H.-W. Snoeck

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

Direct effects of 13-cis and all-trans retinoic acid on normal bone marrow (BM) progenitors: Comparative study on BM mononuclear cells and on isolated CD 34+ BM cells

SummaryThe effects of both 13-cis-and all-trans retinoic acid (RA) on colony formation of normal bone marrow (BM) progenitors were investigated in semi-solid (methylcellulose) assays, using either isolated CD 34+ cells or BM mononuclear cells. Single cell liquid cultures were performed to further discriminate between direct and indirect effects. RA action results in significant decrease of colony forming units (CFUs). This effect is more pronounced starting from CD 34+ progenitors than starting from total BM. This overall decrease in CFUs is due to selective inhibition of CFU-M (macrophage) and erythroid colonies (BFU-E). At the single cell level the CFU-M inhibition is confirmed with — in addition — a significant inhibition of CFU-GM (granulocyte-macrophage) and a marked stimulation of CFU-G(granulocyte)s. Both retinoids exert the above-mentioned effects. All-trans RA, however, is effective at a tenfold lower concentration (10−7M) than 13-cis RA (10−6M). Results on CD 34+ BM fractions (substantially reduced in accessory cells) demonstrate that the described effects can probably be attributed to the direct action of RA on these progenitors; single progenitor (CD 34+) cell liquid cultures further prove this point.

Developmentally regulated responsiveness to transforming growth factor-β is correlated with functional differences between human adult and fetal primitive hematopoietic progenitor cells

Important functional differences exist between primitive CD34++CD38− hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++CD38− cells and used a two-stage culture system to investigate the effects of transforming growth factor-β (TGF-β) and blocking anti-TGF-β antibodies (anti-TGF-β) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-β than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-β, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-β is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-β reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-β.