Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1).

Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.

Stimulation of thrombopoiesis in mice by human recombinant interleukin 6.

To date, testing of various cytokines for the stimulation of blood cell production has not demonstrated a consistent effect on peripheral platelet levels. In this report, we provide evidence that human recombinant IL-6 increased platelet production in mice, as measured by both peripheral platelet levels and [75Se]selenomethionine (75SeM) incorporation into newly forming platelets. Peripheral white blood cell counts also were increased, but only to a modest extent, and hematocrit values were unchanged. A dose-response relationship between the amount of IL-6 administered and platelet count, 75SeM incorporation, and white blood cell count was demonstrated. Detectable megakaryocyte and granulocyte-macrophage colony-forming cells in mice that had received IL-6 also were increased in both bone marrow and spleen. These results demonstrate the ability of a purified, recombinant protein to stimulate platelet production in vivo.

Expression of the macrophage colony-stimulating factor and c-fms genes in human acute myeloblastic leukemia cells.

Macrophage colony-stimulating factor (CSF-1; M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocytes. The effects of CSF-1 are mediated through binding to specific, high-affinity surface receptors encoded by the c-fms gene. CSF-1 and c-fms gene expression was investigated in fresh human acute myeloblastic leukemic cells by Northern blot hybridization using cDNA probes. 4.0-kb CSF-1 transcripts were detected in 10 of 17 cases of acute myeloblastic leukemia (AML), while c-fms transcripts were detected in 7 of 15. Coexpression of CSF-1 and c-fms was observed in five cases, and in five other cases neither gene was expressed. In situ hybridization demonstrated that transcripts for CSF-1 were present in 70-90% of cells in each of three cases studied while c-fms mRNA was detected in 40-70% of cells. The constitutive expression of CSF-1 transcripts was associated with production of CSF-1 protein, although detectable amounts of CSF-1 were not secreted unless the cells were exposed to phorbol ester. These results demonstrate that leukemic myeloblasts from a subset of patients with AML express transcripts for both the CSF-1 and CSF-1 receptor genes, often in the same leukemic cells in vitro.

Human recombinant monocyte chemotactic protein and other C-C chemokines bind and induce directional migration of dendritic cells in vitro.

Because dendritic cells (DC) are the most potent antigen-presenting cells involved in many pathophysiological responses, we investigated the effect of chemokines on the migration of these cells in an effort to determine whether chemokines may contribute to the initiation of immune responses. CD34+ progenitor cells isolated from umbilical cord blood were grown in suspension cultures with cytokines and expanded 50- to 100-fold. A variable proportion of the cells expressed markers consistent with DC. The proportion of CD1a+ DC was increased when the cells were cultured with interleukin-4 (IL-4). These cells expressed specific binding sites for C-C and C-X-C chemokines. Cells cultured with or without IL-4 had similar binding profiles. All C-C chemokines tested, including monocyte chemotactic protein (MCP)-1, MCP-2, MCP-3, macrophage inflammatory protein-1 alpha (MIP1 alpha), MIP-1 beta, and RANTES, induced migration of DC-enriched cells cultured with or without IL-4 with MCP-3 being the most potent chemoattractant. Phenotypic analysis of cell migrating in response to C-C chemokines showed that CD1a+ cells were indeed attracted across the polycarbonate filters, and there was no preferential attraction of contaminating CD14+ monocytes by C-C chemokines. DC-enriched cells also expressed specific binding sites for IL-8 and NAP2, which failed to induce cell migration. Our results suggest that C-C chemokines may participate in the recruitment of DC to amplify host defense.

Expression of granulocyte colony-stimulating factor by human cell lines.

We have isolated and expressed a cDNA clone that encodes a human granulocyte colony-stimulating factor from the MIA PaCa-2 cell line. A genomic clone of this factor has been isolated from the CHU-2 cell line and is reported to encode two alternative transcripts [The EMBO J. 5,575, 1986]; one transcript predicts an amino acid sequence identical to that predicted by our MIA PaCa-2 cDNA clone; the other transcript predicts a similar protein containing a three amino acid residue insertion. To investigate which types of this colony-stimulating factor are produced by other cell lines, we used specific oligonucleotides to determine which types of transcripts were present in MIA PaCa-2, 5637, and LD-1 cells, all of which have been reported to produce a factor that can stimulate the growth of predominantly granulocyte colonies in human bone marrow cell cultures. Northern analysis with these probes revealed MIA PaCa-2-like transcripts in all of these cell lines and failed to detect transcripts that would encode the colony-stimulating factor that contained the three-amino-acid-residue insertion.

