Identification of the erythropoietin receptors of erythroleukemic cells

Erythropoietin (epo) is the physiologic regulator of red blood cell development. We have demonstrated the presence of epo receptors on two erythroleukemia cell lines, IW32 and IW201. IW32 produces epo constitutively while IW201 is a nonproducing cell line. Neither cell lines respond to epo to differentiate. In this study, we have shown that these cells bound 125I-epo specifically. Binding could be displaced by the presence of unlabeled epo but not by insulin or epidermal growth factor. In contrast to that found in epo responsive cells isolated from Friend virus infected mouse spleen, which were reported to contain both "high" and "low" affinity receptors, our finding indicated only a single class of "low" affinity receptors with apparent dissociation constants of 1.7 nM and 2.0 nM for IW32 and IW201 cell lines, respectively. It is suggested that the absence of "high" affinity receptors may account for the lack of responsiveness of these cells to epo.     

Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.     

Erythropoietin causes down regulation of colony-stimulating factor (CSF- 1) receptors on peritoneal exudate macrophages of the mouse

We have shown that erythropoietin (epo), the primary regulator of erythrocyte formation, diminished the binding to peritoneal exudate macrophages (PEM) of the principal macrophage growth regulator, colony- stimulating factor (CSF-1). The effect of epo on 125I-CSF-1 binding was dose-dependent; at a concentration of 1-2 U of epo/ml (10(-10) M), CSF- 1 binding was almost completely suppressed. Erythropoietin did not compete with CSF-1 for occupancy of the latter's receptors. The effect of epo on CSF-1 binding occurred at 37 degrees C but not at 2 degrees C, and during the continuous exposure of PEM to epo at 37 degrees C we found that CSF-1 binding reached a nadir at 1 h and recovered to pre- exposure levels in 7 h. Our novel results are consistent with the notion that specific receptors for epo exist on the cell surface of PEM and that binding of epo sets in motion a series of cellular events resulting in the internalization of CSF-1 receptors. Thus epo causes down regulation of CSF-1 receptors on PEM. We have previously shown that epo causes suppression of CSF-induced granulocyte-macrophage colony formation by mouse bone marrow cells. The results we present here provide a possible mechanism for these results.     

Cloning and expression pattern of the Xenopus erythropoietin receptor

Cytokine signaling plays an important role in the survival and differentiation of vertebrate hematopoietic cells. In red blood cells, erythropoietin is a key component of the differentiation program and maintains the homeostasis of the erythroid compartment. In the adult, anemia stimulates high levels of circulating erythropoietin that drives erythropoiesis to restore normal levels of red blood cells in circulation. Erythropoietin activates the erythropoietin receptor on immature red blood cell precursors to promote their survival and differentiation. Although extensively studied in mammalian systems, a complete understanding of the function of the erythropoietin receptor during primitive erythropoiesis has been lacking. To address this problem, we have cloned the Xenopus laevis erythropoietin receptor in order to further understand the development of primitive erythropoiesis. The amphibian erythropoietin receptor shares 33% amino acid sequence identity with the mammalian erythropoietin receptors and contains the conserved extracellular ligand binding and fibronectin domains, the WSXWS motif common to cytokine receptors, and several tyrosine phosphorylation sites located on the intracellular domain of the receptor. Expression of the erythropoietin receptor is first detected by in situ hybridization in the ventral blood island during tailbud stages.     

Identification of erythropoietin receptors in isolated membranes from Friend virus infected mice spleen cells

It has been shown that radiolabeled erythropoietin (epo) specifically binds to homogeneous epo-responsive spleen cells from mice infected with the anemic variant of the Friend virus. We now report that membranes isolated from these cells retain the ability to specifically bind epo. Spleen cells were swollen in ice-cold 10 mM Tris-Cl pH 7.4 containing 0.2 mM phenylmethyl sulfonylfluoride for 5 minutes, after which the cells were homogenized and centrifuged to remove nuclear fraction. Membranes were collected by centrifuging the supernatant at 75,000 g for 90 min. Utilizing 3H-epo labeled at the terminal sialic acids of the carbohydrate moieties or 125I-epo labeled at the tyrosine residues, it was shown that the isolated membranes contained specific epo binding sites. About 90% of the binding could be inhibited by the presence of unlabeled epo whereas no inhibition was seen with other glycoproteins and growth factors. Equilibrium could be reached in approximately 2.5 hr at 25 degrees C or 1.5 hr at 37 degrees C. Binding could be saturated at an epo concentration of about 3 nM, Scatchard analysis indicated a single class of receptors with a Kd of approximately 1.5 nM.     

