Preliminary Study On The Roles Of Mannan-binding Lectin In Regulating Adaptave Immune Response

Mannan-binding lectin(MBL),a member of the collectin family in the C-type lectin superfamily,is a multimeric protein containing collagen-like sequences.It is synthesized and excreted into blood by hepatocytes.It has the overall 'bundle-of-tulips' structure first described for C1q.The polypeptide of MBL is composed of four domains,a cysteine-rich N-terminal domain,a collagen-like region (CLR) that contains Gly-X-Y repeats(where X is any amino acid,and Y is often hydroxyproline or hydroxylysine),a neck region and a C-terminal carbohydrate-recognition domain(CRD).The MBL polymer is composed of homogenous subunits,which can be as many as six,but only the high polymeric forms of MBL have the biologic activity and can fix complements.MBL can selectively recognize sugars presented on pathogens through the pattern-recognition function.On binding to pathogens,MBL may activate the complement cascade via lectin pathway,leading to the killing of microbes by exerting lysis effect and indirect opsonizing function.At the same time,MBL has a direct opsonization when binding to lectin receptors on the phagocytic cells,and it can also induce the MBL-dependent cell-mediated cytotoxic reaction.MBL also plays an important role in surfactant defenses of mucous membrane.Furtherore,MBL and MBL-mediated lectin pathway have beeen considered as the key molecule and key element in innate immunity, respectively,by such important magazines as Science,Jounnal of Immunology.The basic understanding of the immune system has undergone a substantial paradigm shift in the past decade,as an awareness of the power and influence of the innate immune system has emerged.It is now being recognized that the innate immune system is not only responsible for the induction of acute responses necessary for elimination of pathogens but is also able to activate appropriate adaptive immune responses via ligand interaction and secretion of cytokines and chemokines,and that the nature of the first response to invasion(i.e.,the innate immune response) has significant influence in determining the nature of the subsequent adaptive immune response.Both soluble and membrane bound pattern recognition molecules of the innate immune system assess the level of danger of a particular intrusion and initiate a protection program for the host.When activated by pathogens,monocytes, macrophages and dendritic cells often initiate the synthesis of proinflammatory cytokines by mediating some of these changes.The cytokine environment then influences the subsequent specific immune responses.Conversely,soluble and cellular components of the innate system remove dead or degenerated cells or tissue debris and participate in systems of tissue repair.Thus,in the absence of perceived danger,the innate system seems to be able to remove dying cells and avoid the induction of an adaptive immune response,which would lead to autoimmunity or further tissue damage.Thus,in the absence of perceived danger,the innate system appears to be able to discriminate removal of dying cells and avoid the induction of an adaptive immune response,which would result in autoimmunity and/or further tissue damage,and play roles in regulation of adaptive immune responses.It has been reported that other members of the soluble defensive collagen family, such as surfactant protein A(SP-A),surfactant protein D(SP-D),complement C1q, have some roles in immune regulation by interaction with various cells.For instance, SP-A inhibits the differentiation and maturation of DCs and modulates cellular responses induced by LPS through interacting with CD14;SP-A suppresses TNF-αsecretion from alveolarΜΦstimulated with LPS,and SP-A-deficient mice produce significantly more TNF-αthan wild-type mice after intratracheal LPS-administration. SP-A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response.SP-D enhances bacterial antigen presentation by bone marrow-derived dendritic cells.Complement C1q regulates LPS-induced cytokine production in bone marrow-derived dendritic cells,and can induce maturation of human dendritic cells. As a key soluble pattern recognition molecule in the innate immune system,MBL has been reported to influence the cytokines network after stimulation by various microorganisms.However,so far,little is known concerning the role of MBL in the regulation of adaptive response.The hypothesis that MBL must participate in acquired immune response is based on the following three facts:the structure homology among MBL,SP-A and SP-D,their binding to collectin-receptor,and the findings thatΜΦexpress specific collectin-receptor and mediate lots of immune regulatory processes.A conclusion that MBL may play certain roles in regulating dendritic cell differentiation and maturation has been drawn from Dr.Chen's research work,which suggests that MBL most likely be involved in the regulation of immune responses,considering the key roles of antigen-presenting cells in initiating of adaptive immune response.In the present research,we continued our previous studies to explore the role of MBL in regulating adaptive immune responses.Firstly,we established a convenient procedure for the purification of MBL through the ligand-and monoclonal antibodyaffinity chromatography,and