Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.     

Granulocyte colony-stimulating factor induces neutrophils to secrete macrophage colony-stimulating factor

In this work we provide evidence showing that granulocytes produce macrophage colony-stimulating factor (M-CSF) from the band cell stage and secrete this factor when induced to differentiate into polymorphonuclear cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF). Using an enriched population of myeloid band cells from murine bone marrow, we identified the presence of M-CSF with a chromophore-labelled monoclonal anti-M-CSF antibody. Using ELISA we detected the secretion of M-CSF in the supernatants of cultures of enriched band cells when induced with rhG-CSF to differentiate into mature neutrophils. We also found that M-CSF is the only factor responsible for the colony forming activity in the supernatants and lysates of band cells treated with rhG-CSF.     

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.     

Antitumor effects of human recombinant macrophage colony-stimulating factor against rat brain tumors

The tumoricidal effects of M-CSF were examined using two subcutaneously-transplanted rat brain tumor cell lines, 9L and T9 gliomas. In rats treated with high-dose M-CSF (16 million U/kg administered for 4 days a week for 3 weeks), 9L glioma growth was inhibited by 81.9% following subcutaneous (s.c.) injection and by 70.5% after intraperitoneal (i.p.) injection and T9 glioma growth was inhibited by 69.2% after i.p. injection. After short-term treatment with high-dose M-CSF (32 million U/kg administered s.c. for 6 consecutive days, 9L glioma growth was inhibited by 82.1%. All these inhibitory effects differed significantly compared with the respective untreated control groups. However, treatment with low-dose M-CSF (1.6 million U/kg administered s.c. for 4 days a week for 3 weeks) showed no significant effects against 9L and T9 glioma growth compared with the untreated controls. No significant effects of M-CSF against cell proliferation, measured as PCNA expression, were observed in any group. Significant hematopoietic effects on the leukocyte counts were observed only in the groups treated with high dose M-CSF. These results suggest that M-CSF at a high dose which produces hematopoietic effects on peripheral leukocytes inhibits the growth of gliomas. This inhibitory effect may have been due to a tumoricidal mechanism of M-CSF that depended on the production or release of some hematopoietic soluble factors, but was independent of PCNA expression by the tumors.Abbreviations BBB blood-brain barrier - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - hM-CSF human macrophage colony-stimulating factor - IFN interferon - IL-1 interleukin-1 - IL-6 interleukin-6 - M-CSF macrophage colony-stimulating factor - PCNA proliferating cell nuclear antigen - rhM-CSF recombinant human macrophage colony-stimulating factor - TNF tumor necrosis factor     

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.     

Differential alterations in plasma colony-stimulating factor concentrations after coronary artery bypass graft surgery with extracorporeal circulation

To determine whether colony-stimulating factor (CSF) might participate to the inflammatory response after cardiac surgery, plasma concentrations of granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF) and GM-CSF were measured in 31 patients undergoing coronary artery bypass graft (CABG) surgery with extracorporeal circulation (ECC). Plasma G-CSF and M-CSF concentrations increased after weaning of ECC, reached maximum value at the sixth post-operative hour, and remained elevated at the 24th post-operative hour. In contrast, plasma GM-CSF levels did not change. Plasma M-CSF, G-CSF and GM-CSF values were not different whether patients developed post-operative complications or not. In conclusion, M-CSF and G-CSF are produced after CABG surgery despite the use of high aprotinin doses in hope to abrogate the inflammatory response. G-CSF and M-CSF might play a role in the inflammatory process often observed after CABG surgery.     

The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor.

Two different cDNAs for human granulocyte colony-stimulating factor (G-CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G-CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G-CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G-CSF cDNA and the DNA fragment containing the G-CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G-CSF gene is interrupted by four introns and a comparison of the structures of the two G-CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5' splice donor sequences in the second intron of the G-CSF gene. When the G-CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.     

Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.     

Large-scale preparation and characterization of human colony-stimulating factor

In order to obtain human granulocytic colony-stimulating factor (G-CSF) in large quantities, a large-scale culture system of human G-CSF-producing cells has been established. The cell used for this system was T3M-1, which grew in a monolayered sheet in F-10 synthetic medium supplemented with 10% fetal bovine serum. T3M-1 cells grew in rolling bottles at the velocity of 0.5 r.p.m. with about 22 hr. of population doubling time. When the culture reached confluency, it was incubated in a serum-free medium supplemented with 1% bovine serum albumin. The conditioned medium was harvested every week, concentrated by Amicon PM-10 membrane, and loaded on a Sephadex G-75 column. The molecular weight of G-CSF was estimated at about 30,000. This G-CSF was stable over a pH range of 1.0 to 11.0 at 4°C for 21 hr. The CSF activity was destroyed by either trypsin or chymotrypsin, but resisted to RNase and DNase. A slight decrease in the activity was produced by treatment with neuramidase. G-CSF stimulated granulocytic colony formation of human and mouse marrow cells. By using the roller bottle culture system, we could obtain more than 100 liters of cultured medium in a month, which was able to form about 150,000,000 colonies of human bone marrow cells. The recovery of the human G-CSF activity from gel-filtration column was very high (91.7%), and a large increase of specific activity was obtainable (13.3-fold). This culture system is therefore expected to aid in the large-scale preparation of human G-CSF, thereby facilitating further studies on this granulopoietic factor.     

