Stem Cells, Vol. 15, No. 6, 437-442,
November 1997
© 1997 AlphaMed Press
IL-6 Interferes with Stimulation of HPP-CFC and Large CFU-Mk in Conjunction with Cytokine Combinations from Primitive Murine Marrow Cells
José E. Cardier,
Martin J. Murphy, Jr.,
Connie L. Erickson-Millera
Hipple Cancer Research Center, Dayton, Ohio, USA;
a Current address: SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania, USA
Key Words. Hematopoiesis • Megakaryocytopoiesis • HPP-CFC • CFU-Mk • CFU-Meg • Serum-free
Dr. Connie L. Erickson-Miller, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, UP1455, Collegeville, PA 19426.
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Abstract
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The growth-promoting activities of interleukin 6 (IL-6) in combination with early-acting hematopoietic factors, i.e., stem cell factor (SCF) and interleukin-1
(IL-1), on primitive hematopoietic and megakaryocyte progenitors (high proliferative potential colony-forming cells [HPP-CFC] and colony-forming units-megakaryocyte [CFU-Mk], respectively) from 5-fluorouracil (5-FU)-treated murine bone marrow cells (BMC) were evaluated in serum-free fibrin clot cultures. IL-6 in combination with SCF and IL-1 induced an irregular and abortive hematopoiesis characterized by a reduction in colony size of at least 50% over those stimulated by SCF + IL-1 + IL-3 and an inability to continue growth to day 12. Moreover, IL-6 in combination with the early-acting factors, SCF and IL-1, had no effect on the formation of HPP-CFC. IL-6 is synergistic with SCF + IL-1 on day 7 CFU-Mk but did not stimulate large day 12 CFU-Mk. Our results suggest that, in the absence of serum, IL-6 prevents the continued proliferation of early hematopoietic and megakaryocytic progenitors initiated by SCF + IL-1 + IL-3. Optimization of cytokine combinations for use in ex vivo expansion of marrow progenitors, either for stem cell transplants or gene therapy, must consider not only the number of colonies but their size, as well as the contributions of serum components.
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Introduction
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Hematopoiesis is a hierarchical process in which the proliferation and differentiation of the progenitor cells is highly dependent on the release of growth- and differentiation-promoting molecules. A large body of evidence suggests that the hematopoietic growth factors can be classified into early-, intermediate-, and late-acting factors in relation to the stage of the hematopoiesis in which these factors exert their effect [1-4]. The effects of cytokines on the growth of high proliferative potential colonies (HPP-CFC) derived from the most primitive progenitor cells have assisted in the elucidation of this hierarchy [5].
Megakaryocytopoiesis is also a hierarchical process regulated by several growth factors which act at different levels of megakaryocyte development such as the earliest megakaryocyte progenitors or the more differentiated "colony-forming unit megakaryocyte" (CFU-Mk) [6-13].
Knowledge of the specific protein factors responsible for regulating hematopoiesis and megakaryocytopoiesis can be exploited to increase the number of progenitors ex vivo for subsequent use in stem cell transplants or as a source of replicating stem cells for gene therapy. Optimization of the cytokine combinations needed to induce large numbers of myeloid cells (HPP-CFC) or to increase the number of lineage-specific progenitors (CFU-Mk) is under examination by a number of investigators [14-16]. An understanding of the lineage commitment of either early or late progenitor cells under different cytokine combinations can affect the selection of optimal conditions. For example, previous work demonstrated that erythropoietin (EPO) stimulates CFU-Mk, while GM-CSF is better at stimulating HPP-CFC at the expense of megakaryocytes [17].
Interleukin 6 (IL-6) has been reported to have effects on early hematopoiesis [18-20] and also later in megakaryocytopoiesis [21-23]. To understand these separate effects of IL-6 and how they may regulate the numbers of myeloid and megakaryocyte progenitor cells, we studied the in vitro effects of IL-6 alone or in combination with early-acting hematopoietic factors (i.e., stem cell factor [SCF] and IL-1) on the most primitive hematopoietic colony-forming cells detected in vitro (HPP-CFC) and megakaryocyte progenitors. To more clearly elucidate these effects, a serum-free fibrin clot system was used which allowed us to examine and differentiate the effects of different hematopoietic interleukins without interference by other factors present in serum. In this system, the various cytokine combinations were tested for their ability to stimulate colonies derived from early progenitors of 5-fluorouracil (5-FU)-treated mice.
