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  • Title: Exogenous cytokines enhance survival of macrophages from organ cultured embryonic rat tissues.
    Author: Sorokin SP, McNelly NA, Hoyt RF.
    Journal: Anat Rec; 1994 Nov; 240(3):398-406. PubMed ID: 7825736.
    Abstract:
    BACKGROUND: Macrophage precursors are present in embryonic rats shortly after the onset of hematopoiesis. During organogenesis they soon establish residency in many parts of the body and become convertible into phagocytes, at first gaining morphological characteristics of macrophages and later a range of surface antigens used to characterize subpopulations in adults. Nonetheless, it is uncertain whether representatives of this fetal lineage continue to exist past birth. We investigated the question indirectly by seeing if such cells can be made to survive in vitro to an age equivalent to adulthood and by examining underlying conditions that favor this outcome. METHODS: Fourteen-day embryonic lungs, hearts, and limb buds were organ cultured on a firm serum-containing medium. Fetal macrophages developed within all explants and then migrated out to form a corona of cells surrounding each explant. The lung cultures were selected for subsequent work which mainly used coronal area as the measure of macrophage population size in experimental and control groups. Baseline growth and survival of macrophages were established for cultures grown on standard medium, then effects of the following were examined: indomethacin (10(-6) M) as it influences initial production of macrophages from precursors and later survival of differentiated cells; and macrophage colony-stimulating factor (M-CSF), used alone at moderate dosage (50-100 U), and combined with granulocyte-macrophage CSF (both 200 U), for its importance to long-term survival of the population. Mitogenic influence of M-CSF on differentiated macrophages was demonstrated by uptake of 5-bromo-2'-deoxyuridine. RESULTS: Indomethacin inhibited the formation of macrophages from precursors but enhanced the survival of differentiated cells. M-CSF increased BrdU uptake of differentiated macrophages and permitted coronal growth to continue long past the approximately 30 day limit of controls. Beyond this interval, M-CSF was essential for macrophage survival, since coronas quickly shrank after the cytokine was withdrawn. Administration of the M-CSF/GM-CSF mixture to the 2 oldest M-CSF-exposed cultures between 98 and 127 days in vitro resulted in an increase in the number of coronal macrophages (P < 0.001); withdrawal between 129 and 140 days led to a decrease (P < 0.005). Ultimately a few cells were still surviving at 183 days. CONCLUSIONS: Intrinsic factors promote early formation of macrophages within the explants, but the availability of factors is lessened by the anti-inflammatory action of indomethacin. Its later promotion of macrophage survival may be based on suppression of autogenous prostaglandin (PGE2) synthesis. M-CSF greatly promotes macrophage survival; in context this is sufficient to show that the fetal macrophage line has a clear potential to survive well into adulthood.
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