Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.     

Purification and characterization of the receptor for murine granulocyte colony-stimulating factor.

A receptor for mouse granulocyte colony-stimulating factor (G-CSF) has been found on the cell surface of mouse myeloid leukemia cell line NFS-60. Chemical cross-linking of the receptor with radioiodinated G-CSF, followed by gel electrophoresis in the presence of sodium dodecyl sulfate, has revealed that the G-CSF receptor in the NFS-60 cells is a single polypeptide of Mr approximately 100,000-130,000. The receptor in the membrane fraction of NFS-60 cells were solubilized in an active form with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid. The solubilized receptor was purified approximately 100,000-fold to near homogeneity using a G-CSF affinity gel and gel filtration on a Superose 12 column, as measured by the selective precipitation of the 125I-G-CSF-receptor complex by polyethylene glycol. The purified G-CSF receptor has two classes of binding characteristics, one with an equilibrium dissociation constant (Kd) of 120-360 pM which is comparable with the Kd value for the cell-surface receptor, and the other with a higher Kd value of 2.6-4.2 nM. Analyses of the purified receptor by ligand blotting and sucrose density gradient centrifugation indicated that the low-affinity receptor is the monomer of the Mr 100,000-130,000 protein, whereas the high-affinity receptor consists of oligomers of the protein.     

Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor

Human macrophage differentiation inducing factor (DIF) can induce differentiation of human myeloid leukemic cells into macrophage-like cells in vitro. A procedure is described for purification of DIF from serum-free human monocytic leukemia THP-1 cell-conditioned medium. The procedure included concentration of a conditioned medium by ultrafiltration, lentil lectin-Sepharose affinity chromatography, and fast protein liquid chromatography using Mono S and Mono Q. DIF-A of pI 9.0 and DIF-B of pI 8.8 were obtained after a final purification with a Mono Q column, and both DIF gave a single peak with a molecular weight of approximately 51,000 determined by gel chromatography. NH2-terminal amino acid analysis of DIF-A showed a noticeable homology with murine leukemia inhibitory factor. Human DIF-A was found to induce maturation of human and murine leukemic cells into both macrophage-like cells with nitro blue tetrazolium reducing activity and phagocytic cells, but was found to suppress proliferation of these leukemic cells.     

Expression cloning of a receptor for murine granulocyte colony-stimulating factor

Two cDNAs encoding the receptor for murine granulocyte colony-stimulating factor (G-CSF) were isolated from a CDM8 expression library of mouse myeloid leukemia NFS-60 cells, and their nucleotide sequences were determined. Murine G-CSF receptor expressed in COS cells could bind G-CSF with an affinity and specificity similar to that of the native receptor expressed by mouse NFS-60 cells. The amino acid sequence encoded by the cDNAs has demonstrated that murine G-CSF receptor is an 812 amino acid polypeptide (Mr, 90,814) with a single transmembrane domain. The extracellular domain consists of 601 amino acids with a region of 220 amino acids that shows a remarkable similarity to rat prolactin receptor. The cytoplasmic domain of the G-CSF receptor shows a significant similarity with parts of the cytoplasmic domain of murine interleukin-4 receptor. A 3.7 kb mRNA coding for the G-CSF receptor could be detected in mouse myeloid leukemia NFS-60 and WEHI-3B D+ cells as well as in bone marrow cells.     

Regulation of the differentiation of WEHI-3B D+ leukemia cells by granulocyte colony-stimulating factor receptor

To investigate the role of the G-CSF receptor (G-CSFR) in mediating the action of G-CSF, WEHI-3B D+ murine myelomonocytic leukemia cells were transfected with a plasmid containing the murine G-CSFR gene. Overexpression of G-CSFR in transfected clones was demonstrated by northern blotting, binding of [125I]rhG-CSF and cross-linking experiments. A high level of expression of the G-CSFR did not promote or suppress cellular proliferation or initiate differentiation; however, exposure of transfected cells to G-CSF in suspension culture caused a large percentage of the population to enter a differentiation pathway, as determined by two markers of the mature state, the ability of cells to reduce nitroblue tetrazolium (NBT) and to express the differentiation antigen Mac-1 (CD11b) on the cell surface. Thus, upon treatment with 10 ng/ml of G-CSF, 60% or more of transfected cells exhibited NBT positivity; whereas, in contrast, nontransfected cells exhibited only 6% NBT positivity in response to G-CSF. An eightfold increase in Mac-1 expression over that of the parental line was also observed in transfected cells exposed to G-CSF. The growth rate of the transfected clones was decreased by exposure to G-CSF, presumably due to terminal differentiation. The findings suggest that the predominant function of G-CSF and its receptor in WEHI-3B D+ cells is to mediate differentiation and that the level of the G-CSFR portion of the signal transduction mechanism in this malignant cell line is important for a response to the maturation inducing function of the cytokine.     

