甘露聚糖结合凝集素对人外周血单核细胞分化为调节性树突状细胞的作用及其机制的初步研究
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摘要
甘露聚糖结合凝集素(mannan-binding lectin, MBL)属于血清C型凝集素,是机体天然免疫的重要分子之一,这种高度保守的糖蛋白主要由肝细胞合成并以三聚体亚单位组成的多种寡聚体形式在血清中循环,其寡聚体形式可广泛识别多种病原体表面的糖结构,并通过激活2个MBL相关丝氨酸蛋白酶(MBL associated serine proteases, MASP-1, MASP-2)以不依赖于抗体和Clq的方式激活补体系统,发挥细胞溶破和间接调理功能。目前已知的MBL受体包括吞噬细胞表面胶凝素受体,能通过二者的结合而起直接调理作用,并在调节炎症和启动细胞调亡过程中发挥作用。MBL、Clq和SP-A、SP-D属于可溶性模式识别受体(Patteren Recognition Receptor, PRR),是防御性胶原家族成员。而PRR的配体称为病原体相关分子模式(Pathogen-Associated Molecule Pattern,PAMP),是一组或几大组病原体所共有、对其生存绝对必要且宿主机体没有的保守成分。MBL能够以其球状羧基端识别病原体表面特异性PAMP,引发机体迅速有效的保护性免疫应答。此外,MBL对病原体具有广谱识别作用,可结合免疫球蛋白；参与对凋亡细胞的吞噬以及MBL依赖的Th细胞活化和细胞介导的细胞毒作用。近年发现,大鼠MBL可以刺激肝脏Kupffer细胞对大肠杆菌及金黄色葡萄球的吞噬；重组人MBL可促进单核巨噬细胞株THP-1对大肠杆菌的吞噬。另一方面,细胞因子和抗原提呈细胞在决定感受到危险信号后所发生的免疫应答的类型和强度时具有重要作用。MBL可与单核细胞(Monocyte, Mo)和树突状细胞(Dendritic cells, DC)结合,调节细胞因子的分泌,抑制LPS/CD14诱导的单核细胞释放炎性细胞因子TNF-α,同时上调抑制性细胞因子的分泌；抑制DC-SIGN介导的对T细胞的感染。研究表明,胶凝素家族成员肺表面活性物质相关蛋白SP-A、SP-D亦可与多种免疫细胞如单核细胞、树突状细胞、T细胞结合,在天然免疫和获得性免疫应答中发挥调节功能,如增强吞噬细胞表面受体的表达,调节细胞因子和自由基的释放,清除自身凋亡细胞。
树突状细胞是机体内功能最强的专职抗原提呈细胞,且能够通过分泌特异性的细胞因子和与T、B细胞的相互作用有效启动免疫系统和介导免疫调节。研究者最初对于DC的认识并无抗原摄取功能,但随后发现DC在吞噬活性、迁移能力和介导获得性免疫应答系统方面皆具有其精确的阶段性调控机制,能够有效地刺激T淋巴细胞和B淋巴细胞的活化,从而启动和调节获得性免疫应答,故在免疫应答的诱导中具有独特的地位。但是由于DC在人体内的数量有限且功能复杂多样,对于其体外分化发育、抗原提呈、功能调控等诸多方面的研究存在着许多空白。最初的体外实验发现,人源未成熟DC可以诱导异体抗原反应性Treg的生成,而新近研究表明,在机体处于感染状态时,由病原体释放的Toll样受体(Toll like receptor, TLR)激动剂能够在Mo分化早期抑制其分化为常规未成熟DC,而发展为CD14+CD1a-的调节性细胞亚群。此外,应用Yssel's培养基能够将Mo来源DC诱导为以CDla-产生IL-10而无IL-12分泌为特征的免疫抑制型DC亚群,同时发现,新分离单核细胞的初始24h是改变DC后期表型的重要阶段,此后再加入刺激因素,DC将倾向于向常规方向分化。
其中,CD1家族分子在体内抗微生物感染过程中主要负责脂类抗原提呈,在Mo细胞来源DC的分化早期高表达,是DC分化发育中的标志性表面分子。CDla的表达往往适应于DC的功能需要,细胞捕获抗原时常见CDla高表达,当细胞处于抗原提呈状态时,其表达水平相应下调。病理条件下,CDla往往表达在CD83+的DC亚群中,已有研究证实,CD1蛋白是诱导有效细胞免疫的必要条件,其表达与麻风病分型直接相关。CD1+CD83+单核细胞来源的DC对于鉴别Mo的分化方向、活化CD1限制性T细胞起重要作用,且在临床治疗及预后监测中具有指导性意义。
本课题通过体外分离培养人外周血单核细胞并诱导其分化为DC,在各实验组中加入MBL及其它刺激因素,分别在DC的早期分化阶段、未成熟阶段和成熟阶段连续观察细胞形态,检测细胞表面分子标志、细胞功能及其相关机制,阐明MBL在此过程中的作用。初步揭示了MBL可通过调节单核细胞的分化方向,促进CD14+CD1alow调节性树突状细胞亚群的生成,并影响树突状细胞各发育阶段的表型和功能,从而有效联系机体天然免疫和获得性免疫系统,最终发挥抵抗病原体感染和调节体内免疫应答强度的双重作用。
第一章MBL对人外周血单核细胞诱导为调节性DC的研究
CDla分子是一类具有与MHCI和MHCII高度同源性的兼具抗原肽的识别与提呈功能的非典型抗原提呈分子。多数研究表明,只有郎格罕斯细胞(LC)和某些皮肤DC及胸腺细胞能够表达CDla,此外,CDla的表达水平与DC的成熟状态密切相关,未成熟DC高表达CDla,具且有较强的抗原捕获能力,而随着DC在抗原刺激下的逐步成熟,其抗原提呈能力提高而捕获抗原能力下降,此时CDla的表达也随之减少。
本实验中,取健康人外周血采用密度梯度离心法和磁珠分选获得高纯度单核细胞,联合应用IL-4和GM-CSF成功诱导出大量DC细胞。在单核细胞向DC诱导分化的早期阶段(d0),分别加入MBL和无关蛋白HSA,经常规培养,分别在第2天和第5天收获细胞,采用流式细胞术检测细胞表面Mo标志性分子CD14、CDllb的表达水平,同时检测DC标志性共刺激分子CD80、CD40、CD86、 CDla、CD83以及MHCⅡ类分子HLA-DR的表达,并通过PCR和ELISA分别在基因和蛋白水平检测MBL对细胞因子表达谱的影响。
结果表明,MBL刺激组获得DC细胞数明显少于对照组(F=7.169,P=0.026,多重比较结果P<0.01),且无凋亡现象发生,提示MBL可能以非调亡方式抑制单核细胞向常规DC方向的分化,同时我们观察到,该DC前体细胞持续表达单核细胞标志CD14,上调CD11b表达,而常规DC标志分子CDla低表达,CD80、 CD40和HLA-DR水平均下调(FCD80=0.216, PCD80=0.667, FCD40=0.242, PCD40=0.649,多重比较P CD80<0.05, P CD40<0.05; FHLA-DR/d2=3.582, P HLA-DR/d2=0-095,多重比较P HLA-DR/d2<0.05; FHLA-DR/d5=3.113, PHLA-DR/d5=0.118,多重比较PHLA-DR/d5<0.05),在此检测期内CD83仅少量表达。该细胞亚群以分泌IL-10、IL-6为特征(FIL-10=4.890, PIL-10=0.055, FIL-6=4.111, PIL-6=0.075,多重比较P IL-10<0.01, P IL-6<0.01),几乎无IL-12分泌(FIL-12=2.133, PIL-12=0.200,多重比较P IL-12<0.01),,几乎无IL-12分泌,因此推测在DC分化早期加入MBL,可能诱导出与常规DC表型和功能不同的DC细胞亚群。
此部分工作首先为后续的机制研究提供了充足稳定的MBL蛋白,经鉴定,MBL纯度较高且能够保持其天然生物活性,采用磁珠分选获得了高度纯化的单核细胞并具备良好的细胞活力用于DC细胞体外诱导,该诱导方法稳定、可重复性高,保证了实验所需的DC细胞来源。同时,初步确定了MBL可能诱导早期单核细胞分化为调节性DC亚群,针对这一现象,完成了对DC细胞表型和细胞因子表达特征的检测。
第二章MBL对单核细胞诱导为调节性DC亚群的机制及功能的初步研究
最近研究表明,浆细胞来源DC分泌低水平IL-12并促进T细胞向Th2亚群分化,而单核细胞来源DC则通过产生高水平IL-12诱导T细胞向Thl型分化。由于该研究中的DC细胞来源于不同的细胞前体,而同一细胞群体是否能分化出功能各异并且分泌不同细胞因子的DC亚群尚不得而知。Yssel等发现在诱导人外周血单核细胞向DC分化的常规培养基中加入胰岛素、转铁蛋白、亚麻油酸、油酸及棕榈酸能够促使单核细胞分化为CD14+DC,该细胞亚群即使在LPS和IFN-γ的刺激下仍然表现为CDla-并且不分泌IL-12,研究人员将该类DC细胞定义为mDC2,并且证实,其显著特征为CDla—和缺乏IL-12表达,并且能够调节T细胞的分化方向,促进产生Th0/Th2细胞。髓系DC的分化往往依赖于GM-CSF的激活,GM-CSF通过特异性非受体酪氨酸激酶JAKs和信号传导和激活转录因子STAT,尤其是JAK2和STAT5介导的信号通路发挥作用。
由此,本实验将该细胞亚群与T细胞混合培养,结果表明在分化早期受到MBL刺激的未成熟DC具有抑制T细胞增殖活化,抑制其IFN-y分泌的功能,结果有统计学差异(F1：5=2.280,P1：5=0.183,多重比较P1：5<0.01；F1:5(anti-CD3/CD28):=8.544, P1:5(anti-CD3/CD28)=0.018,多重比较P1:5(anti-CD3/CD28)<0.05, F1:20(anti-CD3/CD28)=3.527, P1:20(anti-CD3/CD28)=0.097,多重比较Pi:20(anti-CD3/CD28)<0-05)。CDla低表达和IL-12缺乏影响这类细胞的抗原提呈功能,但是并不影响其对抗原的摄取能力。同时,通过Westernblot信号通路分析,发现MBL可显著抑制STAT5、JNK表达以及胞外信号调节激酶ERKl磷酸化水平,而刺激PU.1、STAT3活性增强,由此证实了粒细胞巨噬细胞集落刺激因子依赖性STAT5/JAK2通路可能为MBL的作用靶点,从而调控细胞表型及其细胞因子表达谱。此外,实验证实该DC细胞亚群在LPS刺激下仍能够分化为CD83+DC。
第三章MBL对人单核细胞来源DC成熟的影响及机制的初步研究
