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.

scholarly journals Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils

2009 ◽  
Vol 297 (4) ◽  
pp. C886-C897 ◽  
Author(s):  
Phillip C. Eckels ◽  
Anirban Banerjee ◽  
Ernest E. Moore ◽  
Nathan J. D. McLaughlin ◽  
Lynn M. Gries ◽  
...  
Keyword(s):  
Shape Change ◽  
Platelet Activating Factor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Surface Expression ◽  
Cytosolic Calcium ◽  
Polymorphonuclear Neutrophils ◽  
Organ Injury ◽  
Calcium Flux ◽  
Human Neutrophils

Receptor signaling is integral for adhesion, emigration, phagocytosis, and reactive oxygen species production in polymorphonuclear neutrophils (PMNs). Priming is an important part of PMN emigration, but it can also lead to PMN-mediated organ injury in the host. Platelet-activating factor (PAF) primes PMNs through activation of a specific G protein-coupled receptor. We hypothesize that PAF priming of PMNs requires clathrin-mediated endocytosis (CME) of the PAF receptor (PAFr), and, therefore, amantadine, known to inhibit CME, significantly antagonizes PAF signaling. PMNs were isolated by standard techniques to >98% purity and tested for viability. Amantadine (1 mM) significantly inhibited the PAF-mediated changes in the cellular distribution of clathrin and the physical colocalization [fluorescence resonance energy transfer positive (FRET+)] of early endosome antigen-1 and Rab5a, known components of CME and similar to hypertonic saline, a known inhibitor of CME. Furthermore, amantadine had no effect on the PAF-induced cytosolic calcium flux; however, phosphorylation of p38 MAPK was significantly decreased. Amantadine inhibited PAF-mediated changes in PMN physiology, including priming of the NADPH oxidase and shape change with lesser inhibition of increases in CD11b surface expression and elastase release. Furthermore, rimantadine, an amantadine analog, was a more potent inhibitor of PAF priming of the N-formyl-methionyl-leucyl-phenylalanine-activated oxidase. PAF priming of PMNs requires clathrin-mediated endocytosis that is inhibited when PMNs are pretreated with either amantadine or rimantadine. Thus, amantadine and rimantadine have the potential to ameliorate PMN-mediated tissue damage in humans.

scholarly journals Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein.

1989 ◽  
Vol 109 (6) ◽  
pp. 2791-2799 ◽  
Author(s):  
E Särndahl ◽  
M Lindroth ◽  
T Bengtsson ◽  
M Fällman ◽  
J Gustavsson ◽  
...  
Keyword(s):  
G Protein ◽  
Second Messengers ◽  
Human Neutrophils ◽  
Receptor Complex ◽  
Chemotactic Peptide ◽  
Filamentous Actin ◽  
High Concentration ◽  
Intact Cells ◽  
Peptide Receptor ◽  
Receptor Complexes

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand-receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X-100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu-Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G-protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet-Leu-Phe-associated G-protein.

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.

The Signaling Pathways in Nitric Oxide Production by Neutrophils Exposed to N-nitrosodimethylamine

2018 ◽  
Vol 16 (2) ◽  
pp. 194-199
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Ewa Jablonska
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Polymorphonuclear Neutrophils ◽  
Microbial Pathogens ◽  
Human Neutrophils ◽  
No Production ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Background: Polymorphonuclear neutrophils (PMNs) play a crucial role in the innate immune system’s response to microbial pathogens through the release of reactive nitrogen species, including Nitric Oxide (NO). </P><P> Methods: In neutrophils, NO is produced by the inducible Nitric Oxide Synthase (iNOS), which is regulated by various signaling pathways and transcription factors. N-nitrosodimethylamine (NDMA), a potential human carcinogen, affects immune cells. NDMA plays a major part in the growing incidence of cancers. Thanks to the increasing knowledge on the toxicological role of NDMA, the environmental factors that condition the exposure to this compound, especially its precursors- nitrates arouse wide concern. Results: In this article, we present a detailed summary of the molecular mechanisms of NDMA’s effect on the iNOS-dependent NO production in human neutrophils. Conclusion: This research contributes to a more complete understanding of the mechanisms that explain the changes that occur during nonspecific cellular responses to NDMA toxicity.

scholarly journals Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulus-dependent increases in cytoskeletal actin of human neutrophils

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2911-2919 ◽  
Author(s):  
P Kreienbuhl ◽  
H Keller ◽  
V Niggli
Keyword(s):  
Okadaic Acid ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Calyculin A ◽  
Chemotactic Peptide ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Actin Localization ◽  
Shape Changes

