ml)抑制Mo增殖,且具有良好的剂量依赖性,随MBL浓度的增高,增殖抑制作用显著增强。但作用时间不同,高浓度MBL增殖抑制作用也不同。对M-CSF活化的Mo而言,高浓度MBL作用48h后其增殖抑制作用显著(F=8.802P=0.000),而24h和72h,其增殖抑制作用不明显(F=6.135P=0.001; F=7.595P=0.000).对U937细胞而言,高浓度MBL作用24、48、72h后其增殖抑制作用显著(F=18.354P=0.000; F=48.530P=0.000;F=79.501P=0.000)。(2)随着MBL作用浓度的增加和作用时间的延长,U937细胞G0/G1期的细胞百分数逐渐增多(F=5.550P=0.007; F=23.161P=0.002),而G2/M期的细胞百分数逐渐减少(F=14.112P=0.005),表明MBL阻滞U937细胞于G0/G1期,且具有浓度和时间依赖性。我们应用real-time PCR检测细胞周期相关基因mRNA的表达,发现20μg/ml MBL诱导Mo和U937细胞阻滞于G0/G1期可能与p21基因上调(F=13.647P=0.006; F=1135.242P=0.000)和CyqlinDl (F=12.969P=0.007; F=58.886P=0.000)、CyclinD3(F=59.491P=0.000; F=41.07P=0.000)、CDK2(F=91.185P=0.000; F=83.456P=0.000)、CDK4(F=166.65P=0.000；F=39.842P=0.000)基因下调有关,提示高浓度MBL可以诱导Mo和U937细胞阻滞于G0/G1期。(3)随着MBL浓度的增加和作用时间的延长,凋亡细胞占总细胞数的比例不断增加(F=258.709P=0.000;F=1265.444P=0.000),提示高浓度MBL具有诱导U937细胞凋亡的作用,且呈现良好的剂量和时间依赖关系。我们应用real-time PCR检测细胞凋亡相关基因]mRNA表达,发现20μg/ml MBL诱导细胞凋亡可能与caspase-3(F=830.656P=0.000; F=45.956P=0.000)、Fas (F=13,719P=0.006; F=288.811P=0.000)基因上调和Bcl-2基因下调(F=55.86P=0.000；F=196.435P=0.000)有关,提示高浓度MBL可以通过激活死亡受体途径和线粒体途径诱导Mo和U937细胞凋亡。另外我们还发现,20μg/ml MBL作用U937细胞4h或6h后,胞内caspase-3活性显著增强(P=0.003；P=0.031),进一步支持和验证了高浓度MBL可以诱导U937细胞凋亡。以上结果提示,高浓度MBL可有效抑制Mo增殖,可能与其诱导细胞凋亡并使细胞周期阻滞于G0/G1期有关,提示MBL作为一种应急蛋白,具有抗炎作用和免疫调节功能。综上所述,本课题研究发现,MBL可以促进人外周血Mo向M2c型MΦ分化,高浓度MBL还可有效抑制Mo增殖,提示MBL在识别外源性抗原或自身凋亡细胞并清除它们的同时,可能通过单核巨噬细胞向T、B细胞传递抑制信号,避免由于过强的获得性免疫应答造成机体损伤。这充分证明了MBL作为一种应急蛋白,具有抗炎和抑制免疫应答作用。但具体分子机制如Mo表面MBL受体的鉴定,与Mo结合的MBL结构域的确定,MBL对单核巨噬细胞分化信号通路的影响,以及MBL调节Mo分化与增殖,这两者间的关系如何,均需要更深入和系统的研究。下一步工作将明确MBL对单核巨噬细胞分化信号通路的调节作用,并探讨MBL调节Mo分化与增殖的相互关系,以期揭示天然免疫中重要的模式识别分子MBL的新的生物学活性。本研究将为天然免疫指导获得性免疫的基础理论研究提供实验依据,并为MBL失衡和单核吞噬系统功能紊乱有关疾病的防治研究提供新的靶点。更多还原

【Abstract】 The basic understanding of the immune system has undergone a substantial paradigm shift in the past twenty years, since an awareness of the power and influence of the innate immune system emerged in1990s. It is widely believed that the innate immune system is not only responsible for the induction of acute responses necessary for elimination of pathogens but also able to activate appropriate adaptive immune responses via antigen presentation, ligand interaction and secretion of cytokines and chemokines, and that the nature of the first response to invasion (i.e., the innate immune response) has significant influence on determining the nature of the subsequent adaptive immune response. It is also suggested that innate immunity initiates acquired immune response and controls the types of acquired immune response through pattern-recognition between pathogen associated molecular pattern (PAMP) and pattern recognition receptor (PRR). Antigen presenting cells (APCs) play a key role in the process. Professional APC includes macrophages, dendritic cells and B lymphocytes. Today, this field is still frontier and hot of immunology research. However, the majority is only figurative description, the guiding role of natural immunity to the adaptive immune response and its mechanisms need to be further clarified.Mannan-binding lectin (MBL), a member of collectins belonging to C-type lectin superfamily, is a multimeric protein containing collagen-like sequences. As a key soluble PRR of the innate immune system, MBL is synthesized and secreted into the blood by hepatocytes. The MBL polypeptide consists of four regions, a cysteine-rich N-terminal domain, a collagen-like region (CLR) containing Gly-X-Y repeats (where X is any amino acid and Y is often hydroxyproline or hydroxylysine), a neck domain and a C-terminal carbohydrate recognition domain (CRD). The neck region forms an alpha-helical coiled-coil structure which probably promotes trimerization of three polypeptides to form the subunit. The trimer is stabilized by hydrophobic interactions and inter-chain disulphide bonds within the cysteine-rich N-terminal domain. MBL subunits assemble into larger oligomeric structures which is composed of two to six subunits forming a ’bundle-of-tulips’ or sertiform appearance first described for C1q. Only the more highly polymeric forms have biological activity and can fix complement. MBL in serum has been intensively characterized and