在本实验室已有DC研究基础上,本部分实验深入探索MBL对人单核细胞来源DC成熟的影响及其机制。首先常规诱导培养DC,在第5天获得表型和功能稳定的未成熟DC细胞(imDC),继续培养2天,其中实验组加入MBL,在相同培养条件下加入HSA作为无关蛋白组,在第7天收集成熟DC及培养上清,采用流式细胞术、ELISA,在细胞分子水平分别检测MBL对人单核细胞来源的DC成熟的影响,并通过Western Blot在蛋白水平对刺激因素靶向调控的信号通路机制予以证实。结果提示,在天然免疫识别分子MBL存在的条件下,DC表面分子CD83、CD86、CD80、CD40和MHCⅡ类分子HLA-DR的表达均上调,且能促进同种异体非抗原特异性T细胞增殖。与DC前体细胞向未成熟DC分化的作用相反,MBL在未成熟DC向成熟DC发育的过程中刺激STAT5表达,抑制STAT3和SOCS活化,上清中IL-12、表达显著增加(FIL-12=1.468,PIL-12=0.295,多重比较P＜0.05), TNF-α分泌无统计学差异(FTNF-α=1-049, PTNF-α=0.422,多重比较P=0.973)。提示MBL促进未成熟DC向成熟DC的转化,通过影响抗原提呈细胞在其各个发展阶段的功能,调节天然免疫应答强度,最终指导人体的获得性免疫系统的应答。
综上所述,MBL作为识别和清除入侵病原体的重要防御性分子,在发挥宿主天然免疫应答的作用之外,我们的工作证实了MBL能够通过调节APC的分化发育和功能而参与获得性免疫应答,尤其能够在Mo向DC分化早期影响其分化方向,在此特定的发育阶段内促进调节性DC亚群的形成。这类CD14+CD1alowDC细胞以分泌IL-10、IL-6,低水平分泌IL-12为其特征,低表达多种表面共刺激分子,负向调节初始T细胞应答。此外,我们通过对其分子机制和信号传导通路的分析,初步揭示了MBL调节DC发育的双重作用,可能是MBL作为天然免疫防御蛋白在感染等病理条件下参与调控机体的免疫应答强度以及自身免疫性疾病的发生发展的机制之一。
Mannan-binding lectin (MBL) is the member of type C serum lectin superfamily which is recognized as one of the most important molecules for innate immune system. This kind of highly conservative glycosylated protein is mainly synthesized by hepatocytes and subsequently released in peripheral blood. Generally, Serials oligomer of MBL based on the tripolymer composed by3homogeneous peptide chains circled in vivo. MBL is capable of activating2MBL associated serine proteases, as of MASP-1, MASP-2and verified to initiate activity of complement system by lectin pathway through recognizing wide spectrum of carbohydrate moieties ligand on the surface of variety of pathogenic microorganisms. The complex then conduct the cell lysis and opsonophagocytosis indirectly. Receptors for MBL known today is collectin receptor combined with which MBL conduct such modulatory function as direct opsonophagocytosis, inflammation regulation and process of cell apoptosis. Meanwhile, soluble Patteren Recognition Receptor (PRR), including MBL as well as C1q, Pulmonary surfatcant-associated protein A (SP-A) and SP-D are members of defense collagen super family whose ligands are so called Pathogen-Associated Molecule Pattern (PAMP) which are identical to one or even more than one pathogene populations and especially essential for their survival but not for the host. MBL is able to prompt rapid effectively protective immune responses by the combination between globular carboxyl structure and specific PAMP. Additionally, MBL possesses broad spectrum of binding site and structure such as immunoglobulin to conduct phagocytosis of apoptotic cells, MBL-dependent activation of Th cells and cell mediated cytotoxic effects as well. In recent years, it was found that, MBL from rat can enhance the phagocytosis of Escherichia coli and Staphylococcus aureus for Kupffer cells in liver. Artificial reconstructive MBL was also detected to prompt phagocytosis of THP-1towards Escherichia coli. On the other hand, cytokine profile and antigen presenting cell (APC) were believed to play vital roles in determing the type and intensity of immune responses after exposure to dangerous signals. Furthermore, it was reported that MBL possesses ability to bind to both monocytes (Mo) and dendritic cells (DC) and to regulate secretion of cytokine profile leading to inhibition of release of proinflamatory cytokine as TNF-a from LPS/CD14induced Mo and up-regulating expression of cytokines with inhibitory characteristics. T cells are also protected from DC-SIGN mediated infection. Data showed that, SP-A and SP-D were well illustrated to bind to sorts of immune cells, such as Mo, DC and T cells, developing functions as modulators in either innate immune or adaptive immune responses. A enhanced expression of surface receptors on macrophages and release of regulatory cytokines and free radicals as well as clearance of autoapoptotic cells were observed in research on these two members of collectin super family.
DC, as the most potent professional cells for antigen-presenting, are responsible for induction of immune activity and immune modulation in vivo upon activation mainly via cytokines production and interaction with T cells and B cells. Initially, DCs was thought to be poorly phagocytic. Accumulating evidence has been shown on this point that DCs have a potent phagocytic activity which is mostly dedicated to inform and signal the adaptive immune system about peripheral tissue cues through their unique ability to sample tissue antigens. The capability of migration to the draning lymph nodes, presentation extracellular antigens and initiation tissue-specific T cell immunity are also highlighted simultaneously, the way in which adaptive immune responses are activated and regulated. Therefore, DCs were unique and of great importance for effective