Clinical reagents of GM-CSF and IFN-α induce the generation of functional chronic myeloid leukemia dendritic cells in vitro

Dendritic cells (DCs) have been successfully induced in vitro from chronic myeloid leukemia (CML) cells, which may provide a promising immunotherapeutic protocol for CML. To facilitate the optimization of DCs-based vaccination protocols, we investigated the efficiency of in vitro generation of DCs from bone marrow mononuclear cells of CML patients by clinical reagents of GM-CSF and IFN-α. Bone marrow mononuclear cells were isolated from eight CML patients and CML-DCs were generated in the presence of different cytokines (Group A: GM-CSF for research and IL-4 for research; Group B: GM-CSF for injection and IFN-α for injection) in RMPI-1640 medium containing 10% human AB serum. After 8 days, the morphologic features of CML-DCs were observed and their immunophenotypes were analyzed by flow cytometry. The activity of CML-DCs was determined by evaluating their ability to stimulate allogeneic mixed lymphocyte reaction (allo-MLR) and anti-leukemic cytotoxic T lymphocytes (CTLs). The culture protocols were successful in generating functional CML-DCs from all the CML patients as evidenced by the significant upregulation of CD80, CD86, CD83 HLA-DR and CD1a compared to pre-cultured (p < 0.05), and increased allogeneic T cell stimulating proliferation capacity (p < 0.05). CML-DCs could stimulate a specific anti-leukemia response. In summary, we demonstrate that the combination of clinical reagents GM-CSF and IFN-α induced the generation of DCs that have the ability to stimulate a specific anti-leukemia CTLs response in vitro, indicating their feasibility for clinical vaccination protocols for CML patients.     

Karyotype and in vitro-response to GM-CSF. Analysis of bone marrow cultures in leukemia, myelodysplasia and aplastic anemia

The influence of GM-CSF on bone marrow cultures from 13 patients with aplastic anemia, MDS and acute leukemia was studied in a short-term suspension culture system. In each case combined cytogenetic and proliferation analyses were performed with respect to the question, whether chromosome aberrations play a role in the in vitro response to GM-CSF and in order to search for stimulating effects on malignant cells. The responsiveness was compared of aplastic and myelodysplastic cultures on the one hand and of leukemic cells on the other. Our results show that myelodysplastic and aplastic cells display a tendency for reduced susceptibility to GM-CSF as compared to healthy controls, while in leukemic bone marrow the response to the growth factor was significantly enhanced, indicating a leukemia-specific response pattern. In the majority of leukemias analyzed, the presence of cytogenetically abnormal cells in cultures with excessive response to GM-CSF can be taken as a proof for stimulation of malignant cells. The significance of these findings for pathogenesis and prognosis in aplastic anemia, myelodysplasia and leukemia is discussed.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Binding, internalization and degradation of radio-iodinated interleukin 3 and granulocyte-macrophage colony stimulating factor by various hemopoietic cells

The proliferation and differentiation of hemopoietic committed progenitor cells depend on colony stimulating factors (CSF). However, isolated mouse granulocyte-macrophage progenitor cells can still undergo limited proliferation in serum-free cultures after CSF deprivation. To test whether this is due to an accumulated pool of internalized factor, we examined the binding, internalization and degradation of radiolabelled interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) in various hemopoietic cells. We found 20,000 high affinity IL-3 receptors on cells of two IL-3-dependent hemopoietic cell lines, FDC-P1 and FDC-P2 (Kd = 85 and 129 pM). FDC-P1 cells, which also respond to GM-CSF, possess 600 high-affinity GM-CSF receptors (Kd = 64 pM). Cells of both lines internalize IL-3, but only FDC-P1 cells release degraded IL-3 at a rapid rate. Both cell lines have similar dose-response curves for IL-3 and survival kinetics after factor removal. All other cells tested behave like FDC-P1, suggesting that the metabolism of IL-3 by FDC-P2 is exceptional. Our study indicates that transient proliferation of committed progenitor cells in the absence of added factors is apparently not due to a stable pool of internalized CSF but merely represents an intrinsic capability of these cells.     

