Intraphagosomal pH in Alveolar Macrophages Studied with Fluorescein-Labeled Amorphous Silica Particles

Abstract

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.

Phagolysosomal pH in alveolar macrophages.

“…In the measurements using FYP, there was a decrease in pH during the first day after lavage, both in the in vitro and the in vivo procedure, whereas in measurements with FSP, the pH was unchanged during the same period (14,15). Table 3 presents phagolysosomal pH using FYP and FSP in macro- Table 2.…”

“…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).…”

“…For details, see Nilsen etal. (14) and Nyberg et al (15). 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.…”

“…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.…”

Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?

“…Instead, a delayed acidification of the phagosome was observed in neutrophils expressing an activated oxidase, the phagosomal pH remaining initially near-neutral for several minutes (Jankowski et al, 2002).The neutrality of neutrophil phagosomes contrasts with the profound acidification of macrophages phagosomes. Several studies consistently showed that the phagosomal pH of macrophages decreases rapidly and equilibrates around pH 5.0 in 30 min (Nyberg et al, 1989;Lukacs et al, 1990Lukacs et al, , 1991Sturgill-Koszycki et al, 1994;Rybicka et al, 2010). To add to the variability, a phagosomal alkalization was reported in dendritic cells, the phagosomal pH reaching pH 7.6 within 1 hour of phagocytosis in these phagocytes (Savina et al, 2006).…”