| Although studies for innate immunity have been developing rapidly in the recent more than ten years, its essence remains to be revealed and elucidated. In 1996, an article published by Science, whose topic was "The instructive role of innate immunity in the acquired immune response", put forward a new hypothesis that the inbeing of innate immunity is to identify molecular-pattern possessed specially by microorganism. Except for its first-line host defense, innate immunity also guides acquired immunity to response to pathogens and eliminate them in a proper way. Janeway CA Jr et al enriched this hypothesis in the later, suggesting that innate immunity initiates acquired immune response and controls the types of response through pattern-recognition. At present, it has been established that mannan-binding lectin (MBL) is key molecule of innate immune system, and that MBL defection is the most possible etiological factor for unexplained recurrent infections, but its pathologic mechanism is still unknown. Innate immunity pattern-recognition ispressing on towards the central issue of immune response------initiation of responseas well as its control. However, few studies on MBL in this respect have been done.MBL, a member of the collectin subgroup of the C-type lectin superfamily, is a multimeric protein containing collagen-like sequences and has the overall 'bundle-of-tulips' structure first described for C1q. It is mainly synthesized and excreted into blood by hepatocytes, circulates in serum as oligomers of homogeneity trimers subunit, and is able to selectively recognize certain carbohydrate moieties onthe surface of a number of pathogens. MBL activates the lectin complement pathway in a antibody- and C1q-independent way by activating two MBL-associated serine proteases ( MASP-1 and MASP-2) and leads to the killing of microbes by exerting lysis effect and indirect opsonizing function. It also can act directly as an opsonin by enhancing the attachment of microbes to the phagocyte. So MBL is considered as "ante-body" and key pattern-recognition receptor in innate immune system. Recently a study found that some dendritic cells (DCs) express a cell-surface lectin called DC-SIGN. DC-SIGN has been shown to mediate a type of infection called 'trans' infection, where DCs bind human immunodeficiency virus (HIV) and efficiently transfer the virus to T cells. MBL, a soluble lectin that functions as a recognition molecule in innate immunity and that binds to HIV, could block 'trans' infection mediated by DC-SIGN. Therefore, MBL may inhibit DC-SIGN-mediated uptake and spread of HIV. However, few studies have been done on the roles of MBL in immune regulation, and especially so far, little is known about its roles in adaptive immunity.It has been shown that surfactant proteins A and D (SP-A and SP-D), which belong to the collectin subgroup of the C-type lectin superfamily along with MBL together, have a few functions on modulating immune response. For example, SP-A inhibits the differentiation and maturation of dendritic cells (DCs) and modulates cellular responses induced by lipopolysaccharide (LPS) by interaction with CD 14, and SP-D enhances antigen presentation by DCs and promotes an adaptive immune response against bacterial antigens and binds CD14 and alters the CD14-LPS interaction. Therefore, according to the structure homology among MBL, SP-A and SP-D, the fact that they all can bind to collectin-receptor, and the fact that MO and DC express specific collectin-receptor and mediate a number of immune regulatory roles, the conclusion can be deduced that MBL must participate in acquired immune response. DC is the most potential antigen presenting cell (APC) in vivo, compared with other APCs, it can activates naive T cells and initiates adaptive immune response, and so called the initiator of immune response and plays central role in controlling and maintaining immune response. Although T and B lymphocytes are mediators of acquire immune response, however, their functions are controlled by APCs of innate immune sestem.In this study, we firstly established a simple and convenient procedure for thepurification of MBL and proenzyme MASPs, and obtained natural MBL with high purity and bioactivity from freshly frozen human plasma. Then, we respectively explored the possibility of inducing DCs from human peripheral blood monocytes with MBL in vitro as well as effects on the functions of monocyte-derived dendritic cells (MoDC) by MBL. Subsequently, we investigated effects of MBL on the maturation, cytokines releasing, phagocytosis as well as the ability to stimulate the proliferation of allogenic T cells of MoDC induced