Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly enriched population of granulocyte/macrophage colony-forming cells.

We have prepared a population of bone marrow cells that is highly enriched in neutrophil/macrophage progenitor cells (GM-CFC). Four distinct haemopoietic growth factors can stimulate the formation of mature cells from this population, although the proportions of neutrophils and/or macrophages produced varied depending on the growth factor employed: interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulated the formation of colonies containing both neutrophils and macrophages; macrophage colony-stimulating factor (M-CSF) produced predominantly macrophage colonies; and granulocyte colony-stimulating factor (G-CSF) promoted neutrophil colony formation. Combinations of these four growth factors did not lead to any additive or synergistic effect on the number of colonies produced in clonal soft agar assays, indicating the presence of a common set of cells responsive to all four haemopoietic growth factors. These enriched progenitor cells therefore represent an ideal population to study myeloid growth-factor-stimulated survival, proliferation and development. Using this population we have examined the molecular signalling mechanisms associated with progenitor cell proliferation. We have shown that modulation of cyclic AMP levels has no apparent role in GM-CFC proliferation, whereas phorbol esters and/or Ca2+ ionophore can stimulate DNA synthesis, indicating a possible role for protein kinase C activation and increased cytosolic Ca2+ levels in the proliferation of these cells. The lack of ability of all four myeloid growth factors to mobilize intracellular Ca2+ infers that these effects are not achieved via inositol lipid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.     

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.     

Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.     

Clonal analysis of progenitor cell commitment to granulocyte or macrophage production

Granulocyte-macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte-macrophage stimulus, GM-CSF, or by the predominantly macrophage stimulus, M-CSF. After 24 hours, paired daughter cells of granulocyte-macrophage colony-forming cells (GM-CFC) were separated by micromanipulation and one cultured in GM-CSF, the other in M-CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and days 3 clones were cross-transferred to GM-CSF or M-CSF recipient cultures, irreversible commitment was more obvious. Most M-CSF-initiated clones exhibited irreversible commitment to macrophage formation in GM-CSF cultures and a high proportion of GM-CSF-initiated clones continued to produce granulocyte progeny after transfer to M-CSF. The results indicated that GM-CSF and M-CSF can irreversibly commit the progeny of GM-CFC respectively to granulocyte or macrophage production. While for some GM-CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions. Calculations from the data indicated that two-thirds of GM-CFC in adult C57BL marrow are biresponsive and respond to stimulation both by GM-CSF and M-CSF.     

The cloning and expression of the cDNA for ovine stem cell factor (kit-ligand) and characterization of its in vitro haematopoietic activity.

The cDNA encoding the soluble form of ovine stem cell factor (SCF) has been cloned and expressed. The soluble protein is predicted to be 165/166 amino acids in length, one more than the human and murine SCFs with which it shares 87% and 81% identity respectively. Ovine SCF has 98.5%, 95% and 91% identity with cattle, pig and dog SCF, respectively. The recombinant ovine (rov) SCF protein has been expressed in Chinese hamster ovary (CHO) cells, purified, and its biological activity on ovine bone marrow cells compared with that of interleukin 3 (rovIL-3), granulocyte-macrophage colony-stimulating factor (rovGM-CSF), interleukin 5 (rovIL-5), human macrophage colony-stimulating factor (M-CSF) and human erythropoietin (epo). On its own rovSCF supported the development of small numbers of neutrophil, macrophage, eosinophil, granulocyte-macrophage, mixed cell phenotype, haemopoietic blast cell and basophilic granular cell colonies in a soft agar clonogenic assay. In combination with each of the above cytokines rovSCF supported an increase in the number and size of the lineage-specific colony types that were stimulated by the other cytokines on their own. In an assay for precursors of multipotential colony-forming cells (multi-CFC), rovSCF in combination with rovIL-3 (but neither cytokine alone) supported the development of these early haematopoietic progenitor cells.     