found to act as pattern recognition molecules that selectively recognize carbohydrates on the surface of microbial pathogens. Following pathogen recognition, MBL may activate the complement cascade through the lectin pathway, after which microbes are killed by cellular lysis and indirect opsonization. When bound to the collectin receptor of effector cells, MBL can mediate direct opsonization and MBL-dependent, cell-mediated cytotoxicity. MBL is also able to enhance the phagocytosis of cellular debris, apoptotic cells and immune complexes in vitro and in vivo. MBL also plays an important role in surfactant defenses of mucous membrane. A mounting body of evidence suggests that defect opsonophagocytosis resulted from low MBL levels due to gene mutations in MBL coding gene (CGT52TGT, GGC54GAC and GGA57GAA) and promoter as well as5’-untranslated region appear to be more vulnerable to infections in a number of clinical settings, and more susceptible to autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren’s syndrome resulted from phagocytosis defection. On the other hand, exess activation of lectin pathway may result in inflammation disease. MBL is also an acute phase reactant, showing an increase of up to two or three-fold after infection or surgical trauma. Current researches focus on the roles of MBL in innate immunity, mainly in the complement activation and opsonophagocytosis. However, few reports focus on the roles of MBL in adaptive immunity, especially of its regulatory functions. Fraser has claimed that MBL can inhibit IL-1secretion of lipopolysaccharide (LPS)-stimulated monocyte, but promote the secretions of IL-10and IL-6. So it is of great significance in studying the relationship of MBL with adaptive immunity, particularly its regulatory role on monocyte/macrophage differentiation and development.It has been reported that other members of the soluble defensive collagen family such as surfactant protein A (SP-A), surfactant protein D (SP-D) and complement C1q, have roles in immune regulation through interacting with various cells. For instance, SP-A inhibits differentiation and maturation of dendritic cells (DCs) and modulates cellular responses induced by lipopolysaccharide (LPS) through interacting with CD14; SP-A directly interacts with Alveolar macrophages through binding to cell surface receptors Toll-like receptor4(TLR4) and MD-2, resulting in modulation of chemotaxis, phagocytosis, and modified pro-or anti-inflammatory immune responses. SP-D enhances antigen presentation ability of DCs to promote adaptive immune response against bacterial antigens and alters CD14-LPS interaction through binding to CD14. It is reported that both SP-A and SP-D can directly inhibit mitogen-induced T lymphocytes proliferation to avoid lung damages caused by the strong immune response. Clq regulates LPS induced cytokine production in bone marrow-derived DCs and induces maturation of DCs. Therefore, according to the structure homology among MBL, SP-A, SP-D and Clq, the fact that they all can bind to collectin-receptor, and the fact that macrophage and DC express specific collectin-receptor and mediate a number of immune regulatory roles, the conclusion can be deduced that MBL must participate in acquired immune response. Our previous studies showed that MBL could bind to DCs, T and B lymphocytes in a Ca2+-dependent and sugar-sensitive manners through CLR-specific and CRD-specific MBL receptors, of which, the former was also shared with Clq; and that MBL could regulate differentiation and maturation of dendritic cells and reduce DC’s ability to stimulate allogeneic T lymphocytes proliferation; and that MBL could significantly inhibit Raji cells proliferation directly at higher concentrations (10-50μg/ml) in a dose-dependent manner. Further investigation found that MBL may directly regulate T and B cell functions. All these data suggest that MBL may regulate acquired immune responses through APCs, T and B lymphocytes. To date, however, little knowledge has been obtained about its role in the regulation of adaptive immune responses. Moreover, its detailed mechanism has yet to be elaborated by further experiments. The immunomodulatory effects of MBL and its molecular mechanisms elucidation will further reveal the biological significance of MBL served as an important molecule in the innate immune.Therefore, on the basis of existing research, we continued to explore the roles of MBL in regulating the adaptive immune response. As a bridge connecting innate immunity with adaptive immunity, monocytes/macrophages play an important role in the immune response. Our aim in the present study is to explore the effects of MBL on differentiation and proliferation of monocytes/macrophages and their molecular mechanisms. Firstly, we established a convenient procedure for the purification of MBL through the ligand-and monoclonal antibody-affinity chromatography, and obtained natural MBL with high purity and bioactivity from freshly frozen human plasma. Next we investigated the effects of MBL on inducing differentiation of the monocyte-derived macrophages in vitro, including the studies of morphology, surface markers, cytokines, phagocytosis and so on. In addition we also investigated the effects of MBL on the proliferation, apoptosis, cell cycle of monocytes and its possible mechanisms in vitro.In this study, we provided evidences that human natural MBL possibly participates in the adaptive immune response through modulating differentiation and proliferation of monocytes/macrophages. Our study can enrich basic theory of the innate immunity guiding the adaptive immunity. This topic is divided into two parts, it is described as follows.Chapter I Mannan-binding lectin regulates monocytes/macrophages differentiation and developmentAs one of the myeloid precursor cells, monocyte has the potential ability of differentiation to dendritic cell and macrophage in both directions. Monocyte can choose one way under the stimulation of different cytokines. Currently it is known that monocyte development is regulated by cytokines, including IL-10,IL-6, TNF-αand so on. As a key soluble PRR of the innate immune system, MBL is a plasma protein secreted by the liver cells, belonging to the family members of the C-type lectin superfamily. Our previous studies showed that MBL can inhibit monocyte differentiation to dendritic cell, but induce monocyte differentiation to macrophage. Macrophages have long been considered to be important immune effector cells, with great plasticity in their phenotype and function. Under different microenvironment macrophages can be polarized to assume different phenotypes and show different functions. If confirmed MBL can regulate monocyte differentiation to different subtypes of macrophages, there are reasons to speculate that the different subtypes of macrophages may induce different types of adaptive immune response, suggesting that MBL is involved in the regulation of the adaptive immune response. The objectives of this chapter work were to investigate the effects of MBL on regulating differentiation and development of monocytes/macrophages in vitro.Firstly we extracted MBL from human frozen plasma using traditional methods. LPS has polluted MBL in the extraction process. To exclude the interference effect of LPS on the study of MBL regulatory function, the polymyxin B gel column was applied to remove LPS. After removing LPS, MBL identified by ligand binding assays still retains its good mannan binging activity. We obtained human peripheral blood mononuclear cells (PBMC) from peripheral blood of healthy adult volunteers by Ficoll-Hypaque density gradient centrifugation. After purified by MACS, monocytes were analyzed by flow cytometry for molecular maker on cellular surface. CD14+monocytes with a purity of more than95%were obtained. Human peripheral blood monocytes were co-cultured with macrophage colony stimulatory factor (M-CSF) and various concentrations of MBL simultaneously in RPMI1640supplemented with10%fetal bovine serum (FBS) at37℃in5%CO2. During the process of differentiation, it exhibited round cells and spindle cells (or fibroblast-like cells). Round cells were named standard macrophages (s-MΦ), while spindle cells were named fibroblast macrophages (f-MΦ). The confocal microscope was employed to examine MAC-1(CD11b) and CD14expressions on the cell surface and to ensure monocytes differentiation to macrophages (s-MΦ and f-MΦ). In order to further determine the classification of macrophages after treated by MBL, we analyzed the phenotypes and functions of macrophages.(1)MBL binding to monocyte was detected by flow cytometry.(2)The contrast phase microscope was applied to detected macrophage morphological changes.(3)Flow cytometry assay was used to detect the surface markers of macrophage such as HLA-DR, CD86, and CD206.(4) ELISA assay were performed to detect the expression of the cytokines of macrophage. Real-time PCR assay were employed to examine the mRNA expression of the cytokines of macrophage.(5)Flow cytometry and confocal microscopy were used to assay the phagocytosis of macrophage.(6)Macrophages were co-cultured with allogeneic T cells, in order to detect their abilities to stimulate allogeneic T cells into proliferation by CCK-8incorporation assay.Our results revealed that (1) MBL directly combines monocytes, suggesting that MBL receptors of binding proteins may exist on the surface of monocytes.(2) Macrophages presented an enlarged volume, adhensive growth pattern and the puffed cytolymph. MBL at lower concentration (2.5μg/ml) promoted monocytes differentiation into f-MΦ(F=49.2P=0.000), whereas MBL at higher concentration (20μg/ml) promoted monocytes differentiation into s-MO.(3) MBL down-regulated the expression of HLA-DR, CD86and CD206during the process of monocyte differentiation into macrophage (F=3.831P=0.026; F=10.034P=0.001; F=4.524P=0.015). It was presumed that MBL may be involved in the regulation of the adaptive immune response by inhibiting antigen-present ability of macrophage.(4) MBL obviously promoted the anti-inflammatory cytokines IL-10(F=6.985P=0.001; F=25.481P=0.000) and TGF-βsecretions (F=341.411P=0.000; F=8.129P=0.001; F=5.486P=0.007), with IL-12less (F=114.179P=0.000; F=63.346P=0.000), and significantly inhibited the pro-inflammatory cytokines IL-6(F=97.711P=0.000; F=212.528P=0.000; F=28.464P=0.000) and TNF-asecretions (F=10.385P=0.000), and also markedly inhibited macrophage chemoattractant protein-1(MCP-1) secretions (F=471.826P=0.000). The changes of cytokines expression level in macrophage suggested that MBL induced monocyte differentiation into M2c type macrophage.(5) Both s-MΦ and f-MΦ can engulf E.coli or yeast. The phagocytosis of macrophage was not affected by MBL induced and differentiated cells (F=2.674 P=0.076).(6) There was less proliferation of allogeneic T cells co-cultured with macrophages after treated with MBL (F=24.343P=0.000; F=18.604P=0.000; F=6.463P=0.001), suggesting that MBL down-regulated macrophage function to inhibit the adaptive immune response.Our data suggest that MBL may induce differentiation of peripheral blood monocyte into M2c type macrophage, which exhibits a distinct immunosuppressive phenotype and exert a positive role on anti-inflammatory effects. So we suppose that MBL might have anti-inflammatory and immune modulatory effects in pathological states. Thus, MBL not only participates in the innate immune response, but also be involved in the regulation of the adaptive immune response. The study of macrophage differentiation and development in different environments of the body not only helps us better understanding disease mechanisms, but also provides a theoretical basis for new prevention and treatment methods including macrophages as targets.Chapter II Mannan-binding lectin regulates cell proliferation, apoptosis and cycle in human monocytesMonocytes are the key components of innate and adaptive immunity, and are important in the mononuclear phagocytic system. Many studies have indicated that bone marrow monocytes are common precursors for macrophages, which build up the defense system effectively against the invasion of foreign micro-organisms. Meanwhile, these cells play an indispensable role in the removal of senescent and apoptosis