This paper describes an effect of IL-6 on HPP-CFC that has not previously been described in a serum-free system. In addition, the correlation of the effects of cytokine combinations on the myeloid versus the megakaryocyte lineages is presented.
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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 under Institutional Animal Care and Use Committee-approved protocols. The animals were maintained under the American Laboratory Animal Association for Accreditation of Laboratory Animal Care (AAALAC)-approved conditions of our laboratory for at least one week before use.
Reagents
Recombinant murine (rm) SCF, rmIL-3, rmIL-6, and recombinant human (rh) IL-1 were purchased from R&D Systems (Minneapolis, MN) as carrier-free preparations. The cytokines were resuspended in 10% human serum albumin (Sigma; St Louis, MO) before aliquoting. All cytokines were stored at –20°C.
Cell Preparation
Animals were sacrificed by cervical dislocation, and the femurs were removed aseptically. Bone marrow cells were obtained by flushing the femurs with transferrin- and HSA-supplemented serum-free X-vivo medium (BioWhittaker; Walkersville, MD). Single-cell suspensions were prepared by repeated aspiration through a 26-gauge needle. To obtain bone marrow cells enriched in primitive hematopoietic progenitors, a single i.p. injection of 5-FU at 150 mg/kg body weight was administered. Bone marrow cells (BMC) were taken four days after the 5-FU injection (described here as 5-FU BMC).
Hematopoietic Colony Assays
Serum-free murine fibrin clot assays were used to detect hematopoietic colonies (CFC and HPP-CFC) and megakaryocyte colonies (both day 7 and day 12 CFU-Mk). Briefly, marrow cells were resuspended in X-vivo medium. 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 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 in a humidified atmosphere flushed with 5% CO2. After 7 or 12 days of culture, the number of CFC (>40 cells) or HPP-CFC (colonies >0.5 mm in diameter), respectively, were counted. Then, the fibrin clot was dried and stained for acetylcholinesterase (AChE) activity [24] to identify and count megakaryocyte colonies. At day 7, groups of 3-50 AChE+ cells clustered together were scored as megakaryocyte colonies (CFU-Mk). Colonies that contained more than 50 AChE+ cells at day 12 were scored separately and are reported here as large CFU-Mk.
Statistical Analysis
Results are reported as mean ± standard deviation (SD) from triplicate dishes in all assays. All experiments have been repeated at least three times and have been shown to be reproducible. Statistical significance was determined by a two-tailed Student's t-test.
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Results
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Effect of IL-6 Alone or in Combination with Early-Acting Factors on Hematopoiesis
To determine the effect of IL-6 on hematopoiesis alone or in combination with early-acting factors, i. e., SCF and IL-1, the number of CFC from the bone marrow of 5-FU-treated (enriched in primitive hematopoietic progenitors) mice was measured at 7 and 12 days of culture. These effects were compared with those produced by IL-3 alone or combined with SCF + IL-1. IL-6 alone had no effect on hematopoiesis, measured by the number of CFC at 7 and 12 days of culture ( Figs. 1 and 2). Interestingly, while IL-6 in combination with SCF yielded poor colony formation, there was a statistically significant effect on primitive hematopoietic progenitors stimulated by IL-6 + SCF + IL-1 at seven days of culture. This combination induced similar numbers of colonies as those obtained with SCF + IL-1 + IL-3, a combination known for its strong stimulatory effect on primitive hematopoietic cells at day 7 ( Fig. 1). However, although colony numbers were higher, the combination containing IL-6 demonstrated an "abortive hematopoiesis" characterized by: A) at least a 50% reduction in size and number of cells in the colonies over those stimulated by SCF + IL-1 + IL-3 ( Fig. 3) and, B) a failure to support substantial growth of colonies at 12 days ( Fig. 2). On the other hand, the addition of IL-6 to the combination of SCF + IL-3 did not increase the number of colonies more than SCF + IL-3 alone ( Figs. 1 and 2). In our system, neither SCF nor IL-1 alone or in combination had a significant effect on CFC formation from 5-FU BMC.
