Stimulation of uptake of tritiated thymidine into mouse marrow cells in culture by a factor from L-cell conditioned medium

Uptake of ³H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of ³H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The ³H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, ³H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.     

Properties of the mouse embryo conditioned medium factor(s) stimulationg colony formation by mouse bone marrow cells grown in vitro

The properties of the mouse embryo cell conditioned medium (ECM) colony stimulating factor(s) from six day mouse embryo cultures have been examined. The general properties were similar to those described previously for the human urine colony stimulating factor. The ECM colony stimulating activity (CSA) was not lost following treatment with nucleases, glycosidases, phospholipases and proteolytic enzymes with the exception of α-chymotrypsin. ECM CSA was lost following mild periodate treatment. Fractionation of ECM CSA revealed a slight size heterogeneity on gel-filtration and on zone sedimentation in sucrose gradients. There was a discrepancy between the apparent molecular weights determined by gel-filtration (70,000–150,000) and by zone sedimentation (64,000) as has been reported previously for other colony stimulating factors. A gross charge heterogeneity of ECM CSA was apparent on electrophoresis and DEAE-cellulose chromatography, and a heterogeneity of the elution profile on stepwise elution from calcium phosphate gel was observed. This heterogeneity was still apparent in the presence of 6M urea and appeared to be unchanged following re-chromatography on DEAE-cellulose under the usual fractionation conditions. These studies suggested that the heterogeneity was not due to easily reversible combinations of active subunits. The electrophoretic heterogeneity of six day ECM CSA was found to develop gradually from an electrophoretically monodisperse band at day 2 of culture. Experiments in which preparations containing concentrated monodisperse ECM CSA were added back to culture dishes during and after ECM production suggested that the development of heterogeneity was related to the production or release of factor(s) from the cells rather than the action on the colony stimulating factor(s) of an extracellular enzyme in the medium. Alteration of the electrophoretic mobility of six day ECM CSA by incubation with purified sialidase suggested the presence of sialic acid on the active molecules. Purification procedures for the ECM factor(s) were not developed to any large extent primarily in view of the charge heterogeneity. The results of this study suggest that the ECM colony stimulating factor(s) is a glycoprotein(s).     

Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor

An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.     

Purification of human macrophage colony stimulating factor (CSF-1) from medium conditioned by pancreatic carcinoma cells

Colony Stimulating Factor-1 has been purified to apparent homogeneity from the serum-free medium conditioned by cultured human pancreatic carcinoma cells which had been induced with phorbol myristate acetate. The purification scheme consisted of sequential steps of batchwise adsorption to calcium phosphate gel, adsorption to lentil lectin-Sepharose, binding to immobilized antibodies, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography. The purified glycoprotein was found to have a subunit molecular weight corresponding to the smallest of four species (approximately 40,000, 33,000, 28,000 and 23,000) which were observed when less purified preparations were examined.     

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

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

Heterogeneity of molecules with low molecular weight isolated from media conditioned by human leukocytes and capable of stimulating colony formation by human granulopoietic progenitor cells

Molecules of low molecular weight, able to stimulate colony formation by human granulopoietic cells, were prepared from media conditioned by normal and leukemic human peripheral blood leukocytes. When molecules from these sources were studied, heterogeneity was observed in their ability, after radioiodination, to bind and suicide granulopoietic progenitors, in their radiolabelled tryptic digestion products and in their biological activity as stimulators of granulocyte colonies.     

Stimulation of murine bone-marrow colony formation by conditioned medium from human fetal liver cells

Summary A substance that stimulates growth of colonies of mononuclear granulocytic cells derived from the bone marrow of mice was produced by incubating fetal liver cells (conditioned medium). This substance appears to have the same properties described elsehwere as colony-stimulating factor (CSF). The enhanced stimulatory ability of the conditioned medium fromhuman fetal liver cells compared to medium not conditioned suggests that fetal liver is a potent source of colony-stimulating factor.     

