Preliminary Study On The Roles Of Mannan-binding Lectin In Regulating T Cell Function

Innate immunity comprises a sophisticated network of molecules, which recognize pathogens, and effector molecules, working together to establish a quick and efficient immune response to infectious agents. It is considered to be the first line of host by recognizing pathogen associated molecular patterns (PAMPs) through its pattern recognition receptors such as Toll-like receptor (TLR), peptidoglycan recognition molecule, mannan-binding lectin (MBL, also known as mannose-binding lectin), assessing the level of danger, determining the nature and extend of adaptive immune response.The mechanisms of innate immunity in shaping adaptive immunity has been the focus of interest for many research groups around the world.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). MBL can be considered as an 'ante-antibody' because it has an important role against infectious agents by recognizing and binding to conserved carbohydrate motifs, such as D-mannose, L-fucose,N-acetyl-D-glucosamine(GlcNAc), N-acetyl-mannosamine(ManNAc) present on the surface of a variety of microorganisms (bacteria,virus,fungi,parasite), via its carbohydrate recognition domain (CRD) in a calcium-dependent manner, and its biological effect is thought to be mediated by collagen-like region (CLR) through binding with MBL-associated serine protease(MASP).It can trigger immediate cellular lysis or phagocytosis by initiating the lectin pathway of complement activation, or promote opsonophagocytosis by direct binding to cell surface receptors without the involvement of complement. 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 as 5'-untranslated region appear to be more vulnerable to infections in a number of cliical 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.Recent 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 binding to immunoglobulins, recognizing exposed self-antigens during ischemia-reperfusion injury as well as modified self-antigens; binding to autologous apoptotic cells and promoting the clearance of cellular debris.It can also bind to monocytes as well as dendritic cells and regulate cytokine secretion. As a multi-functional molecule in innate immunity, MBL has more functions far beyond innate defense against infection, and may be involved in "crosstalk" between innate and adaptive immunity. So it is of great significance in studying the relationship of MBL with adaptive immunity.However, up to now, few reports focus on the functions of MBL on adaptive immunity, especially of it's regulatory functions. Fraser has claimed that MBL can inhibit IL-1 secretion of lipopolysaccharide (LPS)-stimulated monocyte, but upregulate the secretion of IL-10 and IL-6.Downing et al found that MBL can bind to DC, Mo and B cell, yet the mechanism remains to be evaluated.None of research is about the interation of MBL with T cell.Previous studies by our lab showed that MBL can upregulate the expression of CDla,CD83,CD40,CD80,CD86 and HLA-DR on IL-4+GM-CSF cultured DC, inhibit the uptake of zymosan granules by DCs but enhance the proliferation of naive T cells induced by DCs.While in the presence of LPS, MBL inhibit DC maturation including down-regulate expression of CD83 and CD86 in a dose-dependent manner, enhance the ability to uptake zymozan granules by DCs and decrease the proliferation of naive T cells induced by DCs and inhibit the production of TNF-aand IL-12 p40+p70.These effects are associated with blocking LPS-TLR4 binding to MBL,resulting in decreasing the activity of NF-κB.We also acquired the direct clues of MBL with adaptive immunity by finding that MBL can bind to Raji cell in an Calcium-dependent manner, and high MBL level can inhibit the proliferation of Raji cells, which indicated that under certain situation, MBL concentration in blood and tissue may increase, which lead to the binding of MBL to self immune cells so as to regulate the adaptive immunity.The present study aimed to determine the regulatory roles of MBL on adaptive immunity, especially its regulatory roles on T cell. Firstly, we acquire natural MBL with high purity and bioactivity from freshly frozen human plasma. Then we investigated whether MBL have an effect on ConA-, anti-CD3-mAb-, anti-CD3mAb+anti-CD28 mAb-stimulated proliferative responses and cytokine secretion of human PBMCs as well as purified T cells, and studied preliminarily the mechanisms of its function.PartⅠEffect of MBL on Proliferation and Cytokines Secretion of PBMCsIt is clear that the proteins of collectin family such as surfactant-associated proteins SP-A and SP-D can initiate and shape adaptive immunity.They have been suggested to modulate a variety of immune cell funcions of adaptive immunity beyond binding to pathogens, including Mo, DC, T cell. SP-A and SP-D inhibit lymphocyte proliferation as well as its function, avoiding the lung tissue damage caused by excessive acquire immune response. MBL can also bind to various immune cells and regulate the cytokine secretion.The structural homology of the collectins led us to hypothysize that MBL may have effect on T cells activation that is similar to SP-A and SP-D. In this study, we sought to determine if MBL have functional similarity with SP-A and SP-D. The effects of MBL derived from human plasma on mitogen-stimulated proliferative responses as well as cytokine secretion of human peripheral blood mononuclear cell (PBMC) was examined.PBMCs were isolated from peripheral blood of healthy adult volunteers by Ficoll-Hypaque density gradient centrifugation and cultured with Con A, or anti-CD3 mAb and various concentrations of MBL simultaneously in RPMI 1640 supplemented with 10% fetal bovine serum(FBS) at 37℃in 5% CO2 for 72h. Proliferation of PBMCs were analyzed by CCK-8 incorporation. Supernatants were harvested 24h later and assayed for cytokines secretion.In FCM analysis, PBMCs were stained with CFSE(2.5μM),then cultured with anti-CD3 mAb and MBL for 4d.Cells were labeled with APC-anti-human CD4 and PE-anti-human CD8 and then analyzed the proliferation, especially CD4+T cell