scholarly journals Response of neutrophils to stimulus infusion: differential sensitivity of cytoskeletal activation and oxidant production.

1988 ◽  
Vol 107 (3) ◽  
pp. 951-958 ◽  
Author(s):  
G M Omann ◽  
L A Sklar
Keyword(s):  
Inflammatory Responses ◽  
Receptor Occupancy ◽  
Differential Sensitivity ◽  
Human Neutrophils ◽  
Light Scatter ◽  
Superoxide Generation ◽  
Rate Of Increase ◽  
A Cell ◽  
Oxidant Production ◽  
Formyl Peptides

The response of human neutrophils to N-formyl peptides were studied under conditions where ligand binding was controlled by infusing a cell suspension with the peptide over a time period comparable to the normal half-time for binding. Receptor occupancy was measured in real time with a fluorescently labeled peptide using flow cytometry. This binding was approximated by a simple reversible model using typical on (7 X 10(8) M- min-1) and off (0.35/min) rate constants and the infusion rates (0.02-0.2 nM/min). Under conditions of stimulus infusion intracellular calcium elevation, superoxide generation, and right angle light scatter and F-actin formation were measured. As the infusion rate was decreased into the range of 10 pM/min, lowering the rate of increase of receptor occupancy to approximately 0.5% per min, the calcium and right angle light scatter responses elongated in time and decreased in magnitude. Superoxide generation decreased below infusion rates of approximately 100 pM/min (occupancy increasing at a rate in the range of 5% per min). This behavior could contribute to differences between chemotactic responses, which appear to require low rates of receptor occupancy over long periods, and bactericidal or inflammatory responses (free radical generation and degranulation), which require bursts of occupancy of several percent of the receptors.

scholarly journals Relationship of actin polymerization and depolymerization to light scattering in human neutrophils: dependence on receptor occupancy and intracellular Ca++.

1985 ◽  
Vol 101 (3) ◽  
pp. 1161-1166 ◽  
Author(s):  
L A Sklar ◽  
G M Omann ◽  
R G Painter
Keyword(s):  
Ligand Binding ◽  
Actin Polymerization ◽  
Receptor Occupancy ◽  
Human Neutrophils ◽  
Second Phase ◽  
Light Scatter ◽  
Half Time ◽  
Initial Value ◽  
Intracellular Ca ◽  
Relationship Of

When exposed to the N-formylated chemoattractant peptides, neutrophils undergo a transient ruffling followed by a polarization that involves a redistribution of F-actin (Fechheimer, M., and S. H. Zigmond, 1983, Cell Motil., 3:349-361). The cells also undergo a biphasic right angle light scatter response whose first phase is maximal 10-15 s after exposure to the stimulus, and whose second phase is longer in duration and maximal only after 1 min or more (Yuli, I., and R. Snyderman, 1984, J. Clin. Invest. 73:1408-1417). We now report that the first phase is accompanied by a transient polymerization of actin (monitored by cytometric analysis of phallacidin staining according to the method of Howard, T. H., and W. H. Meyer, 1984, J. Cell Biol., 98:1265-1271) and the second phase is accompanied by a more sustained polymerization of actin. Based on correlated measurements of ligand binding (Sklar, L. A., D. A. Finney, Z. G. Oades, A. J. Jesaitis, R. G. Painter, and C. G. Cochrane, 1984, J. Biol. Chem., 259:5661-5669) and intracellular Ca++ elevation (under conditions where we use the fluorescent Ca++ chelator Quin 2 to modulate intracellular Ca++ levels), we conclude that this first phase requires less than 100 receptors/cell (out of 50,000) and does not require the release of intracellular stores of Ca++. In contrast, the sustained polymerization requires both the occupancy of thousands of receptors (an estimated 10% of the receptors per minute) and may be somewhat sensitive to the availability of intracellular Ca++. When ligand binding is interrupted, F-actin rapidly depolymerizes with a half-time of no greater than approximately 15 s, and the transient light scatter response decays toward its initial value in parallel. Partial disaggregation of the cells follows the recovery of these responses. Based on these observations, we suggest that transient actin polymerization and transient cell ruffling give rise to transient aggregation as long as degranulation is limited.

Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites

2001 ◽  
Vol 358 (2) ◽  
pp. 315-324 ◽  
Author(s):  
Tosti J. MANKELOW ◽  
Lydia M. HENDERSON
Keyword(s):  
Nadph Oxidase ◽  
Human Neutrophils ◽  
Closed Channel ◽  
Superoxide Generation ◽  
Free System ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Target Sites ◽  
A Cell ◽  
Membrane Components

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91phox to function as an H+ pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H+ flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H+ channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67phox and p47phox, to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b558 spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H+ pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.

scholarly journals A sand fly salivary protein acts as a neutrophil chemoattractant

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anderson B. Guimaraes-Costa ◽  
John P. Shannon ◽  
Ingrid Waclawiak ◽  
Jullyanna Oliveira ◽  
Claudio Meneses ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Calcium Influx ◽  
Parasite Burden ◽  
Salivary Protein ◽  
Sand Fly ◽  
Human Neutrophils ◽  
Chemotactic Protein ◽  
The Impact

AbstractApart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

scholarly journals Zinc and magnesium ions synergistically inhibit superoxide generation by cultured human neutrophils—a promising candidate formulation for amnioinfusion fluid

2010 ◽  
Vol 85 (2) ◽  
pp. 209-213 ◽  
Author(s):  
Toshiyuki Uchida ◽  
Hiroaki Itoh ◽  
Yuki Nakamura ◽  
Yukiko Kobayashi ◽  
Kyuya Hirai ◽  
...  
Keyword(s):  
Human Neutrophils ◽  
Magnesium Ions ◽  
Superoxide Generation ◽  
Promising Candidate

A cell-free preparation of human neutrophils catalyzing NADPH-dependent conversion of leukotriene B4

1984 ◽  
Vol 125 (2) ◽  
pp. 615-621 ◽  
Author(s):  
Hideki Sumimoto ◽  
Koichiro Takeshige ◽  
Hironori Sakai ◽  
Shigeki Minakami
Keyword(s):  
Leukotriene B4 ◽  
Human Neutrophils ◽  
A Cell

scholarly journals Electrospun Polydioxanone Loaded With Chloroquine Modulates Template-Induced NET Release and Inflammatory Responses From Human Neutrophils

2021 ◽  
Vol 9 ◽  
Author(s):  
Allison E. Fetz ◽  
Shannon E. Wallace ◽  
Gary L. Bowlin
Keyword(s):  
Growth Factor ◽  
Inflammatory Responses ◽  
Human Peripheral Blood ◽  
Early Time ◽  
Human Neutrophils ◽  
Late Time ◽  
Dependent Manner ◽  
Blood Neutrophils ◽  
Net Release

The implantation of a biomaterial quickly initiates a tissue repair program initially characterized by a neutrophil influx. During the acute inflammatory response, neutrophils release neutrophil extracellular traps (NETs) and secrete soluble signals to modulate the tissue environment. In this work, we evaluated chloroquine diphosphate, an antimalarial with immunomodulatory and antithrombotic effects, as an electrospun biomaterial additive to regulate neutrophil-mediated inflammation. Electrospinning of polydioxanone was optimized for rapid chloroquine elution within 1 h, and acute neutrophil-biomaterial interactions were evaluated in vitro with fresh human peripheral blood neutrophils at 3 and 6 h before quantifying the release of NETs and secretion of inflammatory and regenerative factors. Our results indicate that chloroquine suppresses NET release in a biomaterial surface area–dependent manner at the early time point, whereas it modulates signal secretion at both early and late time points. More specifically, chloroquine elution down-regulates interleukin 8 (IL-8) and matrix metalloproteinase nine secretion while up-regulating hepatocyte growth factor, vascular endothelial growth factor A, and IL-22 secretion, suggesting a potential shift toward a resolving neutrophil phenotype. Our novel repurposing of chloroquine as a biomaterial additive may therefore have synergistic, immunomodulatory effects that are advantageous for biomaterial-guided in situ tissue regeneration applications.

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