scholarly journals Activated Fc receptor supramolecular complex

2021 ◽  
Author(s):  
Veronika Petrenko
Keyword(s):  
Fc Receptor ◽  
Supramolecular Complex ◽  
Dominant Negative ◽  
Immunofluorescent Staining ◽  
Human Neutrophils ◽  
Particle Uptake ◽  
Cumulative Score ◽  
Interaction Partners ◽  
Cell Affinity ◽  
And Control

Phagocytosis is a part of immune response. IgG opsonized particles of greater than 1 um are recognized by Fcy receptors on the surface of professional phagocytes such as macrophages and neutrphils. IgGs are part of the immune system and is a cognate ligand of the Fc receptor. Live Cell Affinity Receptor chromatography (LARC) was used to capture an activated Fcy receptor supramolecular complex from the surface of live human neutrophils, by allowing IgG opsonized microbeads to bind to the cell surface. The cells were burst in PBS, collected and digested along side with controls. Isolated FcyR complex was analysed by LC-MS/MS. Fc and control experiment lists of SEQUEST correlated proteins were screened for a total cumulative score of at least 2400 and a minimum of three different peptides. This served as the basis of protein involvement in the FcyR mediated phagocytosis, which were then searched with iHOP for their interaction partners. Gathered interactions were then exported and Cytoscape, Osprey and String algorithms were used to generate network of interacting proteins. PAKs2-4 and PAK6 were detected with LARC. PAK2 and PAK4 were predicted by algorithms to have a central role in particle uptake. From Western Blotting, endogenous PAKs2-4 and PAK6 were detected in murine macrophages. Immunofluorescent staining was then used to verify the presence of these proteins in the forming phagosome and showed localization of PAKs to the phagosome. The same effect was observed with transfection of GFP constructs of PAKs. Upon transfection with dominant negative PAKs reduction in phagocytosis was observed.

scholarly journals Activated Fc receptor supramolecular complex

2021 ◽  
Author(s):  
Veronika Petrenko
Keyword(s):  
Fc Receptor ◽  
Supramolecular Complex ◽  
Dominant Negative ◽  
Immunofluorescent Staining ◽  
Human Neutrophils ◽  
Particle Uptake ◽  
Cumulative Score ◽  
Interaction Partners ◽  
Cell Affinity ◽  
And Control

Phagocytosis is a part of immune response. IgG opsonized particles of greater than 1 um are recognized by Fcy receptors on the surface of professional phagocytes such as macrophages and neutrphils. IgGs are part of the immune system and is a cognate ligand of the Fc receptor. Live Cell Affinity Receptor chromatography (LARC) was used to capture an activated Fcy receptor supramolecular complex from the surface of live human neutrophils, by allowing IgG opsonized microbeads to bind to the cell surface. The cells were burst in PBS, collected and digested along side with controls. Isolated FcyR complex was analysed by LC-MS/MS. Fc and control experiment lists of SEQUEST correlated proteins were screened for a total cumulative score of at least 2400 and a minimum of three different peptides. This served as the basis of protein involvement in the FcyR mediated phagocytosis, which were then searched with iHOP for their interaction partners. Gathered interactions were then exported and Cytoscape, Osprey and String algorithms were used to generate network of interacting proteins. PAKs2-4 and PAK6 were detected with LARC. PAK2 and PAK4 were predicted by algorithms to have a central role in particle uptake. From Western Blotting, endogenous PAKs2-4 and PAK6 were detected in murine macrophages. Immunofluorescent staining was then used to verify the presence of these proteins in the forming phagosome and showed localization of PAKs to the phagosome. The same effect was observed with transfection of GFP constructs of PAKs. Upon transfection with dominant negative PAKs reduction in phagocytosis was observed.

DARC Regulates Angiogenesis by Mediating Vascular Endothelial Cell Migration

2020 ◽  
Author(s):  
Xiaolin Wang ◽  
Yongqian Bian ◽  
Yuejun Li ◽  
Jing Li ◽  
Congying Zhao ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Vascular Endothelial Cell ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
Immunofluorescent Staining ◽  
Control Group ◽  
Rhoa Activation ◽  
And Control