obtained natural MBL with high purity and bioactivity from freshly frozen human plasma.Then we obtained soluble extracellular-TLR4 protein(sTLR4) by prokaryotic expression system,and investigated possible mechanisms underlying MBL's effects on DC maturation and cytokine expression. Thirdly,we investigated the interaction of MBL with human B cell line Raji cells. Fourthly,we analyzed the role of MBL in the proliferation and cytokine secretion in PHA-,PMA- or PMA/ionomycin-activated human T-cell line Jurkat cells,peripheral blood mononuclear cells(PBMCs),and the freshly isolated peripheral blood CD3⁺ T lymphocytes.Finally,we investigated role of MBL in APC stimulators of allogeneic T cells in mixed lymphocyte reaction(MLR) and in its presentation of tetanus toxin protein C(TTC) antigen to specific T cell lines.â… Purification of MBL from Human Plasma through the Ligand- and Monoclonal Antibody-Affinity ChromatographyMBL 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 patients' serum and preparation of anti-MBL monoclonal antibody.However, the concentration of plasma MBL is quite low(mean 900μg/L,40～3 500μg/L in normal subjects),and the components of the plasma is very complex,therefore,it is quite difficult to obtain MBL with high purity.In order to optimize the method for purifying mannan-binding lectin(MBL) from human plasma,we firstly prepared anti-human MBL-CRD monoclonal antibody-Sepharose 4B affinity chromatography column,then three steps of affinity chromatography on mannan-Sepharose 4B and anti-human MBL-CRD monoclonal antibody-Sepharose 4B were in turn employed and eluateed with EDTA,D-mannose and Gly-HCl(pH 2.4) solution independently to gather the target protein,which was identified by SDS-PAGE,Western blot and ELISA.Our dates showed that the high-purified MBL was found as a functional multimer composed of 28KD and 32KD peptide chains in SDS-PAGE and Western blot,and it was not only able to bind to mannan and to agglutinate baker yeast but also capable of binding to the collectins receptor on U937 cells.Natural MBL protein with high purity and activity was gained from human plasma by combined appfication of ligand affinity chromatography and monoclonal antibody affinity chromatography,laying a solid foundation for the following study.â…¡Preliminary Study on the Role of MBL in Regulating the Differentiation and Function of Dendritic CellsToll-like receptors(TLRs),one of important pattern-recognition receptors (PAMP) of the innate immune system,can distinguish infectious nonself from noninfectious self and control the initiation of adaptive responses,which have been considered as a linkage of innate and adaptive immunity.Among the TLR family, TLR4 is expressed very extensively(e.g.,Monocytes,Macrophages,Immature DCs, T cells and B cells),and plays a critical role in recognition and signaling of bacterial lipopolysaccharid.Dr.Chen of our team found that MBL could regulate dendritic cell maturation and cytokine production induced by lipopolysaccharid.However the regulatory mechanism remains unclear.In this study,we continued our previous studies and evaluated the impact of MBL on LPS/TLR4-mediated signaling pathway.We further investigated the possible mechanistic insight into the inhibitory effect of MBL on DC.We initiallly obtained prokaryotically expressed soluble extracellular-TLR4 protein(sTLR4) identified by SDS-PAGE and Western blot.Then,Western blot and ELISA were used to evaluate whether MBL interact with sTLR4,flow cytometry (FCM) to analyze the binding of MBL to immature dendritic cells(imDCs),and competitive inhibition test to the binding of MBL to TLR4 receptor on the surface of imDCs and its role in the consequent LPS-induced nuclear factor-κB(NF-κB) signaling pathway.Our date showed that MBL could bind to monocyte-derived immature DCs(imMDCs) at physiological calcium concentrations but optimal at superphysiological calcium concentrations.Furthermore,MBL could directly bind to extracellular domain of TLR4 in a dose-dependent manner and attenuate the binding of LPS to cell surface,resulting in decreased LPS-induced nuclear factor-κB(NF-κB) activity in imDCs.All these suggest that MBL could affect the functions of DCs by modifying LPS/TLR-signaling pathways.This study supports an important role of MBL in the regulation of adaptive immune responses.â…¢Characterization of the Action of MBL onto Raji CellsAs a key soluble pattern recognition molecule in the innate immune,MBL can not only recognize pathogens or autologous apoptotic cells,but also bind to healthy autologous cells in a specific and sugar-sensitive manner,and it has been shown that cytokine expression can be altered by interaction of the cells with MBL,suggesting that MBL may play important roles in immune regulation and do some roles in adaptive immune responses.Studies of our previous section showed that MBL be involved in the regulation of DC;however T and B lymphocytes are the mediator of acquired immune responses,thus,it is very informative to investigate whether the soluble defense collagen family could regulate lymphocytes.In this study,we investigated the interaction of MBL with Raji cells by ELISA, and the characteristics of MBL binding to Raji cells by FACS.Our results showed that MBL could bind to Raji cells in a dose-dependent manner.Furthermore,the