Factors influencing fetal macrophage development: III. Immunocytochemical localization of cytokines and time-resolved expression of differentiation markers in organ-cultured rat lungs.
Exogenous TNF alpha, IL-1 beta, M-CSF, and GM-CSF all stimulate growth of macrophages arising in explanted fetal rat lungs. The present study examines the intrinsic availability of these factors in intact and organ-cultured lungs and utilizes expression of cytokines and marker proteins to explore the differentiation pathway followed by phagocytes in vitro.



Factors and markers were localized immunocytochemically in paraffin sections of 14- and 15-day fetal rat lungs and lungs organ-cultured up to 7 days on serum-containing medium solidified with agar. Western analyses for the cytokines were performed on lysates of whole 15-day lungs, and in situ hybridization of M-CSF receptor mRNA was carried out in sections of 14 + 2 day cultured lung.



IL-1 beta, M-CSF, and GM-CSF were demonstrated in the stroma of intact and cultured lungs by immunostaining, results confirmed by Western blotting. TNF alpha appeared to be absent. A few precursors (angular cells) expressed the macrophage lineage marker RM-1 as early as day 14, and immunostaining became stronger and more widespread as the population matured and expanded in cultures. The OX-6 antibody to Ia antigen first reacted with macrophages in 14 + 1 day explants, and within a week 50% of cells were positive. M-CSF and mRNA for its receptor were present at 14 + 2 days, as was PDGF, which had been demonstrated in the stroma and epithelium prior to explantation. Definite reactivity for IL-1 beta and GM-CSF followed at 14 + 4 and 14 + 5 days.



M-CSF, GM-CSF, and IL-1 beta, but not TNF alpha, are available to replicating angular cells before and during their conversion to phagocytes. Fetal lungs thus qualify as a hematopoietic tissue supportive of macrophages. The path of differentiation pursued in organ cultures involves early expression of structural elements (RM-1, Ia antigen) followed by synthesis of cytokines of the TNF alpha cascade. Immunostaining for both RM-1 and OX-6 suggests that fetal lung macrophages share a common heritage with antigen-presenting pulmonary dendritic cells.
Sorokin
S P
SP
Department of Anatomy and Neurobiology, Boston University School of Medicine, Massachusetts 02118-2394, USA.
Hoyt
R F
RF
Reenstra
W R
WR
McNelly
N A
NA
D000818:Animals; D002454:Cell Differentiation; D016207:Cytokines / Q000378:metabolism*; D005865:Gestational Age; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007375:Interleukin-1 / Q000378:metabolism; D008168:Lung / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000166:cytology / Q000378:metabolism*; D009924:Organ Culture Techniques; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D007375:Interleukin-1; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016173:Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor