scholarly journals A recombinant murine granulocyte/macrophage (GM) colony-stimulating factor derived from an inducer T cell line (IH5.5). Functional restriction to GM progenitor cells.

1986 ◽  
Vol 164 (4) ◽  
pp. 1102-1113 ◽  
Author(s):  
S Kajigaya ◽  
T Suda ◽  
J Suda ◽  
M Saito ◽  
Y Miura ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cell ◽  
Cell Line ◽  
Conditioned Medium ◽  
Fetal Liver ◽  
Plasmid Vector ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Blast Cells ◽  
Stimulating Factor

The cDNA for the murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was cloned from a cDNA library obtained from a murine T cell line, IH5.5, by using two synthetic probes that encoded two parts of the GM-CSF from murine lung. The cDNA inserted into the plasmid vector pcDV1 was transfected into monkey COS-1 cells and the conditioned medium was used to investigate the hemopoietic activities of the resultant product, recombinant GM-CSF (rGM-CSF), by means of various colony assays. rGM-CSF stimulated only neutrophil/macrophage colonies in the cultures of murine normal bone marrow and fetal liver cells. No other colony stimulating activities (CSA) were seen in the preparation including burst-promoting activity, eosinophil-CSA, megakaryocyte-CSA and mast cell-CSA. rGM-CSF could not support colony formation of 5-fluorouracil-treated mouse spleen cells, in which only the primitive population of stem cells survived. However, after culture of these cells with PWM-spleen cell-conditioned medium (PWM-SCM), the colonies consisting of blast cells were formed. These blast cells could now be induced to form neutrophil/macrophage colonies in the presence of rGM-CSF. Pure neutrophil colonies, pure macrophage colonies, as well as mixed neutrophil/macrophage colonies, were formed from these single blast cells in the presence of rGM-CSF by micromanipulation. rGM-CSF did not act on pluripotent hemopoietic stem cells, but did act directly and selectively on neutrophil/macrophage progenitors. Moreover, striking heterogeneities were noted in the size of the colonies and the proportion of components. GM-CSF is, therefore, considered to play a noninstructive role in the differentiation of the GM pathway.

scholarly journals A GM-colony-stimulating factor (CSF) activated ribonuclease system transregulates M-CSF receptor expression in the murine FDC-P1/MAC myeloid cell line.

1992 ◽  
Vol 3 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B C Gliniak ◽  
L S Park ◽  
L R Rohrschneider
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Mrna Degradation ◽  
Receptor Expression ◽  
Metabolic Inhibitors ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulating Factor

The murine myeloid precursor cell line FDC-P1/MAC simultaneously expresses receptors for multi-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF. Growth of FDC-P1/MAC cells in either multi-CSF or GM-CSF results in the posttranscriptional suppression of M-CSF receptor (c-fms proto-oncogene) expression. We use the term transregulation to describe this control of receptor expression and have further characterized this regulatory process. The removal of FDC-P1/MAC cells from GM-CSF stimulation resulted in the re-expression of c-fms mRNA independent of M-CSF stimulation and new protein synthesis. Switching FDC-P1/MAC cells from growth in M-CSF to GM-CSF caused the selective degradation of c-fms mRNA within 6 h after factor switching. Blocking protein synthesis or gene transcription with metabolic inhibitors effectively prevented GM-CSF stimulated degradation of c-fms mRNA. These results suggest that the transregulation of c-fms transcripts by GM-CSF requires the transcriptional activation of a selective mRNA degradation factor. In vitro analysis, the use of cytoplasmic cell extracts, provided evidence that a ribonuclease is preferentially active in GM-CSF stimulated cells, although the specificity for mRNA degradation in vitro is broader than seen in vivo. Together, these data suggest that GM-CSF can dominantly transregulate the level of c-fms transcript through the transcriptional activation of a ribonuclease degradation system.

scholarly journals NF-kappa B as inducible transcriptional activator of the granulocyte-macrophage colony-stimulating factor gene.

1990 ◽  
Vol 10 (3) ◽  
pp. 1281-1286 ◽  
Author(s):  
R Schreck ◽  
P A Baeuerle
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Interleukin 2 ◽  
Colony Stimulating Factor ◽  
Nf Kappa B ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The expression of the gene encoding the granulocyte-macrophage colony-stimulating factor (GM-CSF) is induced upon activation of T cells with phytohemagglutinin and active phorbolester and upon expression of tax1, a transactivating protein of the human T-cell leukemia virus type I. The same agents induce transcription from the interleukin-2 receptor alpha-chain and interleukin-2 genes, depending on promoter elements that bind the inducible transcription factor NF-kappa B (or an NF-kappa B-like factor). We therefore tested the possibility that the GM-CSF gene is also regulated by a cognate motif for the NF-kappa B transcription factor. A recent functional analysis by Miyatake et al. (S. Miyatake, M. Seiki, M. Yoshida, and K. Arai, Mol. Cell. Biol. 8:5581-5587, 1988) described a short promoter region in the GM-CSF gene that conferred strong inducibility by T-cell-activating signals and tax1, but no NF-kappa B-binding motifs were identified. Using electrophoretic mobility shift assays, we showed binding of purified human NF-kappa B and of the NF-kappa B activated in Jurkat T cells to an oligonucleotide comprising the GM-CSF promoter element responsible for mediating responsiveness to T-cell-activating signals and tax1. As shown by a methylation interference analysis and oligonucleotide competition experiments, purified NF-kappa B binds at positions -82 to -91 (GGGAACTACC) of the GM-CSF promoter sequence with an affinity similar to that with which it binds to the biologically functional kappa B motif in the beta interferon promoter (GGGAAATTCC). Two kappa B-like motifs at positions -98 to -108 of the GM-CSF promoter were also recognized but with much lower affinities. Our data provide strong evidence that the expression of the GM-CSF gene following T-cell activation is controlled by binding of the NF-kappa B transcription factor to a high-affinity binding site in the GM-CSF promoter.

