Recombinant PDGF enhances megakaryocytopoiesis in vitro

Summary. The effect of recombinant platelet-derived growth factor (PDGF) on both murine and human megakaryocyte colony formation was studied in the plasma clot culture system. PDGF significantly stimulates megakaryocyte colony formation in a dose-dependent manner. The minimum concentration which had a significant stimulating effect on colony forming unit megakaryocyte (CFU-MK) was lOng/ml and maximum stimulation occurred at 50 ng/ml. The effect of PDGF was compared with that of interleukin (IL)-3, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and acid fibroblast growth factor (aFGF) on megakaryocyte colony formation. The results showed that megakaryocyte colony stimulating activity of PDGF was slightly higher than those of GM-CSF and aFGF, but lower than those of IL-3, IL-6 and EPO. The effect of PDGF in combination with IL-3 or IL-6 on megakaryocyte colony formation was also investigated. No synergistic action was found between PDGF and IL-3 or IL-6, but an additive effect was observed with IL-3 plus IL-6. We also studied the effects of PDGF in combination with anti-IL6, anti-IL-3 or anti-GM-CSF antibody. The increase of megakaryocyte colony formation induced by PDGF was partially inhibited by anti-IL-6 or anti-GM-CSF antibody but not by anti-IL-3 antibody. These results indicate that PDGF is a positive regulator for megakaryocytopoiesis in vitro and IL-6 and GM-CSF may play a role in the mechanism whereby PDGF stimulates megakaryocytopoiesis.     

Interleukin-6 enhances murine megakaryocytopoiesis in serum-free culture

We investigated the effect of interleukin-6 (IL-6) on murine megakaryocytopoiesis in a serum-free culture system. The addition of IL-6 to a culture containing interleukin-3 (IL-3) resulted in a significant increase in the number of megakaryocyte colonies by bone marrow cells of normal mice. The megakaryocytic progenitors that survive exposure to 5-fluorouracil (5-FU) exhibited a more significant response to IL-6 and IL-3. Polyclonal anti-IL-6 antibody neutralized the stimulatory effect of IL-6 on megakaryocyte colony growth supported by IL-3. Delayed addition experiments and replating experiments of blast cell colonies showed that megakaryocytic progenitors are supported by IL-3 in the early stage of the development but require IL-6 for their subsequent proliferation and differentiation. In addition, IL-6 increased the size of megakaryocytes in granulocyte-macrophage-megakaryocyte colonies. The combination of granulocyte colony-stimulating factor or granulocyte-macrophage colony stimulating factor with IL-3 resulted in an increase in the granulocyte-macrophage colony growth of bone marrow cells of 5-FU-treated mice or normal mice, respectively, but had little effect on the enhancement of pure and mixed megakaryocyte colony growth. These results suggest that IL-6 plays an important role in murine megakaryocytopoiesis.     

Interleukin-6 supports human megakaryocytic proliferation and differentiation in vitro

The effect of interleukin-6 (IL-6) on cells of human megakaryocyte (MK) lineage from cord blood was explored. In semisolid colony assays containing human plasma, a greater number of both MK colonies and cells per colony was seen in the presence of IL-6 and IL-3 than in the presence of IL-3 alone. This stimulatory effect of IL-6, observed on both small and large MK colonies, was completely eliminated by the addition of anti-IL-6 antibody to the culture. IL-6 alone had no effect on MK colony formation. In the primary culture, MKs showed larger cell size and DNA content in the presence of both IL-3 and IL-6 than IL-3 alone. The replating experiments using immature MKs grown in the presence of IL-3 showed that IL-6 significantly augmented both cell size and DNA content. This effect was also neutralized by an anti-IL-6 antibody. IL-3 had no tangible effect on MK differentiation. Synergism between IL-6 and IL-3 on MK differentiation was not confirmed. These results suggest that IL-6 is a synergistic factor in the proliferation of MK progenitors and a direct effector of differentiation of immature MKs on in vitro human megakaryocytopoiesis.     

Interactions between recombinant human erythropoietin and serum factor(s) on murine megakaryocyte colony formation.

