Study On The Roles Of Mannan-binding Lectin In Regulating Monocytes/Macrophages Differentiation And Development

The basic understanding of the immune system has undergone a substantial paradigm shift in the past twenty years, since an awareness of the power and influence of the innate immune system emerged in1990s. It is widely believed that the innate immune system is not only responsible for the induction of acute responses necessary for elimination of pathogens but also able to activate appropriate adaptive immune responses via antigen presentation, 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 on determining the nature of the subsequent adaptive immune response. It is also suggested that innate immunity initiates acquired immune response and controls the types of acquired immune response through pattern-recognition between pathogen associated molecular pattern (PAMP) and pattern recognition receptor (PRR). Antigen presenting cells (APCs) play a key role in the process. Professional APC includes macrophages, dendritic cells and B lymphocytes. Today, this field is still frontier and hot of immunology research. However, the majority is only figurative description, the guiding role of natural immunity to the adaptive immune response and its mechanisms need to be further clarified.Mannan-binding lectin (MBL), a member of collectins belonging to C-type lectin superfamily, is a multimeric protein containing collagen-like sequences. As a key soluble PRR of the innate immune system, MBL is synthesized and secreted into the blood by hepatocytes. The MBL polypeptide consists of four regions, a cysteine-rich N-terminal domain, a collagen-like region (CLR) containing Gly-X-Y repeats (where X is any amino acid and Y is often hydroxyproline or hydroxylysine), a neck domain and a C-terminal carbohydrate recognition domain (CRD). The neck region forms an alpha-helical coiled-coil structure which probably promotes trimerization of three polypeptides to form the subunit. The trimer is stabilized by hydrophobic interactions and inter-chain disulphide bonds within the cysteine-rich N-terminal domain. MBL subunits assemble into larger oligomeric structures which is composed of two to six subunits forming a ’bundle-of-tulips’ or sertiform appearance first described for C1q. Only the more highly polymeric forms have biological activity and can fix complement. MBL in serum has been intensively characterized and found to act as pattern recognition molecules that selectively recognize carbohydrates on the surface of microbial pathogens. Following pathogen recognition, MBL may activate the complement cascade through the lectin pathway, after which microbes are killed by cellular lysis and indirect opsonization. When bound to the collectin receptor of effector cells, MBL can mediate direct opsonization and MBL-dependent, cell-mediated cytotoxicity. MBL is also able to enhance the phagocytosis of cellular debris, apoptotic cells and immune complexes in vitro and in vivo. MBL also plays an important role in surfactant defenses of mucous membrane. A mounting body of evidence suggests that defect opsonophagocytosis resulted from low MBL levels due to gene mutations in MBL coding gene (CGT52TGT, GGC54GAC and GGA57GAA) and promoter as well as5’-untranslated region appear to be more vulnerable to infections in a number of clinical settings, and more susceptible to autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren’s syndrome resulted from phagocytosis defection. On the other hand, exess activation of lectin pathway may result in inflammation disease. MBL is also an acute phase reactant, showing an increase of up to two or three-fold after infection or surgical trauma. Current researches focus on the roles of MBL in innate immunity, mainly in the complement activation and opsonophagocytosis. However, few reports focus on the roles of MBL in adaptive immunity, especially of its regulatory functions. Fraser has claimed that MBL can inhibit IL-1secretion of lipopolysaccharide (LPS)-stimulated monocyte, but promote the secretions of IL-10and IL-6. So it is of great significance in studying the relationship of MBL with adaptive immunity, particularly its regulatory role on monocyte/macrophage differentiation and development.