Detection of murine bone marrow granulocyte/macrophage progenitor cells (GM-CFU) in serum-free cultures stimulated with purified M-CSF or GM-CSF

Colony formation by mouse granulocyte/macrophage progenitors (GM-CFU) responding to purified colony-stimulating factors (CSF) in serum-free cultures is described. Analysis of the lipid requirements for colony growth stimulated by purified macrophage CSF (M-CSF) demonstrated that cholesterol is essential. Linoleic acid further promoted colony growth only if cholesterol was present, but phospholipid was inhibitory. More colonies were obtained in serum-free cultures, than in serum-supplemented controls. This difference could not be attributed to a change in the range of sensitivity to M-CSF. Stimulation of GM-CFU with granulocyte/macrophage CSF (GM-CSF) required further supplementation with hydrocortisone for optimal expression of colony-forming capacity in serum-free cultures. Hydrocortisone slightly inhibited colony growth stimulated with M-CSF. Under these culture conditions, the number of GM-CFU responding to GM-CSF was twice that obtained with M-CSF.     

An Activated Protein Kinase C α Gives a Differentiation Signal for Hematopoietic Progenitor Cells and Mimicks Macrophage Colony-stimulating Factor–stimulated Signaling Events

Highly enriched, bipotent, hematopoietic granulocyte macrophage colony-forming cells (GM-CFC) require cytokines for their survival, proliferation, and development. GM-CFC will form neutrophils in the presence of the cytokines stem cell factor and granulocyte colony-stimulating factor, whereas macrophage colony-stimulating factor leads to macrophage formation. Previously, we have shown that the commitment to the macrophage lineage is associated with lipid hydrolysis and translocation of protein kinase C α (PKCα) to the nucleus. Here we have transfected freshly prepared GM-CFC with a constitutively activated form of PKCα, namely PKAC, in which the regulatory domain has been truncated. Greater than 95% of the transfected cells showed over a twofold increase in PKCα expression with the protein being located primarily within the nucleus. The expression of PKAC caused macrophage development even in the presence of stimuli that normally promote only neutrophilic development. Thus, M-CSF–stimulated translocation of PKCα to the nucleus is a signal associated with macrophage development in primary mammalian hematopoietic progenitor cells, and this signal can be mimicked by ectopic PKAC, which is also expressed in the nucleus.     

Properties of primary murine stroma induced by macrophage colony-stimulating factor

The ability of purified human macrophage colony-stimulating factor (M-CSF) to accelerate the formation of stromal cells from murine bone marrow cells was investigated. The liquid culture of the marrow cells with M-CSF resulted in the formation of monolayers of macrophages on day 7. When the M-CSF was removed on that day and the residual adherent cells were cultured in the absence of M-CSF for an additional 7 days, many colonies appeared with cells that were morphologically distinguishable from M-CSF-derived macrophages. The appearance of the colonies was dependent on the concentration of M-CSF used at the beginning of the culture. Each colony was isolated as a single clone and analyzed. All clones were negative for esterase staining. These cells did not express M-CSF receptor mRNA and did not show a mitogenic response to M-CSF. On the contrary, these cells could be stimulated to proliferate by fibroblast growth factor and platelet-derived growth factor. The polymerase chain reaction analysis of these cells demonstrated constitutive expression of mRNA for M-CSF, stem cell factor, and interleukin (IL)-1, but not IL-3. Some clones expressed mRNA for granulocyte/M-CSF and IL-6. We also examined the ability of the cells to maintain murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in a coculture system. Most of the clones showed a significant increase in total HPP-CFC numbers after 2 weeks of coculture, although the extent of stimulation differed among clones. These results suggested that the colonies established by M-CSF were composed of functional stromal cells that were phenotypically different from macrophages. J. Cell. Physiol. 173:1–9, 1997. © 1997 Wiley-Liss, Inc.     

Interleukin 2 inhibits in vitro granulocyte-macrophage colony formation

We have previously shown that murine bone marrow cells cultured with interleukin 2 (IL-2) produce interferon-alpha/beta (MuIFN-alpha/beta) and that IFN-alpha/beta can suppress in vitro granulocyte-macrophage colony-forming cell formation (GM-CFC). In this study, IL-2 was directly assessed for its ability to inhibit in vitro granulocyte and/or macrophage colony-forming cell formation (GM-CFC/M-CFC). C57BL/6 bone marrow cells were cultured with different colony-stimulating factors (CSF), i.e., partially purified macrophage-CSF (M-CSF) or recombinant granulocyte and macrophage CSF (GM-CSF) in the presence or absence of different IL-2 preparations. Partially purified mouse IL-2 or recombinant human or mouse IL-2 (rHuIL-2 and rMuIL-2) totally inhibit GM-CFC and M-CFC formation at 7 days of culture. The level of inhibition mediated by IL-2 was concentration-dependent, with as little as 1 U/ml giving total inhibition of colony formation. The ability of IL-2 to inhibit colony formation was completely abolished by treatment with antisera to IL-2. MuIFN-alpha/beta and MuIFN-gamma appeared to play no role in IL-2-induced myelo-suppression in that addition of antisera to these IFN failed to block IL-2-induced suppression. Myelo-suppression mediated by IL-2 was independent of the concentration of CSF used in the bone marrow cultures. Suppression was also not dependent upon the initial presence of T cells or natural killer (NK) cells. Bone marrow cells depleted of Thy-1+, Lyt-1+, Lyt-2+, NK-1.1+, Asialo GM1+, or Qa-5+ cells were as susceptible to IL-2 induced suppression as untreated or complement-treated bone marrow cells. These results suggest that IL-2 may play an important role in regulating different aspects of hematopoiesis.     