The chicken oviduct and embryonic red blood cell transferrin receptors are distinct molecules

We recently described an estrogen-inducible transferrin receptor from the chicken oviduct. We now report on the comparison of the oviduct transferrin receptor with the transferrin receptor obtained from chick embryo red blood cells. Western blot analysis reveals that rabbit polyclonal antibodies raised against one receptor do not cross react with the heterologous receptor. Furthermore, peptide map analyses of either affinity purified, native [125I]-labelled transferrin receptors (dimers) or dissociated, and repurified monomers obtained from oviducts and embryonic red blood cells yield distinct patterns. Therefore, the estrogen-modulated oviduct transferrin receptor appears to be structurally distinct from the iron-modulated red cell transferrin receptor.     

Rosette formation by human thymocytes

A proportion of lymphocytes in human fetal and post-natal thymus, and in blood, formed rosettes with red blood cells from sheep and pig. The count of rosette-forming cells (RFC) among human thymocytes varied widely, from 2–216 per thousand cells, and was higher in fetal than in post-natal life. The count of RFC among human thymocytes was not reduced by specific rabbit anti-human immunoglobulin sera, indicating that the receptor was not of immunoglobulin character; the reaction was inhibited by antithymocyte serum and metabolic poisons and certain enzymes. The receptor may be equivalent to other “non-specific” glycoprotein hemagglutinins in plants and viruses.The importance of species differences in immunological assays is emphasized. Thus human thymocytes gave high counts of RFC only with red blood cells of sheep and pig; moreover thymus lymphocytes from only man and pig, but not several other species including rodents, were highly reactive with sheep red blood cells. The capacity for rosette formation could be a marker for T cells in human blood.     

Linkage between the B-cell specific receptor for monkey red blood cells and HL-A antigens in man-mouse hybrids

Immunogenetics - The established human lymphoid cell lines 6410 and WI-L2 exhibit the recently discovered receptor for monkey red blood cells (MRBC). This receptor is specific for B cells. These...     

Erythropoietin binding and induced differentiation of Rauscher erythroleukemia cell line red 5-1.5

We isolated from the Rauscher erythroleukemia cell line (Red 5), a subclone (Red 5-1.5), which contains erythropoietin (epo) binding sites and demonstrates an epo-dependent erythroid differentiation. One class of high affinity binding sites was detected with a Kd (+/- S.D.) of 0.43 +/- 0.09 nM and a mean density/cell of 1200 +/- 311. The cell-associated 125I-epo was displaced by nonlabeled epo but not by other hormones or factors. The 125I-epo binding to Red 5-1.5 cells was maximal within 3 h at 15 degrees C and 1 h at 37 degrees C and proportional to cell number. The addition of epo increased [3H] uridine incorporation into RNA by 6 h and [3H]thymidine incorporation into DNA by 60 h followed by 59Fe incorporation into protein, cell proliferation, and formation of hemoglobin-containing colonies. The incorporation of 59Fe into protein demonstrated a linear dose response (from 0.002 to 1.5 units of epo/ml) beginning 60 h after addition of the hormone to the cultures, and there was a dose-dependent increase (from 0.1 to 1.0 unit of epo/ml) in the formation of hemoglobin-containing colonies. We concluded that the binding of 125I-epo to Red 5-1.5 suggests the presence of specific epo receptors. The sequence of the epo-induced proliferation and differentiation events is similar to primary erythroid cultures but requires longer epo exposure. Receptor occupancy correlates with the induced biological response.     

Effects of gluconeogenic hormones on insulin binding in intact human red blood cells

The effects of gluconeogenic hormones, adrenaline and cortisol, on insulin binding were studied in intact human red blood cells. Insulin binding was significantly decreased when red blood cells were preincubated with 1.0 microgram . ml-1 adrenaline or cortisol respectively. The Scatchard plot suggested that this was due to a decrease in surface receptor concentration. Furthermore, it showed that adrenaline also increased insulin receptor affinity. The negative co-operativity affinity profile demonstrated that adrenaline caused a rise in only the upper limit average affinity, Ki, of the insulin receptor.     

