Impaired degranulation of secondary and tertiary granules from CF neutrophils and CFTR inhibited cells. HC and CF neutrophils were stimulated with TNF-α (10 ng/2 × 107 cells/mL), and the extracellular supernatants were obtained after 0, 5, 10, and 20 minutes. (A-C) (Top) Representative western blots of supernatants probed for (A) hCAP-18, (B) lactoferrin, and (C) MMP-9. Immunoband intensity was quantified by densitometry, expressed as a multiple of the time zero value of unstimulated cells and illustrated in the corresponding bar graphs (P = .007 at 20-minute time point for hCAP-18; P = .002 at 10-minute time point and P = .005 at 20-minute time point for MMP-9, Student t test, n = 3 subjects per group). Levels of (D) hCAP-18 and (E) lactoferrin in extracellular supernatants were also assessed by enzyme-linked immunosorbent assay (P = .002 and P = .03 at 10 minutes, respectively, Student t test, n=3 subjects per group). Values presented are relative to time 0 of unstimulated cells. (F) HC and CF neutrophils were fixed in 4% (w/v) paraformaldehyde following 0, 5, 10, and 20 minutes of TNF-α stimulation (10 ng/mL) and analyzed by flow cytometry using a fluorescein isothiocyanate-labeled CD66b antibody. Data are represented as the percent increase in mean fluorescence intensity (MFI) from unstimulated control cells (P = .004, P = .01, and P = .004 at 5, 10, and 20 minutes, respectively, Student t test, n=8 subjects per group). (G) HC neutrophils were treated with vehicle control (0.1% v/v DMSO; VC) or the CFTR inhibitors CFTRinh-172 (10 μM; Inh) or GlyH-101 (25 μM; GlyH) and then stimulated with TNF-α for 10 minutes. The level of degranulation was compared by flow cytometry using a fluorescein isothiocyanate-labeled CD66b antibody. Data are represented as increase of MFI relative to vehicle-treated control cells (P = .0001, Student t test, n = 4 subjects per group). All measurements are means ± SEM from biological replicates.
