scholarly journals cAMP attenuates interleukin-1-stimulated macrophage colony-stimulating factor (M-CSF) expression

2000 ◽  
Vol 350 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Pisate J. KAMTHONG ◽  
Fu-mei WU ◽  
Ming-chi WU
Keyword(s):  
Pancreatic Cancer Cell Line ◽  
Human Pancreatic Cancer ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Colony Stimulating Factor ◽  
Rt Pcr ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Camp Elevation

Macrophage colony-stimulating factor (M-CSF) is a multifunctional cytokine attributed with key biological functions beyond the first discovered role in promoting proliferation of myeloid cell lineage. The human pancreatic cancer cell line MIA PaCa-2, from which the M-CSF gene was originally cloned, was used to study regulation of M-CSF expression. Expression of M-CSF was inducible by interleukin-1α (IL-1α), lipopolysaccharide (LPS) and PMA as demonstrated by a biological activity assay, Northern-blot analysis and reverse transcriptase (RT) PCR. Treatment of the cells with forskolin or dibutyryl-cAMP attenuated the expression of M-CSF induced by IL-1α or LPS, but not by PMA. Electromobility shift assays showed that IL-1α predominantly activated nuclear factor κB (NF-κB), while PMA preferentially activated activator protein-1 (AP-1). The activation of NF-κB, but not AP-1, could be attenuated by cAMP elevation. Relative RT-PCR demonstrated that the expression of a 1.6-kb M-CSF mRNA transcript was more effectively induced by IL-1α than a 4.0-kb transcript. By and large the induced expression of both mRNA transcripts could be attenuated by cAMP. M-CSF promoter-driven luciferase reporter-gene assays revealed that cAMP elevation attenuated the IL-1-induced transcription activation of the M-CSF promoter, but it had no effect on PMA-induced transcription. Our findings suggest that cAMP regulates M-CSF gene expression at the transcriptional level and that its inhibitory effect involves NF-κB signalling pathway.

scholarly journals Secretion of cytokines (interleukins-1 alpha, -3, and -6 and granulocyte-macrophage colony-stimulating factor) by normal human bone marrow megakaryocytes

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 685-691 ◽  
Author(s):  
C Wickenhauser ◽  
J Lorenzen ◽  
J Thiele ◽  
A Hillienhof ◽  
K Jungheim ◽  
...  
Keyword(s):  
Plaque Assay ◽  
Secretory Activity ◽  
Colony Stimulating Factor ◽  
Rt Pcr ◽  
Gm Csf ◽  
Clear Cut ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The effects of cytokine stimulation [recombinant human interleukin (rhIL)-1 alpha, rhIL-3, rhIL-6, rhIL-11, and rh granulocyte-macrophage colony-stimulating factor (GM-CSF)] on the secretory activity of normal human megakaryocytes were studied by means of the reverse hemolytic plaque assay (RHPA) in enriched cell preparations. This test facilitates an extremely sensitive determination of cytokine secretion at the single-cell level, together with the clear-cut identification of each immunostained (CD61) secretory active megakaryocyte. Moreover, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of IL-6, IL-6 receptor (IL-6R), IL-9, IL-10, IL-12, and IL-13 mRNA in highly concentrated megakaryocyte preparations. In comparison with the spontaneous secretion rate, stimulation with rhIL-1 alpha, rhIL-6, and rhGM-CSF failed to induce a significant increase in the release of cytokines by CD61+ cells. On the other hand, both rhIL-3 and, in a less pronounced way, rhIL-11 exerted a marked effect on IL-6 secretion. Additionally, after stimulation with rhIL-3, a significant enhancement of the secretion of IL-3 and GM-CSF, but not of IL-1 alpha, could be observed. Using the RT-PCR, a significant induction of IL-6 expression could be appreciated in the enriched megakaryocyte population (60% to 80%) stimulated with rhIL-3. The results of this study provide persuasive evidence that a number of cytokines are synthesized and secreted by human megakaryocytes and not only by hematopoietic stroma cells. These data suggest the existence of autocrine and paracrine mechanisms that may influence maturation and differentiation of megakaryocytes as well as act on various stroma cells to sustain an appropriate hematopoietic micro-environment.

