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The effect of oxygen tensions in the physiological range as an environmental signal on the growth of in vitro murine hemopoietic progenitor cells and the production of hemopoietic growth factors (HGF) from macrophages was investigated. Early (BFU-E) and late (CFU-E) erythroid and granulocyte-macrophage (GM-CFC) progenitor cells were cultured in an atmosphere containing 2%, 3.5%, or 5% oxygen. For both the BFU-E and CFU-E populations, a gas phase containing 3.5% oxygen proved to be optimal, producing greater colony numbers than cultures incubated under 2% or 5% oxygen-tension conditions. For GM-CFC growth, 2% and 3.5% oxygen resulted in a greater stimulation than 5% oxygen. Macrophages derived from unseparated and unstimulated mouse bone marrow cells were cultured on hydrophobic Teflon foils under varying oxygen-tension conditions. The production of erythropoietin (epo), present in the culture supernatants, increased as the oxygen concentration increased from 2% to 3.5%, but then decreased as the oxygen concentration was increased further, from 3.5% to 5%. The presence of a factor demonstrating functional similarity with Interleukin-3 was produced optimally under 5% oxygen-tension conditions. The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) was not significantly affected by changing the oxygen-tension conditions. These results demonstrate that physiological oxygen tension plays an important role not only in the growth of hemopoietic progenitor cells, but also as a physiochemical signal that macrophages can sense and respond to in order to regulate the production of specific secretory products.

Almost pure macrophage populations were obtained when mouse bone marrow cells were cultured under low-oxygen tension on hydrophobic Teflon foils. Macrophage content was determined using nonspecific esterase staining and binding of the mouse, macrophage-specific monoclonal antibody directed against the F4/80 antigen. Using both these techniques, the macrophage content present after 14 days in culture was approximately 98%. This represented an approximate two- to fourfold increase over the initial macrophage content present in primary bone marrow cell suspensions. Granulocytes and erythroblasts were found to be the contaminating cell types. No T-lymphocytes were present at 14 days of culture. The activities of three hemopoietic growth factors (erythropoietin, colony-stimulating factor, and a factor enhancing early erythroid progenitor cells [BFU-E] and stimulating in vitro multipotential stem cells) present in the supernatant were shown to increase in parallel with macrophage content. The results demonstrate that bone-marrow-derived macrophage populations are functionally capable of producing and secreting hemopoietic growth factors. These results form the basis of a hypothesis in which the macrophage is perceived as a regulator cell for hemopoiesis.

Macrophages derived from unstimulated and unseparated mouse bone marrow cells have been shown to release erythropoietin into the extracellular fluid. Additional proof that macrophages can produce the hormone would be a demonstration that the gene is expressed and the mature protein released. In situ hybridization using a 1.2 kb biotinylated erythropoietin DNA probe demonstrates that both cultured macrophages and those present in normal mouse bone marrow express the gene. These results are discussed in terms of the role played by the macrophage in the hemopoietic cellular microenvironment and indicate that a subpopulation is responsible for this function and that cell interactions play an important role in hemopoietic differentiation.

Several functional capacities of the macrophage enable it to act as an "administrator" cell for normal and pathophysiological hemopoietic regulation. Its capacity of sensing and responding to physiso-chemical, cellular and humoral signals indicates that it can regulate myelomonocytopoiesis and erythropoiesis. This occurs by modulating colony stimulating factor and erythropoietin production in response to lactoferrin and oxygen tension respectively. Detection of erythropoietin gene expression in macrophages, both in vitro and in vivo, implies that the macrophage is an "active" member of the hemopoietic cellular microenvironment. Since a subpopulation of macrophages is responsible for this function, a model is proposed in which other hemopoietic regulator molecules may be produced by distinct subpopulations of macrophages under steady-state conditions.

The blood island is found in all hemopoietic organs. It consists of a central macrophage surrounded by other hemopoietic cells, notably developing erythroblasts and cells of the myeloid lineage. In this report, several lines of investigation are combined to discuss the role of the erythroblastic island in erythropoietic differentiation and to question the source and role of circulating erythropoietin (epo). In both cultured and fresh normal bone marrow macrophages, we demonstrate epo gene expression and, simultaneously, localize epo intracellularly. The following observations have also been made: a) red cell production initiated in vitro in mouse fetal liver and normal bone marrow cell suspensions by the addition of horse serum usually occurs in the presence of macrophages that form the blood island functional unit; however, epo can only be detected in the fetal liver supernatants and not in the supernatants derived from bone marrow cultures; b) in 5-fluorouracil-treated mice, circulating epo levels, measured immunologically by ELISA, RIA, and biologically by the in vitro stimulation of mouse bone marrow CFU-E, converged between days 10 and 12 to give epo concentrations that did not correspond with the degree of anemia; c) when bone marrow cells from day 5, 5-fluorouracil-treated mice are placed in culture for 24 hours, an active erythropoiesis is observed in which developing and mature erythrocytes surround macrophages expressing the epo gene (blood islands); at this time immunologically active epo (but little biologically active epo) is present. After 14 days in culture, no erythropoiesis is observed and the culture consists of macrophages, some of which are expressing the epo gene, and biologically, rather than immunologically, active epo is present. These results show that the blood island is a necessary functional unit for active erythropoiesis.