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Neutrophil extracellular traps (NETs) represent an extracellular, spider's web-like structure resulting from cell death of neutrophils. NETs play an important role in innate immunity against microbial infection, but their roles in human pathological processes remain largely unknown. NETs and fibrin meshwork both showing fibrillar structures are observed at the site of fibrinopurulent inflammation, as described in our sister paper [Acta Histochem. Cytochem. 49; 109-116, 2016]. In the present study, immunoelectron microscopic study was performed for visualizing NETs and fibrin fibrils (thick fibrils in our tongue) in formalin-fixed, paraffin-embedded sections of autopsied lung tissue of legionnaire's pneumonia. Lactoferrin and fibrinogen gamma chain were utilized as markers of NETs and fibrin, respectively. Analysis of immuno-scanning electron microscopy indicated that NETs constructed thin fibrils and granular materials were attached onto the NETs fibrils. The smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils. Pre-embedding immunoelectron microscopy demonstrated that lactoferrin immunoreactivities were visible as dots on the fibrils, whereas fibrinogen gamma chain immunoreactivities were homogeneously observed throughout the fibrils. Usefulness of immunoelectron microscopic analysis of NETs and fibrin fibrils should be emphasized.

Neutrophil extracellular traps (NETs) are extracellular fibrillary structures composed of degraded chromatin and granules of neutrophil origin. In fibrinopurulent inflammation such as pneumonia and abscess, deposition of fibrillar eosinophilic material is a common histopathological finding under hematoxylin-eosin staining. Expectedly, not only fibrin fibrils but also NETs consist of the fibrillar material. The aim of the present study is to analyze immunohistochemically how NETs are involved in the inflammatory process. Archival formalin-fixed, paraffin-embedded sections accompanying marked neutrophilic infiltration were the target of analysis. Neutrophil-associated substances (citrullinated histone H3, lactoferrin, myeloperoxidase and neutrophil elastase) were evaluated as NETs markers, while fibrinogen gamma chain was employed as a fibrin marker. Light microscopically, the fibrils were categorized into three types: thin, thick and clustered thick. Lactoferrin represented a good and stable NETs marker. Thin fibrils belonged to NETs. Thick fibrils are composed of either mixed NETs and fibrin or fibrin alone. Clustered thick fibrils were solely composed of fibrin. Neutrophils were entrapped within the fibrilllar meshwork of the thin and thick types. Apoptotic cells immunoreactive to cleaved caspase 3 and cleaved actin were dispersed in the NETs. In conclusion, NETs and fibrin meshwork were consistently recognizable by immunostaining for lactoferrin and fibrinogen gamma chain.

In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders.