Stem Cells, Vol. 15, No. 4, 286-290,
July 1997
© 1997 AlphaMed Press
Differential Effect of Erythropoietin and GM-CSF on Megakaryocytopoiesis from Primitive Bone Marrow Cells in Serum-Free Conditions
José E. Cardier,
Connie L. Erickson-Millera,
Martin J. Murphy, Jr.
Hipple Cancer Research Center, Dayton, Ohio, USA;
a Present address: Molecular Virology and Host Defense Department, SmithKline Beecham, King of Prussia, Philadelphia, USA
Key Words. Erythropoietin (EPO) • GM-CSF • Hematopoiesis • HPP-CFC • CFU-Mk • BFU-Mk
Dr. José E. Cardier, Hipple Cancer Research Center, 4100 South Kettering Boulevard, Dayton, OH 45439-2092, USA.
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Abstract
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In this study we have explored the effect of recombinant human erythropoietin (EPO) and recombinant murine GM-CSF on megakaryocyte progenitors (colony forming units-megakaryocyte [CFU-Mk]) using a serum-free fibrin clot assay and enriched primitive hematopoietic progenitors of marrow cells from day 4 post-5- fluorouracil-treated mice. We have monitored the production of high proliferative potential-colony forming cells ([HPP-CFC]; compact colonies, >0.5 mm) and studied their relationship to CFU-Mk formation. EPO induced the formation of small numbers of megakaryocyte colonies, but acted together with the megakaryocyte-stimulating factors, stem cell factor (SCF) and interleukin (IL-3), to augment the size of CFU-Mk (colonies with >50 megakaryocytes/colony). A strong correlation between the number of CFU-Mk and HPP-CFC formation from 5-fluorouracil bone marrow cells was observed when these cells were stimulated with EPO in the presence of SCF and IL-3. On the other hand, GM-CSF alone had no effect on megakaryocyte colony formation. Moreover, GM-CSF in the presence of SCF and IL-3 potentiates the HPP-CFC formation (i.e., an increase of 3.1-fold compared to the effect induced by SCF + IL-3) with strong inhibitory effects on the number and size of megakaryocyte colonies. Although several studies suggest that EPO and GM-CSF can stimulate megakaryocytopoiesis, our results indicate that neither EPO nor GM-CSF alone are sufficient to stimulate primitive progenitors committed to the megakaryocyte lineage. The fact that EPO can exert a strong effect on the size of CFU-Mk induced by SCF/IL-3 suggests that only those megakaryocyte progenitors previously stimulated by other megakaryocyte stimulating factors are able to respond to EPO. These findings may explain the physiological and clinical observations in which high levels of EPO are often associated with thrombocytosis.
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Introduction
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Megakaryocytopoiesis is a hierarchical process in which the proliferation and differentiation of megakaryocytes are regulated by cytokines [1-7]. Recently, a specific megakaryocytic colony-stimulating factor, thrombopoietin (TPO), has been characterized as a factor able to stimulate both proliferation and maturation of megakaryocytes [8-10]. Although TPO may be the primary regulator of megakaryocytopoiesis and platelet production, the most primitive and mature megakaryocyte progenitors (BFU-megakaryocyte [Mk] and colony forming units [CFU-]Mk, respectively) can also proliferate in vitro in the presence of other stimulating factors (i.e., interleukin 3 [IL-3], stem cell factor [SCF] and IL-6) [6, 7, 11]. Some of these hematopoietic growth factors stimulate proliferation of megakaryocyte progenitors, whereas others appear primarily to affect maturation [1-7].
In addition to its primary role as the principal humoral regulator of erythropoiesis, evidence suggests that erythropoietin (EPO) can affect human and murine megakaryocytopoiesis [12, 13]. There are certain pathological conditions in which high levels of EPO are associated with thrombocytosis, and that a correction of the primary pathological condition returns platelet levels to normal [14]. In contrast, it has been shown that hypoxia or EPO administration to mice does not stimulate thrombopoiesis [15, 16]. Recently, EPO has also been shown to promote differentiation and induce in vitro cytoplasmic process formation in purified murine megakaryocytes, but additional cytokines may be required to stimulate the proliferation of megakaryocyte progenitors in bone marrow [17, 18].
Several laboratories have provided data suggesting that GM-CSF may play an important role in the growth and differentiation of megakaryocytes. It has been shown that GM-CSF synergizes with other megakaryocyte-stimulating factors to induce the proliferation of megakaryocyte progenitors [17, 19]. Moreover, GM-CSF may regulate the DNA replication of purified megakaryocytes [17]. In contrast, other reports have shown that GM-CSF seems to be inactive on megakaryocytopoiesis [20].
The disparity of results obtained with GM-CSF and EPO on megakaryocytopoiesis in vitro may be explained by the facts that: A) heterogeneous cell populations were studied, B) various cell concentration was employed in marrow cultures, and C) the use of fetal bovine serum in cultures provides an additional and immeasurable source of growth factors. Using in vitro conditions which minimize interference, we have attempted to clarify whether GM-CSF and EPO have a direct effect on the growth of megakaryocyte progenitors or whether they can exert their effect(s) through combined action with other megakaryocytic stimulating factors (i.e., SCF, EPO and IL-3). In our system, primitive hematopoietic cells from 5-fluorouracil (5-FU)-treated mice were assayed in serum-free fibrin clot assays in which we explored the effect of GM-CSF and EPO on CFU-Mk. Because both GM-CSF and EPO alone or in different combinations can regulate the growth of different types of progenitor cells (including those that give rise to pure erythroid and granulocyte/macrophage progenitors), we also attempted to identify a correlation between the growth of CFU-Mk and the growth of other hematopoietic colonies in the same cultures. For this purpose, we also monitored total hematopoiesis by measuring the formation of colony forming cells (CFC) and the high proliferative potential-colony forming cells (HPP-CFC).
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Materials and Methods
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Animals
Female B6D2F1 mice, 8-12 weeks of age (Harlan Sprague Dawley; Indianapolis, IN) were used for this study. All animals were maintained under American Association for Accreditation of Laboratory Animal Care conditions.
Reagents
Recombinant murine SCF, IL-3 and GM-CSF were purchased from R&D Systems (Minneapolis, MN) as carrier-free preparations. The cytokines were resuspended in 10% human serum albumin (Sigma Chemical Co.; St. Louis, MO) before aliquoting. All cytokines were stored at –20°C. Recombinant human EPO was purchased from Amgen (Thousand Oaks, CA). Optimal doses for each cytokine was determined from dose-response curves and each used at the following final concentration: SCF at 50 ng/ml; IL-3 at 1 ng/ml; GM-CSF at 25 ng/ml; EPO at 5 U/ml.
Cell Preparation
Animals were sacrificed by cervical dislocation, and the femurs were removed aseptically. Bone marrow cells (BMC) were obtained by flushing the femurs with X-vivo medium (BioWhittaker; Walkersville, MD). Single-cell suspensions were prepared by repeated aspiration through a 26-gauge needle. To obtain a homogeneous progenitor cell population at early stages of maturation, a single i.p. injection of 5-FU at 150 mg/kg body weight was administered. BMC were harvested four days after the 5-FU injection (described here as 5-FUBMC).
Hematopoietic Colony Assays
Serum-free murine fibrin clot assays as developed at Hipple Cancer Research Center were used to evaluate megakaryocytopoiesis (CFU-Mk) and total hematopoiesis (CFC and HPP-CFC) [21, 22]. Unfractionated marrow cells (2 x 104 cells/clot) were placed in 20% bovine fibrinogen (type IV; Sigma) and 10% human plasma thrombin (Sigma) in the presence of different recombinant cytokines. Aliquots (0.4 ml) were placed in the center of 35 mm dishes (Corning 25050-35; Corning, NY) and when the fibrin clots had formed they were bathed with 1.0 ml X-vivo. Dishes were incubated at 37°C flushed with 5% CO2 in humidified air. After seven or twelve days of culture, the number of CFC (>40 cells) or HPP-CFC (colonies greater than 0.5 mm of diameter) [23, 24], respectively, were quantitated microscopically. Subsequently, the fibrin clot was dried and stained for acetylcholinesterase activity [22] to identify and enumerate megakaryocyte colonies. Groups of three or more acetylcholinesterase+ clustered together were scored as CFU-Mk.
Statistical Analysis
Results are reported as mean ± standard deviation from triplicate dishes in all assays. The experimental data were analyzed for statistical significance using analysis of variance (ANOVA) and regression analysis. The level of significance was p < 0.05. All experiments have been repeated at least three times, and have been shown to be reproducible.
