Quantitative evaluation of citrullinated fibrinogen for detection of neutrophil extracellular traps.

Activated neutrophils release neutrophil extracellular traps (NETs) composed of chromatin filaments containing bactericidal proteins and enzymes. This process, known as NETosis, is an innate host defense mechanism. However, NET accumulation can lead to uncontrolled inflammation and organ damage. Therefore, NET detection provides clinically important information for the assessment of inflammatory conditions. We investigated whether quantification of citrullinated fibrinogen (C-Fbg), which is catalyzed by peptidylarginine deiminase (PAD) released during NETosis, can be used to detect NETs. Human neutrophils were stimulated with fibrinogen using phorbol 12-myristate 13-acetate (PMA). The myeloperoxidase (MPO)-DNA complex and C-Fbg concentrations in the culture supernatants were quantified using an enzyme-linked immunosorbent assay. The protein levels of peptidylarginine deiminase 2 and 4 in culture supernatants and mRNA levels in PMA-stimulated neutrophils were also assessed. The levels of the MPO-DNA complex in the supernatants of PMA-stimulated neutrophils increased, indicating NETosis. C-Fbg level also increased, which was suppressed by both NETosis and PAD inhibitors. PAD2 was detected in the culture supernatant; however, PAD4, but not PAD2, mRNA levels increased in PMA-stimulated neutrophils. This study quantitatively demonstrates that fibrinogen is citrullinated by PAD derived from PMA-stimulated neutrophils upon NETosis. Although further studies are needed for clinical application, quantification of C-Fbg in blood may help detect the presence of NETs.