cells to maintain the homeostasis of bodies under physiological conditions. These cells also have the abilities to recognize and destruct tumor cells. Monocytes-derived macrophages, served as one of professional APC, play important roles in immunomodulation and inflammation through secreting a variety of cytokines. Abnormal development of the mononuclear phagocytic system will inevitably lead to the disorder of the immune function and within the environment, and ultimately lead to various diseases of the body. The objectives of work in this chapter were to investigate the effects of MBL on the proliferation, apoptosis, cell cycle of human monocytes in vitro and its possible mechanism.Our results revealed that (1) MBL at lower concentrations (≤4μg/ml) enhanced human monocytes proliferation, whereas MBL at higher concentrations (4-20μg/ml) markedly attenuated cell proliferation (F=6.135P=0.001; F=8.802P=0.000; F=7.595P=0.000). The proliferation effects were examined with the CCK-8assay.(2)In order to investigate the anti-proliferative effect of MBL and its underlying mechanism, human monocytes and monocyte line U937cells were treated with MBL at higher concentrations for24-72h at37℃in a5%CO2incubator. Cell cycle distributions in U937cells stained with propidium iodide were detected by flow cytometry. We further found that MBL-induced growth inhibition was associated with G0/G1arrest (F=5.550P=0.007; F=23.161P=0.002). Cell cycle blockade was manifested by down-regulation of cyclinDl (F=12.969P=0.007; F=58.886P=0.000), cyclinD3(F=59.491P=0.000; F=41.07P=0.000), Cdk2(F=91.185P=0.000; F=83.456P=0.000), Cdk4(F=166.65P=0.000; F=39.842P=0.000) and up-regulation of the Cdk inhibitory protein Cipl/p21(F=13.647P=0.006; F=1135.242P=0.000).(3)Moreover, higher concentrations MBL induced apoptosis (F=258.709P=0.000), as determined by using Annexin V staining assay. Furthermore, higher concentrations MBL triggered the death receptor and mitochondrial apoptotic pathway, as indicated by increasing the expression of Fas (F=13.719P=0.006; F=288.811P=0.000), but decreasing the level of Bcl-2(F=55.86P=0.000; F=196.435P=0.000), resulting in activation of caspase-3(F=830.656P=0.000; F=45.956P=0.000).In this study, our results demonstrated that exposure to higher doses of MBL might effectively inhibit monocytes proliferation through the induction of cell apoptosis and cell cycle arrest at G0/G1phase, suggesting that MBL, as an emergency protein, might have anti-inflammatory effects in pathological states and immunomodulatory functions.In summary, results above have showed that MBL can promote the differentiation of human peripheral blood monocytes to M2c type macrophages. Meanwhile high concentrations of MBL can be effective in inhibition of monocytes proliferation. These data lend further credit to an important physiological role MBL in regulating adaptive immunity. Based on current data, it appears that MBL can dampen lymphocyte responses to exogenous stimuli or apoptotic cells by the monocytes/macrophages passing inhibitory signals to T, B cells and protect the body against collateral immune-mediated damage. This fully proves that the MBL served as an emergency protein has anti-inflammatory effect and inhibits the immune response. Further studies evaluating the specific mechanisms as followed are required: the receptors involved in binding; the effect of MBL on signal transduction; and the effect of MBL on monocytes/macrophages differentiation as well as proliferation. The next step will clear the regulatory role of MBL on monocytes/macrophages differentiation signaling pathways, and explore the relationship between monocytes/macrophages differentiation and proliferation regulated by MBL, in order to reveal MBL served as the innate immune pattern recognition molecule has new biological activity. In this study, the basic theory for the natural immune guiding the acquired immune is powerfully confirmed by experiments. It provides new targets for diseases prevention and control research related to MBL imbalances and mononuclear phagocytic system dysfunction.更多还原