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Inhibition of day-12 spleen colony-forming units by a monoclonal antibody to the murine macrophage/monocyte colony-stimulating factor receptor.

Primitive hematopoietic stem cells differentiate into committed progenitors that are thought to selectively express hematopoietic growth factor receptor(s), thereby acquiring hematopoietic growth factor responsiveness. To assess whether hematopoietic stem cells express hematopoietic growth factor receptors, the progenitor activity of bone marrow (BM) fractions, isolated by expression of receptors for macrophage/monocyte colony-stimulating factor (M-CSF), were examined. Recovery of day-12 spleen colony-forming units (CFU-S) is diminished in both M-CSF receptor-positive (M-CSFR+) and M-CSFR- fractions, indicating antibody inhibition of day-12 CFU-S. Incubation of BM cells with antibody without fractionation inhibits 50% to 60% of day-12 CFU-S. This inhibition is specific (control antibodies have no effect) and reversible by removal of bound antibody at low pH. Incubating BM cells with control or antireceptor antibody does not affect day-8 CFU-S, which are predominantly erythroid. Treating sublethally irradiated mice with antibody inhibits endogenous day-12 CFU-S. These results indicate that some early progenitors express M-CSFRs, and blocking M-CSFRs inhibits the ability of these progenitors to form colonies, possibly because of inactivation caused by prolonged receptor blockade.

Animals↗

Relative roles of osteoclast colony-stimulating factor and macrophage colony-stimulating factor in the course of osteoclast development.

Although recent studies have shown that osteopetrotic (op/op) mice lack macrophage colony-stimulating factor (M-CSF or CSF-1), the precise role of M-CSF in the development of immature osteoclasts remains unknown. Using a recently discovered osteoclast-specific colony-stimulating factor (O-CSF) and in vitro long-term bone marrow culture systems, we investigated the ability of op/op and control marrow stromal cells to support the production of O-CSF-responsive clonogenic osteoclast progenitors (colony-forming unit-osteoclast [CFU-O]) from inoculated normal stem cells. Remarkably, op/op stromal cell cultures produced five times as many nonadherent cells as control cultures throughout the experimental period of 14 weeks; an average of 37% of these cells were nonviable compared with 8% in control cultures. Significantly higher numbers of CFU-O were found in op/op cultures than in control cultures; the CFU-O in op/op and control cultures were proliferating at a similar rate. Higher numbers of calcitonin receptor-bearing cells were found when harvested cells from op/op flasks were cultured with 1,25(OH)2D3. These studies clearly show that op/op marrow stromal cells can support the differentiation and proliferation of osteoclast progenitors from inoculated stem cells and provide the first experimental evidence that M-CSF is not essential for the early stages of osteoclast development. We hypothesize that while O-CSF supports proliferation of osteoclast progenitors, M-CSF plays a role in the later development and maturation of the progenitor as well as in the prevention of cell death.

Animals↗

Glomerular expression of macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in patients with various forms of glomerulonephritis.

Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are major mediators for the differentiation, proliferation, and activation of the macrophage. Recently, M-CSF was documented to play a pivotal role in the development of nephritis via macrophage activation in MRL-lpr mice. The macrophage is also considered to be an important component in the development of human glomerulonephritis. The expression and function of colony-stimulating factors (CSF) in the human kidney have not yet been defined. This study was undertaken to elucidate the various roles of CSF in the development of glomerulonephritis in humans. We examined the glomerular expression of M-CSF and GM-CSF in patients with various forms of glomerulonephritis and in normal subjects using immunohistochemical methods and nonradioisotopic in situ hybridization. The expression of CSF at both the protein and mRNA level in the glomeruli was compared with the degree of mesangial proliferation; the number of macrophages, Ki67-positive cells, and HLA-DR-positive cells; and the degree of alpha-smooth muscle actin-positive area in the glomerulus and clinical data. M-CSF and GM-CSF were expressed mainly on the mesangial cells in the glomerulus. Intraglomerular expressions of CSF at the protein level were increased in cases of IgA nephropathy and lupus nephritis. There was a positive correlation among the M-CSF protein expression and glomerular proliferation, macrophage infiltration, and the degree of proteinuria. M-CSF mRNA expression also was increased in the cases of IgA nephropathy and lupus nephritis. The number of Ki67-positive cells and HLA-DR-positive cells and alpha-smooth muscle actin-positive area in the glomerulus were increased in the cases with enhanced M-CSF expression. These results suggest that the glomerular secretion of M-CSF promotes macrophage infiltration into the glomerulus and activates resident and extraneous macrophages in the mesangial proliferative glomerulonephritis. M-CSF is considered to be a major mediator in the development of mesangial proliferative glomerulonephritis in humans.