Abstract The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to stimulate the incorporation of 32PO4 into many neutrophil proteins several-fold. Calyculin A was effective at 50-fold lower concentrations. In the presence of the inhibitors, the cells exhibited a nonpolar shape and the polarization response induced by chemotactic peptide was inhibited. Both phosphatase inhibitors also induced the association of F-actin with the cell membrane. A steady-state phosphatase activity is thus involved in maintaining shape and F-actin localization of resting cells. Inhibitors alone had no significant effect on the amount of cytoskeleton-associated actin. The increase in cytoskeletal actin observed at 30 minutes of stimulation with phorbol ester or 5 to 30 minutes of stimulation with chemotactic peptide, however, was abolished by okadaic acid or calyculin A, suggesting an important role of a phosphatase. In contrast, the early increase in cytoskeleton-associated actin observed at 1 minute of stimulation with peptide was not affected. This finding indicates that the increased association of actin with the cytoskeleton in the early and the later stages of neutrophil activation may be mediated by different signalling pathways.

scholarly journals Regulation of CD18 expression in human neutrophils as related to shape changes

1993 ◽  
Vol 106 (2) ◽  
pp. 493-501
Author(s):  
A. Volz
Keyword(s):  
Phorbol Myristate Acetate ◽  
Surface Expression ◽  
Cytochalasin D ◽  
Human Neutrophils ◽  
Chemotactic Peptide ◽  
Myristate Acetate ◽  
Surface Distribution ◽  
Quantitative Expression ◽  
High Concentrations ◽  
Shape Changes

The study analyses the distribution and quantitative expression of surface CD18 of neutrophils exposed to distinct stimuli that produce different types of continuous shape changes, including types that are associated with locomotion and others that are not. The chemotactic peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanine, colchicine and nocodazole were used to induce a polarized locomotor morphology, phorbol myristate acetate, 1,2-dioctanoylglycerol and 1-oleoyl-2-acetyl-glycerol to induce non-polar motile cells ruffling all over the surface and 2H2O to induce non-polar cells performing circus movements as have been previously described. Except for colchicine and nocodazole, these stimuli increased surface expression of CD18. Thus, stimulated shape changes are frequently, though not always, associated with increased surface expression of CD18. High concentrations (10(−7) to 10(−5) M) of phorbol myristate acetate but not of chemotactic peptide induced down-regulation of surface CD18. Cytochalasin D (10(−4) M) stimulated CD18 expression even though it inhibited shape changes. The surface distribution of CD18 determined by light microscopy was uniform in unstimulated cells or in various forms of stimulation except for cells treated with 10(−5) M cytochalasin D. Cytochalasin D (10(−5) M) produced CD18 accumulation at the pole opposite the F-actin cap. Experiments with colchicine, nocodazole, 2H2O and cytochalasin D suggest that microtubules as well as microfilaments modulate surface expression of CD18. The results suggest that protein kinase C and phosphatases play a role in regulating surface expression of CD18 in neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Shape oscillations of human neutrophil leukocytes: characterization and relationship to cell motility.

1996 ◽  
Vol 199 (4) ◽  
pp. 741-747
Author(s):  
M U Ehrengruber ◽  
D A Deranleau ◽  
T D Coates
Keyword(s):  
Light Scattering ◽  
Actin Polymerization ◽  
Cellular Migration ◽  
Human Neutrophils ◽  
Filamentous Actin ◽  
Cytoskeletal Component ◽  
Morphological Polarity ◽  
Shape Oscillations ◽  
Shape Changes ◽  
Neutrophil Leukocytes

When neutrophil leukocytes are stimulated by chemotactic factors or by substratum contact, they change their shape. Shape changes are a prerequisite for cellular migration and typically involve the extrusion of thin, veil-like lamellipods and the development of morphological polarity. Stimulation also leads to changes in the neutrophil content of filamentous actin (F-actin), which is the major cytoskeletal component. Suspensions of human neutrophils stimulated with chemoattractants exhibit sinusoidal light-scattering oscillations with a period of approximately 8 s at 37 degrees C. These oscillations arise from periodic fluctuations in the cell body size caused by lamellipod extension and retraction cycles. The light-scattering oscillations are paralleled by corresponding oscillations in F-actin content. This raises the interesting possibility that cyclic actin polymerization constitutes the driving force for shape oscillations of suspended neutrophils. Similar periodic shape changes are present in neutrophils crawling on a surface, suggesting that shape oscillations are important for neutrophil motion. This review summarizes our present knowledge about shape oscillations in suspended and crawling neutrophils and discusses a possible role for these oscillations in neutrophil motility.

scholarly journals Sodium Channel Nav1.3 Is Expressed by Polymorphonuclear Neutrophils during Mouse Heart and Kidney Ischemia InVivo and Regulates Adhesion, Transmigration, and Chemotaxis of Human and Mouse Neutrophils In Vitro

2018 ◽  
Vol 128 (6) ◽  
pp. 1151-1166 ◽  
Author(s):  
Marit Poffers ◽  
Nathalie Bühne ◽  
Christine Herzog ◽  
Anja Thorenz ◽  
Rongjun Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Mouse Heart ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

Abstract Background Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils. Methods Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp. Results Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2. Conclusions Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.

Sign in / Sign up

Export Citation Format

Share Document