induction of immune responses. However, there are far less DCs living inside human body with much functional complexity, by which our understanding of DC differentiation in vitro, antigen presenting and regulation function etc. is greatly limited. The primary in vitro experiment suggested that, human immature DCs could induce the generation of T cells with allogeneic antigen reactivity. Later, Toll like receptor (TLR) agonists derived from invader pathogenes were identified to block conventional differentiation of CD14+Mo at early stage into immature DCs resulting in a deviated phenotype during infection. These TLR induced APCs were characterized by a retained expression of CD14and a lack of CD la, which attracted attention for their regulatory functions. Moreover, it is reported similarly that, culture with Yssel's medium enabled a phenotypically and functionally novel Mo-derived DCs subset that lack IL-12synthesis but produce high levels of IL-10with altered inhibitory characteristic. The first24hours were regarded as the most critical period in determining DC differentiation, because DC is likely to differentiate into conventional phenotype with addition of stimulatory factors after then.
It is worthy to focus on CD1family molecules that are mainly responsible for presenting of most lipid antigen during the process of antimicrobial immunity in vivo. They are normally of high expression at early differentiation of Mo-derived DCs and regarded as surface bio-marker in cell development. The expression of CD1a appears to be dependent on the functional state of the DCs, as CD la expression is high when cells are capturing antigen and is down-regulated when antigen presentation is occurring. Pathologically, the expression of CD la correlates with CD83positive DC subset. There is evidence suggesting that, CD1a is required for induction of potent cell-mediated immunity to Mycobacterium and can be considered clinically to assess the course of leprosy. CD1+CD83+Mo-derived DCs were highly efficient APCs for CD1-restricted T cells activation and for the clinical treatment with further instructional significance on process monitoring.
After isolation and purification of human peripheral blood Monocyte (PBMC), we explored the effect of MBL during differentiation to DCs among different groups by observing cellular morphology on early differentiation, immature stage and mature stage consecutively. Surface marker molecules, cellular function and corresponding mechanism were examined respectively. Here we reported the inhibitory induction of peripheral blood mononuclear cells derived monocytes to conventional dendritic cells when administrated with MBL at very beginning of DC cultivation in vitro and explored the corresponding molecular mechanisms. An alternative phenotype with a reduced expression of CD1a and a retained expression of CD14is presented. It has been observed with up-regulated release of IL-10and IL-6, and functioned as an inhibitor for T-cell proliferation. IL-12was detected at a lower level relatively, but no differences were found in comparative analysis with samples treated with no mannan-banding lectin. Regarding that granulocyte macrophage colony stimulating factor dependent JAK2/STAT5was known as a pivotal transducer during the monocyte-derived dentritic cells development, we therefore examined this pathway and findings showed an attenuated activity of both STAT5and JNK but a stimulated STAT3accumulation in the dentritic cell precursors. Furthermore, phosphorylation of extracellular signal-regulated kinase (ERK) was inhibited as well. Taken together, data indicated that, encounter of Mo with MBL results in an inhibitory differentiation into immature conventional dentritic cells from CD14+monocytes in vitro leading to a deviated phenotype of dentritic cells, which enables mannan-binding lectin to balance the intensity of innate and adaptive immune response indirectly through impeding the eventual generation of monocytes derived dentritic cells. We revealed the possible mechanism that how MBL exert effects on adaptive immunity and elaborated that MBL can probably regulate differentiation of Mo, prompting generation of CD14+CD1alow regulatory DC subset with altered phenotype and function in each stage. Therefore, MBL is conceivable to be regarded as a bridge to integrate innate and adaptive immune responses with novel dual effects both on defense against pathogenes infection and regulation of immune responses intensity.