Binding of 125I-labeled granulocyte colony-stimulating factor to normal murine hemopoietic cells

The binding of granulocyte colony-stimulating factor (G-CSF) to murine bone marrow cells was investigated using a radioiodinated derivative of high specific radioactivity which retained full biological activity. The binding was time- and temperature-dependent, saturable and highly specific. The apparent dissociation constant for the reaction was 60-80 pM at 37 degrees C and 90-110 pM at 4 degrees C, similar to that found for the binding of G-CSF to murine leukemic cells (WEHI-3B D+) and significantly higher than the concentration of G-CSF required to stimulate colony formation in vitro. Autoradiographic analysis confirmed the specificity of binding since granulocytic cells were labeled but lymphocytes, erythroid cells and eosinophils were not. Blast cells and monocytic cells were partially labeled, the latter at low levels. In the neutrophilic granulocyte series, grain counts increased with cell maturity, polymorphs being the most heavily labeled but all cells showed considerable heterogeneity in the degree of labeling. Combination of Scatchard analysis of binding with autoradiographic data indicated that mature granulocytes from murine bone marrow exhibited 50-500 G-CSF receptors per cell.     

Structural studies on the murine granulocyte colony-stimulating factor

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein hemopoietic growth factor which regulates the production of granulocytes and macrophages. Reversed-phase microbore high-performance liquid chromatography was employed to purify a number of tryptic and Staphylococcus aureus V8 proteinase peptides generated from approximately 400 pmol G-CSF purified from medium conditioned by lungs from mice previously injected with endotoxin. N-Terminal amino-acid sequence analyses were performed on the parent polypeptide and on four tryptic peptides and one Staphylococcus aureus V8 protease peptide, yielding 68 unique amino-acid assignments; this corresponds to approximately 38% of the molecule.     

Isolation of a colony-stimulating factor produced by L 1210 murine leukemia cells

Murine L1210 leukemia cells spontaneously produce very low amounts of colony stimulating factor (CSF). CSF production was markedly increased by stimulating L1210 cells with lipopolysaccharide, lectins, and sheep red blood cells. From the conditioned medium of phytohemagglutinin-stimulated L1210 cells we isolated a CSF with an apparent molecular weight of approximately 27,000. This CSF promoted the proliferation and the differentiation of murine GM-CFU showing a weak differentiation-inducing activity on WEHI-3 D (+) cells.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.     

Role of granulocyte/macrophage colony-stimulating factor in the regulation of murine alveolar macrophage proliferation and differentiation

Granulocyte/macrophage (GM)-CSF is one of the hemopoietic growth factors that stimulates neutrophilic granulocyte and macrophage production by bone marrow progenitor cells. In this study, the effect of GM-CSF on the growth and differentiation of murine pulmonary alveolar macrophages (PAM) was investigated. In the presence of GM-CSF, normal murine PAM were induced to proliferate and develop into macrophage colonies with a dose-response curve similar to that of bone marrow GM colony-forming cells. PAM also responded to CSF-1, a lineage-restricted growth factor, but required much higher doses of CSF-1 and a longer incubation time for optimal colony formation. The proliferative response of PAM to CSF-1, however, was greatly enhanced by the concurrent addition of low doses of GM-CSF. In contrast, low doses of CSF-1 failed to potentiate the proliferative response of PAM to GM-CSF. Macrophages derived from GM-CSF cultures were rounder and less stretched and possessed less FcR-mediated phagocytic activity than cells produced in CSF-1 cultures. A study with hydrocortisone-induced monocytopenia showed that nearly one half of lung macrophages may be sustained by local proliferation of PAM without the continuous migration of blood monocytes. This study suggests that GM-CSF may play a major role in the production of PAM by two modes of action, 1) direct stimulation of cell proliferation and 2) enhancement of their responsiveness to CSF-1, thereby producing more mature and functionally competent macrophages.     

