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Phagocytosis of Cryptococcus neoformans by rat alveolar macrophages
1Department of Microbiology and Immunology, Duke University Medical Center, Durham, N.C. 27710.
Abstract:
A radiolabeling method was developed to investigate the interaction in vitro between Cryptococcus neoformans and Lewis rat alveolar macrophages (AM phi). AM phi were harvested by lung lavage, monolayers of adherent cells were established in wells of microtiter plates and [51Cr]-labeled yeast cells were added to the monolayers. After removal of extracellular yeasts, the adherent radioactivity associated with the AM phi was directly proportional both to the number of yeasts added and to the number of yeasts per AM phi as determined by microscopic examination of Giemsa-stained monolayers. Phagocytosis (attachment and/or ingestion) of radiolabeled C. neoformans by AM phi was a sensitive, quantitative and reproducible assay for the evaluation of the AM phi-C. neoformans interaction. AM phi were able to phagocytose encapsulated strains of C. neoformans. The extent of phagocytosis was inversely related to the capsule size. Normal rat serum (NRS) was an excellent source of opsonins for the ingestion. Inactivation of serum complement or depletion of C3 by affinity chromatography removed most of the opsonic activity of NRS. Specific antibodies against C. neoformans did not increase phagocytosis.
Insights
A new radiolabeling method quantifies Cryptococcus neoformans interaction with rat alveolar macrophages. Phagocytosis depends on capsule size and is enhanced by complement opsonins, not antibodies.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Cryptococcus neoformans is a fungal pathogen that can cause life-threatening infections.
- Alveolar macrophages (AM phi) are key immune cells in the lung responsible for clearing inhaled pathogens.
- Understanding the interaction between C. neoformans and AM phi is crucial for developing effective antifungal strategies.
Purpose of the Study:
- To develop and validate a radiolabeling assay for quantifying the in vitro interaction between C. neoformans and Lewis rat alveolar macrophages.
- To investigate the factors influencing phagocytosis of C. neoformans by AM phi, including yeast capsule size and serum opsonins.
Main Methods:
- Lewis rat alveolar macrophages (AM phi) were isolated by lung lavage.
- A novel [51Cr]-radiolabeling method was employed to label C. neoformans yeast cells.
- Radiolabeled yeast cells were co-cultured with AM phi monolayers, and phagocytosis was quantified by measuring adherent radioactivity.
Main Results:
- The radiolabeling assay demonstrated a direct proportionality between added yeast number and measured radioactivity, indicating a sensitive and quantitative method.
- Phagocytosis of C. neoformans by AM phi was inversely correlated with the size of the yeast capsule.
- Normal rat serum significantly enhanced yeast ingestion, with complement component C3 playing a critical role; specific antibodies did not enhance phagocytosis.
Conclusions:
- A robust radiolabeling assay was established for studying C. neoformans-AM phi interactions in vitro.
- Complement-mediated opsonization is essential for efficient phagocytosis of C. neoformans by alveolar macrophages.
- The inverse relationship between capsule size and phagocytosis highlights the capsule's role in immune evasion.