scholarly journals Diethyldithiocarbamate induction of cytokine release in human long-term bone marrow cultures

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1172-1177 ◽  
Author(s):  
CJ East ◽  
CN Abboud ◽  
RF Borch
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Chemotherapeutic Agents ◽  
Tnf Alpha ◽  
Complete Medium ◽  
Maximum Increase ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time- dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)- 6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)- alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF- alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony- stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM- CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.

scholarly journals Diethyldithiocarbamate induction of cytokine release in human long-term bone marrow cultures

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1172-1177
Author(s):  
CJ East ◽  
CN Abboud ◽  
RF Borch
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Chemotherapeutic Agents ◽  
Tnf Alpha ◽  
Complete Medium ◽  
Maximum Increase ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time- dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)- 6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)- alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF- alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony- stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM- CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.

scholarly journals Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma

Blood ◽  
1992 ◽  
Vol 79 (12) ◽  
pp. 3168-3178 ◽  
Author(s):  
EL Kittler ◽  
H McGrath ◽  
D Temeles ◽  
RB Crittenden ◽  
VK Kister ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Stromal Cells ◽  
Pokeweed Mitogen ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Growth Factor Production ◽  
Constitutive Production ◽  
Marrow Cultures

Abstract The “stromal” or adherent cells of long-term murine Dexter explant bone marrow cultures provide the best in vitro model of the bone marrow microenvironment. Colony-stimulating factor-1 (CSF-1) is produced constitutively by these cells and is easily detected, but most investigators have not found constitutive production of the other hemolymphopoietic cytokines. We have previously reported the detection of granulocyte-macrophage-CSF (GM-CSF) in murine stromal cultures and its induction by the lectin Pokeweed mitogen. The present studies analyzing stromal cytokine messenger RNA (mRNA) production by standard Northern blot analysis show constitutive production of mRNAs for CSF-1, GM-CSF, granulocyte-CSF (G-CSF), c-kit ligand (KL), and interleukin-6 (IL-6), but not IL-3, IL-4, or IL-5 by 3-week irradiated or nonirradiated murine Dexter stromal cells. Exposure of stromal cells to Pokeweed mitogen or IL-1 16 hours before RNA harvest induces the messages for GM-CSF, G-CSF, KL, and IL-6, but not IL-3, IL-4, IL-5, or CSF-1. Polymerase chain reaction amplification of cDNA made with reverse transcriptase from stromal RNA using two separate sets of IL-3- specific primers shows the presence of IL-3 message in irradiated stromal cells, which is only detectable with this more sensitive technique. The factor-dependent cell lines FDC-P1 and 32D are supported by the stromal cells without the addition of exogenous growth factors, demonstrating a cytokine activity in these cultures that is inhibited by the addition of anti-IL-3 or anti-GM-CSF antibodies. These data indicate that murine Dexter stromal cells constitutively produce CSF-1, GM-CSF, G-CSF, IL-6, KL, and IL-3. This growth factor production could explain the support of granulocyte, macrophage, and megakaryocyte production and stem cell maintenance in Dexter-type long-term murine bone marrow cultures.

scholarly journals Stromal growth factor production in irradiated lectin exposed long-term murine bone marrow cultures

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1120-1127
Author(s):  
TA Alberico ◽  
JN Ihle ◽  
CM Liang ◽  
HE McGrath ◽  
PJ Quesenberry
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Stromal Cells ◽  
Colony Formation ◽  
Murine Bone Marrow ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Marrow Cultures ◽  
Marrow Cells

Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.

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 Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma

Blood ◽  
1992 ◽  
Vol 79 (12) ◽  
pp. 3168-3178 ◽  
Author(s):  
EL Kittler ◽  
H McGrath ◽  
D Temeles ◽  
RB Crittenden ◽  
VK Kister ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Stromal Cells ◽  
Pokeweed Mitogen ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Growth Factor Production ◽  
Constitutive Production ◽  
Marrow Cultures

The “stromal” or adherent cells of long-term murine Dexter explant bone marrow cultures provide the best in vitro model of the bone marrow microenvironment. Colony-stimulating factor-1 (CSF-1) is produced constitutively by these cells and is easily detected, but most investigators have not found constitutive production of the other hemolymphopoietic cytokines. We have previously reported the detection of granulocyte-macrophage-CSF (GM-CSF) in murine stromal cultures and its induction by the lectin Pokeweed mitogen. The present studies analyzing stromal cytokine messenger RNA (mRNA) production by standard Northern blot analysis show constitutive production of mRNAs for CSF-1, GM-CSF, granulocyte-CSF (G-CSF), c-kit ligand (KL), and interleukin-6 (IL-6), but not IL-3, IL-4, or IL-5 by 3-week irradiated or nonirradiated murine Dexter stromal cells. Exposure of stromal cells to Pokeweed mitogen or IL-1 16 hours before RNA harvest induces the messages for GM-CSF, G-CSF, KL, and IL-6, but not IL-3, IL-4, IL-5, or CSF-1. Polymerase chain reaction amplification of cDNA made with reverse transcriptase from stromal RNA using two separate sets of IL-3- specific primers shows the presence of IL-3 message in irradiated stromal cells, which is only detectable with this more sensitive technique. The factor-dependent cell lines FDC-P1 and 32D are supported by the stromal cells without the addition of exogenous growth factors, demonstrating a cytokine activity in these cultures that is inhibited by the addition of anti-IL-3 or anti-GM-CSF antibodies. These data indicate that murine Dexter stromal cells constitutively produce CSF-1, GM-CSF, G-CSF, IL-6, KL, and IL-3. This growth factor production could explain the support of granulocyte, macrophage, and megakaryocyte production and stem cell maintenance in Dexter-type long-term murine bone marrow cultures.

