Increased Membrane Proteinase 3 Expression on Neutrophils in Patients with Infectious Disease.
Authors: Matsumoto Takeshi , Kaneko Toshihiro , Wada Hideo , Kamikura Yuko , Kobayashi Toshihiko , Nobori Tsutomu , Shiku Hiroshi , Stearns-Kurosawa Deborah J. , Kurosawa Shinichiro 
Published: October 2019

Abstract
Proteinase-3 (PR3) is an abundant serine proteinase stored in azurophilic granules of neutrophils and released to the cell surface upon activation. PR3 expression near a vascular surface likely contributes to local tissue destruction and inflammation. PR3 is generally known as the primary target antigen of PR3-anti-neutrophil cytoplasmic antibodies (ANCA) in Wegener’s granulomatosis (WG), an autoimmune vasculitis. There are many reports about neutrophil PR3 expression in WG, but few reports about PR3 in patients with common inflammatory disorders. Using FCM, we examined membrane PR3 (mPR3) expression on neutrophils from patients with infectious disease (n=54) and from healthy volunteers (n=64). Patients were diagnosed by clinical finding as follows: 24 peumonia, 17 peritonitis, 9 tonsillitis, 2 phlegmon and 2 pyelonephritis. Neutrophil mPR3 expression was determined with and without stimulation by TNF-α and N-formyl-L-methionyl-L-phenylalanine. Correlations between mPR3 expression and clinical condition were evaluated. Previous data demonstrated PR3 at the surface of a subpopulation of freshly isolated neutrophils. The relative distribution of mPR3 high (PR3-high) and low neutrophils (PR3-low) varied among individuals but was extremely stable for each individual over time, consistent with a genetic basis for baseline expressions levels. We confirmed high variability in the percentage of mPR3-high cells from healthy volunteers and patients. However, the percentage of PR3-high cells in patients was significantly higher than in healthy volunteers (72±19% vs 55±20%, P<0.0001). Some cases showed a dramatic increase in the percentage of PR3-high neutrophils, particularly septic patients with a lethal outcome, suggesting that changes in the PR3-high population may be related to inflammation severity. The expression of PR3 was calculated as mean fluorescence intensity (MFI), corrected for non-specific binding of an irrelevant antibody (NSB), in combination with the percentage of PR3-high cells (%PR3-high). Data were expressed as a percentage of the value obtained from the healthy control, according to following formula: Expression Index (EI) = [(MFI-NSB)patientx %PR3-highpatient]/[(MFI-NSB)healthyx %PR3-highhealthy]x100% Both resting and activated neutrophils of patients with infectious disease was significantly higher than in healthy volunteers (P<0.001, P<0.001). There was a significant correlation between mPR3 on resting neutrophils and C-reactive protein levels (r=0.47, n=54, p<0.001). EI of mPR3 on neutrophils activated in vitro and CRP was also significant (r=0.44, n=54, P<0.001). EI of mPR3 on neutrophils either resting or activated in vitro was significantly higher in the patients with SIRS than non-SIRS (P<0.01, P<0.001). The present study demonstrates that both the percentage of PR3-high cells and the absolute level of mPR3 were increased in patients with infectious diseases unrelated to autoimmune vasculitis. These changes were related to C-reactive protein levels. mPR3 expression on neutrophil presents the opportunity to expand inflammation in patients with not only PR3-ANCA positive vasculitis, but also with common inflammatory diseases such as sepsis.