Dendritic cells in vitro development is indipendent from exogeneous gm-csf, il4 and tnf alfa

Dendritic Cells (DC) of myeloid origin are usually obtained from cultures of stern cells of hone marrow or pheripheral blood origin, supplementing the medium with various cytokines, such as GM-CSF, IL4 and TNF alfa Previous observations ( 1,2) have induced us to study the development of DC in a more detailed way. Cells were separated from the peripheral blood of normal subjects. The monocyte fraction was plated in T-flasks and cultured in RPMI 1640, added with 10% PCS and 1% L-glutamine, without any stimulating or differentiation factor(s). After a week, the cells in suspension were plated in two-well chambe; slides with fresh medium supplemented with monocyte-conditioned medium (0.5:1.0 ml).The cells were then cultured for 15 days as an adherent cell population and subsequently stained for morphology, cytochemistry and for typing by APAAP procedure. An immunoenzymatic assay was performed for the quantitative measurement of the cytokines GM-CSF, TNF alfa and IL6 in the culture supernatants. Our results demonstrate [hau a) DC can be obtained easily from circulating precursors without supplementing the medium with exogenous cytokines; b) in the culture supernatants GM-CSF, TNF alfa and IL-6 are detectable. This result suggests that, in our model, the development of DC is induced from precursor cells present in the monocyte fraction (2) stimulated by cytokines physiologically produced in culture. The multiple pathways of monocyte and monocyte-precursors that give rise to different tissue macrophages and the known eterogeneity of DC remain to be explained.