The production of colony stimulating activity by monocyte enriched fractions from murine continuous bone marrow culture adherent layers

The production of colony stimulating activity (GM-CSA) within murine continuous bone marrow cultures was investigated by subjecting either the nonadherent or the adherent layer cells to separation by velocity sedimentation. The presence of GM-CSA in conditioned medium was defined by the support of granulocyte/macrophage colony formation in soft agar culture. Cell free conditioned medium from weekly feedings of intact continuous marrow cultures and medium conditioned by each fraction of velocity sedimentation separated, nonadherent cells did not contain assayable GM-CSA. However, medium conditioned by fractions of adherent layer cells with a modal sedimentation velocity of 8.8 mm/h (range 6.2-10.6 mm/h) contained GM-CSA. Cytochemical studies with Wright's-Giemsa and non-specific esterase stains in addition to immunofluorescent studies with anti-collagen III, anti-collagen IV and monoclonal anti-Mac I antibodies to define fibroblasts, endothelial cells and monocytes, respectively, demonstrated that the cells within the GM-CSA producing fractions were enriched with monocytes/macrophages. Fibroblasts and a small proportion of endothelial cells were also present. GM-CSA is produced within the microenvironment (adherent layer) of murine continuous marrow cultures. Either adherent layer monocytes and/or a monocyte-endothelial cell interaction account for the GM-CSA production.