| Leptin is a 16-kDa nonglycosylated protein with 167 amino acids encoded by the obese (ob) gene, which is located on human chromosome 7 and on mouse chromosome 6. Leptin is an adipocyte hormone secreted mainly by adipocytes. In the early studies, leptin was proved to contribute to the regulation of the balance between food intake and energy expenditure by acting on hypothalamic nuclei. With unceasing development in this field, it was found that leptin receptors are highly expressed in nervous system although expression at functional levels has also been demonstrated in a number of other tissues including liver, lung, testis, etc. So the relationship between leptin and immune response or autoimmune diseases attracts more and more attention in recent years.Human monocytes/macrophages are very important immune cells which play vital roles both in innate immune response and in acquired immune response. In innate immunity, they recognize invading pathogens with pattern recognition receptors on their surfaces, inducing subsequent inflammations and secreting chemokines to recruit inflammatory cells in an effort to control infection. They also mediate phagocytosis of phathogens via mannose receptor (MR), Fc receptor and C3b receptor. In acquired immunity, monocytes/macrophages, as important antigen presenting cells, can present antigen to lymphocytes to promote their activation and differentiation.THP-1 cells were initially induced to differentiate to macrophages with 12-o-tetradecanoylphorbol 13-acetate (PMA). Based on this model, the effects of leptin on phagocytosis, cell proliferation, bactericidal activity and expressions of costimulatory molecules were studied. The mechanisms underlying those effects were also explored. Bacterial phagocytosis is an important function of monocytes/macrophages. THP-1 cells or THP-1-derived macrophages treated with leptin showed more effective bactericidal effect in the context of engulfing Staphylococcus aureus. The increased bactericidal effect may be associated with the production of TNF-a and reactive oxygen species (ROS) in leptin-treated cells, but has not obvious correlation with the production of nitric oxide (NO).Labeled Candida albicans with fluorescein FITC and flow cytometry were used for the evaluation of phagocytosis. The results showed that leptin-treated THP-1 cells or THP-1-derived macrophages exhibited more effective phagocytosis, which may be not associated with upregulation of MR expression. In addition, it was found that leptin is able to stimulate proliferation of THP-1 cells or THP-1-derived macrophages.We also evaluated the effects of leptin on chemotactic capacity of and expressions of costimulatory molecules in monocytes/macrophages and found that leptin induced IL-18 and MCP-1 production and enhanced the chemoattractant activity of THP-1 cells or THP-1-derived macrophages.Expression of costimulatory molecules on the surface of THP-1-derived macrophages and human peripheral blood monocytes (PBMC) was also evaluated. The results indicated that leptin upregulated expression of those costimulatory molecules with potency varying with different cells. PartⅠEffects of leptin on bactericidal activity of human THP-1 cells or THP-1-derived macrophages and the underlying mechanismsAs the important immune cells in innate immunity, monocytes/macrophages possess strong bactericidal activity and can eliminate many kinds of pathogens. The bactericidal activity can be enhanced by the participation of complement, antibody and cytokines. This function is also used to remove aging or scathing cells in a body to keep homeostasis.Based on the literature reported methods, we began with PMA to induce differentiation of THP-1 cells to macrophages. Induced cells exhibited pseudopods and enlarged cell volume, suggesting the success of differentiation. Staphylococcus aureus was used to evaluate bactericidal activity of the macrophages. The results indicated that (greater than 10ng/ml) leptin-treated cells showed stronger bactericidal activity compared with untreated cells. Considering ROS and NO play a vital role in killing pathogens, we assessed the production of ROS and NO in those cells after treatment with leptin. It was observed that leptin (10,50,100ng/ml) could significantly induce the production of ROS, but not NO. TNF-a is also an important factor in controlling inflammation, so we measured TNF-a level in supernatants of leptin-treated cells and found that leptin-treated cells produced higher level of TNF-a compared with untreated cells. These results indicated that leptin enhanced bactericidal activity through the induction of ROS and TNF-a in THP-1 cells or THP-1-derived macrophages.The purpose of research in this part was to evaluate the effects of leptin on bactericidal activity in THP-1 cells or THP-1-derived macrophages. It was observed that leptin enhanced bactericidal activity and induced higher levels of ROS and TNF-a, but not NO, which may be a part of the underlying mechanism of action. PartⅡ. Effects of leptin on pahgocytosis of human THP-1 cells or THP-1-derived macrophages and the underlying mechanismsPhagocytosis is a vital function of monocytes/macrophages in human immunity. It was attempted to confirm whether the treatment with leptin could alter phagocytosis of THP-1 cells or THP-1-derived macrophages. Labeled Candida albicans with FITC and flow cytometry were used to assess phagocytosis. The results showed that (greater than lOng/ml) leptin-treated cells displayed higher phagocytic rate compared with untreated cells, suggesting an increased phagocytosis in leptin-treated cells.MR is a membrane receptor on monocytes/macrophages and believed to be associated with phagocytosis. MR expression was evaluated in leptin-treated THP-1 cells or THP-1-derived macrophages with real-time PCR and no difference was found between leptin-treated cells and untreated ones.The purpose of research in this part was to evaluate the effects of leptin on phagocytosis of THP-1 cells or THP-1-derived macrophages. It was observed that leptin enhanced phagocytosis but did not upregulate the expression of MR, suggesting other mechanisms involved in the enhanced phagocytosis.Part III. Effects of leptin on chemoattractant activity of human THP-1 cells or THP-1-derived macrophages and the underlying mechanismsChemokines have high importance in the inflammatory response to pathogens. Monocytes/macrophages are able to produce a number of chemokines which attract phagocytes to the inflammatory site and eliminate the pathogens in the end. To confirm the effects of leptin on the chemoattractant activity of monocytes/macrophages, two cell lines, THP-1 and HL-60, were utilized to evaluate the effects of leptin on the chemotaxis to monocyte or granulocyte respectively. The results illustrated that (greater than 10ng/ml) leptin-treated cells exhibited higher chemotactic index compared with untreated cells, suggesting an increased chemoattractant activity in leptin-treated cells. Considering IL-8 and MCP-1 are the major chemokines produced by monocytes/macrophages, we measured IL-8 and MCP-1 levels in supernatants of leptin-treated cells and found that leptin-treated cells produced higher levels of IL-8 and MCP-1 compared with untreated cells.The purpose of research in this part was to evaluate the effects of leptin on chemoattractant activity of THP-1 cells or THP-1-derived macrophages. We observed that leptin enhanced the chemoattractant activity and induced higher levels of IL-8 and MCP-1, suggesting that leptin may enhance chemoattractant activity through the induction of IL-8 and MCP-1 in THP-1 cells or THP-1-derived macrophages.PartⅣ. Effects of leptin on expression of costimulatory molecules in human monocytes/macrophages and the underlying mechanismsAs one of the antigen presenting cells, monocytes/macrophages can present foreign antigens to T cells and trigger acquired immune responses. Moreover, IL-1 produced by monocytes/macrophages is essential to the activation of T-helper cells. The efficiency of presentation is associated with the expression of costimulatory molecules, such as CD86 and HLA-DR. We assessed CD86 and HLA-DR expression on the surface of THP-1 cells or PBMC. Higher expression of those costimulatory molecules was observed in leptin-treated cells compared with untreated cells, suggesting an enhanced ability in antigen presentation.The purpose of research in this part was to evaluate the effects of leptin on the expression of costimulatory molecules in THP-1 cells or PBMC. It was found that leptin enhanced CD86 and HLA-DR expression, suggesting that leptin may promote the presenting ability via upregulation of costimulatory molecules expression in THP-1 cells or PBMC. |
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