scholarly journals Application of Western Blot for detection of neutrophil apoptosis-related proteins

2009 ◽  
Vol 57 (1) ◽  
pp. 97-104
Author(s):  
Petr Sláma
Keyword(s):  
Western Blot ◽  
Myeloid Cell ◽  
Neutrophil Apoptosis ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Western Blot Method ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Related Proteins ◽  
Stimulating Factor

The aim of this study was to evaluate suitability of using Western Blot for detection of neutrophil apo­pto­sis and neutrophil apoptosis-related proteins, respectively. Neutrophils were isolated from blood of healthy adult donors and incubated with G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocyte-macrophage colony stimulating factor), ATP (adenosine triphosphate) and FMLP (N-formyl-methionyl-leucyl-phenylalanine). The neutrophils were incubated 4, 8 and 20 hours at 37 °C. In this assay, an expression of Mcl-1 (myeloid cell leukemia 1), XIAP (X-linked inhibitor of apoptosis) and gelsolin was analysed by Western Blot method. The results showed that Western Blot is a suitable method for detection of neutrophil apoptosis-related proteins and detection of neutrophil apoptosis, respectively.

scholarly journals The Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Exists as a Preformed Receptor Complex That Can Be Activated by GM-CSF, Interleukin-3, or Interleukin-5

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3005-3017 ◽  
Author(s):  
Joanna M. Woodcock ◽  
Barbara J. McClure ◽  
Frank C. Stomski ◽  
Michael J. Elliott ◽  
Christopher J. Bagley ◽  
...  
Keyword(s):  
Myeloid Cell ◽  
Cytokine Receptor ◽  
Activation Mechanism ◽  
Receptor Complex ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor α chain (GMRα) and to the common β chain of the GM-CSF, IL-3, and IL-5 receptors (βc ) immunoprecipitated both GMRα and βc from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMRα and βc , as shown by the ability of soluble βc to associate with membrane-anchored GMRα or soluble GMRα. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMRα-associated βc . These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.

scholarly journals M-CSF and GM-CSF Regulation of STAT5 Activation and DNA Binding in Myeloid Cell Differentiation is Disrupted in Nonobese Diabetic Mice

2008 ◽  
Vol 2008 ◽  
pp. 1-8 ◽  
Author(s):  
B. Rumore-Maton ◽  
J. Elf ◽  
N. Belkin ◽  
B. Stutevoss ◽  
F. Seydel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
Myeloid Cell ◽  
Mouse Bone Marrow ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Nonobese Diabetic ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Defects in macrophage colony-stimulating factor (M-CSF) signaling disrupt myeloid cell differentiation in nonobese diabetic (NOD) mice, blocking myeloid maturation into tolerogenic antigen-presenting cells (APCs). In the absence of M-CSF signaling, NOD myeloid cells have abnormally high granulocyte macrophage colony-stimulating factor (GM-CSF) expression, and as a result, persistent activation of signal transducer/activator of transcription 5 (STAT5). Persistent STAT5 phosphorylation found in NOD macrophages is not affected by inhibiting GM-CSF. However, STAT5 phosphorylation in NOD bone marrow cells is diminished if GM-CSF signaling is blocked. Moreover, if M-CSF signaling is inhibited, GM-CSF stimulationin vitrocan promote STAT5 phosphorylation in nonautoimmune C57BL/6 mouse bone marrow cultures to levels seen in the NOD. These findings suggest that excessive GM-CSF production in the NOD bone marrow may interfere with the temporal sequence of GM-CSF and M-CSF signaling needed to mediate normal STAT5 function in myeloid cell differentiation gene regulation.

Human Interleukin-3/Granulocyte Macrophage-Colony Stimulating Factor Knock-In Mice Support Human Myeloid Cell Reconstitution and Human Immune Responses In the Lung.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3789-3789
Author(s):  
Tim Willinger ◽  
Anthony Rongvaux ◽  
Hitoshi Takizawa ◽  
Elizabeth E. Eynon ◽  
Sean Stevens ◽  
...  
Keyword(s):  
Immune Responses ◽  
Myeloid Cell ◽  
Humanized Mice ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Human Immune ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Abstract 3789 Humanized mice, i.e. mice with a functional human immune system, have great potential to study human immunology in vivo and to allow vaccine testing. To this end, mice need to fully support engraftment with human immune cells, allow infection with human pathogens, and mount effective human immune responses to pathogens. A major limitation of current humanized mice is the poor development and function of human myeloid cells. Here we report a novel strategy to overcome this limitation by generating human interleukin-3/granulocyte macrophage colony stimulating factor knock-in (hIL-3/GM-CSF KI) mice to create a better environment for human myeloid cells. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis (PAP) due to elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the PAP syndrome. In addition, these mice showed an enhanced systemic inflammatory response to LPS. Finally, humanization of IL-3 and GM-CSF lead to a stronger innate immune response against influenza virus infection. In summary, hIL-3/GM-CSF KI mice represent a new mouse model to study human immune responses in the lung and against pathogens such as influenza. Disclosures: Stevens: Regeneron Pharmaceuticals: Employment; AnaptysBio Inc: Employment.

scholarly journals Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Y Kanakura ◽  
B Druker ◽  
KW Wood ◽  
HJ Mamon ◽  
K Okuda ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Line ◽  
Human Factor ◽  
Myeloid Cell ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Myeloid Cell Line ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

Autoregulation of interleukin 6 and granulocyte-macrophage colony-stimulating factor in the differentiation of myeloid leukemic cells

