LOCALIZATION OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR IN HUMAN LONG-TERM BONE-MARROW CULTURES - BIOLOGICAL AND IMMUNOCYTOCHEMICAL CHARACTERIZATION

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-C SF 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 pro tein. All antibodies displayed similar patterns of distribution in bot h permeabilised and non-permeabilised stromal cell preparations. Fibro blasts were labelled at their periphery in early cultures and both end othelial 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 t hese cells and may regulate activity of the progenitor cells in the ha emopoietic foci. In contrast, early progenitor cells within the foci d id not stain with any of the anti-GM-CSF antibodies. Adipocytes, which differentiate from fibroblasts in these cultures, showed a diffuse st aining pattern. Two types of macrophage staining were observed in the non-permeabilised cells; those exhibiting only autofluorescence and th ose that bound the antibody. Intracellular staining was apparent in a small sub-population. Generally, the staining persisted up to eight we eks of culture and thereafter declined, becoming virtually undetectabl e after 12 weeks. This correlates with the pattern of GM-CFC productio n in long-term bone marrow cultures.