On the mechanism of erythropoietin-induced differentiation. XII. A cytoplasmic protein mediating induced nuclear RNA synthesis

Erythropoietin, the primary inducer of red blood cell differentiation, has no effect on RNA synthesis by isolated bone marrow nuclei. A cytoplasmic fraction from marrow cells exposed to erythropoietin does, however, stimulate RNA synthesis by such nuclei. This marrow cell cytoplasmic factor (MCF) also stimulates RNA synthesis by liver and kidney nuclei, whereas erythropoietin has no effect on intact kidney or lung cells. MCF appears rapidly in cells after addition of erythropoietin, and its formation does not require protein synthesis. MCF is inactivated by trypsin, but not by ribonuclease. The data suggest that erythropoietin acts on the responsive cells to generate a cytoplasmic protein that mediates the effect of the hormone on nuclear RNA synthesis.     

Early production of a chemotactic factor to T lymphocytes by peritoneal macrophages

Supernatant fluids (SNF) were obtained from peritoneal exudate adherent cells stimulated in vitro with sheep red blood cells (SRBC) or BCG, and SNF collected at 6 and 24 hr were able to induce the migratory responses of rat leukocytes from the spleen and peripheral blood. The production of these SNF was dependent on protein active synthesis upon in vitro antigenic stimulation. The chemotactic activity from 6-hr SNF was inhibited by using several proteolytic enzymes and temperatures. We found the macrophages to be the producer cell of this activity, while the T cells were the target cells. The chemotactic activity from 6-hr SNF was found not to be due to IL-1. Six-hour chemotactic activity has not been reported previously.     

Analysis of rat natural killer cytotoxic factor (NKCF): mechanism of action and relationship to other cytotoxic/cytostatic factors

Natural killer cytotoxic factor (NKCF) has been proposed as one of the factors that mediates lysis induced by natural killer (NK) cells. Recently, an excellent source of NKCF has been found to be the rat large granular lymphocyte (LGL) tumor (RNK) cell line. In this study, the kinetics of lysis of the NK-sensitive, tumor target YAC-1 by the RNK-NKCF was analyzed and found to parallel that seen with NK cell-mediated killing. RNK-NKCF was also capable of killing the NK-resistant target cell, MBL-2, over a longer time period. This study utilized monoclonal antibodies (mAbs) prepared against granule protein, previously termed "anti-NKCF mAbs." These mAbs established the nature of RNK-NKCF as compared to other known cytotoxic factors in combination with studies that show that RNK-NKCF causes both 51Cr release and nuclear degradation. Antibody inhibition experiments have verified that RNK-NKCF is unique from tumor necrosis factor (TNF), leukoregulin, or complement. Anti-NKCF mAbs were capable, however, of neutralizing the RNK cell granule activity against YAC-1 tumor target cells. Based on these results, the ability of anti-NKCF mAbs to neutralize the cytolytic function of pore-forming protein (PFP), a component of these granules, was analyzed. In these experiments, the antibodies were found to inhibit the hemolytic activity of granules. Interestingly, the antibodies were effective in inhibiting the activity of unbound granule proteins as well as those bound to sheep red blood cell (SRBC) targets. Further studies to examine the target lysis requirements demonstrated that in contrast to PFP, the RNK-NKCF was able to lyse the tumor target in the absence of calcium. In addition, treatment of targets with RNA and protein synthesis inhibitors indicated that the mechanism of lysis of NKCF is quite unique from other defined cytotoxic moieties.     

Erythropoietin increases c-myc mRNA by a protein kinase C-dependent pathway

The peptide hormone erythropoietin is a major regulator of red blood cell production. While red blood cell development has been studied intensively, little is known about the intracellular signaling events that follow the binding of erythropoietin to its receptor on the target cell. We report here that erythropoietin-induced activation of the immediate early gene c-myc requires protein kinase C and that the binding of erythropoietin causes rapid phosphorylation of the major protein kinase C substrate, p80. Our results also argue for modulation of activity of a second signal transduction element in addition to protein kinase C.     

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.     

The ligand of the erythrocyte receptor of T lymphocytes: expression on white blood cells and possible involvement in T cell activation

We have recently described a monoclonal antibody (mAb) to sheep erythrocytes, termed L180/1, that blocks the formation of E rosettes between human or sheep T lymphocytes and sheep red blood cells. The cell surface glycoprotein (GP) of 42,000 apparent m.w. recognized by mAb L180/1 was given the preliminary name T11 target structure or T11TS. In the present report, it is shown that T11TS is also expressed on sheep white blood cells, notably on activated T lymphocytes, that are shown to actively synthesize this cell surface GP. In addition, the mixed lymphocyte reaction between outbred sheep is inhibited by mAb L180/1 at an early stage of the response. Together with the known involvement of the E receptor in T lymphocyte activation, these results are taken to suggest that T11 and T11TS are complementary cell interaction molecules involved in regulating T cell proliferation.     

