Granulocyte-macrophage Colony-stimulating Factor Receptor Expression Research Articles

Determination of granulocyte/macrophage-colony-stimulating factor secretion by human melanoma cells and its effects on human melanoma cell proliferation.

Recently, granulocyte/macrophage-colony-stimulating factor (GM-CSF) became available for overcoming chemotherapy-induced granulocytopenia. GM-CSF not only has a prominent role in the regulation of proliferation and differentiation of haematopoietic cells but it is also secreted by a variety of solid tumours and is capable of exerting growth-stimulatory effects. To evaluate the safety of GM-CSF administration in the treatment of malignant melanoma, we investigated GM-CSF secretion, GM-CSF receptor expression and the effect of GM-CSF on the proliferation of human melanoma cells in vitro. A panel of eight human melanoma cell lines and two fresh tumour specimen was studied. GM-CSF protein was not detectable in culture supernatants by ELISA without stimulation. Interleukin-1 and tumour necrosis factor alpha induced GM-CSF secretion in all four melanoma cell lines tested. When biotinylated GM-CSF was used, the corresponding receptor was not detectable by immunohistochemical or FACScan analysis. The proliferation of eight human melanoma cell lines and two fresh melanoma specimens was determined by the MTT test after 4-6 days of growth in the presence of different concentrations of GM-CSF (0.1-1000 U/ml). Neither proliferation nor growth inhibition was observed. Therefore the effect of GM-CSF on residual tumour cells in vivo may not present a problem during clinical use to stimulate marrow regeneration after or during chemotherapy of metastatic malignant melanoma.

GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: evidence for sodium/proton antiporter and PKC mediated activation of acid production.

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.

Human glioblastoma cells produce granulocyte-macrophage colony-stimulating factor in vitro, but not in vivo, without expressing its receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) production and receptor expression by human glioblastomas was studied. Enzyme-linked immunosorbent assay showed four of 10 glioblastoma cell lines spontaneously released GM-CSF (2.9-9.2 pg GM-CSF protein/ml culture medium), which was enhanced by stimulation with tumor necrosis factor-alpha (TNF) (10 U/ml) up to 410 pg/ml. TNF also induced secretion of GM-CSF by another cell line. Northern blot analysis identified increasing GM-CSF gene expression by cells following TNF stimulation. However, no GM-CSF protein was detectable in the cerebrospinal fluid of three malignant glioma patients. Intratumoral administration of TNF in the patients also failed to stimulate GM-CSF levels in the cerebrospinal fluid. A binding assay using flow cytometry with biotinylated GM-CSF and Scatchard analysis using 125I-labeled GM-CSF failed to demonstrate GM-CSF receptor expression on the 13 cell lines. Exogenous GM-CSF stimulation had no effect on production of prostaglandin E2, interleukin-6, or interleukin-8 by glioma cells. Human glioblastoma cells secrete GM-CSF without expressing the receptor in vitro, but there was no evidence of GM-CSF production in vivo.

Endogenous serum levels and surface receptor expression of GM-CSF and IL-3 in patients with myelodysplastic syndromes

The majority of patients suffering from myelodysplastic syndromes (MDS) die of complications due to cytopenia. Clinical trials have demonstrated that in an essential number of MDS patients cytopenia can be ameliorated by exogenously supplied growth factors. We investigated endogenous serum levels of GM-CSF and IL-3 in 15 healthy individuals and 34 patients suffering from MDS. No circulating growth factors were detected in the serum of healthy controls, nor was IL-3 measurable in MDS patients. GM-CSF serum levels, however, were elevated in a significant number of patients (26.5%). Levels did not correlate with FAB classification, leukocyte count or chromosomal abnormalities. No significant differences in GM-CSF or IL-3 receptor expression were detected between healthy individuals and MDS patients. One patient with increased endogenous GM-CSF serum level and normal surface receptor expression responded to exogenously applied GM-CSF. In the light of these results, a funetional alteration of growth factor receptors or disturbances of signal transduction pathways must be discussed for MDS.

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.

