Activation of adenosine A(3) receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells

Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells.     

Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.     

Granulocyte interactions with GM-CSF and G-CSF secretion by endothelial cells and monocytes

We have, in previous studies, characterized the cytokine and cellular regulation of GM-CSF and G-CSF production by monocytes and endothelial cells. In this study, we investigated the regulatory role of granulocytes. The addition of granulocytes to endotoxin-stimulated monocytes dose-dependently decreased both GM-CSF and G-CSF concentrations, presumably by absorbing the cytokines. A similar dose-dependent decrease in GM-CSF concentration was found when granulocytes were added to IL-1-stimulated endothelial cells. In contrast, G-CSF secretion by endothelial cells responded to granulocytes in a biphasic fashion. At low granulocyte concentrations, endothelial cells responded with an increased G-CSF secretion, but at high concentrations of granulocytes G-CSF secretion was down modulated. Our results suggest that there exist two loops between granulocytes and endothelial cells for regulating G-CSF activity. Granulocytes can stimulate G-CSF secretion by activated endothelial cells but can also decrease the biological activity by absorbing the cytokine. These mechanisms might be involved in the regulation of the local and systemic levels of granulocytes.     

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.     

Adenosine potentiates stimulatory effects on granulocyte-macrophage hematopoietic progenitor cells in vitro of IL-3 and SCF, but not those of G-CSF, GM-CSF and IL-11

The aim of the studies was to ascertain if adenosine is able to co-operate with selected hematopoietic growth factors and cytokines, namely with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-11 (IL-11), in inducing the growth of colonies from hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) from normal bone marrow cells in vitro. Adenosine was found not to produce any colonies when present in the cultures as the only potential stimulator. All the tested cytokines and growth factors were observed to induce the growth of distinct numbers of GM-CFC colonies, with the exception of IL-11. When suboptimal concentrations of the evaluated cytokines and growth factors were tested in the cultures in which various concentrations of adenosine were concomitantly present, mutually potentiating effects were found in the case of IL-3 and SCF. These results confirm the role of adenosine in regulation of granulopoiesis and predict IL-3 and SCF as candidates for further in vivo studies of their combined administration with adenosine.     

Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical applications of recombinant human colony-stimulating factors.

The differentiation and maturation of hematopoietic progenitor cells are regulated by certain growth factors. Several of these glycoproteins have been characterized, and their amino acid sequences have been delineated. Modern DNA technology provides sufficient quantities of these hormones for testing in clinical trials. Erythropoietin (EPO) has been shown to increase the hemoglobin level and hematocrit in patients with end-stage renal disease. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) can increase the numbers of neutrophils and monocytes, in a dose-dependent fashion. The function of granulocytes and monocytes is also enhanced. Clinical studies of the toxicity and activity of G-CSF and GM-CSF have been conducted in patients with acquired immune deficiency syndrome, aplastic anemia, myelodysplastic syndromes, and neutropenia due to cancer and chemotherapy. In almost all patients the neutrophil count increased within 24 hours after the start of treatment. Side effects of G-CSF and GM-CSF are infrequent and usually mild. Combinations of CSFs may be even more effective.     

Essential role for cyclin D3 in granulocyte colony-stimulating factor-driven expansion of neutrophil granulocytes

The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G(1) cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation.     

Phagocytosis and oxidative metabolism of granulocytes in mice after stem cell factor (SCF) injection

Stem Cell Factor (SCF) can stimulate the growth and development of primitive multipotential and unipotential hematopoietic stem cells, either alone or in combination with other cytokines such as Granulocyte-Macrophage Colony Stimulating factor (GM-CSF) and Granulocyte-CSF (G-CSF). It was found that these cytokines can also stimulate the function of granulocytes but there is no information concerning SCF influence on the function of these mature cells. SCF was injected into mice subcutaneously during 5 consecutive days in a dose of 1 microgram/kg/d. An examination of the percentage of phagocytic granulocytes and NBT test was performed. The activity of acid phosphatase (AcP), alkaline phosphatase (AP), peroxidase (MPO) and esterase were determined by cytochemistry methods. On the basis of obtained results we can conclude that SCF evidently increases all tested parameters connected with the metabolism of phagocytosis.     

Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation

Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.     

1,25-Dihydroxycholecalciferol-induced differentiation of myelomonocytic leukemic cells unresponsive to colony stimulating factors and phorbol esters

The murine myelomonocytic leukemia cell line WEHI-3B D+, which differentiates in response to granulocyte colony stimulating factor (G-CSF), can also be induced to differentiate into monocyte-macrophages by phorbol myristate acetate (PMA) treatment, whereas the WEHI-3B D- subline, which is unresponsive to G-CSF and PMA, can be induced to differentiate to granulocytes as well as monocytes by 1,25-dihydroxycholecalciferol [1,25-(OH)2 D3], the biologically active metabolite of vitamin D3. A newly developed variant of the WEHI-3B D+ line, named WEHI-3B D+ G, which was responsive to G-CSF but not to PMA, was also differentiated to granulocytes by 1,25-(OH)2 D3. Although vitamin D3 has been reported to induce macrophage differentiation in responsive tumor cells, this is the first demonstration that 1,25-(OH)2 D3 can induce granulocyte differentiation. In both differentiation pathways, cessation of cellular proliferation accompanies changes in morphologic and cytochemical properties of the cells. This suggests that leukemic cell lines unresponsive to differentiation agents acting at the cell surface retain their ability to differentiate in response to agents that do not act via the plasma membrane such as 1,25-(OH)2 D3, which has cytosolic/nuclear receptors. Vitamin D3 could act through different cellular pathways inducing differentiation or by bypassing only the first step of a common differentiation cascade used by agents with cell surface receptors such as CSF. These results suggest that low doses of 1,25-(OH)2 D3 may be useful in combination with hemopoietic growth factors (CSFs) as therapeutic agent to induce leukemic cell differentiation in vivo.     

Cytotoxicity of white blood cells activated by granulocyte-colony-stimulating factor,granulocyte/macrophage-colony-stimulating factor and macrophage-colony-stimulating factor against tumor cells in the presence of various monoclonal antibodies

Unconjugated monoclonal antibodies (mAb) kill tumor cells in vivo by activating immune functions. One of these is ADCC (antibody-dependent cellular cytotoxicity). The efficacy of mAbs might be augmented if the cytotoxic capacity of the effector cells could be increased. In this study the augmenting effect of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage(GM)-CSF and macrophage(M)-CSF was analyzed. Effector cells [peripheral blood mononuclear cells (PBMC) or granulocytes] were activated for 4–6 h by the respective CSF and assayed in an 18-h Cr⁵¹-release assay. Human colorectal, lymphoma, glioma and melanoma cell lines were target cells. Mouse mAbs of different isotypes, as well as chimeric and humanized mAbs, were used. mAbs having the human Fc part of the IgG molecule were the most effective. The killing capacity of PBMC as well as of granulocytes was statistically significantly enhanced when mAbs were added. M-CSF and GM-CSF were the best CSF for augmenting the lytic capacity of PBMC in ADCC. G-CSF had no significant effect on PBMC. Spontaneous cytolysis of PBMC was significantly augmented only by M-CSF. Granulocytes were, in general, significantly less effective than PBMC but may be equally effective killer cells together with mouse or human mAbs of the IgG1 isotype, particularly against melanoma cells. Granulocytes may also be significantly stimulated to increased lytic capacity when activated with G-CSF or GM-CSF. On the basis of the present evaluation, clinical trials in tumor patients are warranted, combining mAbs with GM-CSF or M-CSF. Preference might be given to GM-CSF as this cytokine activates both PBMC and granulocytes.     

