Maturation of human acute myeloid leukaemia in vitro; the response to five recombinant haematopoietic factors in a serum-free system

The abilities of human recombinant IL-3, GM-CSF, G-CSF, M-CSF and Epo to induce maturation in human AML cells in vitro were investigated using cell specimens from 25 AML patients. The experiments were carried out under exactly defined serum-free culture conditions. In the absence of CSFs, monocytic and/or granulocytic maturation was detected in 14/25 cases. IL-3, GM-CSF, G-CSF and M-CSF elevated the proportions of monocyte/macrophages in 3/25, 2/25, 1/25 and 6/25 cases respectively, and increased the percentages of mature granulocytes in 2/25, 1/25, 1/25 and 0/25 cases, and if so only to a limited extent (values below 50%). The 3H-thymidine (3H-TdR) uptake studies revealed that IL-3, GM-CSF, G-CSF and M-CSF were efficient stimulators of DNA synthesis of AML cells in 19, 15, 13 and four of those cases, respectively. Thus, although the cells in most cases responded to CSFs by activation of DNA synthesis, they were unable to give rise to terminally differentiated stages. Provision of CSFs in combination was more frequently effective in enhancing maturation and also increased the magnitude of maturation response. Monocytic versus granulocytic maturation of AML cells after culture did not correlate with the FAB cytology nor with the type of CSF presented; but generally granulocytic maturation was an infrequent phenomenon. Epo stimulated erythroid differentiation and DNA synthesis only in the case of erythroleukaemia, but it had no effect on the cells of 10 other AML cases. Extrapolation of these in vitro findings would suggest that CSFs would have a limited therapeutic utility to induce AML cell maturation in vivo and that hazards of stimulating blast cell proliferation with these factors may be anticipated.     

Interleukin-1, tumor necrosis factor, and the production of colony- stimulating factors by cultured mesenchymal cells

Although the genes for four hematopoietic colony-stimulating factors (CSFs) have been cloned, neither the mechanism of the regulation of their production nor their cellular origins have been established with certainty. Monocytes are known to produce colony-stimulating and burst- promoting activities, as well as several monokines such as interleukin- 1 (IL-1) and tumor necrosis factor (TNF). These monokines indirectly stimulate other mesenchymal cells to produce certain colony-stimulating factors such as granulocyte-macrophage CSF (GM-CSF). To determine whether monocytes produce other CSFs and if so, to compare the mechanism of regulation of production with that of endothelial cells and fibroblasts, we investigated the synthesis of CSFs by monocytes, human umbilical vein endothelial cells, and fibroblasts. We used total cellular RNA blot analysis to determine interleukin-3 (IL-3), GM-CSF, granulocyte CSF (G-CSF), and monocyte CSF (M-CSF) messenger RNA (mRNA) content and immunoprecipitation or bioassay to confirm the presence of the specific secreted proteins. The results indicate that M-CSF mRNA and protein are produced constitutively by all three cell types and their level of expression does not increase after induction. In contrast, GM-CSF and G-CSF mRNAs are barely detectable in uninduced monocytes and show an increase in expression after lipopolysaccharide treatment. Retrovirus-immortalized endothelial cells, unlike primary endothelial cells or both primary and immortalized fibroblasts, produce IL-1 constitutively; this correlates with their constitutive production of GM-CSF and G-CSF. IL-3 mRNA was not detectable in any of these cells either before or after induction. The results indicate that these mesenchymal cells can produce three CSFs: GM-CSF, G-CSF, and M-CSF; furthermore, the data suggest that the mechanism of regulation of M-CSF production is different from that of GM-CSF and G-CSF, and that the latter two inducible CSFs are regulated by different factors in monocytes compared with the other mesenchymal cells.     

Initiation in DNA synthesis by colony-stimulating factors in subsets of human CD34+ marrow cells.

