Actin Polymerization, Calcium-Transients, and Phospholipid Metabolism in Human Neutrophils After Stimulation with Interleukin-8 and N-formyl Peptide

1994 ◽  
Vol 102 (3) ◽  
pp. 310-314 ◽  
Author(s):  
Johannes Norgauer ◽  
Jean Krutmann ◽  
Gustav J. Dobos ◽  
Alexis E. Traynor-Kaplan ◽  
Zenaida G. Oades ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Phospholipid Metabolism ◽  
Interleukin 8 ◽  
Calcium Transients ◽  
Human Neutrophils ◽  
Formyl Peptide

scholarly journals Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils.

1989 ◽  
Vol 109 (3) ◽  
pp. 1133-1140 ◽  
Author(s):  
J Norgauer ◽  
I Just ◽  
K Aktories ◽  
L A Sklar
Keyword(s):  
Actin Polymerization ◽  
Response Characteristic ◽  
Formyl Peptide Receptor ◽  
Human Neutrophils ◽  
Peptide Ligand ◽  
Light Scatter ◽  
Binding Studies ◽  
Rhodamine Phalloidin ◽  
Formyl Peptide ◽  
C2 Toxin

Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.

scholarly journals Actin assembly in electropermeabilized neutrophils: role of intracellular calcium.

1990 ◽  
Vol 110 (6) ◽  
pp. 1975-1982 ◽  
Author(s):  
G P Downey ◽  
C K Chan ◽  
S Trudel ◽  
S Grinstein
Keyword(s):  
Actin Polymerization ◽  
Cytosolic Calcium ◽  
Leukotriene B4 ◽  
Human Neutrophils ◽  
Actin Assembly ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Sequence Of Events ◽  
Formyl Peptide

Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium [( Ca2+]i) in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca2+ ionophores can be known to induce actin assembly, the following observations suggest that an increased [Ca2+]i is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2(+)-free medium, depleted of internal Ca2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2+]i, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 microM free Ca2+, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]i beyond the resting level by direct addition of CaCl2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]i resulted in spontaneous actin assembly. To reconcile these findings with the actin-polymerizing effects of Ca2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]i. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]i, but rather that elevated [Ca2+]i facilitates actin disassembly, an effect possibly mediated by Ca2(+)-sensitive actin filament-severing proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.

scholarly journals Chemical Composition and Immunomodulatory Activity of Essential Oils from Rhododendron albiflorum

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3652
Author(s):  
Igor A. Schepetkin ◽  
Gulmira Özek ◽  
Temel Özek ◽  
Liliya N. Kirpotina ◽  
Andrei I. Khlebnikov ◽  
...  
Keyword(s):  
Chemical Composition ◽  
Essential Oils ◽  
Neutrophil Migration ◽  
Compositional Analysis ◽  
Microglial Cells ◽  
Formyl Peptide Receptor ◽  
Human Neutrophils ◽  
Immunomodulatory Activity ◽  
Hl60 Cells ◽  
Formyl Peptide

Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REOFl) versus leaf (REOLv) essential oils revealed significant differences. REOFl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REOLv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REOLv and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N-formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca2+]i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.

scholarly journals Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.

1993 ◽  
Vol 123 (4) ◽  
pp. 895-907 ◽  
Author(s):  
B A McCormick ◽  
S P Colgan ◽  
C Delp-Archer ◽  
S I Miller ◽  
J L Madara
Keyword(s):  
Epithelial Cells ◽  
Salmonella Typhimurium ◽  
Apical Membrane ◽  
Coli Strain ◽  
Formyl Peptide Receptor ◽  
Human Neutrophils ◽  
Classical Pathway ◽  
Intestinal Epithelial ◽  
Formyl Peptide

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.

The protein kinase C inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion

1990 ◽  
Vol 96 (1) ◽  
pp. 99-106
Author(s):  
H.U. Keller ◽  
V. Niggli ◽  
A. Zimmermann ◽  
R. Portmann
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Kinase C ◽  
Chemotactic Peptides ◽  
Protein Kinase C Inhibitor ◽  
Shape Changes ◽  
Stimulation Of

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.

scholarly journals Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan

1992 ◽  
Vol 282 (2) ◽  
pp. 393-397 ◽  
Author(s):  
J Norgauer ◽  
M Eberle ◽  
H D Lemke ◽  
K Aktories
Keyword(s):  
Pertussis Toxin ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Superoxide Radical ◽  
Human Neutrophils ◽  
Superoxide Anion Production ◽  
Radical Production ◽  
Rapid Formation ◽  
Primary Granules ◽  
Type 3

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.

scholarly journals Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

2008 ◽  
Vol 295 (5) ◽  
pp. C1354-C1365 ◽  
Author(s):  
Troy Mitchell ◽  
Andrea Lo ◽  
Michael R. Logan ◽  
Paige Lacy ◽  
Gary Eitzen
Keyword(s):  
Actin Polymerization ◽  
Microscopic Analysis ◽  
Formyl Peptide Receptor ◽  
Human Neutrophils ◽  
Cell Cortex ◽  
Secretory Cells ◽  
Actin Remodeling ◽  
Granule Exocytosis ◽  
Primary Granule

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (≤10 μM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca2+ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca2+ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

[Ca2+ ]i Changes and Respiratory Burst in Human Neutrophils and Monocytes Induced by NAP-1/Interleukin-8, NAP-2, and gro /MGSA

1991 ◽  
Vol 50 (3) ◽  
pp. 279-286 ◽  
Author(s):  
Alfred Walz ◽  
Federica Meloni ◽  
Ian Clark-Lewis ◽  
Vinzenz von Tscharner ◽  
Marco Baggiolini
Keyword(s):  
Respiratory Burst ◽  
Interleukin 8 ◽  
Human Neutrophils
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