Generation of Functional Human Dendritic Cells From Adherent Peripheral Blood Monocytes by CD40 Ligation in the Absence of Granulocyte-Macrophage Colony-Stimulating Factor

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4238-4247 ◽  
Author(s):  
Peter Brossart ◽  
Frank Grünebach ◽  
Gernot Stuhler ◽  
Volker L. Reichardt ◽  
Robert Möhle ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Colony Stimulating Factor ◽  
Blood Monocytes ◽  
Rt Pcr ◽  
Cd40 Ligation ◽  
Peripheral Blood Monocytes ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Recently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF. Adherent peripheral blood mononuclear cells (PBMNC) were cultured in the presence of different cytokine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mixed lymphocyte reaction (MLR), to present soluble antigens, and to induce primary HIV-peptide–specific cytotoxic T-cell (CTL) responses in vitro. Furthermore, expression of DC-CK1, a recently identified chemokine with specific expression in DC, and of IL-18 (IGIF), a growth and differentiation factor for Th 1 lymphocytes, was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). In our study, Flt3L alone was not sufficient to generate DC and required addition of IL-4. DC generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor- (TNF-) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory molecules as well as increased allogeneic proliferative response. In contrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of mature DC and were potent stimulatory cells in priming of major histocompatibility complex (MHC) class I–restricted CTL responses against an HIV-peptide, whereas their ability to present soluble protein antigens was reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcripts were detected in all generated DC populations, independent of growth factors used. Our findings provide further evidence for the importance of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provide an additional source of DC depending on external stimuli.

Interferon-γ switches monocyte differentiation from dendritic cells to macrophages

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Yves Delneste ◽  
Peggy Charbonnier ◽  
Nathalie Herbault ◽  
Giovanni Magistrelli ◽  
Gersende Caron ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Interferon Γ ◽  
Transcriptional Level ◽  
Colony Stimulating Factor ◽  
Monocyte Differentiation ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Ifn Γ ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Human monocytes differentiate into dendritic cells (DCs) or macrophages according to the nature of environmental signals. Monocytes stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4) yield DCs. We tested here whether interferon-γ (IFN-γ), a potent activator of macrophages, may modulate monocyte differentiation. Addition of IFN-γ to IL-4 plus GM-CSF–stimulated monocytes switches their differentiation from DCs to CD14−CD64+ macrophages. IFN-γ increases macrophage colony-stimulating factor (M-CSF) and IL-6 production by IL-4 plus GM-CSF–stimulated monocytes by acting at the transcriptional level and acts together with IL-4 to up-regulate M-CSF but not IL-6 production. IFN-γ also increases M-CSF receptor internalization. Results from neutralizing experiments show that both M-CSF and IL-6 are involved in the ability of IFN-γ to skew monocyte differentiation from DCs to macrophages. Finally, this effect of IFN-γ is limited to early stages of differentiation. When added to immature DCs, IFN-γ up-regulates IL-6 but not M-CSF production and does not convert them to macrophages, even in the presence of exogenous M-CSF. In conclusion, IFN-γ shifts monocyte differentiation to macrophages rather than DCs through autocrine M-CSF and IL-6 production. These data show that IFN-γ controls the differentiation of antigen-presenting cells and thereby reveals a new mechanism by which IFN-γ orchestrates the outcome of specific immune responses.

scholarly journals 125 CHANGES OF PLASMA MACROPHAGE COLONY-STIMULATING FACTOR LEVELS AND ITS GENE EXPRESSION IN PERIPHERAL WHITE BLOOD CELLS DURING PREGNANCY IN JAPANESE BLACK CATTLE