Reculturing of Cells from Primary CFU-C Colonies

1985 ◽  
Vol 40 (11-12) ◽  
pp. 891-897 ◽  
Author(s):  
G. E. Hübner ◽  
F. Ali-Osman ◽  
M. Kästner ◽  
C. Papadimitriou ◽  
H. R. Maurer
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Primary Cultures ◽  
Fold Increase ◽  
Mouse Lung ◽  
Cell Aggregates ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract This study was aimed at investigating whether cells of CFU-C derived colonies could form secondary colonies. Bone marrow cultures of volumes of agar medium between 25 μl and 75 μl contained in glass capillaries were stimulated with mouse lung-conditioned medium (MLCM) containing granulocyte/macrophage colony-stimulating factor (GM-CSF). Agar gels with colonies of up to ≥ 20 were blown out into identical culture medium, completely dispersed on a whirl-mix to single cell suspensions, and used for establishing secondary agar cultures. In these secondary cultures considerable numbers of secondary granulocytic, mixed granulocytic/macrophage and macrophage colonies as well as numerous clusters arose. In contrast, when single colonies were recultured, only few secondary cell aggregates were formed. When primary cultures containing up to ≥ 20 cell aggregates were used for serial reculture at intermittent intervals of 3 and 4 days, a 2 -7-fold increase of colony-forming cells was found in tertiary cultures as was monitored by 7 day colony counts. And by use of different kinds of CSF-containing media, an over 4-fold increase of secondary over primary colonies was obtained with bovine lung-conditioned medium (BLCM) in primary and L-cell-conditioned medium (LCCM) in secondary 7 day cultures. Primary capillary cultures were found to be devoid of CFU-S. Also, setting up bone marrow cultures in petri dishes and stimulating with MLCM, growth of primary as well as secondary colonies was obtained. The results indicate some self-renewal potential of CFU-C in vitro.

scholarly journals Stromal growth factor production in irradiated lectin exposed long-term murine bone marrow cultures

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1120-1127 ◽  
Author(s):  
TA Alberico ◽  
JN Ihle ◽  
CM Liang ◽  
HE McGrath ◽  
PJ Quesenberry
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Stromal Cells ◽  
Colony Formation ◽  
Murine Bone Marrow ◽  
Gm Csf ◽  
Bone Marrow Cultures ◽  
Marrow Cultures ◽  
Marrow Cells

Abstract Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.

Formation of Multinucleated Cells that Respond to Osteotropic Hormones in Long Term Human Bone Marrow Cultures*

Endocrinology ◽  
1987 ◽  
Vol 120 (6) ◽  
pp. 2326-2333 ◽  
Author(s):  
B. R. MACDONALD ◽  
N. TAKAHASHI ◽  
L. M. MCMANUS ◽  
J. HOLAHAN, ◽  
G. R. MUNDY ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Multinucleated Cells ◽  
Bone Marrow Cultures ◽  
Human Bone Marrow Cultures ◽  
Marrow Cultures

scholarly journals Early pre-B-cell transformation induced by the v-fms oncogene in long-term mouse bone marrow cultures.

1989 ◽  
Vol 9 (9) ◽  
pp. 3973-3981 ◽  
Author(s):  
G V Borzillo ◽  
C J Sherr
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Chain Gene ◽  
Mouse Bone Marrow ◽  
Feeder Layers ◽  
Bone Marrow Cultures ◽  
B Lymphoid ◽  
Marrow Cultures

Murine long-term bone marrow cultures that support B-lymphoid-cell development were infected with a helper-free retrovirus containing the v-fms oncogene. Infection of B-lymphoid cultures resulted in the rapid clonal outgrowth of early pre-B cells, which grew to high cell densities on stromal cell feeder layers, expressed v-fms-coded glycoproteins, and underwent immunoglobulin heavy-chain gene rearrangements. Late-passage cultures gave rise to factor-independent variants that proliferated in the absence of feeder layers, developed resistance to hydrocortisone, and became tumorigenic in syngeneic mice. The v-fms oncogene therefore recapitulates known effects of the v-abl and bcr-abl oncogenes on B-lineage cells. The ability of v-fms to induce transformation of early pre-B cells in vitro underscores the capacity of oncogenic mutants of the colony-stimulating factor-1 receptor to function outside the mononuclear phagocyte lineage.

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