Unregulated activation of STAT-5, ERK1/2 and c-Fos may contribute to the phenotypic transformation from myelodysplastic syndrome to acute leukaemia

Myelodysplastic syndrome (MDS) is characterised by ineffective erythropoiesis and poor progenitor response to erythropoietin (Epo). The aim of this study was to determine the role of the Epo-R mediated signalling in the rise of MDS and whether alteration of signalling pathways contribute to the leukeamogenesis from MDS to acute leukaemia. We analysed Epo and GM-CSF induced ERK1/2 activation, c-Fos expression, STAT-5 and AP-1 DNA binding activities in mononuclear cells of umbilical cord blood (UCBMNC), normal marrow (NBMMNC) or marrow with MDS, AML with prior MDS and de novo AML. In UCBMNC and NBMMNC, Epo and GM-CSF induced the activation of STAT-5 DNA binding and ERK 1/2 activation (n = 6). In contrast, in MDS RA, both signalling pathways were activated only by GM-CSF but not by Epo (n = 7). In acute leukaemia, elevated basal activity of STAT-5 DNA binding appeared in 8/8 cases, which was independent of Epo or GM-CSF treatment. In normal and MDS samples, c-Fos and Egr-1 proteins were not detectable and the expression levels were not increased by Epo or GM-CSF treatment. In contrast, we found an elevated level of c-Fos expression in 5/8 acute leukemia cases, which was not further increased in the presence of Epo or GM-CSF. The elevated c-Fos expression was accompanied by an extremely high blast number in 5/5 cases. These results suggest that impaired ERK/MAPK activation, similarly to impaired STAT-5 activation in Epo-R signalling, may be responsible for the apoptotic process and the block of maturation in MDS RA. The results also suggest that the appearance of the constitutively activated STAT-5 DNA binding and c-Fos expression may be used as a predictor of the blastic transformation.     

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.     

Cytokine receptors: structure and signal transduction.

In the past 2-3 years, a number of cytokine receptors have been partly characterized and the cDNAs for the ligand binding chains cloned. This has revealed that cytokine receptors are complex. Many are known to be multichain receptors (e.g. IL-2) and since their mechanism of signal transduction is not obvious, it is likely that other proteins yet to be defined take part in the signalling process. The cloning of the receptor ligand binding chain has revealed that (unlike cytokines), there are major families of receptors. Some are members of the Ig supergene family (e.g. IL-1 receptor), others are members of the nerve growth factor receptor family (e.g. TNF), but the majority are members of the haematopoietic growth factor family (e.g. IL-3, GM-CSF). Yet other cytokine receptors do not belong to a family, e.g. IFN-gamma.     

Clonal response of murine myelomonocytic leukemia cells (WEHI-3B-Y1) to purified recombinant human hemopoietic factors in serum-free culture

We tested purified recombinant hemopoietic factors for their effects on the proliferation and differentiation of murine myelomonocytic leukemia cells (WEHI-3B-Y1) in serum-free agar culture. We found that purified recombinant human G colony-stimulating factor (CSF) markedly increased the colony number of WEHI-3B-Y1 cells and differentiation-inducing activity. However, at low colony densities [( 100/dish), G-CSF did not induce the differentiation of WEHI-3B-Y1 cells. We conclude that G-CSF does not induce the differentiation of WEHI-3B-Y1 cells directly, but induce the differentiation as a result of the secondary autoinduction of differentiation. Interleukin 3 (IL-3) slightly enhanced but erythropoietin (Epo) did not alter the colony number of WEHI-3B-Y1 cells. GM-CSF or M-CSF decreased the colony number of WEHI-3B-Y1 cells. Such purified recombinant human IL-3, Epo, GM-CSF and M-CSF did not induce morphologically the distinct differentiation of WEHI-3B-Y1 cells.     

Characterization of IL-1 inhibitory factor released from human alveolar macrophages as IL-1 receptor antagonist.

