The action of erythropoietin as the inducer of erythroid differentiation.
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Cell Differentiation↗
Biomedical subjects
E Goldwasser
Publications and source records attributed to E Goldwasser.
Production of erythropoietin by cloned malignant murine erythroid cells.
A specific immunological assay was used to demonstrate that the erythropoietic factor produced by the recently described FMuLV-induced murine erythroleukemic cell line IW32 is an authentic erythropoietin (epo). Several independent virus-induced erythroleukemic and myeloblastic cell lines were tested for epo production. Among six erythroleukemic cell lines induced by FMuLV, another (NN10) was shown to produce epo by biological and immunological assays. Four Friend-virus-induced erythroleukemias and four FMuLV-induced myeloblastic cells were negative. The amounts of epo produced were similar in IW32 and NN10 supernatants after 48 h in culture. The in vitro bioassay gave the highest levels (up to 1000 mU/ml), the in vivo bioassay the lowest, and the radioimmunoassay gave intermediate results. NN10 and IW32 cell lines have been induced by two different FMuLV and were shown to be independent by cytogenetic studies. The molecular weights of IW32 and NN10 epo were close to the molecular weight of mouse plasma epo but elution profiles suggested that some differences might exist between these epos. Cloned IW32 and NN10 cells were shown to retain both the ability for erythroid differentiation after incubation with chemical inducers and the ability to produce epo. This demonstrates that malignant erythroid cells were the source of epo production in these cell lines.
Animals↗
Autosomal dominant polycythemia.
Two families with polycythemia inherited as an autosomal dominant trait are described. Serial hemoglobin determinations in multiple family members and RBC volume measurements in selected affected subjects documented their polycythemia. Measurements of arterial p02s, p50s, and blood oxygen affinity were normal in all affected individuals from each family who were tested. Erythropoietin (EPO) levels were low in affected individuals from family 1 and normal in affected members of family 2. Stimulation of in vitro CFU-E colony growth by low levels of EPO was significantly increased in subjects from family 1, but normal in those affected from family 2. We conclude that although the inheritance pattern for the polycythemia in both of these families appeared to be the same, the biologic defect leading to the disorder in each of these unique families was different. The precise mechanism of the increased EPO sensitivity noted in affected subjects from family 1 awaits elucidation.
Adenosine Triphosphate↗
Specific binding of erythropoietin to spleen cells infected with the anemia strain of Friend virus.
Tritiated erythropoietin with full biological activity has been prepared, and a relatively homogeneous population of enriched progenitor cells that respond to the hormone has been generated by infection of mice with the Friend virus that produces anemia. These cells, obtained from the spleens of infected mice, develop into mature erythroblasts and erythrocytes in the presence of erythropoietin. We have measured the binding of erythropoietin to these target cells; 62% of the binding was inhibited by excess unlabeled erythropoietin, but no inhibition occurred with albumin, serum, or a variety of growth factors and glycoproteins. Apparent equilibrium was reached by 2 hr at 37 degrees C and by 3.5-4 hr at 10 degrees C. The extent of specific binding increased linearly with cell concentration. In binding experiments at 10 degrees C, apparent saturation of specific binding occurred at approximately equal to 8.7 nM. Scatchard analysis showed a single class of binding sites. The dissociation constant is 5.2 nM with an average of 660 binding sites per cell. At 0.06 nM, where most of the cells are induced to terminally differentiate in vitro, an average of only 8 erythropoietin molecules bound per cell. These studies indicate that erythropoietin attaches to specific binding sites, which are most likely receptors since they manifest high affinity and specificity, and that the biologic effect of the hormone may be produced by attachment of a very small number of erythropoietin molecules.
Anemia↗
Metabolism of pure human erythropoietin in the rat.