CHAPER1MBL induces differentiation of Mo into regulatory DCs
The molecule of CD1a is highly homologous with MHCI and MHCII and has similar capability of recognization and presenting of antigen peptide. Most reported shown that, CD la can be only expressed by Langerhans cells (LC) and some DC resided in skin and thymocytes. Besides, the expression of CD1a correlates tightly with maturation of DCs, as immature DCs are more capable on antigen capture than mature DCs with relatively higher expression of CD la. DCs mature rapidly under stimulation of pathogens resulting in increased capability of antigen presenting and down-regulated ability of antigen capture with a reduced expression of CD1a.
We isolated PBMC from fresh heparinized blood of selected healthy donors using Ficoll-Hypaque gradient centrifugation and MACs. High purified Mo were obtained and a large number of Mo-derived DCs were induced using GM-CSF and IL-4simultaneously. At early stage, purified MBL and unrelated protein HSA were added where indicated. Cells were havested at day2and day5respectively aftef routine culture. We examined the expression of surface molecules as markers during Mo development including CD14and CD1a which are regarded as important instructor of Mo differentiation towards DCs by Flow cytometre (FCM). Meanwhile, expression of costimulatory molecules such as CD80, CD40, CD86, CD1a, CD83and class Ⅱ MHC HLA-DR were tested as well to determine maturation of DCs. PCR and ELISA were performed to examine secretion of cytokine profile on protein and gene level respectively.
Data showed that, compared with control culture with GI medium alone, experimental group was observed with a less quantity for cell counting ultimately and poor proliferation stimulated by GM-CSF which was thought to have great importance for maintaining survival and activity of CD14+monocytes. MBL did not induce apoptosis in treated cells. Additionally, expression of co-stimulatory molecules such as CD80, CD40and of HLA-DR decreased as well, whereas cells did not differ in expression of CD86between groups with or without stimulation of MBL. Moreover, there was only a weak expression of CD83among all cases examined. The group added by MBL produced undetectable levels of IL-α, IL-1b, TNF-a and IL-12in gene expression relatively, but were observed with high production of IL-10in contrast to group cultured in GI medium. ELISA was performed for further confirmation. Findings were identical to the observation of genetic test. Besides, the expression of IL-6was stimulated as well by MBL, suggesting that it is probably to obtain an altered DC subset with deviated phenotype and function from conventional DCs with early encounter of MBL
This part of work enables a steady and sufficient supply of MBL protein with high purity and natural biological activity after experimental identification. High purified Monocytes were obtained from MACs isolation and available for DC induction in vitro with high cytoactivity. Being relatively shout-lived, DCThis method applied were completely conceivable and repeatable for subsequent DC research quantitatively. At the same time, we identified a probable regulatory DC subset treated by MBL at early stage of Mo differentiation and finished primarily the characterization on phenotypic analysis and cytokines expression profile of of these DCs.
CHAPER2Primary explorations on mechanism in generation of MBL-induced regulatory DC
Research of recent years showed that, there is low IL-12secretion detected from plasma derived DC which directs Th2differentiation in both human and murine system, whereas, Mo derived DC favors differentiation of Thl cells by producing high levels of IL-12. Because these DC subsets were derived from different cell populations, it remained to be determinded whether the same precursor has the potential to differentiate into DC with different cytokine production profiles. It is discovered by Yssel etc. that, when Mo was induced by Yssel's medium which is IMDM enriched with human transferrin, insulin, linoleic acid, oleic acid and palmitic acid, it will differentiate to CD14+DC. These DCs remains CD1a-and lacks IL-12production even upon stimulation of LPS and IFN-γ, which were designated as mDC2, mDC2is characterized by both lack of IL-12and CD1a negative expression. They are identified by regulating differentiation of T cells and enhancing production of Th0/Th2cells. Myeloid dendritic cell differntiation depends on the activity of GM-CSF, a cytokine that signals through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs), especially JAK2and STAT5.
Based on this, we co-cultured these MBL-induced DCs together with T cells and found that addition of MBL exerted effects on early development of immature DC leading to the inhibition of either proliferation or activation of T cells and their secretion of IFN-γ as well. The lack of IL-12and CD1a expression by these MBL-induced DCs attenuated their capacity of antigen presenting but did not affect their capability of antigen uptake. Meanwhile, significant decreased expression of STAT5and JNK were observed through Westernblot signal pathway analysis. We also examined the extracellular regulatory protein kinases (ERK), finding an decreased activity of phosphorylation and stimulated expression of STAT3, by which way MBL probably regulated Mo differentiation phenotypically and cytokine production profile targeting the pathway of GM-CSF dependent STAT5/JAK2. Moreover, these mDC are verified here that they can be matured into CD83+cells.
CHAPER3Effects on maturation of monocyte-derived dendritic cells by mannan-binding lectin in vitro
Based on the DC research in our lab previously, we explored the further effects of MBL on Mo-derived DC maturation and corresponding mechanism in this part. DC were generated by routine induction culture and harvested at day5. Immature DC cells then can be obtained from this culture system with repeatable classical cellular phenotype. After that, cells were designated into different groups for another2days, among which MBL was added in experimental case and HSA group was considered as control test with unrelated proteins. Mature cells were harvested as DC development at day7and supernatant was collected of each group. FCM and ELISA were performed to analyze effects on maturation of Mo-derived DC of MBL both on cellular and molecular levels. Western Blot was ran to determine the targeting signal pathways controlled by stimulatory factors at protein level. Data showed that, under the condition of existence of innate recognition molecule of MBL, up-regulated changes occurred on such surface markers as CD83, CD86, CD80, CD40and HLA-DR as a molecule of MHCⅡ, resulting in enhancement of proliferation for non antigen specific homologous naive T cells. Contrast to the role in the process of development of DC precursors to immature DCs, MBL stimulated expression of STAT5but attenuated activity of both STAT3and SOCS. Production of IL-12and were increased while nither TNF-a nor IL-6secretion was recorded, which suggested that, MBL possessed the ability to prompt the mature process of immature DC including functional facilitation in each stage of APC development, from which the intensity of innate immune reponses were regulated. Eventually adaptive immune response system was directed through these altered APCs and effecter immune cells.
