Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes

Considerable experimental and clinical evidence suggests a relationship between erythropoiesis and thrombopoiesis. This is supported by observations that erythropoietin (Epo), the primary regulator of erythropoiesis, can affect platelet production when injected into animals. In this study we provide experimental evidence for a direct effect of Epo on thrombopoiesis by demonstrating that 125I-labeled recombinant human Epo binds to rat and mouse bone marrow megakaryocytes. Thus, autoradiographic analysis using cold competition to measure specific binding has been used to demonstrate that Epo binding to megakaryocytes increases with megakaryocyte maturation. When corrected for cell size, Epo binding sites per unit surface area increase from Stage I megakaryoblasts to Stage II megakaryocytes, and then remain approximately constant throughout further megakaryocyte maturation. Receptor density on megakaryocytes is similar to that on pronormoblasts in the rat, and in mice is 60% that on pronormoblasts. No binding of Epo to platelets or to naked megakaryocyte nuclei was detected. Equilibrium binding studies with partially purified rat megakaryocytes (20%-40% pure), where megakaryocytes are the only significant Epo binding cell population, showed a single class of saturable, high-affinity binding sites present on average at 6500 binding sites per megakaryocyte with a KD of 287 pM. Binding of [125I]Epo to rat megakaryocytes was inhibited with an antiserum against murine erythroblasts. These results suggest that the effects of Epo on thrombopoiesis may be directly mediated through specific, high-affinity binding sites for Epo on the surface of maturing megakaryocytes.     

Binding of iodinated erythropoietin to rat bone marrow cells under normal and anemic conditions

Specific binding sites for erythropoietin (Epo) were shown in normal and anemic rat bone marrow cells using [125I]labeled human recombinant Epo. When rats were treated once or several times with phenylhydrazine or malotilate, or by phlebotomy, the serum Epo level determined by RIA began to increase rapidly. Thereafter, both the number of erythroid colony-forming unit (CFU-E)-derived colonies and the Epo binding capacity of bone marrow cells increased almost simultaneously in response to induced anemic states, suggesting that the amount of Epo binding in bone marrow cells may reflect in vivo erythropoiesis. Scatchard analysis of the binding data from normal rats revealed the presence of a single class of binding sites (Kd = 0.18 +/- 0.04 nM, 38 +/- 5 sites/cell). In anemic states, the apparent average receptor number per cell increased (52-62 sites/cell) without changing in binding affinity toward Epo. Furthermore, [125I]Epo was cross-linked to the cell surface molecule of approximately 165 kd in nonreducing conditions and 75 kd in reducing conditions. Autoradiographic analysis indicated that Epo receptors were distributed on immature erythroid cells. Proerythroblasts were the most heavily labeled, whereas orthochromatic erythroblasts and cells of myeloid and lymphoid lineages were not labeled. Calculations based on Scatchard and autoradiographic analysis showed that proerythroblasts have 390 receptor sites per cell, twice as many as basophilic or polychromatophilic erythroblasts have. These results are consistent with the stage-specific action of Epo in physiological differentiation of erythroid cells.     

Down-modulation of high-affinity receptors for erythropoietin on murine erythroblasts by interleukin 3

Erythropoiesis is regulated by the glycoprotein hormone erythropoietin (Epo) and by several other factors including interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor. The possibility that IL-3 and GM-CSF may act by modulating Epo receptor expression was investigated using erythroblasts purified from the spleens of phenylhydrazine-treated mice. AT 37 degrees C, in the presence of sodium azide to inhibit receptor internalization. 125I-labeled human recombinant Epo bound to a single class of high-affinity receptors on splenic erythroblasts (450 sites/cell, Kd = 700 pM). Autoradiographic studies indicated that 94% of specifically bound Epo was associated with erythroblasts, decreased Epo binding being observed with increasing erythroid cell maturation. Whereas recombinant mouse IL-3 and GM-CSF did not compete with 125I-Epo for binding to the Epo receptor, preincubation of cells with IL-3 resulted in a concentration-dependent loss of 125I-Epo binding without altering the affinity of residual receptors for Epo. Complete loss of Epo receptors was effected within 2 h at IL-3 concentrations above 2500 U/ml. Preincubation with recombinant mouse GM-CSF had no effect on binding, even at 100,000 U/ml. In comparison, preincubation of cells with Epo (50 U/ml) caused complete loss of 125I-Epo binding within 30-60 min, an effect not explained by receptor saturation with unlabeled Epo. Thus, in addition to trans-down-modulating growth factor receptors of the granulocyte-macrophage series, IL-3 also trans-down-modulates a growth factor receptor of the erythroid lineage.     

