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.     

Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

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.     

Effects of purified recombinant human interleukin-3 on the growth of human hematopoietic progenitor cells in "long-term" bone marrow culture

Recombinant human interleukin-3 (rhuIL-3) was assessed for its effects on the growth of normal human hematopoietic bone marrow nucleated cells, and on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells in a liquid culture system which allows for the prolonged growth of these cells in vitro. RhuIL-3, at concentrations of 100 and 500 units/mL, significantly enhanced the numbers of nucleated cells, as well as the numbers of supernatant and adherent CFU-GM and BFU-E growing in tissue culture flasks or dishes over a period of 4 to 6 weeks. The results demonstrated the rhuIL-3 has a stimulating effect on the growth of human marrow cells in prolonged culture. This information is consistent with the effects of rhuIL-3 in short-term marrow colony assays in vitro and with the in vivo actions of recombinant murine IL-3 in mice, and may be of relevance to clinical trials that will be assessing the hematopoietic effects of rhuIL-3 in humans.     

Zidovudine (AZT) treatment suppresses granulocyte-monocyte colony stimulating factor receptor type alpha (GM-CSFR alpha) gene expression in murine bone marrow cells

In vitro exposure of murine bone marrow cells to increasing concentrations of zidovudine (AZT, 0.1-50 microM) had a concentration dependent suppressive effect on the growth of granulocyte-monocyte colony forming unit (CFU-GM) derived colonies. In our previous published study, the mechanism of AZT-induced suppression of erythroid colony forming unit (CFU-E) derived colonies was linked to a decrease in erythropoitin receptor (Epo-R) gene expression. In this study, we have observed that AZT exposure also induced a concentration dependent suppressive effect (35-90%) on GM-CSF receptor type alpha (GM-CSFR alpha) gene expression. The suppression of GM-CSFR alpha mRNA expression was specific, since AZT caused a much lower decrease (15-22%) on the IL-3 receptor type alpha (IL-3R alpha) message level, and had an insignificant effect on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and c-myc message levels. Erythropoietin (Epo) therapy has been used for reversal of AZT induced erythroid toxicity. Exposure to increasing concentrations (10-500 U/ml) of GM-CSF was unable to override the suppressive effect of AZT on CFU-GM derived colonies, however, treatment in combination with IL-3 (10-250 U/ml) ameliorated the suppressive effects of AZT on CFU-GM and on GM-CSFR alpha and IL-3R alpha gene expression. These findings suggest a mechanism via which AZT may suppress granulocyte-monocyte specific differentiation in murine bone marrow cells. These data also suggest that a combination of GM-CSF and IL-3 may be a superior therapeutic intervention for AZT-induced neutropenia.     

Target cells for Friend virus-induced erythroid bursts in vitro

Erythropoietin (Epo) acts on mouse bone marrow cells in vitro in plasma clot or methyl cellulose culture systems to induce the formation of single erythroid colonies, or clusters of erythroid colonies termed bursts. Our laboratory has recently reported the observation that infection of mouse bone marrow cells in vitro with the polycythemia-inducing strain of Friend virus (FV) resulted in the formation of erythroid bursts after 5 days in plasma clot culture in the absence of added Epo. We have now used this system to characterize the target cells for this FV-induced erythroid transformation. The greatest number of FV bursts were observed when marrow cells were obtained from mice whose erythropoiesis had been stimulated by bleeding or phenylhydrazine treatment. Bleeding also resulted in an increase in the number of FV bursts following the infection of spleen cells in vitro. Hypertransfusion of mice, which results in decreased erythropoiesis, yielded a reduced number of FV bursts in vitro, as did prior treatment with actinomycin D. Cell separation studies using velocity sedimentation at unit gravity showed that the cells, which give rise to FV bursts, sedimented with a modal sedimentation velocity between 5.1–8.5 mm/hr. The Epo-dependent colony-forming unit erythroid (CFU-E), which gives rise to a single erythroid colony, also sediments with a modal velocity between 5.1–8.5 mm/hr, while the Epo-dependent day 8 burst-forming unit erythroid (day 8 BFU-E) sediments with a modal velocity between 3.0–6.0 mm/hr. A 20 min incubation of marrow cells with high specific activity ³H-thymidine, prior to virus infection, resulted in a 75–80% reduction in the number of FV bursts. Mixing cells from the upper portion of the gradient, which yielded no FV bursts, with cells from an area in which high numbers of FV bursts were observed did not result in the inhibition of burst formation. These experiments indicate that the primary target cells for FV bursts in vitro are most probably erythroid precursor cells that have matured beyond the day 8 BFU-E and are closely related to the CFU-E.     

