Localisation of granulocyte macrophage colony-stimulating factor in human long-term bone marrow cultures Biological and immunocytochemical characterisation

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Abstract
The distribution of granulocyte macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures (HLTBMC) was examined using two monoclonal antibodies raised using purified recombinant GM-CSF and a third commercially available GM-CSF antibody. The antibodies were able to bind to purified recombinant GM-CSF and showed inhibition of GM-CFC colonies in the presence of both recombinant and native protein. All antibodies displayed similar pat-terns of distribution in both permeabilised and non-per-meabilised stromal cell preparations. Fibroblasts were labelled at their periphery in early cultures and both endothelial cells and fibroblasts showed cytoplasmic labelling with anti-GM-CSF. The fact that GM-CSF appears to be sequestered by cells of the bone marrow stroma raises the possibility that it is synthesised by these cells and may regulate activity of the progenitor cells in the haemopoietic foci. In contrast, early progenitor cells within the foci did not stain with any of the anti-GM-CSF antibodies. Adipocytes, which differenti-ate from fibroblasts in these cultures, showed a diffuse staining pattern. Two types of macrophage staining were observed in the non-permeabilised cells; those exhibiting only autofluorescence and those that bound the antibody. Intracellular staining was apparent in a small sub-population. Generally, the staining persisted up to eight weeks of culture and thereafter declined, becoming virtually undetectable after 12 weeks. This correlates with the pattern of GM-CFC production in long-term bone marrow cultures.