Erythropoietin receptor characteristics on primary human erythroid cells.

Erythropoietin (EP) exerts its effects on erythropoiesis by binding to a cell surface receptor. We examined EP receptor expression during normal human erythroid differentiation and maturation from the burst-forming unit-erythroid (BFU-E) to the reticulocyte level. In contrast to previous studies, we assessed EP receptor number and affinity in erythroid precursors immunologically purified from fresh bone marrow aspirates or fetal liver samples and in reticulocytes purified from peripheral blood. EP receptors were quantitated by equilibrium binding experiments with 125I EP. We found that purified primary erythroblasts from both adult and fetal sources exhibited a single high-affinity (kd 100 pmol/L) binding site for EP under our experimental conditions, and 135 or 250 receptors per cell, respectively. Reticulocytes were devoid of EP receptors. We compared these data to in vitro-derived BFU-E progeny at both early and late stages of maturation. Cultured BFU-E progeny also displayed a single class of receptors of slightly lower affinity (210 to 220 pmol/L). Preparations enriched in colony-forming units-erythroid (CFU-E) and proerythroblasts (day 9 BFU-E progeny) displayed approximately 1,100 receptors per cell, whereas populations containing mature erythroblasts (day 14 BFU-E progeny) exhibited approximately 300 receptors per cell. Furthermore, information from binding experiments was complemented by autoradiography in both enriched BFU-E preparations, cultured BFU-E progeny (days 9 and 14), and marrow mononuclear cells. These studies are consistent with a peak in EP receptor expression at the CFU-E/proerythroblast stage and a decrease with further maturation to undetectable levels at the reticulocyte stage. These data examining EP receptor characteristics on freshly isolated erythroid precursor cells complement previous data on EP receptor biology using culture-derived erythroblasts.     

Specific binding of erythropoietin to its receptor on responsive mouse erythroleukemia cells.

Erythropoietin (Epo) is a glycoprotein factor that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Here we describe the isolation of Epo-responsive mouse erythroleukemia cell line SKT6, the characterization of the specific binding of biologically active 125I-labeled human Epo (125I-Epo) to its membrane receptor, and, finally, report information concerning the molecular structure of the receptor. About 75% of erythroid colony-forming precursor cell-like colonies derived from SKT6 cells were hemoglobin-positive after 3- to 4-day exposure to Epo in methylcellulose culture. Radioiodinated Epo bound specifically to SKT6 cells, and Scatchard analysis of the data showed a high affinity for 125I-Epo (Kd = 0.15 nM) but displayed only a small number of specific receptors (approximately equal to 470 per cell). Membrane components that specifically interact with 125I-Epo were identified by covalent crosslinking with disuccinimidyl suberate, and three receptor species with apparent Mr 63,000, 94,000, and 119,000 were found in membrane from SKT6 cells, suggesting the complex structure of the receptor molecules. Specific bindings were also detected in all of the Epo-unresponsive Friend erythroleukemia cells examined, and cross-linking study revealed the presence of only the 63,000 species as a binding site.     

Interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and megakaryocytic-like cells.

We have investigated the interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis. Acidic FGF (aFGF) stimulated the proliferation of murine megakaryocytes and human erythroleukemia (HEL) cells in a concentration-dependent manner. The concentrations of aFGF required to elicit half-maximum and maximum effects were similar for HEL and megakaryocytic colony formation. The effect of aFGF was comparable to that of basic FGF (bFGF) in both cell types. The effect of both FGFs was found to be synergistic with interleukin-3 (IL-3), and was abrogated by a monoclonal anti-IL-6 antibody. A specific cell surface receptor complex of approximately 120 Kd was detected for FGF by crosslinking experiments on HEL cells and total bone marrow (BM) cells. Single-cell autoradiography of megakaryocytes in BM smears and BM cultures showed binding sites for 125I-aFGF. Northern blot analysis of messenger RNA (mRNA) from total BM and HEL cells showed a 4.4-kb mRNA specific for FGF receptors type 1 (flg) and type 2 (bek). This was confirmed by polymerase chain reaction, which also showed the presence of FGF receptor mRNA in megakaryocytic-like cells, normal megakaryocytes, and platelets. Together, these results indicate that FGF is involved in megakaryocytopoiesis and suggest that this interaction may be mediated via FGF receptor type 1 and type 2 located on the megakaryocytic lineage or on accessory cells responsible for the release of megakaryocytic growth-promoting activities.     

