甘露聚糖结合凝集素调节THP1/CD14细胞细胞因子分泌机制的初步探索
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摘要
尽管近年来天然免疫研究进展十分迅速,但是天然免疫的本质亟待揭示和阐明。随着Janeway CA Jr等提出天然免疫通过模式识别作用启动获得性免疫应答并左右其应答类型的假说,模式识别分子成为了免疫学的研究热点之一。模式识别分子是天然免疫的重要分子,根据其存在的形式,可分为可溶性和细胞膜表面两大类。已经发现,膜相模式识别受体中Toll样受体家族(Toll-likereceptors,TLR)、巨噬细胞甘露糖受体(macrophage mannose recetor,MR)和可溶性模式识别分子中的胶凝素(collectins)家族成员肺表面活性物质脱辅基蛋白A和D(surfactant protein A、surfactant protein D,SP-A、SP-D)等是连接天然免疫与获得性免疫的桥梁。天然免疫模式识别作用正直逼免疫应答的核心问题——应答的启动及其调控。
甘露聚糖结合凝集素(mannan-binding lectin,MBL)与SP-A、SP-D同属于胶凝素家族,主要由肝细胞分泌,作为糖蛋白存在于血浆中。MBL的一级结构至高级结构均与补体分子C1q高度同源。MBL的主要功能区有二,即糖识别域(carbohydrate-recognition domain,CRD)和胶原样区(collagen-like region,CLR)。前者是识别功能区,能以Ca~(2+)依赖方式识别多种病原体表面的糖结构。后者为效应功能区,与2个MBL相关丝氨酸蛋白酶(MBL associated serineprotease,MASP-1,MASP-2)结合而激活补体凝集素途径,在天然免疫中发挥防御作用,这是MBL作为补体成分的作用。近年来,对MBL的免疫调节作用亦有一些报道。国外相关研究表明,MBL可抑制DC-SIGN介导的T细胞应答,调节外周血单个核细胞(peripheral blood monoeuclear cell,PBMC)分泌细胞因子。本课题组以前的工作也发现MBL具有免疫调节作用:能抑制LPS刺激DC功能的成熟,抑制LPS刺激不同分化和活化状态的人单核细胞系THP1/CD14细胞产生TNF-α和IL-12。然而,有关MBL免疫调节作用的机制,目前国内外尚未见相关报道。
本研究中,我们以人单核细胞系THP1/CD14为细胞模型,初步研究了MBL与THP1/CD14细胞的结合、结合的特性和MBL与TLR的相互作用。
一、MBL与人单核细胞系THP1/CD14细胞结合
采用Cell-ELISA和流式细胞术,对MBL是否与THP1/CD14细胞结合进行了研究。Cell-ELISA结果显示,MBL能以浓度依赖方式与THP1/CD14细胞结合。以流式细胞术分析发现,在无Ca~(2+)存在的或以EDTA取代CaCl_2的结合缓冲液中,MBL与THP1/CD14细胞几乎不结合;而在含5 mmol/L或10 mmol/LCaCl_2的结合缓冲液中,MBL与THP1/CD14细胞可以结合,且在含10 mmol/LCaCl_2的结合缓冲液中结合能力比较强,提示二者的结合呈Ca~(2+)浓度依赖关系。结果表明,从血浆天然MBL在生理条件下确实可以结合THP1/CD14细胞,且这种结合具有Ca~(2+)浓度依赖性,提示THP1/CD14细胞上可能存在MBL的受体或结合蛋白,且二者结合需要Ca~(2+)参与。
二、MBL与THP1/CD14细胞结合特性的研究
一般认为,MBL可结合单核巨噬细胞表面的胶凝素受体即C1q受体(C1qreceptor,C1qR)。然而,根据上述结果,我们推测MBL可能通过其CRD与细胞表面某些糖蛋白或其他糖结构结合。本课题组已分段表达了重组人MBL的CLR(rhMBL-CLR)和CRD(rhMBL-CRD)蛋白,建立了分泌抗人C1qR单克隆抗体(monoclonal antibody,mAb)的杂交瘤E8,为本部分的实验提供了基础。在本实验中,我们首先制备了抗人C1qR mAb E8、rhMBL-CLR和rhMBL-CRD蛋白,然后采用流式细胞术对MBL结合THP1/CD14细胞的功能区进行了研究,检测了多种糖、rhMBL-CRD、rhMBL-CLR、C1q和E8对THP1/CD14细胞与MBL结合的抑制作用。结果显示,THP1/CD14细胞与MBL的结合能被某些糖抑制;rhMBL-CRD、rhMBL-CLR蛋白单独能部分抑制其结合,两者联合则可达完全抑制效果;但C1q和E8无抑制作用。资料表明,THP1/CD14细胞表达糖敏感的MBL受体或结合蛋白,包括对CLR和CRD特异的两种受体,均与C1qR无关;MBL可能以一种以上的方式结合THP1/CD14细胞。
三、MBL与TLR关系的初步研究
TLR-2是人类TLR家族成员中表达范围最广泛,识别病原微生物及其产物种类最多的分子,也是单核巨噬细胞主要表达的TLR之一。本部分研究中,我们首先采用ELISA、Western blot对TLR-2能否直接与MBL结合进行了分析,结果表明MBL能直接结合真核表达的重组TLR-2胞外段蛋白。对LPS刺激THP1/CD14细胞结合MBL的情况进行研究,ELISA结果显示,MBL与100 ng/LLPS刺激的THP1/CD14细胞结合能力比无LPS刺激者强,且sTLR-2蛋白可部分抑制这种结合;流式细胞术结果同样表明,LPS刺激的THP1/CD14细胞的MBL结合能力增强,且具有LPS浓度依赖性,随着LPS浓度增高,结合逐渐加强。然后,我们采用RT-PCR分析了LPS刺激对细胞TLR-2、TLR-4 mRNA表达的影响,发现LPS刺激2 h后THP1/CD14细胞TLR-2、TLR-4 mRNA表达水平增高;24 h后有所下降,接近与未刺激时水平。最后,提取未刺激的、LPS刺激的和MBL加LPS共同刺激的THP1/CD14细胞的核蛋白,采用Western bolt方法分析和转录因子NF-κB的转位,发现MBL可抑制LPS诱导NF-κB转位,表明MBL调节细胞因子分泌的机制可能与抑制NF-κB转位有关。
上述研究结果表明,THP1/CD14细胞表达Ca~(2+)依赖性、糖敏感的MBL受体或结合蛋白,包括对CLR和CRD特异的两种受体,均与C1qR无关。MBL可与TLR-2胞外段结合;LPS刺激后的THP1/CD14细胞结合MBL能力增强,这与LPS刺激THP1/CD14细胞TLR-2和TLR-4的mRNA表达增高是一致的;MBL与THP1/CD14细胞的结合能被sTLR-2蛋白部分抑制;MBL可抑制LPS诱导THP1/CD14细胞核转录因子NF-κB的转位。因此提出,MBL抑制LPS刺激THP1/CD14细胞产生促炎细胞因子可能与两方面的机制有关:①THP1/CD14细胞在LPS刺激下TLR2和TLR4表达量增加,导致其与MBL的结合力增强、结合量增多;②MBL抑制LPS诱导的核转录因子NF-κB的转位。
Although studies for innate immunity have been developing rapidly in the past few years,its essence remains to be revealed and elucidated.Accompany to the hypothesis that innate immunity initiates acquired immune response and controls the types of response through pattern-recognition put forward by Janeway CA et al, pattern recognition molecules have become one of the hot topics of immunologic researches.As the important molecules in innate immune,pattern recognition molecules can be divided into two categories based on their exsiting forms,including the dissolvability pattern recognition molecules in the humor and pattern recognition receptors expressed in the cell surface.At present,it has been shown that among the dissolvability pattern recognition molecules surfactant proteins A and D(SP-A and SP-D),which belong to the collectin subgroup of the C-type lectin superfamily,can bridge innate immunity and adaptive immunity even regulate adaptive immunity. Among the pattern recognition receptors,macrophage mannose recetor(MMR),a member of C-type lectin receptors(CLRs) family,as well as Toll-like receptors (TLRs) family can also play these functions.Accordingly,innate immunity pattern-recognition is pressing on towards the central issue of immune response, initiation of response as well as its control.