Erythropoietin and retinoic acid,secreted from the epicardium,are required for cardiac myocyte proliferation

We have established a heart slice primary culture, which allows us to mechanically separate distinct cardiac cell populations and assay their relative mitogenic and trophic effects on cardiac myocyte proliferation and survival. Using this system, we have found that a signal(s) from the epicardium, but not the trabeculae and endocardium, is required in embryonic day 10 (E10) chick heart slices for continued cardiac myocyte proliferation and survival. An examination of potential epicardial growth or trophic factors has revealed that blockade of either retinoic acid (RA) or erythopoietin (epo) signaling from the epicardium inhibits cardiac myocyte proliferation and survival. The blockade of cardiac myocyte proliferation following administration of an RA antagonist can be rescued by exogenous epo. Conversely, the blockade of cardiac myocyte proliferation following administration of an anti-epo receptor antisera can be rescued by exogenous RA. Thus, our findings suggest that RA and epo signals work in parallel to support myocardial cell proliferation. In addition, we have found that these factors do not act directly on myocardial cells. Rather, they induce another soluble factor(s) in the epicardium that directly regulates proliferation of cardiac myocytes. We therefore postulate that the epicardium controls normal heart growth in ventricular segments of the embryonic chick heart by secreting a cardiac myocyte mitogen whose expression (or activity) is regulated by both RA and erythropoietin signaling.     

The receptors ensuring Staphylococcus aureus fixation on fibronectin-containing surfaces

The role of protein A and other components of S. aureus cell wall in binding fibronectin on the surface of formulated sheep red blood cells was studied. 41 out of 89 fibronectin-binding clinical isolates lost their capacity for agglutinating fibronectin-sensitized red blood cells after the treatment of such cells with the solution of commercial purified protein A. These strains were also shown to have pronounced direct relationship between the levels of binding of fibronectin and IgG. Other isolates in the collection possessed the protein A-independent receptor capable of binding fibronectin. The receptor was seemingly common for this group of strains, and its presence significantly increased the capacity of staphylococci for binding fibronectin.     

Biological roles of blood group antigens

Recognition and application of blood group differences on human red cells permitted the development of safe procedures for blood transfusion. Blood group antigens are markers on surface-exposed red cell proteins or the sugar moiety of glycoproteins or glycolipids. Apart from their presumed biological function, some antigens have been identified as receptors for host/parasite interactions. Thus, carbohydrates that determine P antigenicity are the binding receptor for certain strains of pyelonephritic coliforms. Other pathogenic coliforms bind to the membrane structure that carries the Dra antigen. A structure associated with Duffy antigens is the attachment receptor for the parasite of Plasmodium vivax malaria, while Plasmodium falciparum parasites bind to structures associated with membrane glycophorins. Structure/function relationships have been established by the finding that lack of Rh protein in red cells of Rhnull phenotype is associated with stomatocytic cell morphology and a hemolytic state. Absence of glycophorin C, and the Gerbich blood group antigens that it carries, is associated with elliptocytic red cells. Absence of Kx antigen protein in the Kell system is associated with the McLeod blood group phenotype, with acanthocytic cell morphology and reduced in vivo survival. McLeod individuals also have late-onset muscular dystrophy and neurological disorders.     

Characterization of mononuclear phagocytes in human CSF using membrane markers.

The purpose of this investigation was to demonstrate those membrane receptor sites on mononuclear phagocytes of human CSF which provide additional evidence for their monocytic origin and function. Using a heterologous system, sheep red blood cells were coated with IgG- and IgM-fraction of the anti-Forssman-antiserum of rabbits. In another series of experiments, sheep red blood cells were additionally sensitized with fresh human serum as a source of complement. The possible inhibitory effect of human IgG on the uptake of red cell antibody complexes was tested. Washed and pretreated sheep red cells were added to different fresh CSF specimens from patients, whose CSF exhibited no conspicious biochemical, serologic or cytologic alterations. The percentage of phagocytizing mononuclear phagocytes was evaluated. When the particular IgG EA reagent described was utilized, most of the mononuclear phagocytes consistently exhibited the IgG- and complement-receptor activity which selectively characterizes blood monocytes and related cells.     

