PMC

a & c, DAPI staining of nuclei of human neutrophils. b, anti-CFTR antibody staining of a human neutrophil revealing a punctate staining pattern. d, isotype-matched antibody staining as a control. e–h, double immunofluorescent staining with rabbit anti-human myeloperoxidase antibody (f) and mouse anti-CFTR antibody (g). The merged image (h) to identify co-localization of CFTR and MPO. i–l, CFTR association with phagocytic vacuoles and phagolysosomes bearing ingested green fluorescence protein-expressing Pseudomonas aeruginosa (GFP-PAO1). DAPI staining of a neutrophil with ingested bacteria (i). Phagocytosed GFP-PAO1 (j). Anti-CFTR immunofluorescent staining of a neutrophil with ingested bacteria (k). Association of internalized GFP-PAO1 bacteria with CFTR (l). Large arrows point to a phagolysosome where CFTR is present on the membrane (j–i). Small arrows point to the CFTR-positive staining granules appearing attached to the phagosomes with ingested GFP-PAO1 (j–i). Arrowheads point to a phagocytic vacuole (j–i). m–p, co-localization of CFTR and lysosomal associated membrane protein-1(LAMP-1) in isolated phagolysosomes. DAPI staining of phagolysosomes with ingested non-fluorescent PAO1 which are stained blue (m). LAMP-1 is localized to the phagolysosomes (n). CFTR is present in the phagolysosomal membranes (o). Merged image shows the co-localization of the two proteins (p).

a, Schematic depiction of the experimental protocol used to quantitatively measure iodination by neutrophils (PMN) of the green fluorescent protein (GFP) expressed in GFP-expressing Pseudomonas aeruginosa. b, Incorporation of ¹²⁵I or ¹⁴C into GFP immunoprecipitated from neutrophils derived from normal donors (n= 4; closed bars) or donors with CF (n=4; open bars). The recovery of ¹⁴C-GFP was similar in both normal and CF cells indicating that the amount of ¹⁴C-labeled GFP-PAO1 phagocytosed by neutrophils and its subsequent recovery were statistically identical. In contrast, the ¹²⁵I content of recovered GFP was about 4.3-fold higher in normal neutrophils than in CF neutrophils. The error bars represent the SEM and the double asterisks represent a P value < 0.05. c, Glybenclamide (GBA), a CFTR channel inhibitor, significantly blocked iodination of bacteria-GFP derived from GFP-PAO1 phagocytosed by normal neutrophils as compared to controls treated with drug vehicle (None) (N=5, P<0.05). In contrast, salicylhydroxamic acid (SHA), an inhibitor of MPO, totally abolished iodination of bacteria-GFP by normal neutrophils relative to controls (N=5, P<0.01). The double asterisks indicate significant differences.

a, schematic flow diagram of the experimental protocol. Neutrophils (PMN) were incubated with green fluorescence protein-expressing Pseudomonas aeruginosa (GFP-PAO1) which had been metabolically prelabeled with ¹³C₉-L-tyrosine. After 60 min at 37° C, the uningested bacteria were removed by low speed centrifugation and the neutrophils containing ingested PAO1 were analyzed for ¹³C₉-3-chlorotyrosine (¹³C₉-3-Cl-Y) and ¹³C₉-L-tyrosine (¹³C₉-Y) as described in the Materials and Methods using the isotope dilution method. ¹³C₆-3-Cl-Y (20 pmol) and ¹³C₆-Y (50 nmol) were used as internal standards. After derivatization, the samples were analyzed by GC/MS using the selected ion mode. b, Representative GC/MS tracings obtained with normal and CF neutrophils. The 289.1 m/z peak eluting at 7.88 min, obtained by monitoring the ion at m/Z 289.1, is associated with PAO1-derived 3-chlorotyrosine. Normal neutrophils show significantly higher levels of chlorotyrosine relative to that seen from CF neutrophils. CF1, CF2, and CF3 are neutrophils (PMN) from 3 different CF donors. c, Ratios of PAO1-derived 3-chlorotyrosine relative to total PAO1-derived tyrosine levels after one hour of incubation with normal or CF patient neutrophils. Error bars indicate the standard error of the mean. Student’s t-test proved a significant difference between normal and CF PMNs (P<0.05, N=4). d, Chlorination of extracellular taurine by HOCl generated by normal and CF neutrophils induced by phorbol-12-myristate-13-acetate (PMA). Statistically, no significant difference was seen between the two groups.

a) RT-PCR to identify CFTR expression in human neutrophils at the mRNA level. Neutrophils were isolated from whole peripheral blood by Percoll-gradients and further purified by a panning procedure taking advantage of neutrophil adherence to plastic surface. Total RNAs were extracted and cDNAs were obtained by reverse-transcription using the CFTR and TBP specific primers. PCR amplification resulted in a CFTR amplicon of 330 bp and TBP 225 bp. Calu-3 is an epithelial cell line derived from airway submucosal gland, which is known to express high levels of CFTR. − RT or + RT represents PCR in the absence or presence of the corresponding RT product. PMN (1 μl) or PMN (2 μl) indicates PCR with 1 μl or 2 μl of PMN RT product to show the dose-dependence of RT. b) Immunoblotting of CFTR of human neutrophils and differentiated HL-60 cells. Neutrophils or HL-60 cells were denatured by TCA to prevent proteolytic degradation by neutrophil-bearing proteases. 7.5% SDS-PAGE was used to resolve the proteins. After being transferred to a nitrocellulose membrane, the membrane was incubated with the CFTR-specific antibody followed by incubation with the goat anti-mouse IgG conjugated with horseradish peroxidase for chemilluminescent detection. ΔF508-wt-CFTR: wild-type CFTR gene-corrected epithelial cell line derived from a ΔF508 CF patient; HL-60: human promyelocytic leukemia cell line. Bands A, B and C represent the three forms of CFTR (Band A: newly synthesized CFTR; Band B: partially glycosylated CFTR; Band C: fully glycosylated mature CFTR). D1–4 indicates times in days after DMSO treatment. c) Immunoblotting of CFTR in subcellular fractions of normal human neutrophils. MPO-rich α granule fractions, vitamin B-12 binding protein-rich β granules and alkaline phosphatase-rich γ fractions were isolated. The γ fractions were further separated into secretory vesicles (SV) and plasma membrane-derived vesicles (PM) by free flow electrophoresis after treated with neuronamidase. 20–50 × 10⁶ cell equivalents of α and β fractions were TCA-precipitated, and the dissolvable proteins were loaded into each lane. For SV and PM, the dissolvable proteins from ~350 × 10⁶ cell equivalents of TCA-precipitated fractions were loaded, respectively. The CFTR expression in the forms of newly synthesized non-glycosylated protein (Band A) and the mature glycosylated protein (Band C) was predominantly detected in the SV fraction.