Granulocyte-macrophage-colony stimulating factor in patients with colorectal cancer

Granulocyte-macrophage-colony stimulating factor (GM-CSF) belongs to the group of glycoproteins called colony-stimulating factors (CSFs). It has been shown that the activity of CSFs is not limited to the hematopoietic cells but can also affect the proliferation of colon carcinoma cell lines. The purpose of this investigation was to compare the serum level of GM-CSF in colorectal cancer patients to a control group, to assess the level of GM-CSF in relation to the level of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), and to define the sensitivity, the specificity and the predictive values of GM-CSF in colorectal cancer. In this study, the serum level of tumour markers was measured in 30 patients with colorectal cancer and in 20 healthy subjects. GM-CSF was assayed using ELISA system, CEA and CA 19-9 were measured by MEIA. The serum levels of CEA, CA 19-9 and GM-CSF were higher in the patients with colorectal cancer than in the control group. The sensitivities of CEA (63%) and CA 19-9 (56%) were lower than the GM-CSF sensitivity (80%). The specificities of tumour markers were 70% (CEA, GM-CSF) and 75% for CA 19-9. The GM-CSF predictive v values were higher than the CEA and CA 19-9 values. These results suggest that GM-CSF may be useful as tumour marker in colorectal cancer, but further studies are needed.     

A recombinant human G-CSF/GM-CSF fusion protein from E. coli showing colony stimulating activity on human bone marrow cells

Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities.     

The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on the synthesis of RNA in liquid cultures of mouse bone marrow, spleen, thymus, peritoneal, peripheral blood leukocytes and lymph node cells was investigated. GM-CSF appeared to stimulate RNA-synthesis in syngeneic bone marrow cells within ten minutes of adding it to the culture. In the presence of GM-CSF bone marrow cultures maintained their initial rate of RNA synthesis for approximately ten hours. GM-CSF had no apparent effect on the uptake of 3H-uridine into bone marrow cells. This stimulation was still observed in the presence of puromycin and cycloheximide, but was abrogated by actinomycin D. The magnitude of the stimulation was not affected by the density of cells between 1 and 20 x 10(6) cells/ml but was slightly smaller at 0.1 and 40 x 10(6) cells/ml. Increasing concentration of GM-CSF (up to 2 X 105 units per ml) led to increased stimulation of RNA synthesis in bone marrow cells, but a significant stimulation could be detected at concentrations as low as 800 units/ml. GM-CSF did not significantly stimulate RNA synthesis in spleen, thymus, mesenteric or subcutaneous lymph node cells. However a small stimulation was observed in peripheral blood leukocytes and peritoneal cells. Autoradiographic studies showed that GM-CSF stimulated RNA synthesis in blast cells, myelocytes, metamyelocytes and polymorphs. Nucleated erythroid cells showed no increased labeling with GM-CFS. Labeling in lymphoid-like cells was highly variable but the level of labeling did not appear to be influenced by GM-CSF.     

Interleukin-6 is the central tumor growth factor in vitro and in vivo in multiple myeloma

When bone-marrow cells from patients with multiple myeloma (MM) were seeded in short-term cultures, a spontaneous proliferation of the myeloma cells occurred for most of the patients with active disease and proliferating myeloma cells in vivo. In all cases, this spontaneous proliferation was inhibited by anti-IL-6 monoclonal antibodies (mabs). Moreover, myeloma cell lines, completely dependent upon exogenous IL-6 for their growth, could be reproducibly established by initially stimulating the myeloma cells with both IL-6 and GM-CSF. These results demonstrate that IL-6 is a major paracrine myeloma-cell growth factor in vitro. High serum IL-6 levels were observed in MM patients with active disease, especially patients with terminal disease. High IL-6 mRNA levels were found in bone-marrow cells of MM patients, mainly in myeloid and monocytic cells, in vivo. The myeloma cells did not express IL-6 mRNA. Injection of anti-IL-6 mabs to MM patients with terminal disease and extramedullary proliferation, completely blocked the myeloma-cell proliferation in vivo and completely inhibited the serum IL-6 bioactivity and the serum CRP levels. One patient with plasma cell leukemia and hypercalcemia was treated for two months with anti-IL-6 mabs and maintain in remission for 2 months without major side effects. Interestingly, the serum calcium levels also decreased in these patients. All these results show that IL-6 is the main cytokine responsible not only for the myeloma-cell proliferation in vivo, but presumably also for the large bone resorption processes observed in human MM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors influencing in vitro production of colony-stimulating factor by mononuclear leukocytes from humans.

