(A) EGFR expression in blood (shown in red) and airway (shown in blue) neutrophils from stable CF patients. FMO, fluorescence minus one. (B) EGFR expression on paired airway and blood neutrophils from CF patients (N = 7, data normalized to blood). (C) Representative Imagestream analysis of phosphorylated EGFR at tyrosine 1086 (Y1086, shown in magenta) in primary CF airway neutrophils (DAPI, shown in blue). (D) Healthy blood neutrophils transmigrated into CFASN for 10 hours upregulate EGFR and its ligands compared with matched blood neutrophils (N = 5). (E) EGFR expression in healthy blood neutrophils before transmigration (blood) and 1 hour and 2 hours after transmigration. (F) Fold increase in expression of surface EGFR between paired healthy blood and CFASN-transmigrated neutrophils (N = 3, data normalized to blood). (G) EGFR expression by ELISA on matched healthy blood and CFASN-transmigrated neutrophils. (H) EGFR phosphorylation in healthy CFASN-transmigrated neutrophils by ELISA. (I) Intracellular calcium levels in transmigrated CF neutrophils treated with EGF. (J and K) Clearance of P. aeruginosa and S. aureus by healthy CFASN-transmigrated neutrophils treated with EGF and/or gefitinib for 2 hours. (L) Quantification of nitrotyrosine in neutrophil lysates upon EGF stimulation and incubation with PAO1 (dots = CF neutrophils transmigrated into CFASN, open square = healthy control neutrophils transmigrated into CFASN). Statistical analysis was performed using Kruskal-Wallis with Dunn’s multiple-comparison test (F and H–K) or by Wilcoxon’s signed-rank test (paired; B, G, and L). *P < 0.05, **P < 0.01.