Blood volume determinations in sheep before and after splenectomy

Using 51Cr labelled RBCs, total blood volume, red cell volume and plasma volume were measured in fifteen adult, female, domestic sheep both before and after splenectomy. Eight of the fifteen animals studied were anemic. Statistical analyses revealed no significant differences in blood volume parameters whether animals were grouped together or separated into normal and anemic groups. We observed: (a) splenectomy produced modest reductions in blood volume parameters in 12 of 15 animals, (b) preoperative variability in blood volume parameters caused by release of sequestered RBCs from the spleen was eliminated after splenectomy, and (c) equilibration of 51Cr required at least 30 minutes in intact animals, but only 10 minutes in splenectomized animals. After volume parameters were normalized to body weight, they were found to agree closely with values reported previously. This study demonstrates the dynamic function of the sheep spleen in the regulation of blood volume.     

Macromolecular insoluble cold globulin (MICG): a novel protein from mouse lymphocytes. III. Relationship to the mixed lymphocyte reaction and effect of anti-MICG antiserum in vivo.

In the present communication we have examined the relationship between the synthesis of macromolecular insoluble cold globulin (MICG) and the mixed lymphocyte reaction (MLR). In addition, we have studied in vivo the effect of antiserum to MICG on the antibody response to sheep red blood cells. The experiments indicate that MICG synthesis compared to either IgM or total protein is selectively stimulated in responder T cells exposed to allogeneic stimulator cells in the MLR. Furthermore, cytotoxicity studies utilizing anti-MICG antiserum demonstrated that T cells bearing MICG on their surface are an essential component of the responder cell population in the MLR. In vivo administration of antiserum to MICG significantly suppressed both the primary and secondary antibody response to sheep red blood cells. A possible mechanism for this suppression is discussed.     

Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Localization of erythropoietin in and around growing cartilage

Erythropoietin (EPO) is known to be a hematopoietic growth factor and a regulator of red blood cell production. Recently, EPO has also been reported to function as a tissue-protective cytokine and as an angiogenesis promoting factor. EPO is mainly regulated by hypoxia through the action of hypoxia inducible factors (HIF-1α and HIF-2α). The localization of the EPO protein and the HIF-2α protein were immunohistochemically analyzed in developing porcine embryos. Both proteins were localized in developing cartilage tissue. HIF-2α and EPO protein were expressed in the peripheral chondrocytes of cartilage anlagen, in the perichondrium and in the cell condensations that will eventually differentiate into cartilage tissue. The results of this study reveal that EPO might play a role as a survival factor or as a mitogen in developing cartilage tissue. Moreover, the presence of both proteins at the same locations supports the hypothesis that EPO expression is regulated by HIF-2α.     

Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors

The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.     

Biogenesis of erythrocyte membrane proteins in vitro studies with rabbit reticulocytes

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [³H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polycrylamide gel electrophoresis of labeled membranes on large (19 mm) gel which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [³H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removal of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Na,K-ATPase isoform expression in sheep red blood cell precursors

Isoform expression of mammalian red cell Na,K-ATPase was analyzed using messenger RNA isolated from red cell precursor-enriched bone marrow of anemic sheep. Expression of the catalytic alpha subunit was analyzed using rat isoform-specific cDNA probes and expression of the beta 1 subunit, using a sheep beta 1-specific cDNA probe. RNA isolated from sheep kidney and brain were analyzed concurrently. In the red cell, as in the kidney, messenger RNA encoding only one isoform (alpha 1) of the catalytic subunit is detected; neither of the other isoforms (alpha 2 or alpha 3) could be detected. This holds true for bone marrow of sheep of either the low potassium or high potassium phenotype. Relative to the expression of alpha 1, beta subunit-specific message (beta 1) was extremely low in the red cell compared to either kidney (less than 5%) or brain (less than 3%). Using a rat cDNA probe specific for a beta 1-like subunit, beta 2, message was detected in brain but not in either kidney or bone marrow.     