Examination of survival, proliferation and cell surface antigen expression of human monocytes exposed to macrophage colony-stimulating factor (M-CSF)

The effects of macrophage colony-stimulating factor (M-CSF or CSF-1) on the survival, proliferation, maturation and activation of human blood monocytes were examined. M-CSF (100-1,000 U/ml) doubled the number of monocytes surviving after eight days in culture and accelerated the usual increase in cell volume. Antiserum to M-CSF abolished both of these effects. There was no sizable increase in 3H-thymidine incorporation in monocytes over this time period. Of various factors tested, including gamma-interferon (gamma-IFN), interleukin (IL) 1 alpha, granulocyte CSF (G-CSF), platelet-derived growth factor (PDGF), and lipopolysaccharide (LPS), only granulocyte-macrophage CSF (GM-CSF) could also enhance survival and augment cell volume. While antiserum to human M-CSF eliminated the increase in survival induced by GM-CSF, it could not ablate the GM-CSF-stimulated increase in monocyte cell volume. Monocyte cell surface markers that increase with maturation (i.e., Fc gamma RIII) or with activation (i.e., Fc gamma RI) were unaffected by incubation with M-CSF.     

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.     

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.     

Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil–granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na₂HPO₄ buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na₂HPO₄ buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique.     

Immunoreactive macrophage colony-stimulating factor is increased in atherosclerotic lesions of watanabe heritable hyperlipidemic rabbits after recombinant human macrophage colony-stimulating factor therapy

Infiltration of monocytes into arteries is an early event in the pathogenesis of atherosclerosis. This recruitment is interpreted as enhancing lesion development, but it could also be a host response limiting lipid accumulation. The ability of macrophages to limit cholesterol uptake, however, can be reduced by the impaired mobility and metabolic activity associated with foam cell development. As lesions enlarge, foam cells die and become the nidus for the necrotic core. Treatments to improve viability might improve foam cell function and promote regression. Macrophage colony-stimulating factor (M-CSF) is vital to monocyte/macrophage differentiation, proliferation, and activation. We found that foam cells of Watanabe heritable hyperlipidemic (WHHL) rabbits had faint staining for M-CSF. Treatment of rabbits with recombinant human M-CSF (rhM-CSF) increased M-CSF staining, which correlated with reduced cholesterol content of these foam cells. Mol Reprod Dev 46:92–95, 1997. © 1997 Wiley-Liss, Inc.     

Enhancement of plasminogen activator activity in cultured endothelial cells by granulocyte colony-stimulating factor

A hitherto unknown function of granulocyte colony-stimulating factor (G-CSF) was found using cultured endothelial cells. G-CSF stimulated activity of plasminogen activator (PA) in both extracellular and intracellular milieus of endothelial cells obtained from bovine carotid and aortic artery. This effect was dependent on the concentration of G-CSF added to the culture medium and on the treatment time. The extracellular activity was enhanced approximately 5-fold at a concentration of 5,000 colony-forming unit (CFU)/ml (2.6 nM) and in about a 15-hr treatment period. Analyses by fibrin and reverse fibrin autography revealed that activity of PA was much more increased than that of PA inhibitor in endothelial cells treated with G-CSF.     

Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor.

Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron-exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.     

A tumor cell line producing granulocyte colony-stimulating factor and an immune suppressive factor

From a patient, both a cell line incapable of secreting granulocyte colony-stimulating factor (G-CSF) (TC873) and a cell line capable of secreting G-CSF (TCM902) were established. The effector cells induced, with TC873 cells showed a high lytic capacity against two types of tumor cells. The effector cells induced by TCM902 cells did not show such capacity. Furthermore, the TCM902 cells excreted a factor suppressing the proliferation of lymphokine activated killer (LAK) cells and the autologous tumor cell lysis of tumor associated lymphocytes. This factor probably is TFG- ₁.Abbreviations CSF colony stimulating factor - ELISA enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IFN interferon - IL interleukin - LAK lymphokine activated killer - M monocyte - MLTC mixed lymphocyte tumor cell culture - TGF transforming growth factor - TILs tumor infiltrating lymphocytes - TNF tumor necrosis factor     

Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli

A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.