Granulocyte/macrophage colony-stimulating factor stimulates monocyte and tissue macrophage proliferation and enhances their responsiveness to macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a specific humoral growth factor that stimulates both neutrophilic granulocyte and macrophage production by bone marrow hematopoietic progenitor cells. GM- CSF also stimulates the proliferation and clonal growth of both tissue macrophages and blood monocytes. Although at low concentrations GM-CSF was unable to support the long-term growth of tissue macrophages, it greatly enhanced their responsiveness to macrophage CSF (M-CSF, or CSF- 1). This effect was also observed by treating macrophages with GM-CSF for a short time. GM-CSF did not compete with M-CSF for binding to M- CSF receptors nor was it inactivated by treatment with anti-M-CSF antiserum. Treatment of tissue macrophages with GM-CSF led to a rapid but transient downregulation of M-CSF receptors; prolonged incubation at 37 degrees C, however, resulted in a restoration and upregulation of M-CSF receptors. Identical effects were observed with both native or recombinant GM-CSF. This study suggests that GM-CSF regulates tissue macrophage production by two modes of action: (a) direct stimulation of macrophage proliferation, and (b) enhancement of their responsiveness to M-CSF.     

Human macrophage colony-stimulating factor induces macrophage colonies after L-phenylalanine methylester treatment of human marrow

Macrophage-colony stimulating factor (M-CSF) has well-known effects on murine bone marrow, but its colony stimulating activity for human bone marrow is controversial. After treatment of human bone marrow with L-phenylalanine methylester (PME), macrophage-colonies (CFU-M) were induced by M-CSF in a dose-dependent fashion. The optimal concentration of recombinant human-macrophage colony stimulating factor (rhM-CSF) was 1,000 U/mL. Purified human urine M-CSF had colony stimulating activity similar to rhM-CSF. Further studies were performed to determine the factors responsible for the enhanced CFU-M formation from PME treated marrow. Compared with nylon wool and carbonyl iron monocyte depletion methods, PME eliminated significantly more monocytes and myeloid cells. This observation suggested that these cells may release hematopoietic inhibitory factors for CFU-M. Low concentrations (1%) but not normal (10%) concentrations of blood monocytes were inhibitory (mean inhibition, 48%) to CFU-M. High concentrations of monocytes (50%) augmented CFU-M colonies. HL-60 conditioned media was used to simulate secretory products of early myeloid cells. HL-60 conditioned media (1%) inhibited CFU-M formation but not granulocyte macrophage or granulocyte colonies. We conclude that M-CSF has colony stimulating activity for human marrow that can be recognized after removal of inhibitory cells by PME treatment.     

Circulating macrophage colony-stimulating factor is not reduced in malignant osteopetrosis.

Malignant osteopetrosis is a disorder characterized by a deficiency in osteoclast number or function. In one animal model of osteopetrosis, the op/op mouse, macrophage colony-stimulating factor (M-CSF) is absent, and the administration of M-CSF corrects the defects. We evaluated the serum of 13 patients with malignant osteopetrosis by an M-CSF radioimmunoassay to determine if a quantitative M-CSF deficiency existed in these patients. All patients had M-CSF present in levels equal to or higher than control serum. In addition, serum from 6 osteopetrotic patients was tested in a bioassay to determine if the M-CSF present is biologically active, and in all cases there was demonstrable activity in these samples. We provide evidence that deficiency of circulating M-CSF is unlikely to be a major contributor to the etiologic basis for the majority of children with malignant osteopetrosis.     

The human macrophage colony-stimulating factor gene is localized at chromosome 1 band p21 and not at 5q33.1

This work was supported in part by a grant from the J. A. Cohen Institute for Radiopathology and Radiation Protection     

Human macrophage colony-stimulating factor levels are elevated in pregnancy and in immune thrombocytopenia.

