scholarly journals Bone marrow stromal fibroblasts secrete interleukin-6 and granulocyte- macrophage colony-stimulating factor in the absence of inflammatory stimulation: demonstration by serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1190-1198 ◽  
Author(s):  
SC Guba ◽  
CI Sartor ◽  
LR Gottschalk ◽  
YH Jing ◽  
T Mulligan ◽  
...  
Keyword(s):  
Human Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stromal Fibroblasts ◽  
Gm Csf ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Polymerase Chain ◽  
Macrophage Colony ◽  
Stimulating Factor

Bone marrow (BM) stromal fibroblasts produce hematopoietic growth factors (HGFs) in response to inflammatory mediators such as tumor necrosis factor-alpha or interleukin-1 alpha (IL-1 alpha). In the absence of such inflammatory stimuli, production of HGFs by BM stromal cells has been problematic and controversial. In vivo, however, basal hematopoiesis maintains blood counts within a normal homeostatic range even in the absence of inflammation, and HGFs are required for progenitor cell differentiation in vitro. To better ascertain the contribution of BM stromal fibroblasts to basal hematopoiesis, we therefore studied HGF production in quiescent BM stromal fibroblasts by three sensitive assays: serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction. Stromal fibroblasts were cultured in the presence or absence of normal human serum to determine if serum factor(s) present in the noninflammatory (basal) state induce secretion of HGFs. Human serum was found to induce or enhance transcription and secretion of granulocyte- macrophage colony-stimulating factor (GM-CSF) and enhance secretion of constitutively expressed IL-6. In contrast, no secretion of either granulocyte-CSF (G-CSF) or IL-3 was found. These data indicate that factors in normal human serum are active in enhancing GM-CSF and IL-6 production by stromal fibroblasts and suggest that these growth factors contribute to the maintainance of normal, basal hematopoiesis in vivo.

scholarly journals Bone marrow stromal fibroblasts secrete interleukin-6 and granulocyte- macrophage colony-stimulating factor in the absence of inflammatory stimulation: demonstration by serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1190-1198 ◽  
Author(s):  
SC Guba ◽  
CI Sartor ◽  
LR Gottschalk ◽  
YH Jing ◽  
T Mulligan ◽  
...  
Keyword(s):  
Human Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stromal Fibroblasts ◽  
Gm Csf ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Polymerase Chain ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Bone marrow (BM) stromal fibroblasts produce hematopoietic growth factors (HGFs) in response to inflammatory mediators such as tumor necrosis factor-alpha or interleukin-1 alpha (IL-1 alpha). In the absence of such inflammatory stimuli, production of HGFs by BM stromal cells has been problematic and controversial. In vivo, however, basal hematopoiesis maintains blood counts within a normal homeostatic range even in the absence of inflammation, and HGFs are required for progenitor cell differentiation in vitro. To better ascertain the contribution of BM stromal fibroblasts to basal hematopoiesis, we therefore studied HGF production in quiescent BM stromal fibroblasts by three sensitive assays: serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction. Stromal fibroblasts were cultured in the presence or absence of normal human serum to determine if serum factor(s) present in the noninflammatory (basal) state induce secretion of HGFs. Human serum was found to induce or enhance transcription and secretion of granulocyte- macrophage colony-stimulating factor (GM-CSF) and enhance secretion of constitutively expressed IL-6. In contrast, no secretion of either granulocyte-CSF (G-CSF) or IL-3 was found. These data indicate that factors in normal human serum are active in enhancing GM-CSF and IL-6 production by stromal fibroblasts and suggest that these growth factors contribute to the maintainance of normal, basal hematopoiesis in vivo.

scholarly journals Treatment with monocyte-derived partially purified GM-CSF but not G-CSF aborts the development of transplanted chloroleukemia in rats

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1077-1080 ◽  
Author(s):  
JJ Jimenez ◽  
AA Yunis
Keyword(s):  
Conditioned Medium ◽  
Molecular Size ◽  
Myelogenous Leukemia ◽  
Rat Lung ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

We have previously demonstrated that cultured rat chloroleukemia cells, MIA C51, will terminally differentiate to macrophages when treated with rat lung-conditioned medium in vitro and in vivo. In the present study we fractionated rat monocyte-conditioned medium by ultrafiltration according to molecular size. The fraction with molecular weight (mol wt) 30 to 50 Kd containing partially purified granulocyte-macrophage colony-stimulating factor (GM-CSF) activity caused the differentiation of C51 cells to macrophages in vitro and in diffusion chambers in vivo. Treatment of young rats with this fraction aborted the development of chloroleukemia from transplanted C51 cells. In contrast, the fraction with mol wt 10 to 30 Kd containing virtually all the G-CSF activity exhibited no differentiation activity either in vitro or in vivo. It is concluded that in this rat myelogenous leukemia model partially purified GM-CSF but not G-CSF contains the effector molecule(s) causing terminal differentiation of C51 cells and tumor cell rejection.

scholarly journals Antibody to Granulocyte-Macrophage Colony-Stimulating Factor Is a Dominant Anti-Cytokine Activity in Human IgG Preparations

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 2054-2061 ◽  
Author(s):  
Morten Svenson ◽  
Morten Bagge Hansen ◽  
Christian Ross ◽  
Marcus Diamant ◽  
Klaus Rieneck ◽  
...  
Keyword(s):  
Pharmaceutical Preparations ◽  
Polymorphonuclear Cells ◽  
High Avidity ◽  
Colony Stimulating Factor ◽  
Human Igg ◽  
Gm Csf ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Pharmaceutical preparations of normal human immunoglobulin (IgG) are known to contain high-avidity and neutralizing antibodies (Ab) to the cytokines interleukin (IL)-1α, IL-6, and interferon (IFN)α. To test for other cytokine Ab, 23 batches of IgG were tested for saturable binding to eight 125I-labeled recombinant cytokines. All batches bound granulocyte-macrophage colony-stimulating factor (GM-CSF) with high avidity (Kav ≈ 10 pmol/L) and capacities of up to 5 μmol GM-CSF/mol IgG. Only 1 of 15 batches bound IL-5, also with high avidity, whereas 13 of 15 batches bound to IL-10 but with lower capacities and avidities. None of the IgG preparations bound IL-1 receptor antagonist (IL-1ra), IL-2, IL-3, IL-4, or G-CSF. Cross-binding and absorption analyses revealed identical or slightly stronger binding of recombinant GM-CSF, IL-5, and IL-10 than their native counterparts. GM-CSF–IgG complexes did not bind to cellular GM-CSF receptors, but Fc-dependent binding occurred to blood polymorphonuclear cells. Increased binding of GM-CSF to patient sera correlated positively with the binding capacities of infused IgG preparations. Patient and normal sera did not interfere with the binding of Ab to GM-CSF. From these and previous experiments, we conclude that pools of normal human IgG contain variable amounts of specific and high-avidity Ab to some cytokines, and that Ab to GM-CSF constitute a dominant anti-cytokine activity in these preparations. These Ab are available for reactionin vivo following IgG therapy.

scholarly journals Recombinant human granulocyte-macrophage colony-stimulating factor in combination with standard induction chemotherapy in de novo acute myeloid leukemia

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 700-711 ◽  
Author(s):  
P Bettelheim ◽  
P Valent ◽  
M Andreeff ◽  
A Tafuri ◽  
J Haimi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Induction Chemotherapy ◽  
De Novo ◽  
Gm Csf ◽  
Cell Counts ◽  
Macrophage Colony Stimulating Factor ◽  
De Novo Aml ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Based on in vitro data suggesting that recombinant human granulocyte- macrophage colony-stimulating factor (rhGM-CSF) is capable of stimulating acute myeloid leukemia (AML) blast cells to become more sensitive to cell-cycle-specific drugs we conducted a phase I/II study in de novo AML patients (pts). rhGM-CSF (250 micrograms/m2/d, continuous intravenous infusion) was administered in 18 pts suffering from de novo AML in combination with standard induction chemotherapy (3 + 7 = daunorubicin 45 mg/m2 days 1 through 3, cytosine-arabinoside [Ara- C] 200 mg/m2 continuous infusion days 1 through 7). GM-CSF was started 48 or 24 hours before chemotherapy (prephase) in 14 pts. In four pts with high white blood cell counts (WBC) rhGM-CSF was started after chemotherapy-induced cell reduction (WBC less than 30,000/mm3). During prephase GM-CSF induced an increase in neutrophil and blast cell counts in 13 of 14 and 10 of 14 pts, respectively. In vivo recruitment of leukemic cells into drug-sensitive phases of the cell cycle could be demonstrated by multiparameter cell-cycle analyses in peripheral blood (n = 7) and bone marrow (n = 4) specimens. On day 14, complete aplasia was evident in 17 of 18 pts. GM-CSF was administered until recovery from chemotherapy-induced myelosuppression (absolute neutrophil counts, [ANC] greater than 500/mm3). Fifteen pts (83%) achieved complete remission, 12 did so with one cycle. A shorter duration of neutropenia was evident in these pts compared with historical controls (n = 39), (ANC greater than 500/mm3, day 22.5 +/- 3.4 v 25.2 +/- 3.7, P less than .05). Three pts achieved complete remission after a second cycle (same combination of rhGM-CSF and 3 + 7). Two pts died during bone marrow aplasia because of invasive pulmonary aspergillosis. Clinical side effects possibly related to GM-CSF, mainly fever, diarrhea, and weight gain were mild and tolerable (World Health Organization toxicity grade less than or equal to 2). Together, rhGM-CSF recruits kinetically quiescient AML cells in vivo to enter drug-sensitive phases of the cell cycle and promotes early myeloid recovery from aplasia after exposure to standard induction chemotherapy for AML.

