|Budget Amount *help
¥5,200,000 (Direct Cost : ¥5,200,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1993 : ¥4,400,000 (Direct Cost : ¥4,400,000)
To establish a large scale culture method for murine and human mast cells, we have examined the effects of various cytokines on the development of mast cells from murine bone marrow cells and human cord blood CD34^+ cells. In murine, the addition of interleukin-3 (IL-3) to cultures of bone marrow cells gives rise to pure cultures of mast cells (bone marrow-derived maust cells : BMMC). Addition of stem cell factor (SCF), IL-4 and IL-10 to culture with IL-3 stimulated the development of BMMC.Although stem cell factor (SCF), IL-4 and IL-10 alone failed to support BMMC development, SCF in combinations with IL-4 or IL-10 significantly stimulated the proliferation of BMMC.In contrast with BMMC,IL-3 alone could not support the proliferation of CTMC,which were purified from mouse peritoneal cells, as well as IL-4 or IL-10. SCF alone supported a small number of colonies from CTMC.IL-3 and IL-4 but not IL-10 enhanced SCF-dependent CTMC proliferation IL-3 stimulated CTMC proliferation in the pres
ence of IL-4 or IL-10.
In human, we obtained both tryptase-positive and chymase-positive mast cells by culturing CD34^+ cells, which were isolated from cord blood mononuclear cells using a cell sortor, in the presense of SCF and IL-6 or IL-11. When CD34^+ cells were cultured with SCF and IL-6 or IL-11, mast cells with tryptase-positive granules were first detected after 2 weeks of culture, and reached to almost 100% purity after 100 days of culture when 20-30% of the cells were immunohistologically stained for chymase. Our cultured human mast cells contained a large amount of histamine and tryptase, and expressed functional IgE receptors on their cell surface. Single cell culture of CD34^+ cells with a clone-sorting method indicated that both tryptase-positive and chymase-positive mast cells were originated from a common hemopoietic progenitor. Phenotipical analyzes of human mast cell progenitors using a clone-sorting system indicated that committed ones were CD34^+CD38^+ and uncommitted ones were CD34^+CD38^-.
Using reverse transcriptase (RT)-PCR,we demonstrated that murine BMMC and cultured CTMC,and cultured human mast cells, which were pretreated with IgE and anti-IgE monoclonal antibodies, express mRNAs of TNF,GM-CSF,IL-3, IL-4, IL-6 and IL-13. These results suggest that both murine and human mast cells can release not only a variety of chemical mediators such as histamine and leukotorienes but also various cytokines. Less