Abstract Background: DARC (The Duffy antigen receptor for chemokines) is a kind of glycosylated membrane protein that binds to members of the CXC chemokine family associated with angiogenesis and has recently been reported to be implicated in diverse normal physiologic processes. This study aimed to investigate the involvement of DARC in angiogenesis, which is known to generate new capillary blood vessels from preexisting ones. Methods: HDMECs (Human dermal microvascular endothelial cells) were divided into two groups (DARC overexpression group, and control group). We used Brdu staining to detect cell proliferation, and wound healing assay to detect cell migration. Then tube formation assay were observed. Also, western blot and immunofluorescent staining were used to estimate the relationship between DARC and RhoA (Ras homolog gene family, member A). Results: HDMECs proliferation, migration, and tube formation were inhibited significantly when DARC was overexpressed intracellular. DARC impaired microfilament dynamics and intercellular connection in migrating cells, and RhoA activation underlay the effect of DARC on endothelial cell. Furthermore, DARC inhibited the formation of new capillaries in vitro. Conclusion: Our findings revealed the role of DARC in the angiogenic process and provided a novel mechanism for RhoA activation during endothelial cell migration and angiogenesis.

scholarly journals Immunohistochemical detection of Fc receptor. I. Light microscopic demonstration of Fc receptor by using soluble immune complexes of peroxidase-antiperoxidase immunoglobulin G.

1977 ◽  
Vol 25 (4) ◽  
pp. 252-258 ◽  
Author(s):  
G Itoh ◽  
S Miura ◽  
I Suzuki
Keyword(s):  
Cell Surface ◽  
Immunoglobulin G ◽  
Immune Complexes ◽  
Surface Characterization ◽  
Fc Receptor ◽  
Frozen Sections ◽  
Microscopic Demonstration ◽  
Lymph Node Cells ◽  
Soluble Immune Complexes ◽  
And Control

The mouse mesenteric lymph node cells (in the cell suspension and frozen sections) were incubated in the soluble immune complexes of peroxidase-antiperoxidase immunoglobulin G. After being washed, they were reacted with diaminobenzidine tetrahydrochloride. Light microscopically brown-colored granules were observed on the cell surface of a proportion of small lymphocytes. In frozen sections, a proportion of small lymphocytes were stained dark brown on the cell surface. Characterization and control experiments suggest that the binding of peroxidase-antiperoxidase immunoglobulin G to the cell surface is mediated by Fc receptor. Peroxidase-antiperoxidase immunoglobulin G, therefore, can be used as in indicator of Fc receptor.

scholarly journals RacG Regulates Morphology, Phagocytosis, and Chemotaxis

2006 ◽  
Vol 5 (10) ◽  
pp. 1648-1663 ◽  
Author(s):  
Baggavalli P. Somesh ◽  
Georgia Vlahou ◽  
Miho Iijima ◽  
Robert H. Insall ◽  
Peter Devreotes ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Free System ◽  
Mild Phenotype ◽  
Particle Uptake ◽  
Phosphoinositide 3 Kinase

ABSTRACTRacG is an unusual member of the complex family of Rho GTPases inDictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or moreDictyosteliumRho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

Modulation of glucocorticoid receptor expression, inflammation, and cell apoptosis in septic guinea pig lungs using methylprednisolone

2008 ◽  
Vol 295 (6) ◽  
pp. L998-L1006 ◽  
Author(s):  
Koki Kamiyama ◽  
Naoyuki Matsuda ◽  
Seiji Yamamoto ◽  
Ken-ichi Takano ◽  
Yasuo Takano ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Cell Apoptosis ◽  
Nuclear Translocation ◽  
Lavage Fluid ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Immunofluorescent Staining ◽  
Terminal Deoxynucleotidyl Transferase ◽  
High Dose ◽  
Mobility Shift

The use of glucocorticoids for treatment of sepsis has waxed and waned during the past several decades, and recent randomized controlled trials have evoked a reassessment of this therapy. Most glucocorticoid actions are mediated by its specific intracellular receptors (GRs). Thus we initially evaluated whether sepsis and high-dose corticosteroid therapy can regulate guinea pig pulmonary expression of GRs: active receptor, GRα, and dominant negative receptor, GRβ. Sepsis induction by LPS injection (300 μg/kg ip) decreased mRNA and protein levels of GRα and increased protein expression of GRβ in lungs. High-dose methylprednisolone (40 mg/kg ip), administered simultaneously with LPS, markedly potentiated the decrease in GRα expression but slightly affected the increase in GRβ expression. Consequently, this led to a significant reduction in GRα nuclear translocation. Nevertheless, methylprednisolone treatment strongly eliminated LPS induction of NF-κB activity, as determined by NF-κB nuclear translocation and by gel mobility shift assays. Furthermore, the LPS-induced increase in inflammatory cells in bronchoalveolar lavage fluid was blunted by administration of the corticosteroid. On the other hand, immunofluorescent staining for cleaved caspase-3 showed a marked increase in this proapoptotic marker in lung sections, and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) represented an enhanced appearance of cell apoptosis in lungs and spleen when methylprednisolone was given together with LPS. Cell apoptosis is now considered to play a role in the pathogenesis of septic syndrome. We thus suggest that the action of glucocorticoids at high doses to accelerate sepsis-induced cell apoptosis may overwhelm their therapeutic advantages in septic shock.