Retrovirus-induced feline pure red cell aplasia: the kinetics of erythroid marrow failure

Cats viremic with feline leukemia virus subgroup C (FeLV-C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU-E) in marrow culture. Normal numbers of early erythroid progenitors (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) remain, suggesting that the maturation of BFU-E to CFU-E is impaired in vivo. We have examined the cell cycle kinetics of BFU-E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV-C infection, yet 6-42 weeks before anemia, the traction of BFU-E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 +/- 4% (normal 23 +/- 2%) while there was no change in the cell cycle kinetics of CFU-GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV-infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV-C but not anemic, a 4-fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU-E decreased, BFU-E no longer responded to FEA/FeLV CM in vitro. BFU-E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU-E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU-E increased 2- to 4-fold although no CFU-E were generated. By 4 to 7 days, CFU-E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.     

Serum-free culture of primitive murine hemopoietic colony-forming cells

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.     

Influence of IL-1 alpha and -1 beta on the survival of human bone marrow cells responding to hematopoietic colony-stimulating factors

Purified recombinant human (rhu) IL-1 alpha and IL-1 beta were evaluated for their effects on the proliferation and survival of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells from normal human bone marrow (BM). Using nonadherent low density T lymphocyte depleted (NALT-) BM cells cultured in the presence or absence of IL-1, CSF-deprivation studies demonstrated that IL-1 alpha or IL-1 beta by itself did not enhance the proliferation of CFU-GM or BFU-E. They did, however, promote the survival of progenitors responding to the delayed addition of media conditioned by the 5637 cell line (5637 conditioned medium), rhu GM-CSF and erythropoietin. The survival promoting effects of IL-1 alpha on CFU-GM and BFU-E were neutralized by anti-IL-1 alpha mAb added to the cultures. The survival promoting effect of IL-1 alpha did not appear to be mediated by CSF, because neither CSF nor erythroid burst promoting activity were detectable in cultures in which NALT- cells were incubated with rhuIL-1 alpha. In addition, suboptimal concentrations of rhu macrophage CSF (CSF-1), G-CSF, GM-CSF, and IL-3, which were just below the levels that would stimulate colony formation, did not enhance progenitor cell survival. Survival of CFU-GM and BFU-E in low density (LD) bone marrow cells did not decrease as drastically as that in NALT- BM cells, and exogenously added IL-1 did not enhance progenitor cell survival of CFU-GM and BFU-E in LD BM cells. However, addition of anti-IL-1 beta decreased survival of CFU-GM and BFU-E in LD BM cells. These results implicate IL-1 in the prolonged survival of human CFU-GM and BFU-E.     

Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell—cell contact

We have previously shown that a fetal liver-derived epithelial cell clone, FHC-4D2, could support hematopoiesis in vitro through its colony-stimulating factor (CSF) activities in a short-term culture. In this study, since FHC-4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC-4D2 could exert hematopoietic supporting activity in a long-term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC-4D2 cells. This clone could maintain the colony-forming unit of granulocytes and macrophages (CFU-GM) of BM for ≥ 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC-4A3, could not support the survival of CFU-GM, even for 1 week. In addition to BM CFU-GM, the FHC-4D2 clone also supported the survival of FL CFU-GM, burst-forming unit of erythroid cells (BFUe), and colony-forming unit of mixed progenitors (CFU-Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC-4D2 cells in the coculture, the comparable number of CFU-GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU-GM were present in both nonadherent and adherent cells to the FHC-4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU-GM. CFU-GM derived from the adherent cells formed larger colonies and contained more bipotential CFU-GM than the nonadherent population. When BM cells from mice given 5-fluorouracil were cocultured with FHC-4D2 cells under the limiting dilution condition, interleukin-3 (IL-3)-responsive CFU-GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL-3. From these data we conclude that the FHC-4D2 clone could generate and maintain IL-3-responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL-3-unresponsive immature hematopoietic cells into CFU-GM (progenitors responsive to IL-3) and in triggering the self-renewal of CFU-GM. © 1994 Wiley-Liss, Inc.     

