Studies On The Mechanism Of IgY-LPS Against Lps Injuries

The barrier function of intestinal tract always descents at different level after burned, including mechanical barrier, biological barrier and immunological barrier. Especially for the sever burn patients,there are some erosion or ulcer on the intestinal mucosa and dysbacteriosis. The level of secretory IgA (sIgA) decents after the immune system function decreases. The endotoxin of the intestine often translocates from the intestine to blood circulation. Additionally, the bacterial on the wound could release LPS into the blood, too. They are the important reasons that systemic infection and multiple organ failure. Currently, some strategies employed to treat injuries caused by LPS are amphotericin B, hemodialysis and specific antibodies. However, none have been effective. Anti-bacterial agents maybe increase the releasing of LPS from bacterial, antibody therapy using monoclonal antibodies against LPS or inflammation mediators were not effective in clinical experiments and failed to decrease the mortality of patients. These unsatisfactory results of monoclonal antibodies may be due to the treatment containing only a single ingredient and no other possibly effective components. Therefore, the use of polyclonal antibodies is being considered as a possible approach.In recent years, it was found that there were abundant IgG sourcing from serum in egg yolk and was named egg yolk antibody. Egg yolk antibody is a type of polyclonal antibody that comes from egg yolks of immunized hens. It can effectively attenuate the biological effects of antigens, such as venoms, and pathogenic microorganisms, by neutralizing the antigens. More interestingly, IgY could recognize more epitopes than mammalian IgG due to the phylogenetic difference. Our previous studies have shown the good effects of IgY against LPS. When orally administrated IgY against LPS to burned mice combined with LPS injuries, the mortality of mice greatly decreased and the level of TNF-a, IL-6 and IL-1βdecreased significantly, too. Some other studies showed that specific IgY against E.coli could protect the mice injuried by intraperitoneal injection of E.coli. when co-incubated IgY against Staphylococcus aureus, Staphylococcus aureus and neutrophil, the phagocytosis of neutrophil to dead Staphylococcus aureus was increased with dependence to IgY concentration. In this study, We hope to obtain IgY against LPS with high purity, and discover some mechanisms of IgY against LPS injuries.Our study showed that using the water solution method followed by salt precipitation and gel chromatographic filtration, pure specific IgY against LPS with high titer could be obtained from the egg yolk of hens immunized with LPS. After incubating LPS with specific IgY, the survival rates of mice which peritoneally injected LPS increased significantly and the injuries were improved greatly. In vitro, specific IgY against LPS could greatly enhance the phagocytizing ability of PMA induced THP-1 cells, and non-immunized IgY had no effect. When intraperitoneally injected specific IgY to mice every day, the ability of peritoneal macrophages to phagocytize fluorescence beads was increased remarkably compare to non-immunized IgY and PBS. For the assays to determine the effect of specific IgY against LPS initiated releasing of TNF-a by PMA induced THP-1 cells, specific IgY could inhibit the releasing of TNF-a significantly. Additionally, when added specific IgY into the reaction system of LPS stimulating THP-1 cells, the TNF-a levels of the system were lower than the reaction system which had no specific IgY added, even the specific IgY was added into the reaction system after one hour later the LPS stimulation. But when the CD14 and TLR4 expressed by PMA induced THP-1 cells were detected by FCS, no positive results were shown, the results meaned that specific IgY could not effect significant changes in expression of CD14 and TLR4 on surface of PMA induced THP-1 cells. However, specific IgY could inhibit the binding between FITC-LPS and PMA induced THP-1 cells. Maybe this was the one reason of specific IgY inhibiting the TNF-a releasing by PMA induced THP-1 cells which stimulated by LPS, for specific IgY prevented the binding of LPS to THP-1 cells.According to the results from this study and our previous works, we could found that specific IgY against LPS had good effects to prevent the LPS injuries. The potential mechanisms maybe include the following three possible ways at least: specific IgY could enhance the phagocytosis of macrophages to clear the causative agent; specific IgY could prevent the binding between LPS and target cells, thereby inhibited the release of inflammation mediators, for example, TNF-a and softened the inflammatory reaction and injuries; specific IgY could affect inflammatory cells directly by some possible ways in secreting inflammatory mediators, although it could not effect significant changes in expression of CD14 and TLR4 on surface of inflammatory cells.