IL-1 possesses pleiotropic properties on various cells and its activity may be stringently regulated in several ways. We have previously reported that both IL-1 and its inhibitory factor are concomitantly released from alveolar macrophages in both healthy subjects and patients with chronic inflammatory lung diseases. An increase in IL-1 activities and a decrease in inhibitory activities are characteristics found in both healthy smokers and patients with interstitial lung diseases. In this study, we further examined the biological properties of IL-1 inhibitory factor. The inhibitor exhibited a dose-dependent specific inhibition of an augmentation by IL-1 of PHA-induced murine thymocyte proliferation, while no inhibition of the augmentation by IL-2, IL-4, IL-6, or tumour necrosis factor (TNF) was found. 125I-labelled IL-1 alpha binding on PHA-stimulated murine thymocytes revealed two types of IL-1 binding sites, 44 sites/cell with a Kd of 2.7 x 10(-10) M and 230 sites/cell with a Kd of 2.5 x 10(-9) M. Alveolar macrophage culture supernatants blocked the binding of labelled IL-1 to the IL-1 receptor in a dose-dependent fashion. Scatchard plot analysis revealed that the inhibitory factor in the supernatants blocked the binding competitively. These results indicate that alveolar macrophages produce a specific IL-1 inhibitory factor, functioning as an IL-1 receptor antagonist.     

Erythropoietin overcomes imatinib-induced apoptosis and induces erythroid differentiation in TF-1/bcr-abl cells

Targeting BCR-ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) on TF-1/bcr-abl (which was generated by transduction of a bcr-abl fusion gene into the TF-1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF-1/bcr-abl cells but not in the parental TF-1 cells. However, GM-CSF, a survival factor of the parental TF-1 cells, protected TF-1/bcr-abl cells from imatinib-induced apoptosis in a dose-dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM-CSF, accompanied by upregulation of Bcl-xL and downregulation of p27/Kip1. In addition, although untreated TF-1/bcr-abl cells had lost responsiveness to both GM-CSF and EPO and showed autonomous growth, GM-CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib-treated TF-1/bcr-abl cells differentiated into hemoglobin-positive cells in the presence of EPO, as in the case for the parental TF-1 cells. Taken together, imatinib-treated CML cells may differentiate into mature cells in the presence of differentiation-inducing cytokines such as EPO.     

The IL-1 system in HIV infection: peripheral concentrations of IL-1β, IL-1 receptor antagonist and soluble IL-1 receptor type II

The proinflammatory cytokine IL-1β is thought to be involved in ongoing HIV disease. Furthermore, its naturally occurring inhibitors soluble IL-1 receptor type II (sIL-1RII) and IL-1 receptor antagonist (IL-1Ra) may play a pivotal role in regulating its biological action. To investigate the involvement of the IL-1 system we determined serum levels of IL-1β, IL-1Ra and sIL-1RII in 90 HIV⁺ patients. The obtained values were compared with markers of disease progression such as CD⁺ count, 5′-neopterin, β₂-microglobulin and soluble tumour necrosis factor receptors (sTNF-R) p55 and p75 and then compared with C-reactive protein (CRP), granulocyte count, lL-6 and TNF-α. While IL-1Ra concentrations increased significantly with progressive CDC disease stages, sIL-1RII and IL-1β were not altered in our cohort. IL-1Ra showed statistical relation to decreasing CD4⁺ lymphocytes and increasing 5′-neopterin, β₂-microglobulin, sTNF-R p55, sTNF-R p75. Furthermore, IL-1Ra correlated positively with serum IL-6, TNF-α, CRP and granulocytes. In contrast, sIL-1RII and IL-1β tended to show an inverse correlation or showed no significant relationship to all these parameters. Il-1β was measurable only in a limited number of samples. IL-1Ra showed a clear relationship to acute inflammatory events as well as to the different disease stages. Our data suggest a dissociation between IL-1Ra and sIL-1RII serum levels which may indicate that the two IL-1 binding proteins have different pathophysiological roles in HIV infection.     

Immunohistochemical detection of GM-CSF, IL-4 and IL-5 in a murine model of allergic bronchopulmonary aspergillosis