Plasma macrophage colony-stimulating factor (M-CSF) levels were measured by enzyme immunosorbent assay (ELISA) using horse and rabbit polyvalent antibodies raised against human M-CSF purified from urine (hM-CSF). Plasma M-CSF levels in nonpregnant female controls were 364 +/- 69 U/mL (mean +/- SD, n = 20). Pregnancy results in significant elevation of circulating M-CSF levels (541 +/- 164 U/mL, n = 46, P < .0005). M-CSF levels were increased by 28 weeks' gestation and did not increase further in later pregnancy. M-CSF levels were also measured in 20 female controls before and after commencing on the oral contraceptive pill. There was no effect of the contraceptive pill on plasma M-CSF levels (364 +/- 69 U/mL before v 373 +/- 66 U/mL after commencing on the pill). In 28 nonpregnant patients with untreated immune thrombocytopenic purpura, (ITP), plasma M-CSF levels were significantly increased (797 +/- 402 U/mL, n = 28, v 364 +/- 69 U/mL in controls, N = 20, P < .0005). Pregnant ITP patients had higher levels of plasma M-CSF (929 +/- 327 U/mL, n = 25) than nonpregnant patients, but this difference was not significant. Elevated levels of M-CSF in ITP may reflect activation of the reticuloendothelial system (RES), which could result in positive feedback to increase the destruction of platelets. The increase in M-CSF associated with pregnancy could contribute to the exacerbation of latent ITP in pregnancy.     

Human granulocyte-macrophage colony-stimulating factor is a growth factor active on a variety of cell types of nonhemopoietic origin.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of a family of glycoprotein hormones that stimulate the proliferation and differentiation of hemopoietic cells in vitro and in vivo. We now report that human GM-CSF can also stimulate the proliferation of two osteogenic sarcoma cell lines, a breast carcinoma cell line, a simian virus 40-transformed marrow stromal cell line, and normal marrow fibroblast precursors. These findings suggest a more general regulatory function of GM-CSF on nonhemopoietic cell types than previously anticipated. They also raise the possibility of adverse side effects of GM-CSF therapy in patients whose malignant cells may be directly stimulated by this molecule and suggest a previously unanticipated role of GM-CSF gene activation in the evolution of solid tumors and in the pathogenesis of myelofibrosis.     

Dynamic modulation of the cell surface expression of the granulocyte-macrophage colony-stimulating factor receptor

Summary. The GM-CSF receptor (GM-CSFR) is composed of α and β subunits. Surface expression of the α chain alone leads to low affinity GM-CSF binding and of both subunits to high affinity binding: the β chain is required for transducing a proliferative signal. Studies of GM-CSFR expression have concentrated largely on static events occurring under conditions of binding equilibrium. We have examined the dynamic regulation of high and low affinity GM-CSFR expression in neutrophils (1100 ± 200 R/cell, K_D 50 ± 15 pm) and a GM-CSF dependent human leukaemic cell line, TF-l (2000 ± 450 R/cell K_D 15 ± 5 PM) and 8600 ± 150 R/cell K_D 1.8 ± 0.3 nm). The addition of GM-CSF to TF-1 cells (350 PM, 4 h at 37°C) caused a reduction in subsequent binding of ¹²⁵ I-GM-CSF at low ligand concentration (100 pm) (following a low pH wash to remove surface bound ligand) to 16 ± 4% and a reduction in binding at high ligand concentration (2 nm ¹²⁵I-GM-CSF) to 36 ± 9% of control. Scatchard analysis showed complete down-regulation of high affinity GM-CSFR and a significant reduction in low affinity GM-CSFR. In neutrophils, concentration-response curves of ligand induced receptor down-regulation at 37°C showed that observed down-modulation was more than 10-fold greater than predicted by static equilibrium binding data and correlated closely with GM-CSF priming of the neutrophil respiratory burst. The addition of IL-3 to TF-1 cells at 37°C reduced 100 PM ¹²⁵I-GM-CSF binding to 18 ± 4% and 2 nm ¹²⁵I-GM-CSF binding to 46 ± 5% of control. TF-1 cells, but not neutrophils, were able to re-express GM-CSFR following removal of GM-CSF from medium. TF-1 proliferation assays showed that pulsed GM-CSF (0.35–3.5 nm) for up to 4 h did not cause a significant increase in ³H-thymidine incorporation which required the continued presence of GM-CSF (control 2875 ± 208 cpm, pulsed GM-CSF 5 ng/ml 49 72 ± 1344. continuous GM-CSF 5 ng/ml 17249 ± 2982). Therefore, proliferation of TF-1 cells required the continued presence of GM-CSF at a time when there was no detectable surface high affinity GM-CSFR. This shows that signal transduction can take place via the GM-CSFR at a time when there is no detectable high affinity GM-CSF binding and introduces a further layer of complexity in the analysis of GM-CSF receptor function.     

