Study Of Immune Enhancement And Antitumor Activities Of Escherichia Coli Maltose-binding Protein

Maltose binding protein (MBP) is a component of the maltose transport system of Escherichia coli. which is responsible for the uptake and catabolism of maltodextrins. Fusion proteins are fused to MBP to improve their yield, facilitate their purification, so it is widely used in the purification of fusion proteins. It is commonly believed that MBP has minimal effects on the bioactivity. so it often used as biological activity of the fusion protein for study. However. more recently. MBP is utilized as a chaperone component in subunit vaccines against pathogenic bacteria. Fusion protein-MBP vaccines can enhance the immunogenicity of the fusion protein in mouse models against pathogens. MBP itself can induce dendritic cell activation and produce proinflammatory cytokines through Toll-like receptor 4. In previous study. we found that MUC1 fusion with MBP can enhance the immunogenicity and induce the immune response. but a study showed that MUC1 fusion with GST can not induce an effective immune response, suggesting that MBP fusion protein as part of the structure, involved in MUC1-specific antitumor activity. We also found that the combined immunization with MBP and Bacillus Calmette-Guerin (BCG) induces antitumor activity in a mouse model of human breast cancer. MBP can promote lymphocyte proliferation and directly induce Th1 cell activation. These studies suggest that MBP has immune enhancement activity and combined with BCG has antitumor activity. Therefore, to further investigate immune enhancement activity of MBP and antitumor mechanism of MBP combined with BCG. We mainly study that MBP alone or combined with BCG induced macrophages and T cells activation in vitro and in vivo. This study was the following aspects:1. Preparation of MBPThe pMAL plasmid was transfected into E. coli DH5α. The MBP was expressed upon induction with IPTG. The MBP was purified by Amylose resin affinity chromatography. LPS was removed by DeToxi-Gel.2. MBP activated mouse peritoneal macrophagesMouse peritoneal macrophages were stimulated with MBP in vitro. The results showed that MBP enhanced the secretion of NO. IL-1βand IL-6 in the dose-dependent and time-dependent manner. MBP increases phagocytosis of mouse peritoneal macrophages. To further observe the activation of macrophages types. The expressions of iNOS and Arg-1 mRNA in mouse peritoneal macrophages were observed by RT-PCR (High expression iNOS mRNA representative of the M1-type macrophages, high expression Arg-1 mRNA representative of the M2-type macrophages). The results showed that iNOS mRNA was highly expressed, but Arg-1 mRNA was not obviously expressed. So MBP induced M1 macrophages. In addition. the mouse was injected intraperitoneally with MBP for stimulating peritoneal macrophages. We found that, when peritoneal macrophages were stimulated without LPS in vitro, the secretion of NO and IL-1βwas low, statistical comparison cannot be done. When peritoneal macrophages were stimulated with LPS in vitro. compared with PBS. MBP enhanced the secretion of NO and IL-1βin the dose-dependent manner. suggesting that MBP increased the sensitivity of macrophage.3. MBP induced Thl cell activationThe splenic mononuclear cells were stimulated with MBP in vitro. The results showed that MBP induced lymphocyte proliferation in the dose-dependent manner. The levels of IFN-y, IL-2 and IL-4 in the cell culture supernatants were detected by ELISA. MBP significantly increased the levels of IFN-y and IL-2, but IL-4 levels were low (< 20 pg/ml). These results indicate that MBP induced Thl activation.4. Immunization and groupMice were randomly divided into the following four groups (five mice per group):PBS group. BCG group. MBP group, and MBP+BCG group. Mice were subcutaneously immunized with PBS. BCG (3 mg/mouse). MBP (50μg/mouse) or a combination of MBP (50μg/mouse) and BCG (3 mg/mouse) two times with a one-week interval. Three days after the last immunization. mice were killed. The spleen from each mouse was harvested and a mononuclear cell suspension was prepared.5. MBP alone or combined with BCG induced lymphocyte proliferationLymphocyte proliferation was assessed using a MTS assay. The results showed that the lymphocyte stimulating index (SI) of the BCG. MBP and MBP+BCG groups was higher than that of the control group, and the SI of MBP+BCG group was the highest. These results suggest that BCG or MBP alone induced lymphocyte proliferation, and that the combination of MBP and BCG significantly induces lymphocyte proliferation.6. MBP alone or combined with BCG induced Thl