Assessment Of Neutrophil Serine Proteinases Activity In Bronchoalveolar Lavage Fluids From Patients With Interstitial Lung Diseases Using Innovative Intramolecular Quenched Substrates

Background: Neutrophil serine proteinases (NSPs): human neutrophil elastase (HNE), proteinase 3 (PR3) and cathepsin G (CG) are released by activated inflammatory cells and are involved in the degradation of elastin fibers during inflammation. Data concerning the potential role of three NSPs in the pathophysiology of interstitial lung diseases (IDL) are lacking. Material and methods: Bronchoalveolar lavage (BAL) collected from 25 patients with IDL: hypersensitivity pneumonitis, HP (n=8), pulmonary fibrosis, PF (n=7) and sarcoidosis (n=10) was analyzed for HNE, PR3 and CG activity by innovative fluorescence resonance energy transfer (FRET) method utilizing highly sensitive, specific substrates for respective NSPs. Total protein content was determined by the Bradford procedure. The NSPs activity were shown as pM/1 ml of recovered BAL. Results: PR3 activity was demonstrated in all analyzed BAL samples (median 0.09 pM, IQR 0.05-0.29; 0.08, 0.03-0.27 and 0.13, 0.06-0.21 for HP, PF and sarcoidosis respectively). Detectable HNE activity was found in 75% HP (0.11 pM, 0.01-0.25), 71% PF (0.10 pM, 0.00-0.20) and 70% sarcoidosis (0.05 pM, 0.00-0.12) BAL samples. CG activity was observed in 38% HP (0.00 pM, 0.00-0.19), 29% PF (0.00 pM, 0.00-0.52) and 20% sarcoidosis (0.00 pM, 0.00-0.00) BAL samples. Conclusions: The innovative intramolecular quenched substrate based method allowed successful detection of individual NSPs activity in BAL samples. Considerable differences observed in theNSPs activity levels might suggest a diverse role of respective NSPs in the pathogenesis of particular ILD. The study is on-going.