The Pathological Spectrum of Neutrophil DNA NET Formation.
Davide Salina, Florian H Pilsczek, Candace Fahey, Francis HY Green, Paul Kubes, Margaret Kelly. University of Calgary, AB, Canada
Background: Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense (Brinkmann et. Al, Science 2004). Neutrophils respond to Staphylococcus aureus and other bacteria via a novel process of NET formation. NET formation has recently shown to be a major player in the pathogenesis of lupus nephritis, small vessel vasculitis, and cystic fibrosis (Marcos et al, Nature Med. 2010). The use of live cell light microscopy and transmission electron microscopy has identified morphological changes of the nuclear envelope integrity during invitro NET formation (submitted J.Immunol.). We include a retrospective observational study to identify the spectrum of NET formation in various human diseases.
Design: Neutrophils were collected from humans, activated with S. aureus and then fixed at various time points. The images obtained from the electron microscope were correlated with live cell imaging. Different cases of purulent inflammation also form NETs, including suppurative appendicitis. We detect NETs using immunoflourescence and immunohistochemistry using antibodies against citrullinated histones. Electron microscopy was performed to compare the ultrastructure to the invitro formed NETs.
Results: NET formation in-vitro is a highly complex process. Initially, the lumen of both the nuclear envelope filled with nuclear chromatin strands. Numerous chromatin containing vesicles budded from the nuclear envelope into the cytoplasm. Eventually nuclear envelope breakdown occurred in a similar fashion to previously published studies (Salina et al, Cell 2002, Vol.108). The vesicles are exocytosed into the extracellular space where they release their contents. Extracellular NETs form fibrillary structures made up of chromatin strands admixed other proteins from granules.This process of NET formation in purulent appendicitis has identical morphological changes, with numerous extracellular DNA containing vesicles and NETs. We are currently trying to analyze a broad spectrum of various human diseases that also involve formations of these NETs.
Conclusions: The neutrophil nucleus is a highly malleable structure with the capability to undergo drastic changes in nuclear morphology when activated by bacteria. Neutrophils form DNA NETs from a highly complex process which includes nuclear envelope breakdown and exocytosis of DNA containing vesicles. This mechanism is an important part of our inflammatory response to acute infection.
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 218, Monday Morning