Granulocyte-macrophage colony-stimulating factor is an endogenous regulator of cell proliferation in juvenile chronic myelogenous leukemia

Juvenile chronic myelogenous leukemia (JCML) is a rare myeloproliferative disorder of early childhood that is clinically and cytogenically distinct from the well-recognized adult type of chronic myeloid leukemia. Unlike the adult disease, growth of hematopoietic progenitors from peripheral blood (PB) occurs in the absence of exogenous stimulus even at low cell densities. This so-called "spontaneous" growth can be abrogated by adherent cell depletion and appears to depend on production of endogenous growth factors. We studied seven children with JCML to determine the nature of endogenous stimulators. With isolated PB mononuclear cells (PBMNCs) and a 3H- thymidine (3H-TdR) incorporation assay, JCML cells were shown to incorporate high levels of 3H-TdR when cultured in the absence of stimulus even at low cell densities. When neutralizing antisera prepared against each of the four known colony-stimulating factors (CSFs), GM-CSF, G-CSF, M-CSF, and interleukin-3 (IL-3), as well as antisera against interleukin-1 (alpha and beta) and tumor necrosis factor (TNF) were added to these cultures, only the antisera against recombinant human GM-CSF (rhGM-CSF) consistently resulted in significant inhibition of cell proliferation, achieving up to 72% inhibition of 3H-TdR incorporation in one case. Monoclonal antibodies (MoAbs) against rhGM-CSF resulted in a similar and highly significant degree of inhibition. A marked inhibitory effect of rhGM-CSF antiserum on "spontaneous" growth of PB CFU-GM derived colonies in semisolid medium was also demonstrated in four of five patients studied (87% to 90% inhibition). Production of growth factors by highly enriched JCML monocytes was variable. When initially studied in five of the seven patients, the monocytes from three of the patients revealed increased release of IL-1-like activities; two patients had levels similar to those of controls. One patient with normal levels when initially studied was later shown to have markedly increased amounts of IL-1-like activities in a second preparation of monocyte-conditioned medium (MCM). High levels of GM-CSF were detected in the initial MCM from one patient, but this may have indirectly reflected elevated IL-1-like activities present in the MCM. IL-3 and M-CSF levels were either low or undetectable in the patients studied as compared with MCM prepared with normal adult monocytes. These results clearly implicate GM-CSF as the primary endogenous regulator of JCML cell proliferation in culture and suggest that this malignant myeloproliferative disease may in part result from paracrine stimulation of marrow progenitor cells by growth factors/cytokines secreted by the malignant monocytes.     

Granulocyte-macrophage colony-stimulating factor synergizes with interleukin-6 in supporting the proliferation of human myeloma cells

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the growth of multiple myeloma (MM) was investigated in 21 patients with MM. In 17 patients with proliferating myeloma cells in vivo, recombinant GM-CSF significantly increased the endogenous-IL-6-mediated spontaneous myeloma cell proliferation occurring in 5-day cultures of tumor cells in vitro (P less than .01). Furthermore, GM-CSF was detected in 5-day culture supernatants of myeloma bone marrow cells. This endogenous GM-CSF was produced by the myeloma bone marrow microenvironment but not by myeloma cells and contributed to the spontaneous myeloma-cell proliferation observed in 5-day cultures. In fact, this proliferation was partially blocked (67%) by anti-GM-CSF monoclonal antibodies. The stimulatory effect of rGM-CSF was mediated through IL-6 because it was abrogated by anti-IL-6 monoclonal antibodies. rGM-CSF did not reproducibly increase the endogenous IL-6 production in short-term cultures of bone marrow cells of MM patients. Using an IL-6-dependent myeloma cell line (XG-1 cell line), rGM-CSF was shown to act directly on myeloma cells stimulating by twofold their IL-6 responsiveness. rGM-CSF did not induce any IL-6 production in XG-1 cells, nor was it able to sustain their growth alone. Although no detectable GM-CSF levels were found in the peripheral or bone marrow blood of MM patients, it is possible that GM-CSF, produced locally by the tumoral environment, enhances the IL-6 responsiveness of myeloma cells in vivo in a way similar to that reported here in vitro.     