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Results
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The Effects of EPO or GM-CSF Alone or in Combination with SCF + IL-3 on Megakaryocytopoiesis from 5-FU-Treated Marrow Cells
We tested whether EPO or GM-CSF alone or with the combination of SCF + IL-3, which is a strong megakaryocytic-stimulatory combination, affects the growth of CFU-Mk from primitive hematopoietic cells obtained from 5-FUBMC. Figure 1 shows that EPO alone induced a small number of CFU-Mk in contrast with GM-CSF which had no effect on CFU-Mk formation from 5-FUBMC. Moreover, neither EPO nor GM-CSF combined with SCF (a known synergistic factor with other megakaryocyte stimulating factors) nor SCF + IL-3 increased the number of CFU-Mk progenitors over those observed with SCF + IL-3. IL-3 alone or combined with SCF showed a strong megakaryocyte stimulatory effect ( Fig. 1). Interestingly, a careful analysis of CFU-Mk at day 10 of culture showed that the number of colonies containing more than 50 megakaryocytes/colony was significantly increased (2.3-fold) when EPO was added to cultures that contained SCF + IL-3 when compared with those induced by SCF + IL-3 ( Table 1). In contrast, when adding GM-CSF to the combination of SCF + IL-3 a significant reduction in the number of large CFU-Mk was observed ( Table 1).
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Figure 1. The effect of EPO or GM-CSF, alone or in combination with other cytokines, on CFU-Mk formation at day 7 from 5-FUBMC. BMC from 5-FU-treated mice were harvested and were plated at 20,000 cells/clot in serum-free medium in the presence of predetermined concentration of EPO, GM-CSF (GM), SCF, and IL-3 (see Material and Methods). The results are the mean ± SD of three experiments. The addition of EPO to the combination of SCF + IL-3 did not induce a statistically significant increase in the number of CFU-Mk compared with the effect induced by SCF + IL-3 (p < 0.05).
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Table 1. Number of large megakaryocyte colonies (CFU-Mk >50 megakaryocyte/colony) and HPP-CFC from 5-FUBMC stimulated with EPO and GM-CSF in combination with SCF and IL-3
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The Effects of EPO or GM-CSF Alone or in Combination with SCF + IL-3 on Hematopoiesis from 5-FU-Treated Marrow Cells
We examined the effect of EPO or GM-CSF alone or in combination with SCF + IL-3 on hematopoiesis through the formation of CFC and HPP-CFC. These colonies are related to the presence of clonogenic cells ranging from less to more immature hematopoietic progenitors (CFC and HPP-CFC, respectively) [23, 24]. Analysis of the growth of CFC and CFU-Mk was performed in order to determine whether the hematopoietic effects of EPO or GM-CSF, in combination with megakaryocyte colony-stimulating factors (i.e., IL-3 and SCF) are correlated with the growth or inhibition of megakaryocyte progenitors. A moderate correlation (r = 0.708) between the number of CFC and CFU-Mk colonies formed from 5-FUBMC stimulated by single and combinations of interleukins ( Fig. 2). The combination of EPO + SCF + IL-3 exerted a strong effect on both hematopoiesis and megakaryocytopoiesis. GM-CSF alone or in combination with SCF or SCF + IL-3 promoted the growth of CFC while concomitantly inhibiting the growth of megakaryocyte colonies.
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Figure 2. Relationship between hematopoietic and megakaryocytic colony formation. Analysis of the CFC and CFU-Mk induced by EPO or GM-CSF, alone or in combination with SCF + IL-3 from 5-FUBMC at day 7 of culture.
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Both EPO and GM-CSF combined with SCF + IL-3 had significant effects on HPP-CFC formation in comparison with the combination of SCF + IL-3 (1.5-fold and 3.1-fold increase, respectively; Table 1). Although the addition of GM-CSF to the combination of SCF + IL-3 was the most effective in stimulating HPP-CFC from 5-FU marrow cells, a significant inhibition in the number and size of megakaryocyte colonies was observed ( Fig. 1 and Table 1, respectively). These studies indicate that GM-CSF in conjunction with SCF and IL-3 has the ability of promoting the growth of primitive hematopoietic cells to HPP-CFC while suppressing the megakaryocyte colony stimulating activity exerted by the combination of SCF and IL-3. Cytological analysis of the colonies stimulated by GM-CSF + SCF + IL-3 showed a predominance of granulocytes and macrophages (data not shown).
