Canine BFU-e progenitors: adaptation of a reproducible assay and anatomical distribution.

In vitro cloning assays are used increasingly in investigative hematotoxicology and in screening candidate compounds for their hematotoxic potential. To expand these applications, a practical cloning assay for erythroid burst-forming units (BFU-e) that uses a microplasma clot (MPC) system was adapted to the dog, a species used extensively in experimental hematology and drug development. This system offers the advantage over the methylcellulose and soft agar culture systems of allowing specimen fixation and, therefore, morphological and cytochemical evaluation. The distribution of BFU-e among various anatomic sites was assessed using the MPC cloning system, which was modified to optimize the BFU-e growth. BFU-e growth required only erythropoietin (Epo) in the culture medium and there was no need for an exogenous source of burst-promoting activity (BPA). The cloning efficiency was linearly proportional to the plating concentrations of Epo and marrow mononuclear cells (MMC) over a range of 0 to 3 U Epo and 1 x 10(5) to 3 x 10(5) MMC per ml of culture, respectively. Increases in concentrations of Epo and MMC beyond these levels were not associated with linear growth. The addition of transferrin and spleen-conditioned medium containing a mixture of growth factors (including BPA) reduced BFU-e growth. The relative concentration of BFU-e was comparable among samples collected from the iliac crest, femur, and humerus. Serial cultures performed on individual dogs were highly reproducible and there was little variation in BFU-e activity among dogs of comparable age. It was concluded that the MPC system is a practical and reproducible cloning system for early (BFU-e), as well as late erythroid colony-forming units (CFU-e) in the dog. The concentration of BFU-e appears comparable throughout the active marrow; therefore, various anatomic sites can be used interchangeably for serial quantitative analysis of this progenitor.     

Effects of five recombinant hematopoietic growth factors on enriched human erythroid progenitors in serum-replaced cultures

Erythroid progenitors from normal human marrow were purified by a two-step immune panning method permitting both the enrichment of erythroid progenitors (plating efficiency up to 10%) and the separation of CFU-E from BFU-E. The purified erythroid progenitors were grown in serum-replaced conditions; in some experiments at an average of one cell per well. Human recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL3), erythroid potentiating activity (EPA), and human erythropoietin (Epo) either recombinant or homogenous native were tested for their effect on CFU-E growth. Epo was an absolute requirement for CFU-E growth and was sufficient to obtain colony formation at the unicellular level whereas GM-CSF and IL3 did not further increase the plating efficiency. EPA potentiated the effect of Epo on this progenitor only in experiments performed at unicellular level. Human recombinant GM-CSF, IL3, Interleukin 1 alpha (IL1 alpha), and Epo were subsequently tested for their ability to promote BFU-E growth. GM-CSF and IL3 supported the growth of erythroid bursts in the presence of Epo, even at the unicellular level. However, IL3 promoted a higher number of bursts than GM-CSF under all conditions tested. These two growth factors have no or very small additive effects when tested in combination. IL1 alpha added to Epo alone had no effect on the growth of BFU-E whereas it potentiated the combined action of IL3 and GM-CSF on the primitive BFU-E. In conclusion, this study confirms at the unicellular level and under serum-free conditions that erythroid progenitors are regulated by multipotential growth factors in early phases of erythropoiesis and become sensitive only to Epo in later phases of differentiation.     

Direct effects of IL-4 on the in vitro differentiation and proliferation of hematopoietic progenitor cells

The purpose of this study was to analyze the effects of recombinant human interleukin 4 (IL-4) on the differentiation and proliferation in vitro of human granulocyte/macrophage (GM) and erythroid progenitors. IL-4 was added to either fetal bovine serum (FBS)-supplemented or to FBS-deprived cultures of unfractionated human marrow cells or marrow cells depleted of adherent and/or T cells. Paradoxical effects similar to those reported in the murine system were detected in these experiments. In FBS-supplemented cultures, IL-4, which had no effect on the growth or erythroid bursts (from burst-forming cells; BFU-E) detected in the presence of Epo alone, decreased by 46% the number of erythroid bursts detected in the presence of Epo and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). In contrast, in FBS-deprived cultures, IL-4 increased by 30-700% the number of erythroid bursts in cultures containing Epo alone or containing Epo, IL-3, and GM-CSF. The stimulatory effect of IL-4 on erythroid burst growth under FBS-deprived conditions was particularly evident when adherent cells were removed. Under the conditions investigated, IL-4 had little effect on the growth of GM colonies. In FBS-deprived suspension cultures of nonadherent, T-cell-depleted marrow cells, IL-4 maintained both the number of BFU-E and CFU-GM for at least 8 days. In these cultures, IL-4 antagonized the capacity of IL-3 to increase the number of BFU-E but IL-4 and IL-3 acted together to maintain the number of CFU-GM. To determine if IL-4 acted directly or indirectly, its effects on the growth of factor-dependent subclones of the murine progenitor cell line 32D were analyzed. Three subclones were studied: the original IL-3-dependent clone 32D cl.3, the Epo-dependent erythroid clone 32D Epo-1, and the G-CSF-dependent myeloid clone 32D G-1. IL-4 alone failed to induce colony growth from these cell lines. However, IL-4 inhibited by 25% the number of colonies formed by 32D cl.3 in the presence of IL-3 while increasing by 25% and 25-50% the number of colonies formed by 32D Epo-1 and 32D G-1 in the presence of Epo or G-CSF, respectively. These results indicate that human IL-4, as its murine counterpart, is a multilineage growth factor with paradoxical effects which are mediated by the direct action of IL-4 on progenitor cells.     

Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development

Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that alpha(4), alpha(5), and beta(1) are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage alpha(4)beta(1)-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha(4)beta(1)-integrin-dependent phase.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02.

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

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.     

Differential effects of human erythroid burst stimulating activity (HuEBSA) on human cord blood burst forming units-erythroid (BFU-Es) as a function of their differentiation state.

An erythroid stimulating activity which promotes the growth of small bursts probably arising from mature burst forming units-erythroid (BFU-Es) of adult human bone marrow cells and called human erythroid burst stimulating activity (HuEBSA), was previously found in media conditioned by a fetal human kidney cell line. In the present work we report that adding HuEBSA to cultures did not increase the burst number but increased the size of bursts from cord blood (CB) cells. A similar observation was made using stem cell factor (SCF). However, a synergistic effect on the burst number was noted when both HuEBSA and SCF were introduced to cultures. We also noticed that CB erythroid progenitors pre-cultured with 5637-Conditioned Medium [as a source of burst promoting activity (BPA)] and erythopoietin (Epo) for 3 days could be stimulated by HuEBSA but not by SCF. Similar results were obtained when interleukin 3 (IL-3) was introduced with Epo to the pre-cultures. These results suggest that two different populations of erythroid progenitors coexist in cord blood, one is Epo- and IL-3-sensitive, the other solely Epo-sensitive. It also seems probable that HuEBSA acts on erythroid progenitors arising from the more immature erythroid population, since its stimulating activity was evident after a 3-day pre-culture of cord blood cells in Epo and IL-3.     

Growth of human normal erythroid progenitors in liquid culture: a comparison with colony growth in semisolid culture

Most studies of erythropoiesis in vitro have employed cloning methods in semisolid medium. We have recently described a two-step liquid culture procedure that supports the proliferation and differentiation of human erythroid progenitors. In the present study, we have modified the procedure to allow large-scale cultures of erythroid cells derived from normal donors. The culture is divided into two phases. In the first phase, which is erythropoietin (Epo) independent, the early erythroid progenitors multiply and differentiate. In the second, Epo-dependent phase, they mature into orthochromatic normoblasts and enucleated erythrocytes. Using this procedure, erythroid cell yield reached 7.5 x 10(6)/ml and a total of 7 x 10(8) cells could be harvested per blood unit. A comparison of the growth of erythroid cells in liquid culture to their colony growth in semisolid culture indicated that cell growth was superior: 1) in liquid culture in terms of cell yield per originally cultured mononuclear cell, 2) per ml culture and per culture surface area and in the purity of the resultant erythroid cell population. In addition, it permits easier manipulation of the culture condition and components and sampling of greater than 1 x 10(7) cells at each maturation stage subsequent to the proerythroblast stage. This liquid culture procedure might provide an important experimental tool for studying erythroid cell development.     

Interleukin-2 (IL-2) induces erythroid differentiation and tyrosine phosphorylation in ELM-I-1 cells transfected with a human IL-2 receptor beta chain cDNA.

The molecular mechanism of erythroid differentiation has been still ill-defined. In this study, we introduced a human interleukin-2 receptor (IL-2R) beta chain cDNA into ELM-I-1 cells which differentiated into hemoglobin-positive cells in the presence of erythropoietin (Epo), and established the transformant which expressed IL-2R beta chain. In this transformant, we revealed that IL-2 induced erythroid differentiation and the same pattern of tyrosine phosphorylation as Epo. These data suggest that tyrosine phosphorylation is involved in signal transduction pathway of erythroid differentiation. It is also implicated that the Epo and IL-2 receptor system share a common signal transduction pathway.     