The metabolism of pure human erythropoietin (EPO) labeled with 125I was studied in the rat. Concentrations of the labeled hormone (125I-EPO) in plasma and urine were measured by both trichloroacetic acid precipitation and gel filtration. During steady-state conditions the metabolic clearance rate of 125I-EPO was slow, averaging 256 +/- 7 microliter. min-1 X kg-1 of which only 19 +/- 2 microliter X min-1 X kg-1 (or 7.4 +/- 0.8% of the metabolic clearance rate) could be accounted for by excretion of the labeled hormone in the urine. Urinary clearance of 125I-EPO amounted to less than 0.3% of the glomerular filtration rate, and there was no detectable arteriovenous concentration difference of 125I-EPO across the kidney. After both pulse injection and constant infusion to equilibrium, disappearance of 125I-EPO from the circulation could be approximated by a single exponential function: plasma half-life was 3.5 +/- 0.2 h in normal rats and was prolonged to 4.4 +/- 0.3 h (P less than 0.05) in animals with ligated renal pedicles. Although kidney homogenates degraded 125I-EPO in vitro (optimum pH 4.5), the hormone did not accumulate in the kidney when injected intravenously. We conclude that EPO metabolism is extremely sluggish compared with that of other polypeptide hormones. Whereas kidney tissue is capable of degrading EPO in vitro, the physicochemical characteristics of this glycoprotein (molecular size, shape, and charge) probably impede its access to degrading sites and therefore account for the limited contribution of renal extraction and excretion to the metabolic clearance of the hormone.
Animals↗
Erythropoietin and its mode of action.
Erythropoietin (Epo) is an unambiguous inducer of red cell differentiation in mammals. Purified Epo, derived from urine of anemic patients, is a glycoprotein of molecular weight about 34,000. There are two forms with the same biological activity and amino acid composition but with differing carbohydrate compositions. All of the carbohydrate is in the form of N-linked complex oligosaccharides. Erythropoietin appears to contain three domains, two protease-resistant regions containing all of the carbohydrate, connected by a protease-sensitive region containing the active site. Cells derived from mice infected with the anemia variant of the Friend leukemia, bind Epo specifically. The receptors have a dissociation constant of about 5 nM and there are only 600-700 per cell. Erythropoietin has a rapid effect on the transcription and processing of the globin genes. Among the enzymes of heme synthesis, it has an early effect only on uroporphyrinogen synthase. Studies with Epo and colony-stimulating factor (CSF) suggest that these inducers compete for the same, or very closely related cells of the hemopoietic system. When marrow cells are put into close apposition by centrifugation, their responses to Epo and CSF and the competitive actions of these inducers increase.
Animals↗
Comparison of allogeneic and self-restricted stimulation of lymphokine production by dual-reactive cloned T cells.
Murine T cell clones were derived that proliferated in response to stimulation by alloantigen or by ovalbumin (OVA) in the presence of irradiated syngeneic spleen cells. Two cloned cell lines of strain B10.BR (H-2k) proliferated in response to alloantigen encoded by I-Ab, whereas the response to OVA was restricted by an element encoded by I-Ak. A cloned cell line of strain B10.A (H-2a) proliferated in response to alloantigen encoded by I-As, whereas the response to OVA was restricted by an element encoded by I-Ak. Cloned cells were stimulated by alloantigen or by OVA to produce lymphokines and to incorporate thymidine. Culture supernatants were collected 24 hr later and were assayed for interleukin 2, colony stimulating factor, interferon, Ia-inducing activity, and interleukin 3; thymidine incorporation was measured 72 hr after stimulation. For each clone tested, stimulation by alloantigen or by OVA led to the production of an identical array of lymphokines. Likewise, the strength of stimulation by alloantigen was approximately equal in magnitude to the strength of stimulation by a particular concentration of OVA. Lymphokine production and thymidine incorporation were co-variant measures of the intensity of stimulation. These data, in combination with data linking OVA reactivity and alloreactivity to identical regions of the major histocompatibility complex, suggest that dual reactivity represents a cross-reaction between alloantigen and self determinants associated with nominal antigen.
Animals↗
Erythropoietin and erythroid differentiation.
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Cell Differentiation↗
Multiple hemopoietic lineages are found after stimulation of mouse bone marrow precursor cells with interleukin 3.
When the murine T-lymphocyte clone L2 is stimulated with concanavalin A, it secretes at least two distinct factors that affect hemopoietic precursor cells, interleukin 3 (IL3) and granulocyte/macrophage colony-stimulating factor (GM-CSF). IL3 accounts for approximately 10% of the colony-stimulating activity in L2-cell-conditioned medium. The IL3 secreted by L2 cells is similar antigenically to the IL3 secreted by WEHI-3 cells. Like the IL3 from WEHI-3 cells, IL3 secreted by L2 cells does not bind to DEAE Sephacel and can be separated from the L2-cell GM-CSF, which does bind to DEAE. By assessment of the functional, morphologic, surface phenotypic, and cytochemical characteristics of bone marrow cells 6 days after stimulation with IL3 in liquid culture, four hemopoietic lineages were found, including macrophage, neutrophilic granulocyte, megakaryocyte, and basophil/mast cell. In addition, when bone marrow cells were stimulated with IL3 in semisolid medium, several types of colonies were found, including mixed colonies containing macrophage, megakaryocyte, and granulocyte lineages.
