Macrophage Colony-stimulating Factor Protein Research Articles

PRODUCTION OF MACROPHAGE COLONY-STIMULATING FACTOR BY MURINE LIVER IN VIVO

In previous reports, the authors demonstrated that M-CSF was produced by primary-cultured non-parenchymal (NPLC) and parenchymal (PLC) liver cells. In order to clarify the biological role of M-CSF produced by the liver, macrophage colony-stimulating factor (M-CSF)-producing cells in vivo were investigated using reverse transcriptase polymerase chain reaction (RT-PCR) dot blot analysis, in situ hybridization and immunohistochemistry, M-CSF mRNA was constantly identified by RT-PCR in the liver, NPLC and PLC, before and after partial hepatectomy. Dot blot analysis showed that fluctuations of M-CSF mRNA level after partial hepatectomy were not statistically significant. In situ hybridization revealed that M-CSF mRNA was expressed mainly in NPLC and vascular endothelial cells (VEC). In addition, a small number of PLC also expressed M-CSF mRNA. Neither the distribution nor the frequency of M-CSF mRNA positive cells in regenerative livers differed significantly from normal livers. M-CSF immunoreactivity was present in NPLC and VEC at all the times before and after partial hepatectomy, while PLC exhibited M-CSF immunostaining 0.5 days after partial hepatectomy. As normal liver expressed M-CSF mRNA to the same degree as regenerative liver, hepatic M-CSF mRNA production in vivo may be related to the physiological function of the liver. However, transient expression of M-CSF protein in PLC at an early state after patrial hepatectomy may be associated with liver regeneration.

Serum levels of cytokines in patients with untreated primary lung cancer: Katsumata N. Eguchi K. Fukuda M. Yamamoto N. Ohe Y. Oshita F. et al. Department Medical Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104. Clin Canc Res 1996;2:553–559

To evaluate the relationships between serum endogenous cytokine levels and their clinical implications in cancer patients, we measured the serum levels of endogenous granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin 6 (IL-6) in patients with untreated primary lung cancer. The serum G-CSF level was measured using a chemiluminescent ELISA, and the other cytokine levels were measured using ELISA. Fifty healthy adults and 183 patients with primary lung cancer were studied. The mean M-CSF level in the lung cancer patients (1106.4 units/ml) was significantly higher than that in the healthy adults (836 units/ml, P = 0.0001). In patients with large cell carcinoma, endogenous G-CSF, M-CSF, and IL-6 levels were significantly higher than those in patients with carcinomas of other cell types (P < 0.05). Univariate analysis showed that survival of 159 non-small cell lung cancer patients with high (more than cutoff level) G-CSF, M-CSF, and IL-6 levels was significantly poorer than that of patients with low levels (Wilcoxon's test, P = 0.018, P < 0. 0001, and P < 0.0001, respectively). Survival of patients with high levels of two or more cytokines was poorer than that of those with high levels of one cytokine or normal cytokine levels (P < 0.0001). Multivariate analysis using Cox's proportional hazards model showed that high M-CSF and C-reactive protein levels correlated significantly with poor survival (P = 0.037 and 0.037, respectively). Our preliminary data suggest that high M-CSF levels in non-small cell lung cancer may be of poor prognostic value.

Low-density lipoprotein stimulates the expression of macrophage colony-stimulating factor in glomerular mesangial cells

Disordered lipoprotein metabolism and the enhanced influx and accumulation of circulating mononuclear leukocytes into vascular tissue are common pathobiological phenomena associated with both atherosclerosis and glomerulosclerosis. Since atherogenic lipoproteins (such as low density lipoprotein, LDL) have been implicated in monocyte migration and proliferation into the glomerular mesangium, we examined the effect of LDL on mesangial cell expression of macrophage colony-stimulating factor (M-CSF), a cytoregulatory peptide associated with monocyte chemoattraction, differentiation and proliferation. Mesangial cell M-CSF gene expression, protein synthesis and secretion, and its biological activity to induce progenitor colony formation and monocyte proliferation were studied in murine mesangial cells. Incubation of either primary cultures or SV-40 transformed murine mesangial cells with LDL (0 to 200 micrograms/ml) induced M-CSF steady-state mRNA expression, in a dose-dependent manner (52 to 183% of control) when Northern blots were analyzed quantitatively by densitometric scanning. Similarly, Western blot analysis showed that LDL-activated SV-40 transformed mesangial cells increased M-CSF protein synthesis and secretion in a dose-dependent manner. The conditioned media obtained by incubating mesangial cells with LDL induced bone marrow progenitor colony formation that could be inhibited by specific neutralizing antibodies against murine M-CSF. Finally, the biological activity of M-CSF secreted by LDL-activated mesangial cells was further confirmed by its enhanced ability to induce monocyte proliferation. These data indicate that LDL, by activating mesangial cells to induce M-CSF and possibly other monocyte chemoattractants, may regulate the migration and proliferation of cells of mononuclear leukocytic origin into the mesangium supporting a pathobiological role for LDL in glomerular injury.

