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pubmed:abstractText |
Human cord blood mononuclear cells were cultured for 35 days in media containing recombinant human interleukin 5 (rhIL-5) supplemented with a fraction of the culture supernatant of phytohemagglutinin (PHA)-stimulated human T lymphocytes from which interleukin 2 (IL-2) was eliminated. Cultured cells were studied by electron microscopy and an immunogold procedure to detect subcellular site(s) of Charcot-Leyden crystal (CLC) protein. The majority of cells (greater than 70%) developing in this system were mature eosinophils, with descending frequency of other cells, including macrophages, mature basophils, eosinophilic myelocytes, and mature neutrophils. Mature eosinophils were characterized by increased numbers of primary granules, small granules, tubulovesicular structures, and decreased secondary granules. These eosinophils showed extensive piecemeal degranulation (PMD) characterized by partially empty and empty secondary granule chambers in the cytoplasm. Small, smooth vesicles were evident within empty granule chambers as well as adjacent to them. Eosinophils formed close associations with phagocytic macrophages that contained both standard-shaped and irregularly shaped CLC within phagolysosomes. Subcellular sites of CLC protein were demonstrated by immunogold in eosinophils and macrophages arising in these cultures. Charcot-Leyden crystal protein was present in the nuclear matrix and extraorganellar cytoplasm of eosinophils. Primary granules and some cytoplasmic vesicles were labeled for CLC protein, but full and empty secondary granules and the extensive network of tubulovesicles were not. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals within phagosomes of macrophages were labeled by the immunogold procedure for CLC protein. These results demonstrate that rhIL-5-supplemented, PHA-stimulated, T-cell-conditioned media induced the development of mature human eosinophils from cord blood cells. These eosinophils underwent PMD of secondary granule contents. Immunogold analysis showed eosinophil CLC protein in the cytoplasm, nucleus, and primary granules of eosinophils. Macrophages also were present in these cultures. They contained CLC protein-containing crystals in their phagosomes, suggesting active sequestration of eosinophil CLC protein by macrophages in vitro.
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