Mannan-binding lectin(MBL),a member of the collectin superfamily(C-type lectin with a collagen-like domain),along with SP-A and SP-D together,is a plasma glycoprotein mainly secreted by liver cells.MBL is a key molecule in innate immume system.MBL insufficiency is one of the most common immunodeficiencies. However,the mechanisms is still not clear.MBL,which has a total structure with C1q is a special molecule.Classical theory believes that the chief biological effect of MBL is that it forms compound with two MBL-associated serine proteases (MASP-1,MASP-2) through its collagen-like region(CLR) and then activates the complement cascade via lectin pathway to play the function of anti-infection in innate immune in the present of Ca~(2+).In the past few years,it has been reported that MBL plays important functions in immunological regulation.Correlated researches abroad demostrated that MBL could inhibit T cell response mediated by DC-SIGN,regulate the cytokines production of peripheral blood monoeuclear cell(PBMC).Previously, our research team found that MBL could inhibit of the maturation of LPS stimulated dendritic cells(DC) and inhibit the production of TNF-αand IL-12 in hunman monocyte line THP1/CD14 cells in different differentiation states.These findings also confirmed the functions of MBL in immunological regulation.However,the mechanisms of MBL in immunological regulation has not been reported yet at home and abroad.
In order to explore the the mechanisms of the functions of MBL in immunological regulation,we selected the hunman monocyte line THP1/CD14 cells as our cell model.We investigated the binding of MBL to THP1/CD14 cells,the characteristics of the binding and the interaction of MBL with TLRs.This research can be divide into three parts as follow.
PartⅠDetection the binding of MBL to hunman monocyte line THP1/CD14 cells
In this part,We investigated whether MBL can bind to THP1/CD14 cells by Cell-ELISA and flow cytometry(FCM).The result of Cell-ELISA revealed that absorptance of 450nm depended on the concentration of MBL in experiment group. The result of FCM showed that MBL could hardly bind to THP1/CD14 cells in the absence of Ca~(2+) or in the presence of EDTA as a substitute of CaCl_2,but it could bind to THP1/CD14 cells in the buffers which contain 5 mmol/L or 10 mmol/L CaCl_2,and the binding capability in the buffer containing 10 mmol/L CaCl_2 is slightly increased compared to in the buffer containing 5 mmol/L CaCl_2,suggesting that the binding of them depended on the Ca~(2+) concentration.These results above indicated that natural MBL purified from plasma could actually bind to THP1/CD14 cells in physiological conditions,and this binding was evidently dependent on Ca~(2+) concentation.Thus there may exist MBL receptors or binding proteins in the cellular membrane surface of THP1/CD14 cells,and the binding of MBL to THP1/CD14 cells needs the participation of Ca~(2+).
PartⅡStudy on the characteristics of MBL binding to THP1/CD14 cells
According to the Ca~(2+) dependent characteristic of MBL binding to THP1/CD14 cells in part one,we presumed that MBL might bind to certain glycoproteins or other glycosylated structure in cellular membrane surface through its carbohydrate-recognition domain(CRD).Classical theory believes MBL binds to C1q receptor (C1qR) in cellular membrane surface of mononuclear macrophage through its collagen-like region(CLR).Previously,our research team have successfully expressed prokaryotic MBL-CRD protein and MBL-CLR protein and prepared cell line E8 which can secrete mouse anti-human C1qR monoclonal antibody(mAb).So,in this part,we firstly resused the cell line E8,immunized mouse to obtain abdominal dropsy,and then purificated the abdominal dropsy obtained this mAb.Secondly,we prepared MBL-CLR protein by Ni~(2+)-NTA agarose chromatography.Subsequently,we focused on the functional domains of MBL binding to THP1/CD14 cells,we detected the inhibitory effect of MBL binding to THP1/CD14 cells,using sugars,prokaryotic rh(recombinant human) MBL-CRD,prokaryotic rhMBL-CLR protein,C1q and mouse anti-C1q mAb as potential inhibitors,by FCM.The results showed that their binding can be partly inhibit by some sugars,rhMBL-CRD alone and rhMBL-CLR alone.And the inhibitory effect was obviously when rhMBL-CRD and rhMBL-CLR were added together.However,C1q and mouse anti-human C1qR mAb had no inhibitory action.These data showed that THP1/CD14 cells can express sugar-sensitive MBL receptors or binding proteins,including two specific categories of receptors against CLR and CRD,and these receptors were unrelated with C1qR. Therefore,this findings reveals that MBL may binding to THP1/CD14 cells through more than one way.
PartⅢPreliminary study of the relationship between MBL and TLRs
Among mammalian TLRs,TLR-2 is expressed very extensively,and mediates recogonition of a wide range of microbial products.TLR-2 is one of the predominant TLRs expressed in mononuclear macrophage.In this part,we focused on whether MBL can directly bind to TLR-2 by ELISA and Western blot.The results of the two methods both showed that MBL can directly bind to the extracellular domain of TLR-2 protein(sTLR-2).In the research of LPS-stimulated THP1/CD14 cells,we investigated the binding capability of LPS-stimulated THP1/CD14 cells with MBL by ELISA and FCM.It was found that the binding capability of LPS(100ng/L) -stimulated THP1/CD14 cells with MBL was strengthened compared to untreated THP1/CD14 cells,statistical analysis showed the difference was significant.The results of FCM also showed the the binding capability of LPS-stimulated THP1/CD14 cells with MBL was strengthened,and the strengthen was in a LPS concentration-dependent manner.The higher concentration of LPS,the more stronger of the binding capability.Subseqently,we analyzed the impact of the stimulation of LPS to the expression of TLR-2 and TLR-4 mRNA in THP1/CD14 cells.RT-PCR analysis showed that the expression of TLR-2 and TLR-4 mRNA in LPS-stimulated THP1/CD14 cells was significantly increased after 2h compared to the non-LPS-stimulated cells,and it was dropped to the approximate level of non-LPS-stimulated cells after 24h.Eventually,we studied the signal transduction. The nulear proteins of THP1/CD14 cells,which were untreated,LPS treated,MBL and LPS treated,were extracted.Then the extracted nulear proteins were essaied by Western bolt.The result of Western bolt showed MBL can inhibit the transposition of transcription factor NF-κB induced by LPS,suggesting the mechanism of MBL in the regulation of cytokines secretion.
The results above indicated that THP1/CD14 cells might epress Ca~(2+)-dependent, sugar-sensitive MBL receptors or binding proteins,including both CLR-specific and CRD-specific,which have nothing to do with C1qR.MBL can directly bind to sTLR-2.The binding capability of LPS-stimulated THP1/CD14 cells was strengthened,and was consistent with the expression of TLR-2 and TLR-4 by RT-PCR in LPS-stimulated THP1/CD14 cells.The binding can be partly inhibit by sTLR-2.MBL can suppress the transposition of transcription factor NF-κB induced by LPS in THP1/CD14 cells.Therefore,we presumed that there might be two aspects of mechanisms about the inhibition of the proinflammatory cytokines produced by LPS-stimulated THP1/CD14 cells,the one is the enhanced expression of TLR-2 and TLR-4 mRNA induced by LPS,which resulted in stronger binding capability and more binding capacity;the other is the suppression of the transposition of transcription factor NF-κB induced by MBL.