by LPS and effects of MBL on TNF-a and IL-12 production in differently differentiated and activated THP1/CD14 cells stimulated by LPS. On the basis of innate immune pattern-recognition, our goal is to explore the events that MBL/collectin receptor mediates antigen up-taking, processing, presenting and induces co-stimulators and cytokines expressing, so as to expound in one side the pathway and mechanism that immune information is delivered from innate immunity to adaptive immunity and thus initiates adaptive immune response and modulates types of immune response. In this study, we provide evidence that human natural MBL possibly participate in the adaptive immune response through modulating functions of APCs, suggesting that MBL may alter cellular responses elicited by bacterial components via interaction with CD 14 and control cytokines secretion. Therefore, our findings provide an novel insight for further investigating the role and mechanism that MBL takes part in adaptive immune response. Future analyses of the properties of MBL regulation, elucidations of the molecular machenism and the signal pathway in which MBL involved will further reveal the biological significance of the key innate molecule.I Purification and Separation of MBL-MASP Complex from Human PlasmaMBL is a central component of the innate immune system. MBL preparation with high purity is required in many practical works such as theory researches concerned with the lectin pathway and its related diseases, measurement of MBL level in clinical disease serum and preparation of anti-MBL monoclonal antibody. However, the concentration of MBL in plasma is quite low, it is between 40-3 500 ug/L in normal persons and approximately 900 ug/L in the average. Furthermore, MBL combines with MASP1 and MASP2 and presents in serum as a large moleculecomplex. Therefore, it is difficult to separate and purify MBL. Purification of MBL relies on its Ca2+-dependent affinity for carbohydrate. At present, two steps of affinity chromatography on mannose-Sepharose is generally employed for extraction of MBL, but this method is susceptible to contamination by anti-carbohydrate antibodies (mainly anti-mannose antibody).In the present study, according to the trait of MBL binding to underivatized Sepharose 4B, two steps of affinity chromatography on underivatized Sepharose 4B was employed to purify MBL-MASP complex from freshly frozen human plasma and followed by gel filtration on a Sephacryl S-300 column to separate MBL from MASPs. All steps were performed at 4°C, pH7.8 or pH5.0 and two proteolytic inhibitors, phenyl-methylsulfonyl fluoride and 1, 10-phenanthroline, were added during affinity chromatography on underivatized Sepharose 4B except the gel-filtration buffer. In this way, preparations of pure MBL and proenzyme MASPs were obtained. SDS-PAGE and Western blot showed that the purified MBL was functional multimer composed of 28KD and 32KD peptide chains, and it had ligand-binding activity, as demonstrated by ligand-binding assay and yeast-coagulating experiment. The assay detecting activating complement activity of the separated products showed that complement could be activated by mannan-MBL-MASP complex, but not by mannan-MBL complex, indicating that purified MBL-MASP complex is bioactive and is separated from each other completely after gel filtration on a Sephacryl S-300 column. Thus, a simple and convenient procedure for the separation and purification of MBL and proenzyme MASPs was established successfully, in which the contamination by anti-carbohydrate antibodies during ligand-affinity purification was also avoid and the cost for purifying MBL was reduced greatly.II Effects on the differentiation and maturation of monocyte-derived dendritic cells by mannan-binding lectin in vitroIn recent years, several investigations showed that SP-A and SP-D, which belong to the collectin subgroup of the C-type lectin superfamily along with MBL together, can modulate the functions of DCs. For example, SP-A inhibits the differentiation and maturation of DCs and SP-D enhances antigen presentation by DCs and promotes anadaptive immune response against bacterial antigens. However, few reports on the immune regulation by MBL have been published, especially so far, little is known about its roles in adaptive immunity. Based on the structural and functional homology among collectins, we speculate that MBL also possibly has some effects on differentiation and function of DC. Thus in the experiment, we explored the possibility of inducing DCs from human peripheral blood monocytes (MoDCs) with MBL in vitro as well as effects of MBL on the functions of MoDCs.PBMC were isolated