概要

Alto Rendimiento de medición de ADN extracelular de salida y la formación neta cuantitativa en neutrófilos humanos<em> In Vitro</em

Published: June 18, 2016
doi:

概要

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Abstract

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation.

Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

Introduction

NET formation is a novel mechanism by which neutrophils fight pathogens.1 The core of NETs is nuclear DNA.1 This DNA network is associated with neutrophil granule proteins and histones.1 The main form of NET formation requires the death of neutrophils characterized by chromatin decondensation, disappearance of granular and nuclear membranes, translocation of neutrophils elastase to the nucleus, citrullination of histones and finally the spill of DNA-based NETs.2 NETs entrap and kill a wide variety of microbes and are an essential part of the innate immune weapon repertoire. Uncontrolled NET formation has, however, been linked to numerous autoinflammatory diseases.3,4 Despite their increasingly established relevance, little is known about the mechanism and regulation of NET release.

Neutrophils dying by releasing NETs are different from apoptotic or necrotic neutrophils.3,5 NET-releasing neutrophils show several features that are characteristic for NET formation. Granule components are associated with DNA in NETs.1 Myeloperoxidase (MPO) and human neutrophil elastase (HNE) are both found in primary granules in resting cells but are translocated to the nucleus to bind to DNA in NETs.1 MPO-DNA and HNE-DNA complexes are specific for NETs, do not occur in apoptotic or necrotic neutrophils.1,3,5 Chromatin decondensation is another feature typical for NETosis.2 NET release also requires citrullination of histones by peptidyl aminidase 4 (PAD4).6 Citrullinated histones are hallmarks of neutrophils that underwent NET release.6

Here three methods are introduced that in combination provide excellent tools to quantitate NETs on a high-throughput scale. The first assay has been used on the field with different changes and quantitates extracellular DNA release in a microplate format. The second and third assays provide confirmation of NETs by measuring NET-specific MPO-DNA and HNE-DNA complexes.

Protocol

La Junta Institucional de la Universidad de Georgia Revisión aprobó el estudio de sujetos humanos para recoger la sangre periférica de voluntarios sanos (UGA # 2012-10769-06). 5,7,8 voluntarios firmaron el consentimiento informado necesario antes de la extracción de sangre. La investigación realizada en este artículo está en conformidad con las directrices éticas para la investigación médica en seres humanos de la Declaración de Helsinki. 1. Aislamiento de los neutrófilos de sangre periféri…

Representative Results

Las cifras en este manuscrito se describe el método de aislamiento de neutrófilos, los procedimientos experimentales, y presentar los resultados representativos con la explicación de análisis de datos. La figura 1 muestra las etapas secuenciales de la preparación de neutrófilos humanos. Este protocolo representa sólo una manera posible de aislamiento de neutrófilos. Se produce grandes cantidades de neutrófilos en reposo capaces de liberar TNE a la estimulación….

Discussion

TNE representan un fascinante nuevo mecanismo por el cual los neutrófilos matar los agentes patógenos. 1 Aunque la literatura de los TNE ha sido cada vez más importante en los últimos diez años desde su descubrimiento, varias cuestiones importantes relacionadas con su papel en la biología, el mecanismo y la regulación siguen sin estar claros. metodología adecuada tiene que ser desarrollado para medir los TNE, este mecanismo antimicrobiano muy singular. Este artículo describe métodos que se pueden ut…

開示

The authors have nothing to disclose.

Acknowledgements

Special thanks to the personnel of the University of Georgia Health Center laboratory for their continuous support of our work on isolating human neutrophils. This work was supported by the start-up fund of Dr. Rada provided by UGA Office of Vice President for Research.

Materials

Anti-Human Neutrophil Elastase  Rabbit Ab  Calbiochem 481001 1:2000X coated
Anti-Myeloperoxidase Ab (Rabbit) Millipore 07-496 1:2000X coated
DNase-1 Roche 10-104-159-001 1ug/ml used for digestion
20mM EGTA/ PBS Sigma-Aldrich E3889-25G
2.5 mM EGTA/PBS Sigma-Aldrich E3889-25G
Cell death detection ELISA Anti-DNA POD Roche 11544675001 1:500X 
Eon Microplate Spectrophotometer Biotek
Gen5 All-in-One microplate software Biotek analytical tool (ELISA)
Sytox orange Life Technology S11368 0.2% final concentration/volume
1 M Hepes Cellgro 25-060-Cl Use 10 mM final concentration.
1 M glucose Sigma Use 5 mM final concentration.
HBSS Corning 21-023-CM
Varioskan Flash Ver.2.4.3 Thermoscientific
PMA Sigma P 8139 100nM final used
ELISA Plate Greiner bio-one 655061
Conical tubes 15ml Thermoscientific 339650
Conical tubes 50ml Thermoscientific 339652
Percoll (pH 8.5-9.5)  Sigma P 1644 Sodium Chloride, Sigma, S7653-250G
Dextran Spectrum D1004
RPMI 1640 media Corning Cellgro 17-105-CV
96 well assay plate black plate clear bottom Costar 3603

参考文献

  1. Brinkmann, V., et al. Neutrophil extracellular traps kill bacteria. Science. 303, 1532-1535 (2004).
  2. Fuchs, T. A., et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol. 176, 231-241 (2007).
  3. Kessenbrock, K., et al. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med. 15, 623-625 (2009).
  4. Leffler, J., et al. Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus. Arthritis Res Ther. 15, R34 (2013).
  5. Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes. Immunol Lett. 160, 186-194 (2014).
  6. Wang, Y., et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 184, 205-213 (2009).
  7. Pang, L., Hayes, C. P., Buac, K., Yoo, D. G., Rada, B. Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps. J Immunol. 190, 6488-6500 (2013).
  8. Yoo, D. G., et al. Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation. J. Immunol. 192, 4728-4738 (2014).
  9. Brinkmann, V., Laube, B., Abu Abed, ., Goosmann, U., Zychlinsky, C., A, Neutrophil extracellular traps, how to generate and visualize them. J. Vis. Exp. , (2010).
  10. Rada, B., et al. Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase. PLoS One. 8, e54205 (2013).
  11. Neumann, A., et al. The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps. Biochem J. 464, 3-11 (2014).
  12. Leon, S. A., Shapiro, B., Sklaroff, D. M., Yaros, M. J. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 37, 646-650 (1977).
  13. Raptis, L., Menard, H. A. Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus. J Clin Invest. 66, 1391-1399 (1980).
  14. Chang, C. P., et al. Elevated cell-free serum DNA detected in patients with myocardial infarction. Clin Chim Acta. 327, 95-101 (2003).

Play Video

記事を引用
Sil, P., Yoo, D., Floyd, M., Gingerich, A., Rada, B. High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro. J. Vis. Exp. (112), e52779, doi:10.3791/52779 (2016).

View Video