Erythropoietin controls heme metabolic enzymes in normal human bone marrow culture

Erythropoietin (Epo) was found to act as a concentration-dependent inducer of aminolevulinic acid (ALA) synthase and porphobilinogen (PBG) deaminase in normal human bone marrow in culture. Epo increased enzymatic activities in individual plated nucleated cells. At a low concentration of Epo, heme oxygenase activity did not change in human bone marrow erythroid progenitor cells. However, Epo at a concentration of 2 U/ml increased heme oxygenase as demonstrated by an increase in both the enzyme protein and its mRNA. In experiments with an inhibitor of heme synthesis, succinylacetone (SA), Epo failed to stimulate erythroid colony-forming unit (CFU-E) growth, but this CFU-E inhibition by SA was completely overcome by the addition of hemin. Epo nevertheless potentiated induction of ALA synthase in the presence of SA. Hemin exerted its regulatory role by negative feedback on ALA synthase in the presence of SA and Epo. Heme potentiated Epo action and resulted in the increase of human marrow erythroid progenitor cell proliferation and differentiation and a concomitant stimulation of ALA synthase and PBG deaminase. The potentiating effects of hemin on CFU-E growth were observed in human bone marrow cells cultured in media supplemented with fetal calf serum or serum-free media with interleukin 3 (IL-3). These results indicate that Epo is a potent inducer of ALA synthase and PBG deaminase in normal human bone marrow. In addition, our results may explain the mechanisms by which heme potentiates Epo or IL-3 enhancement of erythropoiesis. 1) Heme may stimulate the translation of several globin and nonglobin mRNAs, including those of ALA synthase and PBG deaminase; 2) as endogenous cellular heme synthesis reaches optimal levels, heme exerts its regulatory role on ALA synthase by negative feedback inhibition. Additionally, an increase in cellular heme may lead to an increase in its own degradation by induction of heme oxygenase.     

Mast cell growth factor modulates CD36 antigen expression on erythroid progenitors from human bone marrow and peripheral blood associated with ongoing differentiation

To study the differentiation process of erythroid progenitors from normal human bone marrow and peripheral blood, CD34/CD36 sorted cells were cultured in the presence of Erythropoietin (Epo) and Epo plus mast cell growth factor (MGF). The CD34+/CD36- cell fraction from bone marrow supported 74 +/- 33 erythroid burst forming units (BFU-E)/10(4) cells (mean +/- SD, n = 4) in the presence of Epo, which increased 2.1- fold by coculturing with MGF. However, erythroid colony-forming units (CFU-E) were not cultured from the CD34+/CD36- cell fraction. In contrast, the CD34-/CD36+ cell fraction supported CFU-Es in the presence of Epo (152 +/- 115/10(5)) or Epo plus MGF (180 +/- 112/10(5)), whereas BFU-Es were hardly noticed. However, the transition of the BFu-E to CFU-E was observed by incubating CD34+/CD36- cells (10(4)/100 microL) in suspension with Epo plus MGF for 7 days followed by Epo in the colony assay. This was reflected by the appearance of CD34-/CD36+/Glycophorin A+/CD14- cells. In addition high numbers of CFU- Es (1,000 +/- 150, n = 4) were cultured from this cell fraction. In contrast to bone marrow erythroid progenitors, no peripheral blood CFU- Es were cultured from either the CD36+ or CD36- fraction, whereas BFU- Es were predominantly present in the CD36+ fraction. However, the CD34+ progenitor cell from peripheral blood did have intrinsic capacity to differentiate to CFU-Es because CD34+/CD36- cells incubated with Epo plus MGF for 7 days and followed by Epo in the colony assay, supported high numbers of CFU-Es (1,200 +/- 400, n = 3). To study whether additional growth factors have similar effects on erythroid progenitors, experiments were performed with interleukin 1 (IL-1), IL- 3, and IL-6. IL-1 and IL-6 did not modulate the Epo supported proliferation and differentiation. In contrast, IL-3 in the presence of Epo did support CFU-Es, from CD34+/CD36- cells after 7 days in suspension culture. However, flow cytometry analysis showed that Epo plus IL-3 not only supported CD34-/CD36+/Glycophorin A+ cells but also CD36+/CD14+ cells, indicating the differentiation along different cell lineages. In summary, the data show a phenotypic distinction between bone marrow and peripheral blood erythroid progenitors with regard to CD36 expression. In addition, the results suggest that Epo plus MGF or IL-3 and preincubation in suspension culture are prerequisites for the transition of the BFU-E to the CFU-E.     

