Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: III. Correlative Light and Electron Microscopic Study

Abstract

Neutrophil extracellular traps (NETs) released from dead neutrophils at the site of inflammation represent webs of neutrophilic DNA stretches dotted with granule-derived antimicrobial proteins, including lactoferrin, and play important roles in innate immunity against microbial infection. We have shown the coexistence of NETs and fibrin meshwork in varied fibrinopurulent inflammatory lesions at both light and electron microscopic levels. In the present study, correlative light and electron microscopy (CLEM) employing confocal laser scanning microscopy and scanning electron microscopy was performed to bridge light and electron microscopic images of NETs and fibrin fibrils in formalin-fixed, paraffin-embedded, autopsied lung sections of legionnaire's pneumonia. Lactoferrin immunoreactivity and 4'-6-diamidino-2-phenylindole (DAPI) reactivity were used as markers of NETs, and fibrin was probed by fibrinogen gamma chain. Of note is that NETs light microscopically represented as lactoferrin and DAPI-colocalized dots, 2.5 μm in diameter. CLEM gave super-resolution images of NETs and fibrin fibrils: "Dotted" NETs were ultrastructurally composed of fine filaments and masses of 58 nm-sized globular materials. A fibrin fibril consisted of clusters of smooth-surfaced filaments. NETs filaments (26 nm in diameter) were significantly thinner than fibrin filaments (295 nm in diameter). Of note is that CLEM was applicable to formalin-fixed, paraffin-embedded sections of autopsy material.

Keywords: confocal laser scanning microscopy; correlative light and electron microscopy; fibrin; neutrophil extracellular traps; scanning electron microscopy.

Fig. 1.

Localization of LF, FGG and DAPI (DNA) in a paraffin-embedded lung tissue of legionnaire’s pneumonia. Consecutive sections demonstrate HE-stained microscopic features (a), immunoperoxidase staining for Legionella pneumophila, serotype 1 (b), and CLSM images (c–f) of immunofluorescence staining for LF (green) and FGG (red) and DAPI-induced DNA fluorescence (blue). Dual fluorescence illustrates LF and DAPI (c), FGG and DAPI (d) and LF and FGG (e). Triple fluorescence is shown in (f). The brown-stained pathogens are observed in the cytoplasm of neutrophils. Fibrin meshwork is shown not only in HE staining (eosinophilic fibrils) but also with FGG red fluorescence. The cytoplasm and nuclei of neutrophils stain green and blue, respectively. The “dotted” (fine granular) light microscopic localization pattern of LF and DAPI shown here is different from the common NETs-related fibrillary structure. Bars=50 μm.

Fig. 2.

High-powered fluorescent CLSM images of LF, FGG and DAPI (DNA) in NETs and fibrin fibrils. High-powered CLSM images of NETs (a–d), fibrin fibrils (e–h) and the colocalization of NETs and fibrin fibrils (i–l) are shown. NETs are stained with both LF (green) and DAPI (blue), and fibrin fibrils are stained with FGG (red). Two-color merged images are illustrated in (a–c, e–g, i–k). Three-color merged images are seen in (d, h, l). White arrows indicate dotted NETs. Black arrows demonstrate fibrin fibrils. White arrowheads indicate the site of colocalization of NETs and fibrin fibrils: fine NETs-related dots are observed on the fibrin fibrils. Bars=5 μm.

Fig. 3.

Correlation between CLSM and SEM images of NETs and fibrin fibrils. CLSM images (a, d, g) and SEM images (b, e, h) of NETs (a, b), fibrin fibrils (d, e) and the colocalization of NETs and fibrin fibrils (g, h) are shown. NETs are dually stained for LF (green) and DAPI (blue), while fibrin fibrils are stained for FGG (red). Merged features of the CLSM and SEM images are illustrated in (c, f, i). NETs are observed here as a cluster of globular materials (a–c). Note that thick fibrin fibrils are composed of a cluster of smooth-surfaced filaments (d–f). At the site of colocalization, fibrin filaments are attached with globular materials (g–i). Bars=5 μm.

Fig. 4.

Highly magnified SEM images of NETs and fibrin fibrils, in relation to CLEM images. CLEM images illustrate NETs (a), fibrin fibrils (c) and the colocalization of NETs and fibrin fibrils (e). The squared areas in the CLEM images (a, c, e) are observed by SEM at the maximal magnification (b, d, f). White arrowheads in (b) indicate NETs filaments, and globular materials (white arrows) are clustered on the fine filaments. Black arrowheads in (d) demonstrate individual smooth-surfaced fibrin filaments. A transverse section of a single fibrin filament is indicated by black arrowheads in (f). NETs-related globular materials on the fibrin filament are shown by white arrows in (f). Bars=2 μm (a, c, e) and 0.5 μm (b, d, f).