Adult↗

Effect of granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor on prolactin and adrenocorticotropic hormone secretion in rats: dose- and time-response in vivo studies.

The in vivo effect of granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) on the plasma levels of prolactin (PRL) and adrenocorticotropic hormone (ACTH) in rats were studied. The administration of 10 micrograms/kg G-CSF at 45 min (p < 0.05) and 90 min (p < 0.01) or 10 micrograms/kg GM-CSF at 45 and 90 min (p < 0.01) stimulated the secretion of ACTH. Moreover, G-CSF administration only, in doses of 10 micrograms/kg at 45 min (p < 0.05) and 90 min (p < 0.01) augmented PRL secretion into the blood. These experiments suggest that the human colony stimulating factors (GM-CSF and G-CSF) activate the anterior pituitary gland in vivo to ACTH secretion, but only G-CSF positively influenced PRL release in rats.

Adrenocorticotropic Hormone↗

The effect of mouse lung granulocyte-macrophage colony-stimulating factor and other colony-stimulating activities on the proliferation and differentiation of murine bone marrow cells in long-term cultures.

The roles of colony-stimulating factors in long-term bone marrow cultures were studied and compared. After single additions of high concentrations of unpurified colony-stimulating activities to the cultures, rapid deterioration of the cultures was observed. This appears to result from contaminants in the stimulatory preparations. Cultures to which one purified colony-stimulating factor (csf) from endotoxin mouse lung-conditioned medium was added did not run down ten weeks after addition and were found to be the same as the controls. The deterioration of the cultures to which unpurified stimulators were added could not be accounted for by accelerated granulopoiesis leading to subsequent exhaustion of the cultures. The inability of purified CSF to affect the cellularity of the suspension cells did not result from instability or masking of the activity in the cultures, nor did CSF preferentially stimulate the cells within the adherent layer. The suspension cells responded to purified CSF after separation from the adherent cells. The data suggest that if CSFs are marrow stimulators, their effects in turn may be stringently regulated within the marrow.

Animals↗

Helper (OKT4) T cells from human T cell colonies produce potent colony-stimulating activity.

Confluent T cell colonies were grown by culturing blood mononuclear cells in double agar layers containing autologous plasma and phytohemagglutinin (PHA) for one week (37 degrees C, 5% CO2). The plates were then overlaid with serum-free alpha medium which was harvested after 24 h. This medium was demonstrated to have colony-stimulating activity (CSA) of greater potency than conventionally prepared PHA-leukocyte conditioned medium, which was prepared by incubating cells from the same donors. Removal of OKT4-positive cells using a monoclonal antibody and complement abolished CSA production by cells from T cell colonies while the removal of OKT8-positive cells had no effect.

Antibodies, Monoclonal↗

Granulocyte colony-stimulating factor induces neutrophils to secrete macrophage colony-stimulating factor.

In this work we provide evidence showing that granulocytes produce macrophage colony-stimulating factor (M-CSF) from the band cell stage and secrete this factor when induced to differentiate into polymorphonuclear cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF). Using an enriched population of myeloid band cells from murine bone marrow, we identified the presence of M-CSF with a chromophore-labelled monoclonal anti-M-CSF antibody. Using ELISA we detected the secretion of M-CSF in the supernatants of cultures of enriched band cells when induced with rhG-CSF to differentiate into mature neutrophils. We also found that M-CSF is the only factor responsible for the colony forming activity in the supernatants and lysates of band cells treated with rhG-CSF.

