The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release

Bystrzycka, Weronika; Moskalik, Aneta; Sieczkowska, Sandra; Manda-Handzlik, Aneta; Demkow, Urszula; Ciepiela, Olga

doi:10.5114/ceji.2016.58811

Cited by 25 publications

(29 citation statements)

References 14 publications

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“…Subsequently, cells were stimulated with PMA (100 nmol L −1 ) or calcium ionophore A23187 (CI, 4 μmol L −1 ) and incubated for 3 h. Unstimulated cells were used as controls. After incubation, extracellular DNA release was measured fluorometrically using a FLUOstar Omega plate reader (BMG Labtech, Ortenberg, Germany), as described previously …”

Section: Methodsmentioning

confidence: 99%

“…After incubation, extracellular DNA release was measured fluorometrically using a FLUOstar Omega plate reader (BMG Labtech, Ortenberg, Germany), as described previously. 47 To present data as the % of DNA release and easily analyze the fold changes in NETs formation, the data were normalized. Mean fluorescence readout from unstimulated cells from all performed experiments (i.e.…”

Section: Nets Quantificationmentioning

confidence: 99%

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The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

et al. 2018

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Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.

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Section: Methodsmentioning

confidence: 99%

Section: Nets Quantificationmentioning

confidence: 99%

The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

et al. 2018

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“…Neutrophils were isolated by gradient density centrifugation and sedimentation in 1% polyvinyl alcohol, as previously described [19]. To stimulate NETs release, the cells were suspended in RPMI-1640 without phenol red with 10 mM HEPES and seeded at 2.5 × 10 4 cells per well into 8-well Lab-Tek chambers for NETs immunolabeling; 0.5-1 × 10 5 cells per well were seeded into 24-or 96-well plates for extracellular DNA quantification; 2-5 × 10 4 cells per well were seeded into 24-or 48-well plates for live NETs imaging.…”

Section: Neutrophil Isolation and Stimulationmentioning

confidence: 99%

Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps

Manda-Handzlik

Bystrzycka

Cieloch

et al. 2019

Cell. Mol. Life Sci.

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Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions. Keywords Autophagy • Neutrophil extracellular traps • Nitric oxide • Peroxynitrite • Phosphoinositide 3-kinases • Reactive nitrogen species Abbreviations 3-MA 3-Methyladenine ABAH Aminobenzoic acid hydrazide AKT Protein kinase B CGD Chronic granulomatous disease CI Calcium ionophore A23187 DHR 123 Dihydrorhodamine 123 DPI Diphenyleneiodonium ERK Extracellular signal-regulated kinases IL Interleukin LC3 protein Light chain 3 protein L-NAME N-nitroarginine methyl ester LPS Lipopolysaccharide MAPK Mitogen-activated protein kinases MPO Myeloperoxidase NAC N-acetylcysteine NE Neutrophil elastase NEi Neutrophil elastase inhibitor GW 311616A NETs Neutrophil extracellular traps NBT Nitroblue tetrazolium NO Nitric oxide NOS Nitric oxide synthase Cellular and Molecular Life Sciences Malgorzata Wachowska and Urszula Demkow have contributed equally.

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“…3,29,30 Indeed, the bacterial species isolated from patients with AECRS is anaerobic and is similar to those found in CRS. 47 On the F I G U R E 7 A, Extracellular DNA concentration evaluated with dsDNA assay kit in neutrophils unstimulated (cont), neutrophils treated with PMA (cont + PMA), neutrophils treated clarithromycin, azithromycin, josamycin, or dexamethasone. 47 On the F I G U R E 7 A, Extracellular DNA concentration evaluated with dsDNA assay kit in neutrophils unstimulated (cont), neutrophils treated with PMA (cont + PMA), neutrophils treated clarithromycin, azithromycin, josamycin, or dexamethasone.…”

Section: Discussionmentioning

confidence: 99%

“…whereas clindamycin has no influence on NETs release. 47 On the F I G U R E 7 A, Extracellular DNA concentration evaluated with dsDNA assay kit in neutrophils unstimulated (cont), neutrophils treated with PMA (cont + PMA), neutrophils treated clarithromycin, azithromycin, josamycin, or dexamethasone. Data from six independent experiments are presented as means ± SEM and are derived from six individuals (*There is statistical significant difference in the concentration of extracellular DNA between control and dose of macrolides or glucocorticoid, P < 0.05).…”

Section: Bystrzycka Et Al Reported That Amoxicillin Induces Nets Formmentioning

confidence: 99%

Neutrophil extracellular traps in nasal secretions of patients with stable and exacerbated chronic rhinosinusitis and their contribution to induce chemokine secretion and strengthen the epithelial barrier

Hwang

Kim

et al. 2019

Clin Experimental Allergy

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Background: Neutrophil extracellular traps (NETs) participate in innate immunity by trapping microorganisms. Their pathophysiological implications have not been defined in chronic rhinosinusitis (CRS). Objective: We investigated the presence of NETs in nasal secretion of patients with stable or exacerbated CRS and evaluated whether NETs participate in the secretion of chemokines in sinonasal epithelial cells, the epithelial permeability, and transendothelial leucocyte migration, and elucidate whether NETs are released by macrolides and dexamethasone. Methods: The presence of NETs in nasal secretion and the release of NETs from neutrophils stimulated with macrolides or dexamethasone were evaluated by dsDNA Assay kit and fluorescence microscope. The chemokine secretion, epithelial permeability, and transendothelial leucocyte migration were measured in cultured cells incubated with NETs, the supernatant of unstimulated neutrophils (unstim), NETs inhibitor (DPI), or H3Cit, where the expression of junctional complex proteins and ICAM-1 was evaluated by real-time PCR, Western blots, and confocal microscope. Results: The amount of NETs and NETs-forming neutrophils in nasal secretion increased in exacerbated CRS. Epithelial cells treated with NETs or H3Cit secreted chemokines and showed decreased permeability associated with up-regulated junctional complex proteins. Increased transendothelial leucocyte migration associated with up-regulated ICAM-1 was noted in endothelial cells treated with NETs or H3Cit.These findings were not found in cells treated with unstim, or DPI. NETs were released by macrolides, but not by dexamethasone. Conclusions and Clinical Relevance: NETs formation increased in exacerbated CRS,inducing chemokine secretion, strengthening the epithelial barrier, and promoting the neutrophils infiltration. Therefore, the release of NETs in CRS might be beneficial or detrimental to CRS patients. K E Y W O R D Schemokines, dexamethasone, epithelial permeability, macrolides, neutrophil extracellular traps, transendothelial leucocyte migration

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The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release

Cited by 25 publications

References 14 publications

The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps

Neutrophil extracellular traps in nasal secretions of patients with stable and exacerbated chronic rhinosinusitis and their contribution to induce chemokine secretion and strengthen the epithelial barrier

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