UNIPROT:P04141 - FACTA Search

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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dendritic cells (DC) are migratory cells which exhibit complex trafficking properties in vivo, involving interaction with vascular and lymphatic endothelium and extracellular matrix (ECM). The underlying mechanisms involved in these processes are still ill defined. In the present study we have investigated the ability of DC to interact in vitro with human vascular endothelial cells (EC) and ECM. DC were differentiated from monocytes by in vitro exposure to granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days. In adhesion assays a considerable proportion of DC bound to resting EC monolayers: (17% +/- 4%, mean +/- SE of eight experiments). Adhesion to tumor necrosis factor (TNF)-activated EC was increased to 29% +/- 5% (n = 8). Binding to resting EC was strongly inhibited by anti-CD11a and CD11b, but not by CD11c monoclonal antibodies (MoAbs); on TNF-activated EC, anti-VLA-4 in concert with anti-CD18 inhibited adhesion by more than 70%. Binding to a natural ECM, derived from cultured EC, or to purified fibronectin was high: 52% +/- 6% (n = 8) involved VLA-4 and VLA-5 integrins. In a transmigration assay, 10% +/- 2% (n = 6) of input cells were able to cross the EC monolayer. Unlike adhesion, transendothelial migration was significantly reduced by anti-CD31 MoAb. The amount of DC transmigrated through a monolayer of EC was increased twofold to threefold by a defined set of C-C chemokines including RANTES, MIP1alpha, MIP5, and, to a lesser extent, by MIP1beta and MCP-3. Most importantly, in view of the trafficking pattern of these cells, a significant proportion of DC (13% +/- 4% of input cells seeded) was able to migrate across the endothelial basement membrane and, subsequently, across the endothelial barrier (reverse transmigration). The adhesion molecules and chemoattractants characterized herein are likely to underlie the complex trafficking of DC in vivo.
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PMID:Adhesion, transendothelial migration, and reverse transmigration of in vitro cultured dendritic cells. 963 18

It is well known that ozone (O3) causes acute lung inflammation. What is not known is whether there is progression of the inflammatory response in humans with repeated short-term exposures. Our study was designed to test the hypothesis that repeated exposures to a high-ambient concentration of O3 (0.2 ppm) over several days would cause more inflammation than a single exposure. Fifteen healthy volunteers were exposed in random fashion to 0.2 ppm ozone for 4 h on a single day and to 0.2 ppm O3 for 4 h on 4 consecutive days while exercising moderately for 30 min of each hour. Pulmonary function tests were obtained immediately before and after each 4-h exposure. Bronchoscopy was performed 20 h after the completion of each exposure arm to obtain bronchoalveolar lavage (BAL) for measurement of markers of inflammation. Our results show initial progression followed by attenuation of the acute physiologic response to O3 with repeated daily exposures. We found a significant difference in percent change in FEV1, FVC, and specific airway resistance (SRaw) across the single-day exposure when compared with the change across Day 4 of the 4-d exposure. Bronchial fraction (the first 15 ml of BAL return) and BAL were analyzed for the following end points: total and differential cell counts, total protein, lactate dehydrogenase (LDH), fibronectin, interleukin-6 (IL-6), interleukin-8 (IL-8), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In the bronchial fraction the number of polymorphonuclear cells (PMN)s and fibronectin concentration were significantly decreased after 4-d exposure compared with single-day exposure. In BAL, significant decreases in the number of PMNs, fibronectin, and IL-6 were found after 4-d exposure versus single-day exposure. These results suggest that there is attenuation of the O3-induced inflammatory response in both proximal airways and distal lung with repeated daily exposures.
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PMID:Ozone-induced inflammation is attenuated with multiday exposure. 970 Jan 32

We studied the phenotypic characteristics of spontaneously migrated skin dendritic cells (sDC) and monocyte-derived dendritic cells (moDC), generated under different culture conditions, and their interactions with fibronectin (FN) and endothelial cells. Monocyte-derived dendritic cells were obtained after culturing monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) (800 U/ml) and interleukin-4 (IL-4) (500 U/ml) with either 10% fetal bovine serum (FBS) or 10% allogeneic human serum (HS). Regardless of the type of serum used, the majority of moDC expressed human leucocyte antigen-DR (HLA-DR) and CD86. On day 5 of incubation, 20-67% of moDC cultured in the presence of HS (HS-moDC) expressed CD1a, b and c versus 94-97% when cultured in the presence of FBS (FBS-moDC). DC showed a differential gradient of adhesion to FN: FBS-moDC>HS-moDC>sDC approximately monocytes. Both FBS-moDC and HS-moDC were strongly positive for CD49e (alpha5-integrin) and CD29 (beta1-integrin) but negative for CD49d (alpha4-integrin). A monoclonal antibody (mAb) against CD49e blocked the adhesion of both types of moDC to FN. Although both FBS-moDC and HS-moDC attached to endothelium (a 76% and 63% increase, respectively), only HS-moDC were able to migrate through non-activated endothelium. Overall, these results suggest that spontaneously migrated sDC are less adherent to FN than moDC, that HS and FBS induce differences in CD1 expression, that HS-moDC are less adhesive to FN and endothelial cells but more motile than FBS-moDC, and that alpha5beta1-integrin is the molecule involved in moDC adhesion to FN.
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PMID:Interactions of dendritic cells with fibronectin and endothelial cells. 982 88