Animals↗

Colony-forming cells and colony-stimulating activity during listeriosis in genetically resistant or susceptible mice.

Serum colony-forming activity (CSA) and colony-forming cells (CFC) of resistant (C57BL/10 ScSn) and susceptible (BALB/cJ) mice were studied during Listeria monocytogenes infection. Key findings were also checked in susceptible CBA/H mice. Prompt, bacterial dose-dependent increases in serum CSA were observed in all mice following infection. In response to the same challenge dose, serum CSA increased more in susceptible mice, possibly because rapid bacterial proliferation lead to high bacterial numbers. Thus CSA is not a limiting factor which accounts for the differences in Listeria resistance, but is produced in response to bacterial load. In uninfected mice, there were higher numbers of colony-forming cells in the bone marrow and spleen of resistant mice than in susceptible mice. By 24 hr postinfection there was a sharp drop in total cell numbers including CFC, in the bone marrow of resistant C57BL/10 ScSn mice. This coincides with the time when monocytes have been first observed in the blood of infected mice and when differences in bacterial growth between the mouse strains were first observable. Since the superior resistance of C57BL/10 mice has been shown to be radiosensitive, it is probable that this larger, readily mobilized reserve of monocyte/granulocyte precursors in the resistant mice plays an important role in early control of infection. The significance of this is discussed.

Animals↗

Effective object recognition for automated counting of colonies in Petri dishes (automated colony counting).

Determination of the number of colonies (colony forming units, CFU) is a standard method in microbiological analysis to ensure the quality of drinking water. Normally this tedious work is still performed manually. A PC-based method for the automated counting of digitized images of Petri dishes is presented. The method includes highly specific and effective object recognition algorithms that ensure very high detection accuracy. The processing sequence implies internal controls therefore enabling reliable automated evaluations of series of images. Use of the Fuzzy formalism and the high adaptivity of the algorithms lead to an extraordinary user-friendliness. For digitization different devices like flatbed scanners or CCD-cameras can be used. Due to the highly adaptive algorithms samples from the routine standard preparation process in laboratories can be evaluated. The accuracy and quality of the method aim at advancement in objectivity of colony counting and quality control and assurance. The algorithms and the evaluation of the method are presented.

Algorithms↗

Granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells from children contain the same levels of long-term culture-initiating cells producing the same numbers of colony-forming cells as those from adults, but display greater in vitro monocyte/macrophage potential.

Autologous peripheral blood progenitor cell (PBPC) transplantation is now commonly used in children. The ontogenic differences in haematopoiesis published in recent years suggest differences in the categories of mobilized PBPCs between children and adults. We investigated the frequency and distribution of mature progenitor cells (colony-forming cells, CFCs) and primitive progenitor cells [CD34+ CD38- and CD34+ Thy-1+ cells, long-term culture-initiating cells (LTC-ICs)] in children and adults mobilized using granulocyte colony-stimulating factor alone. We found similar proportions of granulocyte colony-forming units (CFU-G) and/or macrophage CFUs (CFU-M), mixed lineage CFUs (CFU-Mix) and megakarocyte CFUs (CFU-Mk), CD34+ CD38- and CD34+ Thy-1+ cells, and LTC-ICs (16.5 +/- 3.5 vs. 10.65 +/- 5 per 104 CD34+ cells), which produced the same number of CFCs (5 +/- 1 vs. 6 +/- 1 CFCs/LTC-ICs) in PB CD34+ cells from children and adults. However, we noted a higher proportion of erythroid blast-forming units (BFU-E) in PB CD34+ cells from adults (x 1.5, P = 0.003). Using cord blood as a third ageing point, we observed an inverse age-related propensity for commitment to the monocyte/macrophage lineage that was still found after normalizing the data per body weight and processed blood mass. This ontogeny-related programming was detected from the LTC-IC level, which produced 1.7 times more CFU-M in children than in adults (P = 0.048). These subtle differences in commitment between children and adults, shown here for the first time, are of interest for the in vitro manipulation of PBPCs and, in particular, for application in adoptive immunotherapy in children.

