| The innate immune system is the first line of defense against pathogenic microbial infection, it is primarily consists of effector molecules and cells which involved in innate immune. The common effector molecules including defensins, the complement system, cytokines and lysozyme;effector cells are phagocytic cells, NK cells and so on. The complexity of the network structure composed by a variety of recognition and effector molecules of innate immune recognition mechanism, through pattern recognition receptors (pattern recognition receptor, PRR) such as mannan-binding lectin (mannan-binding lectin, MBL), peptide poly carbohydrate recognition protein, Toll-like receptors (Toll-like receptor, TLR) recognize pathogen-associated molecular patterns (pathogen associated molecular patterns, PAMPs) recognition of pathogenic microorganisms and play its role, to participate in immune regulation.MBL is one of the plasma protein which synthesized by the liver, it is a key protein of the lectin pathway in the natural immunity of the complement system, and it is a secreted PRR, belong to the family of collectins (C-type lectins with a collagen-like domain). MBL selectively recognizes a variety of the pathogen surface of sugar structure, it can trigger immediate cellular lysis or phagocytosis by initiating the lectin pathway without antibody or C1q activate complement, or promote opsonophagocytosis by direct binding to cell surface receptors without the involvement of complement, and it can clear the immune complexes and apoptotic cells. MBL levels in the normal human plasma between10-5000pg/mL, as an acute phase response protein, stress (e.g., pathogen infection, surgery, etc.), the plasma concentration can be increased to2to3times. It has been found that, other members of the collectins family, such as lung surfactant proteins A and D (surfactant Protein A, the surfactant pool protein D, SP-A, SP-D) and complement Clq, have roles in immune regulation by interacting with various cells. For example, SP-A inhibits the differentiation and maturation of dendritic cells (DCs); Fraser et al reported that MBL can inhibited monocyte macrophages cytokines productions induced by LPS(Lipopolysaccharide, LPS), such as TNF-a.reported that MBL in low concentration (<4μg/ml) can promote human monocyte-derived macrophages secretion of IL-6, TNF-a and IL-lb, while high concentration is inhibiting its secretion. Few studies of neutrophil (Polymorphonuclear leukocyte, PMN), it is necessary to explore the impact of MBL on neutrophil function.In this study, we take advantage of human neutrophil as cell model, to investigate the effects of MBL in human polymorphonuclear cells.Part I MBL extraction and purification of MBL from human plasmaMBL is a plasma protein, it is secreted by liver cells. MBL is one of the key soluble pattern recognition receptors(PRR) of the innate immune system. The concentration of MBL in normal human plasma is low, and it is levels between10-5000μg/ml, and average about900μg/ml. Based on long-term basis for the work of our Lab, we has established a method for the separation and purification of human natural MBL. It has been reported that pyrogen and plasma IgG might interfere with the experiments. We used mannan-Sepharose4B affinity chromatography system to separated MBL. The extract MBL after the LC column and polymyxin B gel column to the removal of IgG and LPS. The purified MBL purity and existing forms were detected by SDS-PAGE and Western blot. Ligand binding activity of the experimental identification of MBL biological activity. For subsequent experiments carried out to provide a stable and credible MBL. The results show that IgG and LPS purified MBL be significantly removed after the LC column and polymyxin B gel columns; Both SDS-PAGE and Western blot analysis showed that the purified MBL mainly in the form of a high polymer (MW>200KDa) exist, after ligand binding experiments show that the purified MBL maintained biological activity. All of these to provide a reliable guarantee for subsequent experiments.Part Ⅱ The effect of MBL on the function of neutrophil with MBL deficient human serumPolymorphonuclear neutrophils (PMN) as important phagocytic cells in the natural Immune system, and plays an important role in the pathogenic microorganism infection. In the role of inflammation in the local pro-inflammatory cytokines and chemokines, PMN can quickly migrate to sites of inflammation, and through the release of inflammatory cytokines, reactive oxygen species and degranulation play the anti-inflammatory effects. Neutrophils are the largest number of phagocytic cells in human body, and play an extremely important role in the innate immune defense. But in the case of immune disorders, neutrophils may exacerbate the inflammatory response allowing the body to produce serious damage, and therefore it is particularly important for the regulation of neutrophil function. At the same time, the MBL is also important effector molecules of the innate immune system. Therefore, We studied the effect of MBL on the function of human neutrophils (PMN). Pathogens binging with MBL and the levels of TNF-α and IL-6in culture supernatants were detected by ELISA; The levels of IL-1β, TNF-α and CD1lb mRNA expressions in PMN were determined by qPCR; Superoxide production was estimated by nitro-blue tetrazolium(NBT) reduction assay. ELISA and Dot-blot results showed that MBL bound to the microorganisms in a dose-dependent manner; MBL had no significant effect on phagocytosis of C. albicans and E.coli by PMNs in the absence of human serum. But in presence of mixed MBL-deficient human sera, MBL promoted the phagocytosis of C. albicans, which could be blocked by mannan; qPCR analysis indicated that MBL can improve neutrophil IL-1β, TNF-α and CD11b mRNA expressions; ELISA results show that MBL can improve neutrophil TNF-α and IL-6production; The NBT results show that MBL can increase Superoxide production by PMN compared with control. These results suggest that the MBL promote secretion of inflammatory cytokines, complement-dependent enhanced neutrophil phagocytosis.Part Ⅲ The effect of MBL on the function of neutrophil without the involvement of complementThe CRD of MBL recognizes carbohydrates on the surface of microbial pathogen, and via CLR binding to the collectin receptor of phagocytes cells, MBL can mediate direct opsonization and MBL-dependent, cell-mediated cytotoxicity without the involvement of complement. Therefore, we studied the effect of MBL on the function of neutrophil without the involvement of complement. The content of TNF-α and IL-6in culture supernatants were detected by ELISA; The levels of IL-6、IL-1β、 TNF-α and apoptosis-associated gene mRNA expressions in PMN were determined by qPCR; Superoxide production was estimated by nitro-blue tetrazolium(NBT) reduction assay; Apoptosis of the PMN was analyzed by staining hochest. ELISA results showed that the low dose of MBL (2.5μg/ml) can significantly promote TNF-α and IL-6secreted by PMN induce by C.albicans, and higher doses of MBL (20μg/ml) compared low-doseMBL (2.5μg/ml), TNF-a secretion by a significant inhibition, while inhibition can be blocked by Mannan; qPCR detection of TNF-a and IL-6mRNA similar results with the ELISA results; ELISA and qPCR results show a high concentration of MBL to inhibit TNF-α secreted by LPS-stimulated PMN; Hochest test results showPMN nuclear and cytoplasmic staining highlights the high concentration of MBL treatment significantly increased compared blank group; qPCR showed that high concentrations of MBL can upregulate the expression of Caspase-3and Fas gene. These results suggest that MBL may have a dual immune function, suggesting MBL regulation of the body’s inflammatory response plays an important role.In summary, we use the purified natural human MBL to invesrigate the effects mannan-bingding lectin(MBL) in human polymorphonuclear cells. Our results suggest that MBL may be affected by the state of the effector cells and the surrounding environment affect vary.MBL role in pathogen-free conditions, the role of proinflammatory may depend on some components of he serum; With Candida albicans or LPS, MBL has a dual effect, low-dose proinflammatory, high doses suppress the expression of inflammatory factors. |
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