Title: Neutrophil Extracellular Traps impair the inhibition of elastase, protease 3 and cathepsin G in neutrophil suspensions from CF patients
Abstract: Nitrated tyrosine (3NT) has been detected in inflammatory diseases including cystic fibrosis (CF).Tyrosine nitration is thought to be mediated by the interaction of the activated oxygen species, superoxide anion radical, with nitric oxide leading to the generation of the nitrating species peroxinitrite.We analysed BAL fluids from patients with asthma and CF for 3NT-positive proteins.Concentrated BAL fluids were incubated with magnetic beads, coated with antibodies against 3NT, and antibody-bound proteins identified after desorption by Western blotting and MALDI-TOF.Human eosinophil peroxidase (EPO) was the only protein identified.3NT staining of various immune cells in lung transplant tissues from CF patients revealed that only eosinophils were positive for 3NT.Four eosinophil granule toxins, including EPO were identified as 3NT-positive and high resolution MS demonstrated the presence of a single surface-exposed nitrosylated tyrosine residues in three of these toxins.However, 3NT-positive eosinophil granule toxins were also present in blood eosinophils from healthy individuals.Further experiments using EPO-/mice, mice with a defect in NADPH oxidase, nitric oxide synthese KO mice and patients with CGD demonstrated that the mechanism of tyrosine nitration is mediated by EPO in the presence of hydrogen peroxide and a minute source of NO or nitrite.These data suggest most if not all of the post-translationally modified 3NT-positive proteins are derived from eosinophils, suggesting that 3NT is not a marker of inflammation but rather a marker of eosinophils in CF airways.