Macrophage development: I. Rationale for using Griffonia simplicifolia isolectin B4 as a marker for the line

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Abstract
The isolectin B4 of Griffonia simplicifolia (GSA I-B4) binds to cell membrane glycoconjugates bearing terminal alpha-D-galactose, which macrophages possess. We have investigated the merits of its use as a marker for cells of this lineage when examining the early origin of macrophage populations in rat embryos, the stages and time scale of transformation from precursor forms to active, matured cells, and the response of precursors and macrophages to colony-stimulating blood factors, the last two studies conducted in organ cultures of prenatal lungs. In the present instance, GSA I-B4 was used either coupled with fluorescein (FITC) for light microscopy of living and fixed cells, or with peroxidase for light or electron microscopy. Control incubations of lung culture-derived macrophages proved that staining resulted from specific binding to galactosyl units on the cell membrane, since it was competitively inhibited by alpha-D-galactose. The lectin binds to few cells in 14-day prenatal lung explants but to a great many macrophages that subsequently develop in the cultures, indicating that it can be relied on for quantitative studies on population growth; however, it is important to provide reagents with good access to the cells. Apart from macrophages and their precursors, virtually no cells in prenatal lung cultures bind this lectin. Granulocytes of adult blood are GSA positive, but they are not yet present in 14-day prenatal explants and do not develop subsequent to culturing; hence they are not a source of confusion for experimental studies using this system. Precursors of granulocytes begin to appear in rat embryos around day 13 and have GSA-positive cell membranes, but like definitive granulocytes they also have conspicuous peroxidase-positive lysosomal granules which serve to distinguish them from early macrophages, particularly when cells are studied at an ultrastructural level. With these objections cleared away, GSA I-B4 emerges as a valuable means to mark cells of the macrophage line, mature or immature.