scholarly journals Chimeric CLL-1 Antibody Fusion Proteins Containing Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2 With Specificity for B-Cell Malignancies Exhibit Enhanced Effector Functions While Retaining Tumor Targeting Properties

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4437-4447 ◽  
Author(s):  
Jason L. Hornick ◽  
Leslie A. Khawli ◽  
Peisheng Hu ◽  
Maureen Lynch ◽  
Peter M. Anderson ◽  
...  
Keyword(s):  
B Cell ◽  
Fusion Proteins ◽  
Tumor Targeting ◽  
B Cell Malignancies ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Although monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman B-cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)-2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioimmunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony formation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent T-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.

125. REGULATION OF STRESS PROTEIN GENES DURING PRE-IMPLANTATION EMBRYO DEVELOPMENT IN MICE BY GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)

2009 ◽  
Vol 21 (9) ◽  
pp. 44
Author(s):  
P. Y. Chin ◽  
A. M. Macpherson ◽  
J. G. Thompson ◽  
M. Lane ◽  
S. A. Robertson
Keyword(s):  
Stress Response ◽  
Embryo Development ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

In vitro culture has been shown to be detrimental for pre-implantation embryo development and this has been associated with culture stress and elevated expression of apoptotic genes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to promote development and survival of both human and mouse pre-implantation embryos. To investigate the mechanism of action of GM-CSF in mouse embryos, gene expression was examined in in vitro cultured blastocysts with and without recombinant mouse GM-CSF (rmGM-CSF) and in vivo blastocysts flushed from Csf2 null mutant and wild-type mice. Microarray analysis of the effect of GM-CSF on transcription profile implicated apoptosis and stress response gene pathways in blastocyst responses to rmGM-CSF in vitro. Groups of 30 blastocysts were collected from in vitro cultured and in vivo developed blastocyst were analysed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis of in vitro blastocysts revealed that addition of rmGM-CSF causes differential expression of several genes associated with apoptosis and cellular stress pathway, including Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5. Immunocytochemical analysis of common proteins of the apoptosis and cellular stress response pathways BAX, BCL2, TRP53 (p53) and HSPA1A/1B (Hsp70) in in vitro blastocysts revealed that HSPA1A/1B and BCL2 proteins were less abundant in embryos cultured in rmGM-CSF, but BAX and TRP53 were unchanged. In in vivo developed blastocysts, Csf2 null mutation resulted in elevated levels of only the heat shock protein Hsph1, suggesting that in vivo, other cytokines can compensate for GM-CSF deficiency as the absence of GM-CSF has a lesser effect on the stress response pathway. We conclude that GM-CSF is a regulator of the apoptosis and cellular stress response pathways influencing mouse pre-implantation embryo development to facilitate embryo growth and survival, and the effects of GM-CSF are particularly evident in in vitro culture media in the absence of other cytokines.

scholarly journals Induction of anti-recombinant human granulocyte-macrophage colony- stimulating factor (Escherichia coli-derived) antibodies and clinical effects in nonimmunocompromised patients