scholarly journals Effects of recombinant GM-CSF on the blast cells of acute myeloblastic leukemia

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 313-316 ◽  
Author(s):  
T Hoang ◽  
N Nara ◽  
G Wong ◽  
S Clark ◽  
MD Minden ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Adherent Cells ◽  
Colony Stimulating Factor ◽  
Response Curves ◽  
Self Renewal ◽  
Gm Csf ◽  
Blast Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The effects of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) were compared to those of media conditioned by the continuous bladder carcinoma line, HTB9 (HTB9-CM), using three criteria. First, both GM-CSF and HTB9-CM stimulated blast colony formation in methylcellulose cultures, patient-to-patient variations were seen in the dose-response curves, and GM-CSF was effective, but less so that HTB9-CM. Second, GM-CSF also enhanced growth of blast progenitors in suspension culture, indicating its capacity to support self-renewal. GM-CSF was as effective as HTB9-CM in the production of adherent cells during the growth of blast cells in suspension, a finding that is interpreted to mean that GM-CSF also supports postdeterministic events in blast differentiation. Finally, colonies growing in the presence of GM-CSF were not phenotypically different than those stimulated by HTB9-CM.

scholarly journals Enhanced expression of the granulocyte-macrophage colony stimulating factor gene in acute myelocytic leukemia cells following in vitro blast cell enrichment

Blood ◽  
1988 ◽  
Vol 72 (4) ◽  
pp. 1329-1332 ◽  
Author(s):  
DC Kaufman ◽  
MR Baer ◽  
XZ Gao ◽  
ZQ Wang ◽  
HD Preisler
Keyword(s):  
T Cell ◽  
Leukemic Cells ◽  
Acute Myelocytic Leukemia ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Myelocytic Leukemia ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Expression of the granulocyte-macrophage colony-stimulating factor (GM- CSF) gene in acute myelocytic leukemia (AML) was assayed by Northern blot analysis. GM-CSF messenger RNA (mRNA) was detected in the freshly obtained mononuclear cells of only one of 48 cases of AML, in contrast with recent reports that GM-CSF mRNA might be detected in half of the cases of AML when RNA is prepared from T-cell- and monocyte-depleted leukemic cells. We did find, however, that expression of the GM-CSF gene was detectable in five of ten cases after in vitro T-cell and monocyte depletion steps. Additional studies suggest that expression of GM-CSF in the bone marrow of the one positive case, rather than being autonomous, was under exogenous control, possibly by a paracrine factor secreted by marrow stromal cells. These studies emphasize the potential for altering in vivo patterns of gene expression by in vitro cell manipulation.

scholarly journals Limiting-dilution analysis of the effects of colony-stimulating factors, phytohemagglutinin, and hydrocortisone on hematopoietic progenitor cell growth

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1611-1618
Author(s):  
Y Takaue ◽  
CL Reading ◽  
AJ Roome ◽  
KA Dicke ◽  
S Tindle ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cell Growth ◽  
Cell Line ◽  
Conditioned Medium ◽  
Hematopoietic Progenitor ◽  
Colony Stimulating Factors ◽  
Gm Csf ◽  
Percoll Gradients ◽  
Limiting Dilution

The effects of colony-stimulating factors (CSFs), phytohemagglutinin (PHA), and hydrocortisone on the growth of human bone marrow hematopoietic progenitor cells (granulocyte-macrophage; GM) were analyzed in a limiting-dilution assay (LDA). Both low-density bone marrow cells separated by discontinuous Percoll gradients and a T cell- depleted and progenitor-enriched cell fraction obtained by the combination of counterflow elutriation centrifugation and Percoll gradients were examined in LDA. GCT (monocytoid cell line-conditioned medium containing GM-CSF), human placenta-conditioned medium, bladder carcinoma cell line 5637-conditioned medium (containing GM- and G-CSF), and recombinant CSF (G-CSF) directly induced proliferation of progenitors with single-hit kinetics. In some instances, however, PHA- stimulated lymphocyte-conditioned medium (containing G- and GM-CSF) showed deviation from single-hit kinetics, which demonstrated the presence of factor(s) suppressive to progenitor growth. In a T cell- depleted, progenitor-enriched fraction, PHA alone was found to suppress progenitor growth at a level as low as 100 ng/mL. The addition of hydrocortisone (10(-6) mol/L) increased the progenitor frequency but suppressed progenitor growth at 10(-4) mol/L. LDA appears to be a valuable method for exploring mechanisms of factors regulating hematopoietic cell growth.

