Molecular and biological properties of a macrophage colony-stimulating factor from mouse yolk sacs

A colony-stimulating factor (M-CSF) has been partially purified and concentrated from mouse yolk sac-conditioned medium (YSCM). M-CSF appeared to preferentially stimulate CBA bone marrow granulocyte-macrophage progenitor cells (GM-CFC) to differentiate to form macrophage colonies in semisolid agar cultures. By comparison, colony-stimulating factor (GM-CSF) from mouse lung-conditioned medium (MLCM) stimulated the formation of granulocytic, mixed granulocytic-macrophage, and pure macrophage colonies. Mixing experiments indicated that both M-CSF and GM-CSF stimulated all of the GM-CFC but that the smaller CFC were more sensitive to GM-CSF and that the larger CFC were more sensitive to M-CSF. Almost all developing "clones" stimulated initially with M-CSF continued to develop when transferred to cultures containing GM-CSF. In the converse situation, only 50% of GM-CSF prestimulated "clones" survived when transferred to cultures containing M-CSF. All clones initially stimulated by M-CSF or transferred to cultures stimulated by M-CSF contained macrophages after 7 days of culture. These results suggest that there is a population of cells (GM-CFC) that are capable of differentiating to form both granulocytes and macrophages, but, once these cells are activated by a specific CSF (e.g. M-CSF), they are committed to a particular differentiation pathway. The pattern of CFC differentiation was not directly related to the rate of proliferation: cultures maximally stimulated by M-CSF produced mostly macrophage colonies, but the presence of small amounts of GM-CSF produced granulocytic cells in 30% of the colonies. Gel filtration, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and affinity chromatography with concanavalin A-Sepharose indicated that M-CSF from yolk sacs was a glycoprotein with an apparent molecular weight of 60,000. There was some heterogeneity of the carbohydrate portion of the molecule as evidenced by chromatography on concanavalin A-Sepharose.     

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.     

Differentiation of Mouse Bone Marrow Precursor Cells into Neutrophil Granulocytes by an Activity Separation from WEHI-3 Cell-Conditioned Medium

Colonies comprised exclusively of neutrophil granulocytes have been obtained by growing mouse bone marrow cells in nutrient semisolid agar cultures. A stimulator of predominantly granulocyte colony formation was present in the breakthrough fraction of preparations of colony-stimulating activity separated on DEAE-Sephadex A. The source of colony-stimulating activity was concentrated conditioned medium of a murine myelomonocytic cell line (WEHI-3), which unfractionated stimulated the growth of colonies of granulocytes, macrophages, megakaryocytes, as well as mixed colony types. After stepwise column chromatography of the conditioned medium, the breakthrough fraction was shown to stimulate predominantly granulocyte colony formation, and the fraction eluted with 1 M NaCl was found to induce primarily macrophage colony growth. Colony morphology was independent of the concentration of eluate used. The morphology of colonies varied with increasing concentrations of the breakthrough fraction. At low concentrations, granulocyte colony formation was almost exclusively observed. With increasing concentrations of this fraction, an increasing proportion of the colonies were found to contain macrophages. The effect of concentration of this activity was in marked contrast to previous findings where the incidence of granulocyte colony formation was inversely related to the concentration of colony-stimulating activity. This differential responsiveness of cell to stimulus has previously been interpreted as low concentrations of a growth and differentiation factor being required for macrophage production and high concentrations of the same factor required for granulocyte formation. Separation of these activities by DEAE Sephadex chromatography, and alteration of the dose-response curve, such that granulocyte colony formation varies directly with the amount of stimulator, indicates that the differentiation of these two cell blood lineages may be controlled by separate entities.     

The effect of mouse lung granulocyte-macrophage colony-stimulating factor and other colony-stimulating activities on the proliferation and differentiation of murine bone marrow cells in long-term cultures

The roles of colony-stimulating factors in long-term bone marrow cultures were studied and compared. After single additions of high concentrations of unpurified colony-stimulating activities to the cultures, rapid deterioration of the cultures was observed. This appears to result from contaminants in the stimulatory preparations. Cultures to which one purified colony-stimulating factor (CSF) from endotoxin mouse lung-conditioned medium was added did not run down ten weeks after addition and were found to be the same as the controls. The deterioration of the cultures to which unpurified stimulators were added could not be accounted for by accelerated granulopoiesis leading to subsequent exhaustion of the cultures. The inability of purified CSF to affect the cellularity of the suspension cells did not result from instability or masking of the activity in the cultures, nor did CSF preferentially stimulate the cells within the adherent layer. The suspension cells responded to purified CSF after separation from the adherent cells. The data suggest that if CSFs are marrow stimulators, their effects in turn may be stringently regulated within the marrow.     

The in vitro differentiation of density sub-populations of colony-forming cells under the influence of different types of colony-stimulating factor.