Decreased Stimulated GM-CSF Production and GM-CSF Gene Expression but Normal Numbers of GM-CSF Receptors in Human Term Newborns Compared with Adults

We investigated cord and adult production of granulocyte-macrophage colony-stimulating factor (GM-CSF), expression of GM-CSF mRNA from unstimulated and activated mononuclear cells, and the affinity and presence of GM-CSF receptors on mature effector cells in an attempt to better understand the underlying pathophysiology of altered neonatal host defense. Utilizing 125I-GM-CSF as a ligand, Scatchard analysis revealed the presence of a single class affinity GM-CSF receptor with similar binding characteristics on both cord and adult peripheral PMN (kd = 44 and 39 pM) for adult and cord, respectively. Additionally, there was no significant difference in the number of GM-CSF receptors on cord versus adult neutrophils. Using a sandwich ELISA for measuring GM-CSF levels, we found nondetectable levels from supernatants of unstimulated cord and adult mononuclear cells and serum from cord and adult peripheral blood. However, there was a significant difference between cord and adult GM-CSF production from stimulated phytohemagglutinin and phorbol-12-myristate-6-acetate mononuclear cells (p less than 0.02). Additionally, GM-CSF mRNA expression from activated cord mononuclear cells was significantly reduced after 6 h of stimulation compared with adults. Nuclear run-on experiments revealed no difference in transcriptional activation from activated cord and adult mononuclear cells. Actinomycin D transcriptional decay studies, however, demonstrated reduced GM-CSF half-life from activated cord versus adult mononuclear cells (t1/2 30 versus 100 min). These results suggest normal affinity and numbers of GM-CSF receptors on peripheral mature effector cells but decreased GM-CSF production and GM-CSF mRNA expression from activated cord versus adult mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor Necrosis Factor Regulates the Expression of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-3 Receptors on Human Acute Myeloid Leukemia Cells

Tumor necrosis factor (TNF) acts as a potent enhancer of granulocyte-macrophage colony-stimulating factor (GM-CSF)- and interleukin-3 (IL-3)-induced human acute myeloid leukemia (AML) growth in vitro. We have analyzed the effects of TNF alpha on the expression of GM-CSF and IL-3 receptors on AML cells. Incubation of blasts from seven patients with AML in serum-free medium with TNF (10(3) U/mL) and subsequent binding studies using 125I-GM-CSF and 125I-IL-3 show that TNF increases the specific binding of GM-CSF (30% to 280%) and IL-3 (40% to 600%) in all cases. From Scatchard plot analysis it appears that TNF upregulates (1) low-affinity GM-CSF binding sites, (2) common high-affinity IL-3/GM-CSF binding sites, and (3) unique (non-GM-CSF binding) IL-3 binding sites. The effect of TNF is dose dependent and is half maximal at a concentration of 100 U/mL, and becomes evident at 18 hours of incubation with TNF at 37 degrees C, but not at 0 degree C. The GM-CSF dose-response curve of AML-colony-forming units plateaus at a higher level in the presence of TNF, which indicates that additional numbers of cells become responsive to GM-CSF. Incubation of AML blasts with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate or formyl-Met-Leu-Phe (protein kinase C activators) does not influence GM-CSF receptor expression, suggesting that receptor upregulation by TNF is not mediated through activation of protein kinase C. On the other hand, the protein synthesis inhibitor cycloheximide abrogates receptor upregulation induced by TNF. In contrast to these findings in AML, TNF does not upregulate GM-CSF receptor numbers on blood granulocytes or monocytes. We conclude that TNF exerts positive effects on growth factor receptor expression of hematopoietic cells.

Tumor necrosis factor regulates the expression of granulocyte- macrophage colony-stimulating factor and interleukin-3 receptors on human acute myeloid leukemia cells

Tumor necrosis factor (TNF) acts as a potent enhancer of granulocyte- macrophage colony-stimulating factor (GM-CSF)- and interleukin-3 (IL-3)- induced human acute myeloid leukemia (AML) growth in vitro. We have analyzed the effects of TNF alpha on the expression of GM-CSF and IL-3 receptors on AML cells. Incubation of blasts from seven patients with AML in serum-free medium with TNF (10(3) U/mL) and subsequent binding studies using 125I-GM-CSF and 125I-IL-3 show that TNF increases the specific binding of GM-CSF (30% to 280%) and IL-3 (40% to 600%) in all cases. From Scatchard plot analysis it appears that TNF upregulates (1) low-affinity GM-CSF binding sites, (2) common high-affinity IL-3/GM-CSF binding sites, and (3) unique (non-GM-CSF binding) IL-3 binding sites. The effect of TNF is dose dependent and is half maximal at a concentration of 100 U/mL, and becomes evident at 18 hours of incubation with TNF at 37 degrees C, but not at 0 degree C. The GM-CSF dose-response curve of AML-colony-forming units plateaus at a higher level in the presence of TNF, which indicates that additional numbers of cells become responsive to GM-CSF. Incubation of AML blasts with the phorbol ester 12–0-tetradecanoylphorbol-13-acetate or formyl-Met-Leu- Phe (protein kinase C activators) does not influence GM-CSF receptor expression, suggesting that receptor upregulation by TNF is not mediated through activation of protein kinase C. On the other hand, the protein synthesis inhibitor cycloheximide abrogates receptor upregulation induced by TNF. In contrast to these findings in AML, TNF does not upregulate GM-CSF receptor numbers on blood granulocytes or monocytes. We conclude that TNF exerts positive effects on growth factor receptor expression of hematopoietic cells.