Background Granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and IL-5 are important in tissue eosinophil accumulation and high IgE production in allergic inflammatory reaction. Objective We examine lung GM-CSF, IL-4 and IL-5 expression in a murine model of allergic bronchopulmonary aspergillosis (ABPA) characterized by eosinophil and lymphocyte lung infiltration and elevated serum IgE level. Methods C57BL/6 mice were intranasally treated three times a week for 1, 2 or 3 week(s) with Aspergillus fumigatus (Af) antigen or saline and were sacrificed on days 7, 14 and 21. Immunohistochemical analyses for GM-CSF, IL-4 and IL-5 were performed on lung sections. Results Af treatment induced a remarkable pulmonary eosinophil influx. Increased numbers of lung T lymphocytes and GM-CSF positive cells were observed on days 14 and 21. IL-4 and IL-5 positive cells were increased significantly only on day 14. Immunostained serial sections showed that most (≥98%) cytokine positive cells were CD3 positive. Few eosinophils (<2% of cytokine positive cells) were immunoreactive for GM-CSF and IL-5. Significant correlations were found between the number of GM-CSF and IL-5 positive cells, and the number of eosinophils in Af-treated lung (r = 0.62, P < 0.05 and r = 0.52, P < 0.05, respectively), and between the number of IL-4 positive cells and the serum total IgE level (r = 0.64, P<0.01). Conclusions Our data suggest a role for T lymphocyte GM-CSF, IL-4 and IL-5 in Af-induced mouse pulmonary eosinophilia and increased serum IgE production and further support the importance of T helper (TH) cells in the pathogenesis of ABPA.     

Cytokine regulation of the interleukin-1 receptor antagonist protein in U937 cells

A naturally occurring receptor-level antagonist of interleukin-1 (IRAPorIL-1 ra) has recently been cloned. To determine what stimuli might regulate this inhibitor, cytokines were tested for their effects on the steady-state level of IRAP mRNA in phorbol ester-differentiated U937 cells. The cytokines tested fell into one of three groups: (a) inducers: granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, (b) weak inducers (<2-fold stimulation): [IL-lα, IL-Iγ, and transforming growth factor-β (TGF-γ)] and (c) cytokines with no effect: (IL-2, platelet-derived growth factor, acidic fibroblast grouth factor, basic fibroblast growth factor, epidermal growth factor, granulocyte colony-stimulating factor, IL-3, IL-5, IL-6, interferon-y, multi-colony stimulating factor, tumor necrosis factor -a and IRAP itself. One hundred U/ml of either GM-CSF or IL-4 was the dose inducing peak IRAP mRNA expression; that peak expression occurred 12 h after addition of cytokine. GM-CSF induced a 34 ±15-fold increase in IRAP mRNA, and IL-4 induced a 15± 6-fold increase. In the same RNA samples, GM-CSF increased IL-ip mRNA 5.9 ± 1.7-fold, but IL-4 decreased IL-Iγ mRNA to half that of control levels (0.45 ± 0.17). Thus, a single stimulus (IL-4) decreased the expression of an agonist (IL-1) while it increased the expression of an antagonist (IRAP). When U937 cells were treated with both IL-4 and GM-CSF, the level of IRAP mRNA induced was additive, suggesting that the cytokines acted differently to increase IRAP mRNA levels. The level of IL-1 mRNA in cells treated with both IL-4 and GM-CSF was intermediate. Dexamethasone and cycloheximide inhibited all mRNA increases and did not reverse IL-4-induced decreases in IL-1 mRNA. These studies have identified two cytokines which induce IRAP in the monocytic cells studied, and have partially characterized the differential regulation of IL-1 and its antagonist, IRAP.     

Thermal Injury Increases the Number of Eosinophil Progenitors in Rat Spleen and Bone Marrow

We have investigated the effects of thermal injury upon myelopoiesis. IL-3, GM-CSF, and IL-5 were used to stimulate myeloid colony formation. IL-3 induces early myeloid progenitors and a more developed myeloid progenitor, the granulocyte-macrophage colony-forming unit (GM-CFU), to multiply and develop into mature myeloid cells. GM-CSF induces GM-CFU to become mature myeloid cells, while IL-5 induces eosinophil progenitors to become mature eosinophils. Stem Cell Factor (SCF) + IL-6 and FLT3 ligand, which have no effect on colony formation by themselves, were used to enhance the effects of IL-3 and GM-CSF, respectively. We found that thermal injury increased the number of early myeloid progenitors and GM-CFU in the spleen with either IL-3 or GM-CSF as a stimulant. Thermal injury increased the number of early myeloid progenitors in the bone marrow when GM-CSF, but not IL-3, was used to stimulate colony growth. Also, thermal injury increased the numbers of eosinophil progenitors in rat spleen and bone marrow and increased splenic levels of IL-5 mRNA.     

Affinity conversion of receptors for colony stimulating factors: properties of solubilized receptors.

