Phagocytosis and Intracellular Killing of Candida albicans by Murine Polymorphonuclear Neutrophils
Polymorphonuclear neutrophils (PMNs) are important phagocytes in the control of Candida infections. The phagocytic contribution of PMNs to host defence can by assessed by various methods, such as microbiological assays. However, assessment and definition
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1. Introduction Antigen-presenting cells such as polymorphonuclear neutrophils (PMNs) constitute an essential component of both innate and adaptive cell-mediated immunity against Candida albicans infections (1, 2). One way to assess the contribution of PMNs to host defence is by means of a microbiological assay. A comparative study of several assays showed that the use of an adherent monolayer of murine exudate peritoneal PMNs is a robust method for distinguishing between the processes of phagocytosis and intracellular killing (3), thus avoiding misinterpretation. Firstly, granulocytes are able to kill Candida yeast both intracellularly (by phagolysosomal fusion) and extracellularly (4, 5), but this is often not taken into account in assays that simply compare the growth rate of C. albicans in the presence of phagocytes with the growth rate of Candida yeast in the absence of phagocytes (6–8). Secondly, such
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_18, © Springer Science+Business Media, LLC 2012
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assays do not distinguish between ingestion and intracellular killing, where apparent differences in “killing” of Candida yeast may actually be due to differences in ongoing phagocytosis or varying phagocytosis efficiency. In addition, the definition of intracellular killing used warrants a cautious approach. Some reports express “killing” as (1 − (CFU after incubation in the presence of phagocytes/CFU of Candida cultured without phagocytes)) × 100 (8), where it may be more appropriate to describe this phenomenon as percentage growth inhibition rather than killing (6). We define the percentage intracellular killing of C. albicans as (1 − (CFU after incubation in the presence of phagocytes/numbers of CFU phagocytosed)) × 100 (2, 9). Furthermore, researchers should be aware that protocol-related issues that influence the outcome of killing assays include the types of mouse and Candida strains used, the choice of eliciting agent (PMNs elicited by the injection of proteose peptone displayed significantly enhanced candidacidal activity compared to those elicited by thioglycollate (10)) and the growth phase of the Candida suspension at the moment of PMN transfer. Here we describe a protocol that allows the unequivocal determination of phagocytosis and killing of C. albicans yeast in an adherent monolayer of murine exudate PMNs and we discuss the issues that can affect the results.
2. Materials 2.1. Preparation at Day = −2
1. Frozen C. albicans aliquot (strain ATCC 10261 MYA-3573 (UC820)). 2. Sabouraud agar Petri dish. 3. Sterile loop (10 μL). 4. Incubator at 37°C.
2.2. Preparation at Day = −1
1. Candida strain on the Sabouraud agar Petri dish. 2. 40 mL Sabouraud broth, prepared according to manufacturer’s prescription. 3. Incubator at 37°C. 4. Orbital shaker. 5. Proteose peptone 10% (Difco Laboratories), 1 mL/mouse. 6. Sterile ice-cold phosphate-buffered saline (PBS) con
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