Kinetics of reduction of tyrosine phenoxyl radicals by glutathione

Erythropoietin (Epo) is crucial for promoting the survival, proliferation, and differentiation of mammalian erythroid progenitors. The central role played by tyrosine phosphorylation of erythropoietin receptor (EpoR) in Epo-cell activation has focused attention on protein tyrosine phosphatases (PTPs) as candidates implicated in the pathogenesis of the resistance to therapy with human recombinant Epo. Prototypic member of the PTP family is PTP1B, which has been implicated in the regulation of EpoR signaling pathways. In previous reports we have shown that PTP1B is reciprocally modulated by Epo in undifferentiated UT-7 cell line. However, no information is available with respect to the modulation of this phosphatase in non-Epo depending cells or at late stages of erythroid differentiation. In order to investigate these issues we induced UT-7 cells to differentiate and studied their PTP1B expression pattern. Simultaneous observations were performed in TF-1 cells which can be cultured either with GM-CSF, IL-3 or Epo. We found that Epo induced PTP1B cleaveage in TF-1 and differentiated UT-7 cells. This pattern of PTP1B modulation may be due to an increased TRPC3/TRPC6 expression ratio which could explain the larger and sustained calcium response to Epo and calpain activation in Epo treated TF-1 and differentiated UT-7 cells.     

Erythroid cells rendered erythropoietin independent by infection with Friend spleen focus-forming virus show constitutive activation of phosphatidylinositol 3-kinase and Akt kinase: involvement of insulin receptor substrate-related adapter proteins

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an effort to understand how SFFV causes Epo independence, we have been examining erythroid cells rendered factor independent by SFFV infection for constitutive activation of signal-transducing molecules. Previous studies from our laboratory showed that various signal-transducing molecules known to be activated by Epo, including Stat proteins and components of the Raf-1/MAP kinase pathway, are constitutively activated in SFFV-infected erythroid cells in the absence of Epo. Since another signal transduction pathway involving activation of phosphatidylinositol 3-kinase (PI 3-kinase) after Epo stimulation plays an important role in erythroid cell proliferation and differentiation, we carried out studies to determine if this pathway was also activated in SFFV-infected cells in the absence of Epo. Our studies show that PI 3-kinase is constitutively activated in erythroid cells rendered factor independent by infection with SFFV and that PI 3-kinase activity, but not Epo receptor tyrosine phosphorylation, is required for the proliferation of these cells in the absence of Epo. We further show that in SFFV-infected erythroid cells grown in the absence of Epo, PI 3-kinase associates with the insulin receptor substrate (IRS)-related adapter molecules IRS-2, Gab1, and Gab2, which are constitutively tyrosine phosphorylated in SFFV-infected cells. Finally, Akt, a protein kinase that is one of the downstream effectors of PI 3-kinase, and SHIP, a lipid phosphatase that is important for Akt activation through PI 3-kinase, are both tyrosine phosphorylated in SFFV-infected cells grown in the absence of Epo. Our results indicate that induction of Epo independence by SFFV requires the activation of PI 3-kinase and suggest that constitutive activation of this kinase in SFFV-infected cells may occur primarily through interaction of PI 3-kinase with constitutively phosphorylated IRS-related adapter molecules.     

Signal transduction in the erythropoietin receptor system

Events relayed via the single transmembrane receptor for erythropoietin (Epo) are essential for the development of committed erythroid progenitor cells beyond the colony-forming unit-erythroid stage, and this clearly involves Epo's inhibition of programmed cell death (PCD). Less well resolved, however, are issues regarding the precise nature of Epo-dependent antiapoptotic mechanisms, the extent to which Epo might also promote mitogenesis and/or terminal erythroid differentiation, and the essential vs modulatory nature of certain Epo receptor cytoplasmic subdomains, signal transducing factors, and downstream pathways. Accordingly, this review focuses on the following aspects of Epo signal transduction: (1) Epo receptor/Jak2 activation mechanisms; (2) the critical vs dispensable nature of (P)Y sites and SH2 domain-encoding effectors in survival, growth, and differentiation responses; (3) primary mechanisms by which Epo inhibits PCD; (4) the integration of signals relayed by coexpressed and possibly directly interacting cytokine receptors; and (5) predictions regarding effector function which are provided by the association of certain primary and familial polycythemias with mutated human Epo receptor forms.     

The effect of bone marrow stromal cells on the cloning of human leukemia/lymphoma cell lines

The cloning of established human leukemia and lymphoma cell lines at limiting dilutions in microwells and soft agar is described. Growth of most cell lines was improved by the addition of human AB serum and irradiated human bone marrow stromal cells. In general, the cloning efficiency in microwells was greater than in soft agar. The effect of bone marrow stromal cells appeared to be caused by a diffusable factor(s), but close cellular interaction could not be excluded since cloning in microwells produced consistent and optimal cell growth compared to growth in soft agar. It was concluded that cloning of leukemia and lymphoma cell lines in microwells was the preferred method and that similar techniques could improve the cloning of fresh leukemia and lymphoma cells.     