Initiation of DNA synthesis by recombinant colony-stimulating factors (CSFs) was assessed in normal human marrow blast cells isolated by expression of CD34 antigen (tritiated thymidine incorporation). Continuous exposure to CSF was required. A mild increase in DNA synthesis was initiated by granulocyte CSF (G-CSF; greater than or equal to 1 ng/ml), to approximately 1.5 times control levels. A greater increase was initiated by granulocyte-macrophage CSF (GM-CSF), with a threshold of approximately 0.1 ng/ml and a plateau increment 2.5 times control levels. CD34+ cells were stimulated by interleukin 3 (IL-3) over a wide concentration range: two times control at 0.1/ml, three times control at 1 ng/ml, and four times control at 10 ng/ml. Overlap between responding populations was analyzed. G-CSF plus GM-CSF induced DNA synthesis greater than GM-CSF alone and supported the growth of much larger granulocyte-monocyte colonies. At saturating IL-3 concentrations, neither G-CSF nor GM-CSF induced additional DNA synthesis; at lower concentrations of IL-3, however, GM-CSF recruited additional cells into DNA synthesis. Using CD10 and CD19 antibodies to separate B-lineage cells, the CD34+ cells responding to CSF were observed to be in the non-B-lineage subset. Therefore 1) the response of CD34+ cell subsets CSFs is IL-3 greater than GM-CSF greater than G-CSF, and the IL-3-responsive population is heterogeneous for dose requirement; 2) a CD34+ subpopulation responding to concurrent G-CSF and GM-CSF includes increased proliferative potential cells; 3) IL-3-responsive cells include GM-CSF- and G-CSF-responsive cells, but cells responding to lower IL-3 concentration do not respond to GM-CSF; and 4) B-cell precursors do not respond to GM-CSF or IL-3 in this assay.     

Myeloid differentiation of purified CD34+ cells after stimulation with recombinant human granulocyte-monocyte colony-stimulating factor (CSF), granulocyte-CSF, monocyte-CSF, and interleukin-3.

CD34+ cells isolated from bone marrow or umbilical cord blood from healthy donors were studied for proliferation and differentiation in liquid cultures in the presence of recombinant human granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), monocyte CSF (M-CSF), and interleukin-3 (IL-3), followed by immunophenotyping for myeloid and myeloid-associated cell surface markers. IL-3, either alone or together with GM-CSF, G-CSF, or M-CSF, induced, on average, 50-fold cell multiplication, GM-CSF five fold to 10-fold, and G-CSF and M-CSF less than fivefold. Cells from cultures stimulated with GM-CSF, G-CSF, or M-CSF alone contained cells with a "broad" myeloid profile, "broader" than observed in cultures with IL-3. However, since IL-3 induced rapid cell multiplication, high numbers of cells expressing early (CD13, CD33) and late myeloid markers (CD14, CD15) were recovered. The presence of other CSFs together with IL-3 did not alter the IL-3-induced effect on the cells. When 5,000 CD34+ cells were cultured with IL-3 alone, the cultures still contained 2,000 to 5,000 CD34+ cells after 14 days of culture, while cells cultured with GM-CSF, G-CSF, or M-CSF contained less than 1,000 CD34+ cells. Furthermore, 1,000 to 3,000 cells were positive for the megakaryocytic lineage marker CD41b after cultures with GM-CSF or IL-3, while cultures with G-CSF or M-CSF did not contain detectable numbers of CD41b+ cells. Finally, erythroid cells could also be generated from purified CD34+ cells. The results show that IL-3 and GM-CSF can induce rapid proliferation of purified CD34+ cells in vitro with differentiation to multiple myeloid lineages, while certain subsets maintain expression of CD34.     

Recombinant gibbon interleukin-3 acts synergistically with recombinant human G-CSF and GM-CSF in vitro

Recombinant gibbon interleukin-3 (IL-3) is a multilineage hematopoietic colony-stimulating factor (CSF) that recently was cloned and found to be highly homologous with human IL-3. Gibbon IL-3, as well as human granulocyte-CSF (G-CSF) and human granulocyte-macrophage CSF (GM-CSF), stimulated normal human bone marrow cells to form myeloid colonies in soft agar in a sigmoidal dose-response manner. When IL-3 was added to increasing concentrations of G-CSF or GM-CSF, synergistic colony formation occurred as compared with the effects of each CSF alone. Synergism was also noted when G-CSF was added with GM-CSF and when all the CSFs were added simultaneously. The combination of IL-3 and GM-CSF was less stimulatory than all the other CSF combinations. At day 11 of culture, IL-3 induced granulocyte-macrophage (38%), eosinophil (30%), granulocyte (18%), and macrophage (14%) colony formation. In summary, gibbon IL-3 is a growth factor that can synergize with other CSFs to enhance proliferation of myeloid-committed progenitors, suggesting that combinations of CSFs may have clinical utility in patients with neutropenia of various etiologies.     

