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Precursors of macrophages in embryonic rat lungs fail to exhibit granulocyte-forming potential
S P Sorokin1, N A McNelly, R F Hoyt
1Department of Anatomy and Neurobiology, Boston University School of Medicine, Massachusetts 02118.
Background:
Mesenchyme-like macrophage (M) precursors called angular cells are present in rat lungs on the thirteenth day of gestation and by then can differentiate into outright macrophages. Based on studies of bone marrow-derived cells, it is widely believed that the macrophage line necessarily proceeds from a colony-forming unit with dual granulocyte-macrophage potential (CFU-GM). In embryos this seems doubtful since macrophages are already scattered throughout the body before the first granulocytes appear. We examined the question in organ cultured 14 day prenatal rat lungs after having shown earlier that the macrophage population developed in explants is increased by exposure to M- and GM-colony-stimulating factors (CSFs) but is unaffected by multi (IL-3)- or granulocyte (G)-CSF. Reportedly retinoic acid (RA) shifts CFU-GM strongly towards granulocytic differentiation and inhibits mitosis of unipotential macrophage precursors but not differentiated cells. Transforming growth factor beta 1 (TGF) inhibits multipotential blood progenitors but allows proliferation of committed precursors, and TGF together with GM-CSF induces granulocytopoiesis from CFU-GM.
Methods:
Lung pairs were grown on a serum-containing medium or one supplemented either by RA, TGF, or TGF/GM-CSF to form a control and three experimental groups. A fourth experiment compared responses to M-CSF exposure and M-CSF/TGF. Macrophage population growth was estimated by measuring the areas of coronas formed by macrophages emerged from the explants. F-actin was stained with fluorescein-labeled phalloidin.
Results:
In all experiments macrophages were produced unmixed with granulocytes. By +8 days they had largely emerged to form coronas about the lungs. In cultures exposed to RA, macrophages were less intensely stained for actin and slower to emerge than controls. At +8 days, however, coronal areas were not significantly different from controls, as was also true for the TGF group. In contrast, coronal areas of cultures grown with TGF/GM-CSF were much larger. At +17 days, mean coronal area of TGF cultures was about half that of controls (P < 0.05), whereas mean coronal area of the TGF/GM-CSF group was 5.4 times greater (P < 0.001). Macrophages from control and TGF-exposed cultures responded to M-CSF by an increase in coronal area which was greater among cultures given M-CSF alone than those given TGF + M-CSF (both P < 0.005).
Conclusions:
Macrophage precursors in embryonic lungs are distinct from CFU-GM.
Insights
Embryonic lung macrophage precursors are distinct from bone marrow-derived CFU-GM. Transforming growth factor beta 1 (TGF) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhanced macrophage growth in embryonic rat lungs.
Area of Science:
- Developmental biology
- Immunology
- Cell biology
Background:
- Mesenchyme-like macrophage precursors, termed angular cells, exist in embryonic rat lungs by day 13 of gestation.
- The conventional view posits that macrophage development originates from a dual granulocyte-macrophage progenitor (CFU-GM), but this is questioned due to macrophages appearing before granulocytes in embryos.
- Previous studies indicated that macrophage populations in organ-cultured embryonic lungs are stimulated by M-CSF and GM-CSF but not IL-3 or G-CSF.
Purpose of the Study:
- To investigate the origin and regulation of macrophage precursors in embryonic rat lungs.
- To determine if embryonic lung macrophages arise from CFU-GM or distinct precursors.
- To examine the effects of retinoic acid (RA) and transforming growth factor beta 1 (TGF) on macrophage development.
Main Methods:
- Organ culture of 14-day prenatal rat lungs.
- Exposure of lung explants to M-CSF, GM-CSF, RA, TGF, or combinations thereof.
- Quantification of macrophage population growth by measuring the area of emergent macrophage coronas.
- F-actin staining using fluorescein-labeled phalloidin.
Main Results:
- Macrophages emerged unmixed with granulocytes in all experimental conditions.
- RA exposure led to slower macrophage emergence and reduced actin staining.
- TGF alone did not significantly alter macrophage growth at 8 days but reduced it by 17 days.
- TGF/GM-CSF significantly increased macrophage coronas by 17 days (5.4-fold increase).
- Both control and TGF-exposed macrophages responded to M-CSF, with a greater response in M-CSF alone cultures.
Conclusions:
- Embryonic lung macrophage precursors are distinct from CFU-GM.
- TGF/GM-CSF promotes significant proliferation of embryonic lung macrophages.
- These findings challenge the universal applicability of the CFU-GM model for all macrophage origins.