甘露聚糖结合凝集素(mannan-binding lectin,MBL)与SP-A、SP-D同属于胶凝素家族,主要由肝细胞分泌,作为糖蛋白存在于血浆中。MBL的一级结构至高级结构均与补体分子C1q高度同源。MBL的主要功能区有二,即糖识别域(carbohydrate-recognition domain,CRD)和胶原样区(collagen-like region,CLR)。前者是识别功能区,能以Ca~(2+)依赖方式识别多种病原体表面的糖结构。后者为效应功能区,与2个MBL相关丝氨酸蛋白酶(MBL associated serineprotease,MASP-1,MASP-2)结合而激活补体凝集素途径,在天然免疫中发挥防御作用,这是MBL作为补体成分的作用。近年来,对MBL的免疫调节作用亦有一些报道。国外相关研究表明,MBL可抑制DC-SIGN介导的T细胞应答,调节外周血单个核细胞(peripheral blood monoeuclear cell,PBMC)分泌细胞因子。本课题组以前的工作也发现MBL具有免疫调节作用:能抑制LPS刺激DC功能的成熟,抑制LPS刺激不同分化和活化状态的人单核细胞系THP1/CD14细胞产生TNF-α和IL-12。然而,有关MBL免疫调节作用的机制,目前国内外尚未见相关报道。
本研究中,我们以人单核细胞系THP1/CD14为细胞模型,初步研究了MBL与THP1/CD14细胞的结合、结合的特性和MBL与TLR的相互作用。
一、MBL与人单核细胞系THP1/CD14细胞结合
采用Cell-ELISA和流式细胞术,对MBL是否与THP1/CD14细胞结合进行了研究。Cell-ELISA结果显示,MBL能以浓度依赖方式与THP1/CD14细胞结合。以流式细胞术分析发现,在无Ca~(2+)存在的或以EDTA取代CaCl_2的结合缓冲液中,MBL与THP1/CD14细胞几乎不结合;而在含5 mmol/L或10 mmol/LCaCl_2的结合缓冲液中,MBL与THP1/CD14细胞可以结合,且在含10 mmol/LCaCl_2的结合缓冲液中结合能力比较强,提示二者的结合呈Ca~(2+)浓度依赖关系。结果表明,从血浆天然MBL在生理条件下确实可以结合THP1/CD14细胞,且这种结合具有Ca~(2+)浓度依赖性,提示THP1/CD14细胞上可能存在MBL的受体或结合蛋白,且二者结合需要Ca~(2+)参与。
二、MBL与THP1/CD14细胞结合特性的研究
一般认为,MBL可结合单核巨噬细胞表面的胶凝素受体即C1q受体(C1qreceptor,C1qR)。然而,根据上述结果,我们推测MBL可能通过其CRD与细胞表面某些糖蛋白或其他糖结构结合。本课题组已分段表达了重组人MBL的CLR(rhMBL-CLR)和CRD(rhMBL-CRD)蛋白,建立了分泌抗人C1qR单克隆抗体(monoclonal antibody,mAb)的杂交瘤E8,为本部分的实验提供了基础。在本实验中,我们首先制备了抗人C1qR mAb E8、rhMBL-CLR和rhMBL-CRD蛋白,然后采用流式细胞术对MBL结合THP1/CD14细胞的功能区进行了研究,检测了多种糖、rhMBL-CRD、rhMBL-CLR、C1q和E8对THP1/CD14细胞与MBL结合的抑制作用。结果显示,THP1/CD14细胞与MBL的结合能被某些糖抑制;rhMBL-CRD、rhMBL-CLR蛋白单独能部分抑制其结合,两者联合则可达完全抑制效果;但C1q和E8无抑制作用。资料表明,THP1/CD14细胞表达糖敏感的MBL受体或结合蛋白,包括对CLR和CRD特异的两种受体,均与C1qR无关;MBL可能以一种以上的方式结合THP1/CD14细胞。
三、MBL与TLR关系的初步研究
TLR-2是人类TLR家族成员中表达范围最广泛,识别病原微生物及其产物种类最多的分子,也是单核巨噬细胞主要表达的TLR之一。本部分研究中,我们首先采用ELISA、Western blot对TLR-2能否直接与MBL结合进行了分析,结果表明MBL能直接结合真核表达的重组TLR-2胞外段蛋白。对LPS刺激THP1/CD14细胞结合MBL的情况进行研究,ELISA结果显示,MBL与100 ng/LLPS刺激的THP1/CD14细胞结合能力比无LPS刺激者强,且sTLR-2蛋白可部分抑制这种结合;流式细胞术结果同样表明,LPS刺激的THP1/CD14细胞的MBL结合能力增强,且具有LPS浓度依赖性,随着LPS浓度增高,结合逐渐加强。然后,我们采用RT-PCR分析了LPS刺激对细胞TLR-2、TLR-4 mRNA表达的影响,发现LPS刺激2 h后THP1/CD14细胞TLR-2、TLR-4 mRNA表达水平增高;24 h后有所下降,接近与未刺激时水平。最后,提取未刺激的、LPS刺激的和MBL加LPS共同刺激的THP1/CD14细胞的核蛋白,采用Western bolt方法分析和转录因子NF-κB的转位,发现MBL可抑制LPS诱导NF-κB转位,表明MBL调节细胞因子分泌的机制可能与抑制NF-κB转位有关。
上述研究结果表明,THP1/CD14细胞表达Ca~(2+)依赖性、糖敏感的MBL受体或结合蛋白,包括对CLR和CRD特异的两种受体,均与C1qR无关。MBL可与TLR-2胞外段结合;LPS刺激后的THP1/CD14细胞结合MBL能力增强,这与LPS刺激THP1/CD14细胞TLR-2和TLR-4的mRNA表达增高是一致的;MBL与THP1/CD14细胞的结合能被sTLR-2蛋白部分抑制;MBL可抑制LPS诱导THP1/CD14细胞核转录因子NF-κB的转位。因此提出,MBL抑制LPS刺激THP1/CD14细胞产生促炎细胞因子可能与两方面的机制有关:①THP1/CD14细胞在LPS刺激下TLR2和TLR4表达量增加,导致其与MBL的结合力增强、结合量增多;②MBL抑制LPS诱导的核转录因子NF-κB的转位。
Although studies for innate immunity have been developing rapidly in the past few years,its essence remains to be revealed and elucidated.Accompany to the hypothesis that innate immunity initiates acquired immune response and controls the types of response through pattern-recognition put forward by Janeway CA et al, pattern recognition molecules have become one of the hot topics of immunologic researches.As the important molecules in innate immune,pattern recognition molecules can be divided into two categories based on their exsiting forms,including the dissolvability pattern recognition molecules in the humor and pattern recognition receptors expressed in the cell surface.At present,it has been shown that among the dissolvability pattern recognition molecules surfactant proteins A and D(SP-A and SP-D),which belong to the collectin subgroup of the C-type lectin superfamily,can bridge innate immunity and adaptive immunity even regulate adaptive immunity. Among the pattern recognition receptors,macrophage mannose recetor(MMR),a member of C-type lectin receptors(CLRs) family,as well as Toll-like receptors (TLRs) family can also play these functions.Accordingly,innate immunity pattern-recognition is pressing on towards the central issue of immune response, initiation of response as well as its control.