The role of interleukin 6 and interleukin 1 in megakaryocyte development.

The effect of human interleukin 6 (IL-6) and interleukin 1 (IL-1) on cells of the megakaryocyte lineage from murine bone marrow was examined. In bone marrow liquid culture, IL-6 but not IL-1 increases the amount of acetylcholinesterase, a megakaryocyte marker. In semisolid colony assays, a low level of interleukin 3 (IL-3) was used as a growth factor, and IL-6 and IL-1 were tested for their ability to potentiate the activity of IL-3 to stimulate megakaryocyte colony formation. IL-6 and/or IL-1 had no effect on megakaryocyte colony formation in the absence of IL-3. However, IL-6 was able to stimulate increased megakaryocyte colonies in the presence of IL-3. IL-1 was able to potentiate colony formation only in the presence of both IL-3 and IL-6.

A new culture and quantitation system for megakaryocyte growth using cord blood CD34+ cells and the GPIIb/IIIa marker.

A new culture and quantitation system has been established for growth of megakaryocyte-lineage cells from human progenitor cells. CD34+ progenitor cells were enriched from umbilical cord blood using an avidin-biotin immunoadsorption process. These cells were preincubated in bulk liquid culture for 3 to 4 days in the presence of the growth factors interleukin-3 (IL-3) and IL-6. The cells were then washed and seeded at 5000 cells/well in 96-well plates that contained a variety of test samples. The plates were incubated for 7 days, and the cells were then washed, transferred to ELISA plates, and fixed. Megakaryocyte growth was determined by an ELISA for the platelet glycoprotein (GP) IIb/IIIa, an abundant membrane protein found on cells committed to the megakaryocyte lineage. The growth factor IL-3 was found to produce a very strong signal in this assay. The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, stem cell factor (SCF), or leukemia inhibitory factor (LIF) to low levels of IL-3 also stimulated megakaryocyte growth, as measured by IIb/IIIa expression. Plasma from patients with aplastic anemia was also stimulatory in this assay, and showed marked synergy with IL-3. This progenitor cell culture system, due to its judicious use of progenitor cells and an automated, 96-well quantitation method, allows for screening large numbers of test samples and multiple combinations and concentrations of growth factors.

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.

Human non-transformed monocyte-derived macrophage cell lines.

We have demonstrated that human blood monocyte-derived macrophages can be passaged from primary cultures and replicate. Passaged cells have typical macrophage characteristics: they are non-specific esterase positive, phagocytic, and respond to 3 days of treatment with interferon-gamma with enhanced production of superoxide on stimulation with PMA. The passaged cells express Fc, CR1, CR3 and FMLP receptors. Both primary and passaged cultures constitutively produce CSF-1 after 3 weeks in culture. Cultures studied between 7 and 16 weeks in culture produce 3712 +/- 478 U of CSF-1 per 10(6) cells. Randomly selected lines were examined to look for cell proliferation by looking at numbers of cells over time and by labelling cells with tritiated thymidine to determine the number of cells synthesizing DNA. In addition, the cells can be frozen at the time of isolation and stored for at least 1 year, and then thawed and shown to retain functional activity. Human monocyte-derived macrophages can be cultured as finite cell lines.

Biological properties and molecular biology of the human macrophage growth factor, CSF-1.

CSF-1 is a growth and differentiation factor for the production of mononuclear phagocytes from undifferentiated bone marrow progenitors. In addition to previously described effects on mature cells, we show here that CSF-1 stimulates the production by monocytes of interferon, tumor necrosis factor, and myeloid CSF that produces mainly mixed neutrophil-macrophage colonies in bone marrow culture. Pretreatment with CSF-1 also promotes resistance to viral infection and tumor cytotoxicity in murine peritoneal macrophages. Based on amino acid sequence data of purified human urinary and murine L cell CSF-1, we have cloned the complementary DNA (cDNA) from messenger RNA (mRNA) of the human CSF-1 producing MIA PaCa cell line. The cDNA specifies a 32 amino acid signal peptide followed by a protein of 224 amino acids. Several facts suggest, however, that one-third of the molecule at the C-terminal end is processed off intracellularly to derive the secreted growth factor. The gene is about 18 kilobases (kb) in length and contains 9 exons. Although there appears to be a single copy gene for CSF-1, cells expressing the factor contain several mRNA species, suggesting that the gene may have several functions or levels of regulation. High level expression of the recombinant protein will allow preclinical testing in several disease models for therapeutic efficacy that has been suggested from in vitro and in vivo biological properties of CSF-1.