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the nasa rotating wall vessel bioreactor

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.     

A novel mechanism of cooperation between c-Kit and erythropoietin receptor. Stem cell factor induces the expression of Stat5 and erythropoietin receptor, resulting in efficient proliferation and survival by erythropoietin

Optimal production of red cells in vivo requires collaboration between c-Kit, erythropoietin receptor (Epo-R), and GATA-1. However, the mechanism(s) of collaboration remain unclear. Utilizing an embryonic stem cell-derived erythroid progenitor cell line from mice deficient in GATA-1, we have examined the role of c-Kit and Epo-R in erythroid cell proliferation, survival, and differentiation. In the absence of GATA-1, we demonstrate an essential role for c-Kit in survival and proliferation of erythroid progenitors via the regulation of Bcl-2 expression. In addition, we demonstrate that Epo-R and Stat5 are regulated by a second, novel mechanism. We demonstrate that c-Kit stimulation by stem cell factor is essential for the maintenance of Epo-R and Stat5 protein expression, which results in significantly enhanced Bcl-x(L) induction and survival of erythroid progenitors in response to Epo stimulation. Restoration of GATA-1 function results in terminal erythroid maturation and up-regulation of Epo-R and Bcl-x(L) expression, leading also to significantly enhanced survival of terminally differentiating erythroid progenitors in the presence of only Epo. These results demonstrate that c-Kit and Epo-R have unique role(s) during distinct phases of erythroid maturation, and both stem cell factor and Epo contribute to the regulation of the Epo-R-Stat5-Bcl-x(L) pathway to ensure optimal survival, proliferation, and differentiation of erythroid progenitors.     

Erythropoietin. Receptor characteristics during the ontogeny of hamster yolk sac erythroid cells

Erythropoietin (Epo) binds specifically to receptors on the surface membrane of responsive erythroid cells. In search of ontogenic changes in Epo receptor behavior, we studied characteristics of specific binding to hamster yolk sac erythroid cells during hamster ontogeny. We detected receptors specific for Epo on these cells throughout the duration of their intravascular existence (hamster gestational days 8 through 13). These receptors are saturable at an Epo concentration of 1.2 nM in the incubation medium. Attainment of equilibrium of binding prior to hormone internalization, a requirement for receptor binding assays, was possible at 10 degrees C but not at 37 degrees C. Hence, all incubations of cells with Epo were carried out at 10 degrees C. Data on specific binding analyzed by the method of Scatchard demonstrated that yolk sac erythroid cells possess a single class of Epo receptors at each stage of gestation examined. Binding affinity and numbers of receptors per cell change as ontogeny progresses: Kd (the dissociation constant) increases, a phenomenon observed in other differentiating cell systems, whereas the number of receptors per cell peaks on gestational day 10. The variability in number of receptors per cell is consonant with up and down regulation controlled by Epo availability. We propose that the progressive increase in Kd might be best explained by ontogenic changes in cell membrane structure contiguous to the receptors themselves.     

Phosphorylation of Bcl-associated death protein (Bad) by erythropoietin-activated c-Jun N-terminal protein kinase 1 contributes to survival of erythropoietin-dependent cells

The glycoprotein erythropoietin (Epo) is a hematopoietic cytokine necessary for the survival of erythrocytes from immature erythroid cells. The mitogen-activated c-Jun N-terminal kinase 1 (JNK1) plays an important role in the proliferation and survival of erythroid cells in response to Epo. However, the precise mechanism of JNK1 activation promoting erythroid cell survival is incompletely understood. Here, we reported that JNK1 is required for Epo-mediated cell survival through phosphorylation and inactivation of the pro-apoptotic, Bcl-2 homology domain 3 (BH3)-only Bcl-associated death protein (Bad). Upon Epo withdrawal, HCD57 cells, a murine Epo-dependent cell line, displayed increased apoptotic cell death that was associated with decreased JNK1 activity. Epo withdrawal-induced apoptosis was promoted by inhibition of JNK1 activity but suppressed by expression of a constitutively active JNK1. Furthermore, Epo-activated JNK1 phosphorylated Bad at threonine 201, thereby inhibiting the association of Bad with the anti-apoptotic molecule B-cell lymphoma-extra large (Bcl-X(L)). Replacement of threonine 201 by alanine in Bad promoted Epo withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK1 contributes to the survival of erythroid cells.