Adult↗

Prognostic value of colony-stimulating and colony-forming cells in peripheral blood in acute non-lymphoblastic leukemia.

Colony-forming cells (CFC) and colony-stimulating activity (CSA) in peripheral blood cells have been studied before and repeatedly during treatment of 30 patients with acute non-lymphoblastic leukemia. WBC obtained after Isopaque-dextran separation were cultured in vitro by a double-layer agar technique. Before treatment 16 patients out of 30 had CSA and 22 out of 29 had CFC; both CSA and CFC were found in 15 patients. In follow-up studies during treatment, CSA was mainly unaffected during the leukopenic phase, while CFC were suppressed. No CFC were found at WBC counts below 900/mm3. This seems to imply that CFC are more sensitive to cytotoxic agents than colony-stimulating cells. Twelve patients entered remission; all of them had CSA and all the 11 who were investigated for CFC had CFC before treatment. Fourteen out of 18 non-responders lacked one or both types of cells. The presence of CSA and CFC in peripheral blood therefore appears to be a sign of favorable prognosis, while the absence of CSA and/or CFC implies lack of response to treatment.

Adult↗

Mixed haematopoietic colony formation via immature blast cell clusters on foetal mesenchymal cell layers distinguishes stem cells from peripheral blood, cord blood, bone marrow and blood stem cells mobilized by granulocyte-macrophage colony-stimulating factor.

Multilineage colony formation was evaluated from healthy donors' bone marrow (BM), peripheral blood (PB) and cord blood (CB) and compared with blood stem cell (BSC) harvests of sarcoma patients mobilized with granulocyte-macrophage colony-stimulating factor (GM-CSF). The test was a modified CFU-blast assay performed with and without an irradiated foetal mesenchymal cell layer (HFFF). These non-transformed mesenchymal cells served as a good source of haematopoietically active stroma cells in that cytokine expression patterns (interleukin (IL)-6, granulocyte (G)-CSF, GM-CSF) and adhesion molecules on HFFF cells were qualitatively identical to BM-derived fibroblasts, but the expression density of adhesion receptors was significantly higher. This HFFF layer stimulated blood stem cells of GM-CSF-treated patients significantly more than a cocktail of exogenous growth factors with IL-1, IL-6, and stem cell factor (SCF). The reverse was true for multilineage colonies from healthy donors' PB, BM, and CB. According to these results, stem cells of GM-CSF-treated patients are functionally distinct due to their dependence on stroma-derived factors and/or matrix-adhesion interactions and can be reproducibly evaluated on these mesenchymal cells.

Blood Cells↗

Colony-stimulating and colony-forming cells in peripheral blood as prognostic markers in acute myelogenous leukemia.

Colony-forming cells (CFC) and colony-stimulating activity (CSA) in peripheral blood cells have been studied before and repeatedly during treatment of 32 patients with acute myelogenous leukemia. WBC obtained after Isopaque-dextran separation were cultured in vitro by a double-layer agar technique. Before treatment 19 patients out of 32 had CSA and all had CFC; both CSA and CFC were found in 19 patients. In follow-up studies during treatment, CSA was mainly unaffected during the leukopenic phase, while CFC were suppressed. No CFC were found at WBC counts below 1,000/mm3. This seems to imply that CFC are more sensitive to chemotherapy than colony-stimulating cells. 14 patients entered remission; all of them had CSA. 16 out of the 18 nonresponders lacked on or both types of cells. The presence of CSA and CFC in peripheral blood therefore appears to be a sign of favorable prognosis, while the absence of CSA and/or CFC implies lack of response to treatment. The conclusion from this study is that the presence of both CFC and CSA in peripheral WBC is a sign of a good prognosis. Absence of CFC and/or CSA in the initial sample indicates that the patient is unlikely to respond to conventional therapy with cytotoxic drugs. The presence or absence of CSA alone has a higher prognostic significance than the presence or absence of CFC.

