CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis.
Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.
Painter
Richard G
RG
Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Valentine
Vincent G
VG
Lanson
Nicholas A
NA
Leidal
Kevin
K
Zhang
Qiang
Q
Lombard
Gisele
G
Thompson
Connie
C
Viswanathan
Anand
A
Nauseef
William M
WM
Wang
Guangdi
G
Wang
Guoshun
G
D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis*; D018922:HL-60 Cells; D006801:Humans; D006997:Hypochlorous Acid / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism* / Q000473:pathology; D010587:Phagocytosis / Q000187:drug effects; D010588:Phagosomes / Q000378:metabolism* / Q000473:pathology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011550:Pseudomonas aeruginosa; D022142:Secretory Vesicles / Q000378:metabolism* / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects
D001426:Bacterial Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006997:Hypochlorous Acid