PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU.1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FCgammaII), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GMCSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages.     

Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages

Murine alveolar macrophages (AM) were shown to have proliferative ability and to form colonies in vitro. The factors in lung-conditioned medium (CM) and L929-CM which stimulate the proliferation of AM were considered to be granulocyte-macrophage colony-stimulating factor (GM-CSF) and CSF-1, respectively, because recombinant murine (rm)GM-CSF and recombinant human (rh)CSF-1 could replace the activities of lung-CM and L929-CM, respectively. The phenotype of the cells in the colonies formed by AM incubated with rmGM-CSF or lung-CM was AM-like; more than 90% of the cells were stained by anti-asialo GM1 but not by FITC-LPS, and had AM-like morphology. Expression of Mac-1 Ag determined by M1/70HL in these cells as well as original AM was low. However, the phenotype of the cells in the colonies formed by AM incubated with rhCSF-1 or L929-CM was peritoneal macrophage (PM)-like; more than 90% of the cells were stained by FITC-LPS and M1/70HL, but not by anti-asialo GM1, and showed PM-like morphology. The cells in the colonies formed by AM incubated with rmGMCSF changed their phenotype after treatment with rhCSF-1; the percentage of cells stained by anti-asialo GM1 decreased, and that of cells stained by FITC-LPS increased. The cells in the colonies formed by AM incubated with rhCSF-1 never changed their phenotype after incubation with rmGM-CSF. In contrast to AM, more than 90% of the cells in all colonies formed by PM incubated with either rmGM-CSF, rhCSF-1, lung-CM, or L929-CM were stained by FITC-LPS but not by anti-asialo GM1. These results show that although AM and PM can proliferate, AM, in contrast to PM, are bipotential cells that can differentiate into two types of macrophages responding to distinct types of CSF, and that one of the molecular mechanisms controlling macrophage heterogeneity may be based on the type of CSF produced at distinct tissues.     

Molecular structure and granulocyte/macrophage colony-stimulating factor activity.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) plays a critical role in myeloid differentiation and in several immune and inflammatory processes. GM-CSF binds to specific cellular receptors (GM-CSFR) which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design and such design depends on a molecular understanding of ligand-receptor interactions. We present our initial studies evaluating the potential active sites of the molecule. The sites on the GM-CSF molecule that were studied represent two alpha-helices predicted to be critical for GM-CSF activity, as implicated by human-murine chimeric molecule studies. These helices are predicted to be adjacent in native GM-CSF. Peptides corresponding to amino acids 17-31 and 78-99 of GM-CSF were synthesized and cross-linked to one another in two different orientations. The ability of anti-GM-CSF to bind the individual and complexed peptides was evaluated by both ELISA and radioimmunoassay. Significant binding to all peptides was demonstrated. A preferred orientation of the two peptides was apparent, and this agreed with the predicted model structures. Antibodies were developed against the coupled peptides, and these demonstrated significant cross-reactivity with recombinant human GM-CSF. Additionally, analyses of anti-peptide antisera binding studies predict these two amino acid sequences to lie in parallel planes to one another in the native human GM-CSF molecule.     

Production of granulocyte/macrophage colony-stimulating factor by cultured astrocytes

We investigated the production of interleukin-3 (IL-3)-like factor by murine astrocytes. Supernatants from lipopolysaccharide (LPS)-stimulated astrocytes induced proliferation of IC-2, an IL-3- and granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent cell line. This activity was completely neutralized by the antibody against GM-CSF but not by the anti-IL-3 monoclonal antibody. Northern blot analysis revealed the expression of GM-CSF mRNA, but not of IL-3 mRNA, in cultured astrocytes. These results indicate that with proper stimuli murine astrocytes produce GM-CSF.     

Interactions among granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and IFN-gamma lead to enhanced proliferation of murine macrophage progenitor cells

This report examines the actions of IFN-gamma on monocytopoiesis in murine liquid and semisolid bone marrow cultures. The proliferative response of bone marrow cells to macrophage CSF and granulocyte-macrophage CSF was assayed by measuring [3H]TdR uptake in a range of mouse strains. No interstrain difference in kinetics was observed for CSF-1 action, but GM-CSF acted significantly more rapidly on C57B1/6, Swiss, and to a lesser extent A/J mice than on BALB/c or CBA. IFN-gamma inhibited [3H]TdR incorporation elicited by CSF-1, and to a much lesser extent, GM-CSF. When the two CSF were added together, the effects were not additive; in fact, the response was the same as that seen with GM-CSF alone. When IFN-gamma was also added, the response was restored to the level seen with CSF-1 alone. In essence, the inhibitory actions of GM-CSF and IFN-gamma were mutually exclusive. The mechanism of these actions was investigated using colony assays. As expected, CSF-1 caused the formation of pure macrophage colonies, whereas GM-CSF stimulated production of macrophage, granulocyte, and mixed granulocyte macrophage colonies. When the two CSF were added in combination, the total colony count was greater than with either alone, but less than additive. The number of pure macrophage colonies was reduced to the number seen with GM-CSF alone. IFN-gamma reduced the number of colonies in the presence of CSF-1, but slightly increased the number with GM-CSF. In the presence of both CSF, IFN-gamma increased the colony count by around 25 to 40%, so that the numbers were greater than the combined total of CSF-1 plus GM-CSF added separately. Similar results were obtained in all mouse strains tested. The results suggest that the thymidine uptake data reflect changes in the number of progenitor cells responding rather than changes in cell cycle time. The results are discussed in terms of the possibility that coadministration of GM-CSF and CSF-1 could ameliorate the myelosuppressive actions of IFN-gamma in vivo, leading to more effective use of this agent as a biologic response modifier.     

