| Objective: To obtain immature dendritic cells cultured with IL-10, assay the cell phenotypes of immature dendritic cells ,and investigate the tolerogeneic properties of immature dendritic cells treated by IL-10 in vitro.Methods: Fresh bone marrow cells (BMC) harvested from rat femurs and tibias were treated with hypotonic buffered tris-ammonium chloride to depleted of red blood cells . BMC containing dendritic cell ( DC ) progenitors were depleted of Fc+ and plastic-adherent cells panning on normal serum-coated dishes. The resulting cells were then cultured in gelatin coated cultrure dishes, using complete culture medium containing granulocyte-macrophage colony-stimulating factor (10ng/ml) andInterleukin-4 ( 8ng/ml ). On the fifth day of cell culture, two groups were classified as IL-10 group (with IL-10 10ng/ml added to the culture medium) and IL-4 group (without IL-10). DCs were harvested on the 13th day. Cell morphology was observed daily by light microscopy . Cell surface molecules including OX6, OX62, CD80 and CD86, were assayed by flow cytometery. The ability of DC capturing antigens was measured by FITC-Dextran endocytosis. Allostimulatory capacity of DC and proliferation of T cells was assayed by 3H-thymidin deoxyribose insertion. Anti-dornor cytotoxic T lymphocyte activity was assayed by 51Cr release test. IL-2 and IL-12 level in culture supernatant were measured by ELISA kit. Results :1. Morphologic features and phenotype of harvested dendritic cells :Compared with mature DC, IL-10-treated DC was smaller and less of obvious sheet like processes and veils. Cells expressing OX62 in IL-10 group were almost the same as IL-4 group ( 79.6% vs 82.3%, P>0.05 ), which indicats the high purity of DC was harvested in both groups . Expression of MHC-II molecule ( OX6 ) in IL-10 treated DC was lower than in mature DC (32.3% vs 61%, P<0.01 ). Expressions of costimulatory molecules , such as CD80 and CD86 in IL-10 treated DC were significantly lower than mature DC (25.3% vs 60.2%, 42.4% vs 72.4% , P<0.01 ) .2. IL-10-treated DC exhibited more powerful ability of endocytosis than mature DC (81.9% vs 36.1%, P<0.01 ). 3. Primary MLR showed that IL-10 treated DC had weaker activity of stimulating proliferation of allogeneic T cells than mature DC at different concentration of stimulating cells (58.4 vs 19.2, 52.1 vs 16.7, 47.3 vs 14.6, P<0.01 ). These immature DC were poorer inducers of allogeneic T cell activation. The T lymphocyte stimulated with immature DC in a primary MLR displayed weak proliferative response in second MLR (9.5 vs 41.2, 8.6 vs 36.7, 7.6 vs 25.6, 5.4 vs 23.8, P<0.01 ) to dornor cells, but normal proliferative response to third party cells. It suggests that hyporesponsiveness of T cells proliferationn was donor-specific. 4. T lymphocyte stimulated by immature DC had decreased cytotoxicity to cells from donor rat ( 5.5% vs 38.4, P<0.01), but showed normal cytotoxicity to cell from the third party rat (12.8%). 5. Immature DC secreted lower level of IL-12 compared with mature DC after seven days culture. IL-2 level in primay MLR supernatant were lower when stimualted with immature DC (1356 vs 245 pg/ml, P<0.01).Conclusion: IL-10 can inhibit maturation of bone marrow-derived dendritc cells, and immature dendritic cells can induce allogeneic-specific immune tolerance in vitro.
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