scholarly journals Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils.

1982 ◽  
Vol 95 (1) ◽  
pp. 234-241 ◽  
Author(s):  
S T Hoffstein ◽  
R S Friedman ◽  
G Weissmann
Keyword(s):  
Light Scattering ◽  
Cell Surface ◽  
Cell Shape ◽  
Shape Change ◽  
Single Cells ◽  
Chemotactic Factor ◽  
Human Neutrophils ◽  
Transient Change ◽  
Membrane Addition ◽  
Induced Aggregation

Neutrophils stimulated by the chemotactic factor formyl-methionyl-leucyl-phenyl-alanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells. With time (3-5 min), small aggregates disappear and single cells reappear. The transient change in adhesiveness is accompanied by a persistent change in cell shape; the cells become polarized and protrude ruffles from one sector of the cell surface. During aggregation the cells adhere to one another with smooth sides together and ruffles pointed outward. During disaggregation the cells dissociate laterally with the simultaneous internalization of membrane in the region opposite the ruffles. Particle bound to the surface by charge (thorotrast, cationized ferritin) are concentrated and internalized in this region. The change in cell shape from round to ruffled occurs within seconds, suggesting that membrane is added to the cell surface from an intracellular store. We therefore quantified surface membrane by electron microscopy morphometry and measured a 25% increase within 10 s of adding FMLP. The source of new membrane appeared to be the specific granule membrane since the kinetics of granule discharge (between 30% and 50% of all release occurs in the first 10 s) correlate with the appearance of new membrane. Furthermore, the amount of membrane that appears at the cell surface at 10 s correlates with that lost from intracellular granules in that time. Chemotaxin-induced aggregation thus begins with granule discharge and membrane addition followed by protrusion of ruffles. Adherence is maximal at 60 s and the gradual loss of adhesiveness that follows is associated with uropod formation and enhanced endocytic activity.

scholarly journals Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils

1992 ◽  
Vol 117 (4) ◽  
pp. 765-774 ◽  
Author(s):  
TD Coates ◽  
RG Watts ◽  
R Hartman ◽  
TH Howard
Keyword(s):  
Shape Change ◽  
Single Cells ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Asymmetric Distribution ◽  
Chemotactic Peptide ◽  
Filamentous Actin ◽  
Cell Asymmetry ◽  
Texas Red ◽  
Cell Thickness

Polymerization of actin has been associated with development of polar shape in human neutrophils (PMN). To examine the relation of filamentous actin (F-actin) distribution to shape change in PMN, we developed a method using computerized video image analysis and fluorescence microscopy to quantify distribution of F-actin in single cells. PMN were labeled with fluorescent probe NBD-phallicidin to measure filamentous actin and Texas red to assess cell thickness. We show that Texas red fluorescence is a reasonable measure of cell thickness and that correction of the NBD-phallicidin image for cell thickness using the Texas red image permits assessment of focal F-actin content. Parameters were derived that quantify total F-actin content, movement of F-actin away from the center of the cell, asymmetry of F-actin distribution, and change from round to polar shape. The sequence of change in F-actin distribution and its relation to development of polar shape in PMN was determined using these parameters. After stimulation with chemotactic peptide at 25 degrees C, F-actin polymerized first at the rim of the PMN. This was followed by development of asymmetry of F-actin distribution and change to polar shape. The dominant pseudopod developed first in the region of lower F-actin concentration followed later by polymerization of actin in the end of the developed pseudopod. Asymmetric F-actin distribution was detected in round PMN before development of polar shape. Based upon these data, asymmetric distribution of F-actin is coincident with and probably precedes development of polar shape in PMN stimulated in suspension by chemotactic peptide.

Roles of calcium in aggregation of rat ascites hepatoma cells by cell surface-associated adhesive factor

1984 ◽  
Vol 66 (1) ◽  
pp. 367-382
Author(s):  
S. Kurano ◽  
M. Ishida ◽  
Y. Ishimaru
Keyword(s):  
Cell Surface ◽  
Single Cells ◽  
Cell Aggregation ◽  
Ascites Hepatoma ◽  
A Cell ◽  
Rat Ascites Hepatoma ◽  
Junctional Complexes ◽  
Adhesive Factor ◽  
Induced Aggregation