scholarly journals Growth and differentiation of a human megakaryoblastic cell line, CMK

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 42-48 ◽  
Author(s):  
N Komatsu ◽  
T Suda ◽  
M Moroi ◽  
N Tokuyama ◽  
Y Sakata ◽  
...  
Keyword(s):  
Cell Line ◽  
Colony Formation ◽  
Culture System ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Hematopoietic Factors ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Recently, a human megakaryoblastic cell line, CMK, was established from the peripheral blood of a megakaryoblastic leukemia patient with Down syndrome. Using this cell line, we studied the proliferation and differentiation of megakaryocytic cells in the presence of highly purified human hematopoietic factors and phorbol 12-myristate-13- acetate (PMA). In a methylcellulose culture system, interleukin-3 (IL- 3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitated colony formation by CMK cells in a dose-dependent manner. The maximum stimulating doses of these factors were 10 and 200 U/mL, respectively. These concentrations were comparable to those that stimulate activity in normal hematopoietic cells. In contrast, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), and erythropoietin (EPO) had no effects on the colony formation of CMK cells. In a liquid culture system, 20% of the CMK cells expressed glycoprotein IIb/IIIa (GPIIb/IIIa) antigen without hematopoietic factors, whereas 40% of the cells expressed GPIIb/IIIa with the addition of IL-3 and GM-CSF. EPO also slightly enhanced expression of GPIIb/IIIa. On the other hand, PMA inhibited growth of CMK cells and induced most of them to express the GPIIb/IIIa antigen. Furthermore, PMA induced CMK cells to produce growth activity toward new inocula of CMK cells. This growth factor (GF) contained colony-stimulating activity (CSA) in normal bone marrow (BM) cells. The activity was believed to be attributable mainly to GM-CSF, since 64% of this activity was neutralized by anti-GM-CSF antibodies and a transcript of GM-CSF was detected in mRNA from PMA-treated CMK cells by Northern blot analysis. These observations suggest that GM-CSF, as well as IL-3, should play an important role in megakaryocytopoiesis.

scholarly journals The immunosuppressant rapamycin blocks in vitro responses to hematopoietic cytokines and inhibits recovering but not steady-state hematopoiesis in vivo

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1543-1552 ◽  
Author(s):  
VF Quesniaux ◽  
S Wehrli ◽  
C Steiner ◽  
J Joergensen ◽  
HJ Schuurman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
T Cell ◽  
T Cell Response ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Hematopoietic Recovery ◽  
Stimulating Factor

Abstract The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.

scholarly journals Inhibition of purinergic P2X receptor 7 (P2X7R) decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in U251 glioblastoma cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthew Drill ◽  
Kim L. Powell ◽  
Liyen Katrina Kan ◽  
Nigel C. Jones ◽  
Terence J. O’Brien ◽  
...  
Keyword(s):  
Cell Line ◽  
P2x Receptor ◽  
Glioblastoma Cell Line ◽  
Glioblastoma Cell ◽  
Colony Stimulating Factor ◽  
U251 Cell ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12–15 months. The P2X receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma.

scholarly journals Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells.

1987 ◽  
Vol 166 (5) ◽  
pp. 1484-1498 ◽  
Author(s):  
M D Witmer-Pack ◽  
W Olivier ◽  
J Valinsky ◽  
G Schuler ◽  
R M Steinman
Keyword(s):  
Dendritic Cells ◽  
Conditioned Medium ◽  
Langerhans Cells ◽  
Cell Mediated Immunity ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Accessory Function ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

A panning method has been developed to enrich Langerhans cells (LC) from murine epidermis. In standard culture media, the enriched populations progressively lose viability over a 3-d interval. When the cultures are supplemented with keratinocyte-conditioned medium, LC viability is improved and the cells increase in size and number of dendritic processes. Accessory function, as monitored by stimulating activity in the mixed lymphocyte reaction (MLR), increases at least 10-20-fold. The conditioned media of stimulated macrophages and T cells also support the viability and maturation of cultured LC. A panel of purified cytokines has been tested, and only granulocyte/macrophage colony-stimulating factor (GM-CSF) substitutes for bulk-conditioned medium. The recombinant molecule exhibits half-maximal activity at 5 pM. Without activity are: IL-1-4; IFN-alpha/beta/gamma; cachectin/TNF; M- and G-CSF. A rabbit anti-GM-CSF specifically neutralizes the capacity of keratinocyte-conditioned medium to generate active LC. However, GM-CSF is not required for LC function during the MLR itself. We conclude that the development of immunologically active LC in culture is mediated by GM-CSF. The observation that these dendritic cells do not respond to lineage-specific G- and M-CSFs suggests that LC represent a distinct myeloid differentiation pathway. Because GM-CSF can be made by nonimmune cells and can mediate the production of active dendritic cells, this cytokine provides a T-independent mechanism for enhancing the sensitization phase of cell-mediated immunity.

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