Identification of neutrophil alkaline phosphatase-inducing factor in cystic fluid of a human squamous cell carcinoma as granulocyte colony-stimulating factor

We have previously shown that a factor termed neutrophil alkaline phosphatase-inducing factor (NAP-IF) has the capacity to induce neutrophil alkaline phosphatase (NAP) in postmitotic granulocytes (PMGs). This factor has characteristics similar to those of granulocyte colony-stimulating factor (G-CSF), suggesting that the two factors assayed by different methods may be attributable to an identical macromolecule. In a preliminary experiment, we showed that purified natural G-CSF (nG-CSF) could induce NAP in vitro in the presence of 10% (v/v) fetal calf serum (FCS). In this study, purified human nG-CSF and recombinant G-CSF (rG-CSF) induced NAP in granulocytes from both normal individuals and patients with chronic myelogenous leukemia in a dose-dependent fashion in serum-free and serum-containing culture conditions. The induction of NAP by G-CSF was detectable at 0.4 ng/ml and became maximal between 10 and 20 ng/ml. Anti-G-CSF serum incubated with either NAP-IF or rG-CSF inhibited induction of NAP. Morphological examinations revealed that granulocytes cultured with G-CSF were more mature than those cultured without G-CSF, indicating that G-CSF promoted maturation of granulocytes in parallel with NAP induction. These results indicate that NAP-IF in the cystic fluid of a human squamous cell carcinoma is identical to G-CSF and that induction of NAP by G-CSF is really a reflection of cell maturation promoted by G-CSF.     

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

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

Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.     

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.     

Identification of a functional domain of human granulocyte colony-stimulating factor using neutralizing monoclonal antibodies.

Human granulocyte colony-stimulating factor (G-CSF) is a hemopoietic growth factor that is being used successfully to treat various forms of neutropenia. To define functionally important regions of G-CSF, we have prepared 37 monoclonal anti-G-CSF antibodies and mapped the regions of G-CSF recognized by different antibody groups. Antibodies recognizing similar epitopes were identified by competition assays, neutralization assays, conformation dependence and cross-reactivity with canine G-CSF. Seven of eight neutralizing antibodies fell into two related epitope groups and were conformation-dependent. The eighth was unrelated and conformation-independent. Peptides of G-CSF were generated by chemical or enzymatic digestion and tested for antibody reactivity. One of the neutralizing antibodies (LMM351) recognized a small, disulfide-bonded peptide from the V8 protease digest (residues 34-46). A synthetic peptide (residues 20-58) was recognized by all the neutralizing antibodies, implicating this disulfide-bonded loop in receptor binding. The epitopes recognized by nonneutralizing antibodies were found throughout G-CSF. Thus, regions of G-CSF that are not involved in receptor binding have also been defined. A CNBr peptide (residues 1-121) had greatly reduced biological activity, indicating that the COOH terminus is required for receptor binding. We predict that residues 20-46 and the COOH terminus bind to the G-CSF receptor.     

A mesoderm-inducing factor produced by WEHI-3 murine myelomonocytic leukemia cells is activin A

The first inductive interaction in amphibian development is mesoderm induction, during which a signal from the vegetal hemisphere of the blastula-staged embryo induces mesoderm from overlying equatorial cells. Recently, a number of 'mesoderm-inducing factors' (MIFs), which may be responsible for this interaction, have been discovered. Examples of these MIFs include members of the fibroblast growth factor family as well as members of the TGF-beta superfamily such as TGF-beta 2. In addition to these purified factors, several new sources of mesoderm-inducing activity have been described. One of the most potent of these is the murine myelomonocytic leukemia cell line WEHI-3. Even at high dilutions, conditioned medium from WEHI-3 cells induces isolated Xenopus animal pole regions to form a variety of mesodermal cell types. In this paper we show by several criteria, including N-terminal amino acid sequencing, Northern blotting and various functional assays, that the WEHI-MIF is activin A. Activins are known to modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and to cause the differentiation of two erythroleukemia cell lines. Our results, along with recent data from other laboratories, indicate that these molecules may also act in early development in the formation of the mesoderm.     

Induction by recombinant human granulocyte colony-stimulating factor of differentiation of mouse myeloid leukemic M1 cells

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on induction of differentiation of mouse myeloid leukemic M1 cells was examined. Purified G-CSF caused dose-dependent induction of phagocytic activity and lysozyme activity in M1 cells. Its half-maximally effective concentration was 10 ng/ml. On treatment of M1 cells with G-CSF (100 ng/ml) for 4 days, 30-50% of the cells differentiated morphologically into macrophage cells; 30-40% of the cells were blast cells and 20-30% of the cells were forms intermediate between blastic cells and mature macrophages.     

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.