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) and complement C1q, have roles in immune regulation through interacting with various cells. For instance, SP-A inhibits differentiation and maturation of dendritic cells (DCs) and modulates cellular responses induced by lipopolysaccharide (LPS) through interacting with CD14; SP-A directly interacts with Alveolar macrophages through binding to cell surface receptors Toll-like receptor4(TLR4) and MD-2, resulting in modulation of chemotaxis, phagocytosis, and modified pro-or anti-inflammatory immune responses. SP-D enhances antigen presentation ability of DCs to promote adaptive immune response against bacterial antigens and alters CD14-LPS interaction through binding to CD14. It is reported that both SP-A and SP-D can directly inhibit mitogen-induced T lymphocytes proliferation to avoid lung damages caused by the strong immune response. Clq regulates LPS induced cytokine production in bone marrow-derived DCs and induces maturation of DCs. Therefore, according to the structure homology among MBL, SP-A, SP-D and Clq, the fact that they all can bind to collectin-receptor, and the fact that macrophage 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. Our previous studies showed that MBL could bind to DCs, T and B lymphocytes in a Ca2+-dependent and sugar-sensitive manners through CLR-specific and CRD-specific MBL receptors, of which, the former was also shared with Clq; and that MBL could regulate differentiation and maturation of dendritic cells and reduce DC’s ability to stimulate allogeneic T lymphocytes proliferation; and that MBL could significantly inhibit Raji cells proliferation directly at higher concentrations (10-50μg/ml) in a dose-dependent manner. Further investigation found that MBL may directly regulate T and B cell functions. All these data suggest that MBL may regulate acquired immune responses through APCs, T and B lymphocytes. To date, however, little knowledge has been obtained about its role in the regulation of adaptive immune responses. Moreover, its detailed mechanism has yet to be elaborated by further experiments. The immunomodulatory effects of MBL and its molecular mechanisms elucidation will further reveal the biological significance of MBL served as an important molecule in the innate immune.Therefore, on the basis of existing research, we continued to explore the roles of MBL in regulating the adaptive immune response. As a bridge connecting innate immunity with adaptive immunity, monocytes/macrophages play an important role in the immune response. Our aim in the present study is to explore the effects of MBL on differentiation and proliferation of monocytes/macrophages and their molecular mechanisms. Firstly, we established a convenient procedure for the purification of MBL through the ligand-and monoclonal antibody-affinity chromatography, and obtained natural MBL with high purity and bioactivity from freshly frozen human plasma. Next we investigated the effects of MBL on inducing differentiation of the monocyte-derived macrophages in vitro, including the studies of morphology, surface markers, cytokines, phagocytosis and so on. In addition we also investigated the effects of MBL on the proliferation, apoptosis, cell cycle of monocytes and its possible mechanisms in vitro.In this study, we provided evidences that human natural MBL possibly participates in the adaptive immune response through modulating differentiation and proliferation of monocytes/macrophages. Our study can enrich basic theory of the innate immunity guiding the adaptive immunity. This topic is divided into two parts, it is described as follows.Chapter I Mannan-binding lectin regulates monocytes/macrophages differentiation and developmentAs one of the myeloid precursor cells, monocyte has the potential ability of differentiation to dendritic cell and macrophage in both directions. Monocyte can choose one way under the stimulation of different cytokines. Currently it is known that monocyte development is regulated by cytokines, including IL-10,IL-6, TNF-αand so on. As a key soluble PRR of the innate immune system, MBL is a plasma protein secreted by the liver cells, belonging to the family members of the C-type lectin superfamily. Our previous studies showed that MBL can inhibit monocyte differentiation to dendritic cell, but induce monocyte differentiation to macrophage. Macrophages have long been considered to be important immune effector cells, with great plasticity in their phenotype and function. Under different microenvironment macrophages can be polarized to assume different phenotypes and show different functions. If confirmed MBL can regulate monocyte differentiation to different subtypes of macrophages, there are reasons to speculate that the different subtypes of macrophages may induce different types of adaptive immune response, suggesting that MBL is involved in the regulation of the adaptive immune response. The objectives of this chapter work were to investigate the effects of MBL on regulating