Animals↗
Characteristics and function of factors affecting erythropoiesis.
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Animals↗
A microassay for colony-stimulating factor based on thymidine incorporation.
A variety of growth factors and lectins were tested; only colony-stimulating factors CSF-1, Interleukin 3, and a T-lymphocyte GM CSF induced colony formation in semisolid medium and stimulated thymidine incorporation in liquid culture. All other growth factors and lectins were inactive in both assays. Factor-stimulated thymidine incorporation was detectable 24 hours after stimulation and reached maximal levels 4-6 days after stimulation. A convenient microassay for measuring CSF activity has been developed, enabling a large number of samples to be screened qualitatively in 2 days and permitting CSF activity to be measured quantitatively in 4-5 days. This microassay can supplement the clonal-cell assay method and be especially useful as an initial screening assay for CSF activity.
Animals↗
Increased immunoreactive erythropoietin in serum and kidney extracts.
Radioimmunoassay of erythropoietin (epo) was performed upon serum samples and kidney extracts from rats that received intrarenal injections of nickel subsulfide (Ni3S2, 20 mg/rat). Serum epo concentrations were significantly increased from 1 to 4 weeks after Ni3S2 injection; the highest values occurred at 3 weeks, when serum epo in 5 Ni3S2-treated rats averaged 230 mU/ml (range = 94 - 370 mU/ml); for comparison, serum epo in 21 vehicle-treated controls averaged 19 mU/ml (range = less than 14 - 56 mU/ml, P less than 0.01). In kidney extracts of 5 rats killed 3 weeks after Ni3S2 injection, epo concentrations averaged 970 mU/g (range = 210 - 1880 mU/g); for comparison, epo in kidney extracts of 6 controls averaged 99 mU/g (range = 40 - 140 mU/g, P less than 0.005). These radioimmunoassays confirm previous bioassays findings that serum epo concentrations are significantly increased in rats with Ni3S2-induced erythrocytosis. Contrary to bioassay findings, this study demonstrates increased immunoreactive epo concentrations in kidney extracts of Ni3S2-treated rats, suggesting that renal epo in Ni3S2-treated rats occurs in a form that lacks biological activity in vivo.
Animals↗
Inhibition of heme synthesis in bone marrow cells by succinylacetone: effect on globin synthesis.
The effects of 4,6-dioxoheptanoic acid (succinylacetone, SA), an inhibitor of delta-aminolevulinic acid dehydratase, on total iron uptake, heme synthesis, and globin synthesis were studied in rat marrow cells in culture in order to examine the coordination of heme and globin synthesis. SA inhibited heme synthesis in both control and erythropoietin-stimulated cells in a dose-dependent fashion; at 10(-3) M, inhibition was complete, whereas at 10(-7) M, there was no significant effect. Inhibition of total iron uptake was also dose-dependent although, at 10(-3) M, it was not complete. The inhibition of heme synthesis by SA was partially overcome by addition of 10(-4) M porphobilinogen or protoporphyrin IX. SA caused an almost complete suppression of globin formation in both erythropoietin-stimulated and unstimulated cells as early as five hours after the addition of the inhibitor. When inhibition of heme synthesis was incomplete, globin synthesis was partially inhibited. These results indicate that heme synthesis is required for erythropoietin-mediated induction of globin synthesis in cultured bone marrow cells.
Animals↗
Purification of a human urinary colony-stimulating factor.
Colony-stimulating factor (CSF), a protein required for the in vitro formation of colonies composed of granulocytes and/or macrophages, was isolated from the urine of anemic patients by using a seven-step procedure. The purified, homogeneous CSF had a specific activity of 1.9 X 10(8) U/absorbance unit at 280 nm (AU). This represents an overall purification of 25,330-fold and a total recovery of 3.8%. Upon iodination of the protein, the radioactivity migrated on sodium dodecyl sulfate (SDS) gel electrophoresis as a single peak with an apparent molecular weight of 46,000; reduction with mercaptoethanol caused dissociation to a single component of molecular weight 23,000. Only the dimer is active in stimulating colony formation. Urinary CSF stimulates formation of colonies comprising only macrophages in the mouse bone marrow cell culture assay. A neutralizing antibody raised against mouse L-cell CSF did not neutralize the activity of the urinary CSF but did bind it. This may indicate that the relative positions of antibody binding sites and the active sites are different in these two glycoproteins.
