Expression of transferrin receptors during erythroid maturation

A monoclonal antibody, F111/2Dl, produced after immunisation of C3H/He mice with the human erythroleukemia cell line, K562, is described. It detects cell surface determinants of similar distribution to those characterised by the OKT-9 monoclonal antibody, which has been shown to identify the transferrin receptor. The F111/2Dl antibody, as well as OKT-9, has been used to investigate the distribution of transferrin receptors during erythroid maturation in normal bone marrow and peripheral blood, and on the K562 cell line during erythroid differentiation, induced in vitro.     

Exposure to chemokines during maturation modulates antigen presenting cell function of mature macrophages

Macrophages generated with macrophage-colony stimulating factor (M-CSF) are defective in antigen presenting cell (APC) function, although they do express major histocompatibility (MHC) class II molecules, numerous accessory molecules, and intercellular adhesion molecules. In the present study, we show evidence that the acquisition of APC function is influenced significantly by microenvironmental condition of development. Macrophages generated from bone marrow progenitor cells with M-CSF and interleukin (IL)-6 were defective in APC function as determined by their ability to induce anti-CD3 monoclonal antibody (mAb)-primed T cell proliferation. Macrophages generated in the presence of some of the CC chemokines such as leukotactin-1, macrophage inflammatory protein (MIP)-1alpha, and RANTES together with M-CSF and IL-6, however, induced proliferation of anti-CD3 mAb-primed T cells. Maximum level of APC function was obtained when developing macrophages were exposed with the chemokines at the late stage of maturation. Enhanced APC function of the macrophages appeared to be correlated with the expression of co-stimulatory molecules and the ability to produce cytokines. These results suggest that the acquisition of APC function of mature macrophage is modulated significantly by the microenvironmental condition during development.     

Autophagy in embryonic erythroid cells: its role in maturation

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.     

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells.

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small.Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis.Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.     

Distribution and shedding of the membrane phosphatidylserine during maturation and aging of erythroid cells

Maturation and aging of erythroid cells are accompanied by extensive remodeling of the membrane and a marked decrease in cell size, processes that are mediated by externalization and shedding of phosphatidylserine (PS). In the present study, we investigated the redistribution of PS in the plasma membrane of erythroid precursors during their maturation and of mature RBCs during senescence, and the involvement of changes in calcium (Ca)-flux in these processes. Maturation was studied by analyzing normal human bone marrow cells as well as cultured human normal erythroid precursors induced by erythropoietin and murine erythroleukemia cells induced by hexamethylene-bisacetamide. Senescence was studied in normal human peripheral RBCs following density fractionation. PS and Ca were determined by flow cytometry using annexin-V and Flu-3, respectively. The outer, inner and shed PS were quantified by a novel two-step binding inhibitory assay. The results indicate a bi-phasic modulation of intracellular Ca and PS externalization/shedding; both of which decreased during maturation and increased during aging. The role of intracellular Ca in PS externalization/shedding was demonstrated by modulating intracellular Ca: Ca was decreased by incubating the cells with an ion chelator (EDTA) or with decreasing concentrations of Ca, whereas treatment with the ionophore A23187 elevated intracellular Ca. The results showed that low Ca resulted in decreased outer and shed PS, whereas high Ca had the opposite effect. The results suggest that PS externalization and shedding are mediated by increased cellular Ca-flux, and that they play an important role in erythroid maturation and RBC senescence.     

Levels of active ubiquitin carrier proteins decline during erythroid maturation

Energy-dependent proteolysis is lost during maturation of rabbit reticulocytes to erythrocytes (Speiser, S., and Etlinger, J.D. (1982) J. Biol Chem. 257, 14122-14127), but nothing is known about the fates of individual components in the multienzyme ATP- and ubiquitin (Ub)-dependent proteolytic pathway during this process. Rabbit reticulocytes contain five low molecular weight carrier proteins (E2s) that form labile Ub adducts in the presence of Ub-activating enzyme (E1) (Pickart, C. M. and Rose, I. A. (1985) J. Biol. Chem. 260, 1573-1581). A method to estimate levels of active E2s in erythroid cells has been developed involving: 1) stepwise anion exchange fractionation of a soluble lysate; 2) addition of purified E1, MgATP, and radioiodinated Ub to the fractions followed by gel electrophoresis of the resulting E2-Ub adducts; and 3) quantitative densitometry of autoradiographs. Levels of active E2s are much lower in (rabbit) erythrocytes than in reticulocytes. Mean -fold decreases are: E235K, 6 x; E2(25K), 11 x; E2(20K), 18 x; E2(17K), not detected in erythrocytes; E2(14K), 12 x. The large decreases in levels of E2(20K) and E2(14K) are consistent with known functions of these proteins in DNA repair and Ub-dependent proteolysis, respectively. Decreases in levels of the other E2s, whose biological roles are presently unknown, suggest diminished requirements, if any, for them in erythrocyte metabolism. The analysis revealed two previously undescribed carrier proteins, one of which has a high molecular weight. Additional catalytic properties of E2(35K) and E2(14K) are reported.     