Decreased Macrophage Colony-Stimulating Factor mRNA Expression From Activated Cord Versus Adult Mononuclear Cells: Altered Posttranscriptional Stability

We have previously shown that protein production and mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and interleukin-3 are decreased in stimulated mononuclear cells (MNCs) from human umbilical cord compared with adult peripheral blood. These deficiencies may contribute to the increased susceptibility of neonates to infection. Macrophage colony-stimulating factor (M-CSF) regulates the proliferation, differentiation, and functional activation of monocytes. In the present study, we compared the regulation of M-CSF gene expression and protein production from stimulated cord and adult MNCs. Upon adhesion to tissue culture flasks, both cord and adult MNCs constitutively expressed M-CSF mRNA. In response to both adhesion and recombinant human GM-CSF (rhGM-CSF) stimulation for 120 hours, radioimmunoassays and bioassays showed that cord MNCs produced twofold to threefold less M-CSF protein compared with adult MNCs. Northern blot analysis also showed a fourfold decrease in M-CSF mRNA expression in both unstimulated and GM-CSF-induced cord versus adult MNCs. M-CSF mRNA expression in both cord and adult MNCs peaked between 16 and 24 hours and decreased to normal levels by 48 hours. We next determined the relative rates of transcription of the M-CSF gene by nuclear run-on assays in both cord and adult MNCs. The basal level signal of the M-CSF gene was similar between cord and adult MNCs. The transcriptional rate after stimulation with rhGM-CSF appeared to increase to a similar extent in both cord and adult MNCs (130% +/- 10% v 150% +/- 15%, C v A, n = 3, mean +/- SD). The comparative stability of M-CSF mRNA from cord versus adult MNCs was next determined by actinomycin D decay studies. The half-life of M-CSF mRNA from stimulated adult MNCs was 70 +/- 7.0 minutes (n = 4) compared with 47 +/- 2.8 minutes (n = 3) from stimulated cord MNCs (mean +/- SD, P < .05). To further determine the involvement of labile protein factors in posttranscriptional regulation, cord and adult MNCs were incubated with cycloheximide (CHX; 10 micrograms/mL). There was a significant increase in the induction of M-CSF mRNA by CHX treatment in both cord and adult MNCs. The increase of M-CSF mRNA induction by CHX was 2.5 times higher in cord MNCs compared with that in adult MNCs. These results suggest that there are one or more labile proteins that regulate M-CSF transcript stability in both cord and adult MNCs.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytokines in the central nervous system: Expression of macrophage colony stimulating factor and its receptor during development

To investigate the potential role of cytokines in the development of the central nervous system, we analyzed the production of cytokine mRNA transcripts by S1-nuclease protection analysis in the brains of Swiss-Webster mice during fetal development and after birth. Cytokines studied were interleukin (IL)-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, macrophage-colony stimulating factor (M-CSF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Only mRNA transcripts for M-CSF were found to be produced constitutively in normal brain tissue. These transcripts were detected continuously from embryonic day (ED) 13 through adulthood. Transcripts encoding the M-CSF receptor (c-fms) were also detected at all of these time points. Despite identification of M-CSF transcripts in the brains of normal mice during development and M-CSF protein in cell cultures, neuropathological examination of the brains of op/op mice, a naturally occuring mouse mutant defective in the production of functional M-CSF, showed no cytoarchitectural abnormalities.

Quantitative analysis of the two macrophage colony-stimulating factor mRNA expressed in a human stromal cell line by reverse transcription-polymerase chain reaction (RT-PCR)

We established a quantitative analysis system for 4.0 kb and 1.6 kb macrophage colony-stimulating factor (M-CSF) mRNA, using reverse transcription-polymerase chain reaction. Using this system, we performed quantitative analysis of the two mRNAs expressed in the human stromal cell line, KM102, in the resting condition and when stimulated by various concentrations of interferon-γ (IFN-γ). The expression of 1.6 kb M-CSF mRNA was more efficiently stimulated by IFN-γ than that of 4.0 kb M-CSF mRNA. The alternative splicing of a single M-CSF gene has been shown to generate several M-CSF proteins with different localization; we believe that molecular analysis of the transcription products by this system is important to better understand the physiological significance of the different species of M-CSF derived from each mRNA.

Constitutive mRNA and protein production of macrophage colony-stimulating factor but not of other cytokines by synovial fibroblasts from rheumatoid arthritis and osteoarthritis patients.