Chromatography↗
Characterization of murine hemopoietic cells using rat anti-mouse monoclonal antibodies.
Three rat anti-mouse monoclonal antibodies reactive with different populations of cells in mouse bone marrow have been extensively characterized with respect to their binding to cells in the lymphohemopoietic system. One antibody identifies lymphocytes committed exclusively to the B lineage. A second antibody identifies cells of the myeloid lineage including granulocytes and macrophages. The third antibody binds to nearly all of the bone marrow cells including erythrocytes, however, quantitative differences in antigenic expression allows the discrimination between early and late erythroid cells and can be used to purify eosinophils. The differential binding of these three monoclonal antibodies to mature cells, immature cells and progenitor cells (CFU-S, CFU-C (GM), BFU-E, and CFU-E) permits the construction of a scheme of lymphohemopoietic differentiation based on the expression of three cell surface antigens.
Animals↗
Biochemical enrichment of lymphokines secreted by a cloned helper T lymphocyte.
The Mls-reactive murine helper T cell clone L2 produces at least 10 lymphokine activities affecting at least five distinct target cells. Culture conditions can be optimized to enable production of high levels of colony-stimulating factors (CSFs) by lectin stimulation of L2 cells under serum-free conditions. Selective enrichment of three lymphokine activities--interleukin 2, CSF, and interferon--can be achieved by using a combination of phenyl-Sepharose and hydroxyapatite chromatography. At least two types of CSF can be separated by concanavalin A-Sepharose chromatography. The CSF that does not bind to concanavalin A-Sepharose can be enriched to a specific activity of at least 5 X 10(8) U/mg of protein by using ion exchange high-pressure liquid chromatography.
Animals↗
Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity.
Interleukin 3 (IL 3) was initially defined as a factor in conditioned media from concanavalin A-stimulated lymphocytes (Con A CM) that induces the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of nu/nu splenic lymphocytes. To determine the spectrum of additional "biologic" activities, IL 3 was purified to homogeneity and its properties were assessed. The protein preparation was judged to be homogeneous IL 3 by the following criteria: 1) elution of a peak of IL 3 with a constant specific activity in the last step of purification, 2) presence of a single protein by SDS-PAGE analysis, 3) receptor-binding activity against IL 3-dependent cell lines, 4) a specific activity of congruent to 0.2 ng/ml required for 50% of maximal biologic activity, and 5) the presence of a single amino terminal sequence. With the use of this preparation of IL 3, the dose-response curves for 20 alpha SDH induction were identical or similar to the dose-response curves for the activities of 1) WEHI-3 growth factor, 2) mast cell growth factor, 3) P cell-stimulating factor, and 4) histamine-producing cell-stimulating factor. In addition, homogeneous IL 3 had colony-stimulating factor activity, although only approximately 2% of the total CSF activity found in Con A CM was associated with IL 3. The major peak of CSF activity could be resolved from IL 3 by DEAE column chromatography and lacked many of the biologic activities associated with IL 3.
Animals↗
Partial purification and characterization of a colony-stimulating factor secreted by a T lymphocyte clone.
The mouse T lymphocyte clone L2 secretes a minimum of 10 lymphokine activities affecting at least 5 different target cells. Large amounts of colony-stimulating factor (CSF) (8.6 X 10(6) U/ml) can be obtained by stimulating L2 cells with concanavalin A. The major CSF activity secreted by L2 cells has been enriched to a specific activity of approximately 2-4 X 10(8) colonies/mg of protein using hydrophobic-interaction, gel-permeation, ion-exchange, and lectin-affinity chromatography. This preparation of CSF contains no detectable interleukin 2, interleukin 3, or interferon. The major L2-cell CSF induces granulocyte/macrophage colonies from bone marrow cells. This GM CSF has an apparent Mr of 22,000 as determined by gel-permeation chromatography. Treatment of L2-cell CSF with proteolytic enzymes abrogates biologic activity.
Animals↗