The effects of GM-CSF and G-CSF in promoting growth of clonogenic cells in acute myeloblastic leukemia

A small subset of leukemic cells from most patients with acute myeloblastic leukemia (AML) have properties of stem cells and can be assayed by colony formation in agar or methylcellulose. Colony formation generally requires the addition of exogenous growth factors, but the exact factors required are incompletely defined. The AML colony- promoting activities of two recombinant human colony-stimulating factors (GM-CSF and G-CSF) were investigated by using blasts from 48 patients with AML. In nine cases, no colonies formed with either CSF. In seven cases colonies formed only in response to G-CSF and in 11 cases only in response to GM-CSF. In 21 cases colonies formed in response to either GM-CSF or G-CSF, and in 12 of these cases there was an additive effect between the two CSFs in determining maximum colony size. For cases responding to both GM- and G-CSF, the total number of colonies formed in response to the combination of both CSFs was almost always less than additive compared with the number of colonies formed in response to the individual CSFs. Further, the AML-CFU responding to either GM-CSF or G-CSF could not be distinguished by surface markers or by the cytochemical staining pattern of the colonies. These results suggest that there is considerable overlap between the GM-CSF- and G- CSF-responsive AML-CFU subpopulations in most cases. For five of seven cases, the combination of GM-CSF and G-CSF could replace a leukocyte feeder layer in providing maximum growth stimulation. These results indicate that GM-CSF and G-CSF are active growth factors for AML cells and are frequently additive in promoting maximum colony size.     

Effects of various recombinant human hematopoietic growth factors on proliferation of blast cells in acute myeloid leukemia in vitro]

In vitro proliferative response of the blast cells from 21 AML patients to hematopoietic growth factors (IL-3, GM-CSF, G-CSF and MCSF) was investigated. Proliferation of AML cells in the majority of cases was induced or promoted by one or more CSFs, among which the stimulation of IL-3 was the most effective. Spontaneous proliferation of the blast cells was also observed in half of the cases and could be inhibited as well as promoted by some CSFs. It is suggested that in vitro proliferation of AML cells varies from patient to patient and that CSF plays important roles in leukemogenesis.     

Mechanism of action of interleukin 1 on the progenitors of blast cells in acute myeloblastic leukemia

We tested the effect of interleukin 1 (IL-1) on the growth of leukemic blast progenitors from patients with acute myeloblastic leukemia (AML). A purified blast cell fraction depleted of both T cells and phagocytic cells was tested at different cell densities. Addition of 1 ng/ml of IL-1 alpha alone enhanced blast colony formation in 10 of 13 cases tested, and the enhancement was prominent when plated cell densities were lowered. The conditioned media (CM) from AML patients contained varied levels of IL-1 activity, and following depletion of phagocytic cells, the levels decreased markedly in all cases tested. Addition of either antiserum against IL-1 alpha or IL-1 beta reduced the IL-1 activity in CM, suggesting that AML blasts produce both IL-1 alpha and IL-1 beta. Addition of IL-1 alpha or IL-1 beta antiserum inhibited blast colony formation in a dose-dependent manner, and a combination of both antisera showed the most marked inhibition. However, the augmentation of blast colony formation was almost completely inhibited by addition of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) serum in all three cases tested. IL-1 is also devoid of this activity when tested in the presence of a combination of granulocyte CSF (G-CSF), GM-CSF, and interleukin 3 (IL-3) at an optimal concentration. These results suggest that blast cells could produce and secrete CSF(s) and/or IL-1, and that the growth-enhancing effect of IL-1 on AML blasts is indirect, via production of CSFs by leukemic cells.     

Regulation of differentiation of murine progenitor cells derived from blast cell colonies under serum-deprived conditions