We investigated the interactions between human erythropoietin (hEpo) and serum factor(s) on murine megakaryocyte (MK) colony formation. Serum-free cultures supported the growth of a large number of murine MK colonies in the presence of murine interleukin-3 (mIL-3). The addition of fetal calf serum (FCS) to mIL-3-containing cultures resulted in only a minimal increase in the number of murine MK colonies. In contrast, hEpo alone had no murine MK colony-stimulating activities in serum-free cultures. hEpo required the presence of FCS, murine serum, or human serum in cultures to promote murine MK colony growth and synergized with these sera to stimulate murine MK colony formation. Furthermore, sera from patients with aplastic anemia showed higher synergistic activities with hEpo than sera from hematologically normal persons (normal human serum). When normal human serum was fractionated by gel-filtration chromatography, two peaks with the synergistic activity were observed in the eluent. However, serum did not show any synergistic effects with hEpo on the growth of murine GM colonies or murine colony-forming unit-erythroid-derived colonies. Although human serum synergized with hEpo to stimulate murine MK colony formation, human cytokines such as IL-3, IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) failed to induce murine MK colony formation in Epo-containing cultures. In cultures containing human IL-1 alpha + human IL-6 + hEpo as well as in cultures containing hEpo, human IL-3 and human GM-CSF failed to show stimulatory effects on murine MK colony formation. Moreover, the synergistic activity of human serum with hEpo could not be neutralized by antibodies such as antihuman IL-1 alpha, antihuman IL-3, antihuman IL-4, antihuman IL-6, antihuman G-CSF, and antihuman GM-CSF. Our data show that serum contains a growth factor(s) that synergizes with Epo to stimulate the proliferation and differentiation of MK precursors, and strongly suggest that this factor(s) is an unique growth factor(s) that is distinct from IL-1 alpha, IL-3, IL-4, IL-6, G-CSF, and GM-CSF.     

New insights into the regulation of megakaryocytopoiesis by haematopoietic and fibroblastic growth factors and transforming growth factor β1

Effects of cytokines on murine megakaryocyte (MK) colony formation from either unfractionated marrow cells or purified early haematopoietic cells were studied. Recombinant interleukin-3 (IL3), interleukin-6 (IL6), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (Epo) and acidic and basic fibroblast growth factor (aFGF and bFGF) each was able to stimulate MK colony growth although they varied somewhat in their potential. IL6 and FGFs, in addition to their effect on MK colony growth, increased the size of individual MK. The combination of IL3 with IL6 or FGF resulted in an additive action. Monoclonal anti-IL6 antibody completely neutralized the activity of mouse IL6 and FGFs but had no effect on human IL6, mouse IL3 and GM-CSF. When using purified lineage negative marrow cells, only IL3 and IL6 promoted MK colony formation. Transforming growth factor beta 1 (TGF-beta 1) at 10-200 pg/ml selectively inhibited IL3-induced MK colony formation, and at 0.2-0.5 ng/ml it still had no obvious effect on the activity of IL6 or GM-CSF but caused an inhibition of FGF-induced MK colony formation. These data suggest that differential mechanisms are involved in the regulation of megakaryocytopoiesis by IL3, IL6, FGFs and GM-CSF, and that TGF-beta 1 negatively regulates MK development mainly by interfering with the action of IL3.     

Cytokine regulation of the human burst-forming unit-megakaryocyte

The human burst-forming unit-megakaryocyte (BFU-MK) is a primitive megakaryocytic progenitor cell. A marrow cell population enriched for BFU-MK (CD34+ DR-) was obtained by monoclonal antibody labeling and fluorescence-activated cell sorting. CD34+DR- cells were assayed in a serum-depleted, fibrin clot culture system. Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant interleukin-3 (rIL-3), and megakaryocyte colony-stimulating factor (MK-CSF), partially purified from human plasma, were each individually capable of promoting BFU-MK-derived colony formation. Recombinant erythropoietin, rG-CSF, rIL-4, rIL-6, and thrombocytopiesis stimulating factor, partially purified from human embryonic kidney cell conditioned media, had no stimulatory effect on BFU-MK-derived colony formation when added alone or in various combinations with either GM-CSF, IL-3, or MK-CSF, GM-CSF and IL-3, GM-CSF and MK-CSF, but not IL-3 and MK-CSF had additive actions in promoting BFU-MK-derived colony formation, rIL-1 alpha had no influence alone on BFU-MK cloning efficiency, but had a dose-dependent, synergistic effect with IL-3, but not with GM-CSF or MK-CSF. The synergistic relationship between IL-1 alpha and IL-3 was abrogated by addition of an IL-1 alpha neutralizing antibody but not by a GM-CSF neutralizing antiserum, suggesting that IL-1 alpha acts directly on the BFU-MK and not by stimulating marrow auxiliary cells to secondarily release additional cytokines. Information presented here indicates that the regulatory influence, acting on the different stages of megakaryocyte development, are stage-specific and accomplished by multiple cytokines.     