The purpose of this study was to identify factors that influence the production of colony-stimulating factor by leukocytes of humans. The use of nonadherent light-density bone marrow cells is semisolid agar cultures to assay the concentrations of colony-stimulating factor in the supernatant of monocyte and mononuclear leukocyte cultures made it possible to distinguish between colony-stimulating factor, which stimulates colony-forming cells directly, and monocyte-dependent stimulating activity, which acts indirectly, by increasing the monocyte production of colony-stimulating factor. Colony-stimulating factor was not detectable in the cytosol of monocytes; that detected in culture must, therefore, have been newly synthesized. Synthesis was enhanced independently by heat-inactivated human serum and by semipurified serum fractions enriched with monocyte-dependent stimulating activity. The kinetics of the production of colony-stimulating factor in the presence and absence of monocyte-dependent stimulating activity indicated that the latter facilitated monocyte production of the former. Factors released from neutrophils were shown to reduce the production of colony-stimulating factor and thr proliferation of colony-forming cells and thus may provide a feedback control mechanism limiting the proliferation of neutrophils.     

Interactions between purified GM-CSF, purified erythropoietin and spleen conditioned medium on hemopoietic colony formation in vitro.

Preincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250-fold excess concentration of purified GM-CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM-CSF were added to agar cultures stimulated by pokeweed mitogen-stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non-erythroid colonies or the non-erythroid content of mixed erythroid colonies. Although neither GM-CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM-CSF was also able to support the survival in vitro of a small proportion of erythroid colony-forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony-forming cells in similar cultures of adult marrow cells. The results do not support the hypothesis that GM-CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM-CSF und EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM-CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM-CSF is unable to stimulate the formation of colonies of these types.     

Effect of proper-myl on humoral interactions between activated lymphocytes and neutrophils in patients with myeloproliferative diseases

The lymphokine synthesizing function of peripheral blood lymphocytes (PBL) was studied in 22 patients with chronic myeloid leukemia (CML), 28 patients with subleukemic myelosis (SLM) and 15 healthy persons. The index of the neutrophil stimulation (INS) in CML (1.73 +/- 0.068) and SLM (1.458 +/- 0.004) was statistically significantly lower than that in the healthy persons (2.6 +/- 0.07). The use of proper-myl in the complex therapy markedly increased the ability of PBL to produce the factor stimulating phagocytic activity of neutrophils (INS 1.94 +/- 0.04 and 1.832 +/- 0.092). On the basis of the findings it was recommended to use proper-myl for prevention and treatment of infectious complications.     

Rapid decline of clonogenic hemopoietic progenitors in semisolid cultures of bone marrow samples derived from patients with chronic myeloid leukemia

Bone marrow samples, from newly diagnosed patients with chronic myeloid leukemia (CML) and normal individuals, were grown in methylcellulose and serially recultured under identical conditions. Specimens derived from normal individuals gave rise to multilineage and megakaryocyte colonies for one to two sequential cultures. Erythroid bursts and granulocyte-macrophage colonies were observed for three to five sequential cultures. Cultures initiated from samples of patients with CML showed a rapid decline of all types of colonies. Colonies were rarely seen for more than two sequential cultures. When pooled colonies and background cells were recloned separately, secondary colonies were mainly seen in cultures of background cells. This observation is consistent with the view that secondary colonies are more likely to arise from dormant clonogenic progenitors, rather than through cells that have formed primary colonies through a self-renewal process.     

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity Together with Polymorphonuclear Leukocytes (PMNs) in Chronic Myeloid Leukemia Patients

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.     

Granulocyte-macrophage colony stimulating factor produced by cloned L3T4a+, class II-restricted T cells induces HT-2 cells to proliferate

Cloned L3T4a+ antigen-specific, class II-restricted T cells can be subdivided by function and by cytokine production. All cloned T cell lines produce T cell growth factors that can be distinguished by the ability of monoclonal antibodies to inhibit the proliferation of cytokine-dependent T cell lines induced by these T cell growth factors. From these types of analyses, it has been shown that all cloned T cells that help hapten-specific B cells secrete immunoglobulin, produce interleukin 4 (IL 4). Those cloned T cells that fail to help for anti-hapten responses produce neither IL 4 nor interleukin 2 (IL 2), yet release an activity that induces the proliferation of the cytokine-dependent T cell line, HT-2. Additional analysis of the HT-2 stimulating activity has shown that it is indistinguishable from granulocyte macrophage-colony stimulating factor (GM-CSF)--this activity being produced by all cloned T cells tested. Thus GM-CSF is a product of all cloned L3T4a+ T cell lines tested thus far, and can serve as a T cell growth factor for HT-2, as well as a co-factor for in vivo derived T cells.     

