Quantitative analysis of phagocytosis of Cryptococcus neoformans by adherent phagocytic cells by fluorescence multi-well plate reader

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Abstract

Macrophages and monocytes are adherent phagocytic cells which play an important role in host defence against the yeast-like fungus Cryptococcus neoformans. Before, phagocytosis by adherent phagocytes could only be measured by means of microscopy or by a radioactive assay, which both have obvious disadvantages. We have developed a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent phagocytes (e.g. alveolar macrophages) using a fluorescence multi-well plate reader. This method allows us to discriminate accurately between adherence and internalisation of C. neoformans by macrophages during long term incubation. In addition, the method was used to study the role of the mannose receptor in phagocytosis of the acapsular yeast in the absence of serum by human monocyte-derived macrophages (MDM).

Introduction

Cryptococcus neoformans is a yeast-like fungus that causes infections in immunocompromised patients, especially in those with AIDS. Cryptococcosis caused by C. neoformans, is worldwide the leading cause of life-threatening mycological disease of the central nervous systems (Levitz, 1991).

In nature, the yeast cells possess minimal capsules and are therefore easily aerosolized. In this state, the yeasts are of a smaller size and can be inhaled to the level of the alveolus (Perfect, 1989). The initial site of infection is the lung, where (alveolar) macrophages represent the first line of host defence (Mitchell and Perfect, 1995). One of the first steps in host defence, therefore, is the interaction between the acapsular C. neoformans and alveolar macrophages. This interaction takes place in the alveolar spaces, where only low concentrations of serum components are present (Mitchell and Perfect, 1995). Cross et al. described that ingestion of acapsular C. neoformans by mice macrophages in the absence of serum occurs via constitutively present mannose- and β-glucan-receptors (Cross and Bancroft, 1995).

Previously, we have described a quantitative analysis of phagocytosis of C. neoformans by peripheral blood mononuclear cells using flow cytometry (Chaka et al., 1995). This method enables us to assess phagocytosis by cells in suspension, but not by adherent cells. As macrophages, like monocytes, strongly tend to adhere to different surfaces, a method is needed to measure phagocytosis by adherent cells. Until now, interaction between adherent phagocytic cells and C. neoformans could be studied by means of microscopy, which has the major disadvantage of being cumbersome and interpretation of results is subjective, or by a radioactive assay. This last method has disadvantages for routine laboratory use involving health hazards and costs associated with radioisotope use and disposal. Phagocytosis of C. neoformans by monocytes and macrophages, measured by these methods, has been described by several investigators (Bolanos and Mitchell, 1989, Reardon et al., 1996).

We describe a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent monocytes and macrophages during short and long term incubation. Fluorescein isothiocyanate (FITC)-labelled C. neoformans were added to adherent phagocytes in a 96-well microtiter plate. At the end of the incubation time, trypan blue was used as a quencher of the non-internalized FITC-labelled cryptococci. Interestingly, the fluorescence of the internalized cryptococci was unaffected. This concept lays the foundation of our novel phagocytosis assay. The fluorescent signal of the FITC-labelled C. neoformans was measured by a fluorescence multi-well plate reader before and after addition of trypan blue. This enabled us to distinguish yeast cells that had been taken up by adherent phagocytes from those that were simply attached.

The new method was compared with microscopic evaluation of phagocytosis of acapsular C. neoformans by monocytes or monocyte derived macrophages (MDM). Finally, the method was used to study the role of the mannose receptors in phagocytosis of the acapsular yeast by human MDM in the absence of serum.

Section snippets

Cryptococcus neoformans preparation and FITC labelling

An acapsular mutant of C. neoformans variety neoformans (NIH B 4131) obtained from the National Institutes of Health (NIH, Bethesda, MD, USA) was used throughout this study. The yeast cells were maintained on Sabouraud dextrose agar (Merck, Darmstadt, Germany) at 4°C. After harvesting, the cryptococci were heat-killed for 30 min at 80°C, the concentration was adjusted to 5×107 cells/ml and the cells were labelled with fluorescein isothiocyanate (FITC, Sigma, St. Louis, MO, USA) at 500 μg/ml in

Association of C. neoformans with elutriated monocytes

In order to study binding and uptake of acapsular C. neoformans by the adherent phagocytes, we pre-opsonized the yeast cells with either 10% normal serum or K1228 serum (including specific rabbit anti-cryptococcal antibodies), or buffer only. Subsequently cryptococci were added to the adherent monocytes for either 3 or 18 h. After 3 h of incubation, the yeast cells which were pre-opsonized with human pooled serum (HPS) showed enhanced binding and uptake by the adherent cells, as compared to the

Discussion

The acapsular form of Cryptococcus neoformans is inhaled by the respiratory tract, and can, because of its size, reach the alveolar spaces. There the yeast will meet the first cells involved in host response: the alveolar macrophages. In vitro these cells, like monocytes, tend to adhere strongly to surfaces. Therefore, to study the first line of host defence against C. neoformans, a system is needed to measure the interaction between cryptococci and adherent phagocytes. A new, rapid and

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