Erythroid colony studies on sickle cell anemia in hypoproliferative crisis

Bone marrow cells, peripheral blood lymphocytes, and sera from patients with sickle-cell anemia in hypoproliferative crisis were studied in the plasma clot culture system in the presence or absence of erythropoietin (Epo). Bone marrow cells from five patients demonstrated a marked ability to form erythroid colonies in the presence of Epo. These studies also suggested that bone marrow cells from some patients may have an increased sensitivity to Epo. The most outstanding observation in the present study was the marked erythroid colony inhibition by serum taken from one patient during crisis. Serum taken from the same patient two months after hypoproliferative crisis had no suppressive effect on erythroid colony formation. Lymphocytes taken from three patients in crisis had a stimulatory effect on erythroid colony formation when included in culture. The conclusion is that the defect of erythropoiesis in sickle-cell anemia during hypoproliferative crisis is not due to the absence of erythroid precursor cells or to the presence of suppressor lymphocytes, but may in some cases be associated with a circulating inhibitor of erythroid maturation.     

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis

A drug that specifically inhibits erythropoiesis would be clinically useful. The erythropoietin (Epo) mutant Epo (R103A) could potentially be used for this purpose. Epo (R103A) has a single amino acid substitution of alanine for arginine at position 103. Because of this mutation, Epo (R103A) is only able to bind to one of the 2 subunits of the erythropoietin receptor (EpoR) homodimer and is thus a competitive inhibitor of Epo activity. To produce large quantities of Epo (R103A) to test in animal models of thalassemia and sickle cell disease, we expressed and purified recombinant Epo (R103A) from the yeast Pichia pastoris. Using this method milligram quantities of highly purified Epo (R103A) are obtained. The yeast-expressed Epo (R103A) is properly processed and glycosylated and specifically inhibits Epo-dependent cell growth and (125)I-Epo binding. Epo (R103A) does not, however, directly induce apoptosis in 32D cells expressing EpoR. Epo (R103A) inhibits erythropoiesis of human CD34(+) hematopoietic cells and completely blocks erythroid burst-forming unit formation in normal human bone marrow colony assays. Yeast-expressed Epo (R103A) is a specific inhibitor of primary erythropoiesis suitable for testing in animal models.     

Characterization of Fc gamma receptors on a human erythroleukemia cell line (HEL).

It has been established that human platelets express a single class of Fc gamma receptors that has been designated Fc gamma RII. However, the function of this receptor on these cells and its regulation are less certain. Studies to further investigate Fc gamma RII on platelets are limited by the inability to culture platelets in vitro. Therefore, identification of a human cell line that expresses Fc gamma RII as its only Fc gamma receptor as well as other platelet characteristics would be of potential importance. To this end, we examined Fc gamma receptor expression by the human erythroleukemia (HEL) cell line, which expresses platelet/megakaryocyte surface proteins. Flow cytometry studies on HEL cells with anti-Fc gamma receptor monoclonal antibodies revealed that, similar to platelets and megakaryocytes, Fc gamma RII is the only Fc gamma receptor expressed on the cell surface. Furthermore, Northern blot analysis revealed that Fc gamma RII is the only Fc gamma receptor mRNA present. Stimulation with dimethylsulfoxide (DMSO) or 12-O-tetradecanoylphorbol-13-acetate (TPA) did not alter Fc gamma RII protein or mRNA expression. Ligand binding studies with [125I]IgG trimer indicated that HEL cells express 92,240 +/- 5030 binding sites per cell, with a kd of 1.94 +/- 0.31 x 10(-8) M. Similar to human platelets, HEL cells preferentially bound oligomeric IgG, and this binding was ionic strength dependent. These observations are similar to those previously observed with Fc gamma RII on human platelets and suggest that the HEL cell Fc gamma receptor is similar, if not identical to the platelet Fc gamma RII receptor. HEL cells may serve as a model for the study of platelet/megakaryocyte Fc gamma RII.     

Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly-devised quantitative flow-cytometric assay

In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine–RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by ¹²⁵I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34⁺CD38⁻ cells (approximately 1600 sites/cell), and decreased in the following order: CD34⁺CD38⁻ cells > CD34⁺CD38⁺ cells > CD34⁻CD38⁺ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34⁺ fraction expressed more EpoR than their CD34⁻ fraction. However, the expression levels of EpoR of these fractions were lower than CD34⁺CD38⁻ cells. These results indicated that EpoR was highly expressed on CD34⁺ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.     

Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential.

The replating capability of human multipotential (colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was assessed in vitro as a potential measure of self-renewal using purified, recombinant (r) human (hu) or murine (mu) mast cell growth factor (MGF), a ligand for the c-kit proto-oncogene receptor. Primary cultures of human umbilical cord blood or adult human bone marrow cells were initiated in methylcellulose with erythropoietin (Epo) alone or in combination with rhu interleukin-3 (IL-3) or MGF. Individual day 14 to 18 CFU-GEMM or BFU-E colonies were removed from primary cultures and reseeded into secondary methylcellulose cultures containing a combination of Epo, MGF, and rhu granulocyte-macrophage colony-stimulating factor (GM-CSF). The data showed a high replating efficiency of cord blood and bone marrow CFU-GEMM in response to Epo + MGF in terms of the percentage of colonies that could be replated and the number of secondary colonies formed per replated primary colony. The average number of hematopoietic colonies and clusters apparent from replated cultures of cord blood or bone marrow CFU-GEMM stimulated by Epo + MGF was greater than with Epo + rhuIL-3 or Epo alone. Replated cord blood CFU-GEMM gave rise to CFU-GEMM, BFU-E, and GM colony-forming units (CFU-GM) in secondary cultures. Replated bone marrow CFU-GEMM gave rise mainly to CFU-GM in secondary cultures. A more limited capacity for replating of cord blood and bone marrow BFU-E was observed. These studies show that CFU-GEMM responding to MGF have an enhanced replating potential, which may be promoted by MGF. These studies also support the concept that MGF acts on more primitive progenitors than IL-3.     

Purification, amplification and characterization of a population of human erythroid progenitors

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.     

BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells

Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo(-/-) and EpoR(-/-) mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210(BCR-ABL) and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR(-/-) mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.     

Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures

In spite of their ability to support myelopoiesis for several months, human long-term marrow cultures (LTMC) are unable to sustain the production of mature erythroid cells for greater than 4 weeks. Because this preference correlates with the presence of myeloid growth factors and possible absence of erythroid factors in LTMC, we studied the effects of the erythroid growth and differentiation factor erythropoietin (Epo) on both erythropoiesis and myelopoiesis in human LTMC. Either natural or recombinant Epo was added weekly to LTMC for 10 weeks, and total cell number, numbers of hemopoietic progenitors (mixed lineage colony-forming units, CFU-MIX; erythroid burst-forming units, BFU-E; erythroid CFU, CFU-E; granulocyte-macrophage CFU (CFU-GM); granulocyte CFU, CFU-G; and macrophage CFU, CFU-M), erythroblasts (early and late), granulocytes, and macrophages were quantitated separately in the adherent and nonadherent layers of the cultures. In the absence of Epo, mature erythroid cells disappeared within the first 3-4 weeks, whereas in cultures supplemented with Epo, erythropoiesis was supported for up to 8 weeks. Results indicate that erythroid maturation is blocked at the BFU-E stage and that exogenous Epo may act on a mature subpopulation of BFU-E located in the nonadherent fraction of the cultures, promoting its maturation into CFU-E, which in turn develop into erythroblasts. However, despite Epo supplementation, erythropoiesis was not restored to in vivo proportions, suggesting that additional factors or conditions necessary for erythropoiesis are lacking in LTMC. Interestingly, we found that exogenous Epo reduced the numbers of presumably more mature (nonadherent) myeloid CFU (CFU-C), granulocytes, and macrophages compared to controls and did not alter the levels of any of the most primitive hemopoietic progenitors measured (CFU-MIX, adherent BFU-E, and adherent CFU-C). Thus the data show that exogenous Epo modulates hemopoiesis in human LTMC, enhancing erythropoiesis and suppressing myelopoiesis, but that its effects appear limited to modulating levels of the nonadherent (more mature) progenitors, leaving the numbers of the adherent (immature) progenitor cells unchanged.     

