Studies of the function and structure of in vitro propagated human granulocytes

In our study, hematopoietic progenitor cells isolated from human umbilical cord blood were grown in an in vitro liquid culture system using the recombinant colony stimulating factors IL-3 plus granulocyte-macrophage colony-stimulatory factor (GM-CSF) or IL-3 plus granulocyte colony stimulating factor (G-CSF). The morphology and function of the cells produced were then studied, and it was demonstrated that continuous exposure to IL-3 plus GM-CSF produced predominantly eosinophilic granulocytes, whereas IL-3 plus G-CSF produced neutrophilic granulocytes. Cells from IL-3/GM-CSF cultures showed progressively increasing oxygen metabolism and locomotive capabilities over time, which became equivalent to peripheral blood neutrophils at wk 4 and 3, respectively. Phagocytic activity of these cells was poor. IL-3/G-CSF cultures produced cells with progressive increases in oxygen metabolism, locomotion, and phagocytosis. These functions never became equivalent to those of peripheral blood neutrophils. Flow cytometric analysis of IL-3/G grown cells showed that they expressed CD11b on their surfaces and that surface expression increased 2-fold after secondary granule secretagogue exposure. Ultrastructurally, the eosinophilic granulocyte nature of the IL-3/GM grown cells was confirmed by immunogold-lectin staining and IL-3/G grown cells were shown to contain antigenic myeloperoxidase. The data demonstrate that human umbilical cord blood mononuclear cells can be used to propagate granulocytes in vitro, that the types of granulocytes produced in this culture system depend on the growth factors used, that the cells produced in vitro develop several of the functional characteristics of peripheral blood granulocytes, and that ultrastructural details of developing human granulocytes can be carefully examined in this model system.