Mannan-binding lectin(MBL),a member of the collectin superfamily(C-type lectin with a collagen-like domain),along with SP-A and SP-D together,is a plasma glycoprotein mainly secreted by liver cells.MBL is a key molecule in innate immume system.MBL insufficiency is one of the most common immunodeficiencies. However,the mechanisms is still not clear.MBL,which has a total structure with C1q is a special molecule.Classical theory believes that the chief biological effect of MBL is that it forms compound with two MBL-associated serine proteases (MASP-1,MASP-2) through its collagen-like region(CLR) and then activates the complement cascade via lectin pathway to play the function of anti-infection in innate immune in the present of Ca~(2+).In the past few years,it has been reported that MBL plays important functions in immunological regulation.Correlated researches abroad demostrated that MBL could inhibit T cell response mediated by DC-SIGN,regulate the cytokines production of peripheral blood monoeuclear cell(PBMC).Previously, our research team found that MBL could inhibit of the maturation of LPS stimulated dendritic cells(DC) and inhibit the production of TNF-αand IL-12 in hunman monocyte line THP1/CD14 cells in different differentiation states.These findings also confirmed the functions of MBL in immunological regulation.However,the mechanisms of MBL in immunological regulation has not been reported yet at home and abroad.
In order to explore the the mechanisms of the functions of MBL in immunological regulation,we selected the hunman monocyte line THP1/CD14 cells as our cell model.We investigated the binding of MBL to THP1/CD14 cells,the characteristics of the binding and the interaction of MBL with TLRs.This research can be divide into three parts as follow.
PartⅠDetection the binding of MBL to hunman monocyte line THP1/CD14 cells
In this part,We investigated whether MBL can bind to THP1/CD14 cells by Cell-ELISA and flow cytometry(FCM).The result of Cell-ELISA revealed that absorptance of 450nm depended on the concentration of MBL in experiment group. The result of FCM showed that MBL could hardly bind to THP1/CD14 cells in the absence of Ca~(2+) or in the presence of EDTA as a substitute of CaCl_2,but it could bind to THP1/CD14 cells in the buffers which contain 5 mmol/L or 10 mmol/L CaCl_2,and the binding capability in the buffer containing 10 mmol/L CaCl_2 is slightly increased compared to in the buffer containing 5 mmol/L CaCl_2,suggesting that the binding of them depended on the Ca~(2+) concentration.These results above indicated that natural MBL purified from plasma could actually bind to THP1/CD14 cells in physiological conditions,and this binding was evidently dependent on Ca~(2+) concentation.Thus there may exist MBL receptors or binding proteins in the cellular membrane surface of THP1/CD14 cells,and the binding of MBL to THP1/CD14 cells needs the participation of Ca~(2+).
PartⅡStudy on the characteristics of MBL binding to THP1/CD14 cells
According to the Ca~(2+) dependent characteristic of MBL binding to THP1/CD14 cells in part one,we presumed that MBL might bind to certain glycoproteins or other glycosylated structure in cellular membrane surface through its carbohydrate-recognition domain(CRD).Classical theory believes MBL binds to C1q receptor (C1qR) in cellular membrane surface of mononuclear macrophage through its collagen-like region(CLR).Previously,our research team have successfully expressed prokaryotic MBL-CRD protein and MBL-CLR protein and prepared cell line E8 which can secrete mouse anti-human C1qR monoclonal antibody(mAb).So,in this part,we firstly resused the cell line E8,immunized mouse to obtain abdominal dropsy,and then purificated the abdominal dropsy obtained this mAb.Secondly,we prepared MBL-CLR protein by Ni~(2+)-NTA agarose chromatography.Subsequently,we focused on the functional domains of MBL binding to THP1/CD14 cells,we detected the inhibitory effect of MBL binding to THP1/CD14 cells,using sugars,prokaryotic rh(recombinant human) MBL-CRD,prokaryotic rhMBL-CLR protein,C1q and mouse anti-C1q mAb as potential inhibitors,by FCM.The results showed that their binding can be partly inhibit by some sugars,rhMBL-CRD alone and rhMBL-CLR alone.And the inhibitory effect was obviously when rhMBL-CRD and rhMBL-CLR were added together.However,C1q and mouse anti-human C1qR mAb had no inhibitory action.These data showed that THP1/CD14 cells can express sugar-sensitive MBL receptors or binding proteins,including two specific categories of receptors against CLR and CRD,and these receptors were unrelated with C1qR. Therefore,this findings reveals that MBL may binding to THP1/CD14 cells through more than one way.
PartⅢPreliminary study of the relationship between MBL and TLRs
Among mammalian TLRs,TLR-2 is expressed very extensively,and mediates recogonition of a wide range of microbial products.TLR-2 is one of the predominant TLRs expressed in mononuclear macrophage.In this part,we focused on whether MBL can directly bind to TLR-2 by ELISA and Western blot.The results of the two methods both showed that MBL can directly bind to the extracellular domain of TLR-2 protein(sTLR-2).In the research of LPS-stimulated THP1/CD14 cells,we investigated the binding capability of LPS-stimulated THP1/CD14 cells with MBL by ELISA and FCM.It was found that the binding capability of LPS(100ng/L) -stimulated THP1/CD14 cells with MBL was strengthened compared to untreated THP1/CD14 cells,statistical analysis showed the difference was significant.The results of FCM also showed the the binding capability of LPS-stimulated THP1/CD14 cells with MBL was strengthened,and the strengthen was in a LPS concentration-dependent manner.The higher concentration of LPS,the more stronger of the binding capability.Subseqently,we analyzed the impact of the stimulation of LPS to the expression of TLR-2 and TLR-4 mRNA in THP1/CD14 cells.RT-PCR analysis showed that the expression of TLR-2 and TLR-4 mRNA in LPS-stimulated THP1/CD14 cells was significantly increased after 2h compared to the non-LPS-stimulated cells,and it was dropped to the approximate level of non-LPS-stimulated cells after 24h.Eventually,we studied the signal transduction. The nulear proteins of THP1/CD14 cells,which were untreated,LPS treated,MBL and LPS treated,were extracted.Then the extracted nulear proteins were essaied by Western bolt.The result of Western bolt showed MBL can inhibit the transposition of transcription factor NF-κB induced by LPS,suggesting the mechanism of MBL in the regulation of cytokines secretion.
The results above indicated that THP1/CD14 cells might epress Ca~(2+)-dependent, sugar-sensitive MBL receptors or binding proteins,including both CLR-specific and CRD-specific,which have nothing to do with C1qR.MBL can directly bind to sTLR-2.The binding capability of LPS-stimulated THP1/CD14 cells was strengthened,and was consistent with the expression of TLR-2 and TLR-4 by RT-PCR in LPS-stimulated THP1/CD14 cells.The binding can be partly inhibit by sTLR-2.MBL can suppress the transposition of transcription factor NF-κB induced by LPS in THP1/CD14 cells.Therefore,we presumed that there might be two aspects of mechanisms about the inhibition of the proinflammatory cytokines produced by LPS-stimulated THP1/CD14 cells,the one is the enhanced expression of TLR-2 and TLR-4 mRNA induced by LPS,which resulted in stronger binding capability and more binding capacity;the other is the suppression of the transposition of transcription factor NF-κB induced by MBL.
引文
[1]Janeway CA Jr,Medzhitov R.Innate immune recognition[J].Annu Rev Immunol,2002,20:197-216.
[2]陈政良.哺乳类C型凝集素超级家族[J].生物化学与生物物理进展,1997,24(6):491-294.
[3]Thompson C.Protein proves to be a key link in innate immunity[J].Science,1995,269(5222):301-2.
[4]Roos A,Bouwman LH,van Gijlswijk-Janssen DJ,et al.Human IgA activates the complement system via the mannan-binding lectin pathway[J].J lmmunol,2001,167(5):2861-8.
[5]陈政良.甘露聚糖结合蛋白[J].国外医学免疫学分册,1997,20(1):16-19.
[6]Ikeda K,Sannoh T,Kawasaki N,et al.Serum lectin with known structure activates complement through the classical pathway[J].J Biol Chem,1987,262(16):7451-4.
[7]Garred P,Madsen HO,Kurtzhals JA,et al.Diallelic polymorphism may explain variation of the blood concentration of mannan-binding protein in Eskimos,but not in black Africans[J].Eur J Immunogenet,1992,19(6):403-12.
