Figure 3.

Pharmacologic CatC inhibition strongly reduces NSPs in neutrophils differentiated from HSC and subsequently reduces neutrophil activation by PR3-ANCA and EC injury. Neutrophils were differentiated from human CD34^pos HSC for 10 days in the presence of buffer control (bu), BI-I, and prednisolone (pred) as indicated. (A) Neutrophil differentiation was assessed using the indicated surface markers. (B) NSP proteins were assessed by immunoblotting and the optical densities (OD) of the NSP bands were quantified. Proteolytic activity was measured by FRET assay. MPO served as a control. (C) Neutrophils differentiated for 10 days were double stained for mPR3 and CD177 and analyzed by flow cytometry. A typical experiment together with the mPR3 and CD177 mean fluorescence intensity are depicted. (D) Superoxide release by neutrophils differentiated for 10 days was assessed using mAbs to PR3 and MPO, human PR3- and MPO-ANCA IgG, and appropriate controls as indicated. (E) mPR3 on viable (AnnV^neg) and apoptotic (Ann^pos) neutrophils differentiated for 10 days was assessed by flow cytometry. (F) The effect of cf-SN from resting and activated neutrophils differentiated for 10 days on EC injury was assessed by phalloidin-FITC (green) and nuclear DAPI staining (blue) and microscopy. *P<0.05 and **P<0.01.