EARLY DEVELOPMENT OF MACROPHAGES IN INTACT AND ORGAN-CULTURED HEARTS OF RAT EMBRYOS

Background: Macrophage precursors are present early in embryonic life, being demonstrable in placental and embryonic connective tissues of rats at the neurula stage and as potential macrophages in the brain, liver, and lungs near the onset of organogenesis. We examined the development of macrophages in the heart and the possibility that they initially appear at sites of programmed cell death (apoptosis). Methods: Precursors were recognized by the binding of peroxidase-coupled Griffonia simplicifolia isolectin B-4 (GSA) on the cell membrane. Their capacity for conversion into macrophages was assayed in organ cultures; confirmation of the progeny as bona fide macrophages was obtained from their responses to particle exposure and macrophage colony-stimulating factor (M-CSF). Results: GSA + cells were first seen on gestational day 12 (4 mm embryos) as 2-3 cycling, nonvacuolated cells located in cardiac tissue outside the blood vessels. This population increased to similar to 12 cells by day 14 (9 mm embryos). Two-thirds were distributed along the bulbus cordis in the jellylike endocardium and a more densely cellular connective tissue closer to the aortic arches where apoptotic sites are expected to develop. Such sites were not found in serial glycol methacrylate sections through our 14-day specimens, although in whole heart explants of this age an area of necrosis developed along the prospective line of bulbar endocardial fusion on the second day of organ culturing, and by then macrophages were fairly abundant. Organ culturing of 13-day embryonic hearts also yielded large, highly vacuolated, GSA+ mononuclear phagocytes. After a few days in culture most of the macrophages migrated onto the medium where they formed a tight corona of cells about the explants. They readily ingested iron oxide particles and concentrated supravitally administered neutral red in their vacuoles. Macrophages from 14-day cultures exposed to M-CSF developed significantly larger coronas than macrophages from explants grown in serum-rich control medium (p < 0.001). In the presence of cytokines, moreover, these cardiac macrophages survived as many as 100 (92 ''postnatal'') days. Conclusions: Macrophage precursors first appear in embryonic rat hearts well before they are needed to clear debris generated by programmed cell death and are capable of rapid conversion into outright phagocytic cells as early as the 13th prenatal day. (C) 1994 Wiley-Liss, Inc.