2005 ◽  
Vol 17 (2) ◽  
pp. 213
Author(s):  
K. Oshima ◽  
K. Yoshihara ◽  
H. Watanabe ◽  
T. Kojima ◽  
M. Komatsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
White Blood Cells ◽  
Colony Stimulating Factor ◽  
Rt Pcr ◽  
Relative Level ◽  
Csf Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Peripheral White Blood Cells ◽  
Stimulating Factor

Macrophage colony-stimulating factor (M-CSF) is a hemopoietic cytokine that plays a primary role in placental physiology. Gene expression of M-CSF in bovine intercaruncular endometrium shows an upward trend in mid-pregnancy. The objective of this study was to determine the plasma M-CSF levels and the M-CSF gene expression levels in maternal peripheral white blood cells (PWBCs) during pregnancy using ELISA and quantitative RT-PCR. In Experiment 1, the plasma M-CSF levels in 112 Japanese Black heifers or cows were determined. Animals were divided into four groups according to pregnancy stage: first- (n = 29), second- (n = 33), third- (n = 26) trimester, and non-pregnant (n = 24). ELISA for bovine M-CSF established by Yoshihara et al. (2003 Vet. Immunol. Immunopathol. 95, 103–111) was used according to their instructions. The absorbance was measured at 405 nm in the Biomek Plate Reader (Beckman Coulter, Fullerton, CA, USA). In Experiment 2, the plasma M-CSF levels and M-CSF gene expression levels in PWBCs during pregnancy were determined. The plasma samples for ELISA were obtained from 8 heifers and 3 cows every 1 and/or 2 weeks. The PWBCs samples for quantitative RT-PCR were obtained from 4 heifers every 1 and/or 4 weeks. All quantitative RT-PCR protocols were carried out according to the previous report (Oshima et al. 2003 Theriogenology 60, 1217–1226). The quantitative PCR assay used an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). Signals were detected according to the manufacturer's instructions. The relative level of M-CSF expression was calculated on the basis of glyceraldehyde-phosphate-dehydrogenase (GAPDH) quantity (in the method of calculation, the relative level = M-CSF quantity/GAPDH quantity). Data were analyzed by Kruskal-Wallis test. In Experiment 1, the plasma M-CSF level in second-trimester cows was significantly higher than those in other stages (P < 0.05). In Experiment 2, the plasma M-CSF levels were significantly higher in gestational age from −4 to 1 weeks compared with the last stage of pregnancy (P < 0.05). The levels decreased until 6 weeks, appeared to temporarily increase, and were relatively constant until 35 weeks. Macrophage colony-stimulating factor genes were expressed in all samples examined; the levels were relatively constant in early pregnancy, and then were widely varied until parturition. These results suggest that plasma M-CSF levels may be related to the maternal condition of pregnancy and to a slight extent to M-CSF gene expression in PWBCs. This work was supported in part by a Grant-in-aid from the Recombinant Cytokine Project (RCP2002-2110), provided by the Ministry of Agriculture, Forestry, and Fisheries, Japan.

scholarly journals Inducible expression of PTX3, a new member of the pentraxin family, in human mononuclear phagocytes

