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Updated: May 17, 2026
12:42
Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator
Published on: April 5, 2017
Chloride transport in functionally active phagosomes isolated from Human neutrophils
Martha L Aiken1, Richard G Painter1, Yun Zhou1
1Department of Microbiology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Free Radical Biology & Medicine
|October 24, 2012
Summary
Chloride transport into neutrophil phagosomes is essential for producing hypochlorous acid (HOCl) to kill microbes. This study identifies multiple chloride channels, including CFTR, responsible for this vital anion supply.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophil phagosomes are crucial for microbial killing via hypochlorous acid (HOCl).
- Chloride anion transport into phagosomes is vital for HOCl production but its mechanism is unclear.
- Understanding chloride transport is key to understanding innate immune responses.
Purpose of the Study:
- To elucidate the molecular mechanisms of chloride anion transport into human neutrophil phagosomes.
- To characterize the ion transport properties of isolated, functional phagosomes.
- To identify specific chloride channels involved in phagosomal function.
Main Methods:
- Isolation of functional human neutrophil phagosomes using paramagnetic latex microspheres and magnetic separation.
- Assessment of phagosome integrity and function using fluorescent markers (FITC-dextran, acridine orange) and enzymatic assays.
- Measurement of HOCl production using a specific fluorescent probe (R19-S) and MPO-mediated iodination.
- Evaluation of chloride channel activity using specific inhibitors (CFTRinh-172, NPPB) and proton flux measurements.
Main Results:
- Isolated phagosomes maintained structural integrity and exhibited proton transport via V-ATPase.
- Phagosomes demonstrated hydrogen peroxide production and HOCl generation.
- Chloride uptake and MPO-mediated iodination were inhibited by chloride channel blockers.
- V-ATPase proton flux was dependent on chloride cotransport, with CFTR identified as a major contributor.
Conclusions:
- The isolated phagosome preparation retains essential ion transport functions.
- Multiple chloride channels, notably the cystic fibrosis transmembrane conductance regulator (CFTR), facilitate chloride supply to neutrophil phagosomes.
- This research clarifies a critical aspect of neutrophil antimicrobial function and opens avenues for therapeutic strategies.
Keywords:
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid4-aminobenzoic acid hydrazide5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone5-nitro-2-(3-phenylpropylamino)benzoic acidABAHAOAp5ACFTRCFTRinh-172Chloride transporterClC-3DFPDHRFree radicalsHepesHypochlorous acidLAMP-1LDHLFMPONPPBNeutrophilsP1,P5-di(adenosine-5′) pentaphosphate.PKAPM-PLSPhagosomesRp-adenosine-3′,5′-cyclic monophosphorothioateRp-cAMPSSODSp-adenosine-3′,5′-cyclic monophosphorothioateSp-cAMPSV-ATPaseacridine orangecatalytic subunit of protein kinase Achloride channel-3cystic fibrosis transmembrane conductance regulatordihydrorhodamine 123diisopropylfluorophosphatelactate dehydrogenaselactoferrinlysosomal-associated membrane protein-1myeloperoxidaseparamagnetic phagolysosomessuperoxide dismutasevacuolar-type ATPase proton pump
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