Localisation of granulocyte macrophage colony-stimulating factor in human long-term bone marrow cultures. Biological and immunocytochemical characterisation

1993 ◽  
Vol 106 (3) ◽  
pp. 761-769
Author(s):  
E. de Wynter ◽  
T. Allen ◽  
L. Coutinho ◽  
D. Flavell ◽  
S.U. Flavell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Marrow Cultures

The distribution of granulocyte macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures (HLTBMC) was examined using two monoclonal antibodies raised using purified recombinant GM-CSF and a third commercially available GM-CSF antibody. The antibodies were able to bind to purified recombinant GM-CSF and showed inhibition of GM-CFC colonies in the presence of both recombinant and native protein. All antibodies displayed similar patterns of distribution in both permeabilised and non-permeabilised stromal cell preparations. Fibroblasts were labelled at their periphery in early cultures and both endothelial cells and fibroblasts showed cytoplasmic labelling with anti-GM-CSF. The fact that GM-CSF appears to be sequestered by cells of the bone marrow stroma raises the possibility that it is synthesized by these cells and may regulate activity of the progenitor cells in the haemopoietic foci. In contrast, early progenitor cells within the foci did not stain with any of the anti-GM-CSF antibodies. Adipocytes, which differentiate from fibroblasts in these cultures, showed a diffuse staining pattern. Two types of macrophage staining were observed in the non-permeabilised cells; those exhibiting only autofluorescence and those that bound the antibody. Intracellular staining was apparent in a small sub-population. Generally, the staining persisted up to eight weeks of culture and thereafter declined, becoming virtually undetectable after 12 weeks. This correlates with the pattern of GM-CFC production in long-term bone marrow cultures.

scholarly journals Granulocyte-macrophage colony-stimulating factor (GM-CSF) in human long- term bone marrow cultures: endogenous production in the adherent layer and effect of exogenous GM-CSF on granulomonopoiesis

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1230-1236 ◽  
Author(s):  
P Charbord ◽  
E Tamayo ◽  
S Saeland ◽  
V Duvert ◽  
J Poulet ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture Medium ◽  
Neutralizing Antibody ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract This study was designed to assess the presence of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) within adherent layers of human Dexter-type cultures and to investigate the effect on granulomonopoiesis of adding exogenous GM-CSF to the culture medium. The presence of GM-CSF was demonstrated using a bioassay, in which adherent layers from normal bone marrows gave rise to endogenous granulocyte-macrophage colony-forming units (CFU-GM) that were specifically inhibited by increasing amounts of an anti-GM-CSF neutralizing antibody. Using an immunoassay, the estimated amounts of GM-CSF were less than or equal to 40 pg per flask in adherent layers, while remaining undetectable in supernatants. The addition of 10 ng or purified recombinant GM-CSF per milliliter of culture medium increased slightly the CFU-GM output over a 5-week culture period. The addition of 50 ng/mL decreased significantly the CFU-GM output after 5 weeks of culture. This decrease was associated with major modifications of the adherent layer cell composition. Large round or ovoid macrophages were generated at the expense of the interdigitated and elongated stromal cells and the extracellular fibronectin network was no longer observed. These studies suggest that GM-CSF production by accessory cells (stromal cells and/or monocytes) is almost equal to its consumption by hematopoietic cells, a situation similar to that found in long-term cultures of murine marrows. They also show that the maintenance of granulomonopoiesis is decreased by adding more than 10 ng/mL of exogenous GM-CSF to the culture medium, which is related to the induction of adherent macrophages, the disappearance of the major smooth-muscle-like stromal cell component of the adherent layer, and that of the fibronectin extracellular matrix.

scholarly journals Biosynthetic (recombinant) human granulocyte-macrophage colony- stimulating factor: effect on normal bone marrow and leukemia cell lines

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 31-36 ◽  
Author(s):  
M Tomonaga ◽  
DW Golde ◽  
JC Gasson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Leukemia Cell ◽  
Colony Stimulating Factor ◽  
Normal Peripheral Blood ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract To examine the biologic properties of the molecule encoded by the human gene for granulocyte-macrophage colony-stimulating factor (GM-CSF), we expressed the cloned complementary DNA (cDNA) in transfected monkey COS cells and purified the resultant protein. Purified biosynthetic human GM-CSF was added to cultures of normal hematopoietic progenitor cells in semisolid media, and the resulting colonies were characterized cytochemically. Non-adherent light-density bone marrow cells from healthy adult volunteers were maximally stimulated with GM-CSF (approximately 250 pmol/L, and four types of colonies were consistently identified by aspirating the individual colonies and staining with a triple stain for specific and nonspecific esterases and eosinophilic granules. Pure neutrophilic granulocyte (G), mixed granulocyte- macrophage (GM), pure macrophage (M), and pure eosinophil (EO) colonies were observed, the mean incidences on day 8 being 70%, 20%, 5%, and 5%, and on day 14, 7.5%, 16.6%, 50.9%, and 25.0%, respectively. In all types of colonies, complete maturation to segmented forms or typical macrophages was detected. GM-CSF did not enhance the growth of BFU-E from normal peripheral blood buffy coat cells in the simultaneous presence of erythropoietin alone or erythropoietin with purified erythroid-potentiating activity. GM-CSF stimulated HL-60 and KG-1 colony formation twofold and fivefold, respectively; consistent differentiation induction towards monocytic and eosinophilic lineages was observed in HL-60 but not in KG-1. These in vitro findings indicate that GM-CSF is a multilineage stimulator for progenitor cells of G, GM, M, and EO colonies.

