scholarly journals β2-Integrins and Acquired Glycoprotein IIb/IIIa (GPIIb/IIIa) Receptors Cooperate in NF-κB Activation of Human Neutrophils

2007 ◽  
Vol 282 (38) ◽  
pp. 27960-27969 ◽  
Author(s):  
Birgit Salanova ◽  
Mira Choi ◽  
Susanne Rolle ◽  
Maren Wellner ◽  
Friedrich C. Luft ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Surface Expression ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Mobility Shift ◽  
Reverse Transcription Pcr ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Β2 Integrins

Microparticles from various cells are generated during inflammation. Platelet-derived microparticles (PMPs) harbor receptors that are not genuinely expressed by neutrophils. We tested whether or not functional glycoprotein IIb/IIIa (GPIIb/IIIa) receptors can be acquired by neutrophils via PMPs and whether these receptors participate in pro-inflammatory signaling. Surface expression was analyzed by flow cytometry and confocal microscopy. NF-κB activation was analyzed by Western blot experiments, electrophoretic mobility shift assays, and reverse transcription-PCR. Cell adhesion and spreading were estimated by myeloperoxidase assay and light microscopy. We found that PMPs transfer GPIIb/IIIa receptors to isolated and whole blood neutrophils via PMPs. We used specific antibodies in granulocyte macrophage colony-stimulating factor-treated neutrophils and observed that acquired GPIIb/IIIa receptors co-localized with β2-integrins and cooperated in NF-κB activation. We show that Src and Syk non-receptor tyrosine kinases, as well as the actin cytoskeleton, control NF-κB activation. In contrast to NF-κB, acquisition of GPIIb/IIIa receptors was not necessary to induce adhesion to fibronectin or phosphatidylinositol 3-kinase/Akt signaling. When granulocyte macrophage colony-stimulating factor-stimulated neutrophils were incubated on fibronectin, strong NF-κB activation was observed, but only after loading with PMPs. Blocking either β2-integrins or GPIIb/IIIa receptors abrogated this effect. Therapeutic GPIIb/IIIa inhibitors were similarly effective. The compounds also inhibited NF-κB-dependent tumor necrosis factor-α mRNA up-regulation. The data implicate GPIIb/IIIa receptors as new therapeutic targets in neutrophil-induced inflammation.

scholarly journals Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 37-45 ◽  
Author(s):  
D Metcalf ◽  
CG Begley ◽  
GR Johnson ◽  
NA Nicola ◽  
MA Vadas ◽  
...  
Keyword(s):  
Colony Formation ◽  
Specific Activity ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Direct Stimulation ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Marrow Cultures

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.

Receptor expression and oxidase activity in human neutrophils: Regulation by granulocyte-macrophage colony-stimulating factor and dependence upon protein biosynthesis

1990 ◽  
Vol 10 (4) ◽  
pp. 393-401 ◽  
Author(s):  
Steven W. Edwards ◽  
Fiona Watson ◽  
Ronald MacLeod ◽  
John Davies
Keyword(s):  
De Novo ◽  
Protein Biosynthesis ◽  
Neutrophil Function ◽  
Membrane Receptors ◽  
Receptor Expression ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Incubation of human bloodstream neutrophils with 50 u/ml recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) “primed” the respiratory burst (as assessed by fMet-Leu-Phe stimulated luminol-dependent chemiluminescence) and resulted in a rapid (within 15 min) upregulation of expression of CD11b and CD18 (as measured by FACS analysis). This rapid “priming” and modulation of receptor expression was not inhibited by cycloheximide and hence appeared to be independent of de novo protein biosynthesis. When neutrophils were incubated for up to 5 h in culture, the fluorescence distributions of CD11b and CD18 declined indicating the loss of expression of these receptors as the neutrophils aged, but in rGM-CSF treated suspensions receptor expression was maintained. When neutrophils were incubated in the presence of cycloheximide, they progressively lost their ability to generate reactive oxidants in response to fMet-Leu-Phe so that by 5 h incubation with this inhibitor they could only generate about 25% of the oxidative response stimulated in untreated cells, and the expression of CD16 and CD18 was grossly impaired. Similar effects were observed in rGM-CSF treated suspensions except that cycloheximide required longer incubation times (typically 4–5 h) before impairment of function or receptor expression occurred. These data show that de novo protein biosynthesis is required for both the maintenance of neutrophil function and also for the continued expression of some plasma membrane receptors.

scholarly journals Characterization of nuclear factors that bind to a critical positive regulatory element of the human granulocyte-macrophage colony- stimulating factor promoter

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2523-2530 ◽  
Author(s):  
JK Fraser ◽  
S Tran ◽  
SD Nimer ◽  
JC Gasson
Keyword(s):  
Regulatory Element ◽  
Point Mutations ◽  
Regulatory Sequence ◽  
Colony Stimulating Factor ◽  
Mobility Shift ◽  
Gm Csf ◽  
Nuclear Factors ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates the proliferation, maturation, and functional activity of myeloid cells in peripheral blood and bone marrow. Expression of GM-CSF is tightly regulated and is limited to cells stimulated directly (T cells, macrophages) or indirectly (fibroblasts, endothelial cells) by immune challenge. Several studies of the transcriptional control of GM-CSF expression have elucidated a region of the GM-CSF promoter that mediates positive regulatory activity in a number of cell types. This region contains a direct repeat of the sequence CATTA/T that extends from nucleotides -37 to -48 upstream of the start of mRNA synthesis. Although specific DNA:protein interactions have been shown within this region, neither the nature nor the number of nuclear factors responsible for these interactions have been characterized. In this study, we use DNase I footprinting analysis to demonstrate that point mutations, which inactivate the GM-CSF promoter, disrupt DNA:protein interactions within this region. By combined electrophoretic mobility shift and ultraviolet cross-linking analysis, we have detected several protein species that bind specifically to the positive regulatory sequence.

Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2539-2553 ◽  
Author(s):  
PD Doshi ◽  
JF DiPersio
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Surface Expression ◽  
Colony Stimulating Factor ◽  
Cos Cells ◽  
Gm Csf ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.

scholarly journals Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter.

1994 ◽  
Vol 14 (9) ◽  
pp. 5975-5985 ◽  
Author(s):  
K M Sakamoto ◽  
J K Fraser ◽  
H J Lee ◽  
E Lehman ◽  
J C Gasson
Keyword(s):  
Binding Site ◽  
Electrophoretic Mobility ◽  
Colony Stimulating Factor ◽  
Mobility Shift ◽  
Response Gene ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Electrophoretic Mobility Shift Assays ◽  
Stimulating Factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

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