[8]Turner MW.Mannose-binding lectin(MBL) in health and disease[J].Immunobiol,1998,199(2):327-39.
[9]陈政良.甘露聚糖结合蛋白基因突变引起的免疫缺损[J].生命的化学,1997,17(4):46-7.
[10]Shepherd VL.Pulmonary surfactant protein D:a novel link between innate and adaptive immunity[J].Am J Physiol Lung Cell Mol Physiol,2001,28(6):L1453-63.
[11]Allavena P,Chieppa M,Monti P,et al.From pattern recognition receptor to regulator of homeostasis:the double-faced macrophage mannose receptor[J].Crit Rev Immunol,2004,24(3):179-92.
[12]Brinker KG,Martin E,Borron P,et al.Surfactant protein D enhances bacterial antigen presentation by bone marrow-derived dendritic cells[J].Am J Physiol Lung Cell Mol Physiol, 2001,281(6): L1453-63.
[13] Brinker KG, Garner H, Wright JR. Surfactant protein A modulates the differentiation of murine bone marrow-derived dendritic cells[J]. Am J Physiol Lung Cell Mol Physiol, 2003,284(1): L232-41.
[14] Borron PJ, Crouch EC, Lewis JF, et al. Recombinant rat surfactant-associated protein D inhibits human T lymphocyte proliferation and IL-2 production. J Immunol[J], 1998,161 (91):4599-603.
[15] Gaynor CD, McCormack FX, Voelker DR,et al. Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages[J]. J Immunol, 1995,155(11):5343—51.
[16] Hartshorn KL, White MR, Shepherd V, et al. Mechanisms of anti-influenza activity of surfactant proteins A and D: comparison with serum collectins[J]. Am J Physiol Lung Cell Mol Physiol, 1997,273(6 Pt 1):L1156-66.
[17] Murakami S, Iwaki D, Mitsuzawa H, et al. Surfactant protein A inhibits peptidoglycan-induced tumor necrosis factor-r secretion in U937 cells and alveolar macrophages by direct interaction with Toll-likereceptor 2[J]. J Biol Chem, 2002,277(9):6830-7.
[18] Sano H, Kuronuma K, Kudo K, et al. Regulation of inflammation and bacterial clearance by lung collectins[J]. Respirology, 2006, 11:S46-50.
[19] Yamada C, Sano H, Shimizu T, et al.Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response[J]. J Biol Chem,2006, 281(31):21771-80.
[20] Ohya M, Nishitani C, Sano H, et al. Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide[J]. Biochemistry, 2006, 45(28): 8657-64.
[21] Guillot L, Balloy V, McCormack FX, et al. Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4[J]. J Immunol, 2002,168(12):5989-92.
[22] Alcorn JF, Wright JR. Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway[J]. Am J Physiol Lung Cell Mol Physiol,2004,286(1):L129-36..
[23]Spear GT,Zariffard MR,Xin J,et al.Inhibition of DC-SIGN-mediated trans infection of T cells by mannose-binding lectin[J].Immunology,2003,110(1):80-5.
[24]Fraser DA,Bohlson SS,Jasinskiene N,et al.C1q and MBL,complement of the innate immune system,influence monocyte cytokine expression[J].J Leukoc Biol,2006,80(1):107-16.
[25]Heggelund L,Mollnes TE,Espevik T,et al.Modulatory effect of mannose -binding lectin on cytokine responses:possible roles in HIV infection[J].Eur J Clin Invest,2005,35(12):765-70.
[26]Saevarsdottir S,Oskarsson OO,Aspelund T et al.Mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk[J].J Exp Med 2005,201(1):117-25.
[27]Ogden CA,deCathelineau A,Hoffmann PR,et al.Clq and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropino-cytosis and uptake of apoptotic cells[J].J Exp Med,2001,194(6):781-95.
[28]陈月,陈政良,左大明,等.MBL对树突状细胞体外分化成熟的影响[J].细胞与分子免疫学杂志,2005,21(2):159-62.
[29]陈月,余新沛,刘莹,等.MBL对LPS诱导树突状细胞成熟的调节作用[J].免疫学杂志,2006,22(4):366-73.
[30]陈月,余新沛,卢晓,等.MBL对LPS诱导不同分化和活化状态THP/CD14细胞产生TNF-a和IL-12的影响[J].中华微生物学与免疫学杂志,2006,26(5):458-62.
[31]Schauvliege R,Janssens S,Beyaert R.Pellino proteins:novel players in TLR and IL-1R signalling[J].J Cell Mol Med,2007,11(3):453-61.
[32]Asea A.Heat shock proteins and toll-like receptors[J].Handb Exp Pharmacol.2008,(183):111-27.
[33]Roelofs MF,Boelens WC,Joosten LA,et al.Identification of small heat shock protein B8(HSP22) as a novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid arthritis[J].J Immunol,2006,176(11):7021-7.
[34]陈月,张丽芸,陈政良.人血浆中MBL-MASP复合物的纯化与分离[J].第一军医大学学报,2004,24(12):1373-7.
[35]Alvarez-Domnguez C,Carrasco-Marin E,Leyva-Cobian F.Role of complement component C1q in phagocytosis of Listeria monocytogenes by murine macrophage-like cell lines[J].Infect Immun,1993,61(9):3664-72.
[36]Vegh Z,Kew RR,Gruber BL,et al.Chemotaxis of human monocyte-derived dendritic cells to complement component C1q is mediated by the receptors gC1qR and cC1qR[J].Mol Immunol,2006,43(9):1402-7.
[37]Borron P,McCormack FX.,Elhalwagi BM,et al.Surfactant protein A inhibits T cell prolifer-ation via its collagenlike tail and a 210-kDa receptor[J].Am J Physiol,1998,275(4 Pt 1):L679-86.
[38]Nadesalingam J,Dodds AW,Reid KB,et al.Mannose-binding lectin recognizes peptidoglycan via the N-Acetyl glucosamine moiety,and inhibits ligand-induced proinflammatory effect and promotes chemokine production by macrophages[J].J Immunol,2005,175(3):1785-94.
[39]Arora M,Munoz E,Tenner AJ.Identification of a site on mannan-binding lectin critical for enhancement of phagocytosis[J].J Biol Chem,2001,276(46):43087-94.
[40]蔡学敏,左大明,赵娜,等.人MBL-CLR表达载体的构建及其在大肠菌中的表达[J].细胞与分子免疫学杂志,2007,23(1):25-8.
[41]Ghiran I,Barbashov SF,Klickstein LB,et al.Complement receptor 1/CD35 is a receptor for mannan-binding lectin[J].J Exp Med.2000,192(12):1797-1808.
[42]Malhotra R,Thiel S,Reid KB,Sim RB.Human leukocyte Clq receptor binds other soluble proteins with collagen domains[J].J Exp Med,1990,172(3):955-9.
[43]Ogden CA,deCathelineau A,Hoffmann PR,et al.C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinoytosis and uptake of apoptotic cells[J].J Exp Med,2001,194(6):781-95.
[44]Vandivier RW,Ogden CA,Fadok V,et al.Role of surfactant A,D,and C1q in the clearance of apoptotic cells in vivo and in vitro;calreticulin and CD91 as a common collectin receptor complex[J].J Immunol,2002,169(7):3978-86.
[45]Bajtay Z,Jozsi M,Banki Z,et al.Mannan-binding lectin and C1q bind to distinct structures and exert differential effects on macrophages[J].Eur J Immunol,2000,30(6):1706-13.
[46] Faure E, Equils 0, Sieling PA, et al. Bacterial lipopolysaccharide activates NF-kB through Toll-like Receptor 4 (TLR-4) in cultured human dermal endothelial cells[J]. Biol Chem, 2000,275(15), 11058-63.
