Neutrophils in Cystic Fibrosis

Guoshun Wang, DVM, PhD, Associate Professor 

 CFTR Expression in Human Neutrophils and 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 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 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, 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.  We have thus generated evidence that CFTR channel expression in neutrophils, and its dysfunction, affects neutrophil chlorination of phagocytosed bacteria.

Development of a novel chloride probe to measure chloride concentrations in phoagolysosomes

Human neutrophils synthesize hypochlorous acid (HOCl) as one of the antimicrobial agents to kill pathogens that they have taken up within phagolysosomes.  The production of HOCl is catalyzed by myeloperoxidase (MPO) using chloride anions (Cl?).  Even though various approaches have been documented to measure cytosolic Cl? concentrations, direct detection of phagolysosomal free Cl? in a real-time fashion is not available. We have recently developed a new dual-labeled fluorescent probe by conjugation of the chloride-sensitive 6-methoxyquinoline and chloride-insensitive tetramethyl rhodamine to zymosan particles (see Figure).  After phagocytosis into neutrophils, the probe was able to monitor intraphagosomal chloride levels authentically.  Human neutrophils in the medium containing 122 mM chloride had a phagolysosomal chloride level of 73.3 mM ± 12.2 (N=5).  In contrast, the neutrophils in a chloride-free iso-osmotic medium had a chloride level of 6.6 mM ± 1.9 (N=5) in the phagolysosomal compartment. These data clearly demonstrated that the extracellular chloride levels affect intraphagolysosomal chloride levels in human neutrophils. Moreover, when the extracellular chloride concentration was switched from high level (122 mM) to low level (0 mM) or vice versa, the probe demonstrated complete reversibility.  This new zymosan-conjugated chloride probe can be used as an indicator for measurement of the free chloride level within the phagolysosomes of neutrophils and other phagocytic cell types. 

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