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Discussion
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Several reports suggest that EPO and GM-CSF can modulate megakaryocytopoiesis [17, 20]. In this study, we used enriched primitive BMC from day 4 post-5-FU-treated mice to examine the effect of EPO and GM-CSF on megakaryocytopoiesis in serum-free conditions. This model uses a homogeneous population of early progenitor cells which survive 5-FU cytoreduction in a system devoid of exogenous growth factors contained in fetal bovine serum.
Several in vitro and in vivo lines of evidence suggest a relationship between erythropoiesis and thrombocytopoiesis [25-28]. There are numerous observations which suggest that EPO may stimulate the growth and differentiation of megakaryocytes [12, 13, 15-18, 25]. Several megakaryoblastic or erythroblastic cell lines, which possess both megakaryocytic and erythroid lineage differentiation markers, have been reported to differentiate into megakaryocytes and erythroid cells following EPO treatment [29]. These effects could be mediated directly by receptors for EPO on megakaryocytes [30]. Alternatively, the partial homology between EPO and the recently cloned TPO and also between their receptors [8-10], suggests that EPO could potentiate megakaryocyte development through nonspecific binding to the TPO receptor. In our in vitro studies, EPO, by itself, induced only a small number of megakaryocyte colonies from primitive hematopoietic cells. However, in the presence of megakaryocyte-stimulating factors (i.e., SCF and IL-3), it exerted a strong effect on the size of CFU-Mk (colonies with >50 megakaryocytes). The stimulatory activity of EPO was also observed on HPP-CFC formation. Since these cells represent very primitive hematopoietic progenitors, our results suggest that EPO can act at early stages of hematopoiesis when presented together with early acting factors (i.e., SCF and IL-3). This, along with the observed effect of EPO in conjunction with SCF and IL-3 on megakaryocytopoiesis, suggests that EPO is capable of affecting the growth of early multipotent progenitors as well as being associated with stimulation of megakaryocyte progenitors in vitro.
Several reports indicate that GM-CSF does not stimulate CFU-Mk formation in vitro, and in vivo studies in mice have demonstrated that GM-CSF has no platelet stimulatory effect [20, 31]. In contrast, other authors have observed a positive effect of GM-CSF in megakaryocytopoiesis [17-19, 31, 32]. Here, we show that although GM-CSF in combination with SCF + IL-3 induced remarkable growth of CFC and HPP-CFC in our culture system, this same combination of cytokines was unable to stimulate CFU-Mk formation. Moreover, while the combination of SCF + IL-3 showed a strong stimulatory activity on megakaryocytopoiesis, the addition of GM-CSF to this combination directed the differentiation of the primitive cultured BMC to the myeloid lineages. Although GM-CSF and IL-3 have significantly broader and overlapping roles in hematopoiesis, our work shows that these two factors exert an opposite effect on megakaryocytopoiesis from primitive hematopoietic progenitors. This effect could be explained by the activation of different signal transducing factors by these hematopoietic cytokines. In fact, different signal transducing factors are activated by these cytokines depending on the differentiation state of the hematopoietic cells [33, 34]. Discrepancies between our results and other reports may be explained by the differences in the culture conditions (serum-containing and serum-free), in the matrix (fibrin clot) and in the number of cells used.
In conclusion, these results suggest that neither EPO nor GM-CSF alone appear to act directly on megakaryocyte progenitor cells. Although EPO had no significant influence on CFU-Mk formation, it potentiates the megakaryocyte-directed activity of the megakaryocyte-stimulating factors SCF and IL-3 on early progenitors. These effects may explain the in vivo observations that EPO influences megakaryocytopoiesis, as suggested by the presence of thrombocytosis associated with erythrocytosis in some clinical states.
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Acknowledgments
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We thank Dr. Emilio Barberá for helpful discussions and critical review. The authors wish to thank Melissa Mogle and Jenny Kwak for their expert technical assistance. We also thank Scott N. West for his assistance in the preparation of this manuscript. We also gratefully acknowledge the support of the Elizabeth Gay Reddig Cancer Research Fund, The Thomas P. Fordham Foundation, The Iddings Foundation and the fellowship support of Consejo Venezolano de Investigaciones Cientificas y Tecnologicas-CONICIT (JEC).
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accepted for publication March 17, 1997.
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Abstract
Introduction