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3483-3493 ◽  
Author(s):  
VV Alles ◽  
B Bottazzi ◽  
G Peri ◽  
J Golay ◽  
M Introna ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Mononuclear Phagocytes ◽  
Transcriptional Level ◽  
Polymorphonuclear Cells ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) are acute-phase proteins produced by liver epithelial cells. PTX3 was recently cloned as an interleukin-1 (IL-1)-inducible gene in endothelial cells, with structural similarities to pentraxins in the C- terminal half of the molecule. The present study was designed to investigate the expression of PTX3 in the human leukocyte populations. Human peripheral blood mononuclear cells exposed to lipopolysaccharide (LPS) or IL-1 beta expressed significant levels of PTX3 mRNA. Tumor necrosis factor-alpha (TNF-alpha) was a less-effective inducer of PTX3, whereas IL-6, monocyte chemotactic protein-1, macrophage colony- stimulating factor, granulocyte-macrophage colony-stimulating factor, and interferon-gamma were inactive. Among leukocytes, only monocytes exposed to inflammatory cytokines or LPS expressed the PTX3 transcript, which was undetectable in resting or stimulated polymorphonuclear cells, T or B lymphocytes, and natural killer cells. PTX3 mRNA was also inducible in in vitro monocyte-derived macrophages, in tumor-associated macrophages, and in the myelomonocytic cell lines HL60, U937, and THP1, but not in GFD8, with the latter possibly representative of earlier stages of myelomonocytic differentiation. T- and B-cell lines had no detectable PTX3. Inhibition of transcription by actinomycin D blocked induction of PTX3 in monocytes and nuclear run-on analysis showed that LPS induces the expression of the PTX3 gene at the transcriptional level in isolated monocytes. Cycloheximide had no effect on PTX3 induction in U937 cells, but was inhibitory on monocytes exposed to LPS or IL-1 beta. Monoclonal antibody against TNF and the IL-1 receptor antagonists did not inhibit induction of PTX3 in monocytes by LPS, thus excluding these cytokines as secondary stimulators of PTX3. IL-4, but not dexamethasone or transforming growth factor-beta, inhibited PTX3 expression in monocytes. Using a PTX3-specific antiserum, release of PTX3 protein was demonstrated for the first time in stimulated monocytes as well as in endothelial and fibroblastic cells. Thus, PTX3, unlike the classical pentraxins CRP and SAP, is expressed and released by cells of the monocyte-macrophage lineage exposed to inflammatory signals.

Inducible expression of PTX3, a new member of the pentraxin family, in human mononuclear phagocytes

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3483-3493 ◽  
Author(s):  
VV Alles ◽  
B Bottazzi ◽  
G Peri ◽  
J Golay ◽  
M Introna ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Mononuclear Phagocytes ◽  
Transcriptional Level ◽  
Polymorphonuclear Cells ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) are acute-phase proteins produced by liver epithelial cells. PTX3 was recently cloned as an interleukin-1 (IL-1)-inducible gene in endothelial cells, with structural similarities to pentraxins in the C- terminal half of the molecule. The present study was designed to investigate the expression of PTX3 in the human leukocyte populations. Human peripheral blood mononuclear cells exposed to lipopolysaccharide (LPS) or IL-1 beta expressed significant levels of PTX3 mRNA. Tumor necrosis factor-alpha (TNF-alpha) was a less-effective inducer of PTX3, whereas IL-6, monocyte chemotactic protein-1, macrophage colony- stimulating factor, granulocyte-macrophage colony-stimulating factor, and interferon-gamma were inactive. Among leukocytes, only monocytes exposed to inflammatory cytokines or LPS expressed the PTX3 transcript, which was undetectable in resting or stimulated polymorphonuclear cells, T or B lymphocytes, and natural killer cells. PTX3 mRNA was also inducible in in vitro monocyte-derived macrophages, in tumor-associated macrophages, and in the myelomonocytic cell lines HL60, U937, and THP1, but not in GFD8, with the latter possibly representative of earlier stages of myelomonocytic differentiation. T- and B-cell lines had no detectable PTX3. Inhibition of transcription by actinomycin D blocked induction of PTX3 in monocytes and nuclear run-on analysis showed that LPS induces the expression of the PTX3 gene at the transcriptional level in isolated monocytes. Cycloheximide had no effect on PTX3 induction in U937 cells, but was inhibitory on monocytes exposed to LPS or IL-1 beta. Monoclonal antibody against TNF and the IL-1 receptor antagonists did not inhibit induction of PTX3 in monocytes by LPS, thus excluding these cytokines as secondary stimulators of PTX3. IL-4, but not dexamethasone or transforming growth factor-beta, inhibited PTX3 expression in monocytes. Using a PTX3-specific antiserum, release of PTX3 protein was demonstrated for the first time in stimulated monocytes as well as in endothelial and fibroblastic cells. Thus, PTX3, unlike the classical pentraxins CRP and SAP, is expressed and released by cells of the monocyte-macrophage lineage exposed to inflammatory signals.

