A method for measurement of phagolysosomal pH in individual alveolar macrophages based on a cytofluorometric technique with fluorescein-labeled silica particles (FSP) as a probe was developed. The size of the FSP, 3.0 or 5.0 microns, did not affect the result of the pH measurements. The average pH values, range 5.1-5.5, of individual particles in macrophages from three rabbits agreed well with the pH values obtained in a macrophage population using a fluorescence spectrometer and the FSP. The variation of pH in phagolysosomes in alveolar macrophages from five rabbits was investigated. Measurements were performed 3, 6, and 24 h after addition of the FSP in cells containing one particle as well as in cells containing two particles. The variation in pH was small, with a coefficient of variation less than 10% in all rabbits at all times. There was a significant correlation between values obtained from two phagolysosomes in the same cell, indicating a cell factor responsible for 10-30% of the total variance. The fact that the variation of pH is small in normal, untreated rabbit alveolar macrophages should be of importance when estimating alveolar clearance of inorganic particles due to dissolution in the acid phagolysosomal milieu.

Intraphagosomal pH in rabbit alveolar macrophages was studied using amorphous silica particles (FSP) and yeast particles (FYP) labeled with fluorescein. The pH was estimated from the quotient between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths 495 and 450 nm. Within a factor of 10, pH was independent of the number of FSP added to macrophages in vitro. In macrophages cultured for 24 h, the pH obtained with FSP and FYP was about 5. Three hours after lavage, pH was the same as after 24 h for the FSP but significantly higher for the FYP, 5.8. Both 3 and 24 h after lavage, more lysosomes were in contact with the FYP-containing phagosomes than with the FSP-containing ones. Most FSP were in tight contact with the phagosomal membrane, while there was a clear zone between most FYP and the phagosomal membrane. The differences in pH and morphology between cells containing FSP and FYP might be explained by the assumptions that the macrophages disintegrate the FYP, which results in higher pH, and that the disintegration of FYP is more efficient at 3 than at 24 h after the lavage. The intraphagosomal pH was lower when the macrophages were allowed to phagocytize the FSP in vivo. The pH values were 5.1 in vitro, 4.9 at 24 h, and 4.5 at 1 week after the FSP had been instilled via trachea. The FSP should be a useful tool for estimation of intraphagosomal pH at basic conditions, i.e., the milieu to which many inhaled inorganic particles will be exposed.

VW tdi phagolysm pH in veobr macrophags (AM) using fluorescein-labeledyead (FYP) amn silica particles (FSP) as probes. Fluorescence intensities from the ingested test particles were measured on populations of AM using fluorescence spectometry and on individual phagolysosomes using fluorescence microscopy. Measurements were performed on rabbit AM, which had been incubated with FYPorFSP (in vitot procedure). ealso insd FYPor FSP via the trachea into rabbit lungs and after 1 day, 1 week, 1 month, and 3 months lavaged thelungs and measured the pH in AM (in vivo procedure). Phagolysosomal pH was independent of the number and size of the fluorescent particles. Measurementsof uadons of AM with fluorescence spec y and of hd ual phagolysosomes with fluorescence microscopy gave similar average pH. For the FYP, pH decreased during the first day after lavage both in the in vit and the in vivo procedures. For the FSP, pH was unchaneddurngthe same period. After 1 daypH was sinilar for both particles. Electron microscopy showed a larger number of lysosomes in contact with phagosomes and a higher pementage ofvacuolated phagosomesforFYPthanforFSP. Inthein uwoprocedure, pH was unchanged at least upto 1 month, and this pH was lower than that in the in vitrv procedure. The difference was probably due to conditions at the time of phagocyosis Particles retained in the lung parenchyma were within AM, and their location within the AM appeared unchanged from 1 week up to 3 months.

The pH in phagolysosomes of rabbit alveolar macrophages was studied using yeast particles labeled with fluorescein isothiocyanate (FITC). The yeast particles were added to the macrophages in vitro a few hours or 1 day after they had been lavaged from the lung and in vivo 3 h or 1 day before the lungs were lavaged. Intracellular pH was estimated from the ratio between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths of 495 and 450 nm. In both the in vitro and in vivo experiments pH decreased significantly during the first hours after lavage, but after a few hours reached almost the day-2 levels, i.e., 4.9-5.4. The decrease in pH was related to time after lavage and not to time after phagocytosis of the particles. It is suggested that intracellular measurements of pH in alveolar macrophages should be combined with determinations of lung clearance of metal particles.