Our previous studies have shown that a cell surface-associated adhesive factor (AF), separated from rat ascites hepatoma AH136B cells of a differentiated type and highly purified by chromatography, induces not only aggregation of dissociated AH136B cells or rat ascites hepatoma AH109A cells of an undifferentiated type but also adhesiveness characterized by the development of junctional complexes; the AF-induced aggregation of the cells was Ca2+-dependent. Further analysis of the roles of Ca2+ in cell aggregation was performed using AH109A cells (present as single cells in vivo). (1) AF clearly enhanced 45Ca uptake by the cells; (2) calmodulin was isolated from the cells; (3) calmodulin inhibitor, W-7 (N-(6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide), strongly inhibited aggregation of the cells; (4) W-7 also inhibited the clustering or capping of AF-binding sites on the cell surface; (5) binding of 125I-labelled AF to the cells was independent of Ca2+ concentration; (6) binding of 125I-labelled AF to AF-conjugated beads was not observed, independently of the presence of Ca2+. These findings suggest that Ca2+ and Ca2+-activated calmodulin may play a key role in the process of aggregation of the cells by controlling the microfilament components and that Ca2+ may not be involved either in the interactions between AF and its cellular receptor or in linkages of AF molecules.

Application of high-resolution field-emission LVSEM, backscatter imaging, immunogold staining to definition of the spatial distributions of two human neutrophil adherence receptors

1993 ◽  
Vol 51 ◽  
pp. 36-37
Author(s):  
Robert D. Nelson ◽  
Sharon R. Hasslen ◽  
Stanley L. Erlandsen
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Three Dimensional ◽  
Human Neutrophils ◽  
Spatial Distributions ◽  
Immunogold Staining ◽  
Functional Control ◽  
Neutrophil Adherence ◽  
Definition Of ◽  
Mode Of Attachment

Receptors are commonly defined in terms of number per cell, affinity for ligand, chemical structure, mode of attachment to the cell surface, and mechanism of signal transduction. We propose to show that knowledge of spatial distribution of receptors on the cell surface can provide additional clues to their function and components of functional control.L-selectin and Mac-1 denote two receptor populations on the neutrophil surface that mediate neutrophil-endothelial cell adherence interactions and provide for targeting of neutrophil recruitment to sites of inflammation. We have studied the spatial distributions of these receptors using LVSEM and backscatter imaging of isolated human neutrophils stained with mouse anti-receptor (primary) antibody and goat anti-mouse (secondary) antibody conjugated to 12 nm colloidal gold. This combination of techniques provides for three-dimensional analysis of the expression of these receptors on different surface membrane domains of the neutrophil: the ruffles and microvilli that project from the cell surface, and the cell body between these projecting structures.

Adenosine Diphosphate-Induced Aggregation of Human Platelets in Flow Through Tubes: III. Shear and Extrinsic Fibrinogen-Dependent Effects

1994 ◽  
Vol 71 (01) ◽  
pp. 078-090 ◽  
Author(s):  
H L Goldsmith ◽  
M M Frojmovic ◽  
Susan Braovac ◽  
Fiona McIntosh ◽  
T Wong
Keyword(s):  
Shear Rate ◽  
Single Cells ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Size Analysis ◽  
Fibrinogen Concentration ◽  
Human Fibrinogen ◽  
Shear Rates ◽  
Rate Dependent ◽  
Induced Aggregation

SummaryThe effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23°C was studied using a previously described double infusion technique and resistive particle counter size analysis (1). Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 × 105 μl−1; (17)] with [fibrinogen] from 0 to 1.2μM, the, rate and extent of aggregation with 0.7 μM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, Ḡ, = 41.9, 335 and 1,335 s−1. As measured by the decrease in singlet concentration, aggregation at 1.2 μM fibrinogen increased with increasing Ḡ up to 1,335 s1, in contrast to that previously reported in citratcd plasma, in which aggregation reached a maximum at Ḡ = 335 s−1. Without added fibrinogen, there was no aggregation at Ḡ = 41.9 s1; at Ḡ = 335 s1, there was significant aggregation but with an initial lag time, aggregation increasing further at Ḡ = 1,335 s−1. Without added fibrinogen, aggregation was abolished at all Ḡ upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab’)2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37°C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab’)2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36 374, and resuspension in Tyrodes-albumin at 5 × 104 μl−1, aggregated with 2 and 5 μM ADP at Ḡ = 335 and 1,335 s−1 in the absence of added fibrinogen. We therefore postulate that a protein such as von Willebrand factor, secreted during platelet isolation or in flow at sufficiently high shear rates, may yield the observed shear-rate dependent aggregation without fibrinogen.