differentiation and development of monocytes/macrophages in vitro.Firstly we extracted MBL from human frozen plasma using traditional methods. LPS has polluted MBL in the extraction process. To exclude the interference effect of LPS on the study of MBL regulatory function, the polymyxin B gel column was applied to remove LPS. After removing LPS, MBL identified by ligand binding assays still retains its good mannan binging activity. We obtained human peripheral blood mononuclear cells (PBMC) from peripheral blood of healthy adult volunteers by Ficoll-Hypaque density gradient centrifugation. After purified by MACS, monocytes were analyzed by flow cytometry for molecular maker on cellular surface. CD14+monocytes with a purity of more than95%were obtained. Human peripheral blood monocytes were co-cultured with macrophage colony stimulatory factor (M-CSF) and various concentrations of MBL simultaneously in RPMI1640supplemented with10%fetal bovine serum (FBS) at37℃in5%CO2. During the process of differentiation, it exhibited round cells and spindle cells (or fibroblast-like cells). Round cells were named standard macrophages (s-MΦ), while spindle cells were named fibroblast macrophages (f-MΦ). The confocal microscope was employed to examine MAC-1(CD11b) and CD14expressions on the cell surface and to ensure monocytes differentiation to macrophages (s-MΦ and f-MΦ). In order to further determine the classification of macrophages after treated by MBL, we analyzed the phenotypes and functions of macrophages.(1)MBL binding to monocyte was detected by flow cytometry.(2)The contrast phase microscope was applied to detected macrophage morphological changes.(3)Flow cytometry assay was used to detect the surface markers of macrophage such as HLA-DR, CD86, and CD206.(4) ELISA assay were performed to detect the expression of the cytokines of macrophage. Real-time PCR assay were employed to examine the mRNA expression of the cytokines of macrophage.(5)Flow cytometry and confocal microscopy were used to assay the phagocytosis of macrophage.(6)Macrophages were co-cultured with allogeneic T cells, in order to detect their abilities to stimulate allogeneic T cells into proliferation by CCK-8incorporation assay.Our results revealed that (1) MBL directly combines monocytes, suggesting that MBL receptors of binding proteins may exist on the surface of monocytes.(2) Macrophages presented an enlarged volume, adhensive growth pattern and the puffed cytolymph. MBL at lower concentration (2.5μg/ml) promoted monocytes differentiation into f-MΦ(F=49.2P=0.000), whereas MBL at higher concentration (20μg/ml) promoted monocytes differentiation into s-MO.(3) MBL down-regulated the expression of HLA-DR, CD86and CD206during the process of monocyte differentiation into macrophage (F=3.831P=0.026; F=10.034P=0.001; F=4.524P=0.015). It was presumed that MBL may be involved in the regulation of the adaptive immune response by inhibiting antigen-present ability of macrophage.(4) MBL obviously promoted the anti-inflammatory cytokines IL-10(F=6.985P=0.001; F=25.481P=0.000) and TGF-βsecretions (F=341.411P=0.000; F=8.129P=0.001; F=5.486P=0.007), with IL-12less (F=114.179P=0.000; F=63.346P=0.000), and significantly inhibited the pro-inflammatory cytokines IL-6(F=97.711P=0.000; F=212.528P=0.000; F=28.464P=0.000) and TNF-asecretions (F=10.385P=0.000), and also markedly inhibited macrophage chemoattractant protein-1(MCP-1) secretions (F=471.826P=0.000). The changes of cytokines expression level in macrophage suggested that MBL induced monocyte differentiation into M2c type macrophage.(5) Both s-MΦ and f-MΦ can engulf E.coli or yeast. The phagocytosis of macrophage was not affected by MBL induced and differentiated cells (F=2.674 P=0.076).(6) There was less proliferation of allogeneic T cells co-cultured with macrophages after treated with MBL (F=24.343P=0.000; F=18.604P=0.000; F=6.463P=0.001), suggesting that MBL down-regulated macrophage function to inhibit the adaptive immune response.Our data suggest that MBL may induce differentiation of peripheral blood monocyte into M2c type macrophage, which exhibits a distinct immunosuppressive phenotype and exert a positive role on anti-inflammatory effects. So we suppose that MBL might have anti-inflammatory and immune modulatory effects in pathological states. Thus, MBL not only participates in the innate immune response, but also be involved in the regulation of the adaptive immune response. The study of macrophage differentiation and development in different environments of the body not only helps us better understanding disease mechanisms, but also provides a theoretical basis for new prevention