| The complement system, which consists of more than thirty proteins, is an important part of innate immune and contributes to many aspects of inflammation and host defense including opsonization of foreign particles, cell lysis, anaphylaxis, chemotaxis, clearance of immune complex and so on. The third component of complement (C3) plays a central role. C3 supports the activation of all the three pathways of complement activation, i.e. the classical, alternative, and lectin pathways. Among the complement proteins, C3 is probably the most versatile and multifunctional molecule and can interact with at least 25 different proteins. In recent years, many researches on the C3 konckout mice (C3-/-) demonstrate that C3 may prime and regulates the acquired immune responses and link innate and acquired immunity. However, the role and the importance of C3 in the cellular immunity or delayed type hypersensitivity (DTH) were still unclear.Mannan-binding lectin (MBL), one of members of the collectin (C-type lectin with a collagen-like domain) family in the superfamily of C-type lectins, is a key component of the innate immune system. On binding to specific carbohydrate structures on the surface of a broad range of microorganisms, MBL activates MBL-associated serine proteases and hence the lectin pathway of complement activation in antibody- and Clq-independent way. Activation of the complement system may lead to killing of the microorganisms by direct lysis effect of membrane attack complex or to phagocytosis of the microorganisms by enhancing the attachment of microbes to the phagocytes. However, It is not clear how important isthe role played by C3 and MBL in the opsonophagocytosis.Therefore, a DTH reaction in the footpad of C3"" mice and C3+/+ mice was induced with ovalbumin (OVA) and the roles of C3 in the reaction were investigated. The effects of C3 and MBL on the phagocytosis of microorganisms by macrophages were also examined by studying the interactions between the microorganisms treated with the sera from C3"'" or C3+/+ mice and/or anti-MBL polyclonal antibody and the macrophage in mice.Objectives:1. To examine the roles of C3 in DTH reaction through inducing the reaction in C3"/"mice and C3+/+mice with OVA.2. To investigate the effects of C3 and MBL on the phagocytosis of microorganisms by mouse macrophages.Methods:1. After a DTH reaction in the footpad of mice was induced with OVA, the thickness of the footpad was measured and compared between C3";" and C3+ + C57BL/6 mice and then, H&E and immunohistochemistry stains were preformed to identify the number and the types of mononuclear cells infiltrated in the footpad tissues. The T lymphocytes were separated from the spleens of mice at day 14 after immunization with OVA and incubated in 96-well plates together with serial dilutions OVA or mitogens and mitomycin C treated macrophages. After three day, the proliferatory reactions were assessed by incorporation of [3H]thymidine.2. The abilities of MBL to bind to several microorganisms were evaluated by a enzyme-linked immunosorbent assay (ELISA). The microorganisms were treated respectively with sera from C3"Aor C3+/+mice and/or anti-MBL polyclonal antibody and used in a phagocytic assay. Macrophages from the peritoneal cavities of C3+/+mice were induced and isolated and added to the system. The changes in the percentage (P%) and the index (PI) of phagocytosis of bacteria by the macrophage were observed and evaluated.Results:1. The footpad thickness of DTH -C3"' mice was significantly lower than that of DTH-C3+/+ mice. After H&E and immunohistochemistry stained, the number of mononuclear cells infiltrated in in the footpad tissue in C3'" mice was notablydecreased, compared with C3+/+ mice, and the type of infiltrated mononuclear cells were mainly CD4+ T lymphocytes. There is no significant difference in the proliferation of splenic T cells stimulated with mitogens between C3"7" mice and C3+/+ mice, but after stimulated with specific antigen, OVA, the proliferation of splenic T cells from C3"'" mice was significantly reduced compared with C3+/+ mice.2. In the binding assay, the binding of MBL to Staphylococcus aureus, group B Streptococcus and Shigella flexneri was strong, that to Escherichia coli, Salmonella typhimurium and pichia yeas was significant and that to type I Streptococcus pneumoniae and type III Streptococcus pneumoniae was weaker. When the microorganisms treated with sera from C3" or C3+/+ mice and/or anti-MBL polyclonal antibody were added to the celiac macrophages of C3+/+ mice in the phagocytosis assay, the P% and PI of the C3"' mice sera-opsonized microorganisms by the celiac macrophages were lower significantly than those of the microorganisms treated with sera from C3+/+mice. After the role of MBL was blocked by anti-MBL polyclonal antibody, the phagocytosis of the microorganisms treated with sera from both Cy' and C3+/+ mice reduced, which were related to the bingding capacities of MBL with the microorganisms, i.e. the phagocytosis of the microorganisms binding strongly MBL by the celiac macrophages reduced much than that of the microorganisms with moderate or low MBL-binding capacities.Conclution:1. The absence of C3 resulted in impaired DTH responses, infiltration of mononuclear cells and proliferation responses of T lymphocytes to specific antigen in vitro, which suggested that C3 plays an important role in the DTH reaction.2. C3 deficiency and blocking the effect of MBL with anti-MBL polyclonal antibodies could inhibit the phagocytosis of microorganisms by magrophages, in which C3 deficiency had greater effects, and the effects of MBL on phagocytosis were associated with its different binding capacities for different microorganisms. These data showed that C3 and MBL are contributors to the opsonophagocytosis, but C3 plays a major role in the opsonophagocytosis.
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