Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver

It has previously been determined that erythroid cells of mice are capable of expressing such cytokines as interleukin (IL) 1 alpha and beta, IL-4, IL-6, interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta). It has been shown that glycophorin A(+) (GlA(+)) and antigen erythroblasts (AG-EB(+)) (both human erythroid cells of embryonic origin) are also capable of producing a series of cytokines such as IL-1 beta, IL-2, IL-4 and IL-6. The aim of this work was to study the capacity of erythroid cells from human embryonic liver to produce such cytokines as IFN-gamma, TGF-beta1, tumour necrosis factor alpha (TNF-alpha) and IL-10. The erythroid cells were isolated by means of antibodies specific to erythroblasts (GlA and AG-EB), as well as those from single erythroid colonies. The production level of some cytokines varies insignificantly under the action of erythropoietin (Epo) and quantitatively differs in GlA(+) and AG-EB(+) erythroid cells. Hence, the erythroid cells express IFN-gamma, TGF-beta1, TNF-alpha and IL-10. The erythroid cells could be involved through the production of these cytokines in the regulation of such processes as self-renewal, proliferation and differentiation of cells of other blood-forming sites.     

Production of cytokines by immature erythroid cells derived from human embryonic liver

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.     

Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.

The target cell specificity of the B19 parvovirus infection was examined by isolating highly enriched hematopoietic progenitor and stem cells from normal human bone marrow. The efficiency of the B19 parvovirus replication in enriched erythroid progenitor cells was approximately 100-fold greater than that in unseparated bone marrow cells. The more-primitive progenitor cells identical to or closely related to the human pluripotent hematopoietic stem cells, on the other hand, did not support viral replication. The B19 progeny virus produced by the enriched erythroid progenitor cells was infectious and strongly suppressed erythropoiesis in vitro. The susceptibility of both the more-primitive erythroid progenitors (burst-forming units-erythroid) and the more-mature erythroid progenitors (CFU-erythroid) to the cytolytic response of the virus and the lack of effect on the myeloid progenitors (CFU-granulocyte-macrophage) further give evidence to the remarkable tropism of the B19 parvovirus for human hematopoietic cells of erythroid lineage.     

Combined action of c-kit and erythropoietin on erythroid progenitor cells.

Mutations at the murine dominant-white spotting locus (W) (c-kit) affect various aspects of hematopoiesis. We have made antibodies against c-Kit with the synthetic peptides deduced from the murine c-kit gene and examined the role of c-Kit in erythropoiesis. The antibody inhibited the stromal cell-dependent large colony formation of the erythroid progenitors. In the culture of erythropoietin-responsive erythroid progenitors of the anemia-inducing Friend virus-infected mouse spleen, the antibody inhibited only proliferation, but not differentiation of the progenitor cells. The inhibition was effective only at the early phase (within 6 hours after erythropoietin addition) before the cells start to proliferate induced by erythropoietin. During the early phase, erythropoietin down-regulated c-kit gene expression. These results suggest a mechanism of combined action of c-Kit with erythropoietin on the lineage-restricted erythroid progenitor cells.     

A murine recombinant retrovirus containing the src oncogene transforms erythroid precursor cells in vitro.

A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.     

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.     

Changes in surface-membrane components during the differentation of rabbit erythroid cells.

The membrane components of rabbit bone-marrow-bound erythroid cells were characterized and compared with those of circulating rabbit erythroid cells. By the criteria of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, radioiodination with lactoperoxidase and binding of radioiodinated lectins, the two circulating forms of erythroid cells (the reticulocyte and erythrocyte) have the same surface components. In contrast, bone-marrow-bound nucleated erythroid cells have a unique set of membrane surface components which are completely different from those found on circulating cells. Of the ten Coomassie-Blue-staining proteins present in nucleated erythroid-cell plasma-membrane preparations, eight are accessible at the extracellular surface, and all of these are lectin-binding glycoproteins. Bone-marrow erythroid cells separated according to age by velocity sedimentation were also studied. The changeover in surface components occurs after the last nucleated stage of the erythroid cells (the orthochromatic normoblast). We discuss the alterations in membrane surface components observed during the differentiation of the erythroid-cell series in relation to the transition from bone-marrow-bound to circulating forms of these cells. We suggest that the change in membrane surface components may be linked to the loss of the nucleus from the normoblast and the entry of the erythroid cell into the circulation.     

Terminal erythroid differentiation in normal and leukemic mouse cells.