GENETIC VARIATION IN THE SHEEP RED BLOOD CELL

1. There are 7 well-established red-cell antigen (blood group) loci. The R-O system has 3 phenotypes, R, O and i, identified by the ‘naturally occurring’ antibodies, anti-R and anti-O. The R and O substances are also present in soluble form in some body secretions. The expression of R and O is controlled by a dominant gene I, epistatic in effect, at an independent locus from that of R. The systems, A, C, M-L, B, D and X-2 are identified by means of ‘immune-type’ antibodies, and several of the loci have multiple alleles. An isoenzymic form of serum alkaline phosphatase is associated with the R-O system. The frequency for the genes at the various loci has been determined in a limited number of breeds. 2. Some sheep red cells have high K+ and low Na+ concentrations (HK type, or Key), others have low K+ and high Na⁺ concentrations (LK type or Kea). Two other rare forms exist; Key type which is HK but with lower than normal K+ values, and Kep type which has approximately equal Na⁺ and K⁺ concentrations. The red cells of foetuses and newborn lambs have high K+ levels irrespective of their potassium genotype. HK cells have 3–4 times greater (Na+-K+)-activated ATPase activity, a 3–4 times increased rate of active K+ transport and a larger number of ouabain-binding sites than LK cells. Antigen M is present on homozygous HK and heterozygous LK red cells, and antigen L is present on homozygous and heterozygous LK red cells. Sensitization of LK cells with anti-L stimulates active K+ transport and ATPase activity and exposes a larger number of ouabain-binding sites in these cells. Anti-M has no effect. The red cells of newborn lambs only show weak L and M antigen activity. It is postulated that L antigen inhibits cation transport in LK cells by masking the pump sites on the membrane. Immature red cells in LK-type sheep have a high rate of active K+ transport and yet have L antigen present. No satisfactory explanation for this has yet been advanced. There is no conclusive evidence that the potassium types have any significance from the point of view of adaptation or sheep breeding. The potassium-gene frequencies are known for a large number of breeds. 3. Two allelic genes, Hb⁴ and Hb^B control 3 haemoglobin phenotypes, A, AB, and B. Foetal haemoglobin (HbF) is present in foetuses and newborn lambs. Sheep with HbA also synthesize small amounts of another haemoglobin (HbC) and under conditions of severe anaemia, synthesis of HbC takes over from that of HbA. No change in HbB is observed in anaemia. A rare haemoglobin (HbD) has been found in 3 Yugoslavian sheep. Hbs A, B, C and F differ in their physicochemical properties; they share the same alpha chains but their non-alpha chains differ in a number of amino acids. HbD differs from HbA in one amino acid in the alpha chain. Certain genetic aspects are discussed. There is some evidence that sheep with HbA are less fertile than those with HbB. The gene frequencies for Hb are known for a large number of breeds. 4. Two isoenzymic forms of carbonic anhydrase are found in red-cell lysates and these are controlled by a pair of allelic autosomal genes, producing 3 phenotypes, CAF, CAFS and CAS. Only a few breeds have been studied but CA^F is apparently quite rare. 5. An unidentified protein, designated ‘X’ is present in electrophoretic separations of haemolysates from some sheep. Its presence is dominant to its absence. Polymorphism at this locus is present in all breeds so far studied. 6. A deficiency of reduced glutathione (GSH) in red cells is found in some sheep and is inherited as an autosomal recessive disorder. Sheep with this deficiency have lower red-cell K+ and Na^f concentrations than normal and it is suggested that the HK GSH-deficient sheep may be Ked type sheep. This deficiency has so far only been found with certainty in one breed of sheep. 7. In sheep twin chimeras, admixture of red-cell antigens, haemoglobin and ‘X’ protein types has been found. Various aspects of chimerism, which occurs only rarely in sheep, are discussed. 8. The significance of the genetic variation is discussed in the light of the physiology and immunology of the red cell and of the sheep itself.     

The erythropoietin receptor transmembrane domain mediates complex formation with viral anemic and polycythemic gp55 proteins

Erythropoietin receptor (EpoR) activation is crucial for mature red blood cell production. The murine EpoR can also be activated by the envelope protein of the polycythemic (P) spleen focus forming virus (SFFV), gp55-P. Due to differences in the TM sequence, gp55 of the anemic (A) strain SFFV, gp55-A, cannot efficiently activate the EpoR. Using antibody-mediated immunofluorescence co-patching, we show that the majority of EpoR forms hetero-oligomers at the cell surface with gp55-P and, surprisingly, with gp55-A. The EpoR TM domain is targeted by gp55-P and -A, as only chimeric receptors containing EpoR TM sequences oligomerized with gp55 proteins. Both gp55-P and gp55-A are homodimers on the cell surface, as shown by co-patching. However, when the homomeric interactions of the isolated TM domains were assayed by TOXCAT bacterial reporter system, only the TM sequence of gp55-P was dimerized. Thus, homo-oligomerization of gp55 proteins is insufficient for full EpoR activation, and a correct conformation of the dimer in the TM region is required. This is supported by the failure of gp55-A-->P, a mutant protein whose TM domain can homo-oligomerize, to fully activate EpoR. As unliganded EpoR forms TM-dependent but inactive homodimers, we propose that the EpoR can be activated to different extents by homodimeric gp55 proteins, depending on the conformation of the gp55 protein dimer in the TM region.     

Interferon action: effects of mouse alpha and beta interferons on rosette formation, phagocytosis, and surface-antigen expression of cells of the macrophage-type line RAW 309Cr.1

Treatment with an essentially pure mouse α or β interferon boosts the binding and phagocytosis of opsonized sheep red blood cells by cells of the murine macrophage-like cell line RAW 309Cr.l. The kinetics and the dose dependence of the effects of the two interferons are very similar. The effects depend on continued RNA and protein synthesis, and they diminish after the removal of interferon from the medium. Studies with agents specifically binding FcRI receptors (i.e., IgG2a) and FcRII receptors (i.e., the Fab fragment of the antireceptor monoclonal antibody 2.4G2) revealed a three- to fivefold increase in the level of FcRI receptors per cell and an about twofold increase in that of FcRII receptors per cell after treatment with interferon. The enhanced binding and phagocytosis of opsonized sheep red blood cells by interferon treatment are apparently a consequence of the increased number of Fc receptors. As revealed by studies involving the binding to the cells of labeled monoclonal antibodies to several cell surface antigens, the level of the H-2D^d surface antigen is also selectively increased three- to fourfold in the cells after exposure to interferon.