Post-chemotherapy and cytokine pretreated marrow stromal cell layers suppress hematopoiesis from normal donor CD34+ cells.

Marrow stromal layers were used to investigate the potential role of negative regulators produced by the marrow microenvironment as one potential cause of hematopoietic suppression after chemotherapy and cytokines. Stromal layers were established from marrow of normal or prechemotherapy donors and breast cancer patients after hematological recovery from one cycle of 5-fluorouracil, leucovorin, doxorubicin, and cyclophosphamide and GM-CSF or PIXY321 (GM-CSF/IL-3 fusion protein). Normal donor CD34+ cells were placed in contact with stromal layers, and the number of colony-forming units for granulocytes and macrophages (CFU-GM) was determined. There were 25-79% fewer CFU-GM in post-chemotherapy stromal layer cocultures than in no chemotherapy cocultures. With neutralizing antibody to TNF-alpha the number of CFU-GM in no chemotherapy and post-chemotherapy stromal cocultures was, respectively, 96 +/- 7% (n = 5) and 142 +/- 8% (n = 5) of the number with no antibody treatment. PIXY321 and GM-CSF pretreated stromal layers also suppressed production of CFU-GM. Anti-TNF-alpha promoted an increase in CFU-GM numbers from GM-CSF, but not PIXY321, pretreated stromal cocultures. The results demonstrate that post-chemotherapy marrow stromal layers were deficient in supporting in vitro hematopoiesis and suggest that negative regulators induced by chemotherapy and cytokines may be one cause for this defect.

Myeloid clonogenic assays for comparison of the in vitro toxicity of alkylating agents.

A battery of clonal assays for myeloid progenitor cells (HPP-CFC, CFU-gemm, CFU-gm, CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine, busulfan, melphalan, carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis, mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine, melphalan, carmustine and lomustine. Generally, there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays, the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents.

Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor, and colony stimulating activity.

CSF-1, a macrophage colony stimulating factor that causes proliferation and differentiation of progenitor cells, may also have effects on mature cells. Human peripheral blood monocytes were used to examine this possibility. Monocytes, separated from normal blood by density centrifugation and adherence, were incubated for 3 days with or without CSF-1 (1,000 U/ml, purified from the MIA PaCa pancreatic carcinoma line). The two groups of cells were then washed and tested for the ability, when induced, to produce several factors. When induced for 2 days with LPS and PMA, the monocytes produced a factor that was cytotoxic to L929 cells, and this factor was completely neutralized by polyclonal antibody to tumor necrosis factor. The cells preincubated with CSF-1 consistently produced an average of 12 times more of this factor than cells not exposed to CSF-1. Monocytes induced with LPS and PMA also produced a colony stimulating activity, as measured by colony formation when using mouse bone marrow. Cells preincubated with CSF-1, washed, and induced with LPS and PMA produced more than three times as much activity compared with control monocytes. When monocytes were induced with poly-I.C, 22-fold higher levels of interferon were produced by the cells exposed to CSF-1. These results show that CSF-1 has direct stimulating effects on mature human monocytes, and suggest that the macrophage growth factor may have clinical application in the treatment of infectious diseases and cancer.

Bone marrow-derived macrophages: development and regulation of differentiation markers by colony-stimulating factor and interferons.

