Effects of IL-3 and SCF on Histamine Production Kinetics and Cell Phenotype in Rat Bone Marrow-derived Mast Cells

Abstract

BackgroundRat mast cells were regarded as a good model for mast cell function in immune response.MethodsRat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of 5×104/ml in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine.ResultsCell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of FcεRI and the mast cell antigen, ganglioside, on culture day 11.ConclusionThese results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrIL-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.