Murine receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and Multi-CSF (interleukin-3) can exist in both high- and low-affinity forms and demonstrate trans-modulation by several different ligands. In contrast the recently cloned human GM-CSF receptor and murine interleukin-3 (IL-3) receptor display only low-affinity binding. To begin to understand the molecular basis of the formation of high- and low-affinity receptors and their trans-modulation we have developed methods for the solubilization and assay of GM-CSF and interleukin-3 receptors so that their binding characteristics can be studied in cell-free solution. Both receptors displayed a single class of high-affinity binding on intact FDC-P1 cells and IL-3 receptors had unaltered binding characteristics in cells, membranes and in detergent solution. However, GM-CSF receptors were converted to a single class of low-affinity binding in detergent solution while both high- and low-affinity forms were evident in membranes. The basis of affinity conversion of GM-CSF receptors was exclusively a change in the kinetic dissociation rate of ligand. Cross-linking experiments suggested that high-affinity receptors for GM-CSF and IL-3 might consist of two different protein species and, if this is so, the data suggest that this association is more stable for IL-3 than for GM-CSF receptors.     

Critical proline residues of the cytoplasmic domain of the IL-5 receptor {alpha} chain and its function in IL-5-mediated activation of JAK kinase and STAT5

The high-affinity receptor (R) for IL-5 consists of a uniquea chain (IL-5Rc) and a ß chain (ßc) thatisshared with the receptors for IL-3 and granulocyte macrophagecolony stimulating factor (GM-CSF). We defined two regions ofIL-5R for the IL-5-induced proliferative response, the expressionof nuclear proto-oncogenes, and the tyrosine phosphorylationof cellular proteins including ßc, SH2/SH3-containingproteins and JAK2 kinase. In the studies described here, wedemonstrate that IL-5, IL-3 or GM-CSF stimulation induces thetyrosine phosphorylation of JAK2, and to a lesser extent JAK1,and of STAT5. Mutational analysis revealed that one of the prolineresidues, particularly Pro352and Pro355, in the membrane-proximalproline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmicdomain of IL-5R is required for cell proliferation, and forboth JAK1 and JAK2 activation. In addition, transfectants expressingchimeric receptors which consist of the extracellular domainof IL-5R and the cytoplasmic domain of ßc respondedtoIL-5 for proliferation and tyrosine phosphorylation of JAK1.Intriguingly, electrophoretic mobility shift assay analysisrevealed that STAT5 was activated in cells showing either JAK1or JAK2 tyrosine phosphorylation. These results indicate thatactivation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-inducedtyrosine phosphorylation and ultimately mitogenesis, and thatPro352 and Pro355 in the proline-rich sequence appear to playmore essential roles in cell growth andin both JAK1/STAT5 andJAK2/STAT5 activation than Pro353 does.     

Monoclonal Antibodies that Specifically Inhibit GM-CSF-and IL-3-Dependent Growth of Human Monocytic Leukemia Cells

Abstract

We describe monoclonal antibodies (mAbs: anti-MaG-1, TGI-1, TGI-5, and TGI-6) that block the proliferation of AML-193 cells in response to GM-CSF or IL-3 and do not affect the proliferation of AML-193 cells in response to G-CSF and IL-2-driven proliferation of Kit 225 cells. However, none of the mAbs tested had any stimulative effect on the proliferation of AML-193 cells. The mAbs (anti-MaG-1, TGI-1, -5, and -6) could inhibit the binding of [¹²⁵I)GM-CSF to AML-193 cells. We were able to purify MaG-1 Ag by anti-MaG-1 affinity chromatography. Thus, the MaG-1 Ag and the Ags recognised by mAbs (TGI-1, -5, and -6) may be associated with the receptor for GM-CSF or IL-3 or a structure close to the receptor for GM-CSF or IL-3.     

Differential binding of human interleukin-1 (IL-1) receptor antagonist to natural and recombinant soluble and cellular IL-1 type I receptors

A recently described factor, interleukin-1 receptor antagonist binding factor (IL-IraBF), in serum of normal individuals is immunologically related to the interleukin-1 receptor type I (IL-1RI). It is presumably a soluble form of the receptor that binds exclusively to interleukin-1 receptor antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5 fibroblasts and the interference afforded by the soluble receptors. The results show that the protein backbones of IL-1raBF and sIL-1RI are of similar size (≈ 35–40 kDa) and that there are differences in the glycosylation of the two molecules. These carbohydrates were necessary for optimal binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble IL-1RI and that IL-1ra binds differently to these molecules and to cellular IL-1RI.     