Granulopoiesis inhibition in acute inflammation: comparative studies in healthy and leukaemic mice

It was demonstrated previously that mice undergoing an inflammatory reaction induced by subcutaneous (SC) implantation of copper rods, produce humoral factors that initially enhance, but subsequently inhibit, diffusion chamber (DC) granulopoiesis. This provided evidence that granulopoiesis is under the control of both humoral stimulators and inhibitors. In order to test the granulopoietic regulatory mechanism in leukaemic mice, we investigated the regulatory role of granulopoietic humoral inhibitors during in vivo granulopoiesis. We noticed that mice suffering from acute myeloid leukaemia (AML) are unable to augment the production of these humoral inhibitory factors when acute inflammation is induced, since no change in DC cell content was observed with or without prior inflammation. Moreover, unlike healthy mice, the serum of leukaemic mice withdrawn during the inhibition phase of acute inflammation did not show any inhibitory activity toward granulocyte-monocyte (GM) colony growth in vitro. Our results also show that increased levels of normal humoral inhibitors do not influence the proliferation and/or differentiation of leukaemic cells implanted in diffusion chamber cultures.     

Dysregulated calcium homeostasis and oxidative stress in chronic myeloid leukemia (CML) cells

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by the oncogenic activity of the Bcr‐Abl protein, a deregulated tyrosine kinase. Calcium may act directly on cellular enzymes and in conjunction with other cellular metabolites, such as cyclic nucleotides, to regulate cell functions. Alteration in the ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation as well as the production of reactive oxygen species (ROS) has been correlates with normal cell proliferation through activation of growth‐related signaling pathways. In this study we evaluated in peripheral blood leukocytes from CML patients the role of the balance between intracellular calcium and oxidative stress in CML disease in order to identify possible therapeutic targets in patients affected by this pathology. Our results demonstrated that peripheral blood mononuclear cells derived from CML patients displayed decreased intracellular calcium [Ca²⁺]_i fluxes both after InsP₃ as well as ATP and ionomycin (IONO) administration. CML cells showed lower levels of superoxide dismutase (SOD) activity and significantly higher malondialdehyde levels (MDA) than peripheral blood mononuclear cells derived from control patients. Finally we showed that resveratrol is able to down‐regulate InsP3 and ATP effects on intracellular calcium [Ca²⁺]_i fluxes as well as the effects of ATP and IONO on oxidative stress in CML cells. J. Cell. Physiol. 224: 443–453, 2010. © 2010 Wiley‐Liss, Inc.     

Circadian rhythms of blood components in dementia senilis

In eleven patients with dementia senilis circadian rhythms of hematocrit, hemoglobin, erythrocytes, leukocytes, serum iron and transferrin were studied by means of the cosinor method. An evident circadian rhythm was observed for all blood components, except leukocytes. There were no significant differences between patients with dementia senilis and healthy aged persons as far as hematocrit, hemoglobin, erythrocytes, and leukocytes are concerned. Acrophase for serum iron and transferrin occurs a little earlier and mesor is lower than in healthy aged persons.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.     

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro

Objective: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). Methods: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. Results: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. Conclusions: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.     

Recombinant murine granulocyte-macrophage (GM) colony-stimulating factor supports formation of GM and multipotential blast cell colonies in culture: comparison with the effects of interleukin-3

We studied the effects of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) on murine hemopoiesis in methylcellulose culture. The GM-CSF was purified from cultures of Saccharomyces cerevisiae transfected with a cloned murine GM-CSF cDNA. In cultures of spleen cells from normal mice, only granulocyte-macrophage (GM) colonies were supported by GM-CSF. Blast cell colonies were the predominant type in cultures of spleen cells from 5-fluorouracil (5-FU)-treated mice. Dose-response studies revealed that maximal GM and blast cell colony formation is achieved with 100 U/ml GM-CSF. Blast cell colonies revealed variable but high replating efficiencies, and the secondary colonies included multilineage colonies. Serial replating of washed blast cell colonies in cultures with GM-CSF provided evidence for the direct effects of GM-CSF on the proliferation of multipotential blast cells. A combination of GM-CSF and interleukin-3 (IL-3) did not increase the number of blast cell colonies over the level supported by IL-3. This observation indicates that the progenitors for blast cell colonies that responded to GM-CSF are a subpopulation of multipotential progenitors that are supported by IL-3. Cytological studies of colonies derived from GM-CSF and/or IL-3 suggest that the eosinophilopoietic ability of murine GM-CSF is less than that of IL-3.