This study analyses the mRNA and protein production and their regulation of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8 and IL-6 by synovial fibroblasts obtained from patients with RA and OA. M-CSF was found to be produced constitutively as opposed to other cytokines. Stimulation of the cells with IL-1 beta caused a marked increase of GM-CSF, IL-8, IL-6 and as well as of M-CSF mRNA levels. In parallel, a time-dependent increase of M-CSF, GM-CSF, IL-8 and IL-6 protein production was observed. Among the cytokine mRNAs examined only that of M-CSF exhibited a pronounced stability in unstimulated synovial fibroblasts, whereas the other cytokines displayed short mRNA half-lives of 1-2 h. Induction by IL-1 beta markedly prolonged IL-8, IL-6 and GM-CSF mRNA half-lives to > 8 h which indicates increased mRNA stability. These findings suggest that among the cytokines that are produced in the inflamed synovium M-CSF may be particularly important for sustaining long-term influx, activation and survival of mononuclear phagocytes. GM-CSF, IL-8 and IL-6, by contrast, may be more involved in more acute cellular responses.

Effects of gamma‐irradiation on the M‐CSF‐promoter linked to a chloramphenicol aminoacyl transferase reporter gene expressed in a clonal murine bone marrow stromal cell line

The effects of cytokines produced by bone marrow stromal cells on closely associated hematopoietic cells constitute a major component of the physiology of the hematopoietic microenvironment. A major cytokine produced by marrow stromal cells is macrophage colony-stimulating factor (M-CSF). To determine the effect of gamma-irradiation on the M-CSF promoter in bone marrow stromal cells, we selected a clonal cell line from the C3H/HeJ mouse marrow stromal cell line D2XRII and stably transfected a reporter construct containing the murine M-CSF-promoter linked to a chloramphenicol aminoacyl transferase (CAT) gene. CAT activity was measured at serial time points after gamma-irradiation in vitro to doses between 500 and 10,000 cGy at a dose rate of 116 cGy/min. D2XRII marrow stromal cells treated with phorbol myristate acetate (40 micrograms/ml, four h), demonstrated a significant two-fold increase in CAT activity. In contrast, CAT activity measured immediately, 24 h, 72 h or 1 week after gamma-irradiation, showed no significant increase or decrease in CAT activity. An increase in CAT activity was detected 48 h after irradiation with cells that received 5,000 cGy. Thus, single fraction gamma-irradiation of plateau phase bone marrow stromal cells did not decrease M-CSF-promoter activity. These results are consistent with prior experimental data demonstrating stable levels of release of M-CSF protein following gamma-irradiation of bone marrow stromal cells and imply that the stability of transcription of the gene for this important cytokine is protected from irradiation.

Production of macrophage colony-stimulating factor (M-CSF) by human monocytes is differentially regulated by GM-CSF, TNFα, and IFN-γ

Macrophage colony-stimulating factor (M-CSF) stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. In this study, the qualitative and relative quantitative ability of various cytokines to induce and to synergize in M-CSF production by monocytes was studied. GM-CSF and the phorbolester PMA were strong inducers of M-CSF m-RNA expression. This was correlated closely with the secretion of M-CSF protein as measured in the murine M-NFS-60 cell line bioassay. Both TNFα and IFN-γ enhanced M-CSF message levels induced by GM-CSF, but only TNFα synergized with GM-CSF in the induction of M-CSF protein secretion. M-CSF transcripts induced by TNFα and IFN-γ were much lower compared to those induced by GM-CSF and PMA and were not accompanied by the secretion of M-CSF protein. In addition, costimulation of cells with TNFa and IFN-γ did not result in M-CSF production. Although M-CSF did not induce its own message, it further enhanced M-CSF transcripts induced by GM-CSF. LPS also failed to induce M-CSF message or secretion. These results show that cytokines differ in their ability to induce or to synergize in the induction of biologically active M-CSF protein. They further demonstrate that M-CSF message expression, induced by cytokines, does not always correlate with M-CSF protein secretion.

Modulation of macrophage colony stimulating factor in cultured human retinal pigment epithelial cells

Steady-state mRNA expression and protein production of macrophage colony stimulating factor were measured in visually confluent monolayers of unstimulated cultured human retinal pigment epithelial cells and after cells were stimulated with recombinant cytokines. Using reverse transcription polymerase chain reaction, macrophage colony stimulating factor mRNA expression was detected in unstimulated cells obtained from each of four separate donors. In these cells, mRNA expression was accompanied by secretion of macrophage colony stimulating factor protein into cell-conditioned medium; 48 hr after cells were switched to fresh medium, the mean (± s.d.) quantity of macrophage colony stimulating factor, measured by enzyme-linked immunoassay, was 5·1±2·3 ng 10 −6 cells. There was a dose- and time-dependent induction of macrophage colony stimulating factor mRNA after cells were exposed to recombinant human interleukin-1 and tumor necrosis factor α. Maximal mRNA induction was observed in cells exposed for 4 hr to interleukin-1β (5U ml −1) or for 4–8 hr to tumor necrosis factor α; under these conditions, macrophage colony stimulating factor mRNA was induced up to 23- and 46-fold after exposure to interleukin-1β and tumor necrosis factor α, respectively. Similarly, macrophage colony stimulating factor protein production was enhanced after cells were exposed to recombinant cytokines. Protein secretion increased 1·3–2·5-fold ( P < 0·001) after exposure to interleukin-1β (5 U ml −1), and 1·2–1·6-fold after exposure to tumor necrosis factor α ( P < 0.03). This cytokine mediated induction of macrophage colony stimulating factor secretion was preceded by an increase in cell-associated macrophage colony stimulating factor measured in cell lysates. The macrophage colony stimulating factor secreted by retinal pigment epithelial cells was biologically active, as determined by a bone marrow stimulation bioassay. The average number of macrophage specific colonies produced (± s.d.) was 190±42 10 −5 bone marrow cells plated. In contrast, colonies were not produced with medium alone. The bioactivity was entirely due to macrophage colony stimulating factor activity, because colony growth was completely suppressed when the assay was conducted in the presence of neutralizing antibody to human macrophage colony stimulating factor. We conclude that human retinal pigment epithelial cells express macrophage colony stimulating factor mRNA, and synthesize and secrete biologically active protein under basal conditions; mRNA expression and protein production are modulated by the inflammatory cytokines interleukin-1β and tumor necrosis factor α.

Epidermal growth factor stimulates macrophage colony-stimulating factor (M-CSF) mRNA expression and M-CSF release in cultured murine stromal cells.

Macrophage colony-stimulating factor (M-CSF) released by stromal cells of the bone marrow microenvironment plays a crucial role in the growth and proliferation of mononuclear cells. Several peptide mitogens including interleukin-1, tumour necrosis factor, platelet-derived growth factor and fibroblast growth factor stimulate the release of M-CSF and may be important in mediating the haematopoietic response to inflammation. Epidermal growth factor (EGF), released from platelets during aggregation, is mitogenic for a variety of cell types and may cause the release of certain cytokines. In this study we used the TC-1 murine stromal cells which constitutively secrete M-CSF as a model to study the regulation of M-CSF in response to EGF. EGF markedly stimulated the steady state expression of M-CSF mRNA with a peak effect observed at 3 h. This was associated with the release of M-CSF protein as determined by radioimmunoassay. EGF also stimulated DNA synthesis in a concentration dependent manner. Although TC-1 cells express GM-CSF mRNA, this was not induced by EGF. These findings suggest that EGF is a key regulatory molecule for M-CSF and may indirectly effect haematopoiesis via the release of M-CSF from stromal cells.

Production of macrophage colony-stimulating factor by human lymphoblastoid cell lines

The in vitro production of macrophage colony-stimulating factor (M-CSF) was studied in 14 human lymphoblastoid cell lines and their lineages were ascertained by surface phenotype analysis. M-CSF gene transcripts were detected in a T-lymphocyte-derived cell line (CCRF-CEM) and 3 B-lymphoblastoid cell lines (IM-9, BALL-1, and CCRF-SB) by Northern-blot analysis. The secretion of M-CSF protein into the culture supernatant by each cell line was also studied using an enzyme-linked immunosorbent assay for M-CSF. The 4 cell lines which expressed the M-CSF gene secreted considerable amounts of M-CSF into their culture supernatants, while the 10 cell lines without M-CSF gene expression did not do so. The cell lines which constitutively produced M-CSF were then subjected to Southern-blot analysis of the M-CSF gene structure, and all 14 cell lines were examined for infection by the Epstein-Barr virus. Neither structural changes nor amplification of the M-CSF gene were detected, and Epstein-Barr virus infection was found to be not directly related to M-CSF production.

Interleukin‐4 regulates mRNA accumulation of macrophage‐colony stimulating factor by fibroblasts: synergism with interleukin‐1β

We demonstrate that macrophage-colony stimulating factor (M-CSF) is expressed in human fibroblasts at the mRNA and protein level. Following activation with both interleukin (IL)-1 beta and IL-4, fibroblasts synthesized M-CSF transcripts detectable by Northern blot analysis with peak expression occurring at 8 h and 12 h, respectively. Exposure of fibroblasts to both cytokines resulted in M-CSF protein release at 60 h of c. 500 U/ml (for IL-1 beta) and 1000 U/ml (for IL-4), relative to a control preparation of recombinant human M-CSF in a murine bone marrow colony assay. Both interleukins synergized to enhance M-CSF mRNA accumulation and their ability to induce M-CSF transcripts could be abolished by treatment with specific neutralizing antibodies. These observations provide support for the idea that fibroblasts may control monocyte/macrophage development and function, and that IL-1 beta and IL-4 are involved in the regulation of this process.