Regulation of megakaryocyte development by interleukin-6

Megakaryocytes develop in densely seeded normal mouse bone marrow (BM) cells cultured in agar or in liquid medium. This formation of megakaryocytes is enhanced by the myeloid differentiation-inducing protein MGI-2, which we have shown to be interleukin-6 (IL-6). Monoclonal antibody (MoAb) that specifically neutralizes mouse IL-6 but not human IL-6 inhibited megakaryocyte development in cells cultured either with or without the addition of mouse IL-6 but did not inhibit megakaryocyte development induced by human IL-6. This MoAb to mouse IL- 6 that does not neutralize mouse IL-3 also inhibited mouse IL-3-induced megakaryocyte development. Antibody to mouse GM-CSF did not inhibit the formation of megakaryocytes. The results show that the induction of megakaryocyte development by IL-3 is due to the production of IL-6 in the BM cultures. The present experiments demonstrate a new property of IL-6 and indicate that IL-6 is a regulatory protein of normal megakaryocyte development.     

Synergism of interferon-gamma and stem cell factor on the development of murine hematopoietic progenitors in serum-free culture

We examined the effects of interferon-gamma (IFN-gamma) on the growth of murine hematopoietic progenitors supported by interleukin-3 (IL-3) or stem cell factor (SCF) in a serum-free culture system. IFN-gamma inhibited IL-3-dependent granulocyte-macrophage colony growth by normal bone marrow cells, but increased the number of pure and mixed megakaryocyte colonies by post-5-fluorouracil bone marrow cells. The addition of IFN-gamma to the culture containing SCF resulted in a synergistic action on the development of primitive hematopoietic progenitors as well as on the development of mature populations. Primitive progenitors responding to SCF + IFN-gamma were suggested to be supported by SCF in the early stage of development and require IFN- gamma for subsequent growth. Replating experiments of blast cell colonies and comparison of total colony growth among SCF + IFN-gamma, SCF + IL-3, and SCF + IFN-gamma + IL-3 suggest that multipotential progenitors supported by SCF + IFN-gamma are a part of those reactive to SCF + IL-3. These findings suggest that IFN-gamma has bifunctional activity on murine hematopoiesis.     

Modulation of megakaryocytopoiesis by thrombopoietin: the c-Mpl ligand

We have further characterized the biological activities, mechanism of action, and target cell populations of recombinant human and murine thrombopoietin (rhTPO and rmTPO) in in vitro human and murine model systems. Alone, hTPO or mTPO stimulated the maturation of immature murine megakaryoblasts as measured in a single cell assay. The combination of hTPO or mTPO and interleukin-6 (IL-6) resulted in a further increase in megakaryocyte differentiation in this system. Murine TPO stimulated mouse megakaryocyte progenitor development. Human megakaryocyte progenitor development was potentiated by hTPO alone and further augmented in the presence of the early-acting cytokines (IL-3) or kit ligand/stem cell factor (KL/SCF). To further define the mechanism of action of TPO, neutralization studies were performed with antisera to IL-3, granulocyte-macrophage colony-stimulating factor (GM- CSF), IL-1 beta, and IL-11. No diminution in TPO activity was observed in the presence of these antisera. Moreover, because adhesive interactions are known to modulate hematopoiesis, we studied whether hTPO might alter such interactions between human bone marrow (BM) megakaryocytes and human BM stromal fibroblasts. No changes were observed in either megakaryocyte expression of the surface molecules lymphocyte function-associated antigen-1, very late activation antigen- 4, or intercellular adhesion molecule-1 or the adhesion of megakaryocytes to stromal fibroblasts after treatment with the growth factor. Furthermore, no induction of secretion of the cytokines IL-1 alpha, IL-1 beta, GM-CSF, IL-6, granulocyte-CSF, tumor necrosis factor- alpha, transforming growth factor-beta 1, or transforming growth factor- beta 2 by primary human BM megakaryocytes was noted after treatment of the cells with hTPO. To address whether TPO affects very primitive hematopoietic progenitors, we studied the residual cells from the BMs of mice treated with high doses of 5-fluorouracil. Although no effect of mTPO alone was noted on the viability or replication of such primitive murine progenitor populations, the triple combination of IL-3 + KL/SCF + TPO stimulated growth of megakaryocyte progenitors. These results indicate that TPO is a highly lineage-specific growth factor whose primary biological effects are likely to be direct modulation of the growth and maturation of committed megakaryocyte precursors and immature megakaryoblasts.     

