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Intraphagolysosomal pH in canine and rat alveolar macrophages: flow cytometric measurements
P Heilmann1, W Beisker, U Miaskowski
1Projekt Inhalation, Gesellschaft für Strahlen- und Umweltforschung mbH München, Neuherberg, Federal Republic of Germany.
Abstract:
Intracellular dissolution of inhaled inorganic particles is an important clearance mechanism of the lung and occurs in phagolysosomal vacuoles of phagocytes. Flow cytometric measurements of intraphagolysosomal pH in alveolar macrophages (AM) obtained from beagle dogs, Wistar rats, and from a baboon were made using fluorescein isothiocyanate-labeled amorphous silica particles (FSP). AM were obtained by bronchoalveolar lavage. FSP were phagocytized by AM in cell suspensions incubated in full media for 24 hr up to 6 days. Dual laser flow cytometry was performed and six-parameter list mode data were recorded from forward scatter, side scatter, and fluorescence intensities at 530 nm excited at 457 nm and 488 nm as well as logarithmic fluorescence intensity at wavelengths 630 nm excited at 488 nm. In this way it was possible to discriminate viable AM with phagocytized FSP from lysing AM with phagocytized FSP and from cells without FSP and from free FSP. Viable cells were distinguished from lysing cells by staining with propidium iodide immediately before the flow cytometric measurement. A calibration curve for the pH value was determined from FSP suspended in buffered media at pH values ranging from 3.5 to 7.5. First flow cytometrical results indicated that after an incubation time of 24 hr, the mean intraphagolysosomal pH of viable AM was 4.7 +/- 0.3 for dogs and 5.1 +/- 0.5 for rats. The intraphagolysosomal pH of the baboon AM was 4.5.
Insights
The intraphagolysosomal pH in alveolar macrophages was measured using flow cytometry. This study reveals the acidic environment within phagolysosomes crucial for particle dissolution in the lungs.
Area of Science:
- Toxicology
- Cell Biology
- Pulmonary Medicine
Background:
- Inhaled inorganic particles are cleared from the lungs via intracellular dissolution within phagolysosomes.
- Alveolar macrophages (AM) are key phagocytes involved in lung clearance mechanisms.
Purpose of the Study:
- To measure the intraphagolysosomal pH in AM from different species after particle ingestion.
- To characterize the intraphagolysosomal environment relevant to particle dissolution and lung clearance.
Main Methods:
- Utilized flow cytometry with fluorescein isothiocyanate-labeled amorphous silica particles (FSP) to assess intraphagolysosomal pH in AM.
- Employed dual-laser flow cytometry and propidium iodide staining to differentiate viable from lysing cells and identify phagocytized FSP.
- Established a pH calibration curve using FSP in buffered media across a pH range of 3.5 to 7.5.
Main Results:
- Determined mean intraphagolysosomal pH values in viable AM after 24-hour incubation: 4.7 ± 0.3 in dogs, 5.1 ± 0.5 in rats, and 4.5 in baboons.
- Successfully discriminated between viable AM with FSP, lysing AM with FSP, cells without FSP, and free FSP.
- Demonstrated the feasibility of using flow cytometry to quantify intraphagolysosomal pH in different species.
Conclusions:
- The intraphagolysosomal environment in AM is acidic, supporting the intracellular dissolution of inhaled particles.
- Flow cytometry is a robust method for assessing intraphagolysosomal pH and cellular responses to particle exposure.
- Findings provide insights into the lung's innate defense mechanisms against inorganic particle inhalation.