A role for the macrophage in normal hemopoiesis. II. Effect of varying physiological oxygen tensions on the release of hemopoietic growth factors from bone-marrow-derived macrophages in vitro.

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.

A role for the macrophage in normal hemopoiesis. I. Functional capacity of bone-marrow-derived macrophages to release hemopoietic growth factors.

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.

The autonomous release of erythropoietic inhibition during long-term in vivo administration of actinomycin D.

In this study, we describe the effect of actinomycin D (Act D), 120 micrograms/kg every 2 days, on hematopoiesis over a 48-day period. Erythropoiesis is almost totally eradicated by day 6. Despite continued Act D treatment, however, the block in differentiation between the early (burst-forming units-erythroid [BFU-E]) and late (colony-forming units-erythroid [CFU-E]) erythropoietic progenitor populations is overcome, giving rise to a synchronized and periodic erythrocytosis in both bone marrow and spleen. The first peak is seen on day 27, followed by a second peak on day 41. Circulating erythropoietin (Epo) levels increase from days 16 to 34, indicating that Act D does not have an effect on Epo production. Increasing endogenous Epo levels by bleeding results in an earlier erythroblast and 59Fe incorporation peak than in unbled animals. The BFU-E population increased in number until the block between BFU-E and CFU-E was overcome. No apparent change occurred in either the day 8 colony-forming units-spleen (CFU-S day 8) or granulocyte-macrophage colony-forming cells (GM-CFC) in the bone marrow, although a dramatic increase in GM-CFC in the spleen was observed. It appears that release from the Act D-induced block in differentiation may, in part, be due to Epo, but the mechanism by which this phenomenon takes place is unclear.

The sensitivity of in vitro erythropoietic progenitor cells to different erythropoietin reagents during development and the role of cell death in culture.

With the availability of several recombinant erythropoietin (Epo) reagents, it has been possible to undertake a systematic study of the relative Epo sensitivity of late erythroid colony-forming units (CFU-E) in 8.5-day embryos, 13.5-day fetal liver, and adult bone marrow of the mouse. All Epo preparations tested, including one from impure sheep and a highly purified human native Epo preparation, produced parallel, but displaced, dose-response curves when Epo concentration was plotted against percent CFU-E response calculated from the optimal Epo concentration. It was found that the CFU-E derived from 8.5-day embryos demonstrated the greatest Epo sensitivity which decreased in fetal liver and adult bone marrow CFU-E populations. Modifications to the culture system allowed CFU-E to be stimulated with as little as 0.003 mU/mL, equivalent to approximately 0.03 fg Epo. Under these culture conditions, no evidence for apoptosis was found, although a normal programmed cell death function cannot be ruled out.

A role for the macrophage in normal hemopoiesis: III. In vitro and in vivo erythropoietin gene expression in macrophages detected by in situ hybridization.

Macrophages derived from unstimulated and unseparated mouse bone marrow cells cultivated on hydrophobic foils can release hemopoietic regulator molecules into the surrounding medium. To prove that one of these regulators exists in macrophages in vitro, in situ hybridization using a 1.2-kb erythropoietin (Epo) gene probe was employed. The probe was biotinylated and the signal developed using a streptavidin-gold reagent. Observation was performed using reflection-contrast microscopy. The results indicate that from a 98% pure population of macrophages, 34% F4/80 (mouse, macrophage-specific antigenic determinant)-positive macrophages exhibited Epo gene expression. The technique was also applied to normal, steady-state mouse bone marrow in which approximately 10% of the cells are F4/80-positive and of which about 3% demonstrated simultaneous Epo gene expression. As positive control, kidneys from anemic mice were hybridized with the biotin-labeled Epo DNA. A second positive control utilized biotin-labeled actin DNA hybridized to cultured macrophages and normal bone marrow cells. The accumulating information, demonstrating that the unstimulated kidney does not express the Epo gene, indicates that Epo is produced in other areas of the body under normal, steady-state conditions. The present results show that 1) macrophages can express the Epo gene, 2) this function is carried out by a subpopulation of macrophages, and 3) bone marrow macrophages in vivo may be responsible for the Epo production-target cell mechanism evoked by short-range and/or cell-to-cell interactions under normal, steady-state conditions.

Rat microglial interleukin-3.