Human serum megakaryocyte colony-stimulating activity appears to be distinct from interleukin-3, granulocyte-macrophage colony-stimulating factor, and lymphocyte-conditioned medium

Sera from patients with bone marrow megakaryocyte aplasia are a rich source of megakaryocyte colony-stimulating activity (Meg-CSA). Other biologic materials exhibiting Meg-CSA include phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM), recombinant interleukin-3 (IL-3), and recombinant granulocyte macrophage colony-stimulating factor (GM-CSF). Neutralizing antisera to both recombinant IL-3 and GM-CSF were used to evaluate the relationship among these sources of Meg-CSA. Varying dilutions of IL-3 and GM-CSF antisera were tested in plasma clot cultures of normal human peripheral blood megakaryocyte progenitors optimally stimulated by either IL-3 (1 U/mL), GM-CSF (1 U/mL), PHA-LCM (2.5% to 5% vol/vol), or aplastic human serum (10% vol/vol). IL-3 antiserum at dilutions up to 1/2,000 totally abrogated megakaryocyte colony growth stimulated by IL-3. A 1/500 dilution of GM-CSF antiserum completely eliminated GM-CSF-induced megakaryocyte colony development. A combination of anti-IL-3 and anti-GM-CSF, each at a 1/500 dilution, inhibited all megakaryocyte colony growth stimulated by optimal concentrations of IL-3 and GM-CSF together. There was no neutralizing crossreactivity between the IL-3 and GM-CSF antisera. At maximally neutralizing concentrations, IL-3 antiserum inhibited 66% of the megakaryocyte colony growth stimulated by PHA-LCM. Residual megakaryocyte colony growth was eliminated by the addition of a 1/500 dilution of anti-GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of megakaryocytopoiesis by human basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) may act to modulate hematopoiesis in addition to its effects on mesenchymal cells. We studied the effects of bFGF on human and murine primary marrow megakaryocytes. bFGF modestly enhanced the size of the human megakaryocyte colony-forming unit (CFU-MK) and cell numbers per colony, in combination with interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF). Adhesion of human megakaryocytes to bone marrow (BM) stromal fibroblasts was enhanced when either stromal fibroblasts or megakaryocytes were treated with bFGF. This resulted in significantly increased proliferation of megakaryocytes. In addition, bFGF augmented secretion of the cytokines tumor necrosis factor alpha and IL-6 by human primary BM megakaryocytes. Immature murine megakaryocytes showed a significant growth response to bFGF as measured by the single cell growth assay. This effect was abrogated by specific antibodies for bFGF and combination of anti-IL-6 and anti-IL-1 beta antibodies. bFGF has no effect on murine CFU-MK formation, but significantly potentiated CFU-MK formation in the presence of IL-3 or GM-CSF. These results indicate that the effect of bFGF on various megakaryocyte populations is different and that bFGF may affect megakaryocytopoiesis via modulation of megakaryocyte-stromal interactions and via augmentation of cytokine secretion from megakaryocytes.     

Synergistic factors for stem cell proliferation: further studies of the target stem cells and the mechanism of stimulation by interleukin-1, interleukin-6, and granulocyte colony-stimulating factor

