Study On The Relationship Between DC-SIGN Expression Regulation And Anti-Tuberculosis Immunity Response

Objective: To screen suitable experimental subject and research the influence of the dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) expression level regulated by different dose of IL-4 on the immune function of DCs, and to analyse the relationship between DC-SIGN expression level and tuberculosis.Methods:①The mononuclear cells from mouse bone marrow, mouse spleen and human peripheral blood were harvested respectively by density gradient centrifugation, and the suspension cells were removaled. The adherent cells were induced into dendritic cells by granulocyte and macrophage colony-stimulus factor (GM-CSF) and interleukin-4 (IL-4). The morphological features were observed by invert optical microscope and the cell surface molecules (CD11c, DC-SIGN, CD80, CD83, CD86 and MHC-Ⅱ(or HLA-DR)) were identified by flow cytometry,②The expression level of DC-SIGN was regulated by different doses of IL-4 (5, 10, 50, 100, 200 ng/ml), and the phenotypes (CD11c, DC-SIGN, CD86, HLA-DR) of DCs were analyzed by flow cytometry.③The DCs were divided into two groups according to the different dose of IL-4, which were IL-4 (5 ng/ml) group and IL-4 (50 ng/ml) group, and were co-cultured with LPS or H37Rv suspension respectively. The phenotypes (CD11c, DC-SIGN, CD83, CD86, HLA-DR) of DCs were analyzed by flow cytometry, the concentration of IL-12 and IL-10 in the DCs culture supernatant was measured by ELISA, and the ability of DCs to stimulate T cell proliferation was detected by mixed leukocytes reaction (MLR).Results:①DCs from mouse bone marrow, mouse spleen and human peripheral blood displayed a typical DC phenotype in morphological analysis at day 7.②The expression levels of DC-SIGN of DCs from mouse bone marrow and spleen were all <2% at day 1, day 3 and day 7, but 52.86% , 87.70% and 83.37% in the DCs from human peripheral blood at day 1, day 3 and day 7.③The expression levels of cell surface costimulatory molecules of DCs from mouse bone marrow such as CD11c, CD80, CD86, and MHC-Ⅱwere 5.42%, 2.95%, 29.19%, and 52.59%. And those of DCs from mouse spleen were 4.01%, 1.19%, 16.16%, 54.89%. The expression level of cell surface costimulatory molecules of DCs from human peripheral blood such as CD11c, CD83, CD86 and HLA-DR were 93.62%, 90.06%, 97.54%, 99.65%.④IL-4 could regulate the expression level of DC-SIGN. The expression level was raised when the dose of IL-4 was larger and was dose-dependent. When the doses of IL-4 increased from 5 ng/ml to 50 ng/ml, the expression level of DC-SIGN rised obviously (P < 0.05). when the doses increased from 50 ng/ml to 200 ng/m, the expression level also rised. But the difference between each group was not statistically significant (P > 0.05).⑤The maturity degree of DCs had no significant difference, although the expression levels of DC-SIGN were different (P > 0.05).⑥There was no significant difference in the concentration of IL-12 in the DCs culture supernatant of all groups (P > 0.05). When co-cultured with LPS , DCs in different expression level of DC-SIGN secreted similar amount of IL-10 (P > 0.05). But when co-cultured with H37Rv, the DC-SIGNhigh-DCs secreted more IL-10 than the DC-SIGNlow-DCs did (P < 0.05).⑦when co-cultured with LPS, DCs in different expression level of DC-SIGN had the similar abilitiy to stimulate allogenic T cell proliferation (P > 0.05). Compared with DCs co-cultured with LPS, the DC-SIGNhigh-DCs, which co-cultured with H37Rv, had similar ability to stimulate allogenic T cell proliferation (P > 0.05). But when co-cultured with H37Rv, the DC-SIGNlow-DCs had lower ability to stimulate allogenic T cell proliferation than other ones did (P < 0.05).Conclusion:①The DCs from mouse bone marrow and spleen are not suitable for post-experiments which have almost none DC-SIGN expression, low costimulatory molecules expression and are low purity. C57BL/6 mouse is not a suitable animal model to sdudy DC-SIGN. On the contrary, the DCs from human peripheral blood are suitable for post-experiments which highly express DC-SIGN and costimulatory molecules and are high purity.②IL-4 could regulate the expression level of DC-SIGN. The expression level was raised when the dose of IL-4 was larger and was dose-dependent.③when the DC-SIGNhigh-DCs co-cultured with H37Rv, they secreted more IL-10. But when the DC-SIGNlow-DCs co-cultured with H37Rv, they had lower ability to stimulate allogenic T cell proliferation. Expression of DC-SIGN in excessive high or low level is disadvantage to the outcome of the tuberculosis immunity.