Acetylation of human fetal hemoglobin occurs throughout erythroid cell maturation

The biosynthesis of human acetylated fetal hemoglobin (Hb F1) has been examined by incubating the following cell types with [3H]leucine: (a) burst-forming unit erythroid cells cultured from umbilical cord mononuclear cells, (b) infant bone marrow, (c) umbilical cord blood, and (d) peripheral blood cells from adults with elevated fetal hemoglobin. Newly synthesized Hb F1 was 18-20% that of Hb F0 in burst-forming unit erythroid cells which were immature, mature, or in an intermediate state of development. In infant marrow and in infant and adult peripheral blood the extant Hb F1 comprised 10.8 +/- 1.8% of the total Hb F. In marrow cells the specific radioactivity (cpm/mg) of Hb F1 was 1.4-2.0-times greater than that of Hb F0. In peripheral blood cells these ratios were slightly greater. [3H]Leucine-labeled infant bone marrow, umbilical cord blood, and adult peripheral blood cells were subjected to density gradient ultracentrifugation. The ratios of specific radioactivity for Hb F1/Hb F0 increased from 1.0-1.8 in the lightest cell zone to 5.2-9.0 in the more dense cells. Thus the biosynthesis of Hb F1 is enhanced in cells which are more mature than those responsible for the bulk of hemoglobin synthesis, and the acetylation of Hb F provides a marker of erythroid cell maturation.     

Direct involvement of surface IA/E molecules during B cell maturation using an antigen-specific B cell clone

TP67.14 is a subclone of a resulting B cell hybridoma established by somatic hybridization between splenic B cells of A/J mice immunized with TNP-LPS and 2.52 M, a HAT medium-sensitive mutant of a B cell line; it expresses IgM, B220, IAk, and IEk on the cell membrane and also possesses a receptor molecule for TNP on its surface derived from TNP-reactive B cells of A/J mice used for cell fusion. As shown previously, TP67.14 could be induced to generate a significant amount of anti-TNP antibodies when treated with TNP-conjugated protein such as TNP-BSA and TNP-keyhole limpet hemocyanin without T cell help as well as LPS. Our study was undertaken to investigate direct involvement of surface MHC class II molecules on B cells during B cell maturation by analysis with this Ag-specific B cell clone. The data demonstrate that mAb against IAk and IEk molecules, but not IAd and H-2k, markedly inhibited the differentiative effects of LPS on TP67.14. In contrast, both antibodies specifically augmented the secretion of anti-TNP antibodies by TP67.14 treated with TNP-BSA, although these antibodies alone failed to induce the generation of anti-TNP antibodies. Interestingly, TP67.14 significantly differentiated into anti-TNP antibody secreting cells when incubated with TNP-conjugated monoclonal anti-IAk or anti-IEk antibodies alone; this differentiative effect was much greater than that of TNP-conjugated anti-IAd mAb or purified mouse IgG under the same conditions. Our result suggests that surface IA/E molecules on B cells may be directly involved in a transductional signal for B cell maturation mediated by the cross-linkage of receptor molecules on B cells with Ag.     

Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells.

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.     

In vitro studies linking induction of spontaneous antibody synthesis to an accessory cell with persisting antigen on its surface

Antibody synthesis was spontaneously induced in lymphoid cell cultures prepared from the draining lymph nodes of immunized rabbits. Induction of this response was attributed to cell-associated persisting antigen. The identity of the cell with which the antigen was associated was sought. Removal of the macrophage-rich adherent cell population did not diminish spontaneous induction. However, removal of a different cell population by the carbonyl iron method markedly inhibited spontaneous antibody synthesis. In addition, treatment of cultured lymph node cells with ethylenediaminetetraacetate, which is known to remove antigen bound to cell surfaces, also inhibited spontaneous induction. Induction of antibody synthesis could be restored in either of these situations by the addition of a suitable quantity of specific antigen. Although removal of the cell type which adhered to carbonyl iron depressed spontaneous antibody synthesis, the treatment had no effect on concanavalin A-mediated lymphocyte transformation. The combined evidence indicates that the persisting antigen responsible for induction of the spontaneous response is bound to the surface of an accessory cell which is probably a sessile macrophage or a dendritic-type cell.     

Transferrin receptor number, synthesis, and endocytosis during erythropoietin-induced maturation of Friend virus-infected erythroid cells

Erythropoietin (EP) responsive Friend virus-infected erythroid cells had 200,000 steady-state binding sites for transferrin at 37 degrees C when isolated from the spleens of Friend virus-infected mice. Upon culture of these cells with EP, the synthesis of transferrin receptors increased 4- to 7-fold and the number of transferrin-binding sites per cell doubled after 24 h. However, the rate of uptake of 59Fe from transferrin remained constant at approximately 35,000 atoms of 59Fe per minute per cell during this period in culture. The amount of 125I-transferrin internalized during the steady-state binding did not change during this culture period while the transferrin bound to the surface increased 3-fold. At all stages of erythroid maturation, the maximum rate of endocytosis was determined to be 18,000 molecules of transferrin per minute per cell, and the interval that 125I-transferrin remains in the interior of the cell was calculated to be 6.9 min. After 48 h of culture with EP, the number of steady-state transferrin-binding sites was reduced in part due to the sequestration of surface receptors within the cell. The uptake of iron from transferrin was limited by the level of endocytosis of transferrin during the initial phase of culture and the number of transferrin receptors at the cell surface during the latter stages of erythroid maturation of these cells.