The Fc receptor-cytoskeleton complex from human neutrophils

2011 ◽  
Vol 75 (2) ◽  
pp. 450-468 ◽  
Author(s):  
Angelica K. Florentinus ◽  
Andy Jankowski ◽  
Veronika Petrenko ◽  
Peter Bowden ◽  
John G. Marshall
Keyword(s):  
Fc Receptor ◽  
Human Neutrophils

scholarly journals Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
PA Ory ◽  
MR Clark ◽  
AS Talhouk ◽  
IM Goldstein
Keyword(s):  
Molecular Mass ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Fc Receptor ◽  
Apparent Molecular Mass ◽  
Human Neutrophils ◽  
Rabbit Reticulocyte Lysate System

Abstract Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

Neutrophil p29 Peroxiredoxin VI (Prdx VI) Enhances Bactericidal Activity and Chemotaxis in Myeloid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1354-1354 ◽  
Author(s):  
Michael Ellison ◽  
Gail Thurman ◽  
Daniel R. Ambruso
Keyword(s):  
Nadph Oxidase ◽  
Bactericidal Activity ◽  
Respiratory Burst ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
And Control ◽  
O2 Production ◽  
In Cells

Abstract Abstract 1354 Poster Board I-377 Introduction We have identified a 29 kDa protein from human neutrophils which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, reconstituted, NADPH oxidase system. The protein was identified as peroxiredoxin VI by sequence and the recombinant molecule was found to have both peroxiredoxin activity and calcium-independent PLA2 activity that is optimum at low pH (aiPLA2). Although p29 Prdx VI is found in many tissues, its role in myeloid cells is not well established. To explore other roles of p29, in addition to its effect on the respiratory burst, a PLB-985 cell line with shRNA mediated knockdown of p29 Prdx VI was established. Chemotaxis, as well as ingestion and killing S. aureus were determined in knockdown and control cells. Methods PLB-985 cells were transfected with a plasmid encoding a p29 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot with no changes in actin or other oxidase components. After maturation of the knockdown and control cells by DMSO for 4 days, each was combined with serum opsonized Staph. aureus in a 2 to1 human cell to bacterial cell ratio, bacterial cells remaining at various times were measured by plating aliquots of the cell mixtures and counting bacterial colonies which grew overnight. To evaluate ingestion, aliquots of the cell mixtures were transferred to slides by cytospin, stained, and examined under a light microscope to determine what proportion of PLB-985 cells had internalized bacteria. To evaluate chemotaxis, distances of migration toward chemo-attractant (opsonized zymosan) in a Boyden chamber were measured for differentiated p29 Prdx VI knockdown and control cells. Results Using stable expression of shRNA p29 protein was reduced to 31+/-18% (SD) of that in non-knockdown control cells. In two separate assays of bactericidal activity, cells without knockdown of p29 Prdx VI had 17 and 13% of initial bacteria surviving at 30 min; cells with p29 Prdx VI knockdown had 30 and 56% of bacteria surviving. This defect in bactericidal activity since ingestion was no different between the two types of cells at 0, 5, 10, and 15 min after addition of the bacteria. In response to zymosan activated serum, stimulated directed migration (distance of leading front in response to zymosan activated serum minus distance of leading front in response to buffer) was greater in cells without knockdown (23.9 ± 3.0 microns, mean ± SEM, n = 4 separate experiments) than movement by cells with knockdown of p29 Prdx VI (18.3 ± 5.3 microns). The difference was significant, p<0.05 by paired t test. Conclusion Optimal O2− production during the respiratory burst in intact myeloid cells is dependent on p29. Deficient bactericidal activity was demonstrated at 30 min; this decrease could not be associated with a difference in ingestion. In addition, directed cell migration was also decreased in cells with decreased amounts of p29 Prdx VI. These results indicate that in addition to its effect on the respiratory burst, p29 Prdx supports multiple functions in neutrophils. Disclosures No relevant conflicts of interest to declare.

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