We have examined the effect of interleukin 3 (IL-3), granulocyte-macrophage (GM)-, granulocyte (G)-, and macrophage (M)-colony-stimulating factors (CSFs) on the induction of GM colonies from highly enriched murine hematopoietic progenitor cells under serum-deprived conditions. Each growth factor was tested alone or in combination with suboptimal concentrations of the others. The effect of each CSF on GM colony growth in fetal bovine serum (FBS)-supplemented cultures of unfractionated marrow cells is reported for comparison. GM-CSF induced GM colony growth in serum-deprived cultures of purified progenitor cells to the same extent as in FBS-supplemented cultures of unfractionated marrow cells. In contrast, IL-3 was only one-tenth as active in promoting the growth of enriched progenitor cells under serum-deprived conditions when compared with its effect on colony growth from unfractionated marrow. M-CSF and G-CSF were almost completely ineffective in both cases. G-CSF induction of GM colony growth from purified progenitor cells was restored by addition of suboptimal concentrations of IL-3 or GM-CSF, suggesting that either IL-3 or GM-CSF is required to observe the effect of G-CSF. Addition of G-CSF to GM-CSF-stimulated cultures did not increase the maximal number of colonies detected, indicating that these two growth factors may act on the same subset of progenitor cells. Addition of GM-CSF or IL-3 to IL-3- or GM-CSF-stimulated cultures, respectively, increased by 40% the maximal number of colonies detected, suggesting that these two factors act on at least partially separate subsets of GM progenitors. These data parallel the recent observations on the control of human GM colony formation under FBS-deprived conditions and support a model for the control of myeloid differentiation that requires the interplay of different growth factors.     

Modulation of tartrate-resistant acid phosphatase expression by calcitriol in CSF-induced macrophage colonies

The osteoclast is thought to be a hemopoietically derived cell, but questions exist about which hemopoietic growth factors are responsible for proliferation of osteoclast precursors. Experiments were thus performed to see if recombinant human colony-stimulating factors (CSFs) influenced the expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast marker enzyme, by monkey bone marrow colonies in vitro. In addition, the effect of 1,25-dihydroxyvitamin D3 (calcitriol) on CSF-induced colony growth and TRAP expression was also determined. Bone marrow was obtained from a single Macaca nemestrina monkey, kept frozen in liquid nitrogen, and aliquots of frozen cells were thawed and placed at 10(5) cells per plate in a standard cell colony-forming unit (CFU-C) assay. The recombinant human CSFs (granulocyte-macrophage CSF, GM-CSF; macrophage CSF, M-CSF; interleukin 3, IL-3; and granulocyte CSF, G-CSF) were added to the cultures at 50 U/ml, and calcitriol was titrated for each CSF from 0.1 to 100 nM. Day-14 colonies were stained to demonstrate TRAP-positive cells in individual colonies. GM-CSF caused an increase (193%, p less than 0.0004) in total colony numbers that was only partially inhibited by calcitriol. IL-3 and M-CSF had less effect, and G-CSF had no effect. GM-CSF also caused a large increase in TRAP-positive macrophage (M) colonies (326%, p less than 0.0001) and changed the relative proportion of TRAP-positive M colonies from 39% to 62% of all M colonies. M-CSF caused less increase in numbers of TRAP-positive M colonies and had no effect on the proportion of TRAP-positive colonies. When GM-CSF was present, calcitriol caused a maximum number of TRAP-positive colonies to appear at 1 nM, and it caused a drastic decrease in TRAP-positive colonies at higher doses. Calcitriol at 10 nM caused TRAP-negative colonies to increase in number and proportion when GM-CSF was present, but in the presence of M-CSF, the same dose of calcitriol caused a decline in numbers of TRAP-negative colonies. These results suggest that GM-CSF may be important in the replication of TRAP-positive mononuclear cells that resemble osteoclast precursors and that myeloid cell development may be weighted toward TRAP-positive or TRAP-negative progeny depending on whether GM-CSF or M-CSF predominates. They further suggest that calcitriol concentration may be critical in this process.     

Secretion of interleukin-1 by acute myeloblastic leukemia cells in vitro induces endothelial cells to secrete colony stimulating factors

The interaction of acute myeloblastic leukemia (AML) cells with stromal cells was investigated by adding AML-conditioned media to cultures of human endothelial cells. This conditioned media contained factors that induced expression of both the granulocyte macrophage colony- stimulating factor (GM-CSF) and granulocyte CSF (G-CSF) genes and release of colony stimulating activity from endothelial cells. The conditioned media contained interleukin-1 (IL-1) bioactivity and the endothelial cell stimulatory activity was partially neutralized by anti- IL-1 antiserum. Constitutive expression of the IL-1-beta gene was detected in ten of 17 AML cases analyzed. These results suggest that the unregulated secretion of IL-1 by AML cells can induce stromal cells in vitro to overproduce CSFs. This could contribute to the unrestricted growth of AML cells.     

