Phagolysosomal pH measurements in alveolar macrophages

Abstract: Background: Mechanical clearance of insoluble particles from the alveolar part of the lung is known to be a slow process in humans, i. e., one or several years. Particles deposited in the alveolar region are phagocytized by alveolar macrophages (AM). During phagocytosis the particles will be enclosed in phagolysosomes. The low pH in these compartments is important for degrading and killing of microorganisms, as well as dissolution of inorganic particles. For many inorganic compounds with low solubility, dissolution by AM in the lung is probably the most important clearance mechanism. Purpose: The aims of this study were to establish methods for measurement of intracellular pH and by these methods estimate the phagolysosomal pH in AM from various species, including humans. Reactions in vitro of rat AM to pathogenic and non-pathogenic yeasts compared to inert silica particles were also investigated. Methods: Alveolar macrophages were obtained by bronchoalveolar lavage of lungs from rabbits, rats, baboons, guinea pigs, beagle dogs, and humans. Yeast particles (S. cerevisiae and C. albicans) and amorphous silica particles were conjugated to fluorescein isothiocyanate (FITC) and added intratracheally or to cultured alveolar macrophages. Assays of phagolysosomal pH, phagocytosis, and oxidative metabolism and electron microscopical examinations on cell samples were performed. Results: The phagolysosomal pH in rabbit AM after in vitro and in vivo phagocytosis of FITC-labelled yeast particles (S. cerevisiae) was found to be around 5. The pH was significantly higher a few hours after lavage than after 24h. Phagolysosomal pH obtained with silica particles after in vitro and in vivo phagocytosis was estimated to be around 5. Neither the size (3 and 5 gm, resp.) nor the number of internalized particles did affect the phagolysosomal pH of AM. The electron-microscopical studies showed more lysosomes in close contact with phagosomes containing yeasts, compared to silica-containing phagosomes. The yeast-containing phagosomes were also more vacuolated. Phagolysosomal pH was found to be similar, around 5, in an interspecies comparison. The variation of pH in the phagolysosomes of rabbit AM after 3, 6, and 24h, using FITC-labelled silica particles, was found to be small (less than 10 %). Phagolysosomal pH of rabbit AM, measured at various time points (24h, I week, I month) after instillation of silica particles into the lung, was similar (about 5). Histological sections from each lung showed that all particles were localized inside AM. A comparison of AM from smokers and nonsmokers gave similar results in phagolysosomal pH, but the morphology of the cells was quite different. Rat alveolar macrophages were challenged with a highly pathogenic yeast (C. albicans), a non-pathogenic yeast (S. cerevisiae), and an inert particle (amorphous silica). After 3 hours of incubation, a fraction of around 10 % with neutral pH was found in the phagolysosomes with the yeast particles, but not in the samples with silica particles. Electron-microscopic examinations revealed narrow passages between phagolysosomes and the cell surface. Both C. albicans and S. cerevisiae were phagocytized at a faster rate compared to the silica particles, mainly due to a faster ingestion process. Both yeasts induced a 3-4-fold increase in oxidative metabolism during phagocytosis and a slightly lower increase 24 hours later. Silica particles only induced a small increase in oxidative metabolism after 24 hours. Conclusions: The phagolysosomal pH in AM estimated with inert silica particles was found to be around 5 for many species, including humans. The phagolysosomal pH was rather constant over time, with a variation less than 10 % within the same animal. pH was affected neither by the size nor by the amount of particles added to the cells. High exposure of combustion products, like cigarette smoke, did not affect the phagolysosomal pH of AM, compared to non-smokers' cells. The yeast cell S. cerevisiae induced a higher phagolysosomal pH in AM after 3 hours, and C. albicans a lower pH after 24 hours, compared to the silica particles. Fractions (around 10 %) of phagolysosomes with neutral pH appeared in samples incubated for 3 hours where either of the yeasts had been phagocytized, but was rarely seen with the silica particles. Both yeasts induced a 3-4-fold increase in the oxidative metabolism in AM during and after 3 hours of phagocytosis compared to inert particles. A smaller increase still persisted after 24 hours. Studies of phagolysosomal pH in alveolar macrophages would probably lead to a better understanding of alveolar clearance. For this purpose it is important to establish methods for pH measurements.