BackgroundCOPD is a progressive disease of the airways that is characterized by neutrophilic inflammation, a condition known to promote the excessive formation of neutrophil extracellular traps (NETs). The presence of large amounts of NETs has recently been demonstrated for a variety of inflammatory lung diseases including cystic fibrosis, asthma and exacerbated COPD.ObjectiveWe test whether excessive NET generation is restricted to exacerbation of COPD or whether it also occurs during stable periods of the disease, and whether NET presence and amount correlates with the severity of airflow limitation.Patients, materials and methodsSputum samples from four study groups were examined: COPD patients during acute exacerbation, patients with stable disease, and smoking and non-smoking controls without airflow limitation. Sputum induction followed the ECLIPSE protocol. Confocal laser microscopy (CLSM) and electron microscopy were used to analyse samples. Immunolabelling and fluorescent DNA staining were applied to trace NETs and related marker proteins. CLSM specimens served for quantitative evaluation.ResultsSputum of COPD patients is clearly characterised by NETs and NET-forming neutrophils. The presence of large amounts of NET is associated with disease severity (p < 0.001): over 90 % in exacerbated COPD, 45 % in stable COPD, and 25 % in smoking controls, but less than 5 % in non-smokers. Quantification of NET-covered areas in sputum preparations confirms these results.ConclusionsNET formation is not confined to exacerbation but also present in stable COPD and correlates with the severity of airflow limitation. We infer that NETs are a major contributor to chronic inflammatory and lung tissue damage in COPD.

Trapping the crevicular bacteria prevents their adhesion to and invasion of the gingiva. The combination of neutrophil extracellular traps and crevicular exudate outflow appears to be a 'novel' defense mechanism for the clearance of crevicular bacteria in chronic periodontitis.

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.

The fate of the neutrophils within the inflammatory exudate in the periodontal crevice and their possible participation in the formation of neutrophil extracellular traps (NETs) are of clinical interest. However, the cytological analysis of clinical samples of inflammatory exudate is restricted by the obtainable quantities, which do not enable employing the routine approaches. Clinical examinations, ACLAR strip sampling, scanning electron microscopy, and confocal laser scanning microscopy were employed to analyze purulent crevicular exudate and gingival crevicular fluid in periodontitis. Bacteria, neutrophil activation, NETosis stages, and NETs were identified by molecular probe, expression of citrullinated histone H3, enzymatic digestion, and ultrastructurally. Crevicular neutrophils, all in diverse NETosis stages marked by the histone citrullination, and an abundance of NETs were found in both purulent crevicular exudate and gingival crevicular fluid. Largely varying quantities of dispersed crevicular bacteria were entrapped by NETs, but no phagocytized bacteria were evident in gingival crevicular fluid. The offered method enables for the first time the demonstration NETs in gingival crevicular fluid. The histone citrullination of all the floating crevicular neutrophils indicates that they all undergo NETosis.

Chlorhexidine, the most used biocide in periodontology, alters the permeability of the bacterial cell membrane. However, the chlorhexidine-induced morphological alterations in the oral biofilm have not been studied. To examine the effects of chlorhexidine on oral biofilm on an electron microscopic level, gingival epithelial cells with attached biofilm were collected from 10 volunteers, subjected to 0.1% chlorhexidine for 1 or 5 min, stained with ruthenium red-tetroxide, and analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM visualized the bacterial glycocalyces and the biofilm matrix on the biofilm surface; however, no chlorhexidine-induced alterations were observed. TEM revealed loss of bacterial membrane integrity and fimbrial disintegration in a few bacteria. In the proximity of these alterations, a restricted matrix disintegration was also observed. However, the chlorhexidine-induced alterations only effected a minor part of the oral biofilm and did not cause its disintegration. These findings suggest the insufficient efficiency of chlorhexidine against oral biofilm.