Interleukin-3 (IL-3, multi-CSF) is a growth factor for a variety of hematopoietic progenitor cells. Recently, microglial cells, the resident macrophages of the central nervous system (CNS) have been shown to proliferate in the presence of IL-3 both in vivo and in culture. Data obtained from cultured astrocytes gave rise to the hypothesis that astrocytes synthesize the microglial growth factor. This is the first report identifying rat microglial cells themselves as a source of IL-3. Culture media conditioned by isolated microglia enhanced microglial proliferation above fresh media controls. IL-3 polypeptide was detected in both conditioned media (CM) and in microglial cells by Western blotting and immunoprecipitation. Furthermore, anti-IL-3 antibodies were able to inhibit microglial proliferation induced by conditioned media. mRNAIL-3 was present in single microglial cells as revealed by in situ hybridization. Total RNA prepared from purified microglia yielded a single PCR amplification product. Identity of the PCR product was confirmed by Southern blot hybridization using a cDNAIL-3 probe and by DNA sequencing. Expression of mRNAIL-3 was observed in both absence and presence of lipopolysaccharide, a bacterial endotoxin, that commonly induces expression of inflammatory cytokines and inhibits microglial proliferation. It is concluded that IL-3 expression in ensuring the recruitment of enhanced numbers of immunocompetent cells at sites of lesion. In the light of weak immune reactions in the brain, it is hypothesized that the expression of a characteristic T cell feature in monocyte-derived microglia may be a partial compensation of T cell functions in brain lesions.

Silent polymorphisms within the coding region of human sodium/hydrogen exchanger isoform-1 cDNA in peripheral blood mononuclear cells of leukemia patients: A comparison with healthy controls.

We have examined the sequence of the cDNA encoding the sodium/hydrogen exchanger isoform 1 (NHE1), from 23 bases upstream of the start codon to 28 bases downstream of the stop codon. Template was prepared from (1) peripheral blood mononuclear cells (PBMC) isolated from 10 healthy unrelated Caucasian volunteers; (2) PBMCs isolated from 6 leukemic patients (acute lymphoblastic leukemia [ALL], n = 3; chronic lymphocytic leukemia [CLL], n = 1; chronic myelogenous leukemia [CML], n = 2); and (3) samples of 4 leukemic cell lines (ALL: CEM, MOLT4; AML: KG1a; CML: K562). NHE1 cDNA in normal PBMCs showed silent polymorphism of nucleotides 112 (N1: T, frequency 0.70; C, frequency 0.30; prevalence of heterozygosity 0.42); 2248 (N2: G, frequency 0.90; A, frequency 0. 10; heterozygosity 0.18); and 2493 (N3: G, frequency 0.90; A, frequency 0.10; heterozygosity 0.18). Deduced primary structure of NHE1 protein in all normal volunteers was identical to that previously published for NHE1 from renal and cardiac tissue. Similar to normal PBMCs, NHE1 cDNA from leukemic cells showed polymorphism of nucleotides N1, N2, and N3, but failed to demonstrate leukemia-specific sequence differences. We conclude that the coding region of NHE1 cDNA shows a greater level of polymorphism than is currently recognized, but that sequence mutation of NHE1 is not a key event in the pathogenesis of leukemia.

Serum erythropoietin and transferrin receptor levels in patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is generally associated with mild anaemia. The role of erythropoietin (EPO) in the pathogenesis of this anaemia of chronic disorders is still a matter of controversy. Therefore, in a multicenter study we investigated the serum EPO concentration in 124 patients with rheumatoid arthritis. METHODS: Patients with uncomplicated iron deficiency and haemolytic anaemia served as the reference group (n = 54). The measurements were performed with a specific and sensitive ELISA: RESULTS: The mean EPO concentration +/- SD of the whole RA group (32.3 +/- 22.2 mU/ml) was elevated above normal. About 40% of the patients had underlying iron deficiency (defined by ferritin values < or = 60 ng/ml) and a significantly higher median EPO concentration than patients with normal iron stores (35.8 mU/ml versus 20.7 mU/ml; p < 0.001). The iron deficiency was associated with lower disease activity, as defined by the C reactive protein. In contrast to the reference group (r = -0.78), there was no significant correlation between EPO and the haematocrit in either RA subgroup, although the values for the RA patients were within the 95% prediction range of the reference group. In addition to the EPO, we investigated the soluble transferrin receptor level as a measure of bone marrow erythropoiesis. The level in iron-replete RA subjects was about 1.6 times higher than in normal persons, reflecting a relatively hypoproliferative erythropoietic activity. CONCLUSION: This study shows that the EPO concentrations in RA are elevated above normal but lower than expected, and that the normal relationship between EPO and the degree of anaemia is impaired.