In conclusion, in this study we focus on the additional capability of MBL besides its conventional role in innate immune response system as a vital recognizer and defense molecule responsible for invading pathogens clearance, confirming that MBL exerts effects on human adaptive immune responses by regulating both development process and function of APCs, especially influent the differentiation towards conventional DC of Mo at early stage. Inhibitory DCs with deviated phenotype were generated as a probable regulatory DC subset at specific stage of development. These CD14+CD1alow cells conducted negative regulation through interaction with naive T cells and characterized by lack of IL-12synthesis, producing medium levels of both IL-10and IL-6. Most co-stimulatory surface molecules were down-regulated. Furthermore, the regulatory mechanisms were revealed primarily on MBL mediated balance between intensity of innate immune and adaptive immune response in the pathological state such as infection by presenting analysis of molecular mechanism and the signal transduction pathways.
树突状细胞是机体内功能最强的专职抗原提呈细胞,且能够通过分泌特异性的细胞因子和与T、B细胞的相互作用有效启动免疫系统和介导免疫调节。研究者最初对于DC的认识并无抗原摄取功能,但随后发现DC在吞噬活性、迁移能力和介导获得性免疫应答系统方面皆具有其精确的阶段性调控机制,能够有效地刺激T淋巴细胞和B淋巴细胞的活化,从而启动和调节获得性免疫应答,故在免疫应答的诱导中具有独特的地位。但是由于DC在人体内的数量有限且功能复杂多样,对于其体外分化发育、抗原提呈、功能调控等诸多方面的研究存在着许多空白。最初的体外实验发现,人源未成熟DC可以诱导异体抗原反应性Treg的生成,而新近研究表明,在机体处于感染状态时,由病原体释放的Toll样受体(Toll like receptor, TLR)激动剂能够在Mo分化早期抑制其分化为常规未成熟DC,而发展为CD14+CD1a-的调节性细胞亚群。此外,应用Yssel's培养基能够将Mo来源DC诱导为以CDla-产生IL-10而无IL-12分泌为特征的免疫抑制型DC亚群,同时发现,新分离单核细胞的初始24h是改变DC后期表型的重要阶段,此后再加入刺激因素,DC将倾向于向常规方向分化。
其中,CD1家族分子在体内抗微生物感染过程中主要负责脂类抗原提呈,在Mo细胞来源DC的分化早期高表达,是DC分化发育中的标志性表面分子。CDla的表达往往适应于DC的功能需要,细胞捕获抗原时常见CDla高表达,当细胞处于抗原提呈状态时,其表达水平相应下调。病理条件下,CDla往往表达在CD83+的DC亚群中,已有研究证实,CD1蛋白是诱导有效细胞免疫的必要条件,其表达与麻风病分型直接相关。CD1+CD83+单核细胞来源的DC对于鉴别Mo的分化方向、活化CD1限制性T细胞起重要作用,且在临床治疗及预后监测中具有指导性意义。
本课题通过体外分离培养人外周血单核细胞并诱导其分化为DC,在各实验组中加入MBL及其它刺激因素,分别在DC的早期分化阶段、未成熟阶段和成熟阶段连续观察细胞形态,检测细胞表面分子标志、细胞功能及其相关机制,阐明MBL在此过程中的作用。初步揭示了MBL可通过调节单核细胞的分化方向,促进CD14+CD1alow调节性树突状细胞亚群的生成,并影响树突状细胞各发育阶段的表型和功能,从而有效联系机体天然免疫和获得性免疫系统,最终发挥抵抗病原体感染和调节体内免疫应答强度的双重作用。
第一章MBL对人外周血单核细胞诱导为调节性DC的研究
CDla分子是一类具有与MHCI和MHCII高度同源性的兼具抗原肽的识别与提呈功能的非典型抗原提呈分子。多数研究表明,只有郎格罕斯细胞(LC)和某些皮肤DC及胸腺细胞能够表达CDla,此外,CDla的表达水平与DC的成熟状态密切相关,未成熟DC高表达CDla,具且有较强的抗原捕获能力,而随着DC在抗原刺激下的逐步成熟,其抗原提呈能力提高而捕获抗原能力下降,此时CDla的表达也随之减少。
本实验中,取健康人外周血采用密度梯度离心法和磁珠分选获得高纯度单核细胞,联合应用IL-4和GM-CSF成功诱导出大量DC细胞。在单核细胞向DC诱导分化的早期阶段(d0),分别加入MBL和无关蛋白HSA,经常规培养,分别在第2天和第5天收获细胞,采用流式细胞术检测细胞表面Mo标志性分子CD14、CDllb的表达水平,同时检测DC标志性共刺激分子CD80、CD40、CD86、 CDla、CD83以及MHCⅡ类分子HLA-DR的表达,并通过PCR和ELISA分别在基因和蛋白水平检测MBL对细胞因子表达谱的影响。
结果表明,MBL刺激组获得DC细胞数明显少于对照组(F=7.169,P=0.026,多重比较结果P<0.01),且无凋亡现象发生,提示MBL可能以非调亡方式抑制单核细胞向常规DC方向的分化,同时我们观察到,该DC前体细胞持续表达单核细胞标志CD14,上调CD11b表达,而常规DC标志分子CDla低表达,CD80、 CD40和HLA-DR水平均下调(FCD80=0.216, PCD80=0.667, FCD40=0.242, PCD40=0.649,多重比较P CD80<0.05, P CD40<0.05; FHLA-DR/d2=3.582, P HLA-DR/d2=0-095,多重比较P HLA-DR/d2<0.05; FHLA-DR/d5=3.113, PHLA-DR/d5=0.118,多重比较PHLA-DR/d5<0.05),在此检测期内CD83仅少量表达。该细胞亚群以分泌IL-10、IL-6为特征(FIL-10=4.890, PIL-10=0.055, FIL-6=4.111, PIL-6=0.075,多重比较P IL-10<0.01, P IL-6<0.01),几乎无IL-12分泌(FIL-12=2.133, PIL-12=0.200,多重比较P IL-12<0.01),,几乎无IL-12分泌,因此推测在DC分化早期加入MBL,可能诱导出与常规DC表型和功能不同的DC细胞亚群。
此部分工作首先为后续的机制研究提供了充足稳定的MBL蛋白,经鉴定,MBL纯度较高且能够保持其天然生物活性,采用磁珠分选获得了高度纯化的单核细胞并具备良好的细胞活力用于DC细胞体外诱导,该诱导方法稳定、可重复性高,保证了实验所需的DC细胞来源。同时,初步确定了MBL可能诱导早期单核细胞分化为调节性DC亚群,针对这一现象,完成了对DC细胞表型和细胞因子表达特征的检测。
第二章MBL对单核细胞诱导为调节性DC亚群的机制及功能的初步研究
最近研究表明,浆细胞来源DC分泌低水平IL-12并促进T细胞向Th2亚群分化,而单核细胞来源DC则通过产生高水平IL-12诱导T细胞向Thl型分化。由于该研究中的DC细胞来源于不同的细胞前体,而同一细胞群体是否能分化出功能各异并且分泌不同细胞因子的DC亚群尚不得而知。Yssel等发现在诱导人外周血单核细胞向DC分化的常规培养基中加入胰岛素、转铁蛋白、亚麻油酸、油酸及棕榈酸能够促使单核细胞分化为CD14+DC,该细胞亚群即使在LPS和IFN-γ的刺激下仍然表现为CDla-并且不分泌IL-12,研究人员将该类DC细胞定义为mDC2,并且证实,其显著特征为CDla—和缺乏IL-12表达,并且能够调节T细胞的分化方向,促进产生Th0/Th2细胞。髓系DC的分化往往依赖于GM-CSF的激活,GM-CSF通过特异性非受体酪氨酸激酶JAKs和信号传导和激活转录因子STAT,尤其是JAK2和STAT5介导的信号通路发挥作用。
由此,本实验将该细胞亚群与T细胞混合培养,结果表明在分化早期受到MBL刺激的未成熟DC具有抑制T细胞增殖活化,抑制其IFN-y分泌的功能,结果有统计学差异(F1：5=2.280,P1：5=0.183,多重比较P1：5<0.01；F1:5(anti-CD3/CD28):=8.544, P1:5(anti-CD3/CD28)=0.018,多重比较P1:5(anti-CD3/CD28)<0.05, F1:20(anti-CD3/CD28)=3.527, P1:20(anti-CD3/CD28)=0.097,多重比较Pi:20(anti-CD3/CD28)<0-05)。CDla低表达和IL-12缺乏影响这类细胞的抗原提呈功能,但是并不影响其对抗原的摄取能力。同时,通过Westernblot信号通路分析,发现MBL可显著抑制STAT5、JNK表达以及胞外信号调节激酶ERKl磷酸化水平,而刺激PU.1、STAT3活性增强,由此证实了粒细胞巨噬细胞集落刺激因子依赖性STAT5/JAK2通路可能为MBL的作用靶点,从而调控细胞表型及其细胞因子表达谱。此外,实验证实该DC细胞亚群在LPS刺激下仍能够分化为CD83+DC。