1989 ◽  
Vol 9 (9) ◽  
pp. 4109-4112
Author(s):  
Y Shabo ◽  
J Lotem ◽  
L Sachs
Keyword(s):  
Gene Expression ◽  
Interleukin 6 ◽  
Myeloid Cell ◽  
Leukemic Cells ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Induction Of Differentiation ◽  
Macrophage Colony ◽  
Stimulating Factor

Induction of differentiation in one type of clone of mouse myeloid leukemic cells by mouse or human interleukin 6 (IL-6) and in another type of clone by mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to be associated with induction of IL-6 and GM-CSF mRNA and protein. The results indicated that IL-6 and GM-CSF could positively autoregulate their own gene expression during myeloid cell differentiation. It is suggested that this autoregulation may serve to enhance and prolong the signal induced by these proteins in cells transiently exposed to IL-6 or GM-CSF.

scholarly journals The diadenosine polyphosphates Ap3A and Ap4A and adenosine triphosphate interact with granulocyte-macrophage colony-stimulating factor to delay neutrophil apoptosis: implications for neutrophil: platelet interactions during inflammation

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3442-3449 ◽  
Author(s):  
L Gasmi ◽  
AG McLennan ◽  
SW Edwards
Keyword(s):  
Adenosine Triphosphate ◽  
Immune Cell ◽  
Cellular Morphology ◽  
Neutrophil Apoptosis ◽  
Colony Stimulating Factor ◽  
Diadenosine Polyphosphates ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Incubation of neutrophils with cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) delays their loss of function and changes in cellular morphology that are characteristic of apoptosis. Adenosine triphosphate (ATP) and the diadenosine polyphosphates Ap4A and AP3A were almost as effective as GM-CSF in delaying neutrophil apoptosis. The nucleotides could thus preserve cellular morphology, protect against chromatin fragmentation, and preserve functions such as NADPH oxidase activity and expression of CD16. Moreover, addition of ATP, AP3A and AP4A together with GM-CSF resulted in more pronounced protection from apoptosis than was observed during incubation with either the cytokine or the nucleotides alone. Because ATP, Ap3A, and AP4A may be secreted from activated platelets, these observations suggest that platelet-derived products, perhaps acting in combination with endothelial-derived or immune cell-derived cytokines, can regulate neutrophil function during certain types of inflammation.

scholarly journals Toxoplasma gondii Induces Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor Secretion by Human Fibroblasts: Implications for Neutrophil Apoptosis

2002 ◽  
Vol 70 (11) ◽  
pp. 6048-6057 ◽  
Author(s):  
Jacqueline Y. Channon ◽  
Kristin A. Miselis ◽  
Laurie A. Minns ◽  
Chaitali Dutta ◽  
Lloyd H. Kasper
Keyword(s):  
Conditioned Medium ◽  
Human Fibroblasts ◽  
Neutrophil Apoptosis ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Soluble Mediator ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

ABSTRACT Human neutrophils are rescued from apoptosis following incubation with once-washed, fibroblast-derived Toxoplasma gondii tachyzoites. Both infected and uninfected neutrophils are rescued, implicating a soluble mediator. In this study we investigated the origin and identity of this soluble mediator. Neutrophils were incubated either with purified tachyzoites or with conditioned medium derived from T. gondii-infected human fibroblasts. Conditioned medium was found to be a potent stimulus that delayed neutrophil apoptosis up to 72 h, whereas purified and extensively washed tachyzoites had no effect. Delayed apoptosis correlated with up-regulation of the neutrophil antiapoptotic protein, Mcl-1, and the neutrophil interleukin 3 receptor α subunit (IL-3Rα), suggesting a role for granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF and granulocyte colony-stimulating factor (G-CSF) were measurable in conditioned medium by enzyme-linked immunosorbent assay. Neutralizing antibodies to GM-CSF and G-CSF were additive in abrogating delayed neutrophil apoptosis induced by conditioned medium. Inhibitors of Src family tyrosine kinases, Gi proteins, phosphatidylinositol 3-kinase, p44 erk1 and p42 erk2 mitogen-activated protein kinases, and Jak2 kinases partially attenuated the effect of conditioned medium, consistent with a role for G-CSF and/or GM-CSF. Hence, delayed neutrophil apoptosis is mediated by GM-CSF and G-CSF secreted by T. gondii-infected human fibroblasts. This enhanced neutrophil survival may contribute to the robust proinflammatory response elicited in the T. gondii-infected host.

scholarly journals Autoregulation of interleukin 6 and granulocyte-macrophage colony-stimulating factor in the differentiation of myeloid leukemic cells.

1989 ◽  
Vol 9 (9) ◽  
pp. 4109-4112 ◽  
Author(s):  
Y Shabo ◽  
J Lotem ◽  
L Sachs
Keyword(s):  
Gene Expression ◽  
Interleukin 6 ◽  
Myeloid Cell ◽  
Leukemic Cells ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Induction Of Differentiation ◽  
Macrophage Colony ◽  
Stimulating Factor

Induction of differentiation in one type of clone of mouse myeloid leukemic cells by mouse or human interleukin 6 (IL-6) and in another type of clone by mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to be associated with induction of IL-6 and GM-CSF mRNA and protein. The results indicated that IL-6 and GM-CSF could positively autoregulate their own gene expression during myeloid cell differentiation. It is suggested that this autoregulation may serve to enhance and prolong the signal induced by these proteins in cells transiently exposed to IL-6 or GM-CSF.

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