and treatment methods including macrophages as targets.Chapter II Mannan-binding lectin regulates cell proliferation, apoptosis and cycle in human monocytesMonocytes are the key components of innate and adaptive immunity, and are important in the mononuclear phagocytic system. Many studies have indicated that bone marrow monocytes are common precursors for macrophages, which build up the defense system effectively against the invasion of foreign micro-organisms. Meanwhile, these cells play an indispensable role in the removal of senescent and apoptosis cells to maintain the homeostasis of bodies under physiological conditions. These cells also have the abilities to recognize and destruct tumor cells. Monocytes-derived macrophages, served as one of professional APC, play important roles in immunomodulation and inflammation through secreting a variety of cytokines. Abnormal development of the mononuclear phagocytic system will inevitably lead to the disorder of the immune function and within the environment, and ultimately lead to various diseases of the body. The objectives of work in this chapter were to investigate the effects of MBL on the proliferation, apoptosis, cell cycle of human monocytes in vitro and its possible mechanism.Our results revealed that (1) MBL at lower concentrations (≤4μg/ml) enhanced human monocytes proliferation, whereas MBL at higher concentrations (4-20μg/ml) markedly attenuated cell proliferation (F=6.135P=0.001; F=8.802P=0.000; F=7.595P=0.000). The proliferation effects were examined with the CCK-8assay.(2)In order to investigate the anti-proliferative effect of MBL and its underlying mechanism, human monocytes and monocyte line U937cells were treated with MBL at higher concentrations for24-72h at37℃in a5%CO2incubator. Cell cycle distributions in U937cells stained with propidium iodide were detected by flow cytometry. We further found that MBL-induced growth inhibition was associated with G0/G1arrest (F=5.550P=0.007; F=23.161P=0.002). Cell cycle blockade was manifested by down-regulation of cyclinDl (F=12.969P=0.007; F=58.886P=0.000), cyclinD3(F=59.491P=0.000; F=41.07P=0.000), Cdk2(F=91.185P=0.000; F=83.456P=0.000), Cdk4(F=166.65P=0.000; F=39.842P=0.000) and up-regulation of the Cdk inhibitory protein Cipl/p21(F=13.647P=0.006; F=1135.242P=0.000).(3)Moreover, higher concentrations MBL induced apoptosis (F=258.709P=0.000), as determined by using Annexin V staining assay. Furthermore, higher concentrations MBL triggered the death receptor and mitochondrial apoptotic pathway, as indicated by increasing the expression of Fas (F=13.719P=0.006; F=288.811P=0.000), but decreasing the level of Bcl-2(F=55.86P=0.000; F=196.435P=0.000), resulting in activation of caspase-3(F=830.656P=0.000; F=45.956P=0.000).In this study, our results demonstrated that exposure to higher doses of MBL might effectively inhibit monocytes proliferation through the induction of cell apoptosis and cell cycle arrest at G0/G1phase, suggesting that MBL, as an emergency protein, might have anti-inflammatory effects in pathological states and immunomodulatory functions.In summary, results above have showed that MBL can promote the differentiation of human peripheral blood monocytes to M2c type macrophages. Meanwhile high concentrations of MBL can be effective in inhibition of monocytes proliferation. These data lend further credit to an important physiological role MBL in regulating adaptive immunity. Based on current data, it appears that MBL can dampen lymphocyte responses to exogenous stimuli or apoptotic cells by the monocytes/macrophages passing inhibitory signals to T, B cells and protect the body against collateral immune-mediated damage. This fully proves that the MBL served as an emergency protein has anti-inflammatory effect and inhibits the immune response. Further studies evaluating the specific mechanisms as followed are required: the receptors involved in binding; the effect of MBL on signal transduction; and the effect of MBL on monocytes/macrophages differentiation as well as proliferation. The next step will clear the regulatory role of MBL on monocytes/macrophages differentiation signaling pathways, and explore the relationship between monocytes/macrophages differentiation and proliferation regulated by MBL, in order to reveal MBL served as the innate immune pattern recognition molecule has new biological activity. In this study, the basic theory for the natural immune guiding the acquired immune is powerfully confirmed by experiments. It provides new targets for diseases prevention and control research related to MBL imbalances and mononuclear phagocytic system dysfunction.