In vitro attachment of mono- and oligosaccharides to surface glycoconjugates of intact cells

We have synthesized glycosylhydrazines of various mono- and oligosaccharides and coupled these to periodate- or galactose oxidase-treated human red cells and K562 erythroleukemia cells. The optimal conditions for this carbohydrate modification of cells have been established. This method makes it possible to specifically elongate oligosaccharide chains of cell surface glycoconjugates with desired carbohydrates. In this way, new antigenic and receptor properties can be conferred to cells, and the functional roles of carbohydrates in cell surface glycoconjugates can be studied. The method has been used to make red cells of blood group O reactive with anti-A and anti-B sera, and in rendering K562 cells or red cells of blood group O agglutinable with the alpha-N-acetylgalactosamine-specific Helix pomatia lectin.     

Characterization of human red blood cell tyrosine kinase

A new tyrosine kinase in human red blood cells has been characterized and partially purified. The major substrate was a protein of molecular weight 93 K which could be phosphorylated both in whole red blood cells incubated with inorganic [32P] orthophosphate and in ghost preparations incubated with [gamma 32P] ATP. This tyrosine kinase displayed an alkaline isoelectric pH (around 8.5), a molecular weight of 32-33 K and does not seem to be autophosphorylable. Some kinetics of the enzyme are reported. This red blood cell tyrosine kinase is unrelated to EGF and insulin or insulin-like receptor subunits. This enzyme may represent a novel class of tyrosine kinases.     

Hydrolysis of phosphatidylserine-exposing red blood cells by secretory phospholipase A2 generates lysophosphatidic acid and results in vascular dysfunction

Secretory phospholipase A(2) (sPLA(2)) type IIa, elevated in inflammation, breaks down membrane phospholipids and generates arachidonic acid. We hypothesized that sPLA(2) will hydrolyze red blood cells that expose phosphatidylserine (PS) and generate lysophosphatidic acid (LPA) from phosphatidic acid that is elevated in PS-exposing red blood cells. In turn, LPA, a powerful lipid mediator, could affect vascular endothelial cell function. Although normal red blood cells were not affected by sPLA(2), at levels of sPLA(2) observed under inflammatory conditions (100 ng/ml) PS-exposing red blood cells hemolyzed and generated LPA (1.2 nM/10(8) RBC). When endothelial cell monolayers were incubated in vitro with LPA, a loss of confluence was noted. Moreover, a dose-dependent increase in hydraulic conductivity was identified in rat mesenteric venules in vivo with 5 microM LPA, and the combination of PS-exposing red blood cells with PLA(2) caused a similar increase in permeability. In the presence of N-palmitoyl L-serine phosphoric acid, a competitive inhibitor for the endothelial LPA receptor, loss of confluence in vitro and the hydraulic permeability caused by 5 microM LPA in vivo were abolished. The present study demonstrates that increased sPLA(2) activity in inflammation in the presence of cells that have lost their membrane phospholipid asymmetry can lead to LPA-mediated endothelial dysfunction and loss of vascular integrity.     

Activation of erythropoietin signaling by receptor dimerization

The hormone erythropoietin (Epo) is essential for red blood cell development. Epo binds a high affinity receptor on the surface of erythroid progenitor cells, stimulating receptor dimerization and activation of the intracellular signal transduction pathways that support erythroid cell survival, proliferation and differentiation. Biochemical and structural analysis of the erythropoietin receptor (EpoR) is revealing the molecular mechanisms of EpoR function, leading the way to the development of small molecule Epo mimetics. This review focuses on the role EpoR dimerization plays in receptor function.     

Unmasking by antimetabolites of receptors for sheep red blood cells on human lymphocytes

The action of antimetabolites (puromycin, cycloheximide) and cold was studied in the human rosette system. We found that the number of detectable receptors for sheep red blood cells on peripheral blood lymphocytes was increased in presence of some concentration of these drugs. A similar finding was noted when the blood lymphocytes were left at 4 °C. The possibility that both cold and antimetabolites, by modifying the cell membrane mobility, increase the receptor affinity and thus the number of detectable receptors is discussed. Another attractive possibility is also presented. We propose that the unmasking effect by antimetabolites is due to inhibition of protein synthesis which is necessary to better express the receptors for sheep red blood cells on human lymphocytes. This concept of decreased protein synthesis affecting the expression of surface receptors may be a more general phenomenon.