Evaluation of phagolysosome fusion in acridine orange stained macrophages infected with Histoplasma capsulatum.

A phagosome-lysosome (PL) fusion was performed in vitro using peritoneal cells from normal BALB/c mice and the J774.2 macrophage cell line infected with the yeast phase of the fungus Histoplasma capsulatum at ratios of 5 x 10(5), 5 x 10(6) or 1 x 10(7) yeasts per 1 x 10(6) macrophages, and phagocytosis was allowed to proceed for 5, 30 and 60 min. Macrophage lysosomes were pre-labelled with acridine orange and the cells were challenged with the parasite. Fusion was evaluated by fluorescence microscopy and the number of macrophages with stained yeast cells was scored. The phagolysosome fusion frequency (PLFF) was calculated by subtracting the specific fusions of infected macrophages from the non-specific fusions of uninfected macrophages and normalizing the total number of bound yeasts. The PLFF was determined using different doses and strains of H. capsulatum. Results showed that PLFF in infected macrophages depends on the infection dose. Inhibition of PL fusion was detected mainly at a high infection ratio (1 x 10(7) yeasts/1 x 10(6) macrophages), and PL fusion varied with phagocytosis time. No significant differences were observed in the fusions when different Histoplasma strains were used. Results with J774.2 cells were similar to peritoneal cells, indicating that both methodology and fusion calculations employed were useful, in spite of the heterogeneity of macrophage populations used.