Serial observations of blast cell colony development from spleen cells of mice treated with 5-fluorouracil (5-FU) four days earlier revealed that either form of human interleukin-1 (IL-1 alpha or IL-1 beta) hastens the emergence of interleukin-3 (IL-3)-dependent blast cell colonies. This activity was essentially indistinguishable from the effect of interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G-CSF) in the same system, an effect that we have ascribed previously to a shortening of the G0 period of the dormant stem cells. We also analyzed the time courses of colony formation from cultures of day-2 post-5-FU marrow cells supported by IL-1 alpha, IL-6, or G-CSF alone or in combination with IL-3. In the presence of IL-3, G-CSF and IL-6 but not IL-1 alpha hastened the development of colonies and increased the numbers of multilineage colonies relative to cultures of IL-3 alone. This observation, together with our previous data from the human system, suggests that the synergistic effect of IL-1 is likely due to induction of secondary growth factors, including IL-6 and G-CSF, by accessory cells in culture. The effect of IL-6 on G0 was confirmed by analysis of the cycling status of progenitor cells in short-term culture. While neither IL-3 nor IL-6 alone had any effect on the cycling status, the combination of factors resulted in a rapid recruitment of quiescent cells into cell cycle (within 48 hours) as represented by a twofold increase in the numbers of multipotential progenitors and a significant increase in the sensitivity of these cells to 3H-thymidine with high specific activity. Combinational testing of all of these synergistic factors revealed that the target cell populations for the IL-1, IL-6, and G-CSF overlap considerably, suggesting that they all may act through a common mechanism. This is further supported by our finding that cells from blast cell colonies grown in the presence of a combination of any one of the synergistic factors with IL-3 replate with higher efficiency and yield more multilineage secondary colonies than those from colonies grown in IL-3 alone. These findings provide further evidence that IL-1, IL-6, and G- CSF serve to integrate the immediate host responses to infection through augmentation of effector cells and antibody production as well as the longer term host responses by recruitment of dormant hemopoietic stem cells into active cell cycling.     

The role of interleukin 6 and interleukin 1 in megakaryocyte development

The effect of human interleukin 6 (IL-6) and interleukin 1 (IL-1) on cells of the megakaryocyte lineage from murine bone marrow was examined. In bone marrow liquid culture, IL-6 but not IL-1 increases the amount of acetylcholinesterase, a megakaryocyte marker. In semisolid colony assays, a low level of interleukin 3 (IL-3) was used as a growth factor, and IL-6 and IL-1 were tested for their ability to potentiate the activity of IL-3 to stimulate megakaryocyte colony formation. IL-6 and/or IL-1 had no effect on megakaryocyte colony formation in the absence of IL-3. However, IL-6 was able to stimulate increased megakaryocyte colonies in the presence of IL-3. IL-1 was able to potentiate colony formation only in the presence of both IL-3 and IL-6.     

Stem cell factor enhances proliferation, but not maturation, of murine megakaryocytic progenitors in serum-free culture.

The effects of recombinant rat stem cell factor (SCF/c-kit ligand) on murine megakaryocytopoiesis were studied using partially purified bone marrow cells derived from normal and 5-fluorouracil (5-FU)-treated mice in a serum-free culture system. SCF alone did not support the formation of megakaryocyte (M) and granulocyte-macrophage-megakaryocyte (GMM) colonies. However, the addition of SCF to cultures containing interleukin-3 (IL-3) resulted in a significant increase in the number of M and GMM colonies formed by bone marrow cells from normal mice, whereas IL-6 augmented only M colony growth. The stimulatory effect of SCF was approximately three to four times as high as that of IL-6 on the primitive progenitors capable of megakaryocytic-lineage expression derived from 5-FU-treated mice. In addition, SCF, but not IL-6, significantly increased the number of constituent cells in the individual M colonies supported by IL-3. On the other hand, SCF did not exert any effect on the size and DNA content of megakaryocytes in IL-3-dependent M and GMM colonies, whereas IL-6 enhanced the maturation of megakaryocytes. These results suggest that SCF stimulates the proliferative process in megakaryocytic progenitors and that the main activity of IL-6 is the promotion of megakaryocyte maturation.     

Circulating megakaryocyte progenitors in myeloproliferative disorders are hypersensitive to interleukin-3

Summary. Previous studies have reported that megakaryocyte progenitors in myeloproliferative disorders (MPD) formed spontaneous megakaryocyte colonies without the addition of megakaryocyte colony-stimulating factor (Meg-CSF). To determine whether this spontaneous colony formation is due to autocrine proliferation of MPD megakaryocyte progenitors or to hypersensitivity to Meg-CSF that might exist in the culture system, we investigated colony-forming unit-megakaryocytes (CFU-Meg) in the peripheral blood of 11 MPD patients, using serum-free cultures. Spontaneous megakaryocyte colonies were observed in serum-free cultures of nonadherent mononuclear cells (NAdMNC) obtained from MPD patients with thrombocytosis, whereas the NAdMNC of MPD patients without thrombocytosis, that of patients with reactive thrombocytosis and normal subjects never formed spontaneous colonies. However, the spontaneous colonies from MPD patients with thrombocytosis disappeared in cultures using highly purified CD34-positive cells as target cells.
To study the hypersensitivity of megakaryocyte progenitors to Meg-CSF, dose-response experiments were performed with interleukin-3 (IL-3). CFU-Meg from MPD patients with thrombocytosis showed maximal growth at the concentrations of IL-3 lower than those for normal subjects. CFU-Meg of MPD patients without thrombocytosis and that of patients with reactive thrombocytosis showed the same colony growth response to IL-3 as that of normal subjects. This result indicates that the CFU-Meg of MPD patients with thrombocytosis are hypersensitive to IL-3. It also suggests that spontaneous colony formation by NAdMNC is not due to the autocrine growth of megakaryocyte progenitors but is due to the hypersensitivity of megakaryocyte progenitors to Meg-CSF, such as IL-3, released by accessory cells. Furthermore, it is possible that such hypersensitivity of CFU-Meg to IL-3 might be a pathogenic factor in MPD with accompanying thrombocytosis.     