[47] Yang JB, Duan ZJ, Yao W, et al. Synergistic transcriptional activation of human Acyl-coenzyme A: cholesterol acyltransterase-1 gene by interferon-gamma and all-trans-retinoic acid THP-1 cells. J Biol Chem, 2001, 276(24):20989-98.
[48] Engering A, Geijtenbeek TB, van Kooyk Y. Immune escape through C-type lectins on dendritic cells[J]. Trends Immunol, 2002, 23(10):480-5.
[49] Gijzen K, Cambi A, Torensma R, et al. C-type lectins on dendritic cells and their interaction with pathogen-derived and endogenous gly-coconjugates[J]. Curr Protein Pep t Sci, 2006, 7(4):283-94.
[50] Gretchen Vogel. Fly development genes lead to immune find[J]. Science, 1998,281(5385):1942-24.
[51] Akira S, Hemmi H. Recognition of pathogen-associated molecular pattern by TLR family[J]. Immunol Lett, 2003, 85(2):85-95.
[52] Underhill DM, Ozinsky A. Toll-like receptors:key mediator of microbe detection[J]. Curr Opin I mmunol, 2002,14(1):103-10.
[53] Caparros E, Munoz P, Filardi E, et al. DC-SIGN ligation on dendritic cells results in ERK and PI3K activation and modulates cytokine production[J].Blood, 2006,107(10):3950-8.
[54] Geijtenbeek TB, van Vliet SJ, EngeringA, et al. Self- and nonself-recognition by C-type lectins on dendritic cells[J]. Annu Rev Immunol, 2004, 22: 33-54.
[55] Chieppa M, Bianchi G, Doni A, et al. Cross-linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immuno-suppressive program[J]. J Immunol, 2003,171(9):4552-60.
[56]Brown GD, Herre J,Williames DL, et al.Dectin-1 mediates the biological effects of the beta-glucans[J]. J Exp Med, 2003,197(9): 1119-24.
[57] Visintin A, Mazzoni A, Spitzer JH, et al. Regulation of Toll-like receptors in human monocytes and dendritic cells[J]. J Immunol, 2001,166(1):249-55.
[58] Liu Y, Wang Y, Yamakuchi M, et al. Upregulation of toll-like receptor 2 gene expression in macrophage response to peptidoglycan and high concentration of lipopolysaccharide is involved in NF-kappa b activation[J]. Infect Immun, 2001, 69(5):2788-96.
[59] Baldwin Jr AS. The NF-kB and I kappaB proteins: new discoveries and insights[J]. Ann Rev Immunol, 1996,14:649-83.
[60] Vincenti MP, Coon CI, Brinckerhoff CE. Nuclear factor kappaB/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-1 beta-stimulated synovial fibroblasts[J]. Arthritis Rheum, 1998,41(11):1987-94.
[61] Bannes PJ. Nuclear factor kappa B: a pivotaltranscription factor in chronic inflammatory diseases[J]. N Enl J Med, 1997,336(15):1066-74.
[62] Haruhiko Ogata and Toshifumi Hibi. Regulation of cytokine signaling and inflammation[J]. Cytokine Growth Factor Rev, 2002,3(4-5):413-21.
[63] Nomura Y. NF-kB activation and I kappaB alpha dynamism involved in iNOS and chemokine induction in astroglial cells[J]. Life Sci, 2001,68(15):1695-701.
[64] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity[J]. Nat Immunol, 2001, 2(8):675-80.
[1]Banchereau J,Briere F,Caux C et al.Immunobiology of dendritic cells[J].Annu Rev Immunol,2000,18:767-811.
[2]Steinman RM,Hawiger D,Nussenzweig MC.Tolerogenic dendritic cells[J].Annu Rev Immunol,2003,21:685-711.
[3]Castellano G,Woltman AM,Schena FP,et al.Dendritic cells and complement:at the cross road of innate and adaptive immunity[J].Mol Immunol,2004,41(2-3):133-40.
[4]K(o|¨)hl J.Self,non-self and danger:a complementary view[J].Adv Exp Med Biol,2006,586:71-94.
[5]Anaphylatoxin CSa induces monocyte recruitment and differentiation into dendritic ceils by TNF-alpha and prostaglandin E2-dependent mechanisms[J].J Immunol,2003,171(5):2631-6.
[6]Csomor E,Bajtay Z,Sandor N,et al.Complement protein C1q induces maturation of human dendritic cells[J].Mol Immunol,2007,44(13):3389-97.
[7]Castellano G,Woltman AM,Nauta AJ,et al.Maturation of dendritic cells abrogates C1q production in vivo and in vitro[J].Blood,2004,103(10):3813-20.
[8]Cao W,Bobryshev YV,Lord RS,et al.Dendritic cells in the arterial wall express C1q:potential significance in atherogenesis[J].Cardiovasc Res,2003,60(1):175-86.
[9]Reis ES,Barbuto JA,Isaac L Human monocyte-derived dendritic cells are a source of several complement proteins[J],Inflamm Res,2006,55(5):179-84.
[10]Reis ES,Barbuto JA,Isaac L.Complement components,regulators and receptors are produced by human monocyte-derived dendritic cells[J].Immunobiol,2007, 212(3):151-7.
[11]Downing I,Koch C,Kilpatrick D C.Immature dendritic cells possess a sugar-sensitive recept -or for human mannan-binding lectin[J].Immunol,2003,109(3):360-4.
[12]Zhou W,Peng Q,Li K,et al.Role of dendritic cell synthesis of complement in the allospecific T cell response[J].Mol Immunol,2007,44(1-3):57-63.
[13]Patel H,Sacks SH,Zhou W,et al.Dendritic cell synthesis of C3 is required for full T cell activeation and development of a Th1 phenotype[J].J Immunol,2006,176(6):3330-41.
[14]Gutzmer R,K(o|¨)ther B,Zwirner J,et al.Human plasmacytoid dendritic cells express receptors for anaphylatoxins c3a and c5a and are chemoattracted to c3a and c5a[J].J Invest Dermatol,2006,126(11):2422-9.
[15]Vegh Z,Goyarts EC,Rozengarten K,et al.Maturation-dependent expression of C1q binding proteins on the cell surfaceof human monocyte-derived dendritic cells[J].Int Immunopharmacol,2003,3:39-51.
[16]Vegh Z,Kew RR,Gruber BL,et al.Chemotaxis of human monocyte-derived dendritic cells to complement component C1q is mediated by the receptors gC1qR and cC1qR[J].Mol Immunol,2006,43(9):1402-7.
[17]陈月,陈政良,左大明,等.MBL对树突状细胞体外分化成熟的影响[J].细胞与分子免疫杂志,2005,21(2):159-62.
[18]Chen Y,Yang C,Jin N,et al.Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1polarization[J].Eur J Immunol,2007,37(1):167-76.
[19]Takahara M,Kang K,Liu L,et al.iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell precursors:a mechanism for transiently decreased dendritic cells in vivo after human skin injury by ultraviolet B[J].J Invest Dermatol,2003,120(5):802-9.
[20]Luo X,Liu L,Tang N,et al.Inhibition of monocyte-derived dendritic cell differentiation and interleukin-12 production by complement iC3b via a mitogen-activated protein kinase signalling pathway[J].Exp Dermatol,2005,14(4):303-10.
[2]陈政良.哺乳类C型凝集素超级家族[J].生物化学与生物物理进展,1997,24(6):491-294.
[3]Thompson C.Protein proves to be a key link in innate immunity[J].Science,1995,269(5222):301-2.
[4]Roos A,Bouwman LH,van Gijlswijk-Janssen DJ,et al.Human IgA activates the complement system via the mannan-binding lectin pathway[J].J lmmunol,2001,167(5):2861-8.