Direct interaction of proteoglycan macrophage colony-stimulating factor and basic fibroblast growth factor

The proteoglycan form of macrophage colony-stimulating factor (PG-M- CSF), but not M-CSF with a molecular weight of 85 kD (85-kD M-CSF), bound to immobilized basic fibroblast growth factor (bFGF), and, conversely, bFGF bound to immobilized PG-M-CSF, but not to the 85-kD M- CSF. PG-M-CSF has an additional amino acid sequence at its carboxyl terminus (part of a precursor sequence that is removed in 85-kD M-CSF by proteolytic processing) and it has one or two chondroitin sulfate glycosaminoglycan chains at the carboxyl terminus. Enzymatic removal of the chondroitin sulfate chain from PG-M-CSF had no effect on the binding between PG-M-CSF and bFGF. Ligand blotting analysis with radioiodinated bFGF showed that bFGF specifically bound to the polypeptide that corresponded to the carboxyl terminus of PG-M-CSF and was produced in Escherichia coli transfected with its gene. The exogeneous addition of heparan sulfate, which has strong affinity for bFGF, efficiently inhibited the binding between PG-M-CSF and bFGF. These results show that PG-M-CSF binds bFGF through its carboxyl terminal peptide and that the binding sites for PG-M-CSF and heparan sulfate on bFGF are located close together. PG-M-CSF also significantly reduced the mitogenic action of bFGF on Balb/c 3T3 mouse fibroblastic cells. Therefore, we conclude that PG-M-CSF not only binds bFGF, but also neutralizes the activity of the growth factor.     

High blood levels of macrophage colony-stimulating factor in preeclampsia

In pregnancy, the decidual cells produce and secrete large amounts of macrophage colony-stimulating factor (M-CSF). M-CSF stimulates the proliferation and differentiation of trophoblasts. In addition, it stimulates them in a dose-dependent manner to produce certain hormones, such as human chorionic gonadotropin and human placental lactogen. Based on these facts, M-CSF is considered to be an essential cytokine for placental maintenance. Because placental dysfunction may sometimes result from preeclampsia, ascertaining blood M-CSF levels in preeclamptic patients would be of interest. The blood was collected from 33 subjects, of whom 19 were normal pregnant women and 14 were preeclamptic patients. The M-CSF level was determined by the sandwich enzyme-linked immunosorbent assay method using three antibodies. The investigators measured peripheral blood M-CSF levels in preeclamptic subjects and compared them with levels in subjects with normal pregnancies. This study showed that peripheral blood M-CSF levels were significantly higher in preeclamptic patients in the 30th and 38th weeks of pregnancy (P < .005). This is the first report concerning high M-CSF blood levels in preeclamptic patients.     

Increased macrophage colony-stimulating factor levels in immune thrombocytopenic purpura

Thrombocytopenia is a dose-limiting toxicity of macrophage colony- stimulating factor (M-CSF) in preclinical and initial phase I trials. Modulation of macrophage-mediated platelet destruction in immune thrombocytopenic purpura (ITP) may be affected by M-CSF activity. In this study, plasma levels of M-CSF were determined by a sensitive radioimmunoassay in 23 patients with ITP. These were compared with control levels measured in 24 healthy subjects. M-CSF levels were significantly higher in the ITP patients than in the control subjects (218 v 179, P < .02); however, there was a great deal of overlap. The highest M-CSF levels (median = 299 U/mL) were observed in three patients with Evan's syndrome. Patients with severe ITP (platelets < 25,000/microL) had intermediate M-CSF levels (median = 231 U/mL) and those with mild thrombocytopenia (> 25,000/microL) had normal levels (median = 173 U/mL). Sixteen patients were treated with corticosteroids: 10 responded and 6 did not. Median M-CSF levels were higher in those who failed to respond compared with responders (272 v 202, P < .05). These findings suggest M-CSF may influence macrophage- mediated platelet destruction in ITP.