Synergistic effects of purified recombinant human and murine B cell growth factor-1/IL-4 on colony formation in vitro by hematopoietic progenitor cells. Multiple actions

Purified recombinant human B cell growth factor-1/IL-4 was evaluated, alone and in combination, with purified preparations of recombinant human (rhu) CSF or erythropoietin (Epo) for effects on colony formation by human bone marrow CFU-GM progenitor cells (GM) and burst forming unit-E progenitor cells. rhu IL-4 synergized with rhu G-CSF to enhance granulocyte colony formation, but had no effect on CFU-GM colony formation stimulated by rhu GM-CSF, rhu IL-3, or rhu CSF-1. Rhu IL-4 synergized with Epo to enhance BFU-E colony formation equal to that of Epo plus either rhu IL-3, rhu GM-CSF, or rhu G-CSF. Removal of adherent cells and T lymphocytes did not influence the synergistic activities of rhu IL-4. Rmu IL-4, synergized with rhu G-CSF, but not with rmu GM-CSF, rmu IL-3, or natural mu CSF-1, to enhance CFU-GM (mainly granulocyte) colony numbers by a greater than 90% pure preparation of murine CFU-GM. Also, rhu IL-4 at low concentrations enhanced release of CSF and at higher concentrations the release also of suppressor molecules from human monocytes and PHA-stimulated human T lymphocytes. Use of specific CSF antibodies suggested that rhu IL-4 was enhancing the release of G-CSF and CSF-1 from monocytes and the release of GM-CSF and possibly G-CSF from PHA-stimulated T lymphocytes. Use of antibodies for TNF-alpha, IFN-gamma, or TNF-beta as well as measurement of TNF and IFN titers suggested that the suppressor molecule(s) released from monocytes were acting with TNF-alpha and those released from PHA-stimulated T lymphocytes were acting with IFN-gamma. These results implicate B cell growth factor-1/IL-4 as a synergistic activity for hematopoietic progenitors and suggest that the actions can be on both progenitor and accessory cells.     

Demonstration of a protector effect of interleukin-1 against hematologic toxicity of azidothymidine (AZT)]

3'-azido-3'-deoxythymidine (Azidothymidine or AZT) has attained wide critical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, treatment with AZT is associated with the development of severe hematopoietic toxicity. The AZT sensitivity of marrow progenitors was different with an IC 50 of 10(-8) M and 10(-6) M for respectively BFU-E and CFU-GM/GEMM. Data reported here show that recombinant human interleukin-1 alpha (IL-1 alpha), a pleiotropic cytokine, was demonstrated to be efficient to protect normal human as well as murine hematopoietic progenitors (CFU-GM, CFU-GEMM and BFU-E) from the toxic effect of AZT. The maximal effect was observed with 30 U/ml (Human cells) or 100 U/ml (murine cells) IL-1 alpha for BFU-E and CFU-GM/GEMM, with a marked effect at 1 U/ml. The results demonstrate that marrow progenitors respond differently to AZT and point out the potential efficacy of IL-1 alpha to enhance the proliferation of hematopoietic stem cells treated with growth factors (IL-3, erythropoietin) and to minimize the hematopoietic toxicity associated with AZT treatment.     

The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma

The influences of human tumor necrosis factor (TNF) (LuKII), recombinant human TNF-alpha, natural human interferon-gamma (HuIFN-gamma), recombinant HuIFN-gamma, and natural HuIFN-alpha were evaluated alone or in combination for their effects in vitro on colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in lowered (5%) O2 tension. TNF (LuKII) and recombinant TNF-alpha caused a similar dose-dependent inhibition of colony formation from CFU-GM, BFU-E, and CFU-GEMM. Day 7 CFU-GM colonies were more sensitive than both day 14 CFU-GM colonies and day 7 CFU-GM clusters to inhibition by TNF. BFU-E colonies and CFU-GEMM colonies were least sensitive to inhibition with TNF. The suppressive effects of TNF (LuKII) and recombinant TNF-alpha were inactivated respectively with hetero-anti-human TNF (LuKII) and monoclonal anti-recombinant human TNF-alpha. The hetero-anti-TNF (LuKII) did not inactivate the suppressive effects of TNF-alpha and the monoclonal anti-recombinant TNF-alpha did not inactivate TNF (LuKII). The suppressive effects of TNF did not appear to be mediated via endogenous T lymphocytes and/or monocytes in the bone marrow preparation, and a pulse exposure of marrow cells with TNF for 60 min resulted in maximal or near maximal inhibition when compared with cells left with TNF for the full culture incubation period. A degree of species specificity was noted in that human TNF were more active against human marrow CFU-GM colonies than against mouse marrow CFU-GM colonies. Samples of bone marrow from patients with non-remission myeloid leukemia were set up in the CFU-GM assay and formed the characteristic abnormal growth pattern of large numbers of small sized clusters. These cluster-forming cells were more sensitive to inhibition by TNF than were the CFU-GM colonies and clusters grown from the bone marrow of normal donors. The sensitivity to TNF of colony formation by CFU-GM of patients with acute myelogenous leukemia in partial or complete remission was comparable with that of normal donors. When combinations of TNF and HuIFN were evaluated together, it was noted that TNF (LuKII) or recombinant TNF synergized with natural or recombinant HuIFN-gamma, but not with HuIFN-alpha, to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM from bone marrow of normal donors at concentrations that had no suppressive effects when molecules were used alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.     