Interleukin-1 stimulates proliferation of acute myeloblastic leukemia cells by induction of granulocyte-macrophage colony-stimulating factor release

In this study, we further established the role of interleukin-1 (IL-1) alpha and IL-1 beta as regulators of proliferation of acute myeloid leukemia (AML) cells. IL-1 stimulated tritiated thymidine (3H-TdR) uptake of AML cells in 13 of 28 cases. Cytogenetic analysis confirmed the leukemic clonality of the IL-1-stimulated cells. Most likely, IL-1 exerted these stimulative effects directly on AML blast cells because IL-1 effectively induced 3H-TdR uptake of CD34-positive AML blasts (separated following cell sorting). Furthermore, adherent cell-depleted AML samples of three patients were more effectively stimulated than nondepleted AML fractions. Cluster and colony formation from adherent cell depleted AML samples could also be stimulated with IL-1, ie, in seven of ten cases analyzed. Subsequent experiments indicated that IL-1 stimulation depended on the release of GM-CSF because (1) induction of DNA synthesis of AML cells by IL-1 could be abrogated with antigranulocyte-macrophage colony-stimulating factor (GM-CSF) antibody, (2) conditioned media (CM) prepared from IL-1 stimulated AML blasts (adherent cell depleted) could stimulate the proliferation of purified normal bone marrow progenitors whereas supernatants from nonstimulated AML blasts did not, and (3) GM-CSF was demonstrated in IL-1/AML-CM with a specific radioimmunoassay, while GM-CSF was not detectable in nonstimulated supernatants. In one case of AML showing significant 3H- TdR uptake in the absence of CSFs, this spontaneous DNA synthesis was found to depend on autocrine IL-1 beta release as it could be suppressed with anti-IL-1 beta antibody or anti-GM-CSF. The blockade by anti-IL-1 beta could be overcome by the addition of high concentrations of IL-1 beta as well as GM-CSF. Thus, in this particular case, endogenously produced IL-1 beta had stimulated the release of GM-CSF which resulted in GM-CSF-dependent proliferation. The results indicate that GM-CSF production by AML blasts is often regulated by IL-1 rather than being constitutive.     

Effect of interleukin-1, tumor necrosis factor-alpha, and interferon-alpha on the blast cells of acute myeloblastic leukemia.

In this study, we further established the role of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-alpha (IFN-alpha) as regulators of proliferation of acute myeloid leukemia (AML) cells. AML cells from 8 of 15 patients incorporated high levels of 3H-thymidine (3H-TdR) in the absence of exogenous growth factors. The spontaneous DNA synthesis could be abrogated with monospecific antibodies directed toward IL-1 alpha, IL-1 beta, or TNF-alpha, as well as with antigranulocyte-macrophage colony-stimulating factor (GM-CSF). Human recombinant GM-CSF reversed the inhibitory action of each of these antibodies and reinduced DNA synthesis in AML cells. Thus, in these cases, constitutively produced IL-1 or TNF-alpha had stimulated the synthesis of GM-CSF, which resulted in GM-CSF-dependent proliferation of AML blasts. Exogenous IL-1 up-regulated the endogenous production of GM-CSF, suggesting a positive regulation of autocrine growth factor production. We also present evidence that TNF-alpha may exert both stimulative as well as inhibitory effects on DNA synthesis in AML cells. The enhancing effect of TNF-alpha was mediated through the induction of GM-CSF production, as stimulation of DNA synthesis in AML blasts could be abrogated with anti-GM-CSF antibody. A concentration-dependent inhibitory effect of TNF-alpha on 3H-TdR incorporation into AML blasts was observed only when these cells were grown in the absence of GM-CSF. Finally, we show that human recombinant IFN-alpha is a potent inhibitor of AML cell proliferation in vitro.     