[5]陈政良.甘露聚糖结合蛋白[J].国外医学免疫学分册,1997,20(1):16-19.
[6]Ikeda K,Sannoh T,Kawasaki N,et al.Serum lectin with known structure activates complement through the classical pathway[J].J Biol Chem,1987,262(16):7451-4.
[7]Garred P,Madsen HO,Kurtzhals JA,et al.Diallelic polymorphism may explain variation of the blood concentration of mannan-binding protein in Eskimos,but not in black Africans[J].Eur J Immunogenet,1992,19(6):403-12.
[8]Turner MW.Mannose-binding lectin(MBL) in health and disease[J].Immunobiol,1998,199(2):327-39.
[9]陈政良.甘露聚糖结合蛋白基因突变引起的免疫缺损[J].生命的化学,1997,17(4):46-7.
[10]Shepherd VL.Pulmonary surfactant protein D:a novel link between innate and adaptive immunity[J].Am J Physiol Lung Cell Mol Physiol,2001,28(6):L1453-63.
[11]Allavena P,Chieppa M,Monti P,et al.From pattern recognition receptor to regulator of homeostasis:the double-faced macrophage mannose receptor[J].Crit Rev Immunol,2004,24(3):179-92.
[12]Brinker KG,Martin E,Borron P,et al.Surfactant protein D enhances bacterial antigen presentation by bone marrow-derived dendritic cells[J].Am J Physiol Lung Cell Mol Physiol, 2001,281(6): L1453-63.
[13] Brinker KG, Garner H, Wright JR. Surfactant protein A modulates the differentiation of murine bone marrow-derived dendritic cells[J]. Am J Physiol Lung Cell Mol Physiol, 2003,284(1): L232-41.
[14] Borron PJ, Crouch EC, Lewis JF, et al. Recombinant rat surfactant-associated protein D inhibits human T lymphocyte proliferation and IL-2 production. J Immunol[J], 1998,161 (91):4599-603.
[15] Gaynor CD, McCormack FX, Voelker DR,et al. Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages[J]. J Immunol, 1995,155(11):5343—51.
[16] Hartshorn KL, White MR, Shepherd V, et al. Mechanisms of anti-influenza activity of surfactant proteins A and D: comparison with serum collectins[J]. Am J Physiol Lung Cell Mol Physiol, 1997,273(6 Pt 1):L1156-66.
[17] Murakami S, Iwaki D, Mitsuzawa H, et al. Surfactant protein A inhibits peptidoglycan-induced tumor necrosis factor-r secretion in U937 cells and alveolar macrophages by direct interaction with Toll-likereceptor 2[J]. J Biol Chem, 2002,277(9):6830-7.
[18] Sano H, Kuronuma K, Kudo K, et al. Regulation of inflammation and bacterial clearance by lung collectins[J]. Respirology, 2006, 11:S46-50.
[19] Yamada C, Sano H, Shimizu T, et al.Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response[J]. J Biol Chem,2006, 281(31):21771-80.
[20] Ohya M, Nishitani C, Sano H, et al. Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide[J]. Biochemistry, 2006, 45(28): 8657-64.
[21] Guillot L, Balloy V, McCormack FX, et al. Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4[J]. J Immunol, 2002,168(12):5989-92.
[22] Alcorn JF, Wright JR. Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway[J]. Am J Physiol Lung Cell Mol Physiol,2004,286(1):L129-36..
[23]Spear GT,Zariffard MR,Xin J,et al.Inhibition of DC-SIGN-mediated trans infection of T cells by mannose-binding lectin[J].Immunology,2003,110(1):80-5.
[24]Fraser DA,Bohlson SS,Jasinskiene N,et al.C1q and MBL,complement of the innate immune system,influence monocyte cytokine expression[J].J Leukoc Biol,2006,80(1):107-16.
[25]Heggelund L,Mollnes TE,Espevik T,et al.Modulatory effect of mannose -binding lectin on cytokine responses:possible roles in HIV infection[J].Eur J Clin Invest,2005,35(12):765-70.
[26]Saevarsdottir S,Oskarsson OO,Aspelund T et al.Mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk[J].J Exp Med 2005,201(1):117-25.
[27]Ogden CA,deCathelineau A,Hoffmann PR,et al.Clq and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropino-cytosis and uptake of apoptotic cells[J].J Exp Med,2001,194(6):781-95.
[28]陈月,陈政良,左大明,等.MBL对树突状细胞体外分化成熟的影响[J].细胞与分子免疫学杂志,2005,21(2):159-62.
[29]陈月,余新沛,刘莹,等.MBL对LPS诱导树突状细胞成熟的调节作用[J].免疫学杂志,2006,22(4):366-73.
[30]陈月,余新沛,卢晓,等.MBL对LPS诱导不同分化和活化状态THP/CD14细胞产生TNF-a和IL-12的影响[J].中华微生物学与免疫学杂志,2006,26(5):458-62.
[31]Schauvliege R,Janssens S,Beyaert R.Pellino proteins:novel players in TLR and IL-1R signalling[J].J Cell Mol Med,2007,11(3):453-61.
[32]Asea A.Heat shock proteins and toll-like receptors[J].Handb Exp Pharmacol.2008,(183):111-27.
[33]Roelofs MF,Boelens WC,Joosten LA,et al.Identification of small heat shock protein B8(HSP22) as a novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid arthritis[J].J Immunol,2006,176(11):7021-7.
[34]陈月,张丽芸,陈政良.人血浆中MBL-MASP复合物的纯化与分离[J].第一军医大学学报,2004,24(12):1373-7.
[35]Alvarez-Domnguez C,Carrasco-Marin E,Leyva-Cobian F.Role of complement component C1q in phagocytosis of Listeria monocytogenes by murine macrophage-like cell lines[J].Infect Immun,1993,61(9):3664-72.
[36]Vegh Z,Kew RR,Gruber BL,et al.Chemotaxis of human monocyte-derived dendritic cells to complement component C1q is mediated by the receptors gC1qR and cC1qR[J].Mol Immunol,2006,43(9):1402-7.
[37]Borron P,McCormack FX.,Elhalwagi BM,et al.Surfactant protein A inhibits T cell prolifer-ation via its collagenlike tail and a 210-kDa receptor[J].Am J Physiol,1998,275(4 Pt 1):L679-86.
[38]Nadesalingam J,Dodds AW,Reid KB,et al.Mannose-binding lectin recognizes peptidoglycan via the N-Acetyl glucosamine moiety,and inhibits ligand-induced proinflammatory effect and promotes chemokine production by macrophages[J].J Immunol,2005,175(3):1785-94.
[39]Arora M,Munoz E,Tenner AJ.Identification of a site on mannan-binding lectin critical for enhancement of phagocytosis[J].J Biol Chem,2001,276(46):43087-94.
[40]蔡学敏,左大明,赵娜,等.人MBL-CLR表达载体的构建及其在大肠菌中的表达[J].细胞与分子免疫学杂志,2007,23(1):25-8.
[41]Ghiran I,Barbashov SF,Klickstein LB,et al.Complement receptor 1/CD35 is a receptor for mannan-binding lectin[J].J Exp Med.2000,192(12):1797-1808.
[42]Malhotra R,Thiel S,Reid KB,Sim RB.Human leukocyte Clq receptor binds other soluble proteins with collagen domains[J].J Exp Med,1990,172(3):955-9.
[43]Ogden CA,deCathelineau A,Hoffmann PR,et al.C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinoytosis and uptake of apoptotic cells[J].J Exp Med,2001,194(6):781-95.
[44]Vandivier RW,Ogden CA,Fadok V,et al.Role of surfactant A,D,and C1q in the clearance of apoptotic cells in vivo and in vitro;calreticulin and CD91 as a common collectin receptor complex[J].J Immunol,2002,169(7):3978-86.