To investigate the role of specific cytokines in the development of the fully mature macrophage, we have employed murine bone marrow cells that were grown in the presence of CSF-1, a colony-stimulating factor that has been shown to induce the proliferation and differentiation of macrophages from their precursor cells. The CSF-1 employed in these studies was partially purified to ensure removal of contaminating interferon (IFN) from the preparations. After 1 to 2 wk in the presence of the partially purified CSF-1, the adherent macrophages were removed from flasks enzymatically and were recultured at known densities in the absence of CSF-1. Cell surface antigens (Mac-1 and Ia) and Fc receptor capacity (as assessed by Fc-mediated phagocytosis) were examined as markers of macrophage differentiation. Basal levels of Fc receptor capacity and Mac-1 antigen were markedly influenced by exposure to CSF-1, and appear to be modulated by CSF-induced, macrophage-derived IFN. When the bone marrow-derived macrophages were exposed to exogenous IFN in the absence of CSF-1, they proved to be extremely inducible with respect to Fc-mediated phagocytosis (IFN-beta and rIFN-gamma) and Ia antigen expression (rIFN-gamma) when compared with thioglycollate-elicited macrophages. Thus, macrophage growth factors, such as CSF-1, promote macrophage maturation by inducing the production of autostimulatory signals, such as macrophage-derived IFN. In addition, exogenous cytokine stimuli, such as IFN-gamma, further amplify the differentiative potential of these cells. Bone marrow-derived macrophages, propagated under well-defined conditions and never exposed to eliciting agents, provide a powerful model for studying the role of cytokines, such as CSF-1 and IFN, in the differentiative pathway of macrophages.

Opposing effects of glucocorticoids on interferon-gamma-induced murine macrophage Fc receptor and Ia antigen expression.

Two macrophage markers associated with differentiation are the Fc receptor (FcR) and the Ia antigen. Expression of these markers is increased with IFN-gamma treatment, although some evidence suggests that the induction pathway for Fc receptor and Ia antigen expression may be dissociable. In this study, the effect of glucocorticoids on basal and IFN-induced levels of Fc-mediated phagocytosis and Ia antigen expression was investigated. Macrophages incubated for 2 days with glucocorticoids alone showed no change in basal levels of Fc-mediated phagocytosis. However, incubation with glucocorticoids plus IFN-gamma resulted in increased Fc-mediated phagocytosis and binding to a much greater extent than IFN-gamma treatment alone. This enhancement was specific for IFN-gamma, because the IFN-beta-induced increase in Fc-mediated phagocytosis and binding was not affected by glucocorticoids. In contrast to the expression of Fc receptor capacity, both basal and IFN-gamma-induced levels of Ia antigen expression were inhibited by glucocorticoids. The glucocorticoid effect on these two markers was not observed with other steroid hormones, nor was it altered by inhibitors of the arachidonic acid pathway. The findings of this study provide additional evidence that induction of Fc receptor and Ia antigen by IFN-gamma occurs by different mechanisms.

CSF-1-induced resistance to viral infection in murine macrophages.

Murine peritoneal thioglycollate-elicited macrophages were cultured for 3 days in the presence or absence of highly purified human macrophage colony stimulating factor (CSF-1). The cells were then challenged with vesicular stomatitis virus (VSV) for 24 hr. Ability to resist viral infection was measured in two ways. First, macrophage viability after infection with VSV was measured by washing to remove dead cells, staining the remaining cells with crystal violet, and reading absorbance. Second, a yield reduction assay was used to measure viral replication in the macrophage cultures. Cells treated with CSF-1 (500 to 2000 U/ml) and infected with VSV looked similar microscopically to uninfected cells and had absorbance values twofold to threefold higher than those of infected cultures not treated with CSF-1. The CSF-1-treated cultures also had a virus titer one log lower than that of the untreated cultures. Treatment with partially purified murine CSF-1 induced a similar reduction in virus titer, whereas other murine CSF tested (purified murine GM-CSF, lung-conditioned medium that contains GM-CSF and G-CSF, and WEHI-3B-conditioned medium as a source of IL 3) had little to no effect on virus titer. Antibody to murine IFN-alpha/beta added to the macrophage cultures inhibited the protective effect of CSF-1, indicating that the CSF-1 effect was due to induction of endogenous IFN. Treatment with lipopolysaccharide (1 ng/ml) had some protective effect, which was blocked with polymyxin B. Polymyxin B did not inhibit the effect of CSF-1.

Expression of the macrophage specific colony-stimulating factor (CSF-1) during human monocytic differentiation.

We and others have previously demonstrated expression of the co-fms proto-oncogene during human monocytic differentiation. The c-fms gene has since been shown to encode for the macrophage specific colony stimulating factor (CSF-1) receptor. The present results demonstrate that both CSF-1 and c-fms transcripts are induced during monocytic differentiation of human HL-60 leukemia cells. The results further demonstrate that normal human monocytes express CSF-1 RNA and that the level of these transcripts increases upon treatment with phorbol ester. Finally, the detection of CSF-1 RNA in HL-60 cells and in monocytes is associated with production of the CSF-1 gene product. These findings would suggest that monocytes are capable of regulating their own survival, growth and differentiation through CSF-1 production.

