Study On Opsonic Activity Of Fish Vitellogenin

Bony fish have a immune system similar to that of higher vertebrates including humans. However, as lower vertebrates, their specific immune mechanism is not perfect, but their non-specific or immate immunity is well-developed. Among innate immunity of fish phagocytes including macrophages play a central role in anti-infection immunity. Some molexcules which can bind antigen nad promote the macrophage phagocytosis is called opsonin, and the process by which the opsonin enhances phagocytosis is named opsonization. The major opsonins found in serum so far are immunoglobulin G (IgG) antibodies and complement fragments C3b. Recently Li et al. (2008; 2009) have demonstrated that fish vitellogenin (Vitellogenin, Vg) also has an opsonin function involved in immune response. Vg from fish H. otakii can act as a multivalent pattern recognition receptor binding to several PAMPs, while with the specific binding of the surface of macrophages, namely, a bridge role between pathogen molecules and macrophages. However, the mechanism of action by which Vg is involved in opsonic phagocytosis remains unknown. The main objective of this study is to demonstrate the underlying mechanism style by which Vg acts as opsonin.First, sea bass(Lateolabrax japonicus) head kidney macrophages were isolated by discontinuous gradient centrigugation. to use Percoll density gradient centrifugation and their phagocytic function were identified by transmission electron microscope. Then phagocytosis assays were performed and two parameters phagocytic ability (PA) and phagocytic index (PI) were used to test the effects of Vg on phagocytosis to draw a kinetic curve macrophages ingesting yeast particle in different time. Then incubating sea bass head kidney macrophages with Hexagrammos otakii Vg coated yeast, Compared with the yeasts pre-incubated with BSA and PBS alone, those pre-incubated with Vg were readily phagocytosed by the macrophages. The PA and PI values of the macrophages phagocytosing Vg-treated yeast were significantly higher than those of cells phagocytosing BSA-treated and untreated microbes (p<0.05). These denoted that H. otakii Vg was able to promote the sea bass macrophage phagocytosis and the opsonin function of Vg does not have a species specific. Next, the opsonin mechanism of Vg is studied. This experiment confirmed that Vg opsonic function can be affected by lavendustin (protein tyrosine kinase PTK specific inhibitor), PP1 (protein tyrosine kinase PTK specific inhibitor), staurosporine (protein kinase C PKC inhibitor); but not by nocodazole (microtubule inhibitor), C3 exoenzyme (Rho GTPases inhibitor). Therefore, we speculated that the opsonin function of Vg may be type-I mechanism. As type I mechanism functions by cell surface receptors FcyRs, stimulating phagocytosis signal transduction pathway. The experiments further verify the FcyRs are involved in the Vg opsonic process. Fluorescein isothiocyanate (FITC)-labeled FcyRs monoclonal antibodys, respectively FcyRI, FcγRⅡand FcγRⅢ, were incubated with macrophages. It is found that FcyRs monoclonal antibodys is able to bind sea bass macrophages, indicating that the surface of macrophages has receptors FcγRⅠ, FcγRⅡand FcγRⅢ. Then these three kinds of monoclonal antibody were used to block the macrophage cell surface receptor, through phagocytosis assays it is found that compared with the control, the blocked receptors can inhibit macrophages ingest Vg coated yeast to varying degrees, indicating FcγRⅠ, FcγRⅡand FcγRⅢare involved in Vg opsonization. In addition, the thesis of the protein tyrosine kinase Sky, and PI3-kinase is involved in Vg opsonization process were studied. The sea bass macrophages were treated with protein tyrosine kinase Sky and PI3-kinase specific inhibitors, respectively piceatannol and wortmannin, after opsonic phagocytosis assays realtime PCR was done to detect inflammatory cytokines TNF-a and IL-1βgene expression changes. It is denoted that compared with the control treatment group, TNF-a and IL-1βexpression of the inhibitor treatment groups were significantly decreased (p<0.05). These results indicate that the protein tyrosine kinase Sky and PI3-kinase may be involved in the process of Vg opsonization signal transduction, affecting production of inflammatory cytokines.In summary, the present study by in vitro experiments demonstrates for the first time that opsonin function of fish Vg is consistent with the type I opsonization. As the type I opsonic phagocytosis is mediated by IgG and CRP(C-reactive protein) can also play as type I phagocytosis opsonin. So it also bolsters the notion that Vg and CRP both function like antibody.