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density subpopulations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post-endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macrophage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Purification and characterization of a colony stimulating factor from human lung.

Conditioned medium prepared from human autopsy lung tissue contains high level activity of colony stimulating factor which stimulates granulocytes and macrophage colony formation in both mouse and human bone marrow. The lung colony stimulating factor has been purified about 2250-fold by methods including hydroxylapatite chromatography, preparative gel electrophoresis, preparative isoelectric focusing, and gel filtration chromatography. The final specific activity was 2.7 X 10(6) units/mg. The purified factor has a molecular weight of 41 000 as determined by gel filtration. It is stable at the pH range of 6.5--10 and 56 degrees C for 30 min but sensitive to protease digestion and periodate oxidation. On polyacrylamide gel electrophoresis, it migrates in the alpha-globulin post-albumin region. Upon isoelectrofocusing lung colony stimulating factor appears heterogeneous with isoelectric points of 3.7--4.3. Treatment with neuraminidase did not affect its activity, but caused a change in electrophoretic mobility and isoelectric point. Antibody produced by immunizing rabbits with partially purified lung colony stimulating factor exerted strong inhibitory activity on the factor from lung as well as on colony stimulating factor from other human sources including serum, urine, and placenta.     

Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.     

T-cell hybridoma secreting hemopoietic regulatory molecules: granulocyte-macrophage and eosinophil colony-stimulating factors.

Murine spleen cells stimulated in vitro with pokeweed mitogen were fused with a HAT-sensitive AKR thymoma (BW5147) to produce T-cell hybridomas secreting hemopoietic colony-stimulating factors (CSFs). A stable cloned T-cell hybridoma has been isolated which expressed the H-2 antigens of both fusion parents, has a median chromosome number of 56 and secretes a factor(s) which stimulates the growth of granulocyte-macrophage and eosinophil colonies. The CSF-secreting hybridoma exhibited only the Thy 1.1 associated with the parent tumor, but no markers normally associated with normal T-cells or macrophages were detected. No CSF was secreted by the parent tumor line, but the hybridoma-conditioned medium, when used at 10% (v/v), contained sufficient CSF to stimulate 10–30 colonies per 10⁵ bone marrow cells. Lipopolysaccharide (1 μg/ml) stimulated the production of CSF by the hybridoma cells 3 fold. CSF production also increased when the cells were held at high density in serum-free medium. The colony-stimulating factor(s) secreted by the hybridoma exhibited similar molecular properties to those produced by pokeweed mitogen-stimulated spleen cells, and both the GM- and EO-CSFs had an apparent molecular weight by gel filtration of approximately 35,000.     

Cellular responsiveness to stimulation in vitro: increased responsiveness to colony stimulating factor of bone marrow colony-forming cells treated with surface-active agents and cyclic 3'5' AMP.

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte-macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10(-8) to 10(-10) M) also enhanced colony numbers although concentrations above 10(-5) M were inhibitory. enhancement was found when marrow cells were pre-treated with these agents and cultured in their absence. The agents did not stimulate colony development in the absence of colony-stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony-stimulating factor which was sub-optimal in terms of maximum colony development. There was no indication of increased colony-stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony-forming cells to colony-stimulating factor.     

Kinetics of the clonal proliferation of granulocytes and macrophages in cultures of mouse bone marrow cells as supported by two distinct types of colony-stimulating factors

Two different types of colony-stimulating factors (CSF) were used to support the clonal growth of myeloid progenitor cells (CFUc) in semi-solid agar or viscous methylcellulose cultures of mouse bone marrow cells. The cultures stimulated for 5 days with RSP-2-P3 cell CSF (CSFRSP) contained mainly granulocyte colonies, whereas the cultures stimulated for 10 days with human urine CSF (CSFhu) contained mainly monocyte/macrophage colonies. Four lines of study were carried out: 1) a kinetic study using combinations of the two types of CSFs in the same culture; 2) a study of transferring CFUc from the initial 3-day cultures to recipient cultures containing the same or different types of CSF; 3) an examination of the morphology over time of colonies that were confined by glass capillaries plunged in agar; and 4) electron microscopic observations on disintegrating granulocytes. The results of all these lines of study suggest that about one third of the CFUc can be stimulated both by CSFRSP and CSFhu while the other two thirds react specifically either with CSFRSP or with CSFhu. The present study also suggests that granulocytes in the culture stop proliferation and disintegrate while macrophages are still growing there. Thus, mixed-type colonies containing both macrophages and granulocytes later become macrophage colonies.     