The macrophage as a production site for hematopoietic regulator molecules: sensing and responding to normal and pathophysiological signals.

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.

Haemopoietic regulation and the role of the macrophage in erythropoietic gene expression.

The macrophage is considered as an "active" component of the haemopoietic cellular microenvironment with respect to erythropoietin (epo) production during embryonic, foetal and adult erythropoiesis. Emphasis is placed on steady-state rather than pathophysiological conditions. In addition, the signals capable of affecting the functional capacity of the macrophage with regard to colony stimulating factor and epo production are also taken into account. Evidence is given demonstrating that a subpopulation of resident macrophages in vitro and in the mouse bone marrow, under normal conditions, can express the epo gene. These results indicate that erythropoiesis can be regulated by short-range or cell-to-cell interactions within the bone marrow.

The effect of 5-fluorouracil on erythropoiesis.

The effects of a single dose (150 mg/kg) of 5-fluorouracil on mature erythroid and erythropoietic and multipotential in vitro precursor populations in the bone marrow and spleen and circulating biologically (erythroid colony forming unit [CFU-E] assay) and immunologically active (enzyme-linked immunosorbent assay) erythropoietin (Epo) are described. All mature erythroid (reticulocytes, erythrocytes) and in vitro erythropoietic precursors (CFU-E, erythroid burst-forming unit [BFU-E]) are severely reduced, if not eradicated. Transient repopulation of the pure BFU-E and CFU-E populations on days 6 and 7, respectively, produces a marked reticulocytosis after day 9. Circulating Epo increases to above normal values by day 2. However, whereas biologically active Epo remains constant at this level until day 9, immunologically active Epo continually increases; by day 12, however, both assays detect circulating Epo levels of about 400 mU/mL. In vitro multipotential stem cells (BFU-E mix) are reduced to 32% on day 1, 7.6% on day 2, and return to normal values between days 4 and 5. The survival and repopulation kinetics of the BFU-E mix imply a stem cell population more mature than the high proliferative potential colony-forming cells. However, the BFU-E mix may be responsible for erythropoiesis repopulating ability.

Primordial germ cells are capable of producing cells of the hematopoietic system in vitro.

The identity of the cells giving rise to the hematopoietic system in the mouse embryo are unknown. The results presented here strongly suggest that hematopoietic cells are derived from a nonhematopoietic cell population that has been previously thought to give rise to the germ cells. These cells are called primordial germ cells (PGCs) and can be recognized as large cells showing blebbing and pseudopodial extrusions on their surface. They are alkaline phosphatase (AP) positive and possess a stage-specific embryonic antigen (SSEA-1) on their surface. They represent a small pool of cells in the extraembryonic mesoderm at the base of the allantois in late day-6 embryos. Primordial germ cells from 7.5- and 8.5-day visceral yolk sac and embryo proper form AP+ and SSEA-1+ colonies within 5 days when grown on an embryonic fibroblast feeder cell layer in the presence of leukemia inhibitory factor (LIF), stem cell factor (SCF), and interleukin-3 (IL-3). Individual colonies taken from day-5 cultures can be shown to differentiate into erythroid lineage cells in secondary methyl cellulose culture and produce secondary and tertiary PGCs in the presence of LIF, SCF, and IL-3. Cells taken from the region of the allantois and primitive streak can form colonies on hydrophilic Teflon (DuPont, Wilmington, DE) foils precoated with collagen and fibronectin. The cells from these colonies were then shown to form cobblestone areas on irradiated adult bone marrow stromal layers, indicating that the most primitive in vitro hematopoietic stem cell, the cobblestone-area forming cell (CAFC), was present. PGC colonies were grown in methyl cellulose in the presence of LIF, SCF, and IL-3 for 5 days, and the colonies were removed and passaged 3 times on pretreated extracellular matrix hydrophilic Teflon foils. After each passage, the cells were assayed for their differentiation capacity and PGC content. After the last passage, the number of CAFCs was also determined. It was found that, under these conditions, the PGC population expanded more than 400-fold and also contained CAFCs. It is postulated that the PGC represents a totipotent stem cell population capable of producing a variety of different cell types including cells of the hematopoietic system.