Earliest origins of macrophage populations in the central nervous system, the liver, and the lungs were studied in rat embryos aged between 10.5-11 days and 14 days of gestation, based on light and electron microscopic identification of macrophages using peroxidase-coupled isolectin B4 of Griffonia simplicifolia (GSA I-B4), which recognizes alpha-D-galactose groups on the cell membrane. During embryonic life macrophages and their precursors are GSA I-B4-positive and generally bereft of peroxidase-positive granules. At 10.5 days the yolk sac and embryonic circulations have just become joined, the brain has five vesicles but nerve cells are little differentiated, the liver exists as a diverticulum of the gut with fingerlike extensions of hepatocytes, and the lungs as a laryngotracheal groove. Macrophages and/or their precursors occurred in small numbers in embryonic mesenchyme and blood vessels but showed no special affinity for either liver or lung rudiments. The developing brain was the first organ to be colonized, beginning on prenatal day 12. The liver followed between days 12 and 13 and was succeeded by the lungs, beginning between days 13 and 14. Dividing macrophages were present in these organs at the outset of colonization and throughout the duration of the embryo series, indicating that from the beginning, replication of resident cells contributes to growth of the local population. Granulocyte precursors were first apparent in the liver around day 13; they are also GSA-positive but are distinguished from macrophages by their content of peroxidase-positive granules. Organ cultures of 13-day liver and lungs, and 14-day brain tissue, indicate that whereas isolated liver fragments support the formation of both granulocytes and macrophages, only the latter develop in brain or lung cultures. A resident population of macrophages evidently is set up very early in these organs, well before white cells colonize the spleen, bone marrow, and other future blood forming regions. The events outlined are seen as stages in an embryo-wide process that leads to establishment of macrophage populations in various organs.
The fate of macrophage precursors residing in 14-day prenatal rat lungs was followed in organ cultures to obtain a detailed, ultrastructurally resolved picture of the sequence and timing of events accompanying their transformation into typical pulmonary macrophages. Cultures were examined at close intervals during the first day (1, 2, 3, 4, 6, 9, 12, 15, 18, and 24 hr) and at wider intervals thereafter (2, 4, 5, 7, 9, and 13 days) to yield a developmental series of cells identified as in the macrophage line based on binding of peroxidase-coupled isolectin B4 of Griffonia simplicifolia (GSA I-B4) to cell membranes and on negligible content of peroxidase-positive granules in the cytoplasm. Organ culturing stimulated virtually all precursors to develop into macrophages. GSA-positive cells in explants occurred outside vessels in pulmonary connective tissue, and at the outset none were typical macrophages: 71% were angular cells, resembling unlabeled mesenchymal cells around them, 16% were undifferentiated leukocytes, and the remainder were irregularly shaped cells with few vacuoles intermediate between the preceding and the macrophages. During the first 12 hr in culture the proportion of angular cells and leukocytes fell to zero, and that of intermediate cells first rose, then receded. In the same interval the proportion of macrophages rose to 87.5%, and by 24 hr all GSA-positive cells were typical macrophages generally engorged with phagocytosed material; about 8 hr appear necessary for converting half the population. Notable ultrastructural changes during this period of transformation involved the centrioles and cytoskeleton, reflecting enhanced cell mobility and phagocytosis. A period of maturation followed, marked by disappearance of cellular debris from phagosomes and an increased prevalence of cells with elaborate lamellipodia. This accords with earlier work showing that macrophage Fc receptor density increases sharply during the first 24 hr, but elevated levels of histochemically demonstrable acid phosphatase appear only later. Mitotic activity was conspicuous in GSA-positive cells throughout both periods. 3H-thymidine labeling indices for precursors and macrophages, determined at six intervals between 1 hr and 24 hr, remained steady at approximately 34%, whereas indices of other categories of lung cells (GSA-negative stromal cells, pleural cells, and airway epithelium) began at this level but rapidly declined, indicating that the GSA-positive cells constitute a single population distinct from others in the lungs. Macrophages found outside the lung cultures after 4-5 days qualify as a mature population, but having migrated away from direct contact with the lung stroma, they survive only a week or two and no longer divide.
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