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Generation of macrophage variants with 5-azacytidine: selection for mannose receptor expression
S Diment1, M S Leech, P D Stahl
1Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri 63110.
Abstract:
Mannose receptors are expressed only in primary macrophages. Established macrophage-derived cell lines, although apparently possessing the potential to synthesize mannose receptors, do not express them on their plasma membranes. Using the drug 5-Azacytidine, mannose receptor expression was induced in the macrophage-derived mouse cell line J774. Receptor positive cells were sorted through a fluorescent activated cell sorter (FACS) prior to cloning. Clones were isolated which continuously express mannose receptors in culture. These macrophages were able to endocytose beta-glucuronidase and phagocytose yeast particles via mannose receptors. Secretion of the lysosomal enzyme beta-hexosaminidase was also reduced in proportion to the degree of mannose receptor expression.
Insights
Researchers induced mannose receptor expression in macrophage cell lines using 5-Azacytidine. This allowed the cells to perform mannose receptor-mediated functions, like endocytosis and phagocytosis.
Area of Science:
- Immunology
- Cell Biology
Background:
- Primary macrophages express mannose receptors, crucial for cellular functions.
- Macrophage-derived cell lines typically lack surface mannose receptor expression, limiting their utility.
- Investigating methods to restore mannose receptor expression in cell lines is important for research.
Purpose of the Study:
- To induce and sustain mannose receptor expression in a macrophage-derived cell line (J774).
- To characterize the functional capabilities of J774 cells with restored mannose receptor expression.
- To explore the impact of mannose receptor expression on lysosomal enzyme secretion.
Main Methods:
- Treatment of J774 cells with 5-Azacytidine to induce gene expression.
- Fluorescence-activated cell sorting (FACS) to isolate receptor-positive cells.
- Cloning of cells to establish stable, receptor-expressing macrophage lines.
- Assays to measure beta-glucuronidase endocytosis and yeast phagocytosis.
- Quantification of beta-hexosaminidase secretion.
Main Results:
- 5-Azacytidine successfully induced mannose receptor expression in J774 cells.
- Sorted and cloned cells demonstrated continuous mannose receptor expression in culture.
- Mannose receptor-positive macrophages effectively endocytosed beta-glucuronidase and phagocytosed yeast.
- Increased mannose receptor expression correlated with reduced secretion of beta-hexosaminidase.
Conclusions:
- Mannose receptor expression can be epigenetically induced in macrophage cell lines.
- Restored mannose receptor expression confers functional endocytic and phagocytic capabilities.
- Mannose receptor expression influences the secretion profile of lysosomal enzymes in macrophages.