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.     

Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro.

We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony-stimulating activity under fetal bovine serum (FBS)-deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) to stimulate colony growth. Colony morphology was determined by the late-acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL-3 and Epo resulted in the largest number of mixed-cell colonies--a larger number than observed with IL-3 and Epo alone even in FBS-supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed-cell colony-forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.     

Phorbol ester-stimulated murine myelopoiesis: role of colony-stimulating factors

Tumor promoting phorbol esters, such as 12-0-tetradecanoyl-phorbol-13-acetate (TPA), stimulate colony formation in vitro by murine granulocyte-macrophage progenitors (GM-CFC) without added colony stimulating factors (CSF). To determine whether TPA induces CSF production in vitro, marrow cells were cultured for 1 to 7 days in liquid medium with or without TPA. No CSF was detected in any sample by a double antibody radioimmunoassay (sensitivity = 2 units/0.1 ml), however, colony-stimulating activity was detected in supernatant fluid from all TPA containing cultures by bioassay. This activity appeared to result from a direct effect of TPA rather than from production of CSF, as equivalent activity was found in TPA-containing medium incubated in the absence of marrow cells. Rabbit antiserum to purified L-cell CSF inhibited colony formation stimulated by L-cell CSF and WEHI-3 CSF, but had no effect on colony formation induced by TPA. Cells from long-term marrow cultures responded to TPA with colony formation, despite culture conditions and cell fractionation procedures that reduced the frequency of CSF-producing macrophages to less than 1.0%. TPA inhibited binding of radioiodinated L-cell CSF to marrow cells, especially if the cells were first exposed to TPA. These results do not support induction of CSF production as the major mechanism of phorbol ester stimulation of myelopoiesis. Phorbol esters may directly stimulate GM-CFC and/or enhance their response to CSF by a mechanism involving CSF binding sites.     

Effects of okadaic acid on mouse hemopoietic cells

Effects of okadaic acid, a potent non-12-O-tetradecanoyl-phorbol-13-acetate(TPA)-type tumor promoter, on mouse hemopoietic cells were investigated. Okadaic acid stimulated mouse bone marrow cells to form granulocyte-macrophage colony-forming unit (CFU-GM) colonies without added colony stimulating factors(CSFs). At the concentration of 1.82 x 10(-8) M, colony formation of 77 +/- 14 colonies/1 x 10(5) bone marrow cells was observed. Observations on the effects of other cells on the CSF induction suggested that okadaic acid primarily stimulated the functions of macrophages, and the CSF production from macrophages might be attributed to the CFU-GM colony formation. On the other hand, the erythroid colony-forming unit(CFU-E) colony formation stimulated by     

Chicken myeloid stem cells infected by retroviruses carrying the v-fps oncogene do not require exogenous growth factors to differentiate in vitro

To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M-CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells.     

The development of macrophages from human CD34+ haematopoietic stem cells in serum-free cultures is optimized by IL-3 and SCF

The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34⁺ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34⁺ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34⁺ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors.     

