scholarly journals The TLR7/8 Agonist CL097 PrimesN-Formyl-Methionyl-Leucyl-Phenylalanine–Stimulated NADPH Oxidase Activation in Human Neutrophils: Critical Role of p47phox Phosphorylation and the Proline Isomerase Pin1

2012 ◽  
Vol 189 (9) ◽  
pp. 4657-4665 ◽  
Author(s):  
Karama Makni-Maalej ◽  
Tarek Boussetta ◽  
Margarita Hurtado-Nedelec ◽  
Sahra Amel Belambri ◽  
Marie-Anne Gougerot-Pocidalo ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Critical Role ◽  
Human Neutrophils ◽  
Nadph Oxidase Activation

Stimulation of the Mineralocorticoid Receptor by Aldosterone Leads to Activation of HL-60 Neutrophils,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3206-3206
Author(s):  
Carlos E Vazquez ◽  
Gregory N Prado ◽  
Enrique R Maldonado ◽  
Gabriela Saca ◽  
Iren M Ortiz ◽  
...  
Keyword(s):  
Western Blot ◽  
Respiratory Burst ◽  
Mineralocorticoid Receptor ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Dead Cell ◽  
Cardiovascular Morbidity And Mortality

Abstract Abstract 3206 Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protein kinase C-β contributes to NADPH oxidase activation in neutrophils

2000 ◽  
Vol 347 (1) ◽  
pp. 285-289 ◽  
Author(s):  
Lodewijk V. DEKKER ◽  
Michael LEITGES ◽  
Gabriel ALTSCHULER ◽  
Nishil MISTRY ◽  
Aileen MCDERMOTT ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Specific Inhibitor ◽  
Human Neutrophils ◽  
Cell Fractionation ◽  
Coated Particles ◽  
Kinase C ◽  
Pkc Β ◽  
Nadph Oxidase Activation

We have analysed the involvement of the β isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-β is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcγ-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-β specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-β, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-β also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-β isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcγ receptors in neutrophils.

Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation

2016 ◽  
Vol 93 ◽  
pp. 190-203 ◽  
Author(s):  
Deepika Awasthi ◽  
Sheela Nagarkoti ◽  
Amit Kumar ◽  
Megha Dubey ◽  
Abhishek Kumar Singh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidized Ldl ◽  
Human Neutrophils ◽  
Trap Formation ◽  
Extracellular Trap ◽  
Nadph Oxidase Activation

scholarly journals Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes

1995 ◽  
Vol 308 (3) ◽  
pp. 991-994 ◽  
Author(s):  
S Dusi ◽  
M Donini ◽  
F Rossi
Keyword(s):  
Nadph Oxidase ◽  
G Protein ◽  
Oxygen Radicals ◽  
Human Neutrophils ◽  
Enzyme Complex ◽  
Granulomatous Disease ◽  
Activated Neutrophils ◽  
Functional Complex ◽  
Flavocytochrome B ◽  
Nadph Oxidase Activation

NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.

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