粒细胞-巨噬细胞集落刺激因子在肺部疾病的研究进展

粒细胞-巨噬细胞集落刺激因子（granulocytemacrophage colony-stimulating factor, GM-CSF）是一种多功能造血因子,属于糖蛋白因子家族。主要由活化的T细胞、树突状细胞、巨噬细胞、角质细胞、内皮细胞和成纤维细胞等分泌,并通过自分泌和旁分泌方式作用于这些细胞,从而刺激粒细胞和巨噬细胞增殖、成熟和分化,具有极重要的免疫调节功能。现就GM—CSF的生理、生化特性及它在肺部疾病中的研究进展作阐述。     

Granulocyte-macrophage colony-stimulating factor augments neutrophil-mediated cartilage degradation and neutrophil adherence

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced in large quantities by synoviocytes in the inflamed arthritic joint and is known to be a neutrophil activator. Neutrophils predominate during acute flares of arthritis and are important mediators of cartilage destruction. In this investigation, we show that treatment of neutrophils with 10-1,000 units/ml of GM-CSF augments their ability to degrade cartilage proteoglycan in vitro. This was associated with increased neutrophil adherence to cartilage and increased release of oxygen-derived reactive species and granule enzymes in response to cartilage. Coating the cartilage with heat-aggregated human immunoglobulin G (AHG) enhanced both neutrophil adherence to the tissue and tissue degradation. GM-CSF, however, augmented these neutrophil effects independently of the presence of AHG. In contrast, neutrophil-mediated inhibition of proteoglycan synthesis was unaffected by GM-CSF.     

Granulocyte-macrophage colony-stimulating factor: signals for its mRNA accumulation

Granulocyte-monocyte colony-stimulating factor (GM-CSF) is an important hematopoietic growth factor. Mesenchymal cells produce abundant GM-CSF in response to tumor necrosis factor alpha (TNF). We wished to determine (1) what cellular pathways enhanced levels of GM-CSF mRNA, and (2) if TNF used any of these pathways. Modulation in levels of GM- CSF mRNA in human fibroblasts (WI-38) was studied by using Northern blot analysis. Markedly increased levels of GM-CSF mRNA occurred in these cells after exposure to sodium fluoride (NaF) and the effect of NaF was slightly enhanced by aluminum chloride; these results suggest that accumulation of GM-CSF mRNA can occur by activating a G-binding protein. Stimulators of protein kinase C dramatically increased levels of GM-CSF mRNA; however, blockade of protein kinase C activity did not attenuate accumulation of GM-CSF mRNA stimulated by TNF and NaF. Exposure to ouabain increased levels of GM-CSF mRNA and this effect was prominently enhanced in the presence of low concentrations of extracellular K+ and was almost abolished in high concentrations of extracellular K+. A monovalent ionophore (monensin) also increased levels of GM-CSF mRNA. Both ouabain and monensin can increase intracellular Ca++ concentration (Cai++) through Na+-Ca++ exchange. A calcium channel blocker (diltiazem) blocked the increased levels of GM- CSF mRNA mediated by ouabain, but could not block the stimulation mediated by TNF alpha. Ca++ ionophores also increased levels of GM-CSF mRNA and rapidly increased levels of Cai++. TNF did not increase Cai++ and, moreover, was able to stimulate accumulation of GM-CSF mRNA in the absence of extracellular Ca++. Taken together, we have found that several different cellular pathways can lead to prominent accumulation of GM-CSF mRNA in mesenchymal cells including (1) activation of protein kinase C, (2) increase in Cai++, and (3) stimulation of G-binding protein. Our studies show that TNF appears to increase levels of GM-CSF mRNA independent of protein kinase C activity or levels of Cai++.     

Granulocyte-macrophage colony-stimulating factor enhances selective effector functions of tissue-derived macrophages

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by a variety of cells at sites of exposure to antigens. GM-CSF has a stimulatory effect on a number of neutrophil functions, but the effect on macrophage function is less clear. We investigated the effect of purified murine recombinant GM-CSF on murine peritoneal macrophage oxidative metabolism, Fc-dependent phagocytosis, anti-Toxoplasma activity, and expression of class II major histocompatibility antigen (Iad). GM-CSF significantly increased phorbol myristate acetate- and zymosan-elicited H2O2 release by resident and thioglycollate-elicited macrophages after 48 hours in vitro. The effect of recombinant GM-CSF was blocked by polyclonal anti-GM-CSF antibody and was not altered by lipopolysaccharide (0.01 to 1.0 microgram/mL). GM-CSF also stimulated Fc-dependent phagocytosis by peritoneal macrophages, although the stimulation of resident macrophages (1.4-fold) was less dramatic than that of thioglycollate-elicited cells (2.1-fold). GM-CSF (at doses up to 100 U/mL) had no effect on macrophage anti-Toxoplasma activity or on expression of Iad. In addition to stimulating macrophage growth, GM-CSF selectively promotes the functional capacity of tissue-derived macrophages.     

Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria by human neutrophils

In order to determine whether human granulocyte-macrophage colony- stimulating factor (GM-CSF) can enhance phagocytosis, neutrophils were combined with Staphylococcus aureus (S aureus), and both the number of bacteria per neutrophil and the percent of neutrophils phagocytizing were assessed in the absence and presence of GM-CSF. Exposure to GM-CSF did not enable neutrophils to ingest unopsonized bacteria. When bacteria were opsonized with serum, both the number of bacteria per neutrophil and the percent of cells phagocytizing were increased by treatment with GM-CSF. Digestion of extracellular organisms by lysostaphin was used to substantiate phagocytosis. These results indicate that another effect of GM-CSF on the mature neutrophil is the enhancement of phagocytosis.     

Granulocyte-macrophage colony-stimulating factor in acute non-lymphocytic leukemia before and after chemotherapy

Summary The introduction of hematopoietic growth factors into the management of leukemia can influence the outcome of treatment in several ways, depending on the sensitivity and the response of normal and leukemic cells. In this paper we report on the effects of the administration ofEscherichia coli-produced, human recombinant granulocyte-macrophage colony-stimulating factor (GMCSF) in 15 adult patients with acute nonlymphocytic leukemia (ANLL) resistant to first-line treatment or in relapse. GM-CSF was given at a dose of 5–10 g/kg/day as a 6-h i.v. infusion, prior to chemotherapy (CHT) (for 7 days) and after CHT (until evidence of failure or of remission). In the pre-CHT period there was a clear trend towards an increase of circulating neutrophils (PMN) and/or blast cell count (median 0.3 vs. 1.0×10⁹/l for PMN, and 0.5 vs. 2.3 for blast cells). After chemotherapy, in the patients who achieved complete remission (CR), the median time to a PMN count > 0.5×10⁹/l and > 1×10⁹/l was 16 days (range 13–27) and 19 days (range 13–42) respectively. The outcome of treatment was CR for 8/15 (53%), death during induction for 3/15 (20%), and failure for 4/15 (27%). All failures occurred in patients with an increase of blast cell count during pre-CHT GM-CSF administration. Toxicity and side effects were minor, apart from an acute respiratory syndrome that developed twice in the same patient, at doses of 10 and 3 g/kg/day. These data suggest that investigation of GM-CSF in the treatment of ANLL is worth pursuing, with special attention to GM-CSF effects prior to chemotherapy.Work supported by MURST (grant 40%–60%) and by CNR     

Granulocyte-macrophage colony-stimulating factor: pleiotropic cytokine with potential clinical usefulness

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 23-kDa glycoprotein with remarkably diverse effects on immune and nonimmune cells. GM-CSF induces differentiation of granulocyte, macrophage, and eosinophil precursor cells. Proliferation of monocyte-macrophages, T lymphocytes, keratinocytes, and endothelial cells is also stimulated by GM-CSF. In addition, GM-CSF alters the functional properties of mature granulocytes, macrophages, eosinophils, and basophils. GM-CSF is produced by T lymphocytes, macrophages, and several cell types in extramedullary sites, where it may act in a paracrine manner to regulate the local response to antigenic challenge. Clinical trials of GM-CSF have been conducted in patients with AIDS, aplastic anemia, myelodysplastic syndromes, and sarcoma and following bone marrow transplantation and accidental radiation exposure. GM-CSF significantly increased circulating numbers of several myeloid cells and produced dose-dependent toxicity consisting primarily of myalgias, fever, fluid retention, and serosal effusions. Additional studies are needed to define the role of GM-CSF in treatment of patients with qualitative and quantitative dysfunction of immune cells.     

