The Effects And Mechanisms Of Anti-MBL MAb1C8on Opsonophagocytosis Of Monocyte Macrophages In Vitro

Mannan binding lectin (MBL) as human natural immune system a important pattern recognition receptor, in recent years has been one of the hot topics in the study of natural immunity. MBL, an evolutionary conserved circulating host defense protein produced mainly by liver, is a member of the family of collectins (C-type lectins with a collagen-like domain).The overall polypeptide structure of MBL includes a cysteine-rich N-terminal domain, a collagen-like region(CLR), the alpha helix consisting of neck region and a C-terminal carbohydrate-recognition domain(CRD). MBL in the natural immunity plays an important role to fight infection, Which can selectively recognize sugars such as D-marmose, N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine and fucose presented on pathogens via the CRD. It is clear that the CRD can recognize a wide range of microbes such as bacteria、viruses、yeast and Leishmania. Classical theory believes that the chief biological effect of MBL is that it forms compound with MBL-associated serine protcases (MASP-1、MASP-2、MASP3) through its collagen-like region (CLR) and then trigger immediate cellular lysis or phagocytosis by initiating the lectin pathway of complement activation. It also can initiate opsonophagocytosis by combining with phagocytes Collectin receptor (C1qR), not rely on the complement. In addition, the binding of the spherical structure of CRD domain and some virus (such as influenza virus A) can neutralize virus activity directly. The MBL in plasma is two to six polymers mixture, only three polymers and above high polymers can effectively identify sugar structure of pathogens surface and then activate complement.In human serum, the content of MBL is very low,the normal average content of which is1mg/L. MBL is also a kind of acute phase reaction protein, in the condition of stress (such as surgery, pathogen infection, etc.), the plasma concentration can increase2-3times. The patients of MBL defection were at high risk of infection, the performance of which was repeated acute or chronic infection of the various kinds pathogens. However, when MBL level is too high, it may excessive activate complement through the lectins way, resulting in inflammatory damage.Some studies reported that the MBL of high levels was related to the inflammation of ischemia reperfusion injury, Diabetic kidney complications and primary biliary cirrhosis. Instead, when the MBL level is low, its ability to activate complement were relative decline, which could reduce the harm of inflammatory mediator to host cell.In addition, the MBL of high levels could promote the intake of fungus by phagocytic cells (monocyte macrophages、neutrophile, etc.), such as mycobacterium tuberculosis and leishmania, which increased the chances of invasion of pathogen [13]. So the normal level of the MBL for the body is particularly important. As is known to all, Monocyte macrophages play an important roles in the natural immune response, and participate in the specific immune response.Under normal circumstances,Monocyte macrophages through swallowing foreign body, bacterium,old and mutated cells as well as immune complex to maintain the body’s immune homeostasis. According to the reports in the literature, human serum rich in the MBL can promote the opsonophagocytosis of monocyte macrophages in vitro [15,16]In view of the above-mentioned facts that MBL is important and the price of commercialization MBL antibody is expensive, so we prepared monoclonal antibody against the MBL. and screened of a functional single1C8strains.Studies have shown that the MBL can enhance the activation of complement and promote the phagocytosis of phagocyte, but the effects of anti-MBL mAb on opsonophagocytosis of monocyte macrophages in vitro has not been reported, so we selected the human monocyte macrophages as our cell model,in order to explore the the effects of anti-MBL mAb1C8on opsonophagocytosis of monocyte macrophages in vitro and its mechanism of action. We first identified the purity of the anti-MBL mAb1C8and the biological activities, it can bind with the commercialized MBL and the natural MBL prepared by our laboratory in the form of a dose dependent, we also preliminary discussed the effects of anti-MBL mAb stimulated with pooled human serum on opsonophagocytosis of monocyte macrophages in vitro and its mechanism.Part Ⅰ Purification and identification of MBL monoclonal antibody1C8Recent work has highlighted that MBL recognizes self-structures, thus extending the role of these molecules beyond the traditional view of first line defense molecules, including recognizing exposed self-antigens during ischemia-reperfusion injury as well as binding to autologous apoptotic cells and promoting the clearance of cellular debris etc,but the reports about anti-MBL mAb is less.At present the monoclonal antibodies of MBL have goods on the market,but its price are prohibitively expensive and the goods is for the MBL of prokaryotic expression, rather than the natural purified MBL.Over the years we has been committing to the research in the MBL of natural purification and the function related, especially the separation and purification technology of the MBL has been mature. In view of this, we prepared monoclonal antibody against the natural purified MBL (1C8, etc). This part mainly is to identify the purity of1C8and its biological activities.First we recovered the hybridoma cell line1C8which has been prepared by our departments