Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2014 Aug;13(4):690-8.
doi: 10.1111/acel.12222. Epub 2014 Apr 30.

VSports - Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals

Jon Hazeldine  1 , Phillipa Harris, Iain L Chapple, Melissa Grant, Hannah Greenwood, Amy Livesey, Elizabeth Sapey, Janet M Lord
Affiliations

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals

Jon Hazeldine et al. Aging Cell. 2014 Aug.

Abstract

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF-α-primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)- and interleukin-8 (IL-8)-induced NET formation exhibits a significant age-related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL-8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol-12-myristate-13-acetate (PMA) showed no age-associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age-related decline in NET and ROS generation. TNF-α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age-matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults VSports手机版. .

Keywords: aging; neutrophil; neutrophil extracellular traps; periodontitis V体育安卓版. .

PubMed Disclaimer

"V体育ios版" Figures

Figure 1
Figure 1
Neutrophil priming significantly increases the NET production and ROS generation. (A) Neutrophils isolated from young adults (n = 5) were cultured for 15 min in the presence (black bars) or absence (white bars) of 10 ng mL−1 TNF-α followed by a 3-h stimulation with 10 ng mL−1 IL-8 or 100 ng mL−1 lipopolysaccharide (LPS). The DNA content of cell-free supernatants was then assessed by fluorometry. Data are presented as arbitrary fluorescence units (AFU) and represent the mean ± SEM. (B) Representative fluorescence images of LPS- and IL-8-induced NET production by resting and primed neutrophils (n = 2). Images were taken at ×20 objective. Arrows point to regions of extracellular DNA. (C) ROS generation by resting (white bars) and TNF-α-primed (black bars) neutrophils in response to 10 ng mL−1 IL-8 or 100 ng mL−1 LPS stimulation was measured over a 60-min period using luminol-based chemiluminescence. Data are presented as area under the curve (AUC) and represent the mean ± SEM of eight experiments performed on neutrophils obtained from young donors.
Figure 2
Figure 2
Effect of age on NET production. Neutrophils isolated from young and old donors were primed with 10 ng mL−1 TNF-α prior to a 3-h stimulation with 10 ng mL−1 IL-8 or 100 ng mL−1 lipopolysaccharide (LPS). NET production was assessed either by measuring the DNA content of cell-free supernatants by fluorometry (A) or by immunofluorescence microscopy (B). For microscopy, images were taken at ×20 objective and are representative of two independent experiments. Arrows point to regions of extracellular DNA. For (A), data are presented as arbitrary fluorescence units (AFU) and represent the mean ± SEM. For IL-8 treatment, data are for 9 young and 10 old donors, and for LPS, data are for 8 young and 11 old donors. Results for young adults are depicted in white bars. Results for older adults are presented as black bars. (C) Neutrophils obtained from 10 young and 8 old donors were stimulated for 3 h with 25 nm PMA, after which NET production was assessed by fluorometry. Data are presented as AFU and are mean ± SEM.
Figure 3
Figure 3
Effect of age on ROS generation. Using luminol-based chemiluminescence, ROS generation by TNF-α-primed neutrophils in response to 10 ng mL−1 IL-8 (A) or 100 ng mL−1 lipopolysaccharide (LPS) (B) challenge or by resting neutrophils treated with 25 nm PMA (C) was measured over a 60-min period. In panels on the left, ROS generation is presented as area under the curve (AUC) and represents the mean ± SEM for 8 young and 8 old subjects for IL-8 and LPS challenge, and 10 young and 8 old donors for PMA treatment. In panels on the right, representative plots of ROS generation, presented as relative light units (RLU), are shown.
Figure 4
Figure 4
Kinetics of p38 mitogen-activated protein kinase (MAPK) activation in TNF-α-primed neutrophils. Neutrophils isolated from young and old donors were primed over a 15-min time period with 10 ng mL−1 TNF-α. At the time points indicated, cell lysates were prepared and p38 activation measured by Western blotting using a phospho-specific anti-p38 antibody. Total p38 expression served as a loading control. (A) Representative Western blot showing p38 activation kinetics in neutrophils isolated from a single young and old donor following TNF-α priming. (B) Densitometric analysis of p38 activation. Data are mean ± SEM for 5 young and 5 old subjects. Results for young adults are depicted in white bars. Results for older adults presented as black bars.
Figure 5
Figure 5
Neutrophil extracellular trap (NET) production by age (< 50 years vs. > 50 years) in periodontitis patients and healthy controls. (A) Peripheral blood neutrophils isolated from young (< 50 years, n = 14) and old (> 50 years, n = 6) subjects diagnosed with chronic periodontal disease were stimulated with 0.75 mm hypochlorous acid (HOCL) or 50 nm PMA and incubated for 3 h (37°C, 5% CO2) prior to quantification of DNA release. (B) Young (n = 14) or old (n = 6) age-matched healthy controls and periodontitis patient neutrophils (young, n = 14; old, n = 6) were stimulated with 0.75 mm HOCL and incubated for 3 h (37°C, 5% CO2) prior to quantification of DNA release. Data are presented as arbitrary fluorescence units (AFU) and are mean ± SEM.
See this image and copyright information in PMC

References

    1. Afford SC, Pongracz J, Stockley RA, Crocker J, Burnett D. The induction by human interleukin-6 of apoptosis in the promonocytic cell line U937 and human neutrophils. J. Biol. Chem. 1992;267:21612–21616. - PubMed
    1. Bianchi M, Hakkim A, Brinkmann V, Siler U, Seger RA, Zychlinsky A, Reichenbach J. Restoration of NET formation by gene therapy in CGD controls aspergillosis. Blood. 2009;114:2619–2622. - PMC (VSports手机版) - PubMed
    1. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–1535. - PubMed (VSports手机版)
    1. Brinkmann V, Laube B, Abu AU, Goosmann C, Zychlinsky A. Neutrophil extracellular traps: how to generate and visualize them. J. Vis. Exp. 2010;36:1724. - V体育ios版 - PMC - PubMed
    1. Butcher SK, Chahal H, Nayak L, Sinclair A, Henriquez NV, Sapey E, O’Mahony D, Lord JM. Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans. J. Leukoc. Biol. 2001;70:881–886. - PubMed

MeSH terms