Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry

Abstract

Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2-7 1-bis(2carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% ؎ 2%, 35% ؎ 14%, 50% ؎ 11%, 51% ؎ 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% ؎ 17%, 22% ؎ 9%, and 40% ؎ 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.

Pulmonary surfactant protein A binds to Cryptococcus neoformans without promoting phagocytosis

“…Opsonized acapsular C. neoformans (100 mL, 10 7 cells mL -1 ) was then incubated with 100 mL of phagocytes, (10 7 cells mL -1 MDMs, HAMs, monocytes or PMNs) for 2 h at 37ЊC. Experiments in our laboratory showed that increasing phagocytosis time (0, 15, 30 60 and 120 min) resulted in enhanced binding and uptake of cryptococci, reaching a maximum after 1 h [6]. In subsequent experiments, a phagocytosis time of 2 h was used in order to ensure optimal phagocytosis [6].…”

“…Phagocytosis of encapsulated and acapsular C. neoformans by alveolar macrophages and other phagocytes has been examined by several investigators [4][5][6]. The deposition of IgG and C3b and their degradative products on C. neoformans are essential for optimal phagocytosis [7].…”

Effects of granulocyte colony-stimulating factor (G-CSF) treatment on granulocyte function and receptor expression in patients with ventilator-dependent pneumonia

“…Using flow cytometry, the killing capacity of isolated neutrophils was determined by the ability of Candida to retain a fluorescent probe. This technique was previously used by our laboratory for Cryptococcus neoformans [19]. Briefly, a clinical isolate of C. albicans was grown overnight in selective medium containing 6 mM 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetomethyl ester (BCECF-AM), washed, and stored at ¹70ЊC in PBS containing 20% glycerol.…”

An Automated Assay to Measure Phagocytosis of Cryptococcus neoformans

“…Assays based on flow cytometry have also been used to assess interactions between C. neoformans and host cells on a larger scale (Chaka et al, 1995;Voelz, Johnston, Rutherford, & May, 2010;Alanio, Desnos-Ollivier, & Dromer 2011;Nicola, Robertson, Chang et al…”