Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation.

The mechanism of piecemeal degranulation by human eosinophils was investigated. Mature eosinophils that developed in rhIL-5-containing conditioned media from cultured human cord blood mononuclear cells were prepared for ultrastructural studies using a combined technique to image eosinophil peroxidase by cytochemistry in the same sections on which postembedding immunogold was used to demonstrate Charcot-Leyden crystal protein. Vesicular transport of eosinophil peroxidase from the specific granule matrix compartment to the cell surface was associated with piecemeal degranulation. This process involved budding of eosinophil peroxidase-loaded vesicles and tubules from specific granules. Some eosinophil peroxidase that was released from eosinophils remained bound to the cell surface; some was free among the cultured cells. Macrophages and basophils bound the released eosinophil peroxidase to their plasma membranes, internalized it in endocytotic vesicles, and stored it in their respective phagolysosomes and secretory granules. Charcot-Leyden crystal protein was diffusely present in the nucleus and cytoplasm of IL-5-stimulated mature eosinophils. Extensive amounts were generally present in granule-poor and subplasma membrane areas of the cytoplasm in contrast to eosinophil peroxidase, which was secreted and bound to the external surface of eosinophil plasma membranes. These studies establish vesicular transport as a mechanism for emptying the specific eosinophil granule matrix compartment during IL-5-associated piecemeal degranulation.     

Ultrastructure of eosinophils and basophils stimulated to develop in human cord blood mononuclear cell cultures containing recombinant human interleukin-5 or interleukin-3

We used recombinant human interleukins (IL) and cultured human cord mononuclear cells to determine the cell lineages supported by the inclusion of the individual interleukins-5, -3, or -4 in the cultured media. Cultures were examined by electron microscopy at 2-, 3-, and 5-week culture intervals. We found that IL-5 primarily supported the eosinophilic lineage with lesser numbers of basophils, that IL-3 initially supported all granulocyte lineages but eventually supported the eosinophilic lineage with lesser numbers of basophils, and that IL-4 did not support the development of granulocyte lineages in these cultures. At early culture intervals in either IL-5 or IL-3, eosinophilic and basophilic myelocytes were seen in variable proportions. Mature cells of these lineages developed later. Both mature eosinophils and basophils showed morphologic evidence of activation similar to those described for tissue eosinophils and basophils in various disorders. Mast cells were absent in all cultures. Recognition of the variable morphologies associated with maturation and activation of human eosinophils and basophils is important for the correct identification of cell lineages that develop in cultures containing human recombinant interleukins.     

The effect of human placental protein 14 (PP14) on the production of interleukin-1 from mitogenically stimulated mononuclear cell cultures.

Twenty-four hours after skin painting mice on the flank with the contact sensitizer fluorescein isothiocyanate (FITC), the number of dendritic cells (DC) increased sharply, not only in draining but also in contralateral (CLN) and distant lymph nodes. High levels of antigen were detected on up to 50% of DC isolated from draining lymph nodes (DLN), and these cells were potent stimulators of naive T cells in vitro. Less than 3% of DC from contralateral and distant lymph nodes carried detectable antigen and did not induce significant T-cell proliferation. A significant number of DC had migrated to draining, contralateral and distant lymph nodes without acquiring detectable antigen. This indicates that there is a systemic signal causing the movement of DC to lymph nodes. This appears to be independent of mature T cells, as the systemic migration of DC also occurred in nude mice.     

Non-major histocompatibility complex-restricted killer cells in human cord blood: generation and cytotoxic activity in recombinant interleukin-2-supplemented cultures.

Interleukin-2 (IL-2)-induced generation of non-major histocompatibility complex (MHC)-restricted killer cells among human cord blood lymphocytes (CBL) was investigated. After 1 week in culture with recombinant (r)IL-2 and human serum (HuSer), the cytotoxicity of CBL against K562 and COLO cells greatly exceeded the cytotoxicity of cultured adult peripheral blood lymphocytes. Culturing of CBL with rIL-2 and HuSer led to preferable generation of CD56+ cells. After 1 month in culture, the number and frequency of CD56+ cells had increased by more than 50 and nine times, respectively. The generation of CD56+ cells in CBL cultures may at least partially be explained by their comparatively strong expression of the IL-2 receptor (IL-2R) beta-chain (p75).     

Ultrastructural identification of human mast cells resembling skin mast cells stimulated to develop in long-term human cord blood mononuclear cells cultured with 3T3 murine skin fibroblasts.

Human mast cells developed in vitro when cord blood mononuclear cells were cocultured for 3 months with 3T3 embryonic mouse skin fibroblasts. The metachromatic cells that arose in these cultures contained histamine, a functional Fc epsilon receptor and granule proteases (tryptase, chymase), and they were definitively identified by the ultrastructural demonstration of crystal granules. We present a detailed ultrastructural analysis of this newly available system for the reliable development of human mast cells in vitro and provide criteria for definitive identification of the mast cell and basophil lineages in humans.