from peripheral blood of healthy adult volunteers by ficoll-hypaque density gradient centrifugation and depleted of nonadherent cells by 3 h plastic adherence of cell suspensions in RPMI 1640 supplemented with 100 ml/L newborn calf serum (NCS) at 37 °C in 50 ml/L CO2 air. The obtained plastic-adherent cell, i.e. monocytes, were cultured in IMDM supplemented with rhGM-CSF and rhBL-4 for 5 days and then cultured for 2 days in presence or absence of rhTNF-a or human natural MBL. DCs' morphology was observed under inverted microscope and the expressions of CD la, CD83, CD40, CD80, CD86 and MHC-DR on DCs were analyzed by FACS. The ability to stimulate the proliferation of allogenic T cells by DCs was detected by 3H-TdR incorporation and the ability to up-take the antigen by DCs was evaluated by zymosan granule phagocytosis test. The levels of IL-12 and TNF-a in culture supernatant of DCs were determined by ELISA. The results showed that GM-CSF + DL-4 couldn't induce complete maturation and activation of DC, CD83 expression on the MoDCs induced for 7 d only reached 22.73% in the average, but the culture system was favorable for amplifying DCs. In presence of MBL, the expressions of CDla, CD83, CD40, CD80, CD86 and MHC-DR on the DCs were up-regulated, the ability to up-take zymosan granules by DCs was weakened but the proliferation of naive T cells induced by DCs was enhanced. MBL stimulated the production of IL-12 by DCs, but had nearly no effect on TNF-a level. These findings provide evidence that MBL can induce differentiation and maturation of DCs in vitro, suggesting that MBL possibly participate in the adaptive immune response through modulation of function of DCs, but the molecular machenism remains to be elucidated.Ill Mannan-binding lectin regulates the maturation of monocyte-deriveddendritic cells induced by lipopolysaccharideLPS is a major component of the outer membrane of gram-negative bacteria. The disorders induced by LPS including sepsis or septic shock are common clinical diseases, and often result in patient death. Studies indicate that LPS is a potent inducer of human DCs maturation and survival and can sufficiently activate DCs in vivo and in vitro. DCs are the most potent APCs and manipulation of DC maturation and activity may provide novel strategies for treatment of LPS-related inflammatory diseases. MBL belongs to the collectin subgroup of the C-type lectin superfamily along with SP-A and SP-D together. It have been shown that SP-A modulates cellular responses induced by LPS by interaction with CD 14 and SP-D binds CD 14 and alters the CD14-LPS interaction. However, whether MBL alter cellular responses induced by LPS by interaction with CD 14 remains to be explored. Thus in this study, we investigated effects of MBL on the maturation, cytokines releasing, phagocytosis as well as the ability to stimulate the proliferation of allogenic T cells of MoDC induced by LPS.The plastic-adherent monocytes were prepared from the peripheral blood of healthy adult volunteers. The MoDCs were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4, and then cultured for 2 days in presence or absence of LPS and human natural MBL at varying concentrations ranging from 10 to 100 mg/L. DCs morphology was observed under inverted microscope, the expression of CD83 and CD86 on DCs analyzed by FACS, the abilities to stimulate the proliferation of allogenic T cells and to up-take the antigen by DCs were evaluated by MTT assay and zymosan granule phagocytosis test respectively, and the levels of TNF-a and IL-12 p40+p70 in culture supernatant of DCs were detected by ELISA. The results showed MBL down-regulated the expression of CD83 and CD86 on the MoDCs induced by LPS in a dose-dependent manner, enhanced the ability to up-take zymozan granules by DCs and decreased the proliferation of naive T cells induced by DCs and inhibited the production of TNF-a and IL-12 p40+p70 induced by LPS. However, MBL at lower concentration (10 mg/L) had no notable effect on the maturation of DCs induced by LPS, therefore the functional significance of above results in physiological conditions remains to be further established. These datashowed that MBL at higher concentrations can inhibit LPS-induced maturation of DCs, suggesting MBL may play a role in controlling LPS-elicited disorders including sepsis or septic shock, can effectively confer protection to patient from LPS-induced lethal endotoxemia through inhibition of proinflammatory cytokine production, and is propitious to hosts to resist acute infection induced by Gram-negative bacteria. Furthermore, as cytokine immunomodulator, MBL might control the occurrence and development of