Human interleukin-4 inhibits proliferation of megakaryocyte progenitor cells in culture

We studied the effects of recombinant human interleukin-4 (rhIL-4) on megakaryocyte colony formation from enriched hematopoietic progenitors. IL-4 strongly inhibited pure and mixed megakaryocyte colony formation in a dose-dependent manner. Formation of erythroid bursts, eosinophil colonies, and erythrocyte-containing mixed colonies was not affected by the addition of IL-4 as reported previously (Sonoda Y, et al; Blood 75:1615, 1990). Delayed addition experiments suggested that IL-4 acts on an early stage of proliferation of megakaryocyte progenitors. Neutralizing antibodies (antisera) prepared against transforming growth factor beta, tumor necrosis factor alpha, interferon alpha (IFN alpha), and IFN gamma did not affect the inhibitory effects of IL-4 on pure and mixed megakaryocyte colony formation. In addition, the inhibitory effects of IL-4 was also seen in serum-free cultures and in cultures containing highly enriched CD34+, HLA-DR+ cells as a target population. These results indicate that IL-4 may function as one of the negative regulators in human megakaryocytopoiesis in vitro.     

Effects of recombinant human thrombopoietin on megakaryocyte colony formation and megakaryocyte ploidy by human CD34+ cells in a serum-free system

In serum-free cultures of human CD34 cells, recombinant human thrombopoietin (TPO) induced megakaryocyte colony formation in a dose-dependent fashion that was further enhanced by the presence of interleukin-3 (IL-3) and stem cell factor (SCF), but not by IL-6, IL-11 or erythropoietin. TPO gave rise to much smaller colonies and at an earlier time than IL-3, indicating that TPO affects predominantly more mature megakaryocytic progenitors. In liquid cultures, TPO increased the percentage and the absolute number of ≥8N megakaryocytes, but it did not shift their modal ploidy from 2N. TPO-induced endomitosis was totally inhibited by the presence of, or previous exposure of cells to, IL-3 and/or SCF. The mechanism by which TPO overcomes in vivo the negative effects of IL-3 and SCF on megakaryocyte ploidy remains unknown.     

Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor.

Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.     

Characterization of the human burst-forming unit-megakaryocyte

Two classes of human marrow megakaryocyte progenitor cells are described. Colony-forming unit-megakaryocyte (CFU-MK)-derived colonies appeared in vitro after 12-day incubation; burst-forming unit-megakaryocyte (BFU-MK)-derived colonies appeared after 21 days. CFU-MK-derived colonies were primarily unifocal and composed of 11.6 +/- 1.2 cells/colony; BFU-MK-derived colonies were composed of 2.3 +/- 0.4 foci and 108.6 +/- 4.4 cells/colony. CFU-MK and BFU-MK were separable by counterflow centrifugal elutriation. CFU-MK colony formation was diminished by exposure to 5-fluorouracil (5-FU); BFU-MK colony formation was unaffected. CFU-MK and BFU-MK were immunologically phenotyped. CFU-MK expressed the human progenitor cell antigen-1 (HPCA-1, CD34, clone My10) and a major histocompatibility class II locus, HLA-DR, and BFU-MK expressed only detectable amounts of CD34. BFU-MK colony formation was entirely dependent on addition of exogenous hematopoietic growth factors. Recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) possessed such colony-stimulating activity, whereas recombinant erythropoietin (Epo), G-CSF, IL-1 alpha, IL-4, and purified thrombocytopoiesis-stimulating factor did not. These studies indicate the existence of a human megakaryocyte progenitor cell, the BFU-MK, which has unique properties allowing it to be distinguished from the CFU-MK.