and CD8+T cell.Results of CCK-8 analysis indicated that, MBL exibit obvious inhibitory functions in anti-CD3 mAb-stimulating PBMCs proliferation.FCM analysis indicated that MBL can inhibit the proliferation of CD4+T cell and CD8+T cell of anti-CD3 mAb-stimulated PBMCs. Analysis of the supernatant of mitogen-stimulated cell cultures treated with MBL showed decreased amounts of interleukin-2(IL-2), IL-10 and IFN-y, compared to mitogen alone,while enhancement of IL-6 was detected. Anti-MBL-MASP mAbs may block the inhibitory roles of MBL on IL-2 and IFN-yproduction. The results mentioned above indicate that MBL may inhibit the proliferation and activation of T cell, laying the foundation that MBL may have direct regulatory roles on purified T cells.Part II Effect of MBL on Proliferation and Cytokine Secretion of T cellsPrevious studies showing that MBL inhibit lymphocyte proliferation were conducted with mixed populations of lymphocyte and monocyte, it was not clear whether MBL were acting directly on T cell or indirectly via a paracrine effect. So the present study sought to address whether MBL act directly on purified T cells independent of accessory cells as well as to determine the cytokine production.PBMCs were isolated from peripheral blood of healthy adult volunteers by Ficoll-Hypaque density gradient centrifugation and further purified into CD3+ and CD4+T cells with MACS, then cultured with Con A and MBL for 72h, followed by analysis with CCK-8 incorporation. In FCM analysis, T cells were stained with CFSE(2.5μM), then cultured with anti-CD3 mAb or anti-CD3 mAb+anti-CD28 mAb for 4d. Proliferation of CD4+T cells and CD8+T cells were analyzed. Cytokine secretion such as Thl(IL-2, IFN-γ) and Th2(IL-6, IL-10), as well as IL-17a were detected by ELISA. CCK-8 analysis indicated that MBL can inhibit the proliferation of T cells induced by Con A.FCM analysis showed that MBL can inhibit proliferation of anti-CD3 mAb, anti-CD3 mAb+anti-CD28 mAb-stimulated T cell proliferation as well as CD4+T cell and CD8+T cell.MBL can inhibit the secretion of IL-2,IFN-γIL-10 and IL-17a induced by various concentrations of ConA and anti-CD3 mAb+anti-CD28 mAb, yet secretion of IL-6 may be up-regulated or down-regulated. MBL can also inhibit Con A induced IFN-ysecretion by CD4+T cells.The results above support an important role of MBL in direct regulation of T cell function.And it remains to be determined:1) whether MBL regulate the T cell function through direct binding to T cell; 2)the domain involved in the recognition;3)effect of MBL T cell activation;4)whether MBL can regulate T cell throuth regulating cytokines secreted by APC.PartⅢMechanisms of regulatory roles of MBL on T cellThe objective of the current study was to characterize preliminarily mechanisms responsible for inhibitory effects of MBL on T cell. We investigated the interation of MBL with purified T cells and Jurkat T cells by cell-ELISA as well as FCM analysis, and the effect of anti-CRD mAb, anti-MBL-MASP mAb on the binding of MBL with T cells. CD69 and HLA-DR expression of T cell activation marker were analyzed by FCM. Exogenous Ca++ was added into the culture in order to investigate the function is Ca++ dependent or not. In blocking assay, MBL was cocultured with anti-MBL-CRD or mannan, and analyze the secretion of IFN-ywas by ELISA. In order to determine whether the inhibitory activity was dependent upon the IL-2, recombinant human IL-2(rhIL-2) was added in the presence of MBL and Con A, and analyze the secretion of IFN-γwas by ELISA. MBL can bind to Jurakt cell and purifiec T cell by cell-ELISA and FCM in a Ca++ independent manner, exogenous Ca++ have no influence on IL-2 and IFN-ysecretion, anti-CRD mAb and mannan have no effect on its inhibitory role.The results presented here suggest that the inhibitory effects exbited by MBL may have been due to the collagen domain rather than CRD. CD69 expression decreased when MBL is added with T cell mitogen, yet there is no change of HLA-DR expression. Exogenous rhIL-2 significantly reverse the IFN-ysecretion inhibited by MBL, indicated that MBL inhibit T cell function through suppressing activation rather than inducing apoptosis.In order to investigate the effect of MBL on cytokines secretion of MBL on APC, MBL was cocultured with LPS-stimulated PBMCs, proinflammatory cytokines (TNF-α, IL-6) and MCP-1 were detected with ELISA. In an in vitro induced DC, MBL was added with LPS or TNF-a, and IL-6 was detected by ELISA.MBL can inhibit the production of TNF-αand IL-6 secretion induced by LPS. IL-6 secretion was down-regulated by MBL in DC culture, which give a clue that MBL may inhibit the proinflammatory cytokine secretion thus to inhibit the T cell function,or inhibit the T cell function followed by inhibition of inflammatory reactions. The mechanisms and biological significance of MBL involved in T cell function remains to be established.In summary, results above have showed that various concentrations of MBL derived from human plasma suppressed proliferation of stimulated PBMCs and purified T cells in comparison to lectin-stimulated cells alone, including concanavalin A, anti-CD3,anti-C3+anti-CD28.Inhibition was associated with a decreased production of cytokines. Acquire direct clue that MBL can interact with T cell.We also mentioned that MBL may regulate T cell function through direct and indirect mechanism. 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 and protect the body against collateral immune-mediated damage. Further studies evaluating the specific mechanisms as followedare required:the receptors involved in binding and the effect of MBL on signal transduction; the effect of MBL on CD4+T cell and CD8+ T cell function as well as differentiation; indirect roles of MBL on T cells mediated by APC. Preliminary results from MBL regulatory functions are encouraging, and extension of the research would provide further credit to an important physiological role of MBL in regulating T cell function through an yet unknown mechanism, and can be used to better understand and treat MBL deficiencies as well as T cell disorder diseases.