Tissue sources of bone marrow colony stimulating factor

Possible tissue sources in C57BL mice of the serum factor stimulating colony formation in vitro by mouse bone marrow cells have been investigated. A reproducible technique employing batch chromatography on calcium phosphate gel was developed for the extraction and assay of material with colony stimulating activity from mouse tissues. Sixteen hematopoietic and non-hematopoietic tissues from C57BL mice were found to vary widely in their content of extractable activity. Characterisation of the colony stimulating factors (CSF's) from these tissues by assay of stepped concentrations of eluate showed that CSF's from most tissues were similar in chromatographic behavior, but all differed significantly from those of serum in being both more disperse and more firmly bound to calcium phosphate gel. Male submaxillary salivary gland gave the richest yield of CSF. CSF from this source displayed a greater dispersity on and affinity to calcium phosphate, a lower electrophoretic mobility and a smaller average sedimentation coefficient than that from any other source investigated. Colony morphology appeared to be identical for all tissue sources investigated.     

Stimulation of murine bone-marrow colony formation by conditioned medium from human fetal liver cells.

A substance that stimulates growth of colonies of mononuclear granulocytic cells derived from the bone marrow of mice was produced by incubating fetal liver cells (conditioned medium). This substance appears to have the same properties described elsewhere as colony-stimulating factor (CSF). The enhanced stimulatory ability of the conditioned medium from human fetal liver cells compared to medium not conditioned suggests that fetal liver is a potent source of colony-stimulating factor.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

Further studies on the factor in lung-conditioned medium stimulating granulocyte and monocyte colony formation in vitro

The CSF's extracted from 6-hour “in vivo” post-endotoxin mouse lung tissue and 48-hour “in vitro” lung-conditioned medium appear to be glyco-proteins, for like human urinary CSF they were inactivated by proteases and altered in charge but not inactivated by sialidase. Neither endotoxin injection nor length of in vitro incubation significantly affected the apparent S_{20 W} (approximately 2.OS) of the bulk of the CSF in mouse lung-conditioned medium. The lower apparent molecular weight of CSF in mouse lung-conditioned medium contrasted with that of monkey lung-conditioned medium (S_{20 W} approximately 3.3S). CSF's in both monkey and mouse lung-conditioned media exhibited a much higher apparent MW on gel nitration than one would expect from their sedimentation behavior, a characteristic generally observed with most unpurified CSF preparations. Mouse lung-conditioned medium CSF was antigenically distinguishable from other CSF's. Failure to demonstrate this form of CSF in the tissue serum or urine of normal or endotoxin-injected mice suggests that this type of CSF might normally lack in vivo significance.     

Production of colony stimulating factor in long-term bone marrow cultures

Previous studies have shown no detectable colony-stimulating factor (CSF) in media harvested from long-term bone marrow cultures. In the present experiments supernatants from long-term cultures established in three laboratories were assayed for CSF by colony assay and by radioimmunoassay (RIA). Most samples were devoid of biologic activity but all contained CSF as judged by RIA. Biologic activity was found in the majority of samples after diafiltration to remove low molecular weight inhibitors or 5-fold concentration by ultrafiltration. Samples that remained inactive in the colony assay were subjected to gel filtration on Sephadex G-150 to remove potential high molecular weight inhibitors. Biologic activity remained lower than that by RIA in two of three samples tested. Thus, most long-term cultures appear to contain biologically active CSF but this activity is masked by various types of inhibitors. In addition some media appear to contain material that is only detected by RIA.     

Characterization of a factor inducing differentiation of mouse myeloid leukemic cells purified from conditioned medium of mouse Ehrlich ascites tumor cells

A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 cells.     

A low molecular weight factor in lung-conditioned medium stimulating granulocyte and monocyte colony formation in vitro

Medium conditioned by excised whole lungs from endotoxin-injected C₅₇BL mice was highly active in stimulating hemopoietic colony formation, particularly of granulocytic type, in agar cultures of mouse bone marrow cells. The colony stimulating factor (CSF) in this material had an α₁-α₂ electrophoretic mobility, was eluted from calcium phosphate gel by 0.04 M phosphate buffer and had an unusually low apparent S₂₀W of 1.9. Sequestered polymor-phonuclear neutrophils were excluded as a major source of this CSF. The high specific activity and ease of preparation of lung conditioned medium make it valuable both for the large scale production of CSF and as a source of an unusual type of CSF.     

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.