Apoptosis of human erythroid colony-forming cells is decreased by stem cell factor and insulin-like growth factor I as well as erythropoietin

To clarify the manner by which erythropoietin (EP), stem cell factor (SCF), or insulin-like growth factor I (IGF-I) regulate erythropoiesis, apoptosis of human erythroid progenitor cells was investigated. Human burst-forming units-erythroid (BFU-E) partially purified from peripheral blood were cultured for 6 days to generate erythroid colony-forming cells (ECFC), which consist mainly of colony-forming units-erythroid (CFU-E). The cells were labeled with [₃H]thymidine, incubated in serum-free liquid media, at 37°C, for 16 h, and the pattern of DNA breakdown was analyzed by agarose gel electrophoresis. When these cells were incubated without EP, 70% of the total cellular DNA was broken down into DNA fragments of less than 5 kilobases and nuclear condensation and fragmentation, characteristic of apoptosis, were evident. While EP greatly reduced the amount of DNA breakdown to 23%, SCF and IGF-I each reduced the amount of DNA breakdown to 38–46% and, when added together, to 24%. Dose-response experiments with SCF and IGF-I showed a dose-dependent reduction in DNA fragmentation at concentrations that stimulate colony formation in serum-free semisolid cultures. Finally, assays of ECFC performed by the plasma clot method, after serum-free liquid culture, at 37°C, for 16 h, demonstrated marked protection of erythroid colony-forming capacity by SCF or IGF-I in the absence of EP, as well as by EP itself. These data indicate that human erythroid progenitor cells undergo apoptosis which is reduced by SCF and IGF-I as well as EP and suggest that the control of apoptosis by each of these factors has a prominent role in the regulation of erythropoiesis. © 1993 Wiley-Liss, Inc.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.     

Activation and proliferation signals in murine macrophages: stimulation of glucose uptake by hemopoietic growth factors and other agents

Purified colony-stimulating factor (CSF-1) (or macrophage colony stimulating factor [M-CSF]) stimulated the glucose uptake of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM) as measured by 3H-2-deoxyglucose (2-DOG) uptake. Similar concentrations of CSF-1 stimulated the 2-DOG uptake and DNA synthesis in BMM. Other purified hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and interleukin-3 (IL-3) (or multi-CSF), and the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though differing in their mitogenic capabilities on BMM, were also stimulators of 2-DOG uptake in BMM and RPM. The nonmitogenic agents, lipopolysaccharide (LPS) and concanavalin A (Con A), were also active. The inhibition by cytochalasin B and by high concentrations of D-glucose suggest that the basal and stimulated 2-DOG uptake occurred via a carrier-facilitated D-glucose transport system. The responses of the two macrophage populations to the hemopoietic growth factors and to the other agents were quite similar, suggesting that events that are important for the induction of DNA synthesis are not tightly coupled to the earlier rise in glucose uptake. For the BMM, the ability of a particular agent to stimulate glucose uptake did not parallel its ability to promote cell survival. However, stimulation of glucose uptake could still be a necessary but insufficient early macrophage response for cell survival and subsequent DNA synthesis.     

Regulation of murine mononuclear phagocyte inflammatory products by macrophage colony-stimulating factor. Lack of IL-1 and prostaglandin E2 production and generation of a specific IL-1 inhibitor

The influence of macrophage (M)-CSF on the production of inflammatory mediators has been examined in murine peritoneal macrophages. Cultures of macrophages treated with up to 30,000 U/ml of human rM-CSF or with 10,000 U/ml of L929-derived M-CSF did not reveal either PGE2, IL-1, or IL-6 secretion. In contrast, LPS, which served as a positive control, stimulated production of significant levels of PGE2, IL-1, and IL-6. Furthermore, Northern blot analysis of macrophage RNA revealed a strong induction of IL-1 alpha and IL-6 mRNA by LPS but not by M-CSF. Conditioned medium from macrophage cultures treated with purified L929 or human rM-CSF in combination with LPS exhibited a significant reduction of IL-1 bioactivity as compared with an LPS challenge alone. To investigate the mechanism involved in this M-CSF-dependent reduction of IL-1 bioactivity, we measured IL-1 alpha gene expression. The addition of M-CSF to LPS-treated macrophages did not affect IL-1 alpha mRNA levels suggesting that M-CSF may regulate production of an IL-1 inhibitor. This hypothesis was shown to be valid because removal of IL-1 alpha from conditioned medium of LPS plus M-CSF-treated cells allowed the detection of a nondialyzable factor that blocked IL-1-dependent thymocyte proliferation. The inhibitor appeared to be specific because it did not inhibit IL-2 and TNF bioactivities. Furthermore, this IL-1 inhibitor, which binds to cells and not to IL-1, competed with the binding of radioactive IL-1 alpha or beta to EL-4.6.1 cells. The results demonstrate that M-CSF alone does not induce proinflammatory mediators and PGE2 as was previously published. The data also suggest that M-CSF may play a role in the down-regulation of inflammatory responses.