septic shock in some extent. Taken together, the results provide theory basis of MBL for therapeutic application in conditions with excessive activation of DCs and replacement of MBL might be as a novel method for treatment of septic shock by modulating differentiation and maturation of DC, and MBL can be used as a new tool to further dissect signaling pathways involved in DC maturation.IV Effects of mannan-binding lectin on TNF-a and IL-12 productionof differently differentiated and activated THP1/CD14 cellsinduced by lipopolysaccharideIn the study of MBL regulation in the maturation of MoDCs induced by lipopolysaccharide, we found that MBL at higher concentrations down-regulated pro-inflammatory cytokines TNF-a and IL-12 production of DC induced by LPS, suggesting MBL possibly provides a signal for inhibiting excessive DC maturation stimulated by LPS and modulating adaptive immune response. However, the process of DC culturing is complex, the price of cytokines which are supplemented with in culture is expensive, and the identifying and counting of DC is also difficult, furthermore, human monocytes can differentiate into DCs with completely different functions according to the used medium and cytokines. Therefore, we selected THP1/CD14 cell, a premonocytic cell line that constitutively express CD14 which is one specific receptor for LPS with high affinity, as the model. From levels of mRNA and protein respectively, effects of MBL on TNF-a and IL-12 production in differently differentiated and activated THP1/CD14 cells induced by LPS were investigated by RT-PCR and ELISA. Our goal is to establish an basis for continuing work in a convenient way to dissect the roles and the mechanism that MBL plays in adaptive immune response regulation.THP1/CD14 cells were grown in IMDM supplemented with 100 ml/L NCS. Inpresence of PMA and/or IFN-y, differently differentiated and activated monocytes were induced, following which the cells were pretreated with human natural MBL at concentrations ranging from 1 to 100 mg/L for 2 h before LPS (lmg/L) stimulation for 24 h. The contents of TNF-a and IL-12 p40+p70 in culture supernatants were detected by ELBA. Cells were harvested for mRNA isolation, and the TNF-a, IL-12 p35 and p40 mRNA expressions were detemined by semiquantitative RT-PCR. ELISA showed that secretion of TNF-a and IL-12 p40+p70 from THP1/CD14 cells in different states can be induced by LPS; the maximal amount of IL-12 p40+p70 is detected in the culture supernatants of THP1/CD14 cells activated by IFN-y and the minimum in those of PMA-differentiated THP1/CD14 cells; the highest level of TNF-a is found in the culture supernatants of THP1/CD14 cells differentiated by PMA and activated by IFN-y and the lowest in those of THP1/CD14 cells without PMA or IFN-y stimulation; the productions of TNF-a and IL-12 p40+p70 by differently differentiated and activated THP1/CD14 cells induced with LPS are profoundly inhibited by MBL at higher concentrations (50~100 mg/L) but not MBL at lower concentrations (1-10 mg/L). RT-PCR analysis also indicated that the mRNA expressions of TNF-a and IL-12 p35 and p40 subunits in THP1/CD14 cells pretreated with MBL at higher concentration are decreased in different degree, compared to the corresponding THP1/CD14 cells stimulated with LPS alone. These data showed that through interaction with CD 14, human natural MBL can inhibit TNF-a and IL-12 production of differently differentiated and activated moncytes induced by LPS, suggesting the interaction with CD14 may be an important property common to the collectin family. Effects of MBL on the secretion of TNF-a and IL-12 protein in THP1/CD14 cells stimulated by LPS is correspond to that of their mRNA expression, indicating that there might be no special regulatory step involved between mRNA and translation. IFN-y can enhance not only IL-12 production in THP1/CD14 cells induced by LPS, but also TNF-a and IL-12 production in PMA-differentiated THP1/CD14 cells stimulated with LPS. Therefore, THP1/CD14 cells can be a appropriate cell-model for further exploring the molecular mechanism of MBL in immune response regulation and the signaling pathways involved in cytokines network.The findings above provide an exciting novel insight for further investigating the roles and mechanism that MBL participates in acquired immune response. At the present time, no reports similar to them are seen. Future analyses of the properties of MBL regulation, elucidations of the molecular machenism and the signal pathway in which MBL involved will further reveal the biological significance of the key innate molecule.
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