Empirical models of the proliferative response of cytokine-dependent hematopoietic cell lines

There is an expanding need for predictive mathematical models to accelerate the optimization of cell therapy culture processes. Here we demonstrate the ability of simple mathematical models to describe quantitatively the cytokine growth-rate dependence of two human hematopoietic cell lines, TF-1 and MO7e. These cells are immortal but depend on either interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF) for their continued survival and maximal proliferation. They are also responsive to interleukin-6 (IL-6) and exhibit saturation kinetics when these cytokines are limiting. A Monod-type relationship consistently failed to fit measured cytokine dose-proliferation response curves while a Hill-type relationship showed a good fit. Cytokine interactions were first modeled by modifying the Hill-function to include an interaction parameter, gamma. This model did not indicate either synergistic or even additive effects between IL-3 and GM-CSF. Based on the reported competition between IL-3 and GM-CSF for their common receptor (beta(c)) subunit, a competitive model was also developed. This model had no new parameters beyond those obtained from single cytokine cultures and provided improved prediction of the growth rates for both cell lines exposed to combinations of IL-3 and GM-CSF over a wide range of concentrations. As expected, the competitive model failed to fit the data for IL-6 in combination with either IL-3 or GM-CSF, since IL-6 signaling does not involve the beta(c) chain of the IL-3/GM-CSF receptors. Interestingly, the cell-specific rates of GM-CSF uptake and cell proliferation were found to be uncoupled processes. Taken together, these results illustrate the utility of appropriately designed empirical models to describe the proliferative responses of hematopoietic cells to cytokine stimulation.     

Hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders: studies of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and Flt-3 ligand (Flt3L)

The levels of hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders were investigated using a variety of approaches. Firstly, serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and Flt3-ligand (Flt3L) were examined in acute leukemia patients with treatment-induced cytopenia and complicating bacterial infections. Increased serum levels of both G-CSF and Flt3-ligand (Flt3L) were detected when these patients developed therapy-induced leukopenia, whereas GM-CSF levels were low or undetectable. Development of complicating bacterial infections then increased the serum levels of both G- and GM-CSF, and the Flt3L levels remained high during the infections. Secondly, release of growth factors was characterized for clonogenic T cells that remained in the circulation of acute leukemia patients with chemotherapy-induced cytopenia. CD4(+) and CD8(+) T cells from these patients released high levels of GM-CSF, relatively low levels of IL-3 secretion having been detected, and only a minority of the clones released detectable amounts of Flt3L. Thus, circulating T cells may contribute to the high systemic growth factor levels in cytopenic patients. Thirdly, plasma levels of GM-CSF and interleukin-3 (IL-3) were examined in patients with malignant disorders who received chemotherapy plus G-CSF for stem cell mobilization. Increased levels of GM-CSF and Flt3L were detected both in the patients' plasma and in the stem cell grafts. Despite the increased growth factor levels in neutropenic patients with complicating infections, the occurrence of febrile neutropenia did not have a major impact on normal hematopoietic reconstitution (i.e. duration of treatment-induced neutropenia) after intensive chemotherapy for acute myelogenous leukemia.     

Molecular modeling of the GM-CSF and IL-3 receptor complexes.

A model for the structure of the cytokine interleukin-3 (IL-3) is presented based on the structural homology of the hematopoietic cytokines and utilizing the crystal structures of interleukin-5 and granulocyte macrophage colony stimulating factor (GM-CSF). In addition, models of the receptor complexes of GM-CSF and IL-3 are presented based on the structural homology of the hematopoietic receptors to growth hormone. Several key interactions between the ligands and their receptors are discovered, some in agreement with previous mutagenesis studies and others that have not yet been the subject of mutagenesis studies. The models provide insights into the binding of GM-CSF and IL-3 to their receptors.     

APCs express DCIR, a novel C-type lectin surface receptor containing an immunoreceptor tyrosine-based inhibitory motif.