第三章MBL对人单核细胞来源DC成熟的影响及机制的初步研究
在本实验室已有DC研究基础上,本部分实验深入探索MBL对人单核细胞来源DC成熟的影响及其机制。首先常规诱导培养DC,在第5天获得表型和功能稳定的未成熟DC细胞(imDC),继续培养2天,其中实验组加入MBL,在相同培养条件下加入HSA作为无关蛋白组,在第7天收集成熟DC及培养上清,采用流式细胞术、ELISA,在细胞分子水平分别检测MBL对人单核细胞来源的DC成熟的影响,并通过Western Blot在蛋白水平对刺激因素靶向调控的信号通路机制予以证实。结果提示,在天然免疫识别分子MBL存在的条件下,DC表面分子CD83、CD86、CD80、CD40和MHCⅡ类分子HLA-DR的表达均上调,且能促进同种异体非抗原特异性T细胞增殖。与DC前体细胞向未成熟DC分化的作用相反,MBL在未成熟DC向成熟DC发育的过程中刺激STAT5表达,抑制STAT3和SOCS活化,上清中IL-12、表达显著增加(FIL-12=1.468,PIL-12=0.295,多重比较P＜0.05), TNF-α分泌无统计学差异(FTNF-α=1-049, PTNF-α=0.422,多重比较P=0.973)。提示MBL促进未成熟DC向成熟DC的转化,通过影响抗原提呈细胞在其各个发展阶段的功能,调节天然免疫应答强度,最终指导人体的获得性免疫系统的应答。
综上所述,MBL作为识别和清除入侵病原体的重要防御性分子,在发挥宿主天然免疫应答的作用之外,我们的工作证实了MBL能够通过调节APC的分化发育和功能而参与获得性免疫应答,尤其能够在Mo向DC分化早期影响其分化方向,在此特定的发育阶段内促进调节性DC亚群的形成。这类CD14+CD1alowDC细胞以分泌IL-10、IL-6,低水平分泌IL-12为其特征,低表达多种表面共刺激分子,负向调节初始T细胞应答。此外,我们通过对其分子机制和信号传导通路的分析,初步揭示了MBL调节DC发育的双重作用,可能是MBL作为天然免疫防御蛋白在感染等病理条件下参与调控机体的免疫应答强度以及自身免疫性疾病的发生发展的机制之一。
Mannan-binding lectin (MBL) is the member of type C serum lectin superfamily which is recognized as one of the most important molecules for innate immune system. This kind of highly conservative glycosylated protein is mainly synthesized by hepatocytes and subsequently released in peripheral blood. Generally, Serials oligomer of MBL based on the tripolymer composed by3homogeneous peptide chains circled in vivo. MBL is capable of activating2MBL associated serine proteases, as of MASP-1, MASP-2and verified to initiate activity of complement system by lectin pathway through recognizing wide spectrum of carbohydrate moieties ligand on the surface of variety of pathogenic microorganisms. The complex then conduct the cell lysis and opsonophagocytosis indirectly. Receptors for MBL known today is collectin receptor combined with which MBL conduct such modulatory function as direct opsonophagocytosis, inflammation regulation and process of cell apoptosis. Meanwhile, soluble Patteren Recognition Receptor (PRR), including MBL as well as C1q, Pulmonary surfatcant-associated protein A (SP-A) and SP-D are members of defense collagen super family whose ligands are so called Pathogen-Associated Molecule Pattern (PAMP) which are identical to one or even more than one pathogene populations and especially essential for their survival but not for the host. MBL is able to prompt rapid effectively protective immune responses by the combination between globular carboxyl structure and specific PAMP. Additionally, MBL possesses broad spectrum of binding site and structure such as immunoglobulin to conduct phagocytosis of apoptotic cells, MBL-dependent activation of Th cells and cell mediated cytotoxic effects as well. In recent years, it was found that, MBL from rat can enhance the phagocytosis of Escherichia coli and Staphylococcus aureus for Kupffer cells in liver. Artificial reconstructive MBL was also detected to prompt phagocytosis of THP-1towards Escherichia coli. On the other hand, cytokine profile and antigen presenting cell (APC) were believed to play vital roles in determing the type and intensity of immune responses after exposure to dangerous signals. Furthermore, it was reported that MBL possesses ability to bind to both monocytes (Mo) and dendritic cells (DC) and to regulate secretion of cytokine profile leading to inhibition of release of proinflamatory cytokine as TNF-a from LPS/CD14induced Mo and up-regulating expression of cytokines with inhibitory characteristics. T cells are also protected from DC-SIGN mediated infection. Data showed that, SP-A and SP-D were well illustrated to bind to sorts of immune cells, such as Mo, DC and T cells, developing functions as modulators in either innate immune or adaptive immune responses. A enhanced expression of surface receptors on macrophages and release of regulatory cytokines and free radicals as well as clearance of autoapoptotic cells were observed in research on these two members of collectin super family.