Antagonistic effect of interferon-beta on the interferon-gamma-induced expression of Ia antigen in murine macrophages.

The cell surface expression of I region-associated (Ia) antigens by murine and human macrophages has been shown by investigators from a number of laboratories to be induced in a dose-dependent fashion by IFN-gamma, which is free of other lymphokines. The experiments described in this report demonstrate that fibroblast-derived IFN-beta exerts an antagonistic effect on IFN-gamma induced Ia expression in murine macrophages. Simultaneous addition of IFN-beta and IFN-gamma to peritoneal exudate macrophages results in decreased Ia expression when compared with macrophages treated with IFN-gamma only. Different sources of highly purified IFN-beta, as well as a recombinant human IFN-alpha (A/D Bgl; shown previously to be as active as IFN-beta in several other murine systems) acted in a similar antagonistic fashion to IFN-gamma-induced Ia induction. The down-regulation of Ia expression by IFN-beta is dose-dependent over a concentration range up to 100 U/ml. Time-course experiments indicated that for IFN-beta to down-regulate IFN-gamma-induced Ia, it had to be present either before stimulation with IFN-gamma or during the first 24 hr of simultaneous stimulation. Further experiments in which a highly specific antibody against IFN-alpha/beta was added to the cultures confirmed the findings of the time-course experiments. Inhibitors of the arachidonic acid pathway failed to reverse the effect of IFN-beta to reduce Ia antigen expression, which suggests that this inhibition is not prostaglandin mediated. Thus, these findings support a role for type I IFN as naturally occurring substances that negatively regulate the expression of class II molecules.

Colony-stimulating factor-induced monocyte survival and differentiation into macrophages in serum-free cultures.

The role of mononuclear phagocyte-specific colony-stimulating factor (CSF-1) in human monocyte to macrophage differentiation was investigated. The addition of 1000 U/ml of CSF-1 to serum-free monocyte cultures resulted in monocyte survival comparable to that in cultures containing 5% AB serum, whereas cells in serum- and CSF-1-free medium lost their viability in 3 to 5 days. The requirement for CSF-1 coincided with the time (40 to 64 hr of culture) when the major changes in morphology and biochemical function took place in monocytes undergoing differentiation into macrophages. If CSF-1 was removed from the cultures before this time, death of the monocytes resulted. In cultures containing CSF-1, as in serum containing cultures, the lysosomal enzyme acid phosphatase was enhanced 10- to 20-fold by day 4 to 5. Superoxide production in response to phorbol myristic acetate was maintained in CSF-1 cultured monocytes, but declined with time in monocytes cultured in serum. The expression of monocyte-macrophage antigens p150.95 (LeuM5), OKM1, LeuM3, Fc receptors (32.2), and HLA-DR had increased in CSF-1 containing cultures at day 4. When antigen expression was analyzed at day 2 to 3, when cell size and 90 degrees scatter characteristics were still identical to control serum-free cultures, only p150.95, HLA-DR and FcR expression were enhanced by CSF-1. Low amounts of lipopolysaccharide (0.1 ng/ml) were found to enhance monocyte survival in the absence of added CSF-1. Lipopolysaccharide-containing cultures were found to produce CSF-1 (up to 450 U/ml, as detected by radioimmunoassay). Lipopolysaccharide (1 microgram/ml), however, did not induce enhanced expression of the maturation-related antigens. Based on these observations we conclude that CSF-1 is enhancing human monocyte survival and is involved in the events leading to the differentiation of monocytes into macrophages.

Expression of the macrophage-specific colony-stimulating factor in human monocytes treated with granulocyte-macrophage colony-stimulating factor.

The macrophage-specific colony-stimulating factor (CSF-1, M-CSF) regulates the survival, growth and differentiation of monocytes. We have recently demonstrated that phorbol ester induces expression of CSF-1 in human monocytes. These findings suggested that activated monocytes are capable of producing their own lineage-specific CSF. The present studies demonstrate that the granulocyte-macrophage colony-stimulating factor (GM-CSF) also induces CSF-1 transcripts in monocytes. Furthermore, we demonstrate that the detection of CSF-1 RNA in GM-CSF-treated monocytes is associated with synthesis of the CSF-1 gene product. The results thus suggest that GM-CSF may indirectly control specific monocyte functions through the regulation of CSF-1 production. These findings indicate another level of interaction between T cells and monocytes.