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Killing of Cryptococcus neoformans by rat alveolar macrophages
1Department of Microbiology and Immunology, Duke University Medical Center, Durham, N.C. 27710.
Abstract:
The addition of [51Cr]-labeled yeast cells of Cryptococcus neoformans to monolayers of Lewis rat alveolar macrophages (AM phi) provided a sensitive and reproducible in vitro assay of phagocytosis. AM phi and yeast cells were incubated in 10% (v:v) normal rat serum for 1 h, non-AM phi associated yeast cells were removed and the AM phi-associated radioactivity (phagocytosis) determined. Replicate wells were replenished with fresh medium and reincubated. At different times, yeast-AM phi monolayers were treated with a non-cryptococcocidal mixture of DNAse and sodium deoxycholate to release the yeast cells from the AM phi. The fate of the yeast cells was critically evaluated by [51Cr]-release and viable plate counts. Killing was detected by plate counts within an hour following phagocytosis and did not increase significantly during the next 5 h. Strains of C. neoformans with small, medium, or large capsules varied in their susceptibility to killing from 10% to 95% but susceptibility to killing was not directly related to capsule size and the extent of phagocytosis. Release of 51Cr did not correlate with viability as determined by culture. The 51Cr was associated with two pools in the yeast cells; one, representing 15-20% of the radiolabel, was easily released and was probably bound to low molecular weight compounds in the cytoplasm. The majority of label was tightly bound to the particulate alkali-soluble cell wall fraction.
Insights
This study developed a new in vitro assay using [51Cr]-labeled Cryptococcus neoformans and rat alveolar macrophages to measure phagocytosis. The assay revealed that yeast cell killing occurs rapidly after phagocytosis, independent of capsule size.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Phagocytosis is a critical immune mechanism for clearing microbial pathogens.
- Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious infections.
- Assessing the interaction between macrophages and Cryptococcus neoformans is vital for understanding host defense.
Purpose of the Study:
- To develop and validate a sensitive in vitro assay for quantifying phagocytosis of Cryptococcus neoformans by alveolar macrophages.
- To investigate the kinetics of Cryptococcus neoformans killing by macrophages post-phagocytosis.
- To explore the relationship between Cryptococcus neoformans capsule size, phagocytosis extent, and macrophage-mediated killing.
Main Methods:
- Utilized [51Cr]-labeled Cryptococcus neoformans yeast cells and Lewis rat alveolar macrophages (AM phi) in a monolayer assay.
- Incubated AM phi with labeled yeast in rat serum, followed by removal of non-phagocytosed yeast.
- Quantified phagocytosis by measuring cell-associated radioactivity and evaluated yeast cell fate using [51Cr]-release and viable plate counts.
Main Results:
- The assay demonstrated sensitivity and reproducibility in measuring phagocytosis.
- Yeast cell killing was detected within one hour post-phagocytosis and did not significantly increase over the next five hours.
- Susceptibility to killing varied widely among C. neoformans strains but was not directly correlated with capsule size or the extent of phagocytosis.
- [51Cr]-release did not accurately reflect yeast viability.
Conclusions:
- A robust in vitro assay for studying Cryptococcus neoformans phagocytosis by alveolar macrophages was established.
- Macrophage-mediated killing of Cryptococcus neoformans occurs rapidly and is not directly influenced by yeast capsule size.
- The [51Cr] labeling method has limitations for assessing yeast viability post-phagocytosis.