scholarly journals A murine cytokine fusion toxin specifically targeting the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on normal committed bone marrow progenitor cells and GM-CSF-dependent tumor cells

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2732-2740 ◽  
Author(s):  
CH Chan ◽  
BR Blazar ◽  
CR Eide ◽  
RJ Kreitman ◽  
DA Vallera
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Leukemia Cells ◽  
Colony Stimulating Factor ◽  
Fusion Toxin ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

A fusion protein was synthesized consisting of the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mGM-CSF hybrid gene was cloned into a vector under the control of an inducible promoter and the protein expressed in Escherichia coli. After induction, a protein was purified from inclusion bodies in accord with the deduced molecular weight of DT390 mGM-CSF. Cell-free studies of the adenosine diphosphate-ribosylating activity of DT390 mGM-CSF showed results that were similar to those of native DT. The DT390 mGM-CSF immunotoxin inhibited FDCP2.1d, a murine myelomonocytic tumor line expressing the GM-CSF receptor with an IC50 (concentration inhibiting 50% activity) of 5 x 10(-11) mol/L. The fusion toxin was specifically cytotoxic and directed by the GM-CSF portion of the molecule because addition of a monoclonal antibody directed against GM-CSF inhibited its ability to kill the cell line. Cell lines that do not express GM-CSF receptor were not inhibited by the fusion toxin. DT390 mGM-CSF was also able to specifically inhibit normal committed bone marrow (BM) progenitor cells as measured in clonal colony-forming unit granulocyte-macrophage assays. Together, these findings indicate that DT390 mGM-CSF may be useful as a novel tool for purging BM of contaminating leukemia cells or in vivo for eliminating residual leukemia cells. Also, it can be used to determine whether committed and/or noncommitted BM progenitor cells express the GM-CSF receptor.

scholarly journals Biosynthetic (recombinant) human granulocyte-macrophage colony- stimulating factor: effect on normal bone marrow and leukemia cell lines

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 31-36
Author(s):  
M Tomonaga ◽  
DW Golde ◽  
JC Gasson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Leukemia Cell ◽  
Colony Stimulating Factor ◽  
Normal Peripheral Blood ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

To examine the biologic properties of the molecule encoded by the human gene for granulocyte-macrophage colony-stimulating factor (GM-CSF), we expressed the cloned complementary DNA (cDNA) in transfected monkey COS cells and purified the resultant protein. Purified biosynthetic human GM-CSF was added to cultures of normal hematopoietic progenitor cells in semisolid media, and the resulting colonies were characterized cytochemically. Non-adherent light-density bone marrow cells from healthy adult volunteers were maximally stimulated with GM-CSF (approximately 250 pmol/L, and four types of colonies were consistently identified by aspirating the individual colonies and staining with a triple stain for specific and nonspecific esterases and eosinophilic granules. Pure neutrophilic granulocyte (G), mixed granulocyte- macrophage (GM), pure macrophage (M), and pure eosinophil (EO) colonies were observed, the mean incidences on day 8 being 70%, 20%, 5%, and 5%, and on day 14, 7.5%, 16.6%, 50.9%, and 25.0%, respectively. In all types of colonies, complete maturation to segmented forms or typical macrophages was detected. GM-CSF did not enhance the growth of BFU-E from normal peripheral blood buffy coat cells in the simultaneous presence of erythropoietin alone or erythropoietin with purified erythroid-potentiating activity. GM-CSF stimulated HL-60 and KG-1 colony formation twofold and fivefold, respectively; consistent differentiation induction towards monocytic and eosinophilic lineages was observed in HL-60 but not in KG-1. These in vitro findings indicate that GM-CSF is a multilineage stimulator for progenitor cells of G, GM, M, and EO colonies.

scholarly journals Granulocyte-macrophage colony-stimulating factor (GM-CSF) in human long- term bone marrow cultures: endogenous production in the adherent layer and effect of exogenous GM-CSF on granulomonopoiesis

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1230-1236
Author(s):  
P Charbord ◽  
E Tamayo ◽  
S Saeland ◽  
V Duvert ◽  
J Poulet ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture Medium ◽  
Neutralizing Antibody ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

This study was designed to assess the presence of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) within adherent layers of human Dexter-type cultures and to investigate the effect on granulomonopoiesis of adding exogenous GM-CSF to the culture medium. The presence of GM-CSF was demonstrated using a bioassay, in which adherent layers from normal bone marrows gave rise to endogenous granulocyte-macrophage colony-forming units (CFU-GM) that were specifically inhibited by increasing amounts of an anti-GM-CSF neutralizing antibody. Using an immunoassay, the estimated amounts of GM-CSF were less than or equal to 40 pg per flask in adherent layers, while remaining undetectable in supernatants. The addition of 10 ng or purified recombinant GM-CSF per milliliter of culture medium increased slightly the CFU-GM output over a 5-week culture period. The addition of 50 ng/mL decreased significantly the CFU-GM output after 5 weeks of culture. This decrease was associated with major modifications of the adherent layer cell composition. Large round or ovoid macrophages were generated at the expense of the interdigitated and elongated stromal cells and the extracellular fibronectin network was no longer observed. These studies suggest that GM-CSF production by accessory cells (stromal cells and/or monocytes) is almost equal to its consumption by hematopoietic cells, a situation similar to that found in long-term cultures of murine marrows. They also show that the maintenance of granulomonopoiesis is decreased by adding more than 10 ng/mL of exogenous GM-CSF to the culture medium, which is related to the induction of adherent macrophages, the disappearance of the major smooth-muscle-like stromal cell component of the adherent layer, and that of the fibronectin extracellular matrix.

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