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4078-4087 ◽  
Author(s):  
P Ragnhammar ◽  
HJ Friesen ◽  
JE Frodin ◽  
AK Lefvert ◽  
M Hassan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibody Response ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clinical Effects ◽  
Immunocompromised Patients ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The pharmacokinetics of recombinant human granulocyte-macrophage colony- stimulating factor (rhGM-CSF), induction of anti-GM-CSF antibodies, and clinical effects related to the induction of the antibodies were analyzed in patients with metastatic colorectal carcinoma (CRC) who were not on chemotherapy (n = 20, nonimmunocompromised patients). rhGM- CSF (250 micrograms/m2/d; Escherichia coli-derived) was administered subcutaneously for 10 days every month for 4 months. Eight patients with multiple myeloma (MM) on intensive chemotherapy followed by rhGM- CSF treatment were also included (immunocompromised patients). After a single injection of GM-CSF at the first cycle in CRC patients, the maximum calculated concentration (Cmax) was 5.24 +/- 0.56 ng/mL; the half life (T1/2) was 2.91 +/- 0.8 hours; and the area under the concentration curve (AUC) was 30.86 +/- 6.03 hours x ng/mL (mean +/- SE). No anti-GM-CSF antibodies were detected. During the subsequent cycles, 95% of the CRC patients developed anti-GM-CSF IgG antibodies, which significantly altered the pharmacokinetics of rhGM-CSF at the third and fourth cycles with decreased Cmax (2.87 +/- 0.57 ng/mL; P < .05), T1/2 (1.57 +/- 0.2 hours; P < .05), and AUC (14.90 +/- 4.10 hours x ng/mL; P < .005). The presence of anti-GM-CSF antibodies significantly reduced the GM-CSF-induced enhancement of granulocytes, and there was a clear tendency for a decreased increment of monocytes. Antibodies diminished systemic side effects of rhGM-CSF. Only 1 of 8 MM patients showed a very low anti-GM-CSF antibody titer after GM-CSF therapy, as shown by enzyme-linked immunosorbent assay and Western blot. Therefore, in nonimmunocompromised patients, exogenous nonglycosylated GM-CSF induced an anti-GM-CSF IgG antibody response in practically all patients, which seemed to be of clinical significance. In immunocompromised patients, virtually no significant antibody response was shown.

scholarly journals Hematopoietic and Lymphopoietic Responses in Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Transgenic Mice Injected With Human GM-CSF

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1031-1038 ◽  
Author(s):  
I. Nishijima ◽  
T. Nakahata ◽  
S. Watanabe ◽  
K. Tsuji ◽  
I. Tanaka ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Blood Cells ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Dose Dependent ◽  
Stimulating Factor

Abstract Using a clonal assay of bone marrow (BM) cells from transgenic mice (Tg-mice) expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGM-CSFR), we found in earlier studies that hGM-CSF alone supported the development not only of granulocyte-macrophage colonies, but also of erythrocytes, megakaryocytes, mast cells, blast cells, and mixed hematopoietic colonies. In this report, we evaluated the in vivo effects of hGM-CSF on hematopoietic and lymphopoietic responses in the hGM-CSFR Tg-mice. Administration of this factor to Tg-mice resulted in dose-dependent increases in numbers of reticulocytes and white blood cells (WBCs) in the peripheral blood. Morphological analysis of WBCs showed that the numbers of all types of the cell, including neutrophils, eosinophils, monocytes, and lymphocytes increased; the most remarkable being in lymphocytes that contained a number of large granular lymphocytes (LGLs) in addition to mature T and B cells. However, total cellularity of the BM of the Tg-mice decreased in a dose-dependent manner when hGM-CSF was injected. In sharp contrast to the BM, spleens of the Tg-mice were grossly enlarged. Although all types of blood cells and hematopoietic progenitors increased in the spleen, erythroid cells and their progenitors showed the most significant increase. Increased numbers of megakaryocytes and LGLs were also observed in spleen and liver of the treated Tg-mice. Flow cytometric analysis showed that LGLs expanded in Tg-mice expressed Mac-1+CD3−NK1.1+. The thymus of Tg-mice treated with hGM-CSF exhibited a dose-dependent shrinkage and a remarkable decrease in CD4+CD8+ cells. Thus, hGM-CSF stimulated not only myelopoiesis but also erythropoiesis and megakaryopoiesis of hGM-CSFR Tg-mice in vivo, in accordance with our reported in vitro findings. In addition, hGM-CSF affected the development of lymphoid cells, including natural killer cells of these Tg-mice.

scholarly journals Identification of functionally distinct domains of human granulocyte- macrophage colony-stimulating factor using monoclonal antibodies

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1033-1043 ◽  
Author(s):  
Y Kanakura ◽  
SA Cannistra ◽  
CB Brown ◽  
M Nakamura ◽  
GF Seelig ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neutralizing Antibodies ◽  
Solid Phase ◽  
Receptor Interaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40–77, 78–94, or 110–127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

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