Erythropoietin enhances immunoglobulin production and proliferation by human plasma cells in a serum-free medium

The effect of recombinant human erythropoietin (Epo) on plasma cells was studied in a serum-free medium, COSMEDIUM-001 (Cosmedium). Epo enhanced both Ig production and thymidine uptake by human plasma cell lines, AF-10 and IM-9. Interleukin-6 (IL-6) enhanced both Ig production and thymidine uptake by AF-10 and IM-9, while other cytokines, including IL-1 beta, IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-alpha (IFN-alpha) or IFN-gamma, failed to do so. However, the Epo effect was specific since Epo-induced enhancement of Ig production and thymidine uptake was blocked by the anti-Epo antibody but not by the anti-IL-6 antibody or the control antibody. Conversely, IL-6-induced enhancement was blocked by the anti-IL-6 antibody but not by the anti-Epo antibody. Epo also enhanced Ig production (IgG, IgM, and IgA) and thymidine uptake by PCA-1+ plasma cells generated in vitro. This enhancement was also blocked by the anti-Epo antibody but not by the anti-IL-6 antibody. Taken together, these results suggest that Epo enhances plasma cell responses by a different mechanism than does IL-6.     

Signal transduction activator of transcription 5 (STAT5) dysfunction in autoimmune monocytes and macrophages

Autocrine granulocyte macrophage-colony stimulating factor (GM-CSF) sequentially activates intracellular components in monocyte/macrophage production of the pro-inflammatory and immunoregulatory prostanoid, prostaglandin E2 (PGE2). GM-CSF first induces STAT5 signaling protein phosphorylation, then prostaglandin synthase 2 (COX2/PGS2) gene expression, and finally IL-10 production, to downregulate the cascade. Without activation, monocytes of at-risk, type 1 diabetic (T1D), and autoimmune thyroid disease (AITD) humans, and macrophages of nonobese diabetic (NOD) mice have aberrantly high GM-CSF, PGS2, and PGE2 expression, but normal levels of IL-10. After GM-CSF stimulation, repressor STAT5A and B isoforms (80-77kDa) in autoimmune human and NOD monocytes and activator STAT5A (96-94kDa) and B (94-92kDa) isoforms in NOD macrophages stay persistently tyrosine phosphorylated. This STAT5 phosphorylation persisted despite treatment in vitro with IL-10, anti-GM-CSF antibody, or the JAK2/3 inhibitor, AG490. Phosphorylated STAT5 repressor isoforms in autoimmune monocytes had diminished DNA binding capacity on GAS sequences found in the PGS2 gene enhancer. In contrast, STAT5 activator isoforms in NOD macrophages retained their DNA binding capacity on these sites much longer than in healthy control strain macrophages. These findings suggest that STAT5 dysfunction may contribute to dysregulation of GM-CSF signaling and gene activation, including PGS2, in autoimmune monocytes and macrophages.     

Binding Sites of Interleukin-3-Mimetic Monoclonal Autoantibodies Derived from a MRL/lpr Mouse

MRL/lpr mouse-derived interleukin-3 (IL-3)-mimetic monoclonal antibodies were examined for their binding sites. One of these five antibodies (B10, F8, F9, F12, H 11), F9 interacted with the IL-3 receptor, as if it were an anti-idiotypic antibody; the IL-3-mimetic activity of F9 was blocked by a neutralizing rat monoclonal anti-IL-3 antibody. IL-3 mRNA was not detected in hybridoma F9, as analyzed by the SI protection assay, Thus, the activity neutralized by the rat antibody is of the F9 antibody itself but not the IL-3 type. Such blocking was not observed with the IL-3-mimetic activity of the other MRL/lpr-derived monoclonal antibodies. On the other hand, the binding of all these monoclonal antibodies to IL-3-depen-dent cells was inhibited by each other and vice versa, as analyzed by two-color flow cytometry. This indicates that the binding sites of the five monoclonal antibodies are located so close to each other that the binding of one would interfere with the binding of any one of the others (since the binding experiment was done on ice, it is unlikely that the inhibition is due to down-modulation of the receptors). Taken together the results obtained by the enzyme digestion study, we discussed that all five IL-3-mimetic monoclonal antibodies are directed to the IL-3 receptor, but only F9 binds to the portion directly responsible for the binding of IL-3 and the other antibodies (B10, F8, F12, H11) bind to different portions, respectively, which are adjacent or overlapping to the binding site of F9.