When the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor was incubated with neutrophils adherent to plastic tissue culture plates or plates coated with extracellular matrix proteins, a rapid (3 min) but transient formation of phosphatidic acid was observed. This stimulation was dependent on the dose of GM-CSF, with an EC50 of 140 pM, and was further enhanced (up to 350%) with the PA phosphatase inhibitor propranolol in a dose-dependent manner. Conversely, GM-CSF was unable to trigger any PA formation in neutrophils maintained in suspension, even in the presence of soluble fibronectin. However, GM-CSF did prime the cells for enhanced PA formation in the presence of a secondary stimulus (fMet-Leu-Phe or PAF). GM-CSF also caused a time-dependent stimulation of diacylglycerol formation in adherent, but not suspended, cells and elicited a time-dependent stimulation of phosphatidylethanol formation, with a concomitant decrease in the formation of PA only at early (< 7 min) times. These observations were consistent with a rapid activation of the enzyme phospholipase D in adherent cells stimulated with GM-CSF. Additional data indicated that the source of DAG was PLD coexisting with PLC, especially at later times ( > 7 min) of stimulation with GM-CSF. Finally, the formation of PA and PEt, and to a minor extent, DAG, were inhibited by the protein tyrosine kinase inhibitor erbstatin in conditions in which tyrosine phosphorylation occurred. Taken together the data indicate that GM-CSF rapidly activates PLD in adherent cells, which is responsible for the generation of PA. Thus, PLD activation is an early event in neutrophil signal transduction following exposure of adherent cells to GM-CSF.
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PMID:Binding of GM-CSF to adherent neutrophils activates phospholipase D. 1035 94

Engagement of integrin receptors during cell adhesion leads to changes in the morphology and the state of activation of cells. We therefore examined whether mast cell adhesion to extracellular matrix proteins affects the synthesis and release of various proinflammatory cytokines. Cells of the human mast cell line HMC-1 were added to fibronectin (FN)-, vitronectin (VN)- or, as a control, bovine serum albumin (BSA)-coated wells and were stimulated with phorbol 12-myristate 13-acetate (PMA) and/or calcium ionophore A23187 (ionophore). Cytokine production was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cell extracts and enzyme-linked immunosorbent assay (ELISA) analysis of cell supernatants. After a 4-hr incubation, mRNA expression of interleukin (IL)-8 (and weakly of IL-6) was up-regulated in matrix-adherent cells, with further increase in the presence of PMA and/or ionophore, compared with unstimulated cells. High-level de novo expression of IL-3 and of granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed mainly in matrix-adherent cells. These changes were paralleled by the secretory pattern of HMC-1 cells after a 24-hr stimulation. Unstimulated cells adherent to FN or VN had already released small amounts of IL-8, and both VN- and FN-adherent cells produced, almost invariably, a higher level of cytokines than BSA-exposed cells after additional stimulation. These results show that mast cell adhesion to matrix proteins by itself has only selected and minor effects, but additional activation of mast cells by secretory stimuli causes significantly enhanced cytokine gene expression and secretion, suggesting that mast cells are far more active in their natural tissue environment than hitherto suggested from data in suspension cultures.
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PMID:Adhesion of human mast cells to extracellular matrix provides a co-stimulatory signal for cytokine production. 1054 Feb 24