Friend virus-transformed murine erythroleukemic cells (FL cells) have been used as an in vitro model for the study of the expression of the genetic program involved in the final stages of erythroid differentiation. Treatment of the FL cells with chemical inducers such as dimethylsulfoxide results in their differentiation from 'pro-erythroblasts' to orthochromatic normoblasts and the appearance of several erythroid markers including hemoglobin, enzymes of the heme pathway, heme, glycophorin, and spectrin. These markers appear in an ordered sequence, suggesting that two genetic programs are involved in the erythroid differentiation of the cells. Preliminary studies with erythropoietin-stimulated fetal liver cultures in vitro suggest that the same is true for normal erythroid differentiation.     

The proliferative capacity of pure red cell aplasia bone marrow cells.

Pure red cell aplasia (PRCA) is a heterogeneous disorder. Immunologic abnormalities have recently been uncovered suggesting that both cell-mediated and humoral immune mechanisms may be of etiological importance in PRCA. Utilizing a technique for the cloning of bone marrow erythroid precursors, we determined the in vitro proliferative capacity of erythroid cells obtained from 21 patients with PRCA. Bone marrow cells from one group of patients produced normal or increased numbers of erythroid colonies while the in vitro proliferative capacity of bone marrow cells from a second group was characterized by subnormal erythroid colony formation. Sera obtained from the former group was frequently associated with demonstrable serum inhibitors of erythropoiesis, while PRCA in the latter group was probably the consequence of intrinsic erythroid stem cell defects or pathologic cellular interactions with nonerythroid regulatory cells. This survey of a relatively large population of patients with PRCA provides evidence for the multiple causative mechanisms that can be operative in the production of PRCA.     

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein.

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.     

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.     

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.     

Antagonism between interleukin 3 and erythropoietin in mice with azidothymidine-induced anemia and in bone marrow endothelial cells

Azidothymidine (AZT)-induced anemia in mice can be reversed by the administration of IGF-IL-3 (fusion protein of insulin-like growth factor II (IGF II) and interleukin 3). Although interleukin 3 (IL-3) and erythropoietin (EPO) are known to act synergistically on hematopoietic cell proliferation in vitro, injection of IGF-IL-3 and EPO in AZT-treated mice resulted in a reduction of red cells and an increase of plasma EPO levels as compared to animals treated with IGF-IL-3 or EPO alone. We tested the hypothesis that the antagonistic effect of IL-3 and EPO on erythroid cells may be mediated by endothelial cells. Bovine liver erythroid cells were cultured on monolayers of human bone marrow endothelial cells previously treated with EPO and IGF-IL-3. There was a significant reduction of thymidine incorporation into both erythroid and endothelial cells in cultures pre-treated with IGF-IL-3 and EPO. Endothelial cell culture supernatants separated by ultrafiltration and ultracentrifugation from cells treated with EPO and IL-3 significantly reduced thymidine incorporation into erythroid cells as compared to identical fractions obtained from the media of cells cultured with EPO alone. These results suggest that endothelial cells treated simultaneously with EPO and IL-3 have a negative effect on erythroid cell production.     

Specific erythroid differentiation of mouse erythroleukemia cells by activins and its enhancement by retinoic acids.

Activin A has been shown to induce hemoglobin production in various hematopoietic cells. Such activities of three structurally distinct activins (activin A, activin AB, and activin B) were compared using F5-5 mouse erythroleukemia cells. Activin A and AB had similarly potent inducing activities whereas that of activin B was much lower. The erythroid inducing activity of activins was suppressed by follistatin, an activin-binding protein but not by inhibin A and inhibin B. Retinoic acids (both all-trans and 13-cis) had weak erythroid differentiation activity. In addition, clear synergistic erythroid induction occurred when retinoic acid and activin A were mixed together. These results indicate that retinoic acid may modulate activin-induced erythropoiesis in vivo.     

Terminal differentiation of yolk-sac erythroid cells of the Syrian hamster in vitro

Summary The developmental fate of Syrian hamster yolk-sac (primitive) erythroid cells was examined in vitro. Highly purified yolk-sac erythroid cells at the polychromatophilic stage, obtained from the peripheral blood of embryos at day 10 of gestation, showed morphological and biochemical changes in our modified semi-solid culture system. Several morphological changes observed in the primitive erythroid cell cultures, such as nuclear condensation, approach of nuclei to the periphery of cells, development by cells of an extended pear-like shape, enucleation, and an increase in haemoglobin content, were quite similar to those of the terminal differentiation of fetal liver or adult bone marrow (definitive) erythroid cells. In addition, the transition of molecular species of haemoglobin from the embryonic to the fetal/adult pattern was also observed in our culture system. Thus we provide evidence, by the in vitro culture of yolk-sac erythroid cells, that primitive erythroid cells undergo terminal differentiation in a manner similar to that of definitive erythroid cells.