Comparison of the effects of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity

Cultured human monocytes undergo a process of differentiation and maturation lasting 5 to 10 days that ultimately leads to the appearance of large macrophage-like cells. This differentiation is growth factor dependent: of all the cytokines tested, only macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3 proved capable of supporting the differentiation and the long term survival of the macrophage-like cells. Although all three cytokines yield cells with macrophage characteristics, cells developed in M-CSF have features distinct from those matured in either IL-3 or GM-CSF. At the morphologic level, the M-CSF-supported monocyte cultures yield elongated, spindle-shaped cells whereas those supported with IL-3 or GM-CSF yielded round cells with distinct nuclei. All three macrophage populations expressed similar levels of HLA-DR, CD11b, and CD11c, but the M-CSF-treated cultures yielded more CD14+ and CD16+ (Fc gamma RIII) cells. All three cell populations developed capacity for antibody-dependent cellular cytotoxicity (ADCC) as well as antibody-independent cytotoxicity with peak activity achieved after 8 to 12 days in culture. ADCC capacity developed earliest and the level of activity was usually greatest in the M-CSF-treated cultures, possibly correlating with the higher level of expression of CD16. Our findings indicate that any of these cytokines, but particularly M-CSF, may be useful clinically in enhancing the tumoricidal capacity of tumor-specific mAb through augmentation of macrophage capacity for ADCC.     

Occurrence of a monocyte/macrophage colony-stimulating factor in the continuous ambulatory peritoneal dialysis fluids and its chromatographic behaviors and antigenicity compared with human urinary colony-stimulating factor

Continuous ambulatory peritoneal dialysis (CAPD) fluid from three patients with chronic renal failure exhibited the activity of colony-stimulating factor (CSF) in amounts varying from 5 to 40 units per ml. Like the CSF obtained from normal human urine, the peritoneal CSF predominantly produced monocyte/macrophage colonies in soft-agar culture of mouse bone marrow cells. Semipurified peritoneal CSF showed its isoelectric point at pH 3.6 and 4.9 before and after the treatment with neuraminidase. Under the same conditions, the urinary CSF was focused at pH 3.1 and 4.6. The position of elution of the peritoneal and urinary CSF in ordinary gel-filtration chromatography corresponded to a molecular weight of 62,000 and 117,000, whereas both CSFs exhibited a molecular weight of 28,000 upon gel-filtration in the presence of 6 M guanidine HCl. Furthermore, the two CSFs from the human sources were neutralized by antimouse L cell CSF serum in the same manner. We conclude that the peritoneal CSF is a sialoglycoprotein which is nearly identical with the urinary CSF despite processing of the latter through kidneys.     

Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.     

Dioxin induces a novel nuclear factor, DIF-3, that is implicated in spermatogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), a member of a class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans, is one of the most toxic artificial compounds ever developed. In this study, we identified a novel TCDD target gene, DIF-3 (dioxin inducible factor-3), by cDNA representational difference analysis. DIF-3 protein is a nuclear factor and possesses a zinc-finger motif at its N-terminus. High DIF-3 mRNA expression in the testes was demonstrated by Northern blot analysis and abundant DIF-3 protein was detected during spermatogenesis. Thus, these results suggest that DIF-3 may be a target gene mediating the reproductive toxicity induced by TCDD.     

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.     

c-fps/fes protein-tyrosine kinase is implicated in a signaling pathway triggered by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) are hematopoietic growth factors which stimulate the proliferation and differentiation of myeloid progenitor cells. There is a considerable degree of overlap in target cell specificity and the functional effects of GM-CSF and IL-3. GM-CSF and IL-3 induce a nearly identical pattern of protein-tyrosine phosphorylation in certain cell lines, although their receptors have no kinase domains. Furthermore, their receptor complexes share one subunit (designated as beta). These observations raise the possibility that GM-CSF and IL-3 have a common signaling pathway. Here we show that both GM-CSF and IL-3 induce tyrosine phosphorylation and kinase activity of the c-fps/fes proto-oncogene product (p92c-fes), a non-receptor protein-tyrosine kinase, in a human erythro-leukemia cell line, TF-1, which requires GM-CSF or IL-3 for growth. In addition, GM-CSF induces physical association between p92c-fes and the beta chain of the GM-CSF receptor. p92c-fes is therefore a possible signal transducer of several hematopoietic growth factors including GM-CSF and IL-3 through the common beta chain.     