Platelet Aggregation Caused by Dithiothreitol

1984 ◽  
Vol 51 (01) ◽  
pp. 119-124 ◽  
Author(s):  
M B Zucker ◽  
N C Masiello
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Electrophoretic Patterns ◽  
Discernible Effect ◽  
Variable Extent ◽  
Triton X ◽  
Induced Aggregation ◽  
Platelet Shape

SummaryMacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DTT- induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0° C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to “expose” the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I- labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

The Inhibitory Effect of Heparin and Related Glycosaminoglycans on Neutrophil Chemotaxis

1984 ◽  
Vol 52 (02) ◽  
pp. 134-137 ◽  
Author(s):  
Yaacov Matzner ◽  
Gerard Marx ◽  
Ruth Drexler ◽  
Amiram Eldor
Keyword(s):  
Specific Binding ◽  
Anticoagulant Activity ◽  
Neutrophil Migration ◽  
Inhibitory Effect ◽  
Chemotactic Factor ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
Neutrophil Chemotaxis ◽  
Inhibitory Effects ◽  
Chemotactic Factors

SummaryClinical observations have shown that heparin has antiinflammatory activities. The effect of heparin on neutrophil chemotaxis was evaluated in vitro in the Boyden Chamber. This method enabled differentiation between the direct effects of heparin on neutrophil migration and locomotion, and its effects on chemotactic factors. Heparin inhibited both the random migration and directed locomotion of human neutrophils toward zymosan-activated serum (ZAS) and F-met-leu-phe (FMLP). Inhibition was found to be dependent on the concentrations of the heparin and of the chemotactic factors. No specific binding of heparin to the neutrophils could be demonstrated, and heparin’s inhibitory effects were eliminated by simple washing of the cells. When added directly to the chamber containing chemotactic factor, heparin inhibited the chemotactic activity of ZAS but not that of FMLP, suggesting a direct inhibitory effect against C5a, the principal chemotactic factor in ZAS.Experiments performed with low-molecular-weight heparin, N-desulfated heparin, dextran sulfate, chondroitin sulfate and dextran indicated that the inhibitory effects of heparin on neutrophil chemotaxis are not related to its anticoagulant activity, but probably depend on the degree of sulfation of the heparin molecule.

scholarly journals Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonas Mattisson ◽  
Marcus Danielsson ◽  
Maria Hammond ◽  
Hanna Davies ◽  
Caroline J. Gallant ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Cells ◽  
Surface Protein ◽  
Protein Abundance ◽  
Blood Leukocytes ◽  
Chromosome Y ◽  
Increased Risk ◽  
A Cell ◽  
Pseudoautosomal Regions ◽  
Male Specific

AbstractMosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.

scholarly journals Mapping Surface Charge Distribution of Single-Cell via Charged Nanoparticle

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1519
Author(s):  
Leixin Ouyang ◽  
Rubia Shaik ◽  
Ruiting Xu ◽  
Ge Zhang ◽  
Jiang Zhe
Keyword(s):  
Cell Surface ◽  
Charge Density ◽  
Surface Charge ◽  
Charge Distribution ◽  
Surface Charge Density ◽  
Single Cells ◽  
Fluorescent Light ◽  
Fluorescent Nanoparticles ◽  
Cell Surface Charge ◽  
Surface Charge Distribution

Many bio-functions of cells can be regulated by their surface charge characteristics. Mapping surface charge density in a single cell’s surface is vital to advance the understanding of cell behaviors. This article demonstrates a method of cell surface charge mapping via electrostatic cell–nanoparticle (NP) interactions. Fluorescent nanoparticles (NPs) were used as the marker to investigate single cells’ surface charge distribution. The nanoparticles with opposite charges were electrostatically bonded to the cell surface; a stack of fluorescence distribution on a cell’s surface at a series of vertical distances was imaged and analyzed. By establishing a relationship between fluorescent light intensity and number of nanoparticles, cells’ surface charge distribution was quantified from the fluorescence distribution. Two types of cells, human umbilical vein endothelial cells (HUVECs) and HeLa cells, were tested. From the measured surface charge density of a group of single cells, the average zeta potentials of the two types of cells were obtained, which are in good agreement with the standard electrophoretic light scattering measurement. This method can be used for rapid surface charge mapping of single particles or cells, and can advance cell-surface-charge characterization applications in many biomedical fields.

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