cell activationThe levels of IFN-y. IL-2 and IL-4 in the cell culture supernatants were detected by ELISA. The results showed that The BCG group and MBP+BCG group showed high levels of IFN-y compared with the PBS group. Furthermore. the levels of the MBP+BCG group were significantly higher than those of the BCG group. but IFN- secretion was not detectable in the MBP group. High levels of IL-2 secretion were found in the BCG, MBP and MBP+BCG groups. However, IL-4 levels were low for all groups (< 20 pg/ml). These results indicate that BCG alone induced Thl activation. and that the combination of MBP and BCG enhanced BCG-induced Thl activation. To further analyze whether MBP induces Thl activation, splenic mononuclear cells were incubated with MBP in vitro for 24 h. and IFN-γsecretion was assessed by sensitive ELISPOT. We found that MBP nonspecifically activates Thl cells and obviously enhances the activities of BCG-induced Thl cells.7. MBP alone or combined with BCG increased splenic CD4+ and CD8+ T cell subsetsCD4+ and CD8+T cell subsets were analyzed by flow cytometry. We found that the number of CD4+ T and CD8+ T cells in MBP group. BCG group and MBP+BCG group increased significantly compared with the PBS group.8. The combination of MBP and BCG increased the percentage of activated splenic macrophagesCD14 and CD68 are macrophage cell surface markers, and CD86 is highly expressed on the surface of activated macrophages. The expression of CD14. CD68 or CD86 was analyzed using flow cytometry. The results showed that the expression of CD 14 in the BCG. MBP and MBP+BCG groups was significantly up-regulated compared with the PBS group. The expression of CD86 in the MBP+BCG group was significantly increased compared with the PBS group or single treatment groups. but all groups did not show a significant increase in the expression of CD68. These data indicate that the combination of MBP and BCG increased the percentage of activate(?) macrophages in the spleen. 9. MBP alone or combined with BCG activated mouse peritoneal macrophagesTheir pinocytic activity was examined with a neutral red assay. We found that the BCG or MBP group alone increased phagocytic activity compared with the PBS group. The MBP+BCG group significantly increased phagocytic activity compared with the PBS group or single treatment groups. These data indicate that MBP or BCG alone activated peritoneal macrophages. and that combination of MBP and BCG induced a synergistic effect on macrophage activity.10. MBP alone or combined with BCG induced NK cell cytotoxicityThe splenic NK cell activity was detected using a LDH-release assay. YAC-1 cells were used as target cells and the splenic mononuclear cells were used as effector cells. NK cell activity of the BCG or MBP group alone was higher than that of the PBS group at an E/T ratio of 100:1. NK cell activity in the MBP+BCG group was significantly higher than that of the PBS group or single treatment groups at E/T ratios of 100:1 and 50:1. These results suggest that MBP or BCG alone induced NK cell cytotoxicity, and that the combination of MBP and BCG had a synergistic effect on NK cell cytotoxicity.11. The combination of MBP and BCG induced antitumor activityA mouse Lewis lung carcinoma model was established. For prophylactic treatment, mice were immunized two times with a one-week interval and inoculated with LLC1 cells on day 3 after the last immunization. The results showed that tumor volumes were not significantly different between the BCG or MBP group alone and the PBS group:whereas the MBP+BCG group had a smaller tumor volume compared with the PBS. For therapeutic treatment. mice were inoculated with LLC1 cells. then mice were immunized two times with a one-week interval. Similar results were also observed in the therapeutic experiment. In addition, a mouse B16 model was established, Similar results were also observed. These findings indicate that the combination of MBP and BCG, but not BCG or MBP alone, can inhibit tumor growth.In conclusion. MBP can activate mouse peritoneal macrophages in vitro. And MBP induced M1 macrophages. MBP induced Thl cell activation. In addition, MBP alone induced lymphocyte proliferation. enhanced the activities of Thl cells, increased the number of CD4+ and CD8+ T cells. activated splenic and peritoneal macrophage. and activated NK cells in vivo. and that the combination of MBP and BCG significantly enhanced these immune-stimulating activities. In addition. we used Lewis lung carcinoma and B16 melanoma tumor model. we found that MBP can not inhibit tumor growth. when the combination of MBP and BCG. which can significantly inhibit tumor growth.