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.     

TNF-alpha-converting enzyme cleaves the macrophage colony-stimulating factor receptor in macrophages undergoing activation

We previously reported that macrophage activators such as LPS, IL-2, and IL-4 down-modulate the M-CSFR via a mechanism involving protein kinase C and phospholipase C. In this study, we showed that M-CSFR is shed from macrophage surface and identified the protease responsible for M-CSFR cleavage and down-modulation. The shedding of M-CSFR elicited by phorbol esters (tetradecanoylphorbol myristate acetate (TPA)) or LPS in murine BAC.1-2F5 macrophages was prevented by cation chelators, as well as hydroxamate-based competitive inhibitors of metalloproteases. We found that the protease cleaving M-CSFR is a transmembrane enzyme and that its expression is controlled by furin-like serine endoproteases, which selectively process transmembrane metalloproteases. M-CSFR down-modulation was inhibited by treating cells in vivo, before TPA stimulation, with an Ab raised against the extracellular, catalytic domain of proTNF-converting enzyme (TACE). TACE expression was confirmed in BAC.1-2F5 cells and found inhibited after blocking furin-dependent processing. Using TACE-negative murine Dexter-ras-myc cell monocytes, we found that in these cells TPA is unable to down-modulate M-CSFR expression. These data indicated that TACE is required for the TPA-induced M-CSFR cleavage. The possibility that the cleavage is indirectly driven by TACE via the release of TNF was excluded by treating cells in vivo with anti-TNF Ab. Thus, we concluded that TACE is the protease responsible for M-CSFR shedding and down-modulation in mononuclear phagocytes undergoing activation. The possible physiological relevance of this mechanism is discussed.     

Fibroblast-secreted macrophage colony-stimulating factor is responsible for generation of biphenotypic B/macrophage cells from a subset of mouse B lymphocytes.

Normal and malignant CD5+ B lymphocytes can develop macrophage-like characteristics. One stimulus of this phenotypic shift is culture of normal mouse splenic B lymphocytes with splenic fibroblasts or their conditioned media. These biphenotypic B/macrophage (B/M phi) cells simultaneously display macrophage characteristics, such as phagocytosis and F4/80 expression, while retaining B cell features, including expression of surface Ig, CD5, B220, and rearranged Ig genes. The present study investigated the fibroblast-secreted factor that promotes this phenotypic change from B cell to B/M phi cell. RT-PCR analysis demonstrated that mRNA for M-CSF is produced by splenic fibroblasts. Recombinant M-CSF (CSF-1) could replace fibroblast-conditioned medium to elicit the development and survival of B/M phi cells from splenic B lymphocytes. In addition, neutralization of fibroblast-secreted M-CSF with specific mAbs abrogated the ability of conditioned supernatants to promote outgrowth of B/M phi cells. The transition from B lymphocyte to B/M phi cell was marked by the kinetic appearance of mRNA for the M-CSF receptor, c-fms, at day 3 following culture initiation. These results demonstrate that M-CSF is important in the development and physiology of mouse B/M phi cells and potentially in the growth of human biphenotypic hematological malignancies. Interestingly, the presence of IFN-gamma in splenic B lymphocyte cultures abrogated the effect of fibroblast-conditioned medium or M-CSF on outgrowth of B/M phi cells. Furthermore, these findings suggest that a Th1 microenvironment favored by typical macrophages is detrimental to the outgrowth of B/M phi cells.     

Sortilin,a novel APOE receptor implicated in Alzheimer disease

In the brain, apolipoprotein E (APOE) delivers cholesterol-rich lipoproteins to neurons to support synaptogenesis and maintenance of synaptic connections. Three APOE alleles exist in the human population with ε4 being an Alzheimer disease (AD) risk gene and ε2 being protective relative to the common ε3 variant. Many hypotheses have been advanced concerning allele-specific effects of APOE on neurodegeneration including effects on Aβ clearance, synaptic transmission, or neurotoxicity. Central to most proposed APOE functions is its interaction with receptors that mediate cellular uptake of this ligand. Several members of the LDL receptor gene family have been implicated as APOE receptors in the (patho)physiology of APOE in the brain, yet their specific modes of action in AD remain controversial. Recently, the pro-neurotrophin receptor sortilin has been identified as a novel APOE receptor in neurons. Ablation of sortilin expression in mice results in accumulation of APOE and Aβ in the brain. Moreover, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/Aβ complexes. Despite increased brain APOE levels, sortilin-deficient animals recapitulate anomalies in brain lipid homeostasis seen in APOE null mice, indicating functional deficiency in APOE uptake pathways. Taken together, these findings suggest a link between Aβ catabolism and pro-neurotrophin signaling converging on this receptor pathway.     