and preserved in liquid nitrogen, and then prepared the monoclonal antibody ascites, the monoclonal antibody of ascites was purified by CS-AS, we further determined the purity of1C8and specificity by SDS-PAGE and Western-blot respectively, and compared to the commercialization antibody HYB131-11of the MBL by enzyme-linked immunosorbent assay in order to detect the sensitivity of 1C8.According to the results,1C8can not only best bind to the natural active of the MBL prepared by our departments but also can combine with the commercialization of the MBL,the comparison with antibody HYB131-11were not significantly difference. Above results showed that1C8has a good combination of the biological activity of the MBL, which provide experiment basis for the application of the1C8.Part Ⅱ The effects of anti-MBL mAb1C8on opsonophagocytosis of Monocytes/macrophages in vitroResearch has shown that human serum rich in the MBL can promote the phagocytosis of phagocyte and enhance the activation of complement, but the strong lectin pathway leads to inflammatory injury, monocyte macrophages as a full-time antigen presenting cell, it maintain the steady state of the body by phagocytosing old cells and pathogens. This part mainly is to separate and cultivate human monocyte macrophages, in order to explores the effects of anti-MBL mAb1C8stimulated with pooled human serum on opsonophagocytosis of monocyte macrophages in vitro.Sterile separation of human peripheral blood mononuclear cells (PBMC), using flow cytometry (FCM) analysis, we take1C8/IgG、10%inactivated/not inactivated pooled human serum and FITC-C. albicans in advance incubation for30minutes,then join them to PBMC, tabling incubation for30minutes at37℃, thus we detected the effects of anti-MBL mAb1C8on opsonophagocytosis of FITC-C. albicans by CD14+monocyte macrophages in vitro.By fluorescence microscope observing the effects of anti-MBL mAb1C8on opsonophagocytosis of FITC-C. albicans of macrophages in vitro,After PBMC adherent2hours, adding to M-CSF50ng/ml and cultivating9days. TheFCM results showed that1C8could significantly inhibit the opsonophagocytosis of FITC-C. albicans by monocytes stimulated with pooled human serum and could reduce to the inactivated serum levels. By fluorescence microscope observing the effects of anti-MBL mAb1C8on opsonophagocytosis of FITC-C. albicans of macrophages in vitro,the results are consistent with the FCM, the influence in group1C8was that the number of FITC-C albicans phagocytosed by macrophages was reduced.These results provide experiment basis that1C8directly regulates the lectin pathway mediated by MBL. The problem still need to be solved is:the binding site of MBL with1C8; the effects of anti-MBL mAb1C8on other immune cells.Part Ⅲ The mechanisms of regulatory roles of1C8on monocytes/macrophagesIn order to discuss the mechanisms of regulatory roles of1C8on monocyte macrophages,first we expressed the recombinant protein MBL-CRD (recom-binant human MBL-CRD, rhMBL-CRD), and identificated the structure and function of rhMBL-CRD by SDS-PAGE and Western-blot. The binding of1C8with recom-binant human proteins rhMBL-CRD、MASP-1C、MASP-2C and MASP-2N by ELISA, we also analysed the specificity of1C8binding with rhMBL-CRD and rhMBL-CLR by SDS-PAGE and Western-blot.The effects of anti-MBL mAb1C8on rhMBL-CRD binding with mannan、mannose、D-galactose、D-galactosamine、 D-mannosamine and D-glucosamine were analysed by direct ELISA. We also observed the effects of anti-MBL mAb (1C8) on FITC-E. coli and FITC-C. albicans agglutination induced by rhMBL-CRD through agglutination test.The results by Western-blot showed that rhMBL-CRD could best bind with anti MBL-CRD mAb, and had obvious stripe in the43KD.The binding of rhMBL-CRD with1C8were concentration dependent,but1C8could not binding with MASP-1C、MASP-2C and MASP-2N by ELISA, these results were consistent with the results of Western-blot.The results of direct ELISA showed that the binding of rhMBL-CRD with1C8were blocked by mannan, mannose, D-galactose, D-galactosamine, D-mannosamine and D-glucosamine, with the concentration of six sugar decreasing, the binding of1C8with rhMBL-CRD was enhanced.We observed the agglutination of FITC-E. coli and FITC-C. albicans under fluorescence microscope,the results showed that the binding of rhMBL-CRD with FITC-E. coli and FITC-C. albicans were blocked by 1C8,but the group of IgG had no difference.These results indicated that1C8could block the lectin pathway mediated by MBL through binding with the carbohydrate recognition domain (CRD) or competiting binding with the sugar structure on the surface of pathogens.In conclusion, we found that the anti-MBL antibody1C8not only could best bind with the natural active MBL and the commercialization of the MBL, but also had no difference from the HYB131-11. By detecting the effects of anti-MBL mAb1C8on opsonophagocytosis of monocyte macrophages in vitro and the binding site of MBL with1C8,we obtained the evidence of application value about1C8.But the effects of anti-MBL mAb1C8on the secretion of cytokine and proliferation of monocyte macrophages in vitro and the effects of anti-MBL mAb1C8on other immune cells need to be further study.Next step we will continue to explore the effects of1C8on the secretion of inflammatory cytokine and apoptosis of monocyte macrophages, in addition we will discuss the effects of1C8on T cells and NK cells, etc. Our study will provide experimental basis for the1C8subsequent applications, and provide new theoretical basis and targets for the diaease relate to the high level of MBL.