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Figure 1. Number of colonies formed on day 7 by BMC from 5-FU mice stimulated with IL-6 alone or in combinations with other cytokines. Bone marrow cells from 5-FU-treated mice were harvested and plated at 20,000 cells/clot in serum-free medium. The results are the mean ± SD of three experiments.
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Figure 2. Number of colonies formed on day 12 by BMC from 5-FU mice stimulated with IL-6 alone or in combination with other cytokines. Bone marrow cells from 5-FU-treated mice were harvested and plated at 20,000 cells/clot in serum-free medium. The results are the mean ± SD of three experiments.
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Figure 3. Abortive hematopoiesis induced by IL-6 in combination with SCF and IL-1 from 5-FU BMC after 7 days of culture. The abortive hematopoiesis is signified by a 50% decrease in the size and number of cells of the colonies and by an inability to continue growth to day 12. (A) HPP-CFC stimulated by SCF + IL-1 + IL-3. (B) Colony stimulated by SCF + IL-1 + IL-6. (x 35 magnification).
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To determine whether the abortive hematopoiesis induced by IL-6 in combination with SCF + IL-1 was due to a failure in the stimulation of primitive hematopoietic cells to divide, we investigated the effect of this combination on the formation of HPP-CFC, which represents a primitive cell population, detected in vitro, closely related to hematopoietic stem cells [1, 5, 25]. This population of cells is characterized by: A) its in vitro capacity to produce very large colonies (>0.5 mm) and an average of 5 x 104 cells/colony; B) their ability to survive in vivo treatment with 5-FU, and C) their capacity to repopulate the bone marrow of lethally irradiated mice [5, 25]. IL-6 was tested for its capacity to stimulate the growth of HPP-CFC colonies from 5-FU BMC in the presence of SCF and IL-1. Table 1 shows that IL-3 in combination with SCF or SCF + IL-1 significantly stimulated HPP-CFC formation. The SCF + IL-3 + IL-1 combination was most effective in producing classic HPP-CFC from 5-FU BMC. These results also show that IL-6 in combination with SCF and IL-1 did not induce HPP-CFC formation and had no effect on the HPP-CFC stimulated by SCF + IL-3. Examination of these colonies stained with Wright-Giemsa revealed a variety of cell lineages in the HPP-CFC at 12 days of culture (data not shown).
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Table 1. Number of HPP-CFC formed from 5-FU bone marrow cells stimulated with IL-6 in combination with early-acting hematopoietic factors (i.e., SCF and IL-1)
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The Effect of IL-6 Alone or in Combination with Early-Acting Factors on Megakaryocytopoiesis
Since IL-6 also acts as a late-acting factor in megakaryocytopoiesis, it was of interest to determine whether decreases in hematopoietic growth induced by IL-6 in combination with SCF + IL-1 affected the growth of megakaryocyte progenitors. Figure 4 shows that IL-6 alone had very little effect on CFU-Mk formation on 5-FU marrow cells at seven days. However, IL-6 in combination with SCF + IL-1 showed a synergistic effect in contrast with each cytokine alone. Other combinations that included IL-6 did not increase the number of megakaryocyte colonies over those observed in a similar combination containing IL-3 (which is known for its strong megakaryocyte-stimulatory effect). The effect of IL-6 on large, day 12 CFU-Mk is shown in Table 2. As seen with its effect on HPP-CFC, IL-6 alone or combined with SCF or SCF + IL-1 did not stimulate the growth of early megakaryocyte progenitors at 12 days of culture. However, in contrast to the HPP-CFC results, the addition of IL-6 to cultures containing SCF + IL-3 actually inhibited the number of large CFU-Mk stimulated by the combination of SCF + IL-3 ( Table 2). Likewise, the other early-acting factor, IL-1, inhibited large CFU-Mk when added to the SCF + IL-3. In these assays, neither SCF nor IL-1, alone or in combination, had any significant megakaryocyte clonogenic capacity, and even under optimal conditions (SCF + IL-3 + IL-1), few large CFU-Mk were grown.
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Figure 4. The effect of IL-6 alone or in combination with other cytokines on megakaryocyte formation at day 7 from 5-FU BMC. BMC from 5-FU-treated mice were harvested and plated at 20,000 cells/clot in serum-free medium. The results are the mean ± SD of three experiments.
