Abstract

Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.

Highlights

  • Neutrophil extracellular traps (NETs) are web-like extracellular structures generated by activated polymorphonuclear neutrophils (PMNs) in a distinct process of cell death termed NETosis, involving the extrusion of nuclear DNA after chromatin decondensation

  • NETosis can be initiated by a variety of molecular signals that bind to neutrophil surface receptors, among them bacterial breakdown products and endogenous pro-inflammatory inducers such as the chemokine IL

  • Somewhat surprisingly, staining for citrullinated histone H3 (CitH3) in early activated stages was more common at cytoplasmic sites than at nuclear sites (Figs. 3C,D), but was usually co-localised with the chromatin of already extruded neutrophil extracellular traps (NETs) (Figs. 2B,C)

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Summary

Introduction

Neutrophil extracellular traps (NETs) are web-like extracellular structures generated by activated polymorphonuclear neutrophils (PMNs) in a distinct process of cell death termed NETosis, involving the extrusion of nuclear DNA after chromatin decondensation. They consist of a scaffold of DNA strands of varying thickness laced with histones, neutrophil elastase (NE) and other antimicrobial, and potentially cytotoxic, molecules from the neutrophil cytoplasm [1,2,3]. The antimicrobial activity appears to depend on the enzymes and histones, and on the integrity of the DNA network, as it can be inhibited by DNase treatment [1,13] This is a mechanism devised by bacteria as part of their strategies for escaping from NETs [14]. Recent evidence from a bacterial infection model in mouse skin suggests that NET release in vivo occurs while the PMNs still move around by pseudopodial crawling, thereby casting large areas of NETs [15]

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