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.     

Effect of tumor necrosis factor and granulocyte/macrophage colony-stimulating factor on neutrophil degranulation

Both TNF and and granulocyte/macrophage CSF (GM-CSF) can activate neutrophils. The aim of this work was to determine the effect of these cytokines on neutrophil degranulation. The secretion of lactoferrin of secondary granules and myeloperoxidase (MPO) of primary granules from single adherent human neutrophils was assayed by use of a reverse hemolytic plaque assay. Both rTNF and rGM-CSF caused secretion of lactoferrin in a dose-dependent manner. Both agents also caused secretion of MPO, but only in the presence of cytochalasin B. Preincubation with pertussis toxin inhibited rGM-CSF-induced secretion of both lactoferrin and MPO. rTNF-induced MPO secretion was also blocked by pertussis toxin, whereas lactoferrin secretion was only slightly affected. Neither rTNF nor rGM-CSF caused any detectable changes in the concentration of cytoplasmic free Ca2+ in fura-2-loaded cells. However, when neutrophils were loaded with increasing concentrations of quin-2 to buffer any local, not detectable, changes in the concentration of cytoplasmic Ca2+, both rTNF- and rGM-CSF-induced secretion of lactoferrin and MPO were almost totally abolished at a relatively low quin-2 concentration. These results suggest a role of a regulatory G-protein and minute local changes in the concentration of cytoplasmic Ca2+ in TNF- and GM-CSF-induced neutrophil degranulation.     

Intraclonal diversity of fibrosarcoma cells for the production of macrophage colony-stimulating factor and granulocyte colony-stimulating factor

A new cell line was established from fibrosarcoma that had spontaneously developed in a mouse. The cells were maintained growing in culture for two years and constantly produced both macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). Cloning of the cells by anchorage-independent colony formation gave subclones showing the activity of producing M-CSF and G-CSF in different proportions, whereas no subclone produced G-CSF without producing M-CSF simultaneously. Recloning of the bipotential subclones again gave clonal derivatives producing two types of CSF in various proportions. The observed heterogeneity of the cloned cells seems to be an epigenetic phenomenon, because the cells resumed the G-CSF producing activity in the absence of cell proliferation. After equilibrium was achieved, all of the subclones produced both M-CSF and G-CSF nearly in equal proportions. Tumorigenic and leukocytosis-inducing activity of the cloned cells was nearly comparable with the activity of the original tumor cells.     

Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

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.     

Properties of voltage-gated potassium currents of microglia differentiated with granulocyte/macrophage colony-stimulating factor

Voltage-gated whole-cell currents were recorded from cultured microglial cells which had been developed in the presence of the macrophage/microglial growth factor granulocyte/macrophage colony-stimulating factor. Outward K⁺ currents (I _K) were most prominent in these cells. I _Kcould be activated at potentials more positive than –40 mV. Half-maximal activation of I _Kwas achieved at –13.8 mV and half-maximal inactivation of I _Kwas determined at –33.8 mV. The recovery of I _Kfrom inactivation was described by a time constant of 7.9 sec. For a tenfold change in extracellular K⁺ concentration the reversal potential of I _Kshifted by 54 mV.Extracellularly applied 10 mm tetraethylammonium chloride reduced I _K by about 50%, while 5 mm 4-aminopyridine almost completely abolished I _K. Several divalent cations (Ba²⁺, Cd²⁺, Co²⁺, Zn²⁺) reduced current amplitudes and shifted the activation curve of I _Kto more positive values. Charybdotoxin (IC₅₀ = 1.14 nm) and noxiustoxin (IC₅₀=0.89 nm) blocked I _Kin a concentration-dependent manner, whereas dendrotoxin and mast cell degranulating peptide had no effect on the current amplitudes.     

The expression of granulocyte/macrophage colony-stimulating factor in activated mouse lymphocytes declines with age

The expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) was studied in spleen lymphocytes isolated from male C57BL/6J mice of 6, 20, and 29 months of age. GM-CSF expression (biological activity and mRNA level) was maximum after culturing the lymphocytes for 45 hr with concanavalin A and phorbol myristate acetate. The induction of both GM-CSF activity and mRNA levels was observed to decline over 60% between 6 and 29 months of age. The age-related decline in the level of GM-CSF paralleled the age-related decline in the mRNA levels of interleukin-2 and interleukin-3.     

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.     

Cultivation of HL-60 cells in a serum-free medium containing granulocyte/macrophage colony-stimulating factor.

In order to develop a defined cultivation medium for HL-60 cells, we cultivated these cells in a serum-free suspension medium and tested the effect of various growth factors. Of the factors tested, granulocyte/macrophage colony-stimulating factor was most active in growth stimulation. A much lower effect was obtained with granulocyte colony-stimulating factor and transferrin. No effect was found with interleukin-3 and insulin. Granulocyte colony-stimulating factor was the only growth factor tested that also induced differentiation as judged by the nitroblue tetrazolium test. Growth of HL-60 cells in medium containing granulocyte/macrophage colony-stimulating factor (125 U/ml) and transferrin (5 micrograms/ml) as the only protein factors was similar to growth in medium containing 10% serum. No increase in spontaneous differentiation of HL-60 cells in this defined medium was observed. Physiological concentrations of retinol bound to retinol-binding protein and retinyl ester in chylomicron remnants reduced proliferation as well as the level of c-myc oncoprotein and induced differentiation of HL-60 cells cultivated in defined medium. Hence, this defined medium may be useful when studying the function of retinoids in HL-60 cells.