Adolescent↗

Macrophage colony development: properties of colony stimulating factors from murine embryo and pregnant uterus.

Extracts from embryonic and uterine tissue of mice, operationally defined as colony stimulating factor (CSF), promoted the growth of macrophage-granulocyte colonies in vitro. Uterine CSF focusses from pH 5.15 to 6.00 and embryonic CSF from pH 3.60 to 5.20, although both forms have similar biological activity. CSF is relatively resistant to denaturation but it is inactivated by periodate and dithiothreitol. Gel filtration indicates a molecular weight of 45,000 which is unchanged following treatment with insolubilized trypsin, a procedure which affords a useful purification (240-fold). Trypsin-sensitive material in CSF preparations modifies colonial form under certain conditions of culture, probably by increasing the motility of macrophages. Diaminoethane derivatives of CSF were prepared and retained biological activity at isoelectric points above pH 9.0. These derivatives may be covalently linked to Sepharose providing an insolubilized form of CSF to study interactions of CSF with the cell surface.

Animals↗

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor.

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.

Biological Products↗

High serum colony-stimulating activity of leukocytopenic patients after intravenous infusions of human urinary colony-stimulating factor.

Eight adult patients suffering from leukocytopenia have been injected by droplet intravenous infusions with partially purified human urinary colony-stimulating factor (CSFHU), which stimulated human monocytes to produce colony-stimulating activity (CSA) in vitro. Three of eight patients were injected with low-dose CSFHU and five with high-dose CSFHU. The infusion of high-dose CSFHU led to a slightly earlier rise in absolute neutrophil counts and a higher CSA level in the serum, as compared to noninjected leukocytopenic patients. There was little toxicity associated with it. Human urinary colony-stimulating factor used in the clinical studies did not have any CSA in vitro in the presence or absence of the patients' sera. These data may suggest that the intravenous infusion of CSFHU increased the serum CSA level, probably through the stimulation of CSA-producing cells in vivo.

Clinical Trials as Topic↗

Colony assay using collagen gel: a better technique for studying the cell morphology of colonies.

Collagen gel was used as a substance for the assay of human granulocyte-macrophage colony-forming units (CFUc). The number of colonies formed in collagen gel was consistently higher than that in a conventional soft-agar system. In six out of eight experiments, the difference was statistically significant. Whole permanent preparations of the plates were made easily and were stained by Giemsa solution with minimum background staining. Preservation of cellular morphology in the preparations was excellent, and neutrophil, macrophage, and eosinophil colonies could be differentiated by their nuclear shape, cytoplasmic color, and specific granules.

Agar↗

Cyclic hematopoiesis: effects of lithium on colony-forming cells and colony-stimulating activity in grey collie dogs.

The cycling of blood cell counts in grey collie dogs with cyclic hematopoiesis can be eliminated by treatment with oral lithium carbonate. To explore the mechanism by which lithium alters this stem cell disorder, studies of bone marrow granulocyte-macrophage progenitor cells (CFU-C), neutrophil colony-forming cells (neutrophilic CFU-C), and colony-stimulating activity (CSA) were performed. In untreated dogs, the proportions of CFU-C were found to fluctuate cyclically, but the cyclic fluctuations in neutrophil colony-forming cells were even more marked, with numbers decreasing to undetectable levels during each period of neutrophilia. Dogs on lithium, however, did not cycle the numbers of total or neutrophilic CFU-C. Tritiated thymidine suicide rates were not altered by treatment with lithium. Serum CSA levels and bone marrow cell elaboration of CSA were not increased by lithium. These studies suggest that lithium corrects cyclic neutropenia by a direct effect on the differentiation and proliferation of CFU-C; normalization of the proportion of CFU-C that enter neutrophilopoiesis appears to be an important effect of the lithium therapy.

Animals↗