Isolation of Human Neutrophils From Venous Blood

Nauseef, William M.

doi:10.1007/978-1-59745-467-4_2

Cited by 158 publications

(90 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

Section: Human Neutrophil Isolationmentioning

confidence: 99%

“…Neutrophils were isolated from heparinized human blood by centrifugation on FicollPaque (GE Healthcare, Munich, Germany), dextran sedimentation, and hypotonic lysis, as previously described [38]. Cells were resuspended at 5 × 10 6 /mL in complete medium: RPMI 1640 (HyClone Laboratories, Logan, UT, USA) supplemented with 10% FBS (Gibco Invitrogen, Carlsbad, CA, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL).…”

Section: Human Neutrophil Isolationmentioning

confidence: 99%

See 1 more Smart Citation

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

et al. 2013

View full text Add to dashboard Cite

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS).Interleukin-1 beta (IL-1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1β processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1β; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1β out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1β secretion might constitute a physiological mechanism to control IL-1β-dependent inflammatory processes where neutrophils play a crucial role.Keywords: Caspase-1 r IL-1β r NADPH-oxidase r Neutrophil r Reactive oxygen species Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionNeutrophils represent the first line of defense against bacterial and fungal infections. Their pivotal role is highlighted by recurCorrespondence: Dr. Analía S. Trevani e-mail: analiatrevani@yahoo.com.ar rent infections in individuals suffering from neutrophil functional disorders or neutropenia [1]. Neutrophils kill microbes by the release of destructive molecules such as proteases and highly reactive oxygen species (ROS), and can also produce a variety of proteins, including cytokines, chemotactic molecules, and other mediators that are involved in their effector functions [2]. However, the microbicidal effectiveness of neutrophils is sometimes obscured by the damage suffered by adjacent healthy tissues; this is a price that has to be paid to contain potentially C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2013. 43: 3324-3335 Innate Immunity 3325 life-threatening situations [3]. Although infections are the major triggers of neutrophil recruitment, many different sterile stimuli, including mechanical trauma, ischemia, toxins, minerals, crystals, and chemicals also lead to neutrophil accumulation in the tissues. In these situations, neutrophils may contribute to inflict collateral damage on otherwise healthy cells [3]. Interleukin-1 beta (IL-1β) is a key cytokine involved in the development of neutrophilic inflammation induced either by microbial or sterile inflammatory stimuli. It is a potent multifunctional proinflammatory cytokine, whose activity is controlled at the le...

show abstract

Section: Human Neutrophil Isolationmentioning

confidence: 99%

Section: Human Neutrophil Isolationmentioning

confidence: 99%

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Buffy coats were purchased from the North London Blood Transfusion Centre (Colindale, U.K.). Neutrophils were isolated as described previously (14). Briefly, RBCs were sedimented with 3% dextran (Fisher Scientific, Leicestershire, U.K.) at room temperature for 40 min.…”

Section: Neutrophil Isolationmentioning

confidence: 99%

Attenuated, Oncolytic, but Not Wild-Type Measles Virus Infection Has Pleiotropic Effects on Human Neutrophil Function

Zhang

Patel

Dey

et al. 2012

The Journal of Immunology

View full text Add to dashboard Cite

We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.

show abstract

“…Isolation of Neutrophils and Co-culture with A549 CellsHuman neutrophils were isolated from venous blood by buoyant density centrifugation (21), and their purity was determined by analysis of a cytospin slide stained with Diff-Quick (Thermo Fisher Scientific). Confluent monolayers of A549 cells, 10 6 isolated human neutrophils, or both in co-culture were incubated in 2 ml of HBSS ϩ in 6-well tissue culture plates at 37°C.…”

Section: Induction Of Mpo In Macrophages With Lipopolysaccharide (Lps)mentioning

confidence: 99%

Myeloperoxidase-induced Genomic DNA-centered Radicals

Gomez-Mejiba

Zhai

Giménez

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.Activation of neutrophils can initiate chemical mutagenesis and carcinogenesis by producing oxidative damage to the genome in the inflammatory environment (1-3). Myeloperoxidase (MPO, 4 donor hydrogen peroxide, oxidoreductase, EC 1.11.1.7), is a hemeprotein found in the azurophilic granules of neutrophils (4 -6). MPO uses H 2 O 2 to oxidize chloride ions, converting them into the powerful oxidant hypochlorous acid (HOCl/OCl Ϫ , pK a ϭ 7.46) (5). Although other mammalian peroxidases can oxidize a number of halides and pseudohalides to hypohalous and pseudohypohalous acids, MPO is the only mammalian enzyme that produces HOCl under physiological conditions (5). In sites of inflammation, H 2 O 2 used in the MPOcatalyzed oxidation of chloride is produced by dismutation of superoxide radical anion (O 2 . ). It is noteworthy that DNA is negatively charged and MPO is a cationic protein (5), which suggests that they can bind each other by electrostatic interactions. MPO is known to be taken up by cells surrounding an inflammatory site (7). It might be anticipated that this would make them highly vulnerable to DNA damage induced by H 2 O 2 . DNA-chloramines, which are less reactive than HOCl, are produced when HOCl reacts with DNA at its heterocyclic (ring) amino groups of guanosine and thymine groups and with exocyclic amino groups of guanosine, adenosine, and cytidine (8). Once formed, DNA-chloramines appear to undergo both one-and two-electron decay to produce DNA nitrogen-centered radicals, a process that is catalyzed by reduced metals and is promoted by UV irradiation and high temperatures (8 -9). Importantly, nitrogen-and carboncentered radicals can be trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form radic...

show abstract

Isolation of Human Neutrophils From Venous Blood

Cited by 158 publications

References 2 publications

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

Attenuated, Oncolytic, but Not Wild-Type Measles Virus Infection Has Pleiotropic Effects on Human Neutrophil Function

Myeloperoxidase-induced Genomic DNA-centered Radicals

Contact Info

Product

Resources

About