We have identified a novel member of the calcium-dependent (C-type) lectin family. This molecule, designated DCIR (for dendritic cell (DC) immunoreceptor), is a type II membrane glycoprotein of 237 aa with a single carbohydrate recognition domain (CRD), closest in homology to those of the macrophage lectin and hepatic asialoglycoprotein receptors. The intracellular domain of DCIR contains a consensus immunoreceptor tyrosine-based inhibitory motif. A mouse cDNA, encoding a homologous protein has been identified. Northern blot analysis showed DCIR mRNA to be predominantly transcribed in hematopoietic tissues. The gene encoding human DCIR was localized to chromosome 12p13, in a region close to the NK gene complex. Unlike members of this complex, DCIR displays a typical lectin CRD rather than an NK cell type extracellular domain, and was expressed on DC, monocytes, macrophages, B lymphocytes, and granulocytes, but not detected on NK and T cells. DCIR was strongly expressed by DC derived from blood monocytes cultured with GM-CSF and IL-4. DCIR was mostly expressed by monocyte-related rather than Langerhans cell related DC obtained from CD34+ progenitor cells. Finally, DCIR expression was down-regulated by signals inducing DC maturation such as CD40 ligand, LPS, or TNF-alpha. Thus, DCIR is differentially expressed on DC depending on their origin and stage of maturation/activation. DCIR represents a novel surface molecule expressed by Ag presenting cells, and of potential importance in regulation of DC function.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells

We have investigated, by semiquantitative RT-PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum-free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL-3)], or erythroid [SCF + IL-3 + erythropoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulating factor (G-CSF)] differentiation. The GF receptor genes investigated were the α and β subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin receptor, the G-CSF receptor, and c-Fms, the receptor for macrophage colony-stimulating factor (M-CSF). The expression of Gata1 and α- and β-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage-negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (≥17% of which reconstitute long-term hematopoiesis in recipient mice) and into Rho-bright (which are as capable as Rho-dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the α subunit of the IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over time in culture. The β subunits of the IL-3 and GM-CSF receptor, the α subunit of the GM-CSF receptor, the Epo and G-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho-bright cells but was below the level of detection in Rho-dull cells. Rho-dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, α- and β-globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the α subunit of the IL-3 receptor) are expressed at low levels, if any, in purified Rho-bright or Rho-dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor,in particular, is activated both in erythroid (α- and β-globin positive) and in myeloid (α- and β-globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis. J. Cell. Physiol. 171:343–356, 1997. © 1997 Wiley-Liss, Inc.     

Synergistic effects of purified recombinant human and murine B cell growth factor-1/IL-4 on colony formation in vitro by hematopoietic progenitor cells. Multiple actions

Purified recombinant human B cell growth factor-1/IL-4 was evaluated, alone and in combination, with purified preparations of recombinant human (rhu) CSF or erythropoietin (Epo) for effects on colony formation by human bone marrow CFU-GM progenitor cells (GM) and burst forming unit-E progenitor cells. rhu IL-4 synergized with rhu G-CSF to enhance granulocyte colony formation, but had no effect on CFU-GM colony formation stimulated by rhu GM-CSF, rhu IL-3, or rhu CSF-1. Rhu IL-4 synergized with Epo to enhance BFU-E colony formation equal to that of Epo plus either rhu IL-3, rhu GM-CSF, or rhu G-CSF. Removal of adherent cells and T lymphocytes did not influence the synergistic activities of rhu IL-4. Rmu IL-4, synergized with rhu G-CSF, but not with rmu GM-CSF, rmu IL-3, or natural mu CSF-1, to enhance CFU-GM (mainly granulocyte) colony numbers by a greater than 90% pure preparation of murine CFU-GM. Also, rhu IL-4 at low concentrations enhanced release of CSF and at higher concentrations the release also of suppressor molecules from human monocytes and PHA-stimulated human T lymphocytes. Use of specific CSF antibodies suggested that rhu IL-4 was enhancing the release of G-CSF and CSF-1 from monocytes and the release of GM-CSF and possibly G-CSF from PHA-stimulated T lymphocytes. Use of antibodies for TNF-alpha, IFN-gamma, or TNF-beta as well as measurement of TNF and IFN titers suggested that the suppressor molecule(s) released from monocytes were acting with TNF-alpha and those released from PHA-stimulated T lymphocytes were acting with IFN-gamma. These results implicate B cell growth factor-1/IL-4 as a synergistic activity for hematopoietic progenitors and suggest that the actions can be on both progenitor and accessory cells.