title: Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils.
abstract: Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.
authors.affiliation: Department of Microbiology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhou
authors.forename: Kejing
authors.initials: K
authors.lastname: Song
authors.forename: Richard G
authors.initials: RG
authors.lastname: Painter
authors.forename: Martha
authors.initials: M
authors.lastname: Aiken
authors.forename: Jakob
authors.initials: J
authors.lastname: Reiser
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Stanton
authors.forename: William M
authors.initials: WM
authors.lastname: Nauseef
authors.forename: Guoshun
authors.initials: G
authors.lastname: Wang
mesh_terms: D002460:Cell Line; D002712:Chlorides / Q000276:immunology* / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000276:immunology* / Q000378:metabolism; D006997:Hypochlorous Acid / Q000276:immunology* / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects / Q000276:immunology; D051907:Lysosomal Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D019255:NADPH Oxidases / Q000276:immunology / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D010588:Phagosomes / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006861:Hydrogen Peroxide; D006997:Hypochlorous Acid; D051907:Lysosomal Membrane Proteins; D019255:NADPH Oxidases; D010879:Piperazines; D011799:Quinazolines; C507255:4-cyclohexyloxy-2-(1-(4-(4-methoxy-benzenesulfonyl)piperazin-1-yl)ethyl)quinazoline; C505032:CFTR protein, human; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1