Granulocyte-macrophage colony-stimulating factor binding sites and oxidative metabolism in human granulocytes

Summary We investigated the interaction between GM-CSF and its receptor on human granulocytes and on several human tumor cell lines. Specific high-affinity binding for GM-CSF was characterized by Scatchard plot analysis. The specific radioactivity of the ¹²⁵I-labeled derivative of rH. GM-CSF was determined by self-displacement analysis and calculated to be 30 Ci/g. The maximum concentration of binding sites (B max) in granulocytes was 40 fmol/mg protein (2,200 molecules GM-CSF bound/cell) and the dissociation constant (K_D) was 0.42 nM. No binding sites for GM-CSF were found in two lung cancer cell lines, SCLC-16HV and NCI-N417 or in the urinary bladder carcinoma cell line 5637, whereas the promyelocytic leukemia cell line HL60 was positive for GM-CSF binding. Time course experiments showed maximum binding of GM-CSF in granulocytes after an incubation period of 60 min and a decrease in binding after an incubation period of 2 h. In parallel, we found a maximum biological signal when granulocytes were preincubated for 90 min with GM-CSF, and a decrease after an incubation time of 120 min. Preincubation of the cells with rH. GM-CSF induced an enhancement of the production of activated oxygen species by the cells in response to PMA.Abbreviations CSF colony stimulating factor - rH.GM-CSF recombinant human granulocyte-macrophage colony-stimulating factor - PMNs polymorphonuclears - ADCC antibody dependent cell mediated cytotoxicity - PMA phorbol myristate acetate - SCLC small cell lung cancer - DAG diacylglycerol - PIP ₂ phosphatidylinositol-biphosphate     

Granulocyte-macrophage colony-stimulating factor from cultured normal rat kidney cell line

Serum-free medium conditioned by the normal rat kidney (NRK) cell line contains colony-stimulating factors (CSF) that stimulate the in vitro formation of granulocyte and macrophage colonies from rat, mouse, and human marrow. There are two types of CSF: NRK-CSF I stimulates rat and mouse marrow, while NRK-CSF II stimulates the human marrow. The NRK-CSF I has been partially purified to a specific activity of 5 X 10(7) units/mg by employing methods such as preparative isoelectrofocusing, gel filtration chromatography, and preparative gel electrophoresis. It has an isoelectric point of 5.1 and an apparent molecular weight of 35,000 daltons as estimated by gel filtration. It is stable at 50 degrees C for 30 min and relatively resistant to papain, but highly sensitive to trypsin, chymotrypsin, and subtilisin. The secretion of CSF by NRK cells is inhibited by actinomycin D (0.5 micrograms/ml) and cycloheximide (0.5 micrograms/ml), but not by cytosine arabinoside (5 micrograms/ml). Although the CSF activity is inactivated by periodate oxidation (5 mM), thus suggesting its glycoprotein nature, treatment of the CSF with neuraminidase and other glycosidases has no effect on its activity. Anti-NRK-CSF I antibody inhibits the rat/mouse-active CSF from all rat sources, but has no effect on the human-active CSF. Comparative studies with CSF from media conditioned by the transformed cell line (442) and from rat lung and spleen have shown that CSF I from different rat sources share similar isoelectric points, molecular weights, and immunological properties.     

Granulocyte-macrophage colony-stimulating factor augments human monocyte fungicidal activity for Candida albicans

The ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to augment the fungicidal activity of human monocytes for Candida albicans was evaluated. Purified human monocytes cultured with [3H]leucine-labeled C. albicans caused a dose-dependent release of the [3H]leucine. The amount of [3H]leucine released correlated with a decrease in the number of viable yeast colonies. Monocyte cytotoxicity for C. albicans was reduced by superoxide dismutase and catalase and by inhibitors of myeloperoxidase and scavengers of hydroxyl radical and single oxygen, consistent with monocyte candidacidal activity being partly dependent upon products of oxidative metabolism. Monocytes incubated with rhGM-CSF produced more superoxide anion (O2-) spontaneously and after stimulation than control monocytes (P less than .05). Enhanced O2- production was dose-dependent and specific for rhGM-CSF and could be inhibited by antibody to rhGM-CSF. In association with rhGM-CSF-induced production of O2-, the cytokine enhanced cytotoxic activity for C. albicans. These findings indicate that rhGM-CSF stimulates human monocyte fungicidal activity for C. albicans.