Estimation of pH in Individual Alveolar Macrophage Phagolysosomes

Abstract

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.

Phagolysosomal pH in alveolar macrophages.

“…Table 2 compares phagolysosomal pH obtained from measurements ofmacrophage populations by fluorescence spectrometry with measurements on single particles in phagolysosomes by fluorescence spectrometry in samples from the same rabbits. Table 2 shows a good agreement between the pH values obtained with the two methods, apparently due to the rather small variation in pH among phagolysosomes in macrophages from the same cell sample with a coefficient of variation below 10%, often only around 5% (16).…”

“…Figure 1 show standard curves for FYP and FSP obtained from fluorescense measurements ofthe particles suspended in series of Hank's balanced salt solution (BSS) with various pHs. (14) and Nyberg et al (15,16).…”

“…Table 3 presents phagolysosomal pH using FYP and FSP in macro- Table 2. Phagolysosomal pH in samples from three rabbits estimated from measurements on individual phagolysosomes in a fluorescence microscope and on populations of macrophages in a fluorescence spectrometer (16 the pH was unchanged during the same period (14,15). Table 3 presents phagolysosomal pH using FYP and FSP in macrophages cultured for 3 and 24 hr.…”

“…In cell populations, using a fluorescence spectrometer, they estimated lysosomal pH from the relationship between the ratio of the fluorescenceat wavelength 519 nm and excitation wavelengths 495 and 450 nm and pH. We used this principle for measurements of phagolysosomal pH in alveolar macrophages with fluoresceinlabeled yeastparticles (FYP) or fluorescein-labeled amorphous silicaparticles (FSP) as probes (14)(15)(16)(17). The presentpaper reviews our studies with these particles concerning methodological aspects and includes examination of the influence of particle number, size, and material on pH, comparisons ofpH obtained from the testparticles in vitro and in vivo, as well as after various times after the administration ofparticles.…”

Intracellular Particle Dissolution in Alveolar Macrophages

“…range of 5 (45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60), it is plausible to conclude that the intracellular mechanism of particle dissolution is controlled by these two parameters. However, at this moment we cannot exclude other mechanisms of IPD resulting from proteolytic enzymes, oxygen radicals, chelators, precipitators, etc., that are contained in the phagolysosomal plasma (20).…”