Recycling of the epidermal growth factor receptor is mediated by a novel form of the clathrin adaptor protein Eps15

Levels of the epidermal growth factor receptor (EGFR) at the cell surface are tightly regulated by a complex endocytic machinery. Following internalization, EGFR is either recycled back to the cell surface or transported to the late endosome/lysosome for degradation. Currently, the molecular machinery that regulates this sorting pathway is only partially defined. Eps15 (EGFR pathway substrate 15) is an endocytic adaptor protein that is well known to support clathrin-mediated internalization of EGFR at the plasma membrane. Using RT-PCR, we have identified a novel short form of Eps15 (Eps15S) from rat liver that lacks the 111 C-terminal amino acids present in the traditional Eps15 form. The goal of this study was to define the functional role of the novel Eps15S form in EGFR trafficking. Overexpression of a mutant form of Eps15S (Eps15S ΔEH2/EH3) did not block EGFR internalization but reduced its recycling to the cell surface. After knockdown of all Eps15 forms, re-expression of Eps15S significantly reduced EGFR degradation while promoting recycling back to the cell surface. In contrast, re-expression of Eps15 did not potentiate receptor recycling. Furthermore, overexpression of the mutant Eps15S substantially reduced cell proliferation, linking EGFR recycling to downstream mitogenic effects. Finally, we found that Eps15S is localized to the Rab11-positive recycling endosome that is disrupted in cells expressing the Eps15S mutant, leading to an accumulation of the EGFR in early endosomes. These findings suggest that distinct forms of Eps15 direct EGFR to either the late endosome/lysosome for degradation (Eps15) or to the recycling endosome for transit back to the cell surface (Eps15S).     

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.     

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.     

Granulocyte-macrophage colony-stimulating factor is a major macrophage fusion factor present in conditioned medium of concanavalin A-stimulated spleen cell cultures

In 1983, we reported that the conditioned medium (CM) of spleen cell cultures treated with Con A greatly induced fusion of mouse alveolar macrophages within 2 to 3 days at a very high rate of more than 80% (Proc. Natl. Acad. Sci. USA 80:5583, 1983). In the course of examining macrophage fusion factors (MFF) present in Con A-CM, we found that IL-4 induced fusion of alveolar macrophages with a time course similar to that induced by Con A-CM. However, the maximal fusion rate induced by IL-4 (4 ng/ml) was about 35%. Furthermore, the fusion induced by Con A-CM was blocked only partially by adding IL-4 antibody, indicating that there are unknown MFF other than in Con A-CM. Of several other cytokines produced by Con A-stimulated spleen cells, IL-6 (20 ng/ml), IFN-gamma (45 ng/ml) and granulocyte-macrophage (GM)-CSF (10 ng/ml) greatly potentiated the fusion induced by 4 ng/ml of IL-4. The assay of these cytokines in Con A-CM proved that it contained 0.44 +/- 0.04 ng/ml of IL-4, 1.0 +/- 0.24 ng/ml of IL-6, 9.1 +/- 0.07 ng/ml of IFN-gamma, and 11.6 +/- 1.66 ng/ml of GM-CSF. When the potentiating effects of IL-6, IFN-gamma and GM-CSF on macrophage fusion were examined in the presence of 0.4 ng/ml of IL-4, only GM-CSF increased the fusion rate to the maximal level induced by Con A-CM at its physiologic concentration (10 ng/ml). The macrophage fusion induced by Con A-CM was greatly suppressed by adding antibody against GM-CSF. GM-CSF had a biphasic effect on growth and fusion, depending on its dose levels used: 0.01 to 0.1 ng/ml increased proliferation without inducing fusion and 10 ng/ml preferentially induced fusion. There was a negative relationship between macrophage growth and fusion. IL-4 was a potent inhibitor of proliferation of macrophages induced by GM-CSF. These results clearly indicate that GM-CSF is a major MFF present in Con A-CM.