PHAGOCYTOSIS OF VIABLE CANDIDA-ALBICANS BY ALVEOLAR MACROPHAGES - FLOW CYTOMETRIC QUANTIFICATION

The phagocytic capacity of blood leukocytes may be assessed by flow cy tometric techniques using fluorochrome-labeled particles including via ble microorganisms. Application of this approach to alveolar macrophag es (AM) is hampered or even rendered impossible by the strong autofluo rescence of this cell type, superimposing the fluorescence intensity o f the labeled phagocytic targets. Viable Candida albicans were loaded with the membrane-permeable fluorescent dye carboxy-seminaphtorhodaflu or 2/acetoxymethylester (carboxy-SNARF 2-AM), which is cleaved intrace llularly to generate the membrane-impermeable derivative carboxy-SNARF 2. Fluorescence was excited with the 488-nm line of an argon-ion lase r, and the emission peak at 633 nm was used for quantification of dye- associated fluorescence. Rabbit and human AM were labeled with fluores cein isothiocyanate-coupled monoclonal mouse anti-macrophage antibodie s. After coincubation of macrophages and yeast, 4% paraformaldehyde pl us 0.5% EDTA in phosphate-buffered saline was used to stop the phagocy tic process and detach adherent yeast from the AM surface. Macrophages loaded with yeast displayed a shift from monochromatic (green) to dua l (green and red) fluorescence. The percentage of yeast-positive AM an d red fluorescence intensity of phagocytosing macrophages were quantif ied. Yeast opsonization with serum or anti-Candida immunoglobulins was a prerequisite for phagocytosis. Under optimized conditions (0.5-10% serum; 60 min yeast-AM incubation; yeast-AM ratio 8:1 to 12:1), 71-91% of the AM were involved in the phagocytic process. Yeast engulfment w as completely inhibited by N-ethylmaleimide and iodoacetic acid. We co nclude that the phagocytosis of viable Candida albicans by AM can he q uantified feasibly on a single-cell level by use of a flow cytometric technique designed to circumvent the interfering autofluorescence of t his cell type.