Regulatory mechanisms governing adhesion of hematopoietic progenitor cells to the stromal nische are poorly understood. Growth factors such as stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor, and thrombopoietin were reported to upregulate the adhesion of hematopoietic progenitors to immobilized fibronectin through activation of integrin alpha4beta1 and alpha5beta1. Macrophage inflammatory protein (MIP)-1alpha is a C-C chemokine that suppresses colony formation by stem/progenitor cells in vitro. We asked if MIP-1alpha would modulate the adhesive phenotype of colony-forming cells (CFCs) obtained from healthy donor bone marrow (BM), cord blood (CB), and mobilized peripheral blood (mPB) CD34+ cells, in comparison with SCF, using immobilized fibronectin. SCF significantly increased the level of adhesion of CFCs from BM, CB, and mPB. On the other hand, MIP-1alpha significantly increased the level of adhesion of CFCs from BM and CB, but less so from mPB. The effects of MIP-1alpha were inhibited by blocking antibodies to integrin alpha4, alpha5, or beta1, and polymerization plus rearrangement of F-actin were observed in affected cells by labeling with rhodamine-conjugated phalloidine. These data indicate that the effect of MIP-1alpha on the adhesive phenotype of CFCs is mediated by modulation of the organization of integrin. The amount of MIP-1alpha receptor on mPB was less than for BM or CB, which may explain the distinct characteristics in the adhesive response induced by MIP-1alpha. We suggest that hematopoietic progenitor cells from different sources may be heterogeneous with respect to maturation, integrin affinity, MIP-1alpha receptor expression, and regulation of MIP-1alpha signaling. Our data indicate that MIP-1alpha may affect migration, homing, and mobilization of hematopoietic progenitors by modulating the adhesive phenotype of these cells.
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PMID:Macrophage inflammatory protein 1alpha enhances in a different manner adhesion of hematopoietic progenitor cells from bone marrow, cord blood, and mobilized peripheral blood. 1056 Sep 11

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is composed of two chains that belong to the superfamily of cytokine receptors typified by the growth hormone receptor. A common structural element found in cytokine receptors is a module of two fibronectin-like domains, each characterized by seven beta-strands denoted A-G and A'-G', respectively. The alpha-chain (GMRalpha) confers low affinity GM-CSF binding (K(d) = 1-5 nM), whereas the beta-chain (beta(c)) does not bind GM-CSF by itself but confers high affinity binding when associated with alpha (K(d) = 40-100 pM). In the present study, we define the molecular determinants required for ligand recognition and for stabilization of the complex through a convergence of several approaches, including the construction of chimeric receptors, the molecular dynamics of our three-dimensional model of the GM.GMR complex, and site-directed mutagenesis. The functional importance of individual residues was then investigated through ligand binding studies at equilibrium and through determination of the kinetic constants of the GM.GMR complex. Critical to this tripartite complex is the establishment of four noncovalent bonds, three that determine the nature of the ligand recognition process involving residues Arg(280) and Tyr(226) of the alpha-chain and residue Tyr(365) of the beta-chain, since mutations of either one of these residues resulted in a significant decrease in the association rate. Finally, residue Tyr(365) of beta(c) serves a dual function in that it cooperates with another residue of beta(c), Tyr(421) to stabilize the complex since mutation of Tyr(365) and Tyr(421) result in a drastic increase in the dissociation rate (Koff). Interestingly, these four residues are located at the B'-C' and F'-G' loops of GMRalpha and of beta(c), thus establishing a functional symmetry within an apparently asymmetrical heterodimeric structure.
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PMID:Molecular determinants of the granulocyte-macrophage colony-stimulating factor receptor complex assembly. 1056 87

The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand-specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine-dependent and factor-independent activation of the receptor.
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PMID:The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5. 1073 32

Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)
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PMID:Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta-chain bound to an antagonist. 1075 26

Eosinophilia is a feature of airway inflammation associated with asthma. Leukotriene antagonists provide therapeutic benefit in asthma, but their potential antiinflammatory actions have not been fully explored. We have examined the role of eosinophil-derived cysteinyl leukotrienes in the maintenance of eosinophil survival, and the involvement of leukotrienes in the paracrine stimulation of eosinophil survival by mast cells and lymphocytes. We obtained eosinophils and autologous lymphocytes from peripheral blood of asthmatic subjects. Leukotriene (LT)-B(4), LTC(4) and LTD(4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fibronectin promoted eosinophil survival. LTD(4) (10(-)(6) M) was as effective as GM-CSF (5 ng/ml) and fibronectin (400 ng/ml) in promoting survival. Lymphocytes and conditioned medium from a human mast cell line (HMC-1) induced eosinophil survival. Blockade of cysteinyl leukotriene receptors with SKF 104353 (pobilukast, 3 nM), and inhibition of 5-lipoxygenase (5-LO) with BW A4C (1 microM) and of 5-LO activating protein with MK 886 (1 microM), all increased basal rates of eosinophil apoptosis and reversed GM-CSF-induced eosinophil survival. Fifty percent reversal of GM-CSF- induced survival was achieved with SKF 104353 at 0.3 nM. The potency of SKF 104353 was two orders of magnitude greater than that of the LTB(4) receptor antagonist SB 201146. Mast cell- and lymphocyte-induced eosinophil survival were completely reversed by SB 201146, SKF 104353, BW A4C, and MK 886. These findings provide evidence for the involvement of an autocrine cysteinyl leukotriene pathway that supports eosinophil survival in response to a range of survival stimuli. They also suggest that LTB(4) could act as a paracrine stimulus of eosinophil survival.
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PMID:Leukotriene receptor antagonists and synthesis inhibitors reverse survival in eosinophils of asthmatic individuals. 1085 61

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