DC, as the most potent professional cells for antigen-presenting, are responsible for induction of immune activity and immune modulation in vivo upon activation mainly via cytokines production and interaction with T cells and B cells. Initially, DCs was thought to be poorly phagocytic. Accumulating evidence has been shown on this point that DCs have a potent phagocytic activity which is mostly dedicated to inform and signal the adaptive immune system about peripheral tissue cues through their unique ability to sample tissue antigens. The capability of migration to the draning lymph nodes, presentation extracellular antigens and initiation tissue-specific T cell immunity are also highlighted simultaneously, the way in which adaptive immune responses are activated and regulated. Therefore, DCs were unique and of great importance for effective induction of immune responses. However, there are far less DCs living inside human body with much functional complexity, by which our understanding of DC differentiation in vitro, antigen presenting and regulation function etc. is greatly limited. The primary in vitro experiment suggested that, human immature DCs could induce the generation of T cells with allogeneic antigen reactivity. Later, Toll like receptor (TLR) agonists derived from invader pathogenes were identified to block conventional differentiation of CD14+Mo at early stage into immature DCs resulting in a deviated phenotype during infection. These TLR induced APCs were characterized by a retained expression of CD14and a lack of CD la, which attracted attention for their regulatory functions. Moreover, it is reported similarly that, culture with Yssel's medium enabled a phenotypically and functionally novel Mo-derived DCs subset that lack IL-12synthesis but produce high levels of IL-10with altered inhibitory characteristic. The first24hours were regarded as the most critical period in determining DC differentiation, because DC is likely to differentiate into conventional phenotype with addition of stimulatory factors after then.
It is worthy to focus on CD1family molecules that are mainly responsible for presenting of most lipid antigen during the process of antimicrobial immunity in vivo. They are normally of high expression at early differentiation of Mo-derived DCs and regarded as surface bio-marker in cell development. The expression of CD1a appears to be dependent on the functional state of the DCs, as CD la expression is high when cells are capturing antigen and is down-regulated when antigen presentation is occurring. Pathologically, the expression of CD la correlates with CD83positive DC subset. There is evidence suggesting that, CD1a is required for induction of potent cell-mediated immunity to Mycobacterium and can be considered clinically to assess the course of leprosy. CD1+CD83+Mo-derived DCs were highly efficient APCs for CD1-restricted T cells activation and for the clinical treatment with further instructional significance on process monitoring.
After isolation and purification of human peripheral blood Monocyte (PBMC), we explored the effect of MBL during differentiation to DCs among different groups by observing cellular morphology on early differentiation, immature stage and mature stage consecutively. Surface marker molecules, cellular function and corresponding mechanism were examined respectively. Here we reported the inhibitory induction of peripheral blood mononuclear cells derived monocytes to conventional dendritic cells when administrated with MBL at very beginning of DC cultivation in vitro and explored the corresponding molecular mechanisms. An alternative phenotype with a reduced expression of CD1a and a retained expression of CD14is presented. It has been observed with up-regulated release of IL-10and IL-6, and functioned as an inhibitor for T-cell proliferation. IL-12was detected at a lower level relatively, but no differences were found in comparative analysis with samples treated with no mannan-banding lectin. Regarding that granulocyte macrophage colony stimulating factor dependent JAK2/STAT5was known as a pivotal transducer during the monocyte-derived dentritic cells development, we therefore examined this pathway and findings showed an attenuated activity of both STAT5and JNK but a stimulated STAT3accumulation in the dentritic cell precursors. Furthermore, phosphorylation of extracellular signal-regulated kinase (ERK) was inhibited as well. Taken together, data indicated that, encounter of Mo with MBL results in an inhibitory differentiation into immature conventional dentritic cells from CD14+monocytes in vitro leading to a deviated phenotype of dentritic cells, which enables mannan-binding lectin to balance the intensity of innate and adaptive immune response indirectly through impeding the eventual generation of monocytes derived dentritic cells. We revealed the possible mechanism that how MBL exert effects on adaptive immunity and elaborated that MBL can probably regulate differentiation of Mo, prompting generation of CD14+CD1alow regulatory DC subset with altered phenotype and function in each stage. Therefore, MBL is conceivable to be regarded as a bridge to integrate innate and adaptive immune responses with novel dual effects both on defense against pathogenes infection and regulation of immune responses intensity.
CHAPER1MBL induces differentiation of Mo into regulatory DCs
The molecule of CD1a is highly homologous with MHCI and MHCII and has similar capability of recognization and presenting of antigen peptide. Most reported shown that, CD la can be only expressed by Langerhans cells (LC) and some DC resided in skin and thymocytes. Besides, the expression of CD1a correlates tightly with maturation of DCs, as immature DCs are more capable on antigen capture than mature DCs with relatively higher expression of CD la. DCs mature rapidly under stimulation of pathogens resulting in increased capability of antigen presenting and down-regulated ability of antigen capture with a reduced expression of CD1a.