Generation of Functional Human Dendritic Cells From Adherent Peripheral Blood Monocytes by CD40 Ligation in the Absence of Granulocyte-Macrophage Colony-Stimulating Factor

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4238-4247 ◽  
Author(s):  
Peter Brossart ◽  
Frank Grünebach ◽  
Gernot Stuhler ◽  
Volker L. Reichardt ◽  
Robert Möhle ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Colony Stimulating Factor ◽  
Blood Monocytes ◽  
Rt Pcr ◽  
Cd40 Ligation ◽  
Peripheral Blood Monocytes ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Recently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF. Adherent peripheral blood mononuclear cells (PBMNC) were cultured in the presence of different cytokine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mixed lymphocyte reaction (MLR), to present soluble antigens, and to induce primary HIV-peptide–specific cytotoxic T-cell (CTL) responses in vitro. Furthermore, expression of DC-CK1, a recently identified chemokine with specific expression in DC, and of IL-18 (IGIF), a growth and differentiation factor for Th 1 lymphocytes, was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). In our study, Flt3L alone was not sufficient to generate DC and required addition of IL-4. DC generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor- (TNF-) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory molecules as well as increased allogeneic proliferative response. In contrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of mature DC and were potent stimulatory cells in priming of major histocompatibility complex (MHC) class I–restricted CTL responses against an HIV-peptide, whereas their ability to present soluble protein antigens was reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcripts were detected in all generated DC populations, independent of growth factors used. Our findings provide further evidence for the importance of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provide an additional source of DC depending on external stimuli.

Identification of granulocyte-macrophage colony stimulating factor in the rat paneth cells by singie cell RT-PCR technique and cDNA array

2001 ◽  
Vol 120 (5) ◽  
pp. A697-A698
Author(s):  
Hiroaki Fukuzawa ◽  
Mitsutaka Sawada ◽  
Shigeo Takaishi ◽  
Hiroshi Seno ◽  
Takahisa Kayahara ◽  
...  
Keyword(s):  
Cdna Array ◽  
Paneth Cells ◽  
Colony Stimulating Factor ◽  
Rt Pcr ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Vitamin C inhibits granulocyte macrophage–colony-stimulating factor–induced signaling pathways

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3205-3212 ◽  
Author(s):  
Juan M. Cárcamo ◽  
Oriana Bórquez-Ojeda ◽  
David W. Golde
Keyword(s):  
Vitamin C ◽  
Map Kinase ◽  
Luciferase Reporter ◽  
Enzymatic Reactions ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Vitamin C is present in the cytosol as ascorbic acid, functioning primarily as a cofactor for enzymatic reactions and as an antioxidant to scavenge free radicals. Human granulocyte macrophage–colony-stimulating factor (GM-CSF) induces an increase in reactive oxygen species (ROS) and uses ROS for some signaling functions. We therefore investigated the effect of vitamin C on GM-CSF–mediated responses. Loading U937 cells with vitamin C decreased intracellular levels of ROS and inhibited the production of ROS induced by GM-CSF. Vitamin C suppressed GM-CSF–dependent phosphorylation of the signal transducer and activator of transcription 5 (Stat-5) and mitogen-activated protein (MAP) kinase (Erk1 and Erk2) in a dose-dependent manner as was phosphorylation of MAP kinase induced by both interleukin 3 (IL-3) and GM-CSF in HL-60 cells. In 293T cells transfected with alpha and beta GM-CSF receptor subunits (αGMR and βGMR), GM-CSF–induced phosphorylation of βGMR and Jak-2 activation was suppressed by vitamin C loading. GM-CSF–mediated transcriptional activation of a luciferase reporter construct containing STAT-binding sites was also inhibited by vitamin C. These results substantiate the importance of ROS in GM-CSF signaling and indicate a role for vitamin C in downmodulating GM-CSF signaling responses. Our findings point to vitamin C as a regulator of cytokine redox-signal transduction in host defense cells and a possible role in controlling inflammatory responses.

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