Mechanism of synergy between granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in colony formation from human marrow cells in vitro

The synergy of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human granulocyte colony-stimulating factor (G-CSF) in the colony formation derived from human marrow cells was studied. The colony formation stimulated by GM-CSF plus G-CSF was dependent on the dose of each CSF, with the plateau for the number of GM colonies being higher than the sum of the individual plateaus by GM-CSF or G-CSF. Analysis of the colonies formed by GM-CSF plus G-CSF revealed efficient formation of neutrophil and monocyte colonies. To study the effect of GM-CSF and G-CSF on the maintenance of the progenitors that respond to the synergy of the CSFs, addition of each CSF to the medium of clonal cell culture was delayed. The progenitors that formed colonies on day 7 due to synergy of the CSFs were perfectly maintained by GM-CSF for at least 72 h and the progenitors that formed colonies on day 14 due to synergy of the CSFs were partly maintained by G-CSF or GM-CSF. The DNA synthetic rate of the progenitor cells that respond to GM-CSF plus G-CSF was significantly lower than those that respond to GM-CSF or G-CSF. According to light scatter analysis of phagocyte-depleted marrow mononuclear cells (PD-MMCs) using a flow cytometer, the peak population of progenitors that respond to GM-CSF plus G-CSF was in the smaller part of the PD-MMCs than those to GM-CSF or G-CSF. These results indicated that the progenitors to the synergy of GM-CSF and G-CSF are in a different proliferative state than those to each CSF. The synergy of GM-CSF and G-CSF depends on each CSF maintaining the viability of a different population of GM progenitors that can form GM colonies by both CSFs together.     

Stem cell factor stimulates the in vitro growth of bone marrow cells from aplastic anemia patients.

Aplastic anemia (AA) is a rare human bone marrow disorder of unknown etiology manifested by a strongly impaired growth of hematopoietic precursors. In this study, we examined the ability of recombinant human stem cell factor (SCF) to stimulate proliferation in vitro of bone marrow cells from 15 AA patients. All patients had been previously treated with antilymphocyte globulin (ALG). SCF, in combination with erythropoietin (Epo), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), increased the number of hematopoietic colonies formed in a semisolid medium by AA marrows. Maximal colony numbers reached 30% of the numbers observed with normal bone marrow cells. Proliferation of AA cells cultured in a liquid medium containing SCF together with Epo, IL-3, GM-CSF, and G-CSF approached 70% of the control level, as measured by 3H-thymidine incorporation. The effect of the combination of SCF with the other growth factors was more than 10 times stronger than that of the growth factors alone. The most marked effect of SCF was on the generation of erythroid colonies by precursor cells. The results demonstrate synergism between CSF and other hematopoietic growth factors, resulting in the most efficient stimulation of the in vitro growth of AA bone marrow cells described to date. Use of SCF, either alone or in combination with other factors, may be of potential value in treatment of AA.     

Independent regulation of M-CSF and G-CSF gene expression in human monocytes

The macrophage and granulocyte colony-stimulating factors, M-CSF and G- CSF, act in vitro to induce proliferation and differentiation of monocyte and granulocyte progenitor cells, respectively. We show here that both of these CSFs can be produced by stimulated human blood monocytes, but the M-CSF and G-CSF genes are independently regulated. Recombinant human interleukin-3 (IL-3) and GM-CSF primarily induce expression of the M-CSF gene and secretion of M-CSF, whereas bacterial lipopolysaccharide primarily induces expression of the G-CSF gene and secretion of G-CSF. These results suggest that under different conditions of in vitro stimulation the monocyte secretes factors that could lead selectively to either granulocyte or monocyte production.     

Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells

Nonadherent low density T-lymphocyte depleted (NALT-) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My10 and phycoerythrin conjugated anti HLA-DR monoclonal antibodies in order to obtain enriched populations of colony forming unit-megakaryocyte (CFU-MK). The CFU-MK cloning efficiency (CE) was 1.1 +/- 0.5% for cells expressing both high densities of My10 and low densities of HLA-DR (My10 DR+). This procedure resulted in an 18-fold increase in CE over NALT- cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1 alpha (IL-1 alpha), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF or CSF-1) and interleukin MK colony formation by My10 DR+ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF-1, IL-1 alpha nor G-CSF alone augmented MK colony formation above baseline (2.5 +/- 0.8/5 x 10(3) My10 DR+ cells plated). In contrast, the addition of GM-CSF and IL-3 each increased both CFU-MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL-3 and 25 units/ml of GM-CSF. At maximal concentration, IL-3 had a greater ability to promote megakaryocyte colony formation than GM-CSF. The stimulatory effects of GM-CSF and IL-3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU-MK appears, therefore, to express HPCA-1 and HLA-DR antigens. These studies also indicate that GM-CSF and IL-3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My10 DR+ cells are largely devoid of these accessory cells.