[45]Bajtay Z,Jozsi M,Banki Z,et al.Mannan-binding lectin and C1q bind to distinct structures and exert differential effects on macrophages[J].Eur J Immunol,2000,30(6):1706-13.
[46] Faure E, Equils 0, Sieling PA, et al. Bacterial lipopolysaccharide activates NF-kB through Toll-like Receptor 4 (TLR-4) in cultured human dermal endothelial cells[J]. Biol Chem, 2000,275(15), 11058-63.
[47] Yang JB, Duan ZJ, Yao W, et al. Synergistic transcriptional activation of human Acyl-coenzyme A: cholesterol acyltransterase-1 gene by interferon-gamma and all-trans-retinoic acid THP-1 cells. J Biol Chem, 2001, 276(24):20989-98.
[48] Engering A, Geijtenbeek TB, van Kooyk Y. Immune escape through C-type lectins on dendritic cells[J]. Trends Immunol, 2002, 23(10):480-5.
[49] Gijzen K, Cambi A, Torensma R, et al. C-type lectins on dendritic cells and their interaction with pathogen-derived and endogenous gly-coconjugates[J]. Curr Protein Pep t Sci, 2006, 7(4):283-94.
[50] Gretchen Vogel. Fly development genes lead to immune find[J]. Science, 1998,281(5385):1942-24.
[51] Akira S, Hemmi H. Recognition of pathogen-associated molecular pattern by TLR family[J]. Immunol Lett, 2003, 85(2):85-95.
[52] Underhill DM, Ozinsky A. Toll-like receptors:key mediator of microbe detection[J]. Curr Opin I mmunol, 2002,14(1):103-10.
[53] Caparros E, Munoz P, Filardi E, et al. DC-SIGN ligation on dendritic cells results in ERK and PI3K activation and modulates cytokine production[J].Blood, 2006,107(10):3950-8.
[54] Geijtenbeek TB, van Vliet SJ, EngeringA, et al. Self- and nonself-recognition by C-type lectins on dendritic cells[J]. Annu Rev Immunol, 2004, 22: 33-54.
[55] Chieppa M, Bianchi G, Doni A, et al. Cross-linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immuno-suppressive program[J]. J Immunol, 2003,171(9):4552-60.
[56]Brown GD, Herre J,Williames DL, et al.Dectin-1 mediates the biological effects of the beta-glucans[J]. J Exp Med, 2003,197(9): 1119-24.
[57] Visintin A, Mazzoni A, Spitzer JH, et al. Regulation of Toll-like receptors in human monocytes and dendritic cells[J]. J Immunol, 2001,166(1):249-55.
[58] Liu Y, Wang Y, Yamakuchi M, et al. Upregulation of toll-like receptor 2 gene expression in macrophage response to peptidoglycan and high concentration of lipopolysaccharide is involved in NF-kappa b activation[J]. Infect Immun, 2001, 69(5):2788-96.
[59] Baldwin Jr AS. The NF-kB and I kappaB proteins: new discoveries and insights[J]. Ann Rev Immunol, 1996,14:649-83.
[60] Vincenti MP, Coon CI, Brinckerhoff CE. Nuclear factor kappaB/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-1 beta-stimulated synovial fibroblasts[J]. Arthritis Rheum, 1998,41(11):1987-94.
[61] Bannes PJ. Nuclear factor kappa B: a pivotaltranscription factor in chronic inflammatory diseases[J]. N Enl J Med, 1997,336(15):1066-74.
[62] Haruhiko Ogata and Toshifumi Hibi. Regulation of cytokine signaling and inflammation[J]. Cytokine Growth Factor Rev, 2002,3(4-5):413-21.
[63] Nomura Y. NF-kB activation and I kappaB alpha dynamism involved in iNOS and chemokine induction in astroglial cells[J]. Life Sci, 2001,68(15):1695-701.
[64] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity[J]. Nat Immunol, 2001, 2(8):675-80.
[1]Banchereau J,Briere F,Caux C et al.Immunobiology of dendritic cells[J].Annu Rev Immunol,2000,18:767-811.
[2]Steinman RM,Hawiger D,Nussenzweig MC.Tolerogenic dendritic cells[J].Annu Rev Immunol,2003,21:685-711.
[3]Castellano G,Woltman AM,Schena FP,et al.Dendritic cells and complement:at the cross road of innate and adaptive immunity[J].Mol Immunol,2004,41(2-3):133-40.
[4]K(o|¨)hl J.Self,non-self and danger:a complementary view[J].Adv Exp Med Biol,2006,586:71-94.
[5]Anaphylatoxin CSa induces monocyte recruitment and differentiation into dendritic ceils by TNF-alpha and prostaglandin E2-dependent mechanisms[J].J Immunol,2003,171(5):2631-6.
[6]Csomor E,Bajtay Z,Sandor N,et al.Complement protein C1q induces maturation of human dendritic cells[J].Mol Immunol,2007,44(13):3389-97.
[7]Castellano G,Woltman AM,Nauta AJ,et al.Maturation of dendritic cells abrogates C1q production in vivo and in vitro[J].Blood,2004,103(10):3813-20.
[8]Cao W,Bobryshev YV,Lord RS,et al.Dendritic cells in the arterial wall express C1q:potential significance in atherogenesis[J].Cardiovasc Res,2003,60(1):175-86.
[9]Reis ES,Barbuto JA,Isaac L Human monocyte-derived dendritic cells are a source of several complement proteins[J],Inflamm Res,2006,55(5):179-84.
[10]Reis ES,Barbuto JA,Isaac L.Complement components,regulators and receptors are produced by human monocyte-derived dendritic cells[J].Immunobiol,2007, 212(3):151-7.
[11]Downing I,Koch C,Kilpatrick D C.Immature dendritic cells possess a sugar-sensitive recept -or for human mannan-binding lectin[J].Immunol,2003,109(3):360-4.
[12]Zhou W,Peng Q,Li K,et al.Role of dendritic cell synthesis of complement in the allospecific T cell response[J].Mol Immunol,2007,44(1-3):57-63.
[13]Patel H,Sacks SH,Zhou W,et al.Dendritic cell synthesis of C3 is required for full T cell activeation and development of a Th1 phenotype[J].J Immunol,2006,176(6):3330-41.
[14]Gutzmer R,K(o|¨)ther B,Zwirner J,et al.Human plasmacytoid dendritic cells express receptors for anaphylatoxins c3a and c5a and are chemoattracted to c3a and c5a[J].J Invest Dermatol,2006,126(11):2422-9.
[15]Vegh Z,Goyarts EC,Rozengarten K,et al.Maturation-dependent expression of C1q binding proteins on the cell surfaceof human monocyte-derived dendritic cells[J].Int Immunopharmacol,2003,3:39-51.
[16]Vegh Z,Kew RR,Gruber BL,et al.Chemotaxis of human monocyte-derived dendritic cells to complement component C1q is mediated by the receptors gC1qR and cC1qR[J].Mol Immunol,2006,43(9):1402-7.
[17]陈月,陈政良,左大明,等.MBL对树突状细胞体外分化成熟的影响[J].细胞与分子免疫杂志,2005,21(2):159-62.
[18]Chen Y,Yang C,Jin N,et al.Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1polarization[J].Eur J Immunol,2007,37(1):167-76.
[19]Takahara M,Kang K,Liu L,et al.iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell precursors:a mechanism for transiently decreased dendritic cells in vivo after human skin injury by ultraviolet B[J].J Invest Dermatol,2003,120(5):802-9.
[20]Luo X,Liu L,Tang N,et al.Inhibition of monocyte-derived dendritic cell differentiation and interleukin-12 production by complement iC3b via a mitogen-activated protein kinase signalling pathway[J].Exp Dermatol,2005,14(4):303-10.