Maturation of granulocytes (i.e. neutrophils) from stem cells is believed to be directed by specific factors which control proliferation and differentiation. In the murine system, Interleukin-3(IL-3), granulocyte-macrophage CSF(GM-CSF) and granulocyte-CSF(G-CSF) have been shown to produce granulocytes from progenitor cells in vitro. The long term goals of this proposal are to develop a system to culture large numbers of granulocytes from bone marrow progenitors, to examine their functional and cell surface phenotypes using multiparameter flow cytometry, and to explicitly define the maturational sequence for granulocytes based on these phenotypes. The functional capacities include the production of H2O2, formation of the NADPH pool and its utilization, membrane depolarization, phagocytosis, and degranulation. The physical properties include Coulter volume, light scatter (low angle and 90 degrees) and axial light loss, the ligands include fluorescent chemotactic factors, aggregated Ig to identify Fc receptors and a panel of monoclonal antibodies. The first goal is to develop an in vitro culture system to produce large numbers of mature granulocytes in a liquid culture system where these cells are readily accessible for functional analysis. Multiparameter flow cytometry will be used to determine whether cells produced in this system are functionally and phenotypically comparable to bone marrow and mature peripheral blood granulocytes. The second goal is to determine if the growth and differentiation of these cells can be modulated by agents which have been shown to influence the differentiation and cell lineage of myeloid tumor cells. The third goal is to determine whether GM-CSF or IL-3 will modulate the oxidative functional capacity of mature granulocytes and if these factors will act additively or synergistically with chemotactic factors or endotoxin to modulate the activation of granulocytes. The fourth goal is to use multiparameter flow cytometry to evaluate the maturation of progenitor cells and define the developmental sequence for expression of functional activity, receptors for chemotactic factors, opsonins, and surface phenotypes as determined with monoclonal antibodies. The fifth goal is to determine the relationship between proliferative capacity and development of functional activity in maturing bone marrow granulocytes.