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Table 2. The effects of IL-6 alone or in combination with early-acting hematopoietic factors (i.e., SCF and IL-1) on the formation of large, day 12, CFU-Meg from 5-FU BMC
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Discussion
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The need for increased numbers of marrow progenitors for use in stem cell transplants or as a source of replicating stem cells for gene therapy has stimulated interest in the optimal combination of cytokines required to induce large numbers of myeloid cells (HPP-CFC). The stimulation of specific lineages, such as megakaryocytes, would provide cells to meet the specific needs of patients; thus, experiments to clarify the effects of specific cytokines used in such combinations on early and late progenitors may elucidate the commitment to certain lineages. 5-FU-treated murine marrow was chosen for this work because the reduced number of accessory cells and mature progenitors in marrow would enable the delineation of effects only on early progenitors.
IL-6, in particular, has been described as acting both early and late in hematopoiesis [6, 18, 26-28]. Its actions include a synergistic action with IL-3 and SCF to stimulate the growth of multipotential hematopoietic progenitors in marrow prepared from mice injected with 5-FU [1, 5, 25]. In contrast, the work described here showed that replacement of IL-3 by IL-6 in the IL-3 + SCF + IL-1 combination induced, from 5-FU marrow cells, the same number of colonies as observed with IL-3 + SCF + IL-1, but with a dramatic reduction of more than 50% in the size of these colonies observed at seven days of culture. Moreover, the combination of IL-6 with SCF + IL-1 or SCF alone had only a limited ability to sustain long-term colony formation to 12 days of culture. Data in Table 1 show no effect of IL-6 on HPP-CFC and suggest that an intermediate-acting factor such as IL-3 is required to continue the proliferation of these cells.
IL-6 has been shown to act directly to induce proliferation of Lin– Sca+ cells [20], and numerous reports describe a stimulatory effect of IL-6 in combination with other cytokines on colony formation [10, 19, 23]. However, these experiments were performed in the presence of serum [29]. The results using serum-free conditions described in this paper confirm other reports that there are elements in serum that act to sustain the proliferation initiated by IL-6.
Optimal stimulation of the megakaryocyte lineage using combinations of cytokines is under extensive study [14, 15, 29]. In this serum-free system, the combination of IL-6 with early-acting hematopoietic factors did not support the growth of large CFU-Mk, from 5-FU marrow at 12 days of culture, suggesting that early megakaryocyte progenitors were unable to develop when this cytokine was present in these combinations. IL-1 had a similar effect, suggesting that serum factors may be necessary for sustaining proliferation of megakaryocytic progenitors in the presence of either IL-1 or IL-6. However, although IL-6 was a poor stimulant of megakaryocytopoiesis, the megakaryocyte colonies induced by IL-6 alone or in combination with SCF + IL-1 contained larger and more mature individual megakaryocytes (data not shown), which is consistent with results obtained by other investigators who suggest that IL-6 promotes cell maturation during later stages of megakaryocytopoiesis [21, 30].
In our system, the optimal combination of cytokines to induce classical HPP-CFC was SCF + IL-3 + IL-1, while SCF + IL-3 was best at inducing CFU-Mk at day 12 from 5-FU marrow. In conclusion, this study shows that IL-6 in combination with early-acting hematopoietic factors, i.e., SCF + IL-1, produced abortive hematopoiesis on primitive hematopoietic cells from 5-FU marrow cells. Our results suggest that the more primitive cells with higher proliferative capacity, both myeloid (HPP-CFC) and megakaryocytic (large, day 12, CFU-Mk), do not respond to IL-6 alone or in combination with SCF and IL-1. These experiments also demonstrate that serum-free systems provide different results that may be a more accurate reflection of the true contribution of the individual cytokines to in vitro proliferation and differentiation.
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Acknowledgments
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The authors wish to thank Melissa Mogle, Jenny Kwak, and Tony Lombardo for technical assistance. They also gratefully acknowledge the support of the Elizabeth Gay Reddig Cancer Research Fund, The Thomas P. Fordham Foundation, and The Iddings Foundation. The authors are grateful to Consejo Venezolano de Investigaciones Cientificas y Tecnologicas-CONICIT (JEC) and the Sigma Beta International Cancer Research Fellowship (CLEM) for their support.
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accepted for publication August 22, 1997.
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Abstract
Introduction