We isolated PBMC from fresh heparinized blood of selected healthy donors using Ficoll-Hypaque gradient centrifugation and MACs. High purified Mo were obtained and a large number of Mo-derived DCs were induced using GM-CSF and IL-4simultaneously. At early stage, purified MBL and unrelated protein HSA were added where indicated. Cells were havested at day2and day5respectively aftef routine culture. We examined the expression of surface molecules as markers during Mo development including CD14and CD1a which are regarded as important instructor of Mo differentiation towards DCs by Flow cytometre (FCM). Meanwhile, expression of costimulatory molecules such as CD80, CD40, CD86, CD1a, CD83and class Ⅱ MHC HLA-DR were tested as well to determine maturation of DCs. PCR and ELISA were performed to examine secretion of cytokine profile on protein and gene level respectively.
Data showed that, compared with control culture with GI medium alone, experimental group was observed with a less quantity for cell counting ultimately and poor proliferation stimulated by GM-CSF which was thought to have great importance for maintaining survival and activity of CD14+monocytes. MBL did not induce apoptosis in treated cells. Additionally, expression of co-stimulatory molecules such as CD80, CD40and of HLA-DR decreased as well, whereas cells did not differ in expression of CD86between groups with or without stimulation of MBL. Moreover, there was only a weak expression of CD83among all cases examined. The group added by MBL produced undetectable levels of IL-α, IL-1b, TNF-a and IL-12in gene expression relatively, but were observed with high production of IL-10in contrast to group cultured in GI medium. ELISA was performed for further confirmation. Findings were identical to the observation of genetic test. Besides, the expression of IL-6was stimulated as well by MBL, suggesting that it is probably to obtain an altered DC subset with deviated phenotype and function from conventional DCs with early encounter of MBL
This part of work enables a steady and sufficient supply of MBL protein with high purity and natural biological activity after experimental identification. High purified Monocytes were obtained from MACs isolation and available for DC induction in vitro with high cytoactivity. Being relatively shout-lived, DCThis method applied were completely conceivable and repeatable for subsequent DC research quantitatively. At the same time, we identified a probable regulatory DC subset treated by MBL at early stage of Mo differentiation and finished primarily the characterization on phenotypic analysis and cytokines expression profile of of these DCs.
CHAPER2Primary explorations on mechanism in generation of MBL-induced regulatory DC
Research of recent years showed that, there is low IL-12secretion detected from plasma derived DC which directs Th2differentiation in both human and murine system, whereas, Mo derived DC favors differentiation of Thl cells by producing high levels of IL-12. Because these DC subsets were derived from different cell populations, it remained to be determinded whether the same precursor has the potential to differentiate into DC with different cytokine production profiles. It is discovered by Yssel etc. that, when Mo was induced by Yssel's medium which is IMDM enriched with human transferrin, insulin, linoleic acid, oleic acid and palmitic acid, it will differentiate to CD14+DC. These DCs remains CD1a-and lacks IL-12production even upon stimulation of LPS and IFN-γ, which were designated as mDC2, mDC2is characterized by both lack of IL-12and CD1a negative expression. They are identified by regulating differentiation of T cells and enhancing production of Th0/Th2cells. Myeloid dendritic cell differntiation depends on the activity of GM-CSF, a cytokine that signals through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs), especially JAK2and STAT5.
Based on this, we co-cultured these MBL-induced DCs together with T cells and found that addition of MBL exerted effects on early development of immature DC leading to the inhibition of either proliferation or activation of T cells and their secretion of IFN-γ as well. The lack of IL-12and CD1a expression by these MBL-induced DCs attenuated their capacity of antigen presenting but did not affect their capability of antigen uptake. Meanwhile, significant decreased expression of STAT5and JNK were observed through Westernblot signal pathway analysis. We also examined the extracellular regulatory protein kinases (ERK), finding an decreased activity of phosphorylation and stimulated expression of STAT3, by which way MBL probably regulated Mo differentiation phenotypically and cytokine production profile targeting the pathway of GM-CSF dependent STAT5/JAK2. Moreover, these mDC are verified here that they can be matured into CD83+cells.
CHAPER3Effects on maturation of monocyte-derived dendritic cells by mannan-binding lectin in vitro
Based on the DC research in our lab previously, we explored the further effects of MBL on Mo-derived DC maturation and corresponding mechanism in this part. DC were generated by routine induction culture and harvested at day5. Immature DC cells then can be obtained from this culture system with repeatable classical cellular phenotype. After that, cells were designated into different groups for another2days, among which MBL was added in experimental case and HSA group was considered as control test with unrelated proteins. Mature cells were harvested as DC development at day7and supernatant was collected of each group. FCM and ELISA were performed to analyze effects on maturation of Mo-derived DC of MBL both on cellular and molecular levels. Western Blot was ran to determine the targeting signal pathways controlled by stimulatory factors at protein level. Data showed that, under the condition of existence of innate recognition molecule of MBL, up-regulated changes occurred on such surface markers as CD83, CD86, CD80, CD40and HLA-DR as a molecule of MHCⅡ, resulting in enhancement of proliferation for non antigen specific homologous naive T cells. Contrast to the role in the process of development of DC precursors to immature DCs, MBL stimulated expression of STAT5but attenuated activity of both STAT3and SOCS. Production of IL-12and were increased while nither TNF-a nor IL-6secretion was recorded, which suggested that, MBL possessed the ability to prompt the mature process of immature DC including functional facilitation in each stage of APC development, from which the intensity of innate immune reponses were regulated. Eventually adaptive immune response system was directed through these altered APCs and effecter immune cells.
In conclusion, in this study we focus on the additional capability of MBL besides its conventional role in innate immune response system as a vital recognizer and defense molecule responsible for invading pathogens clearance, confirming that MBL exerts effects on human adaptive immune responses by regulating both development process and function of APCs, especially influent the differentiation towards conventional DC of Mo at early stage. Inhibitory DCs with deviated phenotype were generated as a probable regulatory DC subset at specific stage of development. These CD14+CD1alow cells conducted negative regulation through interaction with naive T cells and characterized by lack of IL-12synthesis, producing medium levels of both IL-10and IL-6. Most co-stimulatory surface molecules were down-regulated. Furthermore, the regulatory mechanisms were revealed primarily on MBL mediated balance between intensity of innate immune and adaptive immune response in the pathological state such as infection by presenting analysis of molecular mechanism and the signal transduction pathways.
引文
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