binding of MBL to Raji cells was evident in a Ca²⁺-dependent manner,which was partially inhibited by some saccharides(mannose,glucose,or N-acetylglucosamine), C1q and anti-C1qR mAb.Similarly,it was also incompletely inhibited by recombinant human MBL carbohydrate recognition domains(rhMBL-CRD) protein and recombinant human MBL collagen-like region(rhMBL-CLR) protein, respectively,but completely inhibited by both.These data indicated that Raji cells express Ca²⁺-dependent and sugar-sensitive CLR-specific and CRD-specific MBL receptors,of which,the former was also shared with C1q.Further investigation showed that MBL could significantly inhibit Raji cell proliferation directly at higher concentrations(10～50 mg/L) in a dose-dependent manner,suggesting that MBL might also play certain roles in adaptive immune responses.â…£Preliminary Study on the Role of MBL in Regulation of T lymphocytesIt is reported that both SP-A and SP-D can not only inhibit mitogen-induced T cell proliferation,but also attenuate interleukin-2(IL-2) secretion from these cells in a dose-dependent manner.The inhibitory effects of SP-A on T cell proliferation occurs at 24 hours after the treatment of lymphocytes with mitogen.The functions of DC can also be regulated by SP-A through its inhibitory effect on DC differentiation and maturation,and by SP-D through enhancing DC's antigen-presenting capacity,which promotes its acquired immune response to infection.However,so far,little is known about the role of MBL in the regulation of lymphocytes,especially in its direct action on T cells.We speculate that MBL might have regulatory roles in T lymphocytes based on the structural homology among MBL,SP-A and SP-D.In the present study, we explored the feasibility of the in vitro regulation of MBL in T lymphocytes.Tetanus toxin(TT) has been widely used as a model antigen in the study of CD4+ T cell-mediated immune responses due to its activation of CD4+ T lymphocytes.However,the traditional tetanus toxoid(TToid) is detoxified tetanus bacilli by formaldehyde.Since tetanus bacilli can form spores with high toxicity,the preparation of TToid appears to be dangerous to certain extent.Furthermore,it is easy to cause pollution during processing TT by formaldehyde,and the toxicity of TToid can be recovered sometimes.Therefore,the application of TToid in researches subjects to certain restrictions.TTC can not only stimulate the body to produce sufficient neutralizing antibodies against toxin-mediated attacks,but also activate CD4⁺ T cells,which has been considered as a safe and optimal tetanus subunit vaccine candidate.In the present study,we used TTC as a model antigen to investigate the role of MBL in regulation of T cell-mediated acquired immune response.We investigated the possibility of MBL in regulating T lymphocytes in vitro. Firstly,we cloned,expressed and analysed the immunogenicity of TTC and obtained TTC with high purity and immunogenicity,which provides an ideal model antigen for studying antigen presentation and immune responses.Then,PBMCs were isolated by sequential centrifugation on Ficoll-Hypaque and Percoll gradients from blood drawn from normal adult donors in our laboratory and CD3⁺ T lymphocytes were obtained from PBMCs by passing a nylon wool column.Jurkat cells,PBMCs and CD3⁺ T lymphocytes were activated by non-specific stimulation of phytohemagglutinin (PHA),PMA or PMA/ionomycin.The binding of MBL to PHA- or PMA- activated Jurkat cells and CD3⁺ T lymphocytes were measured by FCM.WST-1 and ELISA were usued to analyse the impact of MBL on the proliferation and IL-2 or IFN-γsecretion in PHA-,PMA- or PMA/ionomycin- activated Jurkat cells,CD3⁺ T lymphocytes and PBMCs.And finally,we investigated the role of MBL in APC stimulators of allogeneic T cells in mixed lymphocyte reaction(MLR) and presentation of TTC antigen to specific T cell lines.Our date showed that MBL could bind to PHA- or PMA- activated Jurkat cells and CD3⁺ T lymphocytes;that MBL could inhibit the proliferation and IL-2 or IFN-γsecretion in a concentration-dependent manner in PHA-,PMA- or PMA/ionomycinactivated cell;and that MBL could reduce APC's ability to stimulate allogeneic T lymphocyte proliferation and DC's ability to present TTC to specific T cell lines.All these date suggest that MBL could down-regulate T cell-mediated acquired immune responses.The findings mentioned above indicate that MBL could directly interact with TLR4 expressed on DCs,and that MBL could alter the downstream of NF-κB signaling pathway,suggesting that MBL and TLR might establish a large network for host defense and regulate the subsequent immune responses.In addition,MBL can directly regulate the functions of T and B lymphocytes,and down-regulate the antigen-presenting ability of APC,suggesting that MBL may regulate acquired immune responses through T cells,B cells and APCs.So far,no similar researches have been reported,although the detailed mechanism remains to be elucidated.What we found in this study proposed a novel idea for MBL's role in acquired immune responses and its mechanism.The interpretation of the future analyses of the properties of MBL regulation,elucidations of the molecular mechanism and the signal pathway in which MBL is involved will further reveal the biological significance of this key innate molecule,MBL.