Development of Multipotential Haemopoietic Stem Cells to Neutrophils is Associated with Increased Expression of Receptors for Granulocyte Macrophage Colony-Stimulating Factor: Altered Biological Responses to GM-CSF during Development

Interleukin-3 (IL-3) dependent multipotent haemopoietic stem cells FDCP-Mix A4 (A4) were induced to differentiate and develop into mature neutrophils in response to Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) plus granulocyte CSF (G-CSF). This resulted in an increase in cell number over seven days of culture, following which the cells lost the ability to undergo further proliferation. The effect of GM-CSF on these cells has been assessed at various stages of development. Clonogenic cells, able to respond to GM-CSF, were generated only at days 3, 4 post-induction. From day 5 onwards, mature post-mitotic neutrophils are produced and clonogenic cells are lost. Loss of proliferative potential, in response to GM-CSF, was confirmed using [3H]-thymidine incorporation. Receptors for GM-CSF, were also measured during development using [125I]-GM-CSF binding assays. Although the dissociation constant for GM-CSF binding sites did not vary considerably, the number of such sites increased dramatically from about 20 (day 0, when the cells have a primitive morphology) to about 1000 by day 6 (when the cells are predominantly mature neutrophils). GM-CSF-stimulated Na+/H+ antiport activation was also determined. Although few GM-CSF receptors are expressed at day 0, there is a significant response (63% of maximal) to GM-CSF in terms of intracellular alkalinisation: this response increased markedly until, by day 4 (700 GM-CSF binding sites/cell), there is a maximal activation of the antiport by GM-CSF. By day 7 (greater than 900 GM-CSF binding sites/cell), however, there is significant reduction in activation of the Na+/H+ antiport by GM-CSF. Nonetheless, increased viability of these mature cells is still seen in response to GM-CSF. These results suggest that not only does expression of GM-CSF receptors alter during development of multipotential cells to mature neutrophils, but that these receptors are coupled to different intracellular effector mechanisms as the cells progressively mature.

Expression and modulation of IL‐3 and GM‐CSF receptors in human growth factor dependent leukaemic cells

From the interleukin-3 (IL-3) responsive human myeloid cell line M-07 we derived a subclone, named M-07e, which is fully dependent for growth and survival on either granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-3. In this paper the expression, specificity and modulation of GM-CSF and IL-3 receptors on M-07e cells are described. The specificity of the binding was demonstrated by the failure of other cytokines to compete, at 4 degrees C, with GM-CSF or IL-3 receptors. In addition, IL-3 was found to compete as well as GM-CSF for GM-CSF receptors while GM-CSF was a weak competitor for IL-3 receptors. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high-affinity GM-CSF binding sites (405 +/- 27.4 sites per cell, dissociation constant at the equilibrium 52 +/- 20 pM) and the presence of high and low-affinity regions for IL-3 binding sites (27 +/- 12 and 416 +/- 92 sites per cell for the high and low affinity regions respectively, dissociation constant at the equilibrium, 58 +/- 22 pM and 5.7 +/- 2.0 nM respectively). Finally, in agreement with the hierarchical down-modulation model, we found that IL-3 can down-modulate both IL-3 and GM-CSF receptors while GM-CSF can down-modulate only its own receptors. The present results suggest that M-07e cells, in spite of their neoplastic nature, share, with murine bone marrow cells, similar growth factor receptor regulatory mechanisms. This cell line may be regarded as a candidate model to investigate the physiological events triggered by growth factors binding to human haemopoietic cells.

Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells.

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors (Kd = 30-100 pM). By using radiolabeled GM-CSF with high specific activity, we have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations (95% +/- 1%, 34% +/- 5%, 48% +/- 8% receptor down-regulation, respectively). GM-CSF also down-regulated its own receptor on monocytes and myeloid progenitor cells. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, we also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.

Interleukin-3, GM-CSF, and G-CSF Receptor Expression on Cell Lines and Primary Leukemia Cells: Receptor Heterogeneity and Relationship to Growth Factor Responsiveness

AbstractRecombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 12*l were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre-B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.

Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness

Recombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 125I were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre- B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.

Nonhematopoietic tumor cells express functional GM-CSF receptors

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the colony growth of myeloid progenitors in semisolid media, and enhances the function of mature effector cells, including neutrophils, monocytes, and eosinophils. Small cell carcinoma lines (SCCL) have properties of amine precursor uptake and decarboxylation (APUD) cells and express high levels of the enzyme, L-aromatic amino acid decarboxylase. We looked for possible expression of GM-CSF receptors on nonhematopoietic cells and found specific high-affinity binding of human GM-CSF to SCCL and to the SV40-transformed African green monkey kidney cell line, COS. The small cell carcinoma lines responded to GM-CSF with enhanced proliferation, and both small cells and COS cells were found to express authentic 84,000 dalton GM-CSF receptor protein. These findings indicate that nonhematopoietic cells can bind and respond to GM-CSF, suggesting additional biological activities as well as the possibility of tumor responses when GM-CSF is used therapeutically in humans. Since preliminary clinical trials using CSFs as adjunctive treatment in patients with solid tumors are underway, it will be important to consider the possible responsiveness of nonhematopoietic tumor cells to CSFs.

Nonhematopoietic Tumor Cells Express Functional GM-CSF Receptors

AbstractHuman granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the colony growth of myeloid progenitors in semisolid media, and enhances the function of mature effector cells, including neutrophils, monocytes, and eosinophils. Small cell carcinoma lines (SCCL) have properties of amine precursor uptake and decarboxylation (APUD) cells and express high levels of the enzyme, L-aromatic amino acid decarboxylase. We looked for possible expression of GM-CSF receptors on nonhematopoietic cells and found specific high-affinity binding of human GM-CSF to SCCL and to the SV40-transformed African green monkey kidney cell line, COS. The small cell carci- noma lines responded to GM-CSF with enhanced proliferation, and both small cells and COS cells were found to express authentic 84,000 dalton GM-CSF receptor protein. These findings indicate that nonhematopoietic cells can bind and respond to GM-CSF, suggesting additional biological activities as well as the possibility of tumor responses when GM-CSF is used therapeutically in humans. Since preliminary clinical trials using CSFs as adjunctive treatment in patients with solid tumors are underway, it will be important to consider the possible responsiveness of non-hematopoietic tumor cells to CSFs.

The kinetics of S phase entry by FMP2.1: effect of IL-3 and GM-CSF receptor expression and ligand affinity.

FMP2.1, a cloned cell line which has morphological characteristics of mast/basophil cells, requires either interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for both survival and proliferation. IL-3 and GM-CSF were equally effective as proliferative stimuli. FMP2.1 cells were sensitive to growth factor stimulation in the G1 phase, which has a duration of 9.5 h. G1 cells were selected from FMP2.1 in log phase growth on the basis of Hoechst 33324 staining using a fluorescence activated cell sorter (FACS). It was found that G1 phase cells had to be exposed to either IL-3 or GM-CSF for approximately 1 h for cells to enter S (greater than 20%); without growth factor, FMP2.1 remained in G1 unable to progress into S. Receptor expression was analyzed to further understand this rapid activation of FMP2.1 into cycle. Autoradiography using either 125I-IL-3 or 125I-GM-CSF showed that most cells express both receptor types. In the presence of saturating concentrations of IL-3, FMP2.1 have a relatively high number of IL-3 receptors (42,000/cell) compared to other cell lines (e.g., 32D cl23; 13,000 receptors/cell), and far outnumber GM-CSF receptors on the same cells (600 receptors/cell). Although average IL-3 receptor expression differed for FMP2.1- and IL-3-dependent 32D cl23, the concentration-dependent proliferative response to IL-3 was essentially identical for both cell types. Scatchard plot analysis for 125I-IL-3 and 125I-GM-CSF binding to FMP2.1 cells at 4 degrees C revealed a single type of binding site for both ligands, with dissociation constants (Kd) of approximately 1 nM for GM-CSF and 8 pM for IL-3. The relatively high affinity IL-3 binding to a large number of available IL-3 receptors was associated with a shallow dose response of the FMP2.1 cells to IL-3, compared to the steep GM-CSF dose response which was mediated through fewer receptor sites of relatively low affinity. Mitogenic stimulation of G1 phase cells was observed with either IL-3 or GM-CSF, and appeared to be unaffected by differences in receptor number or binding affinity.