| The interaction between splenic stromal cell and dendritic cell (DC) was observed in our study on the basis of the establishment of and epithelial-like splenic stromal cell line. The differentiation of DC from hematopoietic progenitors and regulation of DC phenotype, growth and antigen presentation were investigated in the stable stroma model in vitro.As a specialized antigen presenting cell, DC is commonly regarded as an end stage cell type differentiated from CD34+ progenitors or monocytes. After loading and delivering antigen messages to the spleen or lymph node and presenting them to T cells, DC died, and the new one is born again in the bone marrow. This is the fate of DC speculated by nearly all the researchers. Indeed, some researchers have found that DC undergo activation induced apoptosis after IFN-ct or MHC class II stimulation in vitro. We also found there is a growth arrest and followed cell death on DCs cultured in vitro at 10-15 days in the presence of GM-CSF and IL-4. Immune system is a complex adaptive system in vivo, immune cells such as T cells, B cells and DC interact with and affect each other and compose a dynamic network. Besides the interaction between immune cells, the influence of immune microenvironment must be considered if the fate of DC is speculated. The fact is that nearly all the immune responses occur in the secondary lymph organs under the influence of immune microenvironment.Immune microenvironment of secondary lymph organs commonly composed of stromal cells and extracellular matrix molecules, provides a homostatic microenvironment to the immune response and influences the fate of immune cells. Some researchers have done some work on the splenic stromal cells and the development of DC in vitro using originally cultured splenic stromal cells.For further investigation,we established an epithelial-like splenic stromalcell line of C57BL/6 mouse origin named MSSC-1 and a steady stroma/DC interaction model and then the influence of the stroma on the differentiation and functional regulation of DC would be well evaluated.Our experiments show that this stroma layer can support the growth and proliferation of c-kit+ bone marrow progenitors and directly differentiation to DC-like cells. Surprising to us, mature DC (purified using CD lie magnetic beads) seeded on the monolayer of the MSSC-1 stromal cell re-proliferate and form colonies, as described in the following. Bone marrow mononeuclear cells , prepared from the mouse femurs by depletion of red cells, were cultured in the presence of 10ng/ml recombinant murine GM-CSF and Ing /ml murine IL-4. The nonadherent cells were gently washed off after 3 days' culture, the remained clusters were cultured for 4 days; on day 8, CDllc+ mature DC were positively selected using CDllc magnetic microbeads (Mylteni ). These mature DCs express high levels of MHC class II, CD80, CD86, CD40 molecules and have poor ability of OVA-FITC phagocytosis. Then the MSSC-1 cells were dispersed and seeded in 24-well culture plate at a concentration of 5xl03/lml per well and cultured in RPMI1640 complete medium containing 10% fetal calf serum at 37C in fully-humidified atmosphere with 5% CO2 in air. The selected CDllc+ mature DCs were seeded on the monolayer at a concentration of Ixl05/lml per well when the stromal cells reached 50% confluence. The culture medium was replaced by RPMI1640 supplemented with 5% FBS to surpress the growth of stromal cells. With fresh medium supplement every 4 days, the mixed cells can grow for at least 3 weeks with no necessary of passaging. Separate the proliferative DCs separated from the stroma using 5mM EDTA , and DNA contents were determined by propidium iodide staining and subsequent FACS analysis. The phenotype of DC and its phagocytosis were also determined by FACS. Data show that the induced proliferative DCs are of higher level of S and G2/M phase, with down-regulated expression of MHC class II and CD86, up-regulated of FcR and CDllb, and enhanced phagocytosis ability compared with mature DCs. The expression o...
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