Evidence suggesting a stimulatory role for interleukin-10 in erythropoiesis in vitro

Interleukin-10 (IL-10) has been shown to exert anti-inflammatory effects by suppressing macrophage proliferation and inhibiting cytokine production. In this study we show that in the presence of erythropoietin (EPO), the addition of IL-10 results in a significant dose-dependent increase in both Burst Forming Unit-Erythroid (BFU-E) and Colony Forming Unit-Erythroid (CFU-E) colony growth in both serum-containing and serum-free murine cultures in vitro. IL-10 acts at the later stages of erythroid cell proliferation and differentiation as the increase in colony number was greater in CFU-E than in BFU-E, and was similar when IL-10 was added to BFU-E cultures at the time of culture initiation as when its addition to culture was delayed for 7 days. Furthermore, no increase in BFU-E colony number was noted when IL-10, added at the time of culture initiation, was neutralized by the addition to culture of a monoclonal anti-IL-10 antibody up to 7 days later. The increases in BFU-E by IL-10 addition were not the result of prolongation of BFU-E colony lifespan, which was not significantly different in IL-10 treated and control cultures, respectively. Rather IL-10 stimulated the proliferation of erythroid clusters that were now large enough to be recognized as colonies. IL-10-induced stimulation of erythropoiesis appeared to be independent of its inhibitory effects on macrophage function, as stimulation of erythroid colony growth was similar in macrophage-containing and depleted cultures. Studies to determine if the IL-10 effect was direct or indirect yielded equivocal results. A limiting dilution assay suggested a direct effect. However, a log/log dose response curve with IL-10 did not pass through the origin suggesting an indirect effect. These studies indicate that IL-10 acts synergistically with EPO to significantly increase stimulation of erythroid differentiation and proliferation in vitro and may be involved in the regulation of normal erythropoiesis in vivo. © 1996 Wiley-Liss, Inc.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

The in vitro growth of murine high proliferative potential-colony forming cells is not enhanced by growth in a low oxygen atmosphere

The growth of primitive murine hematopoietic progenitors, high proliferative potential colony-forming cells (HPP-CFC), has been reported to be improved in low O2 tension cultures. In this report we investigated the growth of HPP-CFC stimulated by combinations of interleukin (IL)-1, IL-6, kit-ligand (KL), granulocyte (G) colony-stimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and IL-3 in clonal cultures incubated at 7% or 21% O2 tension. Neither the numbers of HPP-CFC colonies nor the number of cells per HPP-CFC colony differed significantly between cultures grown under 7% or 21% O2 tension. The mean number of cells per HPP-CFC colony was found to range from 3.9 x 10(4) to 2.2 x 10(5). The smallest HPP-CFC colonies were stimulated by the cytokine combination IL-1 + IL-6 + KL, whereas the largest colonies were stimulated by a combination of all seven cytokines tested. The growth of erythroid colonies from murine or human bone marrow did, however, show some enhancement when cultured at a lower O2 tension. These results demonstrate that the growth of murine HPP-CFC was not compromised when cultured at ambient O2 concentration.     

The effect of recombinant erythropoietin on murine megakaryocyte colony formation

We investigated the effect of a recombinant human erythropoietin preparation (recombinant Epo) on murine megakaryocyte (MK) colony formation in serum-free and serum-containing culture systems, in order to study the relationship between Epo and megakaryopoiesis. Pokeweed mitogen spleen-conditioned medium (PWM-SCM), a standard source of MK colony stimulator, dose-dependently stimulated MK colony formation in the two culture systems. The plating efficiency of serum-free cultures was almost equal to that of cultures containing serum. Recombinant Epo also dose dependently stimulated MK colony formation in serum-containing cultures. However, in serum-free cultures recombinant Epo alone did not stimulate the growth of MK colonies; with the addition of fetal calf serum (FCS) to the serum-free cultures, recombinant Epo induced the growth of MK colonies. Furthermore, recombinant Epo enhanced MK colony formation through the stimulation of PWM-SCM or murine interleukin 3 (IL-3) in serum-free cultures. Our data show that Epo can act as a stimulator of megakaryopoiesis in collaboration with a factor in serum, or with an MK colony stimulator such as IL-3.