Mononuclear phagocytes including monocytes play a major role in inflammation, infection and various immunologic reactions. Bone marrow is considered the main source of circulating monocytes. In this study, we plan to isolate and characterize different classes of monocyte precursors in bone marrow. This will allow us to study the factors that control and regulate the supply of monocytes and macrophages under normal steady state and pathological conditions. Three classes of cells belonging to monocyte lineage have been morphologically identified. They are bone marrow monocyte, promonocyte and monoblasts. However, cells less differentiated than monoblasts are not well characterized. Recent studies have shown that about 4-6% of nucleated bone marrow cells bind to a class of colony-stimulating factor specific for mouse mononuclear phagocytes (CSF-1). We will produce a monoclonal antibody against CSF-1. A rat is hyperimmunized with purified CSF-1. The spleen cells are then fused with mouse myeloma cells. Monoclonal antibody specific for CSF-1 is selected and used for subsequent studies. Monocyte precursors in bone marrow are first isolated by indirect immunofluorescent technique using anti-CSF-1 antibody as the first antibody and fluorescent-activated cell sorter. These CSF-1-positive cells are then analyzed according to their size, morphology, enzymatic activity the presence of receptors (FcR, CR and CR2) and ability to replicate in tissue culture. We will also develop monoclonal antibodies reactive against blood monocytes and other mononuclear phagocytes. These antibodies will be used to isolate monocyte precursors and study their surface properties.
