We investigated the effects of transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on very primitive CD34++CD38- and on more mature CD34++CD38+ human hematopoietic progenitor cells by means of a two stage pre-colony-forming cell (pre-CFC) assay. The first (liquid) stage of this assay allows evaluation of the effects of TGF-beta and MIP-1 alpha on the "primary" proliferation of the progenitors under study and on the generation of "secondary" colony-forming cells (CFC, cells for which a second stage semisolid clonogenic assay was used as a read-out). TGF-beta inhibited the proliferation and CFC generation of CD34++CD38- and CD34+CD38+ cells, showing the strongest inhibitory activity on CD34++CD38- cells. MIP-1 alpha exerted a weaker inhibitory activity on CD34+2CD38- cells, whereas it enhanced the primary proliferation of CD34+CD38+ cells and generation of secondary CFC in this subpopulation. Thus, TGF-beta, and MIP-1 alpha both inhibit very primitive CD34+2)CD38- cells, but they are not equally potent. The effects of TGF-beta and MIP-1 alpha on more mature progenitor cells are more complex. Our results and data from the literature indicate that, as progenitor cells mature, they reach a "pivotal point" at a certain stage in their differentiation pathway, depending on the inhibitor, where they are no longer inhibited or where they may even be stimulated by the former inhibitor to proliferate.

Since tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta have all been shown to be specific inhibitors of early human hematopoiesis, we wanted to investigate the interactions of these three cytokines on very primitive human adult bone marrow CD34++CD38- hematopoietic progenitor cells, using a pre-colony-forming cell (pre-CFC) assay, which detects the effects of these cytokines on the initial phases of the differentiation of these primitive progenitors, which are unresponsive to interleukin (IL) 3 alone. Surprisingly, TNF-alpha was a very potent stimulator of the proliferation of CD34++CD38- cells and was the most potent synergistic factor for the IL-3-induced proliferation of these cells of all cytokines tested (IL-1, IL-6, granulocyte colony-stimulating factor, kit ligand). TNF-alpha was the only cytokine that, as a single added factor, induced substantial proliferation in CD34++CD38- cells in the presence of IL-3, except for kit ligand, which induced very limited proliferation. TNF-alpha, moreover, induced a high degree of resistance to the inhibitory effects of TGF-beta in a dose-dependent way. The inhibitory effects of IFN-gamma, however, were not affected by the presence of TNF-alpha. We hypothesize that in situations of the hematopoietic stress, TNF-alpha may abrogate the inhibitory effect of ambient TGF-beta in the bone marrow microenvironment to allow primitive stem cells to proliferate and differentiate in response to an increased demand for mature blood cells.

We have previously shown that interleukin 4 (IL-4) and interferon gamma (INF-gamma) reciprocally regulate the production of granulocytes and monocytes from mature monopotential hematopoietic progenitor cells, while at the level of the very primitive stem cells IFN-gamma is a selective inhibitor of proliferation and differentiation, and IL-4 has weak stimulatory effects. We investigated the effects of IL-4 and IFN-gamma on the expansion in suspension culture of myeloid colony-forming cells (CFCs) induced by either IL-3 or IL-1+IL-3, using on the one hand more differentiated CD34+HLA-DR strongly positive (HLA-DR++) and on the other hand more primitive Cd34+HLA-DR weakly positive (HLA-DR+/-) human bone marrow cells. It is shown that both IL-4 and IFN-gamma stimulate the IL-3- and IL-3+IL-1-induced expansion of the number of CFCs in the HLA-DR+/- population. In the presence, but not in the absence of IL-1, additive effects of IL-4 and IFN-gamma were seen. We could not demonstrate any IL-3-like effect by IL-4 on early human hematopoietic progenitors. No expansion of CFC number was seen in the HLA-DR++ population. Based on these data and on data which we have published previously, a model for the regulation of myelopoiesis by IL-4 and IFN-gamma is proposed. In this model, IL-4 and IFN-gamma, which are both immune recognition induced inflammatory cytokines, both stimulate the expansion and recruitment of early myeloid progenitors, whereas at the level of their terminal differentiation, the balance between both cytokines determines whether preferentially monocytes/macrophages (IFN-gamma) or granulocytes (IL-4) are being produced. At the level of the most primitive cells, the inhibitory action of IFN-gamma might prevent differentiative exhaustion of the stem cell compartment in situations of hematopoietic stress.

We investigated the cell kinetic effects of retinoic acid (RA) and the functional interaction between RA and TGF-beta on normal human bone marrow progenitor cells (CD34+). Cell cycle progression throughout the first three consecutive cell cycles and alterations in cell kinetic responses were measured using the BrdU-Hoechst quenching technique. RA stimulates the IL-3-induced growth by additionally recruiting quiescent stem and progenitor cells out of the G0/G1-phase and by increasing the cell cycle traverse rate. In contrast, TGF-beta addition resulted in a significant decrease in the number of proliferating cells. Simultaneous addition of RA and TGF-beta resulted in a stronger inhibition compared to addition of TGF-beta alone. Preincubation experiments further showed that RA is capable of sensitizing the progenitors to the inhibitory action of TGF-beta: the inhibitory effect of TGF-beta was significantly increased when cells were pretreated with RA. These data show that, in combination with IL-3, RA additionally stimulates quiescent bone marrow progenitors in a simultaneous way, and that it increases sensitivity of the progenitors to the inhibitory action of TGF-beta. The combination of RA and TGF-beta on normal and leukemic hematopoiesis has to be further investigated, since this combination may possibly provide additional therapeutic benefit.