Table 1.
Characterisation of study groups.
Figure 1.
Semi-quantitative evaluation of NET abundance in induced sputa from hospitalized COPD patients with acute exacerbations and from controls.
Images depict characteristic microscopic appearance conforming to the categories ‘large amounts’ (A) and ‘minor traces’ (B).
Figure 2.
CLSM images of NETs in COPD sputum.
Immunolabelling for neutrophil elastase (NE, green) or citrullinated Histone H3 (citH3, green), DNA stained with propidium iodide (PI, red). A–C: The sputum matrix contains PMNs (open arrows), citH3-positive granules and areas of condensed NETs (asterisks). D–F: Less condensed matrix traversed by thin NET strands associated with bacteria (arrowheads), non-activated PMNs (open arrow) and NETotic PMNs (solid arrow); asterisks indicate fully spread NETs. G–I: COPD sputum treated with DNAse. The DNA meshwork is dissolved while PMNs and citH3 positive granules persist.
Figure 3.
Individual PMNs in various stages of NETosis.
COPD sputa immunolabelled for NE (green: A,B), citH3 (green: C–F), and PAD4 (red: F). DNA stained with either PI (red: A–D) or DAPI (blue: F). A: Non-activated stage with still lobulated nucleus, NE confined to cytoplasm. B: advanced stage with highly swollen nucleus, NE colocalised with chromatin (staining to the right extracellular). C–E: Stages during nuclear swelling. C: Nuclear lobulation still visible, part of chromatin stains for citH3. D: Staining for citH3 most intense in perinuclear cytoplasm. E: Chromatin extrusion. F: CitH3 and PAD4 colocalised in cytoplasm and nucleus. Scale bars are all at 5 µm and valid for each photograph within the individual combinations A–F.
Figure 4.
Electron microscopic characterisation of NET components.
SEM (A,C) and TEM (B,D–H) micrographs from COPD sputum (A–E,H) and in vitro induced NETs (F,G). A: Fibrous strands diverge from a PMN cell body with a sculptured surface and attached granulae and merge into loosely structured texture. B: Motif corresponding to that of A. Thinly spread areas (inset) exhibit a fibrous texture with attached globules (arrows). C: Bacterium (open arrow) entrapped by NET fibres with globular protrusions. D,E: Sections of COPD sputum. D: Bacterium (open arrow) surrounded by a fibrous network that embeds spherical granules with amorphous content (arrow). E: Undisturbed PMN with normal nuclear chromatin and amorphous vesicular inclusions (arrows). F–H: Sections of in vitro induced NETs. F: NETs attached to the remnants of their cell of origin. NET fibres exhibit less accretions than those from COPD sputum NETs (cf. E); arrows indicate neutrophilic granula. G: Fibres and neutrophilic granula of the NETs shown in F. H: Membrane coated in COPD sputum vesicle containing NET-like structures.
Figure 5.
Ultrastructure of in vitro generated NETs.
TEM images of in vitro generated NETs immunogold stained for citH3. A: Thick NET trajectory with attached electron-dense substance and small branches giving rise to loosely structured fibrous meshwork. Frames depict details shown in B–D. B: Patch of dense substance adhering to a thick NET strand with parallel fibrous subarchitecture (black arrowhead). C,D: Examples of NET strand disintegration into a network of randomly oriented small fibres with diameters down to about 2 nm (arrowheads). Some accretions at fibre intersections and along the strands are labelled for citH3 (arrows).
Figure 6.
Detailed TEM analysis of NETs from COPD sputum (A–C,F) and in vitro generated NETs (D,E,G,H).
A,D: Immunogold staining for NE. Amorphous attachments and ‘grid-point’ aggregates of both COPD NETs (A) and in vitro NETs (D) are all strongly labelled. B,E: Immunogold staining for citH3. Only some of the aggregates adhered to COPD NETs (B) and in vitro NETs (E) are stained (white arrow). Arrowheads indicate fibres consisting of single DNA double-helices. C,F: Sectioned COPD sputum. C: NET attachment to epithelial cell surface. F: Individual NET fibres (arrowheads) and bundles of such fibres in parallel alignment (asterisk) interspersed with granules and pieces of cell debris. The bundles are abundantly clotted with amorphous substance. G,H: Structure of fully spread NETs. Bundles of parallel fibres (black arrowheads) provide a scaffold for an irregular network of much finer fibres with diameters down to about 2 nm (white arrowheads) some of which are equidistantly decorated with protrusions of about 10 nm, likely corresponding to nucleosome cores (black arrows in H). Immunolabelling for citH3 recognises sites along the fibrous bundles (white arrows) and on detached substance (open arrowheads in G), but in no case the presumed nucleosomes.