| The complement system, a key and conserved system of innate immunity, provides a rapid and efficient means of deleting invading microorganisms. Pathogenic microorganism and inflammation mediators can promote complement activation and assembly membrane attack complex (MAC), C5b-9. C5b-9 is a multieffect immune factor. It can lytic target cells through inserting cell membrane when bulk activation of complement system. Recently, this view was challenged because of the interaction between membrane attack complex and nucleated cell. Previous investigations indicated that nucleated cells could clear membrane attack complex deposited on their membrane through vesiculation and endocytosis, in that way they could protect themselves from being attacked by complement. We call this membrane attack complex (MAC) sublytic membrane attack complex (sMAC). Recently, the new researches indicated sublytic membrane attack complex could stimulate and activate nucleated cells as a pro-inflammatory mediator in addition to inducing cell death. Sublytic membrane attack complex can stimulate nucleated cells to secrete inflammatory mediators and cytokines and induce the expression of adhesion molecules and some functional proteins. In addition to direct killing, activation of complement system results in generation of numerous split products, which bind to complement receptors on various cells of the immune system, thereby modulating inflammation and mounting an immune response. Thus, the complement system acts as a link between innate and acquired immunity.Dendritic cells (DCs) are characterized by their ability of efficiently presenting antigens, and are unique to stimulate na?ve T cell response. Immature dendritic cells acquire antigens in peripheral tissues, and then migrate to T-lymphocyte dependent areas of lymph nodes. Moreover, there are some interactions between dendritic cells and B lymphocytes and NK cells. Thus, dendritic cells act as a bridge between innate and acquired immunity. Dendritic cells play a critical role in immune response. Dendritic cells exist in heterogeneous groups, which include several subsets with different roles in the regulation of immune response and maintenance of immune homeostasis. The ability of dendritic cells to activate T cells depends on their maturation, which can be induced by endogenous stimulators such as inflammatory cytokines, exogenous factors including viral products or bacterial components. Therefore, it is very important to identify factors affecting the process of DC maturation. In view of the common property of dendritic cells and complement, namely their ability of link innate and acquired immunity, but there are no reports about dendritic cells and complement interaction.Blood dendritic cells are found to carry viable terminal complement complex (TCC) on their surface, even after several days of culture. It remains to be examined if this is due to an increased resistance or recovery from complement membrane attack and if elimination or shedding of TCC complexes occurs together with ongoing complement activation. Since membrane-bound but non-lytic TCC has been shown to exhibit manifold biological functions, its presence on the surface may play a role in the differentiation or function of the carrying cells. Therefore, we propose that complement may regulate the differentiation, maturation and immunological function of DC, and then regulate immune response. In order to confirm this hypothesis, we decided to study the interaction between the sublytic membrane attack complex C5b-9 and dendritic cells. We hope that our study will provide us with more comprehensive knowledge of the mechanisms of immune regulation and the roles that dendritic cell and complement play in innate and acquired immunity, as well as to establish a foundation for further exploration of the roles dendritic cell plays in antigen-specific immune response and immune tolerance with a new perspective.We focused on the following aspects:Part one, inducing monocytes from peripheral blood of healthy volunteers into immature dendritic cell (imDC). Human peripheral blood monouclear (PBMC) cells were isolated from the buffy coats of 38 healthy volunteers using Ficoll Paque density gradient centrifugation. Hunman monocytes isolate from PBMC by adherence to plastic for 2 h. ImDC were induced from monocytes by incubating in standard medium supplemented with 50ng/mL GM-CSF and 20ng/mL IL-4 for 5 days. On the 5 days, observe the cell morphous using invert microscope and analyze the surface markers by flow cytometry. Data showed that expression of CD1a was upregulation while the CD14 was down. This indicated we successfully get the imDC.Part two, establishing sublytic model of dendritic cells in vivo. On day 5, imDCs were washed three times with serum-free medium and then assembled C5b-9 on their suface. The formation of C5b-9 on the membrane of imDCs was verified by laser scanning confocal microscopy (LSCM), membrane integrity was determined by measuring the release of LDH, and cell vigour was analyzed by flow cytometry. Data indicated C5b-9 assembled on the membrane and cell vigour was not affected.Part three, analyzing the effection of sublytic C5b-9 on dendritic cells. ImDC was stimulated with sublytic C5b-9 for three days, and then differentiation and maturation marker, costimulatory molecules, HLA relative antigen and the capacity of antigen uptake were analyzed by flow cytometry. Cytokine secretion was detected using ELISA. T cell stimulating activity was analyzed by allo-MLR. Compared with the control, some muturation markers of C5b-9 treated DC were significant up-regulated. Cytokine secretion was increased while antigen uptake activity and relative molecules were decreased. The capability of promoting CD4~+T cells activation and proliferation was increased, at the same time IL-2 and IFN-γsecretion of CD4~+T cells was promoted. CD4~+CD45RA~+T cells cocultured with sublytic C5b-9 treated DC secrete more Th1 and less Th2 cytokine than cocultured with untreated DC. However, CD8~+T cells stimulated by C5b-9 treated DC have not affected.In conclusion, our study revealed that monocytes can differentiate into imDC in vitro with rhGM-CSF combination rhIL-4 and then to maturation stimulated with LPS. ImDC stimulated with sublytic C5b-9 undergo maturation, downregulate the capacity of antigen uptake, increase cytokine secretion and antigen presention, and induce Th1 polarization. Therefore, in spite of its identity as a component of innate immune system, the complement system also plays central roles in the regulation of dendritic cells which shuttle between the innate and acquired immune system.
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