title: Chloride transport in functionally active phagosomes isolated from Human neutrophils.
abstract: Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC-dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes.
authors.affiliation: Department of Microbiology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
authors.forename: Martha L
authors.initials: ML
authors.lastname: Aiken
authors.affiliation: Department of Microbiology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Painter
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.affiliation: Department of Microbiology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA; Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA; Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA. Electronic address: gwang@lsuhsc.edu.
authors.forename: Guoshun
authors.initials: G
authors.lastname: Wang
mesh_terms: D001692:Biological Transport; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D054879:Halogenation; D006801:Humans; D006997:Hypochlorous Acid / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006997:Hypochlorous Acid; D009195:Peroxidase; D019255:NADPH Oxidases; D025262:Vacuolar Proton-Translocating ATPases
keywords: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 4-aminobenzoic acid hydrazide; 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone; 5-nitro-2-(3-phenylpropylamino)benzoic acid; ABAH; AO; Ap5A; CFTR; CFTRinh-172; Chloride transporter; ClC-3; DFP; DHR; Free radicals; Hepes; Hypochlorous acid; LAMP-1; LDH; LF; MPO; NPPB; Neutrophils; P1,P5-di(adenosine-5′) pentaphosphate.; PKA; PM-PLS; Phagosomes; Rp-adenosine-3′,5′-cyclic monophosphorothioate; Rp-cAMPS; SOD; Sp-adenosine-3′,5′-cyclic monophosphorothioate; Sp-cAMPS; V-ATPase; acridine orange; catalytic subunit of protein kinase A; chloride channel-3; cystic fibrosis transmembrane conductance regulator; dihydrorhodamine 123; diisopropylfluorophosphate; lactate dehydrogenase; lactoferrin; lysosomal-associated membrane protein-1; myeloperoxidase; paramagnetic phagolysosomes; superoxide dismutase; vacuolar-type ATPase proton pump