Formation of eosinophilic-like granulocytic colonies by mouse bone marrow cells in vitro

Formation of granulocytic and macrophage colonies in agar cultures of mouse marrow or spleen cells was stimulated by the addition of medium from pokeweed mitogen-stimulated cultures of mouse spleen cells (PKW-CM). Approximately 5% of the colonies developing were large, dispersed granulocytic colonies (DG-colonies) composed of cells with eosinophilic cytoplasmic granules. The capacity to stimulate DG-colonies was shown by media conditioned by PKW-treated lymphoid and peritoneal cells but not by other cells or organ fragments. Velocity sedimentation studies indicated that cells generating DG-colonies were separable from cells generating regular granulocytic or macrophage colonies. DG-colonies did not survive if transfered to cultures containing other forms of CSF. The active colony stimulating factor in pokeweed mitogen-conditioned medium which stimulates DG-colony formation was antigenically distinct from the factor stimulating granulocytic and macrophage colony formation, was separable electrophoretically from the latter factor and on gel filtration had an apparent molecular weight of 50,000. Although the cells in DG-colonies have not been established to be eosinophils, DG-colonies represent an interesting new system for analysing further aspects of the control of growth and differentiation in hemopoietic populations.     

Serum of lipopolysacharide-treated mice contains two types of colony-stimulating factor,separable by affinity chromatography

Serum from mice traated with bacterial lipopolysaccharide (LPS) was fractionated by Con A-Sepharose affinity chromatography, and assayed in vitro for colony-stimulating factor (CSF) using mouse bone marrow cells. The CSF failing to bind to concanavalin A-Sepharose (pool A) had similar biological properties to the unfractionated serum, i.e., it stimulated the formation of about equal numbers of granulocytic, mixed granulocyte-macrophage and macrophage colonies. The fraction eluted from the Con A-Sepharose column with α-methyl-D-glucopyranoside (pool B) had a steeper dose-response curve than either the unfractionated serum or the pool A CSF and most of the colonies were composed of macrophages. A mixture of the pool A and pool B CSFs stimulated colonies in a similar way as unfractionated serum and pool A. The apparent molecular weights of the two types of CSF were determined by two different gel-filtration procedures. Sephacryl S-200 gel-filtration suggested an apparent molecular weight of 85,000 for pool A CSF and 180,000 for pool B CSF. Gel-filtration on Sepharose CL-6B in the presence of guanidine hydrochloride (6M) yielded an apparent molecular weight of approximately 23,000 for pool A CSF and 33,000 for pool B CSF. The colony-forming cells (CFC) responding to pool B CSF were found to have a relatively high sedimentation velocity (peak sedimentation velocity 5.6–6.2 mm/hr) compared to the CFC responding to mouse-lung conditioned medium (MLCM) whose peak sedimentation velocity was between 4.0–4.5 mm/hour. The CFC responding to pool A CSF had an intermediate sedimentation velocity (peak 4.6–5.2 mm/hour). A time-course analysis of the morphology of clones or colonies in cultures stimulated with either MLCM or pool B CSF showed that the proporation of different colony types depends significantly on the incubation period and suggested that pool B CSF induced an early commitment of CFC towards macrophage differentiation.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Heterogeneity of in vitro colony- and cluster-forming cells in the mouse marrow: segregation by velocity sedimentation.

C57BL bone marrow cells were separated on the basis of their sedimentation velocity at unit gravity and cell fractions cultured in agar using three types of colony stimulating factor (CSF). Colony-forming cells separated as a single peak (s equal 4.4 mm/hr) in cultures stimulated by mouse lung conditioned medium (CSFMLCM) or endotoxin serum (CSFES). Cluster-forming cells were separable into two peaks and the majority were larger than colony-forming cells (s equal 5.7 mm/hr). Partial segregation of colony-forming cells was observed according to the morphological types of colonies generated, large cells tending to generate macrophage colonies and small cells, granulocytic colonies. Large colony-forming cells were more responsive to stimulation by CSF than small cells. Human urine (CSFHU) appeared unable to proliferation of most small colony-forming cells. Colony-forming cells appear to be a highly heterogeneous population with intrinsic differences in responsiveness to CSF and with differing capacities to generate colonies whose cells differentiate to granulocytes of macrophages.     

A quantitative assay for mouse granulocyte (CFU-g) and macrophage (CFU-m) precursors using plasma clots

Cytochemical procedures were used to identify and quantitate granulocyte and macrophage precursors from mouse bone marrow cells in plasma clot cultures. Excellent clonal morphology and cellular enzyme activity were obtained when using plasma clots as the support matrix and buffered formalin acetone as the fixative. For cytochemical identification, naphthol AS acetate esterase staining was used for macrophages and peroxidase for granulocytes. These enzyme properties were confirmed by inactivation studies with a variety of inhibitors, group specific chemical modifications, and pinocytotic affinity for horseradish peroxidase. When mouse bone marrow cells (3 X 10(4) cells/dish) were cultured in plasma clots with human placental or L-cell-conditioned medium, 70 to 110 colonies were produced. Both pure granulocyte (CFU-g) and pure macrophage colonies (CFU-m) were observed, but approximately 5% of the total colony number was composed of mixed granulocyte/macrophage colonies (CFU-gm). The number of plated cells correlated strongly with the colony number (0.990 less than r less than 0.999).     

Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells

A procedure is described for purification of a factor (D-factor)-inducing differentiation of mouse myeloid leukemic cells (M1) into macrophages from serum-free mouse L929 cell-conditioned medium. The procedure included ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-200 and phenyl-Sepharose column chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel-filtration column. The purified factor gave a single band of protein with a molecular weight of 62,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with biological activity. Its half-maximal concentration for inducing differentiation of M1 cells into macrophages was 1.7 X 10(-11) M. Even at 2.6 X 10(-9) M, it did not induce colony formation of normal bone marrow cells, suggesting that it was distinct from the growth factor for normal precursors of macrophages and/or granulocytes.     

Purification of a colony-stimulating factor from cultured pancreatic carcinoma cells.

Serum-free conditioned medium prepared from an established line of human pancreatic carcinoma (MIA PaCa-2) provides a rich source of colony-stimulating factor (CSF). Two activities distinctly separable by isoelectrofocusing have been identified: a high molecular weight CSF exhibiting greater activity in mouse bone marrow and a low molecular weight CSF more active in human bone marrow. The high molecular weight CSF has been purified 1000-fold to apparent homogeneity by a two-step procedure including isoelectrofocusing and gel filtration chromatography. The purified CSF has a molecular weight of 50,000 and an isoelectric point of 3.7 to 4.6. It is a glycoprotein as shown by periodic acid-Schiff stain and exhibits greater activity in mouse marrow than in human marrow.     

Characterization of a human basophil-like cell promoting activity

Biologic and biochemical properties of a human basophil-like cell promoting activity (BaPA), which induces growth of metachromatically staining cells from normal bone marrow cells in a liquid culture system have been examined. In order to study this T cell factor, an assay was developed based on the intracellular histamine content of the cultured human bone marrow cells. Many lymphokines, including granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin 1 alpha and 1 beta, interleukin 2, and interferon-alpha and gamma, did not exhibit any significant activity in the assay. By employing this assay, BaPA was purified approximately 500-fold from lectin-stimulated spleen cell-conditioned medium. BaPA has a molecular weight of 23,000 on high performance liquid chromatography gel filtration and displays isoelectric points between 5.8 and 7.3. It is heat stable up to 80 degrees C for 30 min and resistant to 6 M guanidine hydrochloride, whereas it is rather sensitive to sulfhydryl reagents. BaPA has no stimulating activity on mouse bone marrow cells.     

Properties and purification of a colony-stimulating factor of granulocytes and macrophages produced by mouse spleen cells

Mouse splenocytes are induced by pokeweed mitogen to secrete a factor that stimulates mouse hemopoetic (spelling per Nomina Histologica in the Nomina Anatomica, 5th edition, 1983, Williams and Wilkins, Baltimore) progenitor cells to undergo proliferation and differentiation into granulocytes and macrophages in a semi-solid culture system. The granulocyte and macrophage colony-stimulating factor (GM-CSF) was purified with a four-step procedure that includes ultrafiltration, chromatography on DEAE-agarose, Sephacryl S-200, and chromatofocusing gel. The isoelectric point (pI) of 4.2 of the GM-CSF was determined by analytical isoelectrofocusing gel electrophoresis. The sensitivity of the biological activity of GM-CSF to digestion by trypsin and neuraminidase suggests that GM-CSF is a glycoprotein with its sugar moieties at the active site. The GM-CSF is also sensitive to heat denaturation at 60 degrees C or higher suggesting that a three-dimensional conformation is required for its biological activity. The molecular weight of GM-CSF is approximately 57,000 Daltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Characterization of recombinant human granulocyte-colony-stimulating factor produced in mouse cells.

Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus DNA and human granulocyte-colony-stimulating factor (G-CSF) cDNA placed under the control of the SV40 early promoter. The transformed cells secreted constitutively a high level of human G-CSF, 10-20 micrograms/ml in a low-serum medium. The secreted G-CSF has been purified to homogeneity by a two-step procedure including gel filtration and hydrophobic column chromatography. The purified recombinant G-CSF runs as a single band with an apparent Mr of 19,000 on a polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value corresponds to that of the native human G-CSF purified from the medium conditioned by human carcinoma CHU-2 cells. The recombinant human G-CSF was as active as native G-CSF in vitro in supporting proliferation of mouse NFS-60 cells and stimulating colony formation from human as well as mouse bone marrow cells. When the recombinant human G-CSF was subcutaneously administrated into mice, a remarkable stimulation of granulopoiesis and splenomegaly was observed.