Interleukin-4 suppresses the interleukin-3 dependent erythroid colony formation from normal human bone marrow cells

Human recombinant interleukin 4 (IL-4) was studied for its effects on the erythroid burst forming unit (BFU-E) from human bone marrow cells. IL-4 alone neither supports nor suppresses the erythropoietin (Epo)-dependent colony formation. Different results were obtained when IL-4 was combined with interleukin-3 (IL-3) in the presence of Epo. IL-4 suppressed the IL-3 supported erythroid colony formation in all cases (an increase of 58 +/- 8% with IL-3 versus an increase of 14 +/- 7% with IL-3 plus IL-4, n = 8). This antagonizing effect was dependent on the continuous presence of IL-4 in the culture medium, but was independent of adherent cells, B-, T-cells, or the presence of serum in the culture medium. Finally, the effects of IL-4 and IL-3 were studied on the 'Epo-independent' BFU-E by adding Epo on day 3. A decline of the IL-3 supported BFU-E was observed in the presence of IL-4 but the degree of reduction was equivalent to the results obtained when Epo was supplied at day 0. These findings indicate that IL-4 acts as suppressive growth factor for the IL-3 supported erythroid colony formation from human bone marrow cells.     

Expression and modulation of IL-3 and GM-CSF receptors in human growth factor dependent leukaemic cells

From the interleukin-3 (IL-3) responsive human myeloid cell line M-07 we derived a subclone, named M-07e, which is fully dependent for growth and survival on either granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-3. In this paper the expression, specificity and modulation of GM-CSF and IL-3 receptors on M-07e cells are described. The specificity of the binding was demonstrated by the failure of other cytokines to compete, at 4 degrees C, with GM-CSF or IL-3 receptors. In addition, IL-3 was found to compete as well as GM-CSF for GM-CSF receptors while GM-CSF was a weak competitor for IL-3 receptors. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high-affinity GM-CSF binding sites (405 +/- 27.4 sites per cell, dissociation constant at the equilibrium 52 +/- 20 pM) and the presence of high and low-affinity regions for IL-3 binding sites (27 +/- 12 and 416 +/- 92 sites per cell for the high and low affinity regions respectively, dissociation constant at the equilibrium, 58 +/- 22 pM and 5.7 +/- 2.0 nM respectively). Finally, in agreement with the hierarchical down-modulation model, we found that IL-3 can down-modulate both IL-3 and GM-CSF receptors while GM-CSF can down-modulate only its own receptors. The present results suggest that M-07e cells, in spite of their neoplastic nature, share, with murine bone marrow cells, similar growth factor receptor regulatory mechanisms. This cell line may be regarded as a candidate model to investigate the physiological events triggered by growth factors binding to human haemopoietic cells.     

Isolation and in vitro differentiation of human erythroid precursor cells.

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.     

Ultrastructural localization of the transferrin receptor and transferrin on marrow cell surfaces

The monoclonal antibody OKT9 (a known transferrin receptor antibody) and a monoclonal antibody to transferrin (ATfn) were used to localize the transferrin receptor and transferrin on marrow cells. After incubation of cell suspensions with the antibody, the cells were reacted with an affinity purified Fab fragment of goat anti-mouse IgG conjugated to horseradish peroxidase (GAM-HRP), which was in turn visualized by reaction with 3,3'-diaminobenzidine (DAB). Erythroblast cell surfaces stained intensely with OKT9-GAM-HRP-DAB, weaker staining was observed on reticulocyte surfaces, whereas erythrocyte surfaces lacked staining. Staining was present on surface caveolae, which often contained endogenous ferritin particles. Moderate to strong OKT9 staining was observed on less than 10% of presumed lymphoid cells. Monocytes, macrophages, promyelocytes, granulocytes, megakaryocytes and platelets consistently lacked OKT9 staining. ATfn-GAM-HRP-DAB staining of erythroid cells was similar to that observed with OKT9 staining; however, in contrast to the latter staining, ATfn stained the surfaces of megakaryocytes, platelets, monocytes and most lymphocytes. Promyelocytes stained weakly, whereas late granulocytes lacked staining. These results indicate that the T9 transferrin receptor (1) is largely confined to erythroid cells in marrow, (2) is diffusely distributed on the surface of early erythroid cells, (3) decreases with cell maturation, and (4) is lost when haemoglobin synthesis is completed. Transferrin appears in a similar distribution on erythroid cell surfaces but also appears to bind to some cell surfaces lacking the T9 receptor.