Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: antagonistic effects on IL-4 and IFN-gamma and selective involvement of TFN-alpha receptor-1

We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two-stage culture system in which, besides granulocyte–macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α), stem-cell factor (SCF) was added during the first 5 days, while interleukin-4 (IL-4) and/or interferon-γ (IFN-γ) were added during the secondary culture period of 9 days. Addition of IL-4 favoured the outgrowth of CD1a+, HLA-DR+, CD4+, CD40+, CD80+ but CD14− cells with dendritic morphology and strong antigen-presenting capacity. Addition of IFN-γ selectively induced HLA-DR and CD86 but did not up-regulate CD1a expression or antigen-presenting capacity of the differentiated cells. An antagonism between IL-4 and IFN-γ could further be confirmed in that, as compared with IL-4 alone, the simultaneous addition of IL-4 and IFN-γ to GM-CSF plus TNF-α during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor-specific TNF-α mutants, we investigated the relative involvement of TNF-α receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA-DR, as well as the increase in allostimulatory capacity were dependent on TNF-R1 triggering, whereas triggering through TNF-R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two-stage culture assay using SCF, GM-CSF, TNF-α and IL-4; second, that the effect of TNF-α in DC generation involves signalling via the TNF-R1 receptor; and third, that IFN-γ counteracts some of the effects of IL-4 in DC generation.